Probiotic in Bakery: Ana Paula Zapelini de Melo Thais de Oliveira

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Methods and Protocols
in Food Science
Adriano Gomes da Cruz · Marcia Cristina Silva

Tatiana Colombo Pimentel
Erick Almeida Esmerino · Silvani Verruck
Editors
Probiotic Foods
and Beverages
Technologies and Protocols
METHODS AND PROTOCOLS IN FOOD SCIENCE
Series Editor
Anderson S. Sant’Ana
University of Campinas
Campinas, Brazil
For further volumes:

http://www.springer.com/series/16556
Methods and Protocols in Food Science series is devoted to the publication of research
protocols and methodologies in all fields of food science.
Volumes and chapters will be organized by field and presented in such way that the
readers will be able to reproduce the experiments in a step-by-step style. Each protocol will
be characterized by a brief introductory section, followed by a short aims section, in which
the precise purpose of the protocol will be clarified.
Probiotic Foods and Beverages
Technologies and Protocols
Edited by
Adriano Gomes da Cruz

Department of Food, Federal Institute of Science, Rio de Janeiro, Brazil
Marcia Cristina Silva

Department of Food, Federal Institute of Science, Rio de Janeiro, Brazil
Tatiana Colombo Pimentel

Federal University of Paraná, Paranavaí, Brazil
Erick Almeida Esmerino

Department of Food Technology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Silvani Verruck
Department of Food Science and Technology, Federal University of Santa Catarina, Florianópolis, Brazil
Editors
Adriano Gomes da Cruz Marcia Cristina Silva
Department of Food Department of Food
Federal Institute of Science Federal Institute of Science
Rio de Janeiro, Brazil Rio de Janeiro, Brazil
Tatiana Colombo Pimentel Erick Almeida Esmerino

Federal University of Paraná Department of Food Technology
Paranavaı́, Brazil Federal University of Rio de Janeiro
Rio de Janeiro, Brazil
Silvani Verruck
Department of Food Science
and Technology
Federal University of Santa Catarina
Florianópolis, Brazil
ISSN 2662-950X ISSN 2662-9518 (electronic)

Methods and Protocols in Food Science
ISBN 978-1-0716-3186-7 ISBN 978-1-0716-3187-4 (eBook)
https://doi.org/10.1007/978-1-0716-3187-4
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part
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Preface to the Series
Methods and Protocols in Food Science series is devoted to the publication of research
protocols and methodologies in all fields of food science. The series is unique as it includes
protocols developed, validated and used by food and related scientists, as well as theoretical
basis are provided for each protocol. Aspects related to improvements in the protocols,
adaptations and further developments in the protocols may also be approached.
Methods and Protocols in Food Science series aims to bring the most recent developments
in research protocols in the field as well as very well established methods. As such the series
targets undergraduate, graduate and researchers in the field of food science and correlated
areas. The protocols documented in the series will be highly useful for scientific inquiries in
the field of food sciences, presented in such way that the readers will be able to reproduce the
experiments in a step-by-step style.
Each protocol will be characterized by a brief introductory section, followed by a short
aims section, in which the precise purpose of the protocol is clarified. Then, an in-depth list
of materials and reagents required for employing the protocol is presented, followed by a
comprehensive and step-by-step procedures on how to perform that experiment. The next
section brings the do’s and don’ts when carrying out the protocol, followed by the main
pitfalls faced and how to troubleshoot them. Finally, template results will be presented and
their meaning/conclusions addressed.
The Methods and Protocols in Food Science series will fill an important gap, addressing a
common complain of food scientists, regarding the difficulties in repeating experiments
detailed in scientific papers. With this, the series has a potential to become a reference
material in food science laboratories of research centers and universities throughout the
world.
Campinas, Brazil Anderson S. Sant’Ana
v
Preface
The Food and Agriculture Organization of the United Nations (FAO) and the World
Health Organization (WHO) have defined probiotics as live microorganisms that, when
administered in adequate amounts, confer a health benefit on the host. The global probio-
tics market was valued at USD 58.17 billion in 2021 and is expected to expand at a
compound annual growth rate (CAGR) of 7.5% from 2021 to 2030. The health promotion
provided by these microorganisms has been the main driving force of this market niche.
Also, an emerging functional food discipline in this field is using postbiotics and parapro-
biotics in food and beverages. Paraprobiotics and postbiotics can express health benefits in
addition to the inherent viability of probiotics, proving that not all mechanisms, nor clinical
effects, are directly related to viable bacteria and broadening the current concept of what
probiotics are. Furthermore, paraprobiotics and postbiotics have valuable potential for
developing biotechnological products with functional ingredients and are more stable,
allowing for easier use on an industrial scale.
Protocols in Technology of Probiotic Foods and Beverages is a book that addresses the latest
relevant state-of-the-art protocols to manufacture functional probiotic foods and beverages.
In addition, this book combines, as comprehensibly as possible, well-established protocols
and procedures used by many laboratories in academia and industry.
Regarding dairy products, Chap. 1 provides information about the material, main
processing procedure, and packaging steps for processing fermented milks. At the same
time, Chap. 2 discusses probiotic strains used to manufacture different cheese types and the
survival of those probiotics, regarding actions taken to increase their viability. The limita-
tions from research to industrial limitations and the main factors to consider for appropriate
probiotic strain selection for industrial application are pointed out. Chapter 3 is a practical
guidance for probiotic ice cream manufacture, presenting the steps and amount of probiotic
addition into ice cream production. Finally, Chap. 4 is a practical guidance for probiotic
butter manufacture, discussing ways of adding probiotics.
Regarding non-dairy products, Chap. 5 deals with plant-based beverages, demonstrat-
ing the process of obtaining soy, oat, and rice extracts and the fermentation process to obtain
probiotic beverages. At the same time, Chap. 6 describes the process of obtaining probiotic
plant-based cheeses, such as pea cheese, tofu, soy-based cream cheese, and chickpea petit
Suisse cheese. Chapter 7 describes a method incorporating probiotic bacteria encapsulated
in an alginate matrix using an emulsification process as a pretreatment into fruit juices.
Furthermore, techniques for morphological analysis by scanning electron microscopy, as
well as the characterization of the juice and the evaluation of cell viability against simulated
gastric conditions, are provided. Chapter 8 describes the process of obtaining probiotic-
fermented vegetables, such as pickles, sauerkraut, and natto. Chapter 9 describes two
preparation methods of Kombucha using a symbiotic culture of bacteria and yeast or a
synthetic microbial community as a starter. Moreover, the determination of bioactive
compounds, including organic acids, sugars, and catechins, has been introduced.
Chapter 10 provides a guideline on preparing a probiotic beer that can be used for
vii
viii Preface
researching new probiotic microorganisms and highlights essential points to be considered

when developing probiotic beers. Chapter 11 describes a protocol for probiotic Friolano-
type sausage. Furthermore, the possible sources of defects in producing probiotic salami and
the best alternatives to overcome them are presented. Chapter 12 proposes the design of two
independent protocols for the delivery of probiotics through bakery products: (I) a probi-
otic bread by adding microorganisms directly to the dough and (II) an edible probiotic film
based on sodium caseinate and chia mucilage for application in bread surface. Furthermore,
for both protocols, the function of each reagent/ingredient and the chemical reactions
involved are described in detail, indicating the possible issues, sources, and the best alter-
natives to overcome them. Finally, Chap. 13 has up-to-date and detailed information on the
production of different probiotics and synbiotic chocolate.
Regarding proposals for increasing probiotic survival in food products, Chap. 14
describes in detail the main methods of encapsulation of probiotics, including emulsion,
extrusion, and spray-drying techniques.
However, in recent years, researchers have observed that viability may not be necessary
for some health effects, and products with inactivated microorganisms have been developed.
In this way, Chaps. 15 and 16 provide detailed protocols for obtaining potential parapro-
biotics and postbiotics for use in food and beverages.
Finally, following new health effects associated with probiotic cultures, Chap. 17
describes protocols for elaborating on a food product with psychobiotic potential in detail.
In addition, the most used behavioral tests for preclinical trials that can be applied to confirm
the psychobiotic effect are also discussed.
Rio de Janeiro, Brazil Adriano Gomes da Cruz

Rio de Janeiro, Brazil Marcia Cristina Silva
Paranavaı́, Brazil Tatiana Colombo Pimentel
Rio de Janeiro, Brazil Erick Almeida Esmerino
Florianopolis, Brazil Silvani Verruck
Contents
Preface to the Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1 Probiotic Fermented Milk. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Shibo Ma, J. K. Vidanarachchi, and Chaminda Senaka Ranadheera
2 Probiotic Cheeses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Celso Fasura Balthazar, Julien Chamberland,
and Marie-Claude Gentès
3 Probiotic Ice Creams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Vanessa Cortina Zanetti, Celso Fasura Balthazar,
Callebe Camelo-Silva, and Silvani Verruck
4 Probiotic Butter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Patrı́cia Blumer Zacarchenco, Leila Maria Spadoti,
Adriana Torres Silva e Alves, Vanessa Cortina Zanetti,
and Silvani Verruck
5 Probiotic Plant-Based Beverages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Tahis R. Baú, Danielle C. B. H. Ferreira, Cintia L. Handa,
Fernando S. de Lima, and Tatiana Colombo Pimentel
6 Probiotic Plant-Based Cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
7 Probiotic Fruit Juices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Oscar O. Romero-Chapol, Abigail Varela-Pérez,
Ana G. Castillo-Olmos, Hugo S. Garcı́a,
and Cynthia Cano-Sarmiento
8 Probiotic Fermented Vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Hadi Pourjafar, Tatiana Colombo Pimentel, and Tahis R. Baú
9 Kombucha Production and Its Bioactive Compounds Analysis. . . . . . . . . . . . . . . . 133
Chun Zou, Yong-Quan Xu, Yi-Bin Huang, and Jun-Feng Yin
10 Probiotic Beer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Fernanda Meybom, Bárbara Mortl, and Alan Ambrosi
11 Probiotic Fermented Sausage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Claudio M. E. Malaghini, Ana Paula Zapelini de Melo,
and Silvani Verruck
12 Probiotic in Bakery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Ana Paula Zapelini de Melo, Thais de Oliveira,
Pedro Luiz Manique Barreto, and Silvani Verruck
13 Probiotic and Synbiotic Chocolate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Milena Dutra Pierezan, Callebe Camelo-Silva, Alan Ambrosi,
Marco Di Luccio, and Silvani Verruck
ix
x Contents
14 Microencapsulation of Probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

Callebe Camelo-Silva, Lais Leite Figueredo, Vanessa Cortina Zanetti,
Alan Ambrosi, Marco Di Luccio, and Silvani Verruck
15 Paraprobiotics Preparation for Use in Food and Beverages . . . . . . . . . . . . . . . . . . . 213
Cássia Pereira Barros, Cynthia Manassi, Silvani Verruck,
Marcia Cristina Silva, Erick A. Esmerino, Monica Q. Freitas,
and Adriano Gomes da Cruz
16 Postbiotics Preparation for Use in Food and Beverages . . . . . . . . . . . . . . . . . . . . . . 223
Jonas de Toledo Guimarães, Cássia Barros, Houshmand Sharafi,
Mehran Moradi, Erick A. Esmerino, and Adriano Gomes da Cruz
17 Psychobiotic Carried by Food and Beverage. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Cássia Pereira Barros, Erick A. Esmerino, Roberto Laureano Melo,
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Contributors
ALAN AMBROSI • Graduate Program in Food Engineering, Federal University of Santa

Catarina, Florianopolis, SC, Brazil; Graduate Program in Food Science, Federal
University of Santa Catarina, Florianopolis, SC, Brazil
CELSO FASURA BALTHAZAR • Department of Food Science and Nutrition, Faculty of Food
Engineering, University of Campinas, Campinas, SP, Brazil
PEDRO LUIZ MANIQUE BARRETO • Graduate Program in Food Science, Federal University of
Santa Catarina, Florianopolis, SC, Brazil
CÁSSIA PEREIRA BARROS • Department of Food Technology, Faculty of Veterinary Medicine,
Federal Fluminense University (UFF), Niteroi, Rio de Janeiro, Brazil
TAHIS R. BAÚ • Federal Institute of Santa Catarina, Campus São Miguel do Oeste, São
Miguel do Oeste, Santa Catarina, Brazil
CALLEBE CAMELO-SILVA • Graduate Program in Food Science, Federal University of Santa
Catarina, Florianopolis, SC, Brazil; Graduate Program in Food Engineering, Federal
CYNTHIA CANO-SARMIENTO • CONACYT—Tecnologico Nacional de México/Instituto
Tecnologico de Veracruz, Unidad de Investigacion y Desarrollo en Alimentos, Veracruz,
Mexico
ANA G. CASTILLO-OLMOS • CONACYT—Tecnologico Nacional de México/Instituto
Mexico
JULIEN CHAMBERLAND • STELA Dairy Research Center, Institute of Nutrition and
Functional Foods (INAF), Department of Food Science and Nutrition, Faculty of
Agricultural Science and Food, University Laval, Québec City, QC, Canada
ADRIANO GOMES DA CRUZ • Department of Food Science and Technology, Agrarian Science
Center, Federal University of Santa Catarina (UFSC), Florianopolis, SC, Brazil;
Department of Food, Federal Institute of Education, Science and Technology of Rio de
Janeiro (IFRJ), Rio de Janeiro, Brazil
FERNANDO S. DE LIMA • Federal Institute of Paraná, Campus Paranavaı́, Paraná, Brazil
ANA PAULA ZAPELINI DE MELO • Graduate Program in Food Science, Federal University of
THAIS DE OLIVEIRA • Graduate Program in Food Science, Federal University of Santa
Catarina, Florianopolis, SC, Brazil
JONAS DE TOLEDO GUIMARÃES • Department of Food Technology, Faculty of Veterinary,
Federal Fluminense University, Niteroi, RJ, Brazil
MARCO DI LUCCIO • Graduate Program in Food Engineering, Federal University of Santa
Catarina, Florianopolis, SC, Brazil; Graduate Program in Food Science, Federal
ERICK A. ESMERINO • Department of Food Technology, Faculty of Veterinary Medicine,
Federal Fluminense University (UFF), Niteroi, Rio de Janeiro, Brazil
DANIELLE C. B. H. FERREIRA • Federal Institute of Paraná, Campus Jaguariaı́va, Paraná,
Brazil
LAIS LEITE FIGUEREDO • Graduate Program in Food Engineering, Federal University of
xi
xii Contributors
MONICA Q. FREITAS • Department of Food Science and Technology, Agrarian Science Center,
Federal University of Santa Catarina (UFSC), Florianopolis, SC, Brazil
HUGO S. GARCÍA • CONACYT—Tecnologico Nacional de México/Instituto Tecnologico de
Veracruz, Unidad de Investigacion y Desarrollo en Alimentos, Veracruz, Mexico
MARIE-CLAUDE GENTÈS • Saint-Hyacinthe Development and Research Centre, Agriculture
and Agri-Food Canada, Saint-Hyacinthe, QC, Canada
CINTIA L. HANDA • Federal Institute of Paraná, Campus Pitanga, Paraná, Brazil
YI-BIN HUANG • Tea Research Institute Chinese Academy of Agricultural Sciences,
Hangzhou, China
CLAUDIO M. E. MALAGHINI • Graduate Program in Food Science, Federal University of
CYNTHIA MANASSI • Department of Food Science and Technology, Agrarian Science Center,
Federal University of Santa Catarina (UFSC), Florianopolis, SC, Brazil
SHIBO MA • School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences,
University of Melbourne, Parkville, VIC, Australia
ROBERTO LAUREANO MELO • Behavioral Physiopharmacology Laboratory, Barra Mansa
Center University, Rio de Janeiro, Brazil; Department of Food Technology, Behavioral
Physiopharmacology Laboratory, Barra Mansa Center University, Rio de Janeiro, Brazil;
Institute of Technology, Federal Rural University of Rio de Janeiro, Rio de Janeiro, Brazil
FERNANDA MEYBOM • Regional Council of Engineering and Agronomy of Santa Catarina
(CREA), Florianopolis, SC, Brazil; Graduate Program in Food Engineering, Federal
MEHRAN MORADI • Department of Food Hygiene and Quality Control, Faculty of Veterinary
Medicine, Urmia University, Urmia, Iran
BÁRBARA MORTL • Kairos Brewery, Florianopolis, SC, Brazil
MILENA DUTRA PIEREZAN • Graduate Program in Food Science, Federal University of Santa
Catarina, Florianopolis, SC, Brazil
TATIANA COLOMBO PIMENTEL • Federal Institute of Paraná, Campus Paranavaı́, Paraná,
Brazil
HADI POURJAFAR • Alborz University of Medical Sciences, Dietary Supplements, and Probiotic
Research Center, Karaj, Iran
CHAMINDA SENAKA RANADHEERA • School of Agriculture and Food, Faculty of Veterinary and
Agricultural Sciences, University of Melbourne, Parkville, VIC, Australia
OSCAR O. ROMERO-CHAPOL • CONACYT—Tecnologico Nacional de México/Instituto
Mexico
HOUSHMAND SHARAFI • Department of Food Hygiene and Quality Control, Faculty of
Veterinary Medicine, Urmia University, Urmia, Iran
ADRIANA TORRES SILVA E ALVES • Institute of Food Technology ITAL, São Paulo, SP, Brazil
MARCIA CRISTINA SILVA • Department of Food, Federal Institute of Education, Science and
Technology of Rio de Janeiro (IFRJ), Rio de Janeiro, Brazil
LEILA MARIA SPADOTI • Institute of Food Technology ITAL, São Paulo, SP, Brazil
ABIGAIL VARELA-PÉREZ • CONACYT—Tecnologico Nacional de México/Instituto
Mexico
Contributors xiii
SILVANI VERRUCK • Graduate Program in Food Science, Federal University of Santa

Catarina, Florianopolis, SC, Brazil; Department of Food Science and Technology,
Agrarian Science Center, Federal University of Santa Catarina (UFSC), Florianopolis,
SC, Brazil
J. K. VIDANARACHCHI • Department of Animal Science, Faculty of Agriculture, University of
Peradeniya, Peradeniya, Sri Lanka
YONG-QUAN XU • Tea Research Institute Chinese Academy of Agricultural Sciences,
Hangzhou, China
JUN-FENG YIN • Tea Research Institute Chinese Academy of Agricultural Sciences,
Hangzhou, China
PATRÍCIA BLUMER ZACARCHENCO • Institute of Food Technology ITAL, São Paulo, SP, Brazil
VANESSA CORTINA ZANETTI • Graduate Program in Food Science, Federal University of Santa
Catarina, Florianopolis, SC, Brazil; Graduate Program in Food Science and Technology,
Federal University of Santa Catarina, Florianopolis, SC, Brazil
CHUN ZOU • Tea Research Institute Chinese Academy of Agricultural Sciences, Hangzhou,
China
Chapter 1
Probiotic Fermented Milk

Shibo Ma, J. K. Vidanarachchi, and Chaminda Senaka Ranadheera
Abstract
Probiotic fermented milk is a product made by appropriate microbial growth using milk as the substrate
which contains mainly live microorganisms. Fermented milk has been consumed for thousands of years
worldwide, and the incorporation of probiotics has pushed it in a novel direction. The substrate selection
includes cows, buffalo, goats, sheep, yak, horses, camel, and others’ milk. The various substrate has their
uniqueness, and typical traditional products, including kefir, koumiss, etc., are made from them. Further,
the range of probiotics is vast, and commonly used genera contain Lactobacillus and Bifidobacterium. The
primarily incorporated method is to inoculate it into the starter culture to co-ferment substrate with
traditional fermentation culture. Other methods include fermenting substrate directly or adding it back
into the product. The typical products include ambient-temperature fermented milk or probiotic fermented
milk beverage. The basic processing method of probiotic fermented milk is similar to traditional fermented
milk, where the incorporation of probiotics into the fermented milk product is unique due to the special
incubation requirement of each probiotic. Commonly seen additives include sweetener, thickener
(thickening technology), and prebiotics which were introduced in this chapter, which could give a compre-
hensive vision of the current fermented milk production and the indication of applying these additives to
the fermented milk considering the existence of probiotics. Some novel and popular fermented milk
products and their manufacturing methods were briefly introduced in this chapter, such as ambient-
temperature fermented milk, roasted flavor fermented milk, and probiotic fermented milk beverage.
General products’ quality issues and legal compliance were also mentioned. Still, the most critical way to
determine the manufacturing procedure and parameter is by running a pilot test based on the designation of
the product, which could give a clear indication of the material, method, and post-manufacturing issues.
Key words Probiotic fermented milk, Manufacture process, Probiotics, Special milk, Sweetener,
Prebiotics, Thickening technology
1 Introduction
Probiotic fermented milk is a product derived from traditional

fermented milk. Fermented milk is a milk product made via appro-
priate microbial growth and/or enzymatic conversions of milk
[1]. Here, the probiotic fermented milk should go further, where
it requires the existence of probiotics in the fermented milk. It was
recognized that probiotic fermented milk should contain live
Adriano Gomes da Cruz et al. (eds.), Probiotic Foods and Beverages: Technologies and Protocols,
Methods and Protocols in Food Science, https://doi.org/10.1007/978-1-0716-3187-4_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2023
1
2 Shibo Ma et al.
microorganisms [2]. However, the recent research regarding

parabiotics and postbiotics broadened the scope of the products
[3], where the importance of the viability of probiotics had been
assimilated. In this chapter, probiotic fermented milk, referred in a
broad sense, is a cluster of fermented milk products containing
probiotic strains, live or not. More detailed introduction about
parabiotics and postbiotics will be given in Chaps. 15 and 16.
Fermented milk has been consumed for thousands of years. It was
originated from various places, such as Mongolia, Egypt, Caucasian
areas, etc., where multiple products were developed to fulfill the
local requirements. For a clear written record, Greek and Roman
are the first to mention this type of product (yogurt) in their
history, around 100 BC [4]. For probiotics, its health effect had
been realized and applied for dozens of centuries, combined with
fermented milk consumption [5]. However, its mystery hadn’t
been revealed until modern times for their existence and taxonomy.
In 1857, Pasteur discovered lactic acid bacteria (LAB) for their role
in the fermentation of yogurt. In 1908, Elie Metchnikov proposed
the idea of probiotics’ health effect in his book The prolongation
of life: optimistic studies (where the word “probiotic” haven’t been
proposed yet) [5, 6]. In 1953, German scientist Werner Kollath
proposed the term “probiotic,” which has been further used
[7]. For currently admitted and used probiotic definition and
effect, it was determined and published by FAO/WHO in 2001
and slightly modified in 2014 by Hill et al. [8] who confirmed that
the probiotic should be “live microorganisms which could confer a
health benefit on the host, when being administrated in appropriate
amount.” This definition differed the probiotic fermented milk
from other traditional fermented milk (relatively different, tradi-
tionally used microorganisms for fermentation were sometimes
regarded as probiotic in some situations), where the probiotic in
the products should be capable of conferring benefit to humans
after consumption. Firstly, the probiotic should tolerate gastric,
bile, and intestinal fluid, and could colonize and proliferate in the
gastrointestinal tract (GI tract). The safety and viability of probio-
tics are critical to the selection criteria, where the evaluation proce-
dure has been clarified recently. China has published a new Group
Standard names Probiotic Food by China National Food Industry
Association (CNFIA) to define the requirement of probiotics used
in food and the evaluation procedure to evaluate their safety and
viability (T/CNFIA 131–2021) (see Notes 1 and 2) [9]. The stan-
dard also requires the precise strain number and source, and the
completion of whole genome sequencing and random clinical trial
to support its efficacy based on scientific articles. Other scholars
also believe the probiotics used in the fermented milk (food) should
exist in the GI tract originally, and genetically modified
(GM) strain/species should not be used [10]. Meanwhile, there
are a lot of strains or species that were tested and claimed to possess
probiotic potential. Still, the authorities did not have explicit
Probiotic Fermented Milk 3
consensuses to determine which strain/species or groups can be

regarded as probiotics. For example, China and Canada had a list
showing the possibility of adding these species into foods as pro-
biotics (Table 1).
Table 1
The list of microbial strains available to be used in foods in China and other countries [89–92]
Genera Species
Bifidobacterium Bifidobacterium adolescentis*,^,#
Bifidobacterium animalis subsp. animalis *,^,#
Bifidobacterium animalis subsp. lactis*,^,#
Bifidobacterium bifidum*,^,#
Bifidobacterium breve*,^,#
Bifidobacterium longum*,^,#
Bifidobacterium longum subsp. Longum*,^,#!
Bifidobacterium longum subsp. Infantis
Bifidobacterium longum subsp. Suis*,^,#!
Bacillus (Assessed case-by-case in AU) Bacillus subtilis^!
Bacillus cereus^!
Companilactobacillus Companilactobacillus farciminis #!
Debaryomyce% Debaryomyces hansenii#!
Enterococcus (Assessed case-by-case in AU) Enterococcus faecium^!
Enterococcus faecalis^!
Fructilactobacillus Fructilactobacillus sanfranciscensis #!
Lacticaseibacillus Lacticaseibacillus casei#
Lacticaseibacillus paracasei #
Lacticaseibacillus rhamnosus#
Lactiplantibacillus Lactiplantibacillus paraplantarum #!
Lactiplantibacillus plantarum#
Lactobacillus Lactobacillus acidophilus*,^,#
Lactobacillus amylolyticus*,^,#!
Lactobacillus crispatus*,^,#
Lactobacillus delbrueckii#!
Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus
bulgaricus)#
Lactobacillus delbrueckii subsp. Delbrueckii#!
Lactobacillus delbrueckii subsp. Lactis#
Lactobacillus gallinarum#!
Lactobacillus gasseri#
Lactobacillus helveticus#
Lactobacillus johnsonii#
Lactobacillus kefiranofaciens subsp. kefiranofaciens#
Streptococcus Streptococcus salivarius subsp. thermophilus
Lactococcus Lactococcus Lactis subsp. lactis
Lactococcus cremoris
Lactococcus Lactis subsp. Lactis biovar diacetylactis
Latilactobacillus Latilactobacillus curvatus #
Latilactobacillus sakei
(continued)
4 Shibo Ma et al.
Table 1
(continued)
Genera Species
Lentilactobacillus Lentilactobacillus buchneri #!
Lentilactobacillus hilgardii#!
Lentilactobacillus kefiri #!
Propionibacterium Propionibacterium freudenreichii subsp. Shermanii^,#
Propionibacterium freudenreichii^,#!
Acidipropionibacterium Acidipropionibacterium acidipropionici #
Leuconostoc Leuconostoc mesenteroides subsp. Mesenteroides#
Leuconostoc citreum#!
Leuconostoc lactis#!
Leuconostoc pseudomesenteroides#!
Levilactobacillus Levilactobacillus brevis#!
Ligilactobacillus Ligilactobacillus salivarius#
Limosilactobacillus Limosilactobacillus fermentum ^,#
Limosilactobacillus mucosae #!
Limosilactobacillus panis#!
Limosilactobacillus pontis#!
Limosilactobacillus reuteri *,#
Loigolactobacillus Loigolactobacillus coryniformis #!
Mammaliicoccus Mammaliicoccus vitulinus
Oenococcus Oenococcus oeni#!
Kluyveromyces% Kluyveromyces lactis#!
Kluyveromyces marxianus#
Pediococcus Pediococcus acidilactici#
Pediococcus pentosaceus#
Staphylococcus Staphylococcus xylosus
Staphylococcus carnosus
Saccharomyces% Saccharomyces bayanus#!
Saccharomyces boulardii*,#!
Saccharomyces cerevisiae#!
Saccharomyces pastorianus#!
Schizosaccharomyces% Schizosaccharomyces pombe#!
Weizmanni Weizmannia coagulans
Xanthophyllomyces% Xanthophyllomyces dendrorhous#!
*: genera or species available in foods as probiotic (or showing health effect) in USA, symbols marked at species column,
separated with other symbols using comma (,)
^: genera or species available in foods as probiotic (or showing health effect) in Australia (includes those that were not
authorized by China, which was marked as ^!), symbols marked at species column, separated with other symbols using
comma (,).
#: genera or species available in foods as probiotic (or showing health effect) in Canada (includes those that were not
authorized by China, which was marked as #!), symbols marked at species column, separated with other symbols using
comma (,).
%: yeast, marked at genera column.
Table 2
The approximate composition of various typical probiotic fermented milk products [47, 93–96]
Ymer (Denmark Skyr (Iceland

Yogurt Kefir Koumiss product) product)
Protein, % 5 3 2.2 5–6 12.7
Fat, % 7.5 0.2 1.9 3.5 0.2
Acidity, % 0.8 1
Total solids, % 18.5 10.6–14.9 14.5 17.5
Carbohydrate, % 6 2.8 3.5 3.9
Alcohol, % 1 2.2 0.3–0.5
Ash, % 0.7 0.8
Others 1.97 g/L CO2
Many fermented milk could contain probiotics, such as yogurt,

kefir, koumiss (kumys, kumis, kumiss, coomys), sour cream,
and fermented buttermilk. Besides these traditional probiotic fer-
mented milk products, some novel fermented dairy beverages con-
taining probiotics have been developed recently, and the most
famous one is YakultⓇ. The main difference among them is the
product status (fluidity) and intrinsic microbial environment
(multi vs. single strain) (see Note 3). They have different substrates,
processing procedures, and storage conditions, where the most
important is their proximate composition (Table 2). By the time
of quality detection, the parameter measured had been regulated by
the authorities from various countries. Table 3 summarizes the
regulation parameters and numbers of the parameters which the
products should achieve.
As mentioned above, the strict definition of probiotic fermen-
ted milk should contain live microorganisms in their product matri-
ces. However, recent product development has combined the
inactivation of live cells into the processing procedure to extend
the shelf life or more stable quality, such as ambient-temperature
yogurt (pasteurized fermented milk) and other products. They
apply various live-cell inactivation methods to limit or eliminate
the activity of viable microorganisms in the products to prolong the
shelf life of the products for a farther distribution range or more
manageable storage conditions. The inactivation methods include
radiation, heating, high pressure, etc. (see Note 4). There are also
coupled designs for these sterilized products about packaging
material and style. General packaging uses plastic cups/containers
(set) or bottles (stirred) to package fermented milk. For premium
products, the glass jar is acceptable to package the product as well.
6
Table 3
The compilation of fermented milk standards from various countries [27, 38, 97–100]
Codex Alimentarius China USA Canada Australia

Shibo Ma et al.
acd b a e ab
Fat, % ≤10 , 15 3.1 , 2.5 ≥3.25 —— ——
a ab b e f
Non-fat-solid, % —— 8.1 ≥8.25 ≥9.5 , 7.6 , 6.5 ——
Protein, % Min 2.7abcd 2.9a, 2.3e —— ≥2.8b, 2.2f ≥3a (cow’s milk)
Acidity, % Min 0.3a, 0.6bc, 0.7d —— ≥0.5a, 0.7b ≥0.7b ≤pH 4.5a
(or ≤ pH 4.6)
Acidity, °T —— 70 —— —— ——
6, ab 6, ae 7,a 7,b
Microbial load, cfu/g(mL) Min 10 (total), ≥10 ≥10 ≥10 ≥106, a
104, cd (yeast)
Ethanol, %vol./w Min 0.5d —— —— —— ——
Document number CXS 243–2003 GB19302–2010 FDA-21 CFR National Dairy Australia New Zealand
131.112, 131.200 Code, Part III Food Standards 2.5.3,
F2015L00413
a
Fermented milk/Cultured milk.
b
Yogurt, alternate culture yogurt, acidophilus milk (Yogurt: fermented milk using Lactobacillus bulgaricus and Streptococcus thermophilus as culture; alternate culture yogurt: using
Streptococcus thermophilus and any Lactobacillus species; acidophilus milk: using Lactobacillus acidophilus as culture).
c
Kefir.
d
Kumys.
e
Flavored fermented milk (with sugar or fruit component addition).
f
Yogurt drink (drinkable fermented milk).
——: Not mentioned or required by such standards.
However, novel tetra packaging was developed to comply with the

requirement of ambient-temperature fermented milk to assist its
prolonged storage time. The shelf life of regular fermented milk
(with or without probiotics) is around 21–28 days. For plastic
packaged products, some of them can be shortened to 14 days
(it is worth noting that the shelf life does not have a severe relation-
ship with the preservation ability of LAB or the health effect of
probiotics). The optimal storage condition of such products is
around 4 °C, requiring fully cold-chain logistics. For ambient-
temperature fermented milk, the shelf life can be extended to
6 months at ambient temperature (around 25 °C).
Moreover, there are vast amounts of products commercially
available in the market. Still, they can be characterized according
to several criteria, such as matrix status (set/stirred), product addi-
tive (natural, sweetened (flavored), nutritionally enhanced), post-
fermentation processing (condensed, frozen, carbonized, spray-
dried), fat content (full-fat, partially skimmed, skimmed, and
Greek yogurt) [11]. Nevertheless, their material, main processing
procedure, and packaging step are very similar, with a slight differ-
ence in additive, post-fermentation, and packaging steps. These will
be described in detail in Part III.
2 Material
Materials used for probiotic fermented milk production can be

divided into several groups: raw milk and milk substrate, starter
culture and probiotic strains, sweetener and additive. They have
different effects on the probiotic fermented milk, which should be
focused on during processing.
2.1 Raw Milk and The substrate and primary material of probiotic fermented milk
Milk Substrate should be various milk originating from multiple breeds or species
of mammals. Commonly seen dairy animal species include cows,
goats, sheep, buffalo, donkeys, and camels, where cows are the
most used for raw milk production. Bovine milk is the most con-
sumed milk by humans. Various cattle breeds have been domesti-
cated by humans for milk production (some of the breeds are for
both milk and beef). These temperate breeds include Ayrshire,
Guernsey, Brown Swiss, Shorthorn, Jersey, and Holstein Friesian.
Among them, Holstein Friesian is the only most important breed
for milk production. Holstein Friesian originated from the Nether-
lands and had been exported widely to the world due to its adapt-
ability. It has excellent milk production capability, where its average
milk yield is 25–35 kg/day [11]. This yield is far from other dairy
breeds. Holstein Friesian has a lower milk fat content than other
temperate breeds except for Shorthorn [11]. The typical appear-
ance of Holstein Friesian is black and white color. Besides, other
8 Shibo Ma et al.
species have their characteristics, such as Jersey has a high milk fat
(4.95%) content and dry matter (14.54%) content with low yield
(19–25 kg/day), and shorthorn has a high protein-fat ratio but low
yield as well (17–25 kg/day) [11]. Therefore, the selection of raw
milk sources would significantly affect the final product’s quality.
Notably, the quality of raw milk produced by different animals
can be affected by various factors. Of which the most important and
controllable are milking season, feeding (water and fodder), and
equipment. The raw milk composition could be varied significantly
following the milking season (lactation season) change, but the
lactose in the milk could be stable. Protein and milk fat have a
solid response to season change, where the lowest content occurred
in summer (3.21% for protein, 4.1% for fat) and the highest content
occurred in winter (3.38% for protein, 4.57% for fat), respectively
[12]. It had been reported that the raw milk yield and composition
were negatively related to environmental temperature [13–17].
This phenomenon is reasonable and explainable due to the Hol-
stein Friesian originating from a cool area, which has a stress
reaction to heat. Heat stress is one of the most significant issues
in cows, especially Holstein Friesian husbandry. Lactation season
could also affect raw milk yield and composition, whereas Holstein
Friesian’s lactation season could be over 200 days. Raw milk yield
and composition have fluctuated over a long period [17]. The raw
milk yield increases and reaches a peak during the early lactation
period but goes lower following the lactation period [17]. The fat
content has a real controversial tendency compared with yield
[17]. It went lower at the beginning of the lactation period and
turned to increase, accompanied by lactation progress [17]. Milk
protein also has higher content at the beginning of the lactation
period [17].
Water and forage feed could be crucial factors that impact the
raw milk quality, where the contaminant and odor components,
such as heavy metals, animal drugs, and toxins, could be transmit-
ted to the milk through cow’s milk secretion [18]. The type and
quality of forage could also affect the milk fat content and compo-
sition, where the involvement of phytochemical composition in the
forage attracts attention [19]. The feeding method could influence
the quality of raw milk as well. Grazing cows have lower raw milk
yield than feedlot cows, but the fat content in grazing cows’ milk is
higher than in feedlot cows’ milk. The difference between the
protein content is negligible [18, 20]. It is worth noting that the
fatty acid composition in the milk produced from grazing or feedlot
cow is also different. In summary, it is wise to determine the source
of raw milk regarding the abovementioned factors before adopting
it in fermented milk production for better product quality.
Apart from species, breed, and lactation season, and feeding
material quality and method, milking sanitation and equipment are
also critical to raw milk quality, especially microbial load. Essential
sanitation of the cows’ udder (or other dairy animals) is necessary as

the microbe in the raw milk strongly correlates with teat skin
sanitation. Research proved that the microbial composition is sig-
nificantly different between raw milk and teat skin due to the both-
way contamination. However, 92.1% of the bacteria in the raw milk
come from the teats’ skin (genetically connected) [21]. An efficient
way to sanitize the udder is teat dipping (pre and post), in which the
teat was sanitized via iodine solution. The same research also
revealed that the microbial composition of teat skin is significantly
similar to raw milk, which means the both-way contaminations
were halted, and the microbial was only transferred from raw milk
to teat [21]. This result proved that iodine sanitation is an efficient
way to intercept teat-raw milk contamination. Sanitation of milk
equipment is also a pivotal step in ensuring the quality of raw milk.
Research indicates that appropriate sanitized equipment could
reduce raw milk’s thermophilic spore load [22]. Other factors
that have relationships with low spore load include farming envi-
ronment, husbandry scale, regular udder massage, and others
[22]. These factors also confirmed that appropriate farming meth-
ods, feeding (fodder and silage), housing conditions, and even the
cow’s mood influence the raw milk quality, which needs attention.
Milking is an essential step for raw milk collection, where the
equipment evolution has served this step well. Machine milking has
far higher efficiency than manual milking, which has improved the
raw milk yield significantly [11]. Recently, automatic milking
equipment (robotic milking) was developed to avoid excess stress
on cows and save human labor. This equipment ensures the animal
welfare of cows and eases their nervousness, anxiety or other nega-
tive moods to prevent low-quality raw milk. Usually, the cows were
tagged and managed via ear tag, where the information of each cow
can be collected when they enter the milking robot for milk tracing.
The cost of milking also decreased compared with traditional milk-
ing. This automated milking machine has attracted the attention of
farmers from developed countries, such as the USA, Australia, The
Netherlands, and New Zealand, to apply this system for better raw
milk production.
After milking, the raw milk should be tested before production.
Some standards or codes require the quality of raw milk. The most
crucial parameters are microbial load and somatic cell count (SCC).
For microbial load, the USA requires that the raw milk for direct
consumption should not contain more than 15,000 total viable
bacteria/mL and < 10 coliform bacteria/mL [23]; China has a
2 × 106/mL total viable microbial count limitation of raw milk,
whereas the EU limited the total viable microbial count to 1 × 106/
mL [24]. For somatic cells, it is not required by China, but the USA
and EU had limited the count below 6 × 106 and 4 × 106 cells/mL,
respectively [23, 25]. Somatic cell count (SCC) is vital for raw milk
quality. It indicates the health status of cows or other dairy animals.
10 Shibo Ma et al.
SCC was influenced by parities, calving season, and lactation

period, and the yield will drop dramatically when the SCC goes
higher [26]. Research proved that the composition of raw milk
reached the lowest amount when the SCC exceeded 5 × 106/mL;
hence the researcher recommended that the SCC in raw milk
should not be above 5 × 106 cell/mL [26].
Besides the microbial count and SCC, many other parameters
should be satisfied, including fat, protein, and non-fat milk solids in
many countries. For industry raw milk collection, many essential
tests need to be performed to ensure the quality of raw milk and
perspective products. These include sensory tests, ethanol tests,
clot-on-boil tests, titratable acidity, density test, microbial (dye
reduction methods)/somatic cell/antibiotic test, composition
determination, and adulterant tests [11]. Among them, the ethanol
test is a rapid method to determine whether the raw milk is fresh or
not, based on the acidity of raw milk [11]. This is a very fast and
easy method to be applied in the industry due to the simple phe-
nomenon, equipment, and indicative capability. For fresh raw milk,
there will be no phenomenon when ethanol (68%, 70%, 72%) is
added to the raw milk, where the coagulation of casein (protein)
will occur when the raw milk deteriorates [11, 18]. Notably, a
microbial/somatic cell/antibiotic test is necessary, especially for
fermented milk production. Besides the microbial count, excess
antibiotic in the raw milk is crucial for fermented milk production
due to their inhibitory effect on the starter culture cultivation and
growth, especially probiotic, which requires a strict environment.
The source of antibiotics is vast, but it may come from cattle disease
treatment, fodder additive residue, and milking contamination
[18]. Addition of antibiotics purposely is rarely seen, but it affects
the quality significantly, which needs strict regulation. Developed
countries require that antibiotics should not be tested in raw milk
[18]. However, a trace amount of antibiotics is still allowed in
developing countries [27], indicating that raw milk should be
appropriately tested and treated when applied to produce fermen-
ted milk in these countries.
After collection, pre-treatment should be performed to ensure
the quality of raw milk for further production. Usually,
pre-treatment includes filtration, purification, cooling,
pre-pasteurization (optional), and deaeration (optional) [11]. Fil-
tration and purification could efficiently remove physical contami-
nants and excessive microbial and somatic cells to reduce
observable contaminants by the naked eye. However, rapid cooling
is essential for the stable quality of raw milk during storage before
processing. Usually, freshly collected milk has cow’s body tempera-
ture, which should be cooled around 4–6 °C as soon as possible.
The growth of microbes could be attenuated or inhibited at this
temperature. If its temperature could be cooled down to 2–3 °C,
the growth of the microorganism could be near completely halted,
and it can be stored for about 7 days [11]. Pre-pasteurization

should be performed if the raw milk is not used immediately to
avoid quality deterioration.
2.2 Starter Culture Starter culture is essential for probiotic fermented milk production.
and Probiotic Strains It usually contained lactic acid and polysaccharides producers, such
as Lactobacillus (L) and Streptococcus spp. (S). The ratio of L/S is
around 1:1 or 1:2, where the overwhelming of Lactobacillus will
result in excess lactic acid content and unacceptable flavor
[11]. Detailed starter culture production will not be mentioned
here. Still, the type of starter culture and production of starter
culture are described in Table 4 and Fig. 1, respectively. It is
worth noting that adding probiotics as a starter culture is the
main method to incorporate probiotics into fermented milk.
Hence, the cultivation of probiotics needs further attention. The
synergistic or antagonistic bio-relationship between conventional
starter culture (Lactobacillus & Streptococcus (L&S)) and probiotic
could affect the success of fermentation [28]. For example, the
difference between the growth rate of L&S and probiotic leads to
desired microorganism cultivation failure, or the metabolites of
each species could promote or inhibit the growth of other species
(hydrogen peroxide, oxygen content, carbonized, etc.) [28–34].
Table 4
Various types of starter cultures used in fermented milk production [11, 40, 47]
Type of
Classification criteria starter culture Notes
Preparation of Starter LAB pure culture Primary strains included in the culture (step 1)
cultures Mother starter culture Proliferation of primary strains (step 2)
Bulk starter culture Proliferation of mother culture, used for
manufacture directly (step 3)
Strain composition of Mixed strains starter Contains more than one strains for synergistic
Starter cultures culture fermentation
Single strain starter Contains only one strain, mixed when applying
culture
Supplemented strains Contains one or more strains for special purpose,
starter culture includes exopolysaccharides production, aroma
component production, and probiotics
Status of Starter culture Liquid starter culture Easy to operate and cheap, but the viability can be
weakened
Powder starter culture Better viability and stability than liquid form
Frozen starter culture Highly concentrated, highest viability, direct usage
LAB lactic acid bacteria
Steps 1, 2, 3: The steps required for starter culture application during production procedure. These steps were performed
according to factories in situ application.
12 Shibo Ma et al.
Substrate
Cooling2 Pure Culture3
preparation1
LAB pure Activity

Cultivation6 Inoculation5
culture restoration4
Reinoculation Mother Inoculation & Bulk starter

Cooling10 LAB pure culture
& Incubation7 starter culture Proliferation8 culture
Mother starter culture
Substrate Storage & Bulk starter culture

preparation9 Cooling
Application
Fig. 1 The flow chart of starter culture preparation [11, 40]. (1) Reconstituted skimmed milk (10–12% solid),
heated 90–95 °C for 30–40 min or 121 °C for 15 min. (2) Mesophilic culture: 20–30 °C; theromophilic culture:
42–45 °C. (3) 0–4 °C storage, subculture every 1–2 weeks; random purification is needed. (4) Restoration for
2–3 times. (5) 1–2% addition amount. (6) Temperature determination according to strain
characteristics; Time: 3–20 h. (7) Same condition or 2–3 times. (8) At 42 °C, stop when acidity >0.8%.
(9) Same substrate treatment condition, but using product raw material as substrate, 1–2% of total raw
material. (10) Use within 6 h: 10–20 °C; use after 6 h: 4–5 °C
Due to the growth rate, the time and form of probiotic addition are
crucial. As for the preservation of the viability of probiotics, many
ways are used to protect probiotics and assist them in reaching the
GI tract without severe weakening due to lactic acid in fermented
milk or harsh condition in the GI tract. Encapsulation is a com-
monly used method to protect probiotics. Probiotics can be
encapsulated (usually microencapsulated) in different wall materials
or matrices to maintain viability (see Chap. 14 for more details).
Different wall material has various properties, such as protection,
lyse, texture alteration, etc. There is a study that showed that the
addition of microencapsulated probiotics could affect the texture of
yogurt (smoothness), which needs attention (alginate-starch as wall
material, which can affect the texture) [35]. Other materials used
for microencapsulation include whey protein (an useful by-product
of cheese production), gellan gum (polysaccharides), etc. The
microencapsulation method includes drop-out, emulsification,
extrusion, coacervation, and others. Compared with extrusion,
emulsification has a higher encapsulation rate [36]. Microencapsu-
lated probiotics can shorten the fermentation time of fermented
milk as well [36], but this phenomenon needs further clarification
to differentiate between bacteria synergistic effect or microencap-
sulation promotion. Besides, the strong buffer capacity of the
substrate (neutralized pH) or the firm texture of fermented milk
(gel) (prevents acid contact with probiotics) can protect probiotics
efficiently as well [28].
2.3 Sweetener and Many additives can be used in probiotic fermented milk, where the
Additives sweetener is the most important one. Sweeteners could provide a
sweet taste to the consumer to assimilate or cover the harsh taste of
lactic acid in the fermented milk. A commonly used sweetener is
sugar (sucrose), which is accepted by most consumers. Recently,
artificial sweeteners, such as sucralose and aspartame, were used to
provide a more intense sweet taste and reduce cost. However, the
health requirement of customers had forced the producer to replace
artificial sweeteners with natural sweeteners, hence stevia, erythri-
tol, and mogroside have come into sight of the producers. These
selections have broadened the horizon of sweeteners from a health
perspective and increased the acceptability and functionality of
fermented milk. Besides, there are other additives, such as fruit
components (jam, crushed or pulp), thickener/stabilizer/emulsi-
fier, essence, pigment/colorant, etc. [11], that can be added into
the fermented milk in accordance with local regulations.
It is worth noting that some unique carbohydrates, such as
dietary fiber, resistance starch, oligosaccharides, and inulin, were
added to the probiotic fermented milk to acquire its health benefit
and probiotic promoting capability (synbiotic ability). These sub-
stances are called as prebiotics. Prebiotics is a type of food compo-
nent that could not be digested by the endogenous host enzymes
yet could exert benefit on the host by modulating gut microflora
[37]. In this case, the type, purity, chain length, percentage of
prebiotic, target probiotic/microflora, product formula and char-
acteristic, and storage conditions need to be considered when
applying prebiotic in probiotic fermented milk [37]. Prebiotics
can significantly affect the probiotic viability and the physiochem-
ical (texture and rheology), organoleptic and functional properties
of the products [37]. However, the effect (positive, negative, or
neutral) is still under debate, which needs more attention when
utilizing them in the products [37]. More detailed availably of
thickener (thickening technology) and prebiotic selection will be
discussed in Notes 5 and 6.
3 Method
The production method of probiotic fermented milk is similar to

yogurt production, which involves pre-treatment (standardization,
pre-heating, homogenization, heating, cooling), inoculation, fer-
mentation, additive addition, and packaging. The flow chart of the
processing procedure is shown in Fig. 2. Here, it is notable that the
order of fermentation, packaging, and additive use is different
between set-fermented milk and stirred-fermented milk. Detailed
order is shown in Fig. 2 as well. In the following paragraph, each
step will be discussed separately, and the combination of such steps
should be performed as per product and in situ requirements.
14 Shibo Ma et al.
Standardization1 Homogenization Heat treatment2 Cooling Pure culture
Pre-
heat
Cooling Fermentation6 Packaging5 Inoculation4 Bulk culture3
Flavoring Set
component7 fermented
milk
Stirring & Fermentation6

Storage Packaging5 Stirred
Cooling
fermented
milk
Fig. 2 The flow chart of fermented milk processing [11, 40, 47]. (1) Milk solid, includes protein, cream,
thickener, sweeteners were added here; filtration may be applicable for unsolved substances, critical control
point 2 (CCP 2) for both set and stirred fermented milk. (2) Significantly important for product quality control,
CCP3 for both set and stirred fermented milk. (3) The hygienic condition of starter culture is important, CCP4
for both set and stirred fermented milk. (4) The control of hygienic condition and relative parameter is critical
for this step, CCP5 for both set and stirred fermented milk. (5) The hygienic condition of environment and
packaging container is critical, CCP6 for set fermented milk, CCP 7 for stirred fermented milk. (6) The
fermentation temperature and time are critical for the success of products processing, CCP 7 for set-
fermented milk, CCP 6 for stirred fermented milk. (7) Includes fruit component (pulp or jam), essence
substances, etc
3.1 Pre-Treatment Pre-treatment includes raw milk standardization, homogenization,

heat treatment, and inoculation steps. Firstly, the raw milk pumped
from the storage tank should be standardized to fulfill the require-
ment of local regulations where the factory resides, or the product
will sell. In general, any product should satisfy the requirement of
FAO/WHO regulation [38] for global distribution and retail sell-
ing. Fat and protein content should be less than 10% and more than
2.7%, respectively. Hence, any raw milk that does not meet this
requirement should be standardized to achieve this limitation.
Usually, the fermented milk fat content is between 0.5–3.0% [11],
depending on whether it is skim or not, where the addition of
cream is necessary to adjust this content to not only fulfill the
regulation but also to guarantee the sensation of such product.
Besides, the non-fat-solid of milk will be fortified, if necessary,
whereas the skimmed milk powder should be used here. These
components (cream, skimmed milk powder) can be provided
within the factory from other product lines to utilize the
by-product and make the best value of it. The sugar and stabilizer
should be added here to favor the growth and fermentation of
starter culture and possess desired texture of the product. Detailed

additive addition will be discussed in the Subheading 3.4.
After standardization, milk should be pumped into the heater
to pre-heat for homogenization. Appropriate heating could stabi-
lize the fat globule in the milk for homogenization in case any
undesired consequences occurred, such as fat separation (creaming)
or incomplete homogenization. Homogenization aims to shrink
the size of fat globules to prevent cream separation and unify fat
distribution. Hence, the stability and consistency of the fermented
milk could be improved. Further, this step could mix the ingredi-
ents added during standardization well to enhance the texture and
mouthfeel of the final product [11, 39]. This step does not affect
the growth of probiotics but could increase viable cell count
[39]. In general, appropriate pressure should be 20–25 MPa at
60–65 °C [11]. However, slight modification should be applied
in accordance with in situ, such as higher pressure for a higher
amount of stabilizer or thickener. The time for homogenization
varied significantly, which depends on the volume of milk to be
homogenized.
The homogenization will not reduce the temperature of milk
significantly, where it should be followed by further heating to
sterilize the milk for fermentation. Any living microorganism will
be killed during this step, but the spore may not be eliminated due
to its heat resistance. However, the fermentation and growth of
starter culture (including probiotic) could occupy the niche for
spore growth, which make the product consumable. Meanwhile,
heating could inactivate intrinsic antimicrobial components, such
as some antimicrobial peptides or proteins, to favor the growth of
starter culture [11]. Further, heating could denature whey protein
to modify its tissue to improve viscosity and prevent whey separa-
tion [11]. Usually, an appropriate heating condition should be
90–95 °C for 5 min [40], where 120 °C for 3–5 s is acceptable,
such as in Ultra High Temperature processed milk (UHT).
Scalding milk should be gradually and immediately cooled to
~40 °C for inoculation. Traditionally, yogurt fermentation uses 43 °
C for fermentation, with a starter culture addition of 2–4%
[11, 40]. However, this temperature should be modified when
probiotics are incorporated into the starter culture for fermenta-
tion. The synergistic and antagonistic effect among bacteria or
yeasts should be considered to obtain the best probiotic growth
with acceptable product quality. Mostly, the optimal growth condi-
tion of probiotics is around 37 °C for many genera, such as Lacto-
bacilli, Bifidobacteria [41], and the optimal condition of
Propionibacterium is around 30 °C [42]. However, the starter
culture bacteria (Lactobacillus delbrueckii subsp. Bulgaricus, and
Streptococcus thermophilus) perform badly under this condition
(lower lactic acid, volatile component, polysaccharides production,
etc.), hence appropriate fermentation temperature modification
16 Shibo Ma et al.
should be determined previously during the pilot plant test before

larger scale production, as well as the amount of addition, if the
starter culture was developed by the fermented milk producer itself.
Otherwise, sticking to starter culture instruction provided by the
starter culture manufacturer (if applicable) is a wise decision to
guarantee the success of fermentation. Previous thorough agitation
is recommended for starter culture before addition for better per-
formance [11]. Notably, the sterile operation is crucial for this step
due to processing demand. There will be no more sterilization or
pasteurization involved (generally, but there is ambient-
temperature fermented milk available in the market, which is dis-
cussed in Note 4), where any contaminant (bacteria, yeast, mold,
bacteriophage, etc.) introduced into the product will affect the
quality of probiotic fermented milk significantly, hence causing
severe consequences or results. This step and fruit pulp or jam
addition (discussed in Subheading 3.4) are both critical control
points of fermented milk production, which requires complete
and careful administration.
3.2 Fermentation Fermentation is the most critical step of fermented milk processing
to obtain desired flavor and texture. The order of fermentation and
packaging is decided by the desired fermented milk texture (set,
stirred, or drinking). Here, we discussed fermentation firstly, then
packaging, but the order can be changed. In general, starter culture
contains Lactobacillus delbrueckii subsp. bulgaricus (L) and Strepto-
coccus thermophilus (S) and requires a fermentation temperature
around 41–42 °C for 2.5–4.0 h fermentation time (2–4% addition)
[11]. However, introducing probiotics into the starter culture
altered the appropriate fermentation temperature. As mentioned
above, probiotic strains have the best performance when the tem-
perature reaches 37 °C, but L&S cannot grow well at this tempera-
ture. Even the antimicrobial properties of probiotics could inhibit
the growth of L&S, and the fermentation fails. Also, probiotic
requires a longer fermentation time, from 8–9 h to 48 h, even
some requires 72 h [42–45], this had led to a more difficult deter-
mination of fermentation time. Hence, an appropriate adjustment
should be performed for fermentation conditions to facilitate the
growth of both L&S and probiotic. For example, two-step fermen-
tation is a practical way to ferment milk containing complicated
microbial environments, such as kefir. Yoo et al. [46] developed a
two-step fermentation method, which applied 37 °C for 9 h at the
first step, and then 24 °C for 15 h for the second step. This method
had acquired better sensory acceptance. Therefore, appropriate
adjustment or separation of such fermented time or temperature
could be applied to fit the growth of all the strains. Some probiotic
strains can also grow at 40 °C, which is strain-specific, but this
could also provide chances for producers to ferment milk at this
temperature.
For set-fermented milk, the milk and starter culture mixture are
packaged into the container firstly, which is plastic or glass, but the
hygienic and aseptic conditions should be guaranteed before pack-
aging. The packaged (sealed) products are placed in a warm room
(fermentation room) which has appropriate spaces between the
containers for better airflow [11, 40]. Stable temperature and
shaking avoidance should be monitored during the whole proce-
dure in case tissue breakdown or fermentation quality deteriorates
[47]. The fermentation should be stopped when pH arrived at 4.6
and appropriate curdling happen in the container. At this time,
immediate cooling is essential for controlling the acid content in
the fermented milk. Generally, the temperature should be cooled
down to 35 °C within 30 min, then 18–20 °C in the next
30–40 min, then 5 °C as soon as possible [11] and wait for
distribution.
For stirred fermented milk, this fermentation step is carried out
in a fermentation tank, which requires uniform temperature distri-
bution in the tank due to the tank size. The upper and lower tank
temperature difference should not exceed 1.5 °C [11]. When the
fermentation is stopped (pH 4.2–4.5), immediate cooling is
required to avoid excess acid production or flavor deterioration
(see Note 7). However, cooling down should not be too fast,
which could lead to curd shrink, and whey may be squeezed out
of the curd [11]. The stirred fermented milk is agitated, in which
mechanical force is involved in agitation. Appropriate control of
such process is needed to maintain the tissue structure. Slow stir-
ring, medium stirring temperature (10–25 °C), and desired pH
(below 4.7) should be affirmed to maintain the tissue structure
and avoid whey isolation due to mechanical force [11, 40]. Mechan-
ical force may also occur when pumping the fermented milk for
transportation due to turbulence, so slow pumping is needed to
prevent any undesired results. The flow rate should be maintained
below 0.5 m/s [11]. However, any parameters mentioned here are
adjustable in accordance with the actual textural and other sensory
properties of fermented milk products, where the pilot test is
significantly essential to obtain the best parameter to favor the
production.
3.3 Packaging and Packaging material can be plastic or glass, with different container
Storage shapes. Bottles, cups, bowls, or jars are all acceptable. It depends on
the product or consumer demand. For set-fermented milk, the milk
and starter culture mixture is packaged into retail containers/cups,
with or without additives, to prepare for fermentation. However,
for stirred-fermented milk, the fermented milk is packaged at
15–22 °C when mixed with additive or not [11, 40]. It is signifi-
cantly vital to ensure aseptic and sterile condition during any pack-
aging step, especially air cleanness. This is the rare step where
products are exposed to and have contact with the outer environ-
ment, hence complete cleanness needs to be focused on to avoid
introducing contaminants into the products.
18 Shibo Ma et al.
Besides traditional packaging, the novel product pushed the

development of packaging. There is a pasteurized fermented milk
product commercially available in China, which uses Tetra PrismaⓇ
Aseptic to ensure the shelf life (6 months) (see Fig. 3). Other types
Fig. 3 (a) The photos of Tetra PrismaⓇ Aseptic package, (b) plastic bottle for
ambient-temperature fermented milk, and (c) EcoleanⓇ package. *optional.
(1) Skimmed milk powder is dissolved at 50–55 °C warm water where steriliza-
tion of such substrate is optional. (2) Lactobacillus casei Shirota as the culture
seed was added into the substrate for incubation at 37 °C, and stopped when
appropriate parameters were detected. (3) Culture base (fermented substrate)
were stored at 5 °C after sweetened by syrup. (4): The sweetened culture base
was mixed with sterilized water for better fluidity. (5): Bottles were made by
food-grade polystyrene and transported with clean air for following selection
step. Bottle selector makes the bottle oriented to the same direction for
decorating (printing). The logo was printed on the bottle using instant-dry red
ink (for sugar-reduced version, it is blue ink with more complicated decoration).
(6) The content of each Yakult bottle is 100 mL, and the cap was made by
aluminum foil which is easily opened. (7) The product was stored at 5 °C for
following distribution, but it should be maintained at this temperature when
selling and at home until consumption
of packaging are also available for this particular product, such as

plastic bottles, which are rarely seen in traditional fermented milk
packaging. EcoleanⓇ air is also an available packaging for fresh
stirred fermented milk due to its low weight, unique handle
(air-filled), and suitability for straw use. The detailed production
method of ambient-temperature fermented milk will be discussed
in Note 4.
The storage condition of fermented milk products should be
around 4–5 °C, where the storage step begins after packaging
(stirred) or fermentation (set), depending on their production
procedure. Usually, the storage time (shelf life) for traditionally
fermented milk (yogurt) is around 28 days at 5 °C. However,
research revealed that the Lactobacillus acidophilus could drop
significantly after 21 days of storage at 5 °C, where Lacticaseibacil-
lus casei have the highest viability retention [48]. These results
indicate the incorporation of probiotics should be considered
when examining shelf life to maintain the essential viability of
probiotics.
3.4 Additive Addition There are many additives available for fermented milk production.
Common additives include fruit flavor components (mainly pulp or
jam), sweetener, thickener/stabilizer, or other flavor ingredients,
such as nuts or raisins. Detailed additive selection and commercially
used novel additives will be discussed in Notes 4–6. Here, the
procedure operation will be mainly introduced. As mentioned
above, the milk-solid enhancers (protein, fat, etc.) are added at
the beginning of production at the pre-treatment stage [11, 40,
47]. Protein is usually stored and sold as solid statues, where it
needs to be added and solved into milk. Appropriate agitation is
important to maintain the uniform milk texture and nutritious
component distribution in the milk. Conversely, milk fat is usually
added in liquid form (milk cream), which does not require long-
time agitation. Excessive agitation or stirring would isolate fat and
induce quality deterioration, such as unpleasant mouth sensation or
lack of aroma. Sugars and other stabilizers are added at this stage for
a better solution. Sugar (sucrose) is essential for certain microbial
growth as well, making it a pivotal component to favor the growth
of probiotics, especially for those non-lactose fermenters.
In general, fruit components or other flavor ingredients are
added just before packaging [47] due to heat treatment could
cause unexpected fouling in pipe or component degradation. How-
ever, this raised the hygiene issue when adding these ingredients.
There is no more heat treatment following this addition, and the
possibility of introducing contaminants needs to be controlled. As
mentioned above, this is a critical control point of the whole
processing procedure. Hence, complete sterilization of such ingre-
dients should be guaranteed to ensure the safety and quality of
desired products.
20 Shibo Ma et al.
4 Notes
1. Several probiotics can be applied as starter cultures, where the

commonly used probiotic species are listed in Table 5. It should
be noted that the health effect of probiotics is strain-specific,
hence the claim of strains on the label is necessary for legal
compliance. However, as mentioned above, there is no precise
list of probiotics in most countries, therefore a thorough eval-
uation of probiotics needs to be performed, especially for novel
probiotic strains to ensure the availability of the strains. China
has published a new probiotic standard (Probiotic Food,
T/CNFIA 131–2021) which gives a good indication of the
evaluation procedure applied to probiotic food. This evaluation
Table 5
Predominantly used thickeners/stabilizers, probiotics, prebiotics, and sweeteners in fermented milk
products at present
Additive type Name of the additive Additive type Name of the additive
Thickener Acetylated distarch adipate Sweetener Acesulfame potassium
Acetylated distarch phosphatea Aspartame
Agara Erythritola
Carob bean gum Isomaltose (palatinose)
Diacetyl tartaric acid ester of mono Jamb
(di) glycerides (DATEM)
Gelatin Lactaseb
Gellan guma Maltitol & maltitol syrup
Guar gum Mogroside
Hydroxypropyl distarch phosphate Neotame
Lactic and fatty acid esters of glycerol Steviol glycosidesa
Pectina Sucralose
Starch Sucrosea
Sweetened condensed milkb
Probiotics Bifidobacterium Xylitol
Lacticaseibacillus paracasei (Formerly
Lactobacillus paracasei)
Lacticaseibacillus rhamnosus (Formerly Prebiotic Plant powder
Lactobacillus rhamnosus)
Lactobacillus acidophilus Inulin
Lactobacillus delbrueckii subsp. Polydextrose
Bulgaricus (Lactobacillus bulgaricus)
Lactococcus Lactis subsp. Cremoris Resistance dextrin
Lactococcus Lactis subsp. Diacetylactis Fructo-oligosaccharides
Lactococcus Lactis subsp. Lactis
Leuconostoc mesenteroides subsp.
Mesenteroides
Streptococcus thermophilus
a
could be used in ambient-temperature fermented milk
b
the additive can provide sweetness but not a sweet additive (sweetener)
includes (1) probiotic species and strain identification with

clear and well-known sources, (2) probiotic preservation
method and safety evaluation, (3) whole-genome sequencing,
which is peer-reviewed, and (4) in vivo or randomized clinical
trial which supports its health effect [9].
2. Probiotic fermented milk has the potential to be claimed as
food for a special purpose, but appropriate legal appliance and
related clinical trial needs to be performed. For example, foods
for health purposes need to be registered in China, and clearly
label the registration number on the packaging bottle or other
forms of packaging. Other countries have their regulations,
where the individual analysis of local law, regulation, and policy
is significantly vital for such claims. It should be mentioned that
some products claim to provide nutrients to >3-year kids, but
they are normal foods instead of foods for special purposes. The
targeted population of the products limits the type of such
product, which needs to be considered when developing
products.
3. Dairy-based probiotic fermented beverage (probiotic bever-
age) is an alternative product that contains live probiotics but
is more drinkable than traditional fermented milk. YakultⓇ is a
popular product that is a representative of such products. It has
a special and patented probiotic, Lacticaseibacillus casei Shir-
ota, which was isolated by Minoru Shirota in 1930. The phe-
notype of this strain is similar to other Lactobacillus spp., and it
is worth noting that it could grow at temperatures 15 °C–41 °
C, but the optimal temperature is 37 °C [49], as mentioned
above. Lacticaseibacillus casei Shirota is a sucrose fermenter
[49], where possible sugar addition may involve in the produc-
tion procedure, just before fermentation for better lactic acid
production, but this depends on in situ application, not com-
pulsory. The brief manufacturing procedure includes milk
reconstitution, sterilization, fermentation, homogenization,
flavoring, balancing (adding sterilized water to dilute the con-
centrated product), packaging, and further storage [49] (see
Fig. 4). The uniqueness here is that probiotic beverages use
skimmed milk powder to reconstituted milk as their fermenta-
tion substrate to minimize the effect of milk fat (fat isolation, as
mentioned in Note 6) and control cost. Moreover, the sterili-
zation procedure conferred brown color to the substrate, just
like roasted fermented milk (see Note 4), due to such high
temperature and time. Meanwhile, the fermentation tempera-
ture is maintained at 37 °C for better growth of the Lacticasei-
bacillus casei Shirota. This temperature differs from traditional
fermented milk due to the simplified microbial environment
(multi strains vs. individual strain).
22 Shibo Ma et al.
Substrate Bottle
Fermentation2 Storage3
preparation1 production5
Syrup
Bottle
Sterilization* Culture Seed2 Mixing4
Selection5
Sterilised
water
Filling, capping Bottle

Storage7 Packaging
& sealing6 decorating5
Bottle production line Main manufacture line Extra addition step
Fig. 4 The manufacturing flow chart of Yakult® [49]. *: optional (1) Skim milk powder is dissolved at 50–55 °C
warm water where sterilisation of such substrate is optional. (2) Lactobacillus casei Shirota as the culture
seed was added into the substrate for incubation at 37 °C, and stopped when appropriate parameters were
detected. (3) Culture base (fermented substrate) were stored at 5 °C after sweetened by syrup. (4) The
sweetened culture base was mixed with sterilised water for better fluidity. (5) Bottles were made by food-
grade polystyrene and transported with clean air for following selection step. Bottle selector makes the bottle
oriented to the same direction for decorating (printing). The logo was printed to the bottle using instant-dry red
ink (for sugar-reduced version, it is blue ink with more complicated decoration). (6) The content of each Yakult
bottle is 100 mL, and the cap was made by aluminum foil which is easily opened. (7) The product was stored
at 5for following distribution, but it should be maintained at this temperature when selling and home storage
until consumption
Additionally, balancing is a particular step for probiotic

beverage production. This step added sterilized water into the
product to dilute it and confer higher fluidity, making it more
drinkable to mimic beverage status. This step differentiated
YakultⓇ from traditional fermented milk products. Interest-
ingly, the bottle of YakultⓇ is produced at the same factory by
molding food-grade polystyrene [49]. This design made the
packaging bottle controllable, but the YakultⓇ product should
be stored avoiding light and kept at 4 °C due to the semi-
transparent properties of the bottle. Other probiotic beverages
have a very similar procedure, where the difference is the strain
used. Lacticaseibacillus paracasei is the most used species for
other probiotic beverages. However, the commercialization
potential of Lacticaseibacillus casei Zhang is growing and has
performed good ability to be utilized in yogurt and some
health effect on rats [50–52].
4. As mentioned above, some unique products are commercially
available in China with high sales. One of the most popular
products is pasteurized or sterilized fermented milk. It is also

called ambient-temperature fermented milk, which indicates its
most valued characteristic. It can be stored under ambient
temperature with no quality deterioration for 6 months. The
unusual step involved for this long storage is the following
sterilization or pasteurization after fermentation
[53, 54]. This step killed all available live microorganisms in
the fermented milk, which stopped continuing fermentation
usually occurring in unsterilized or unpasteurized fermented
milk (traditional fermented milk). The pasteurization or sterili-
zation method occurs by heating (72–121 °C, 4 s–20 min,
usually 75 °C, 20 min) and high pressure (600–680 MPa,
10–40 min) [54]. However, inappropriate temperature or
time for heating may cause color change or protein matrix
breakdown, hence novel methods have been developed to
perform this step, such as radiation and microwave
[55, 56]. They all perform well for sterilization or second
pasteurization (pasteurization is performed before fermenta-
tion). Meanwhile, the centrifugation in pre-treatment is
another critical control point to remove the spores in the raw
milk to avoid quality deterioration after packaging, just like
traditional fermented milk production, where the parameters
of the temperature and rotation speed is 50–65 °C and
4200–6300/min, respectively [54].
Despite having no live microorganisms in the fermented
milk (including probiotics), the health effect of such a product
may not be eliminated. Recently, the studies regarding post-
biotics and parabiotics have been getting popular, and they are
inactivated or killed probiotics [3], just like the microorganism
in the pasteurized or sterilized fermented milk. Therefore, the
health effect of postbiotics and parabiotics products cannot be
determined, as not comparable with products containing live
probiotics but needs more profound research to reveal the
functional properties. The balance of nutritional values and
the sale of the products need to be considered by
manufacturers.
Another popular product, roasted-flavor fermented milk,
has been developed based on Maillard reaction. This step was
performed before homogenization, with the addition of glu-
cose to promote the Maillard reaction to obtain the brown
color and roasted flavor of raw milk [57]. Meanwhile, some
concerns about this reaction are raised due to the production of
harmful by-products, such as 5-hydroxymethylfurfural (HMF),
glyoxal (GO), and methylglyoxal [58]. However, the harmful
by-products all can be controlled after appropriate modifica-
tion, such as keeping the product under 4 °C or setting a short
shelf life [58]. Therefore, controlling such harmful by-products
24 Shibo Ma et al.
must be considered when developing new products using new

technology or additives.
5. There are many thickening methods in fermented milk produc-
tion. Thickening is significantly important due to its wide
application in thickened yoghurt production in both stirred
and set types. The easiest way to improve or thicken the fer-
mented milk texture is by adding thickener into the product at
the beginning of the milk treatment. Usually, this kind of
additive needs thorough mixing to have a better solution;
hence homogenisation could be a good step to achieve this
under such high pressure, and heat treatment can be done
simultaneously. The rationale for thickener is that it could
absorb or bind more water to enhance the protein matrix’s
strength and improve texture [59]. Therefore, appropriate
agents that could absorb more water or strengthen the protein
matrix are selected to improve texture, such as polysaccharides
or proteins or both.
In general, most thickeners do not influence the viability or
the survival rate of probiotics. However, natural ingredients
and naturally produced additives have been selected to replace
artificial thickeners to improve the health value of the product,
where their prebiotic potential has been revealed for these
substances. Carob bean gum and chia seed mucilage have
been proven to be able to enhance fermented milk’s texture,
but have no significant impact on probiotic growth
[60, 61]. The bitter almond gum exudate and its conjugates
with sodium caseinate (SBAG-SC) had performed preservative
ability on the viability of Lactobacillus acidophilus, La-5, but
possess lower prebiotic potential compared with inulin, as well
as a comparable ability for preventing phase separation to fer-
mented milk compared with carboxymethylcellulose (CMC)
[62]. In fact, multi-types of polysaccharides have been adopted
as encapsulated wall material for the protection of probiotics.
This trend indicates that the dispersion of such polysaccharides,
such as alginate, xanthan gum, gum arabic, and maltodextrin,
could not only enhance the firmness of the fermented milk but
also protect probiotics from the digestion of the human stom-
ach to reach the intestinal tract efficiently, via the matrix and gel
formed by them [63]. Probiotic encapsulation using polysac-
charides could have texture modification ability as well. Low
methoxyl pectin encapsulated Bifidobacterium breve could
improve the viscosity of yogurt and the hardness when the
capsule was applied before fermentation [64]. This result
shows that polysaccharides’ preservation and texture modifica-
tion ability can co-exist, but their effect may vary when applied
under different forms.
Meanwhile, a starch-pectin blend, in which the ingredients

were both commonly used in fermented milk as texture modi-
fiers, could form resistant starch and slow-digestible starch via
their interaction (starch and pectin), possessing a synergistic
effect on probiotics growth [65]. This capacity has broadened
the horizon that the impact of thickeners may not act alone,
but the interaction between various thickeners could benefit
the growth or viability of probiotics. In addition, some pre-
biotics (inulin, tragacanth gum, gellan gum) could improve the
rheological properties of fermented milk, such as firmness and
apparent viscosity, and weaken the syneresis as biopolymers
[66]. The application of prebiotics to improve the viability or
count of probiotics should consider their texture enhancement
effect for better performance, or avoid undesired quality
deterioration.
Some intrinsic components of the milk, especially fortified
milk protein, could enhance the texture of fermented milk
through crosslink formation with polysaccharides by the starter
culture. Polymerized whey protein could be a good thickener
for set fermented goat milk due to its good performance with
the adjunction of pectin (PWP) [67]. PWP has properties like
low syneresis, desirable viscosity and hardness, but its retention
ability for the population of probiotics has added value to this
mixture [67]. PWP could retain the viable cell of Lactobacillus
acidophilus above 106 cfu/mL for 4 weeks, which proves its
effect on probiotic retention [67]. Milk protein concentration
can be regarded as a protecting agent for encapsulating Lacti-
caseibacillus paracasei, combined with gellan-caseinate [68]
(Kia et al., 2018). This shell material could reduce syneresis as
well [68]. This phenomenon shows its capability to maintain
the viability of probiotics during storage alongside the eleva-
tion of textural quality.
Recently, many physical technologies have been developed
and applied to realize this target. They remove an appropriate
amount of water or whey to thicken the milk without adding
anything extra to provide a “thick” mouth sensation to con-
sumers. These technologies include flash evaporation [69],
freeze concentration [70], centrifugation concentration, and
membrane filtration [71]. Their core mechanism is that they
could improve the content of dry matter to provide a thick
sensation to the consumers, whereas the higher dry matter
content could benefit the viability of probiotics as well [32].
6. Sweetener is another vital ingredient in fermented milk due to
the harsh sensation of lactic acid and other organic acids. It
could reduce and balance such sensations by providing sweet-
ness to consumers. Some sweeteners could also promote the
growth of probiotics. Traditionally, sucrose (sugar) has been
26 Shibo Ma et al.
used as a sweetener with the highest acceptance among other

sweeteners, where the growth of probiotics can also be main-
tained and controlled. However, the recent trend of fermented
milk requires healthier ingredients, where the calorie needs to
be controlled. Hence, artificial sweeteners and natural sweet-
eners (low glycemic index) have been developed to replace
sugar as sweeteners. Most of the commonly used artificial
sweeteners, such as aspartame, neotame, sucralose, sorbitol,
and polynols (xylitol, erythritol, maltitol and isomalt), have
no influence on the growth of probiotics[72–77], but their
health concern mainly resides at metabolism aspects. For natu-
ral sweeteners, honey is a popular natural sweetener that con-
sumers welcome and accept widely when replacing sugar. It is
shown that the addition of honey does not affect the viability
of Streptococcus in starter culture and could improve the viabil-
ity retention of Bifidobacterium animalis BB-12, which show
the suitability of honey to be used as a healthier natural sweet-
ener compared with sugar[78]. Stevia (steviol glycoside) is a
leaf extract of Stevia rebaudiana, a popular natural and
low-calorie sweetener. It has been proved that it could enhance
gel matrix and probiotic growth (lactobacillus acidophilus) with
no harm to the sensory properties of fermented milk [79]. Ste-
via could also maintain the survival rate of lacticaseibacillus
casei above 9 logs CFU/ml for 28 days of storage. Fermentable
fibre addition (red beetroot) could assist its prebiotic perfor-
mance[80], which was mainly observed in lacticaseibacillus
casei’s growth promotion[81]. However, some researchers
reported that stevia has a bitter aftertaste[82], which makes
its usage need further attention. Iso-maltulose, also known as
palatinose, is a product of an enzymatic reaction (glucosyltrans-
ferases) from sucrose[83]. It has both sweetness and prebiotic
potential, which could favour the growth of probiotics, includ-
ing lactobacillus acidophilus, lactococcus sp. and bifidobacterium
animalis, with preservation of their biofunctions[83, 84]. This
characteristic has broadened the horizon of this sweetener and
makes it possible to be regarded as a prebiotic sweetener. This
multifunctional property could reduce the ingredients added
to the fermented milk and favour the growth of probiotics for
better performance.
As mentioned above, prebiotics is a kind of non-digestible
component of the host by endogenous enzymes that benefits
the modulation of intestinal microflora. Prebiotics usually
appear as a carbohydrate that does not digest in the small
intestine but is fermented in the colon. Sweetener is typically
a kind of carbohydrate as well, which makes it possess the high
prebiotic potential to benefit the growth of probiotics. Mogro-
side is an extract from Siraitia grosvenorii (monk fruit) with
high sweetness intensity. It is claimed that it has no effect on
the fermented camel milk’s organoleptic properties and could

modulate the gut microbiota [85, 86]. Recent research
revealed that the enzymatically modified mogroside combined
with galactooligosaccharides (produced from mogroside and
lactose combination by β-galactosidase) could improve the
growth of gut microbiota, which includes Bifidobacterium,
Bacteroides, Enterococcus, and Clostridium coccoides
[87]. These examples indicate that using natural sweeteners
has the advantages of low-calorie, prebiotic potential and
high sweetness intensity, which makes them suitable to be
used in many products and situations, especially probiotic fer-
mented milk.
7. Many quality deteriorations may occur after product
manufacturing and during storage. The deteriorations include
texture, flavor, and color changes due to many factors. Here,
we will briefly discuss some typical quality decline to give a
comprehensive vision of quality control, but specified issue
solution needs to be considered individually according to the
situation. One of the most critical issues here is post-
acidification. This phenomenon is mainly due to the growth
of microorganisms during storage. Post-acidification can be
affected by many factors, such as type of starter cultures, milk
composition, temperature, and pH, homogenization and stir-
ring, packaging material, and pre- and probiotics [88]. Here,
the pre- and probiotic effects must be focused on due to their
contradictory effect. Probiotics could metabolize some micro-
bial inhibitory substances, such as bacteriocins or anti-
microbial peptides, which could inhibit the growth of lactic
acid producers. Hence, post-acidification can be assimilated.
Further, the temperature set for probiotic growth may not
favor the growth of lactic acid producers, where the pH
dropping rate may be slowed under this condition, as men-
tioned above. However, prebiotics could accelerate the growth
of many microorganisms, including lactic acid producers, as a
promoting substance for microbial growth [88]. Even probio-
tics can produce more acid than usual [88]. Hence, controlling
the production of such components is essential to ameliorate
the post-acid effect. Other controlling methods mostly involve
killing (or partially killing) the microorganisms in the fermen-
ted milk, but this does not meet some criteria of local regula-
tions [88]. Hence, maybe the future direction could
(1) genetically modify the microorganisms for reduced post-
acidify capability [88] and (2) add live microorganisms (espe-
cially probiotics) back to the product after killing the intrinsic
live cells and maintain the storage temperature at 4–5 °C for
slowing the growth of such added microorganisms. However,
the safety evaluation (such as antibiotic resistance, horizontal
28 Shibo Ma et al.
gene transfer, etc.) would be more crucial to understanding the

benefit of such products.
Other quality deterioration that can be discussed is based
on the product. For example, set-fermented milk has issues
with curd texture, whey isolation, undesired flavor, mold
growth, and bad mouthfeel (sandy texture) [11]. For the
curd texture and whey isolation, the main reason is the curd
structure breakdown or inappropriate structure. The curd
structure is affected by protein and polysaccharides cross-link.
The protein content, protein quality (milk quality and compo-
sition), acid content, and microbial growth (polysaccharides
production) may affect the structure. Among them, phage
contamination may cause a significant quality issue by inhibit-
ing the growth of microorganisms [11], which affect not only
the curd but also other aspects, such as acid production, aroma
component, etc. Phage is a type of virus with specificity to
particular microorganism. It could lyse the microbial cell and
kill them by leaching. Hence, the hygienic condition of the
starter culture is important. Also, starter culture replacement
and strain mixture are optional methods to avoid phage attacks
due to their specificity [11]. The rest issues include undesired
flavor, mold growth, and bad mouthfeel, possibly due to
microbial contamination and raw milk quality deterioration
(even mastitis milk has been used for manufacturing fermented
milk products) [11]. Excessive hygienic practices and raw milk
tests could prevent these issues.
Stirred fermented milk also has very similar issues to the
set-counterpart. It is worth noting that stirring may introduce
air into the product, which raises the possibility of whey isola-
tion (due to air stratification) and microbial contamination
(unclean air introduces yeast or mold into the product, espe-
cially the cross-contamination from other production lines
which contain such contaminants) [11].
For probiotic beverages, the quality issues include live cell
count, precipitation, fat isolation, flavoring ingredient quality,
and microbial contamination [11]. Precipitation is a particular
issue in probiotic beverages because observable precipitation in
fermented milk is acceptable but not for beverages, hence
appropriate methods to solve this issue are needed. Homoge-
nization is an excellent way to mix ingredients and break pro-
tein (the main component of precipitate) to obtain a uniform
beverage [11, 40]. This step is also used in Yakult® production
[49]. Stabilizers and sugar are also available to facilitate
homogenization [11, 40].
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Chapter 2
Probiotic Cheeses
Celso Fasura Balthazar, Julien Chamberland, and Marie-Claude Gentès
Abstract
Chemical and microbiological stresses experienced by probiotic bacteria in certain fermented milks,
especially in low-pH products, led to loss of viability in commercial products in a strain- and product-
dependent manner. In this scenario, cheeses appeared as a valid alternative for delivering probiotic bacteria.
A large number of scientific reports showed the suitability of different kind of cheeses for protecting cell
viability due to special characteristics of this food matrix, especially in fresh and semi-hard cheeses. Yet,
certain technological characteristics of cheese manufacture should still be considered when designing a
probiotic cheese such as salt content, heating of the curd, temperature during ripening, or cheese shelf life
in order to improve the cell viability and the sensory features of the product. For the moment, the market
success of probiotic cheese is still far behind that of probiotic fermented milks. The suitability of cheese for
the inclusion of probiotics should be highlighted, leading to the development of novel functional cheeses.
As well, there is no single recipe for selecting the appropriate probiotic for large-scale application. A deep
overview of the process and factors to ensure viability of the probiotic throughout cheese processing and
shelf life has to be performed, as well, consumer’s acceptability, because probiotic selection should have
minimal impact on taste and texture. Sensory testing and quality control plan are tools that the dairy
industry can rely on to assess these changes and as a decision-making aid.
Key words Bacteria, Cheese, Dairy, Functional, Healthy, Probiotic
1 Introduction
Cheese production involves complex interactions between milk

components, coagulant enzymes, and a wide diversity of microor-
ganisms. Among them, lactic acid bacteria (LAB) in starter culture
play a crucial role during all phases of cheese making and ripening
processes. When LAB grow in milk, they convert lactose to lactic
acid, acidifying the media and promoting curd syneresis through its
demineralization. During ripening, LAB also influence the devel-
opment of flavor, aroma, texture, and eye formation, depending on
the starter selected and the cheese type produced. Moreover,
health-promoting bacteria, so-called probiotic can be included in
some starter cultures to provide additional functionality to the
35
36 Celso Fasura Balthazar et al.
cheese. New cheese varieties, for example, those containing high

levels of probiotic bacteria, have resulted from further development
of starter cultures. While probiotic bacteria are best known in
fermented milk and yoghurt, the interest in adding probiotics to
cheese is increasing [1].
Probiotic is defined as “live microorganisms which when admi-
nistered in adequate amounts confer a health benefit on the host”
[2]. In order to provide a beneficial health effect, probiotics should
be consumed daily and remain viable throughout the gastrointesti-
nal tract. The recommended intake is around 8–9 log10 colony
forming units per gram (log10 CFU/g1). The health benefits of
probiotic bacteria include: (a) improving intestinal tract health;
(b) enhancing the immune system; (c) synthesizing and enhancing
the bioavailability of nutrients; (d) reducing symptoms of lactose
intolerance; (e) decreasing the prevalence of allergy in susceptible
individuals; and (f) reducing the risk of certain cancers. In this
sense, the most extensively studied and widely used probiotic bac-
teria are from genera Bifidobacterium and Lactobacillus [3].
Cheese as a delivery matrix for probiotic bacteria is based on the
fact that cheese is universally accepted in many diets around the
world [4]. It is worthy to note that the efficacy of probiotic intake
on health is highly linked to the frequency of consumption. There is
growing consciousness about the importance of the dose of probi-
otic bacteria, frequency, and duration of treatment required for
different conditions in different population groups [1]. Then it is
reasonable to choose those foods as a vehicle of probiotics that are
regularly consumed. As well in technological terms, cheeses are
good carriers to deliver probiotic bacteria to human due to its
generally lower acidic content, higher solid content, lower O2
concentration than traditional dairy probiotic carriers such as
yogurt and fermented milk. The higher buffering capacity of cheese
is also beneficial to protect the probiotic bacteria in the gastric juice.
The challenge in producing probiotic cheese is survival of the
probiotic organisms during the long shelf life of the product.
Survival in a cheese matrix is strain dependent, and the selected
probiotic bacteria should have a high acid- and salt-tolerance and
be compatible with the cheese starter culture [5]. Furthermore,
survival depends on the processing conditions, product matrix, and
storage conditions. A low cooking temperature during cheese
making, a high pH at the end of the curd acidification, a low oxygen
and salt content in the cheese combined with low storage tempera-
tures during maturation might be applied to maximize the survival
rate of the probiotic bacteria [5].
An in vitro simulation of gastric digestion (3 h, pH 2, and 3) of
Argentinean fresh cheese containing probiotic Lactobacillus aci-
dophilus, Lacticaseibacillus casei, and Bifidobacterium bifidum [6]
showed that the food matrix conferred a significant protection of
cell viability compared to cells digested as pure cultures. Cell counts
Probiotic Cheeses 37
were around 7 log10 CFU/mL for Bifidobacteria and around

4 log10 CFU/mL for L. acidophilus and L. casei at pH 2 whereas
after exposure of 3 h at pH 3, counts of probiotic bacteria were
above 7 log10 CFU/mL. Sharp et al. [7] compared the survival
capacity of a strain of L. casei in yogurt and in low-fat Cheddar
cheese submitted to gastric conditions at pH 2. The authors con-
cluded that in terms of exposure to acidic conditions, Cheddar
cheese could be a better dairy probiotic carrier, as L. casei viability
decreased by around 2 log10 CFU/mL after 30 min and dropped
by only 1 log10 CFU/mL after 2 h of exposure (final concentration
of 4 log10 CFU/mL). In yogurt, viable counts diminished to less
than 1 log10 CFU/mL just after 30 min of exposure
[7]. M€akel€ainen et al. [8] published that the probiotic Lacticasei-
bacillus rhamnosus HN001 and L. acidophilus NCFM included in a
semi-soft Gouda cheese survived in a model of the gastrointestinal
tract. However, the cheese matrix did not appear to affect the
probiotic survival because the probiotics in the cheese and in
freezed-dried powders exhibited similar survival levels, although
differences were observed in the magnitude of the stimulating
effects on cell lines [9].
Thus, probiotic bacteria are normally added together with the
starter culture. During cheese manufacturing, the concentration of
milk components, namely casein and fat, allows for a lower inocu-
lation rate if the selected strain and manufacturing parameters are
optimal. The introduction of DVS cultures for direct inoculation of
the cheese milk has allowed culture producers to launch new cul-
ture blends consisting of both thermophilic and mesophilic strains
designed for special cheese types as well as special cultures for the
production of probiotic cheese [5].
This chapter discusses about probiotic strain used to manufac-
ture different cheese types and the survival of those probiotics,
regarding actions taken to increase their viability. A section will
also describe the limitations from research to industrial limitations
as well as point out the main factors to consider for appropriate
probiotic strain selection for industrial application with the aim to
spread the development of probiotic cheese.
2 Material and Methods
2.1 Cheese Cheeses are made with two essential steps: (1) gelation of milk
Classification through enzymatic hydrolysis of κ-casein or acidification to
pH 4.6 (isoelectric point of caseins); and (2) whey drainage of the
resulting curd. However, differences in milk types and key cheese
manufacturing parameters such as the use of different starter
and/or adjuncts, fermentation conditions, renneting parameters,
scalding temperature, salting method, ripening conditions, or even
cheese shape contribute to explain the diversity in textures,
Fig. 1 Processing steps of major cheese types, based on their moisture content on a free fat basis (MFFB)
functional properties, flavors, and aroma of the many cheese types

produced around the world [10]. These factors collectively influ-
ence final cheese composition and sensory characteristics.
Cheeses have been classified in relation to their chemical com-
position, the manufacturing processes, the presence of particular
ripening microorganisms, maturity indices, etc. [11]. A classifica-
tion based on the moisture content on a free fat basis (MFFB,
Eq. 1) is particularly relevant when determining which cheese
type should be manufactured to improve cell viability, because
MFFB refers indirectly to the water activity.
%Moisture
MFFB = × 100 ð1Þ
100 - %Fat
Cheeses can be classified as soft (MFFB >67%), semi-soft
(MFFB between 61 and 69%), semi-hard (MFFB between 54 and
62%), hard (MFFB between and extra hard (MFFB content
between 49 and 56%) and extra hard cheeses (MFFB <47%) (see
Fig. 1) [12].
2.2 Cheese As mentioned previously, in the initial stage of cheese production, a

Manufacturing gel is formed from milk either by acid coagulation or by enzymatic
Process coagulation (see Fig. 1). For the enzymatic coagulation, coagulat-
ing enzymes are used (e.g., chymosin) to hydrolyze the κ-casein
fraction of casein micelles (breaking of the Phe105-Met106 peptide
bond). The release of glycomacropeptide—a highly hydrophilyc

fragment—destabilizes casein micelles, which results in the aggre-
gation, and gelation of resulting casein aggregates. Once the gel
formed is cut, many operating parameters (e.g., cutting size or
firmness, duration of stirring steps, scalding time, and temperature)
are selected to control the moisture loss of the resulting curd,
depending on its moisture-content target. In fact, the cheese
matrix, also known as paracaseinate matrix, is naturally prone to
syneresis (whey expulsion). Consequently, from fresh to hard
cheeses, the major difference between the manufacturing processes
is that those for harder cheeses promote syneresis to reduce their
moisture content. Syneresis is notably promoted by cutting a softer
gel in smaller particles, by increasing scalding temperatures or by
accelerating the acidification of the curd. The end of the draining
step gives rise to the cheeses.
At this time, salting is carried out by means of brine or dry
salting. Dry salting (salt being spread on the external surface of the
product) is recommended for more humid cheeses, especially when
promoting halotolerant microorganisms (e.g., Penicillium camem-
berti). Salting in saturated brine at >20% (w/w) NaCl is recom-
mended for most cheeses, with the determination of the immersion
period varying according to the type of cheese to be produced.
Salted cheeses can be consumed fresh, or stored in controlled
environment for cheese maturation that evidence the action of
proteolytic or lipolytic enzymes from milk, coagulant, starters and
ripening microorganisms commercial cultures, or introduced from
the dairy environment, which provide the formation of unique
flavor of each type of cheese [13].
Acid coagulation is generally achieved biologically through the
production of lactic acid by LAB, but direct acidification with the
addition of organic acids is possible. This coagulation type is used in
a limited number of soft (fresh) cheese types (MFFB generally
higher than 80%) such as cream cheese, quark (Tvorog), or labneh.
The pH drops to around 4.6 (between 5 and 20 h), depending on
the starter selected and the type of cheese produced. The following
microorganisms are normally used: mesophiles Lactococcus lactis
subsp. lactis and L. lactis subsp. cremoris, L. lactis subsp. lactis
biovar. Diacetylactis, and Leuconostoc mesenteroides; and thermo-
philic Streptococcus thermophilus and L. delbrueckii subsp. bulgaricus
[13]. The acid gel (or lactic curd) cannot undergo intense draining
steps as the enzymatic gel, because it is far less resistant to mechani-
cal stresses due to its extensive decalcification. Lactic gels are con-
centrated by simple molding with ladles (or cheese draining bags) at
the artisan scale, or by means of centrifugation or ultrafiltration at
the industrial scale.
2.3 Incorporation of With regard to the time of incorporation of probiotics into cheese
Probiotic Bacteria into during its manufacture, this generally occurs along with the addi-
Soft Cheese tion of the starter cultures. In some cases, probiotics could be
added after whey is drained from curd in order not to lose probiotic
cells in whey [14]. For the following sections, manufacturing pro-
cesses are presented for different cheese types.
2.3.1 Probiotic Cottage Probiotic cottage cheese could be produced by addition of fermen-
Cheese ted cream at the end of the cheese manufacture and has the advan-
tage to avoid cell loss in drained whey and the exposure of viable
cells to high cooking temperatures. Some studies [15–17] used this
strategy to add Bifidobacteria to cottage cheese. Cream (14% fat
content with or without 1.8% salt) was inoculated with freeze-dried
B. infantis ATCC 27920G and fermented at 37 °C until pH 4.5
was reached. The cultured dressing contained high levels of viable
cells (ca. 8.5 log10 CFU/g), when salt was not present during
fermentation but added once the desired pH was achieved. For
cheese making, curd was formed at 30–32 °C from pasteurized
skim milk inoculated with starter and supplemented with CaCl2
solution (0.02% final concentration). Chymosin (100 μL/L milk)
was added 1 to 1.5 h after starter addition and the curd was cut
when pH reached 4.7. The curd was cooked at a rate to reach 50 °C
to 55 °C in 1.5 h. Whey was drained and curd was washed with
water at 21 °C and 5 °C. Curd was salted (0.6% w/w) and dressed
with cream dressing fermented by Bifidobacteria to obtain approxi-
mately 4.5% fat (w/w) in the final cheese. The moisture of the
cheeses produced ranged from 79.5 to 81.6%. However, the par-
ticular strain used did not adapt well to the food matrix since after
28 days of storage counts of viable cells were lower than 1 log10
CFU/g. However, after 14 days of storage at 4 °C, losses in cell
viability around 2 to 3 log cycles were noted. Other types of soft
cheeses can be elaborated using probiotic strains together with
starter cultures or mixed with rennet [18].
2.3.2 Probiotic Minas Minas is a typical Brazilian fresh unripened cheese with high mois-
Frescal ture, low salt content, and absence of preservatives. These charac-
teristics offer excellent conditions for survival and growth of
probiotic strains [19]. The traditional procedure employed by Bra-
zilian dairies for the manufacture of Minas fresh cheese implies the
addition of mesophilic homofermentative lactic culture consisting
of Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris.
Probiotic strains as Lacticaseibacillus paracasei subsp. paracasei
LBC 82 could be added [20]. An alternative procedure employed
by some Brazilian dairies is the direct acidification with lactic acid
and without any addition of starter cultures. Pasteurized milk is
inoculated with freeze-dried commercial cultures for direct vat
inoculation. As soon as acidity reached about 20–22 °D, commer-
cial rennet (3 g/L milk) and calcium chloride (0.2 g/L milk) were
added at 36 °C. After 40 min, the gel was cut gently into cubes
(1 cm2), allowed to drain, and placed in perforated circular molds.
Cheeses were immersed in saturated brine for 30 min. After salting,
cheeses were packaged in sealed plastic bags and stored at 5 °C for
21 days [19–21].
2.3.3 Probiotic Fresh According to Roy et al. [22], fresh cheese was elaborated using
Cheese ultrafiltered skim milk standardized at 30% solids and 20% fat,
homogenized at 300 bar and pasteurized, followed by cooling
milk up to 30 °C and starter and Bifidobacteria cultures were
inoculated. Lyophilized cultures of B. breve R070 and B. longum
R175 were used. Then milk was incubated at 30 °C for 10 h until
pH reached a value of 4.6 and traditional fresh cheese procedure
was performed. The product manufactured was kept at 4 or 12 °C
for 57 days. The survival of Bifidobacteria at 4 or 12 °C was above
6 log10 CFU/g until day 15 of storage and gradually lost viability.
After 22 days, probiotic bacteria counts were below the claim to be
considered probiotic, and by day 50 of storage, Bifidobacteria were
no longer detected. In another study with Argentinean fresh cheese
containing L. casei, L. acidophilus, and Bifidobacteria, the cells’
quantification decreased after 60 days of storage at 5 °C to less
than 1.2 log10 CFU/g [6].
2.3.4 Probiotic Crescenza is a soft, rindless, Italian cheese (MFFB ~88%) with a
Crescenza short ripening time. Whole bovine milk was pasteurized and cooled
to 35 °C. A direct-to-vat, freeze-dried Streptococcus thermophilus
culture was added along with B. bifidum, B. infantis, and
B. longum, initial concentrations of 6 and 7.5 log CFU/mL,
respectively for starter and probiotics. Liquid calf rennet (3 mL,
20% pepsin, 80% chymosin) was immediately added and curd was
formed in approximately 25 min to be cut to a size of 1.5 to 2.0 cm.
After a 60-min holding period, the curd was then cut to a final size
of ca. 0.5 to 1.0 cm and warmed at 35 °C for 150 min. The cheese
was salted by immersion in 16 to 18% (w/w) NaCl brine for 1 h at
15 °C. The cheese was ripened for 10 days at 5 °C and subsequently
stored at 6 °C for 4 days, which corresponded to commercial
storage for this type of cheese. At day 14, the moisture content
ranged from 61.7 to 63.0% and initial counts of Bifidobacteria were
above 7 log CFU/g as in the production day [23].
2.3.5 Probiotic Turkish Turkish Beyaz (MFFB ~73%), Kasar (MFFB ~74%) and Tulum
Cheese (MFFB ~73%) cheeses are also considered good matrices for probi-
otic bacteria delivery. Studies indicated that CaCl2 addition to
pasteurized milk inoculated with Limosilactobacillus fermentum
(AB5–18 and AK4–120) and Lactiplantibacillus plantarum
(AB16–65 and AC18–82) probiotic strains with commercial starter
mix consisting of L. lactis subsp. cremoris and L. lactis subsp. lactis
(1% according to the manufacturer) were incubated until the pH
reached ca. 5.9–6.3, then chymosin (20 mL) was added to milk to
allow coagulation for 60 min. After that, curd was cut and rested in
the whey for 5–10 min, followed by drainage without pressure for
30 min, and pressed (40 kg weights for 100 L milk) for 180 min.
When the curd reached the appropriate strength, the cheese cloths
were opened and the cheeses were cut into cubes. The cubes were
brine-salted. Then, cheeses were kept at 37 °C for 12–18 h and the
brined cheese blocks were packed in plastic bags containing brine
and sealed with heat. Cheeses were ripened at 4 °C for 120 days,
when moisture content ranged from 58.6 to 68.2%. Counts of total
Lactobacilli remained up to 8 log10 CFU/g [24] (see Note 1).
2.4 Incorporation of Festivo cheese is a semi-soft Finnish cheese with high moisture
Probiotic Bacteria into content (~62% w/w), in which pasteurized milk is inoculated at
Semi-Soft Cheese 32 °C with starter cultures containing Lactococci, L. acidophilus,
and Bifidobacterium sp.. After 30 min of coagulation action, the
curd is cut and part of the whey removed, followed by temperature
raise up to 35 °C and curd stir. The whey is drainage and the cheese
is pressed for 3 h and further rests in brine overnight. During this
procedure the pH drops around one unit (from 6.45 to 5.20) and
cheese is wrapped in plastic for storage about 10 °C for 4 months.
L. acidophilus and Bifidobacterium spp. probiotic strains lost viabil-
ity by ca. half log cycle compared to the initial levels of inoculation
during cheese manufacture [25].
White cheese is a common semi-soft cheese manufactured in
dairy industries, in which some studies [25–28] mixed probiotic
strains with mesophilic starter and chymosin (amount varies
according to manufacturers). Coagulation of milk takes place in
60 min and coagulum is cut with a knife into small cubes for curd to
be allowed to stand in the whey for 5 to 10 min. Then, the curd is
transferred to mold at pH 6.4 and the surfaces of the cheese is
covered with cheesecloth, drained without pressure for 20 min, and
pressed for 2.5 h. The cheese mass is cut into cubes (7 × 7 × 7 cm)
for brine-salting (13% [w/w] NaCl) for 13 h. In sequence the
cheese is kept at room temperature for 6 h to rest and, finally
being ripened at 4 °C for 90 days. The probiotic inoculum should
achieve initial counts of log 9 CFU/g, because during brining and
storage at 4 °C, probiotic cells lose viability approximately by 2 to
3 log10. Thus that procedure assures probiotic levels until the end
of shelf life. The authors found that the decrease in the colony
counts of Bifidobacterium and L. acidophilus, La-5, was faster dur-
ing the first 30 days of storage [25–28].
Pategrás cheese is an Argentinean semi-soft cheese produced
with pasteurized cow’s milk, which is cooled to 37 °C for calcium
chloride and lyophilized starter (S. thermophilus) addition. In this
moment of manufacture, probiotic cultures (L. acidophilus,
L. paracasei and B. lactis) are added and after 15 min, there is
addition of chymosin for proper formation of curd in appropriate
strength. The curd is cut and stirred in whey under heat at the rate
of 1 °C/min until 45 °C for 15–20 min to reduce the moisture
content in the final product. Then, the curd is separated from whey,
molded and pressed during 24 h. Young cheeses are salted in
saturated brine 20% (w/w), pH 5.40, for 24 h and ripened for
2 months at 12 °C. Both probiotic strains are able to grow approxi-
mately 1.5 log cycles during cheese manufacture and counts remain
above 8 log10 during the 60 days of refrigerated storage [29–31].
Feta cheese is a semi-soft, white cheese, usually ripened in
brine, which is originally produced in Greece using ewes’ or
goats’ milk, or both, by coagulation using only rennet [32]. Probi-
otic Feta cheese could be obtained by pasteurized ewes’ milk added
with L. casei ATCC 393 and rennet. The inoculated milk is left
undisturbed for 2 h for curd formation. The curd is cut and cloth-
filtered overnight at room temperature (18 to 22 °C). The effect of
salt addition on cheese quality characteristics was studied by
rubbing 10 g of salt per 100 g of cheese on the surface. Ripening
of the cheeses was monitored at 4 to 6 °C for 71 days. The probiotic
strain used showed a satisfactory survival in Feta cheese either in the
products with or without salt added: above log 6 CFU/g [32].
A mix of L. acidophilus, B. longum, and B. lactis were
incorporated directly into lamb rennet paste as an approach to not
modify the traditional step in pasta filata cheese production
[33]. Pasta filata cheese is produced by thermiziation and textur-
ization process, cooking curd in hot water (or whey) by mechani-
cally mixing to achieve plastic consistency and extruded into
different shapes and size. The method resulted in higher structural
uniformity, lower friability, and higher creaminess and graininess
cheese, presenting high levels of probiotic cells, in particular Bifi-
dobacteria, throughout 2 weeks of ripening. Furthermore, the
peptide profile of the pasta filata ewe ripened cheese highlighted
specific peptides derived from the presence and activity of probiotic
bacteria. In particular, the presence of bioactive sequences in long-
ripened cheeses demonstrated the ability of probiotics to control
and enhance the proteolytic process and generate peptides in the
cheese matrix that could be delivered upon cheese
consumption [33].
As described above, many studies reported successful produc-
tion of probiotic semi-soft cheeses in adequate amount of viable
probiotic cells in the end of storage, despite losses during salting
and/or ripening (see Note 2).
2.5 Incorporation of For semi-hard and hard cheeses, probiotic strains are added with
Probiotic Bacteria into starter during cheese manufacture. Some time is usually required
Semi-Hard and Hard for strains to be activated and grown and acidify the media at
Cheeses temperatures around 30 °C, depending on the type of cheese.
Then the procedure follows each type of cheese production estab-
lished standards (see Note 3).
During cheddar cheese manufacture, mesophilic starter LAB

(L. lactis subsp. lactis and L. lactis subsp. cremoris) are added to
pasteurized cow’s milk followed by rennetting. For probiotic ched-
dar cheese production, probiotic strains are added together with
starter cultures in general. The mixture is allowed to set and form
curds at a temperature of around 29–31 °C for 30 to 40 min. The
curd is cut and allowed to stand for approximately 15 min. Then,
the curd is cooked around 39 °C for 20–60 min; when the whey
acidity is about pH 6.1–6.4 at the end of cooking phase, it has to be
drained. Cheddaring is a unique step in cheddar cheese making to
give the typical cheddar flavor. Thus, loaves of curds are cut about
15 cm wide along each side of the vat. After 10 min, the loaves are
turned and stacked every 10 min. This process is complete when the
pH is around 5.3–5.4. A curd mill is used to cut the loaves into
cubes during constant stirring and salt is added up to 1 to 3%
(w/w). Following, the curds are placed into molds and pressed to
form blocks of cheddar cheese. Then, ripening takes place for differ-
ent periods depending on the extent of ripening desired. The major-
ity of the reports suggest that Cheddar cheese was successful in
relation to the maintenance of cell viability for relatively long periods
such as 9 or 15 months. An appropriate selection of starter/probiotic
culture combination and adequate control of cheese and storage
variables are a prerequisite for the successful incorporation of
probiotics into cheese [34, 35] (see Notes 4 and 5).
To produce a probiotic caprine semi-hard cheese, goat milk is
pasteurized and cooled to 32 °C for CaCl2, a mixed-mesophilic
starter culture, B. lactis and L. acidophilus addition. Double-
strength calf rennet is added and curd formed in a min. The curd
is cut and temperature is raised slowly to 38 °C within 20 min. The
first whey drain occurs and the resulting curd is maintained at 38 °C
for additional 20 min. After the second whey drainage, the curd is
placed in hoops and held at 40 °C throughout pressing and held for
3 h) for then being brined. Cheese is ripened for 70 days at 6 °C.
The resulting salt and moisture contents are about 3.5% and 37.6 to
43.2%, respectively, and cheese storage is until refrigeration. A
significant loss in cell viability of 1.5 and 1 log10 CFU/g was
observed for B. lactis and L. acidophilus after storage [36].
Canestrato Pugliese cheese is an Italian semi-hard cheese, for
which sheep milk is used. The probiotic version was elaborated
adding B. bifidum Bb02 and B. longum Bb46 to starter
S. thermophilus and L. delbrueckii subsp. bulgaricus. The traditional
protocol was modified in order to improve probiotic cell viability.
Instead of heating the curd in hot whey at 80 °C for 30 s, the
probiotic curd was kept to 50 °C for 2 min, and then held at 40 °C
for 5 h to limit acidification by the starters. The salting was carried
out by spreading salt over the surface and cheeses were ripened at
12 °C for 3 months. By month 2 of storage, Bifidobacteria counts
decreased by 1.7 to 2.8 log10 cycles, indicating rather poor viability
of these strains in this particular product.
Etchepare et al. [37] and Mirzaei et al. [38] have used resistant
starch to microcapsulate probiotic bacteria (L. acidophilus, and
B. bifidum, L. casei, and/or B. lactis, respectively) in cheeses, which

helped avoid viability losses during long-period storage and
provided thermal stability improvement and protection against
simulated gastrointestinal stress. Also the encapsulation of probio-
tics into rennet paste was investigated on Pecorino cheese manu-
factured from Gentile di Puglia ewe milk at different maturation
times. The probiotic-containing alginate beads underwent a pro-
gressive disaggregation process, leading to the liberation of live
microorganisms. In this study, after 60 days of ripening, the beads
disappeared, and the probiotic cells remained at a level of about
7 log10 CFU/g of cheese up to 120 days of ripening [39]. There-
fore, encapsulation may sustain the production of long-ripened
cheese, maintaining high levels of live cells throughout the matur-
ing process [40].
Probiotic strains in cheese reach levels above 6 log10 CFU/g,
not modifying gross composition, nitrogen fractions, lipolysis, fatty
acid profiles including conjugated linoleic acids (CLA) and volatile
profile. The sensorial profile of the probiotic ovine cheese after
45 days of ripening was also influenced, showing lower humidity
and gumminess and higher ratings for salty and pungent attributes
compared to the control cheese [41]. The ability of L. acidophilus
to enrich the cheese matrix with a great amount of free fatty acids
(FFA) and CLA was further confirmed in a study of probiotic
Pecorino foggiano cheese [42, 43]. Bifidobacteria added in cheese
was able to produce high levels of linoleic acid as well as to induce
greater proteolysis that was associated with lower cheese hardness.
A study highlighted that probiotic bacteria may not survive in high
numbers when freely incorporated into dairy products, and encap-
sulation was proposed as a method to increase the survival and
delivery of probiotics [44, 45].
2.6 From Research Despite the numerous literature on probiotic cheese, only a few are
Laboratory to Large- available regarding the key elements to consider to scale-up from
Scale Production laboratory research to large-scale production. Commercial probi-
otic cheeses are already sold in market around the world proven the
feasibility.
This practical guide will then focus on the various points to
consider before developing probiotic cheeses. Nevertheless, there is
no one-size-fits-all probiotic cheese recipe. Selection of the appro-
priate strain must take into account cheese processing, biocompati-
bility of probiotic strain with other cultures, desired sensory
properties and use once in the consumer’s hand.
At laboratory scale, it is possible to control and easily modify
the environment which is much more challenging under industrial
reality. Minimal change on process increases the success of viable
probiotic application. As previously stated, different cheese types
have been tested for probiotic survival (Table 1). To select the
appropriate probiotics for the right application, the first step is to
review the cheese process and point out the possible steps that
Table 1
Probiotic strains used in different types of cheese
Cheese type Probiotic Viability/Storage Reference

Soft Mina Frescal Lacticaseibacillus casei 01 8 log10 CFU/g / Sperry et al. [19]
cheeses cheese 28 days
Fresh cheese Bifidobacterium breve R070 6 log10 CFU/g / Roy et al. [22]
B. longum R175 15 days
Beyaz, Kasar, Limosilactobacillus fermentum 8 log10 CFU/g / Kılıç et al. [24]
and Tulum (AB5–18 and AK4–120) 120 days
cheeses Lactiplantibacillus
plantarum (AB16–65 and
AC18–82)
Boursin cheese B. animalis subsp. lactis BB-12 >7log10 CFU/g/ Martins et al. [56]
Lacticaseibacillus rhamnosus 35 days
LRB 10 C109721A
Soft goat L. plantarum 564 8.82 log10 CFU/g/ Radulović et al.
cheese 42 days [57]
Semi-soft Pategrás L. paracasei subsp. paracasei 8 log10 CFU/g / Bergamini et al.
cheeses cheese B. lactis BB-12 60 days [31]
Argentineanm Lactobacillus acidophilus LA-5 6 log10 CFU/g / Perotti et al. [58]
cheese B. lactis BB-12 15 days
Feta cheese Propionibacterium 9 log10 CFU/g / Angelopoulou
freudenreichii subsp. 60 days et al. [59]
shermanii LMG 16424
Coalho goat L. mucosae CNPC007 >8 log10 CFU/g / Moraes et al. [60]
cheese 28 days
Scamorza B. longum 46, B. lactis BB-12, L. acidophilus: 7.5 Albenzio et al.
cheese L. acidophilus LA-5 log10 CFU/g / [33]
15 days
B. longum and
B. lactis: 9.9 log10
CFU/g /15 days
Semi-hard Cheese L. rhamnosus EM1107 Rodrigues et al.
cheeses [61]
Low-fat L. casei 334e 7 log10 CFU/g/ Sharp et al. [7]
cheddar 90 days
cheese
Low-fat B. animalis subsp. lactis >4 log10 CFU/g/ Demers-Mathieu
cheddar L. rhamnosus 120 days et al. [54]
cheese L. paracasei/
L. plantarum
Cheedar L. rhamnosus DPC7102 >7 log10 CFU/g/ Leeuwendaal
cheese 180 days et al. [55]
Hard Ovine cheese L. acidophilus LA-5, B. longum 7 log10 CFU/g/ Santillo &
cheeses and B. lactis BB-12 45 days Albenzio [41]
Pecorino L. acidophilus, B. longum, and L. acidophilus: Santillo &
cheese B. lactis no strain number? 8 log10 CFU/g / Albenzio [42]
60 days Santillo et al. [43]
B. longum and
B. lactis: 9 log10
CFU/g/60 days
(continued)
Table 1
(continued)
Cheese type Probiotic Viability/Storage Reference

Pecorino L. acidophilus, B. longum, and L. acidophilus: Albenzio et al.
cheese B. lactis no strain number? 8 log10 CFU/g/ [62]
30 days
B. longum and
B. lactis: 9 log10
CFU/g/30 days
Pecorino L. acidophilus, B. longum, and L. acidophilus: 7.52 Santillo et al. [39]
cheese B. lactis no strain number? log10 CFU/g/
120 days
B. longum and
B. lactis: 6.84
log10 CFUg/
120 days
Canestrato B. bifidum Bb02 > 6 log10 CFUg/ Corbo et al. [63]
Pugliesa B. longum Bb46 56 days
cheese
might affect the probiotic viability such as cooking, salting, storage

time, and temperature [46]. Probiotic has to be heat-, acid- or salt-
tolerant depending on the process. For instance, heat-resistant
probiotics might be more adapted for pasta filata which involved a
stretching curd step at high temperature (>80 °C) [47]. In addi-
tion, the cheese might be used at the consumer’s hand for grating
dish or pizza topping where high temperatures are used. Bacillus
coagulans, a known heat-resistant probiotic strains, have been suc-
cessfully added during curd fusion (>90 °C) of processed
cheese [48].
The final pH and salt level of the cheese are also important to
take into consideration [5]. Salt-resistant probiotic strain has to be
selected for brined-cheese [28]. As the pH of acid curd is lower
than 4.6, an acid-resistant probiotic strain should be selected.
Another important element to consider is the biocompatibility of
probiotics with the starter or the other adjunct cultures. Microor-
ganisms can release metabolites that impart the growth of others.
Though, an in vitro test can be performed to assess the different
combinations of cultures/probiotics prior to large-scale test
[49]. The method consists of simulating a Cheddar cheese fermen-
tation process with the first culture (starter). A second fermentation
is carried out with the other cultures (or probiotics) in the fermen-
ted whey obtained during the first fermentation. Growth kinetic is
monitored by spectrophotometry. The biocompatibility of starter
(Lactococci) and various commercial probiotics (Lacticaseibacillus
rhamnosus GR-1, L. rhamnosus GG, L. rhamnosus R0011, Lactoba-
cillus helveticus R0052, L. acidophilus LA-5 and B. animalis ssp.
lactis BB-12) was successfully evaluated by this method as selection

tool prior to Cheddar cheesemaking at pilot plant scale [50].
From a technical point of view, the expertise of the quality
control staff to monitor probiotic population throughout the
shelf life as well as the required material to perform enumeration
analysis has to be in place. Additional cost for training and equip-
ment (e.g., autoclave to sterilize the culture media) has to be taken
in consideration when developing probiotic cheese (see Note 6).
From consumer’s point of view, the most important criterion
for acceptability on functional food is taste [51]. Even though there
is an added benefit to cheese, it has to taste good or even better
than the traditional version. It is important to select probiotic that
will not modify cheese sensory properties. For instance, Bifidobac-
teria sp. produces acetic acid that could impart an unpleasant or
unusual flavor in cheese [52]. Sensorial evaluation testing is then
critical. Performing laboratory-scale sensory testing is not straight-
forward due to ethical considerations. Research approval from
ethical committee is required to conduct professional sensory test-
ing [53]. At the opposite, the dairy plant has quality control plan in
place to produce safe cheese according to strict regulations.
Another key element is regarding assessing the functionality once
in consumer’s hand. In other words, it is expected that a
low-moisture mozzarella will be used on pizza topping. The viabil-
ity of the probiotic has also to be evaluated under these conditions
to ensure the required minimal concentrations after this
cooking step.
To summarize, there is no single recipe for selecting the appro-
priate probiotic for large-scale application. A deep overview of the
process and factors to ensure viability of the probiotic throughout
cheese processing and shelf life has to be performed. Consumer’s
acceptability is also key. Probiotic selection should have minimal
impact on taste and texture. Sensory testing and quality control
plan are tools that the dairy industry can rely on to assess these
changes and as a decision-making aid (see Note 7).
3 Notes
1. Fresh cheeses are good matrix to deliver probiotics due to their

high-water activity, pH below 5.0 (4.5–4.6), low salt content,
absence of preservatives, and because they are not ripened.
These cheeses are thus stored at refrigeration temperatures
(<4 °C) and are consumed rapidly.
2. In regard to semi-soft cheeses, emulsion and extrusion micro-
encapsulation techniques might be an effective way to improve
probiotic cells’ viability and to minimize losses during salting
and/or ripening period.
3. The manufacture of probiotic semi-hard or hard cheeses must
consider the selection of the most robust probiotic strains that
are capable of surviving the salting step and long ripening
periods. It is recommended to reduce the salt content of the

cheese, or to use encapsulated probiotics.
4. Sharp et al. [7], Demers-Mathieu et al. [54] and Leeuwendaal
et al. [55] indicated Cheddar cheese as a good food matrix to
deliver probiotic bacteria to the host. Probiotic bacteria
showed good viability after 90 (7 log10 CFU/g), 120 (>
7 log10 CFU/g), and 180 (> 4 log10 CFU/g) days of ripening,
respectively.
5. Sharp et al. [7] and Leeuwendaal et al. [55] also presented
Cheddar cheese as protective matrix for probiotic delivery to
host, being those viable at 4 log10 and 6 log10 CFU/g after
simulated digestion, respectively.
6. The obstacle to manufacture probiotic cheeses, especially for
semi-hard and hard cheeses, is to keep the adequate number of
viable probiotic bacteria in cheese to be claimed as probiotic
food due to be more challenging for probiotic bacteria survival
in low moisture environment such as hard cheeses. Thus, pro-
biotic concentrations in these cheeses have to be raised in the
future in order to assure minimal daily intake of probiotics for
nutritional health.
7. Since it is not easy to modify or to introduce new and persistent
dietary habits in consumers, the success of the incorporation of
probiotic bacteria into cheeses should be done by informing
consumers about the advantages of these new products. The
practical guide aimed to shed light on the key elements to
consider for large-scale development from technical point of
view to consumer acceptability in order to help widespread
commercial probiotic cheese application.
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1854
Chapter 3
Probiotic Ice Creams

Vanessa Cortina Zanetti, Celso Fasura Balthazar, Callebe Camelo-Silva,
and Silvani Verruck
Abstract
The growing consumer demand for healthier and more functional foods has led to the introduction of new
ingredients in ice cream formulations with nutritional and physiological properties, such as probiotics.
Incorporating probiotic bacteria into an ice cream should not affect the quality of the product. Therefore,
its quality parameters such as air incorporation, melting rate, and sensory characteristics must be the same or
better when compared to conventional ice cream. This chapter is a practical guidance for probiotic ice cream
manufacture, presenting the steps and amount of probiotic addition into ice cream production.
Key words Ice cream, Probiotic, Functional food, Health
1 Introduction
Dairy products such as ice cream and frozen desserts can serve as
vehicles for delivering probiotics to humans. In addition, ice cream
can be kept in storage for longer time than other dairy products
[9]. In this context, consumers’ perception of healthy and func-
tional foods led to the introduction, in the manufacture of ice
cream, of ingredients with nutritional and physiological properties,
such as probiotics [2, 24], dietary fibers [6–8], and synbiotics
[9]. Ice cream is a complex colloidal system consisting of air cells,
ice crystals, and partially destabilized fat globules dispersed in a
continuous aqueous phase within polysaccharides, lactose, sugars
and mineral salts [15]. As well the high level of total solids in ice
cream provides protection to probiotic bacteria [20].
Probiotic ice cream is an acidified dairy frozen dessert of par-
tially frozen structures. Acidification of the ice cream mixture can
be carried out through direct inoculation of probiotic cultures, for
example, Bifidobacterium spp. and Lactobacillus spp., the mixture
of acidified milk or probiotic yogurt mixed with ice cream [3]. The
therapeutic value of probiotic bacteria usually depends on the
53
54 Vanessa Cortina Zanetti et al.
viability of these bacteria. Therefore, the International Dairy Fed-

eration [21] suggested that a minimum of 7 log10 probiotic bacte-
rial colony forming units should be viable at the time of
consumption per gram of the product (CFU/g), which is in accor-
dance with the latest International Scientific Association for Pro-
biotics and Prebiotics consensus statement [19]. The viability of
probiotic bacteria in frozen dairy desserts is limited due to intrinsic
environmental parameters, such as high redox value, oxygen toxic-
ity, rupture of bacterial cell membranes during the freezing process,
and the vulnerability of bacteria to acidic conditions [10]. There-
fore, the efficiency in adding the probiotic depends on the inocu-
lated dose, temperature, type of dairy foods, and the presence of air,
and its viability must be maintained throughout the shelf life and
intestinal environment, in addition to resisting at gastric pH [9].
Akin et al. [2] investigated the effects of inulin on the viability
of probiotic bacteria in ice cream and found that their survival was
higher in samples with inulin, probably due to the effect of the
prebiotic. L. acidophilus and B. lactis counts were less than 5 log10
CFU/g in the control samples, while in the inulin-supplemented
samples they were 5 log10 CFU/g. These results suggested that the
addition of inulin stimulated the growth and improved the viability
of those probiotic bacteria. Indeed, similar results were also verified
by Balthazar et al. [9] in fermented sheep milk ice cream, in which
synbiotic ice cream presented 7.61 log10 CFU/g and 5.18 log10
CFU/g against 6.89 log10 CFU/g and 5.02 log10 CFU/g from
probiotic version viability after 150 days of frozen storage and
in vitro simulated digestion, respectively, explained by inulin pro-
tection during storage and in vitro simulated digestion.
Homayouni et al. [20] verified in ice cream with microencap-
sulated or non-microencapsulated probiotics that there was a loss of
only 0.7 and 0.4 log10 CFU/g of Lacticaseibacillus casei and
B. lactis in the free state, respectively, and 0.3 and 0.2 log10 CFU/-
gin the encapsulated state during the first month of storage. In the
following five months, probiotic counts remained with a loss of 2.7
and 2.5 log10 CFU/g for the free state and 1.1 and 0.5 log10
CFU/g for the encapsulated state, and after the sixth month, the
final drop was 3 0.4 and 2.9 log10 CFU/g in the free state of L. casei
and B. lactis, respectively, against 1.4 and 0.7 log10 CFU/g in the
encapsulated state. The numbers of viable probiotic bacteria in all
types of ice cream were between 8 and 9 log10 CFU/g after three
months of storage, the normal shelf life of ice cream. Pandiyan et al.
[22] noticed that the melting rate of probiotic and symbiotic ice
cream is faster and this behavior was attributed to the technological
characteristics of the product such as freezing point and viscosity.
This chapter is a practical guidance for probiotic ice cream manu-
facture, presenting the steps and amount of probiotic addition into
ice cream production.
Probiotic Ice Creams 55
2 Materials
2.1 Ice Cream Ice cream can be processed with a variety of ingredients, including:
Ingredients
– Milk.
– Yogurt (see Note 1).
– Fat (see Note 2).
– Protein (see Note 3).
– Milk solids-not-fat (see Note 4).
– Water.
– Sweeteners (see Note 5).
– Stabilizers (see Note 6).
– Emulsifiers (see Note 7).
– Flavoring (see Note 8).
– Coloring.
– Probiotics—examples Table 1.
– Fruits.
– Nuts.
– Bakery pieces.
– Candy pieces.
Table 1 shows a traditional ice cream formulation. This formu-
lation can be adapted with higher or lower values than those shown
in Table 1, in addition to the addition of others, for specific
purposes.
2.2 Ice Cream The main equipment needed to produce ice cream are described
Equipment below, along with suggestions for equipment to be purchased.
– Doser: Ingredient Doser A3 (Tetra Pak).
– Mixer: High shear blender (Bredoo Likwifier); High shear
mixer B200–300 A (Tetra Pak).
– Pasteurizer: Pasteurizer D (Tetra Pak).
– Homogenizer: Industrial five-piston homogenizer (Tetra Pak);
Homogenizer 250 (Tetra Pak).
– Maturator: Incubator (cooled Incubator ILW 115, POL-EKO-
APARATURA.
– Freezer: Tetra Hoyer Frigus-KF freezer (TetraPak), WCB Ice
Cream freezer (WCB Ice Cream); Ice Cream Machine (Delon-
ghi, Il gelato, ICK5000), Ice Cream Machine ( 5 C; L/30–3,
SEVEL Cooling INC.); Continuous Freezer S300 M2
(Tetra Pak).
Table 1
Traditional ice cream composition
Composition (%) Amount (%)

Milk solids-not-fat 9.0–11.0
Milk fat 10.0–16.0
Sucrose 10.0–16.0
Corn syrup solids 2.0–5.0
Stabilizer 0.15–0.35
Emulsifier 0.10–0.15
Total solids 36.0–42.0
Water 58.0–64.0
Source: Adapted from [7]
– Filler: Rotary-type filler for cups and round nested containers

(Huhtamaki, Inc.); In-line filler for square-round packages
(TD Sawvel Co., Inc.); Ice Cream Smart Filler A1 (Tetra Pak).
– Wrapper: Ice Cream Wrapper A2 (Tetra Pak).
– Hardening: Super Deep Chest Freezer LY450LD ( 35 C)
(Snow-MY).
2.3 Probiotic Strains Inoculation of probiotics can be performed by Direct Vat Set
(DVS) or by Propagation. The DVS method promotes the use of
standardized freeze-dried cultures, with low amounts sufficient for
inoculation. The propagation method is carried out by cell cultiva-
tion in a specific medium, purification of the culture, and
subsequent incorporation into the product. Table 2 presents the
species of probiotics used in the preparation of ice cream and
describes the inoculation method used.
3 Methods
The processing of ice cream is divided into two distinct stages, the
production of the mixture and the freezing operations. To produce
quality ice cream, the steps must be carried out in a controlled
manner, adapting the desired final characteristics. The elaboration
steps include mixing, heat treatment, homogenization, maturation,
freezing, packaging, and hardening (see Fig. 1).
1. Mix the ingredients, as described in Table 1, in a tank with
agitation and heating, heating them to 50 C to facilitate
solubilization (see Note 9) [12].
2. After the complete incorporation of the ingredients, pasteurize
the mixture at 70–85 C from 30 s to 30 min (see Note 10) [5].
Table 2
Information about probiotic strains added to ice cream formulations
Inoculation Inoculation Probiotic viability

dose temperature Storage after storage
Probiotic strain (CFU/mL) Inoculation method ( C) (days) (CFU/mL) Reference
Lactobacillus acidophilus 9.0 90% of the milk was transferred into two sterile 4.0 60 7.37 [4]
La-5® jars, and the milk samples were inoculated with
freeze-dried L. acidophillus La-5® and
incubated at 37 C/4 hours until the pH
reached 4.7.
Bifidobacterium bifidum 7.56–7.60 Freeze-dried Bifidobacteria was prepared in 4.0 60 6.20–6.28 [5]
200 mL of reconstituted skimmed milk (10%
w/w), and it was incubated at 37 C/24 hours.
The probiotic was reactivated into 300 mL of
milk and incubated at 37 C until the pH
became 4.6.
Lactobacillus acidophilus 8.20 The L. acidophilus La-5 was inoculated in the ice 37 60 7.25 [13]
La-5® cream mix and incubated at 37 C until the pH
reached 5.80.
Lactiplantibacillus 6.0–7.0 About 1% of L. plantarum subsp. plantarum 40 60 >7.46 [16]
plantarum subsp. inoculum was cultured in sterile man, Rogosa,
plantarum ATCC 8014 and sharp broth and incubated overnight at
37 C. after that, the cells were centrifugated
and washed twice with sterile peptone water.
Approximately 1 mL of L. plantarum subsp.
plantarum after 20 h of incubation was added
to 150 mL of pasteurized full cream milk.
Saccharomyces boulardii 7.34 The S. boulardii was incubated in YPD broth for 37 28 6.2 [26]
Lacticaseibacillus rhamnosus 10.11 48 h, and the L. rhamnosus was incubated in 9.2
GG MRS broth for 24 h at 37 C. both strains were

grown in 5 L volumes to obtain them at desired
levels before the inoculation of the ice cream
57
(continued)
58
Table 2
(continued)

dose temperature Storage after storage
Probiotic strain (CFU/mL) Inoculation method ( C) (days) (CFU/mL) Reference
mix. After the incubation, the cultures were
centrifuged, and obtained pellets were washed
twice with PBS and inoculated to the
pasteurized ice cream mix to 37 C before and
after the aging steps.
Vanessa Cortina Zanetti et al.
Lacticaseibacillus paracasei 9.0 The starter probiotic cultures were previously 37 21 10.18–10.17 [17]
subsp. Paracasei L-26 inoculated in milk at 40 C for 5 hours. 5% of 9.88–9.85
Lacticaseibacillus casei 431 inoculum was added to the ice mixes. 8.83–9.68
Lactobacillus acidophilus
La-5®
Bifidobacterium lactis 9.0 The culture was dissolved into UHT milk and 2 120 7.16 [14]
(Bl-04) activated to obtain 109 CFU/mL of bacteria
cells in MRS broth.
Lacticaseibacillus paracasei 8.0–9.0 The probiotic cultures were inoculated in the ice 37 120 8.19 [1]
subsp. Paracasei L-26 cream mix by the commercial company’s 8.15
Bifidobacterium longum + recommendations. The inoculated mixtures
Bifidobacterium bifidum were left to incubate at 37 C, which was carried
B-94 out until the pH values reached 4.8–4.9.
Lactiplantibacillus 8.50–9.0 An aliquot of each probiotic culture was 37 60 >7.50 [25]
plantarum subsp. individually transferred into MRS broth and
plantarum LP299v placed in an incubator at 37 C for 24 hours.
Lacticaseibacillus casei After, the 10 mL culture was diluted to 100 mL
ATCC 393 and re-incubated at 37 C/48 h. it was
centrifuged to isolate the probiotics from the
MRS.
Lactobacillus acidophilus 8.0 Freeze-dried cultures of probiotic strains were 37 180 5.95 [18]
Bifidobacterium lactis inoculated separately in glass tubes containing >6.0
MRS broth and incubated at 37 C/24 h under
aerobic conditions. Then they were
centrifuged, and washed twice with sterile
saline. The resulting pellet was diluted in sterile
saline.
Lacticaseibacillus casei 01 6.0 100 mg (w/w) of freeze driedLactobacillus casei- 37.0 150 > 7.0 [9]
01 in 1 L (v/v) of skimmed sheep milk (w/v)
for 6-h incubation. Subsequently, fermented
sheep milk was added by sheep milk fat and
skimmed sheep milk to totalize 2 L of mix
added with inulin, sugar, and stabilizer/
emulsifier.
59
Mixing
(milk, sweeteners, stabilizer, emulsifier)
Heat Treatment
(70.0 - 85.0˚C/30 s - 30 min)
Homogenization
(15.5 - 18.9 MPa/3.4 MPa)
Cooling
(4.0˚C)
Probiotic Incorporation
(DVS or Propagation)
Maturation
(4.0˚C/4 - 24 h)
Freezing
(-5.0 - -6.0˚C/10 - 20 min)
Packaging
Hardening
(-18.0˚C)
Fig. 1 Flowchart of ice cream making
3. Then homogenize the pasteurized mixture in two steps: at high

pressure (15.5–18.9 MPa) and after low pressure (3.4 MPa)
(see Note 11) [23].
4. After homogenization, incorporate probiotic microorganisms
into the mixture (see Note 12) and cool until you reach 4 C to
start the maturation stage.
5. Perform maturation by stirring the mixture at 4 C tempera-
ture for a time of 4–24 h (see Note 13). After complete matu-
ration, the mixture proceeds to freeze.
6. Freeze the ice cream in equipment containing a rotary stirrer,
for air incorporation, up to an overrun of 50%, at a temperature
of 5 to 6 C for 10–20 min (see Notes 14, 15, and 16).
7. Then fill the ice cream and proceed to the final hardening at
30 C or lower (see Note 17), with subsequent storage at
18 C (see Note 18).
4 Notes
1. Yogurt is used in the preparation of frozen yogurt.

2. Fat sources can come from milk fat, such as cream milk, butter,
and butter oil, fats, and oils from plants, such as corn, sun-
flower, canola, and peanut, and blends of oils [15].
3. Protein sources may or may not be milk. Whey proteins and
caseins are used, in addition to soy proteins and nuts [15]. Pro-
teins are also used to give ice structuring.
4. The milk solids-not-fat contribute to the flavor and texture.
The industry usually uses concentrated milks, dried skim and
whole milk, milk power blends, and whey products [15].
5. Sweetening sources may be corn sweeteners, maple sugar,
honey, invert sugar, fructose, molasses, malt syrup, brown
sugar, lactose, sugar alcohols, sorbitol, mannitol, xylitol, and
other nonnutritive sweeteners such as saccharin, aspartame,
and sucralose [15].
6. Examples of stabilizers used: carob gum, guar gum, xanthan
gum, sodium carboxymethylcellulose, sodium alginate, micro-
crystalline cellulose, carrageenan, gelatin, and pectin [23].
7. The emulsifiers added in ice cream formulations are of two
types: mono- and diglycerides and sorbitan esters, as polysor-
bate 80. Some factories also use eggs or egg yolk.
8. The most used flavors are chocolate, vanilla, and strawberry.
But, neopolitan, lemon, nut, pear, rum and raisin, cookies and
cream, and others can also be added.
9. Automatic dosing pumps or tanks in load cells can add liquid
ingredients. Dry ingredients are added by pumping at high
speed or with high shear mixers to prevent the formation of
lumps. Dry ingredients should be incorporated into the mix-
ture at a temperature below 30 C [12, 15].
10. The four methods to pasteurize ice cream can be
low-temperature long-time (LTLT—69 C/30 min), high-
temperature short-time (HTST—83 C/15 s), higher-heat
shorter-time (HHST—90 C/1–3 s), or ultra-high tempera-
ture (UHT—138 C/2 s) [11]. Batch pasteurization uses
double-shirt tanks, in which the mixture is heated, with steam
circulation or hot water inside, performing heat changes. Con-
tinuous pasteurization is performed in heat exchangers, and
there may be a preheating of the mixture between 30 and 40 C
to mix the ingredients.
11. Homogenization is performed in two stages so that in the first
stage fat globules tend to group and form agglutinated. In the
second stage, the adsorption of proteins occurs on the fat
surface, avoiding further regrouping, and making the emulsion
more stable.
12. The cultures can be added to ice cream in several ways, of type
DVS (direct vat set), for direct addition of the product in a
pasteurized mixture, or in the use of milk as a substrate for
fermentation [4].
The freeze-dried probiotic culture can be added to the
mixture before maturation and freezing, which presents advan-
tages related to the easy insertion of the same in the mixture;
however, as it is not in its active form, it may be that the
probiotic remains inactive. In addition to freeze-dried culture,
there is the possibility of incorporating probiotic biomass into
the mixture before maturation [5]. The added biomass fer-
ments the mixture, which then proceeds to maturation.
When milk is used as substrate for probiotic incorporation,
the step can be performed by means of a partial mixture of milk
with probiotic, so that 10 to 30% of milk proceeds to a fermen-
tation stage with probiotic culture, at 37 C, for up to 12 hours
in anaerobiosis, and then that fermented milk is incorporated
into the rest of the mixture before freezing. This partial fer-
mentation of milk promotes the activation of probiotic culture,
besides not significantly altering the organoleptic characteris-
tics of ice cream [4].
Also, from the use of milk, it can be entirely fermented by
probiotic culture and, after, the other ingredients are added.
However, this type of process ends up resulting in more acidic
sensory characteristics due to the high production of lactic
acid [4].
13. In the maturation stage, the addition of flavorings and dyes
that are sensitive to the heat of thermal processing is carried out
aseptically. The additives added, both in the mixing stage and
in maturation, cannot interfere with the action of the probi-
otic, nor cause any kind of damage. At this stage occurs the
development of the sensory characteristics of flavor and aroma
of the product.
14. The freezing stage is one of the main parts of the preparation of
ice cream because there is the incorporation of air in the ice
cream, also known as overrun, a step that gives the characteris-
tic of the body and texture of the ice cream. The amount of
overrun should be between 2.5 to 3 times the total solids of the
ice cream. The presence of this stage in the preparation of ice
cream causes risks to the survival of probiotics, due to oxygen
toxicity, and the use of aerotolerant strains is necessary. In
addition, the size of the particles should be monitored, and
the ice crystals should have sizes of 30 to 50 um, air bubbles
from 20 to 80 um, the agglomerated fat globules from 2 to
20 um, and the isolated fat globules of 0.1 to 15 um [23].
15. The freezing step takes place in two stages. The first step takes
place by passing the mixture in a high-beat shaved surface heat
exchanger to allow extensive nucleation of ice crystals and air
incorporation. The second step is freezing the ice cream packed
in reduced time to prevent the formation of large crystals [23].
16. The probiotic cultures are usually sensitive to freezing, so the
incorporation of air and the reduction at shallow temperatures
become a lethal medium for these bacteria. The use of cryo-
protection, such as sugars, fats, or proteins, promotes the
improvement of the resistance of these bacteria to the frozen
environment, considering freezing time. The encapsulation of
probiotics with these cryoprotectants, in addition to protecting
against freezing, also has improved viability during passage in
the gastrointestinal tract.
17. During storage, it is extremely important that there is no great
variation in temperature, to the point of releasing water in the
ice cream, because this failure promotes recrystallization, with
the increase of ice crystals, leading to an unpleasant texture in
the product. In ice cream, small and quite numerous crystals
are sought, the opposite of this is considered a manufacturing
defect [23].
18. The formation of ice crystals occurs in two stages. Nucleation is
the step that occurs on the wall of the heat exchanger, with
small and numerous ice crystals. The low temperature during
hardening promotes the continuous growth of the formed
crystals. When hardening is slow, the remaining water in the
ice cream migrates to the crystals already formed, causing large
crystals, which promote the disruption of the cell membrane of
probiotics and lead to their inactivation [23].
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Chapter 4
Probiotic Butter
Patrı́cia Blumer Zacarchenco , Leila Maria Spadoti,
Adriana Torres Silva e Alves, Vanessa Cortina Zanetti, and Silvani Verruck
Abstract
In recent years butter has seen a growth in its consumption because of the understanding of the physiologi-
cal effects of dairy fat. The small-scale batch production of butter depends on the operator’s hands-on skill
and experience about cream aging, ending point of churning, and other parameters at each of the
manufacturing stages. Large-scale processes are automated and allow better control to produce butter
with constant characteristics. While the principles of butter making have not changed significantly over
many decades, with greater understanding, control of key parameters during cream preparation and
processing conditions have improved. In recent years there has been a lot of products from butter post-
churning, mixing new ingredients like probiotics, flavors, and spices that will be beneficial in attracting and
engaging consumers. Butter has shown to be a good matrix for adding probiotics and maintaining its
viability throughout storage due to the fat protection effect. There are several ways of adding probiotics in
butter, such as in microencapsulated form, together with traditional starters during cream fermentation or
during the working step. Thus, this chapter is a practical guidance for probiotic butter manufacture.
Key words Milk fat, Butter, Probiotic, Functional food, Health
1 Introduction
The amount of fat in milk and the fatty acids content can be
influenced by many parameters such as the diet of the cows, num-
ber of pregnancies, animal health, breed, and stage of lactation. The
type of feed or pasture consumed by the animal has been used to
modify the fatty acid profile of dairy fat to obtain beneficial nutri-
ents as the increased polyunsaturated content. However, all
changes in the percentage and types of fatty acids in milk can
influence the processing and characteristics of manufactured pro-
ducts. The lipid fraction of milk corresponds to 4.2% of dairy solids
and is composed of about 98% of triglycerides and other compo-
nents in smaller amounts, such as phospholipids, sterols, lipopro-
67
68 Patrı́cia Blumer Zacarchenco et al.
teins, and vitamins. It is important to remember that milk is a liquid

emulsion of oil in water, which is very nutritious because it is a
source of several essential compounds [1].
The butter production has grown worldwide [2] while the
price of butter has doubled in the international market and its
consumption grows by about 4% per year [3]. Around 40% of the
consumers consulted on the research stated that they started to buy
more butter because it is a healthier option [3]. This change in
demand for butter is in function of changes in consumer percep-
tions of dairy fat. While in the past dairy fat has been associated with
an increased risk of cardiovascular disease, there is growing evi-
dence to suggest that regular consumption of dairy products with
a regular fat content is not associated with an increased risk of these
diseases [4]. In addition, some benefits of consuming dairy pro-
ducts with a regular fat content have been demonstrated in terms of
the presence of bioactive nutrients and anti-inflammatory proper-
ties [5]. Butter is a complex of at least 400 types of fatty acids, the
consumption of which, combined with a balanced diet and a
healthy lifestyle, can protect against certain types of cancer, and
reduce the risk of cardiovascular disease. Due to this new perspec-
tive on milk fat and its effect on human health, research in ruminant
nutrition has generated results that demonstrate the production of
bovine milk from tropical grasses can increase the level of beneficial
fats. One of these fatty acids, known by the acronym CLA (conju-
gated linoleic acid), from its chemical name, has proven anti-
carcinogenic properties. Several works carried out by [3] have
been demonstrating that cows fed with fresh elephant grass
increased the CLA content in the milk, therefore, also in the butter.
Butter is one of the oldest dairy products that is still being
made. Butter is obtained by concentrating milk fat in the form of
cream, which is then churned until the oil-in-water emulsion is
converted to water-in-oil emulsion. There are criteria for the com-
position of the product classified as butter. According to Codex
Alimentarius [6] butter is a fatty product derived exclusively from
milk that must contain at least 80% fat, a maximum of 16% mois-
ture, and 2% defatted milk solids (proteins, lactose, and minerals).
USDA [7] also defines this minimum fat content, in addition to
other parameters, and indicates a three-level classification (AA, A,
and B) based on the sensory attributes of the butter. In Brazil, there
are also standards defined in the legislation to regulate the mini-
mum fat content in butter, in addition to the maximum moisture
content, sodium chloride and acidity, among other physical–chem-
ical and microbiological parameters [8, 9].
Butter can be produced from sweet cream or fermented cream,
with and without salt. It is used in several culinary preparations,
such as an ingredient in the chocolate, confectionery, and bakery
industries, in addition to the dairy industry. In cream fermentation,
different probiotic or non-probiotic lactic ferments can be used.
Probiotic Butter 69
The ferment can be defined as a microbial preparation containing a

large number of cells of at least one microorganism that is added to
a raw material (in this case, cream) to produce the fermented food
and it can speed up and control the fermentation process [10].
The group of lactic acid bacteria (LAB) plays an important role
in this process and has a long history of safe application and con-
sumption in fermented foods and beverages. These bacteria gener-
ate rapid acidification of the raw material through the production of
organic acids, mainly lactic acid, but also acetic acid, ethanol,
aromatic compounds, bacteriocins, exopolysaccharides, and several
important enzymes. With this, these bacteria increase shelf life,
improve texture, and contribute to pleasant sensory characteristics
in the final product [10]. Most of the lactic acid bacteria belonged
to the genus Lactobacillus, which was recently reclassified [11]. The
member species were distributed in 25 genera, including those that
remained in the original genus Lactobacillus. Thus, species of pro-
biotic lactic acid bacteria, or not, applied in butter studies may have
been reclassified to other genera and have their gender designation
changed [12]. Several LAB strains are recognized as probiotics.
Probiotics are defined as “live microorganisms that, when adminis-
tered in adequate amounts, confer a health benefit on the host”
[13] indicating that viability is a necessary requirement to ensure
the expected benefits. In turn, paraprobiotics, defined as non-viable
cells, and postbiotics, which are substances generated by bacteria,
respectively [14–16], have been studied and the health benefits are
not all necessarily related only to viable cells. Thus, probiotics, but
also paraprobiotics and postbiotics, can be present in butter, adding
additional beneficial characteristics to the bioactivity of naturally
present nutrients such as CLA, phospholipids, and vitamins. In this
sense, this chapter’s purpose is to describe the butter
manufacturing and possible steps where probiotics can be added
during the process.
2 Material and Methods
There are different butter varieties mainly involving the presence or

absence of salt and primary fermentation of the cream. The type of
ferment used in this fermentation will influence the flavor of the
final butter (see Note 1). There are also different methods of
making butter which include emulsification and the more well-
known churning method. Figure 1 shows the process of making
salted butter from cream using churning method. In addition, the
main stages in the manufacture of butter will be presented so it can
be verified in which stages the probiotics can be added.
Fig. 1 Steps of the process of making salted butter
2.1 Cream The cream can be extracted from milk kept at rest if it has not been
Preparation treated in the double-piston homogenizer, as it separates naturally
due to the fat particles being less dense and floating in the upper
portion of the tank. It is more applied, however, the separation
using centrifugation with the milk heated above 40 °C so that less
damage occurs to the milk fat globule membrane (MFGM) since
the fat will be in liquid form. An optimal temperature condition is
63 °C, for a cream with 35 to 40% fat or 45 to 48%, depending on
the equipment and process [1]. There is a batch equipment called
skimmer used to separate the cream from the milk and continuous
separators for larger industrial volumes. After separating the cream,
it undergoes thermal treatment in plate heat exchangers with time
and temperature binomials between 85 and 110 °C for 10 to
30 seconds to eliminate pathogenic microorganisms and reduce
the counts of deteriorating microorganisms (see Fig. 1). At this
stage, vacuum can be applied to remove volatile compounds with
unpleasant odors that can influence the sensory characteristics of
the final product. After the thermal treatment, the cream is cooled
to 4–5 °C so the maturation stage takes place during a minimum
period of 4 hours or “overnight” when the milk fat crystallizes (see
Note 2).
For the cream maturation, there are different combinations of
time/temperature that influence the shape of the crystals that
appear in the cream and it will affect characteristics as consistency
and spreadability of the butter after churning. The greater efficiency
of the different cream maturation time and temperature binomials
Probiotic Butter 71
are related to factors such as the season of the year and the levels of
unsaturated fatty acids present [17, 18] (see Note 3). Slow and
rapid cooling of the cream can result in rheological properties and
crystals with similar polymorphic forms (α- and β‘-), but with
differences in their microstructure (see Note 4). Unlike butter
produced from slowly cooled cream, rapid cooling results in butter
with more uniformly sized crystals (see Note 5). When the amount
of low melting fat is reduced (iodine number less than 35 I2/100g)
the butter will have a firm texture if the cream is cooled in stages. In
these cases, the application of the “cool/heat/cool” procedure
called “Alnarp method” can be beneficial for the butter final tex-
ture. Cooling to 8 °C after heat treatment, keeping at this temper-
ature for 2 hours, heating to 20 °C, keeping for 2 to 3 hours at this
temperature, and finally cooling to room temperature (see Note 6).
Finally, the cream is cooled down to the churning temperature.
There are several temperature sequences for chilling the cream
before churning to separate the butter grains. For example, a
combination is cool to 8 °C, raise to 18 °C and cool to 12 °C or
cool to 8 °C, raise to 22 °C and cool again to 12 °C (see Note 7).
One of the ways of adding probiotics to butter is through their
direct inoculation into the cream with or without a fermentation
step (see Note 8). Several probiotic strains are available for use as
DVS (Direct vat set) inoculums in butter production. The main
cultures already used in probiotic butter and cell viability after
storage are summarized in Table 1. The cell amount to be added
into the butter varies between different strains to ensure the health
benefits. The amounts of cells added by [19, 20], and others are
adequate to keep cells active until the end of storage.
2.2 Batch Butter 1. The pre-treated cream is transferred to the mixer (see Note 9)
Making Process where it is stirred at high speed to break the emulsion, and the
grains of butter emerge as the drained buttermilk separates.
2. The temperature range for churning is 8 to 12 °C.
3. After the emulsion has broken, the buttermilk is drained from
the tank and can be filtered to remove fat lumps.
4. Add fresh water to the churner and whisk to wash the grains
and remove buttermilk residues until the water runs clear.
5. The working (mechanical treatment) of the butter takes place
at low speed with the tank valve open to drain the buttermilk
expelled from it. When the amount of water is sufficiently low,
the valve is closed and, if necessary, salt is added.
6. As the working time is longer in the batch process compared to
the continuous one, the salting process can take place by dry,
wet, or brine salting (see Note 10).
7. When the butter appears to be dry, the working is stopped and
the moisture content is measured and adjusted, continuing the
mechanical work until all the free water has been absorbed.
8. Then, the butter is packaged.
Table 1 72
Information about probiotic strains added to butter formulations

dose (log temperature Storage after storage
Probiotic strain CFU/g) Inoculation method (°C) (days) (CFU/g) Reference
Lactobacillus acidophilus 10.63 The microorganisms were encapsulated using the 10.0 60 8.90 [19]
La-14 technique of extrusion and ionic gelation. The
encapsulating matrix used was low viscosity sodium
alginate at a concentration of 10 g/L, dripped onto
the covering material, 0.1 Mol/L calcium chloride
dihydrate. The capsules had an average diameter of
1.8 mm. The culture was lyophilized.
Bifidobacterium animalis 8.98 The spray-dried formulation was prepared by a – – 5.09–8.22 [20, 21]
subsp. lactis ATCC combination of probiotic microorganisms, in a
Patrı́cia Blumer Zacarchenco et al.
27536 conjugated whey protein hydrolysate (WPH10-

Lactobacillus acidophilus maltodextrin) matrix (1:1). Pure freeze-dried
ATCC 4356 probiotic cultures were obtained from ATCC. Fresh
cultures were obtained after two transfers in de man,
Rogosa, and Sharpe broth (MRS) supplemented
with 0.05% (w/v) L-cysteine at 37 °C for 72 h,
under anaerobic conditions. Propagation of the
cultures was continued, and the cells were harvested
in their late log phase by centrifugation at 7000 × g
for 10 min at 4 °C. the cell pellets were washed twice
in phosphate-buffered saline (PBS) and suspended
to achieve cell suspensions of 10 log CFU/mL.
Lacticaseibacillus casei 8.2–8.7 The probiotic culture was pre-activated in pasteurized 19.0 1.42 >6.0 [22]
Lactiplantibacillus milk for 15 h at 35 °C, with an initial concentration
plantarum subsp. of 0.5% (w/v) of milk. Then, the pre-activated
plantarum inoculum, with a probiotic level of 8.2–8.7 log
Lacticaseibacillus CFU/mL was used to inoculate the cream applied to
paracasei subsp. produce probiotic butter samples.
paracasei
Lyofast CPR1
Lacticaseibacillus casei 7.0 After obtaining butter, the formulations with probiotic 4.0 90 4.81–5.80 [23]
LAFTI L 26; CSL3 bacteria were supplemented using 0.1% of each
probiotic (7 log CFU/g of butter).
Lactobacillus acidophilus 6.0–8.0 The culture was used for fermenting cream. It was 20.0–37.0 42 >6.0 [24]
La-5 added to the cream at fermentation temperature,
which varied according to the characteristics of the
seasons. On I group the fermentation took place at
30 °C and physical maturing at 7 °C. on II group the
fermentation took place at 37 °C and physical
maturing at 7 °C. on III group to simulate autumn-
winter season cream fermentation took place after
physical maturing, at 20 °C. to simulate spring-
summer the cream fermentation took place before
physical fermentation, at 20 °C. IV group
introduced the fermenting cultures in butter kernel.
The samples were kept at 9 °C for 3 days for the
increase of acid-creating activity.
Bifidobacterium bifidum 6.60–6.66 The cream was pasteurized, cooled to 18–20 °C, and 18.0–20.0 60 6.57–6.68 [25]
ATCC 29521 divided in three parts for each replicate. All batches
Lactobacillus acidophilus were inoculated with direct vat set culture (freeze-
ATCC 4356 dried) at a level of 15 g/500 L.
Bifidobacterium lactis >9.0 The butters were prepared in seven repetitions by – 28 >7.4 [26]
B100.6 churning the pasteurized cream (30% (v/v) fat) with
the probiotic bacteria. The prepared butters were
stored at 6 °C for 4 weeks.
Probiotic Butter
73
2.3 Continuous Most continuous butter production processes are based on the
Butter Making Process “Fritz method,” the German scientist who designed the first equip-
ment for this purpose. The current equipment have some points of
difference in relation to the originally built one, but follow its
operating principles containing a churning section, separation sec-
tion, and working sections containing a vacuum chamber (see Note
11). At the output of the equipment, the butter is packaged.
2.3.1 Churning The heat-treated cream is transferred to the churning section via a
pump. There are studies carried out on the production of butter
with probiotics that involve the addition of microencapsulated
probiotics to the butter in the churning stage (Table 1). In this
step, the cream is churned in a horizontal cylinder with an adjus-
table speed rotary beater. The phase inversion occurs in a few
seconds and the speed of the beater regulates the size of the grains
formed, which influences the fat loss to the buttermilk (see Note
12). Churning temperature is particularly important as phase inver-
sion will only take place if there is enough liquid fat (see Note 13).
In general, the churning temperature is around 10 to 12 °C. The fat
content in the buttermilk should not be higher than 0.3 to 0.5% if
there is an optimized churning (see Note 14).
2.3.2 Buttermilk After phase separation, the buttermilk and buttermilk mixture are
Remotion carried from the churning section to the separation section. This
section consists of a horizontal, slow-rotating sieve drum with
adjustable speed where the butter grains are retained, while the
buttermilk passes through a finely meshed wire screen (see Note
15).
2.3.3 Working The butter grains, now gathered in larger lumps, are then trans-
ferred into the first working section in which a pair of parallel
contra-rotating endless screw transports the butter forward and
“squeeze” most of the remaining buttermilk out of the product
(see Note 16). This step allows for the water content to be adjusted
so that probiotic culture addition and/or salting can be carried out
if required without exceeding the limit of 16 g/100 g water in the
final product. If the cream used in butter manufacturing has not
been inoculated with probiotics, they can also be added at this stage
(Table 1). Some butter machines are equipped with a couple of
working units consisting of perforated plates interspersed with
mixing vanes where the intensive working of the butter is per-
formed. Another approach is to install a medium-shear mixer after
the first working section for the same purpose. From the first
working section the butter is conveyed either directly or indirectly
via a butter pump to the second working section, where the final
working takes place (see Note 17). The working temperature must
be kept low (14–16 °C), as this temperature determines the size
and the composition of the continuous fat phase, and thereby the
extent of the three-dimensional crystal network (see Note 18).
Probiotic Butter 75
2.3.4 Salting Salt can be added in the last part of the first working section by a
dosage pump with adjustable capacity and mixed into the butter by
the working units. Subsequent working of the butter is accom-
plished in a short time that is insufficient for dissolving large salt
grains (see Note 19).
3 Notes
1. The buttermilk resulting from the production of butter made

with fermented cream is acidic, which restricts the possibilities
of application compared to non-fermented one.
2. The temperature of the cooling step can be adjusted to achieve
those apropriated to the multiplication of the probiotic or
non-lactic ferment that wants to add. Instead, the probiotic
can be added as an ingredient (without multiplying in the
cream or butter) in the kneading step.
3. Studies such as those by [27] found that butter produced with
sweet cream (without fermenting) from milk from cows raised
on pasture had a softer texture and different melting properties
than that produced from milk cream from stabled cows fed
with feed. Milk from cattle on pasture has advantages in terms
of increasing the content of polyunsaturated fatty acids such as
omega 3, vaccenic acid, and CLA while reducing the levels of
omega 6 and palmitic acid [28]. During the summer when
cows are on pasture, dairy fat is less saturated and softer than
winter cream, which contains higher levels of saturated fatty
acids, resulting in harder fat. In general, the rapid cooling of
the cream leads to the formation of crystals with low levels of
liquid-free fat, which makes the texture of the butter harder
and with lower spreadability characteristics. This characteristic
can be improved with the method of maturation of the cream
where it has its temperature increased in the initial stage and
then reduced in two distinct stages. This technique involves
cooling the cream to 20 °C, after heat treatment, keeping it for
a few hours at this temperature, cooling it to 16 °C, keeping it
for 2 to 3 hours, and, finally, cooling it down to the churning
temperature. This maturation method leads to the separation
of triacylglycerols with a high melting point from those with a
low melting point, forming crystals that have laminated struc-
tures with the first ones in the center and layers of the second
ones that are formed with the decrease in temperature. Butter
produced with cream cooled in stages has a higher liquid fat
content than that produced with cream cooled quickly and
therefore has a softer and more spreadable consistency.
4. There may be differences in the polymorphism of milk fat

crystals as a function of the cream’s cooling speed [29]. It is
noteworthy that the polymorphism is the phenomenon in
which milk fat (solid) crystallizes in more than one form, pre-
senting three-dimensional structures of crystalline packaging,
giving it a distinct property.
5. Rapid cooling of the cream does not cause changes in the
rheological profile and microstructure of the butter. However,
slow cooling of aged cream will result in butter with a firm and
brittleness texture, due to the formation of a denser crystal
network. Butters produced from unripened creams are mainly
formed by α- and β‘- crystals and by few crystals in the β- form.
Maturation results in a transition of crystals from the α- to β‘-
form and to the β- form which is formed by the recrystallization
of fat. This alteration in the structure of the cream facilitates the
next stage of obtaining butter, which consists of churning the
cream [30]. The chemical composition and the mechanical
treatment employed in obtaining butter can also affect the fat
crystal network. Fat crystals are present both in the continuous
fat phase and within milk fat globules [29].
6. This method favors the formation of crystals with a laminated
structure, increases the liquid fat content, and reduces the
firmness of the butter compared to the “heat–cool–cool”
method. If the objective is to churn sweet cream, a frequently
used cooling procedure is to start by chilling at 8 °C for at least
2 hours, regardless of the dairy fat composition. The cream is
then heated to the temperature indicated by the composition of
the fat considering the iodine index, and the higher this index,
the lower the intermediate heating temperature in the cream
maturation process.
7. These temperature variations are made to achieve adequate
crystallization of the milk fat and to facilitate and/or optimize
the separation of the butter grains. Different temperature com-
binations can be suitable for creams produced in summer or
winter.
8. The addition of probiotics has also been widely discussed in
terms of food safety since some strains can be used as protective
cultures (bioprotection). This characteristic is associated with
some species of Lactobacilli, Streptococci, Enterococci, Lacto-
cocci, and Bifidobacteria having a long history of safe use,
proven antimicrobial properties, ability to naturally dominate
the microflora, and occupy the ecological niche during the
storage of products [31].
9. The process and equipment used to manufacture butter can be
dependent on many factors, including production volume. For
small-scale operations and artisanal production, the butter
maker or churn is better adaptable and requires less investment.
Probiotic Butter 77
In general, 100 L of raw milk yields 4 to 5 kg of butter. There

are butter churns of different sizes ranging from those for
around 5 kg of cream at 40% fat to those for processing several
hundred kilos of cream. The typical mixer consists of a hori-
zontal stainless-steel barrel that rotates around a shaft or a
cylindrical tank with a rotating rod or paddle inside.
10. In wet salting, salt is moistened with water before being added
to butter as a dough. This method leads to the rapid solubili-
zation of salt in butter. Salting in brine requires the amount of
water in the butter to be low. In this stage of working, it is
common to use vacuum (20 kPa). After adjusting the moisture
content, working continues, but with less vacuum intensity, as
excess can lead to the migration of liquid fat and the presence
of free oil droplets in the butter.
11. The capacities of these continuous equipment range from
500 to 15,000 kg/h.
12. Both very high and very low speeds will increase fat loss. The
general rule is that the lowest speeds should be used and will
result in grains with a diameter of 2 to 4 mm. The optimum
churning speed depends on the fat content and temperature of
the cream. Lower speeds are used for higher fat content and
higher temperature.
13. If the liquid fat content is very low, high rotational speeds are
needed to increase the temperature of the cream until there is
enough liquid fat. However, raising the temperature by
mechanical agitation consumes a lot of energy. However, if
the churning temperature is too high, there is a loss of fat in
the buttermilk and an increase in butter moisture.
14. If the sweet buttermilk (from unfermented cream butter) is to
be reused in other dairy products such as cheese or powdered
milk, the fat present in it will also be reused.
15. It is especially important to keep the temperature of the butter
low through the entire process, and an efficient way to do that
is to cool the butter grains in the separation section before they
gather into larger lumps. This cooling can be achieved by
spraying the butter grains either with chilly water or even
better with recirculated cooled buttermilk, which will not
cause dilution. Another way of controlling the temperature of
the butter grains is circulating chilled water in the jacket of the
separating section, but this is not as efficient as spraying the
butter grains.
16. Different parameters influence the water content such as
low-fat content of the cream, increased churning temperature,
inadequate churning speed, size of butter grains, and inade-
quate working temperature. It should be noted that the inter-
action between these parameters is very strong, so careful
adjustment of their effect must be done.
17. It is important that the working intensity is high enough to

ensure a homogeneous texture in the butter. An intensity that
is too low will result in loose or free moisture in the product,
and an intensity that is too high will result in a greasy and sticky
consistency.
18. The working temperature can be controlled by circulating
chilled water in the jacket of the two working sections.
19. Undissolved grains will attract moisture during storage, which
will result in free water droplets in the butter and reduced
keeping quality. It is therefore necessary to use very fine-
grained salt (average particle size around 15 mm), which
could be added as a suspension (e.g., 100 g salt in 100 g
water). It is critical that the salt is not contaminated, especially
with copper and iron, as this will reduce dramatically the oxi-
dative stability of the butter.
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Chapter 5
Probiotic Plant-Based Beverages

Abstract
Vegetable matrices are suitable substrates for obtaining probiotic plant-based beverages. The development
of these products aims to serve a new segment of consumers who prefer plant-based foods or have
restrictions on consuming dairy products. Here we will describe the process of obtaining soy, oat, and
rice extracts. Next, we will discuss the fermentation process to obtain probiotic beverages.
Key words Fermentation, Soy, Oat, Rice, Functional food, Plant-based drinks, Milk alternatives
1 Introduction
Demand for probiotic plant-based beverages has grown worldwide

among dairy and non-dairy consumers. Consuming probiotic bev-
erages to improve gut health is not new [1]. However, the demand
for these products also includes rising vegetarianism and emerging
veganism, lactose intolerance, allergenicity for dairy products, dys-
lipidemia, and consumer demand for differentiated products [2, 3].
Non-dairy plant-based food matrices such as aqueous extracts
of plant-based cereals and legumes have successfully been used to
produce probiotic beverages. These substrates contain nutrients
easily assimilated by probiotics, stimulating the growth of single
and mixed cultures during fermentation [3–5] and providing better
food product digestibility. The primary sources used to develop
probiotic plant-based beverages are soybean, malt, wheat, barley,
tree nuts, rice, and oat, which are suitable substrates for microbial
growth [3].
Soy extract (commonly known as soymilk) is rich in com-
pounds important for nutrition and beneficial for health [6]. Soy
extract has a similar appearance and chemical composition to animal
milk and can be used as a substrate for fermentation by lactic acid
bacteria [7]. Soy extract has 3.0% protein, 2.0% lipids, and 2.0%
81
82 Tahis R. Baú et al.
carbohydrates, mainly sucrose, raffinose, and stachyose [8]. This

product is an alternative to milk due to its protein quality, absence
of cholesterol and lactose, and functional properties. However,
consumption of this product is limited due to the characteristic
flavor and astringent potential of soybeans due to the presence of
lipoxygenase enzymes and non-digestible oligosaccharides [9].
Oat (Avena sativa L.) is a gluten-free cereal with significant
nutritional and functional values, mainly due to its high content of
β-glucans. Intake of these soluble fibers has been associated with
reduced serum cholesterol and risk of cardiovascular disease
[10]. The utilization of this information is authorized by the
U.S. Food and Drug Administration (FDA) and European Food
and Safety Agency (EFSA) to appear on food labels that contain the
minimum amount of β-glucan required for such a health claim.
Other oat constituents, such as avenanthramides, tocols, sterols,
phytic acid, and avenacosides, have also demonstrated diverse
health benefits, including anticarcinogenic and antihyperglycemic
activities and improvements in gastrointestinal disorders [11].
Rice is a basic and important food for many of the world’s
population, and the Oryza sativa L. is the most widely grown.
The rice grain has a high concentration of carbohydrates, mainly
starch, and lower concentrations of vitamins and minerals [12]. The
rice grain has potential health benefits so that it can be used as a
matrix for plant-based extracts and a vehicle for producing probi-
otic foods [2]. Plant-based rice extract is a non-dairy beverage
extracted from ground rice grain in water followed by homogeni-
zation, obtaining emulsions in the water phase of the components
present in the rice grain [13–15]. The emulsion formed is a colloi-
dal system constituted by a continuous phase (water) and a dis-
persed phase (particles). Fraction protein, starch granules, solid
parts of rice matrices, and lipid droplets are particles that may be
present in the dispersed phase [16, 17]. Plant-based rice extract has
been considered an excellent alternative to cow’s milk due to health
concerns [18–20], is cheaper, and is environmentally friendly
[13, 17]. However, plant-based rice extract has different sensory
characteristics, stability, and nutritional composition than cow’s
milk [15].
The aqueous extracts of plant materials, which form the base
for probiotic plant-based beverages, are prepared by cleaning,
dehulling, soaking, and milling the raw materials in water. Alterna-
tively, it can also be dry milled and subsequently solubilized in
water. Then, the slurry is filtered to separate the residues
[1, 21]. As a result, these products have been noted for having
adequate sensory characteristics, minimum recommended viable
probiotic numbers, and a high level of beneficial substances in
human nutrition, such as vitamins, minerals, antioxidants, dietary
fibers, and natural prebiotic compounds [2, 4, 22]. However, the
food matrix is significantly changed during the fermentation
Probiotic Plant-Based Beverages 83
process due to the production of acids, volatile compounds, and

other transformations that alter the sensory characteristics of the
beverage. Therefore, preparing probiotic plant-based beverages
requires they possess desirable sensory, physical, and chemical char-
acteristics and meet probiotic requirements.
Plant-based fermented beverages can be produced through
microbial fermentation processes that can occur by the addition
of starter cultures (culture-dependent ferments) or naturally (spon-
taneous ferments) [23]. Industrially produced probiotic products
often use starter cultures to ensure product standardization. Indus-
trial microbial cultures include liquid, frozen, freeze-dried, or ato-
mized concentrated microorganisms for starter culture propagation
and subsequent inoculation or the use of readily soluble cultures
that allow direct vat set (DVS) inoculation [24]. The DVS inocula-
tion of probiotics is convenient for storage and commercial applica-
tions. At the same time, freeze-dried concentrated microorganisms
are widely used due to their intense fermentation activity and low
storage and transportation costs [25, 26].
The probiotic strains used, besides having GRAS (Generally
Recognized As Safe) and QPS (Qualified Presumption of Safety)
status, recognized by the European Safety Authority—EFSA, must
comply with the requirements to be considered probiotic and
technological criteria to be employed in beverages, such as: grow
in the food matrix, resist the technological processing steps, not
produce undesirable sensory compounds, tolerate storage condi-
tions, and resist marketing conditions [24]. Different species of
Lactobacilli and Bifidobacteria have been reported in probiotic
plant-based beverages, such as Limosilactobacillus fermentum,
Limosilactobacillus reuteri, Lactobacillus acidophilus, Lactiplantiba-
cillus plantarum, Lacticaseibacillus rhamnosus, Lacticaseibacillus
casei, and Bifidobacterium sp. In addition, other studies of mixed
plant-based substrates and probiotic strains are being developed to
produce beverages of particular flavor characteristics [3]. This chap-
ter describes the processes for obtaining soy (see Subheading 2.1),
oat (see Subheading 2.2), and rice extract (see Subheading 2.3).
After obtaining one of the extracts, the fermentation procedure
can be carried out according to Subheading 3.
2 Plant-Based Aqueous Extract
The processes for obtaining soy (see Subheading 2.1), oat (see
Subheading 2.2), and rice extracts (see Subheading 2.3) will be
described.
2.1 Soymilk 1. Food-grade whole soybeans, full-fat soy flakes, or full-fat soy-
bean flour can be used (see Note 1).
2.1.1 Materials
2. Drinking water.
3. Container or tank for soaking soybean.
4. Stainless steel semi-industrial blender or grinder.
5. Filtration system.
6. System for heat treatment.
2.1.2 Methods The soy extract can be obtained from the following steps:
1. Selection and removal of major and minor dirt from soybeans
through sieving (see Note 2).
2. Soak soybeans in drinking water at a ratio of 1:3 soy: water, for
3 to 15 h, depending on conditions (see Note 3).
3. Grind the wet soybeans in a stone mill or hammer mill with
additional fresh water. The ratio of water to soybeans is usually
6:1 to 10:1 (see Note 4).
4. Filter the slurry to separate the soybean extract from the insol-
uble fiber (okara) through a sieve, cloth, or pressing bag, with
or without a wooden press. The slurry also can be filtered on
one or more scraped filters in batch or semicontinuous mode or
by continuous centrifugation filtration. The okara is usually
washed once or twice with cold or hot water, stirred, and
re-pressed to maximize soymilk yield. The total volume of the
combined filtrate (raw soymilk) is about 6–10 times the origi-
nal soybean volume.
5. Carry out the heat treatment followed by rapid cooling of the
soymilk before fermentation. Heat treatment can be carried out
by pasteurization or sterilization using ultra-high temperature
(UHT). At the pasteurization, soybean extract is boiled for
10 min (95–98 °C) from the start of boiling, continuously
stirring. The processing by UHT of soymilk is usually done
by using UHT directly with steam injection, followed by a
short holding time of about 5 s at about 145 °C, followed by
flashing to remove unpleasant odors and excess steam.
2.1.3 Notes 1. For a better result, you should preferably use clear hilum soy-
beans to produce soy extract with whiter color, higher yield,
and better overall quality.
2. At selection, discard the broken soybeans because the enzy-
matic reactions that cause the off flavor have already taken
place. After the selection, optionally, the soybean can be
dehulled to improve the flavor of the soy extract by removing
bitter and astringent compounds from the husk.
3. Before soaking, the beans should be rinsed under running

water, careful not to damage them. After soaking, the water
can be removed and replaced with fresh process water. Soaking
the soybeans in cold water causes little or no lipoxygenase
activity. However, it requires more time than soaking in warm
or hot water. Soaking in hot water has the advantage of remov-
ing all adverse enzyme reactions very quickly. Therefore, many
processes include a hot blanching step for 15 to 20 min in 3–5
volumes of water at 85 to 90 °C. In addition, soaking in
bicarbonate solution plays an essential role in softening the
structure of soybeans, which will later create a finer slurry. As
a result, soy extraction yield is higher because a large portion of
the soybeans can seep through the filter cloth, resulting in
higher protein content.
4. If heating has not yet taken place before grinding, hot water is
used, or steam is injected to increase the temperature of the
soybean slurry as quickly as possible. In addition to inactivating
the enzyme, increasing the cooking temperature of soybeans
will reduce the viscosity of the oil in soy extract and make it
easier for the oil cells to break down. This phenomenon allows
oil to be released and further increases the crude fat content of
soy extract.
2.2 Oat Extract 1. Whole meal, hulled, or flakes oats.

2.2.1 Materials 2. Drinking water.
3. Container or tank for soaking oat.
5. Water bath.
6. Optional application of thermostable endo-acting enzymes:
α-amylase (120 KNU-T/g) and β-glucanase (1 U/mg), both
food grades.
7. Filtration system with mesh opening size up to 20 μm.
2.2.2 Methods The preparation of oat extract, popularly known as oat “milk,” can
be obtained from the following steps:
6. Select and wash whole or hulled oats to remove dirt and
unwanted material.
7. Soak whole meal, hulled, or flakes oats in drinking water at a
ratio of 4 to 8% (w/v) at room temperature for 12 h (see
Note 1).
8. Grind the oat with their soaking water in a stainless-steel
blender or grinder for up to 5 min (see Note 2). Food indus-
tries generally do not perform the soaking step due to the long
time required. In this case, the oat can be ground with hot
water at 80–90 °C for up to 5 min to adequately extract

proteins, β-glucan, vitamins, minerals, etc.
9. If the objective is to reduce beverage viscosity and/or increase
protein extraction, add 0.15% α-amylase (w/w; enzyme/oat)
and 0.04% β-glucanase (w/w; enzyme/oat) to the slurry and
keep it in a water bath at 80 °C for 2 h under moderate
agitation (see Note 3).
10. Cool the slurry to 30 °C and then filter or centrifuge it to pass
through a mesh opening up to 20 μm (see Note 4). The
material retained in the mesh can be applied for the elabora-
tion of bakery products.
11. This oat extract must be pasteurized in a water bath, discon-
tinuous or continuous heat exchanger, before inoculating the
probiotic culture (see Subheading 3).
2.2.3 Notes 1. The ratio of 4 to 8% (w/v; oat/water) is suitable for extracting

a significant amount of β-glucan and making the beverage
prebiotic. However, oat extract with high amounts of this
fiber can show undesirable sensory properties to consumers
due to its high viscosity. Therefore, carrying out previous
tests with consumers is important to assess the drink’s
acceptability.
2. High grinding time contributes to greater β-glucan extraction,
but its impact on beverage viscosity must be investigated.
3. Termamyl® (Novozymes) is an endo-acting α-amylase used to
liquefy oat starch and produces oat-based drinks with moderate
viscosity. Beerzym BGHK4® (Erbslöh Geisenheim GmbH) is a
β-glucanase that also reduces the viscosity of the beverage
through the hydrolysis of β-glucan.
4. High pressure during filtration can make it easier for the slurry
compounds to pass through the mesh, especially β-glucan and
proteins.
2.3 Rice Extract 1. Rice grain (fully milled or partially milled), milled broken rice,
or rice flour.
2.3.1 Materials
2. Drinking water.
3. Container or tank for soaking rice.
5. Enzymes: α-amylase and glucoamylase (Glucozyme).
2.3.2 Methods The process of extracting the rice extract (“rice milk”) follows the
following steps:
1. Prepare the grains, passing them through cleaning, selection,

classification, and washing steps. The rice grains can be fully or
partially milled (see Note 1).
2. Hydrate the grains in water in the proportion of 1:2 (w/v) at
2 °C for 5 h.
3. Grind the rice with the hydration water using the stainless-steel
blender or grinder for about 3 min until a homogeneous
mixture is obtained. Then, for liquefaction, add 0.1% enzyme
α-amylase at 90 °C for 30 min in the homogeneous mixture
and use 0.1% glucoamylase (Glucozyme) at 55 °C for 12 h to
reduce the beverage’s viscosity and convert rice extract’s starch
to simple sugars for consumption in the fermentation process
of plant-based rice extract. Saccharification can also be carried
out simultaneously with the fermentation step, when applica-
ble, by adding 0.1% glucoamylase (Glucozyme), a fermenting
microorganism, to 200 g of rice extract (see Note 2).
4. Remove coarse particles by filtration, decanting, or centrifuga-
tion. These larger or coarse particles can be used in other food
products. Next, extract the soluble phase (“rice milk” or rice
extract).
5. Add other necessary ingredients to improve the chemical and
sensory characteristics of the beverage (see Note 3).
6. Pasteurize the rice extract before inoculation of the probiotic
culture. Pasteurization can occur in a water bath, discontinu-
ous, or continuous heat exchanger.
2.3.3 Notes 1. The process of rice extract involves grain preparation, hydra-
tion, and breakdown (size reduction) of grain extracted in
water, treated with enzymes to partially break down the starch
and facilitate a suspension mixture, filtered, and thermic treat-
ment. Fully milled indicates that the husk, germ, and bran have
been removed, and only the endosperm (white rice) remains,
while the partially milled only husk has been removed. Rice
flour can also be used as a raw material. Fully milled grain may
result in better texture in the slurry (paste), but it has high
starch content and low content of nutrients, fiber, vitamins,
and bioactive components. Several rice grains can be used in
plant-based rice processing, but polished rice is the most used
raw material. Brown rice is also sometimes used. When rice
flour is used to extract the beverage, there will be no need for a
milling step, but an effective mixing solution is recommended
to create a uniform slurry.
2. Cereals and pseudocereals have a high proportion of starch and
form a thick paste when heated above the gelatinization tem-
perature (55–65 °C). Therefore, to prevent and avoid problems
in the later stages of processing, the use of the α-amylase

enzyme is required.
3. After obtaining the desired viscosity (thickness), other ingre-
dients can be added [13, 14]. Protein, calcium, and vitamins
are examples of essential nutrients required in the rice extract
once they are limiting nutrients [13, 27].
3 Fermentation
The general process of obtaining probiotic plant-based beverages

using DVS culture will be described. However, depending on the
culture used and the substrate, it will probably be necessary to adapt
the inoculation and fermentation conditions. In some cases, it is
necessary to reactivate the culture, which can be purchased in
liquid, freeze-dried, or concentrate-frozen form, before inocula-
tion, so follow the manufacturer’s instructions.
3.1 Materials 1. The aqueous extract was obtained according to Subheading 2.

2. Microbial culture: add 2% to 3% of the microbial culture or use
the manufacturer’s recommendations (see Note 1).
3. Optional ingredients: Sucrose (6 to12%), thickeners (0.5%),
flavorings (0.1%), and other optional ingredients such as
pulps, fruits, and prebiotics are recommended.
4. Flasks and incubator oven or industrial fermentation tank.
5. Cooling heating system for beverages.
3.2 Methods The preparation of probiotic plant-based beverages can be obtained

from the following steps:
1. Add the non-volatile or thermolabile ingredients and homoge-
nize for solubilization (aqueous extract, sucrose, thickeners,
and others) (see Note 2).
2. Heat treat the mixture of aqueous extract and other ingredients
before receiving the inoculum for fermentation (see Note 3). It
is suggested to use 95 °C for at least 5 min (see Note 4).
3. Cool the aqueous extract rapidly to fermentation temperature
(usually between 25 °C and 43 °C), according to the manu-
facturer’s recommendations (see Note 5).
4. Add the inoculum, homogenizing with the help of sanitized or
sterile utensils.
5. Ferment at the temperature indicated for the microorganism
(usually between 25 °C and 43 °C) until the desired final pH
(see Note 6).
6. Cool to 5 °C for 12 h for stabilization and then homogenize,

preferably without incorporating air (see Note 7).
7. Add the flavorings and other thermolabile ingredients.
8. Aseptically fill in appropriate packaging and, preferably, store
under refrigeration.
3.3 Notes 1. Check the manufacturer’s instructions for use in volumes lower
than the recommended in the microbial culture envelope.
Generally, for mixed culture envelopes, you should sterilize
1 L of the fermentation substrate at 121 °C for 15 min. After
cooling down (~10 °C), add the mixed culture (1 envelope of
5 UC for 1 L of the substrate), and homogenize with sterile
utensils. Then distribute into sterile 10 mL containers and
freeze quickly at -18 °C until use. In this example, each
container will contain enough microbial culture to be used in
5 L of beverage. However, other proportions can be used
according to the production scale.
2. Flavoring ingredients or ingredients with thermal instability
can be added aseptically after heat treatment. The sanitary
quality of these ingredients must be checked beforehand or
ensured by the manufacturer through technical reports.
Sucrose is usually added to improve flavor and consistency
and is used as a fermentation substrate by some starter cultures.
In addition, thickeners, fruit pulp or juice, and other com-
pounds can improve the product’s stability and acceptability.
3. The aqueous extract must have a typical taste and aroma, an
absence of spoiling microorganisms, pathogens, and an
absence of fermentation inhibiting substances. In addition,
the heat treatment must be carried out in such a way as to
guarantee the safety of the product.
4. If the heating is done in an open tank, at a lower temperature
and longer time, an increase in the solids content of the aque-
ous extract will occur. The same effect can be observed with
heating at 95 °C for 5 min in a plate heat exchanger under a
partial vacuum, where part of the water is evaporated.
5. Heating and cooling operations can be performed on a plate
heat exchanger.
6. Generally, aqueous extracts are fermented to a pH of about 4.3.
The fermentation time depends on the characteristics of the
culture and temperature employed and typically ranges from
4 to 30 h. Some probiotic microorganisms, such as Bifidobac-
teria, can accumulate compounds undesirable for the product
during fermentation, such as acetic acid. Therefore, in some
cases, the probiotic may not participate in the fermentation
process. In this case, the highly concentrated culture is added at

the end of the process, followed by cooling and packaging.
7. Most probiotic microorganisms are anaerobic, and mixing can
lead to the incorporation of oxygen into the beverage, reducing
the probiotics’ viability.
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Chapter 6
Probiotic Plant-Based Cheese

Abstract
Due to several factors, the demand for plant-based cheese has increased. However, formulating products
with characteristics similar to cheese made with animal milk is still a challenge for researchers and industries.
Here we will describe the process of obtaining probiotic pea cheese, probiotic tofu, probiotic soy-based
cream cheese, and probiotic chickpea petit suisse cheese.
Key words Pea cheese probiotic, Probiotic tofu, Chickpea probiotic petit suisse cheese, Functional
food
1 Introduction
Probiotic plant-based cheeses are products made from vegetable

sources, usually legumes, including non-dairy fats or proteins,
which result in a cheese analogous to that made with animal milk.
It has benefits for the health of the consumer. Ethical reasons,
sustainability, animal welfare, and health, such as lactose intoler-
ance, milk allergy, and cholesterol issues, are the main consumer
concerns that drive interest in plant-based dairy alternatives
[1, 2]. The increased production and demand for plant-based
cheese alternatives are also due to the increasing number of people
following vegan diets [3].
The cheeses of vegetable origin differ sensorially from the
cheeses of animal origin due to their composition. The
plant-based cheese does not present the physical and sensory char-
acteristics of dairy-based cheese, whereas the comparisons between
flavor, taste, aroma, mouthfeel, and meltability limit consumer
acceptability [2]. In cheese made with animal milk, casein-casein
interactions promote stretch and flow functionality, which provides
structure to the cheese matrix. In plant-based cheese, the formation
of compact gel networks does not occur in the same way as with
93
casein. As rennet does not induce coagulation in plant milk, other

protein coagulation methods should be employed [4–6]. Therefore,
applying enzymes, acids, lactic acid fermentation, or heat treat-
ments are technologies that need to be considered [7]. This is
because plant proteins have larger molecule sizes and more complex
quaternary structures than milk proteins, in addition to having
properties such as cross-linking and hydrophobicity [8]. Thus,
these products are formed by lipids embedded in polysaccharides
or protein matrices, forming a colloidal dispersion [6].
Many types of plant-based cheeses are formulated using com-
binations of oils (e.g., coconut or palm oil) and starches (e.g.,
potato or tapioca starch) [1]. The main ingredients for the formu-
lation of a plant-based cheese are water, lipids, and vegetable pro-
teins, which can be included as stabilizers, emulsifying salts,
acidifying agents, preservatives, and flavors [9, 10]. In addition, a
combination of ingredients has been used to provide a product with
structure, viscosity, and melting similar to traditional cheese
[5]. Although most of the time, a combination of ingredients is
necessary, the primary raw materials used in producing plant-based
cheeses are soybeans, peas, cashew nuts, coconut oil, oats, almonds,
palm fruit oil, and corn zein [3, 11].
Peas represent a good alternative to produce plant-based
cheeses as they have a low production cost and high protein con-
tent, which also stand out for their excellent gel formation. The gel
is mainly formed by the presence of globulins, such as legumin and
vicilins, representing 70–80% of the total protein content
[11, 12]. In addition, pea cheese proved to be a suitable substrate
for fermentation, and protein gels can be produced with 10%
protein content and 10% olive oil levels without compromising
gel hardness [11].
Soybean is a food cholesterol-free, low in sodium, a good
source of nutrients, and a suitable medium for probiotic growth
[13, 14]. Tofu is a plant-based cheese made from soy milk, one of
the most important and popular foods in Asian countries, and is
widely accepted worldwide. It is gaining popularity in Western
countries due to its health benefits [15]. The fermentation increases
the nutritional value and helps remove soy’s taste, which many
consumers do not accept [16]. Probiotic tofu can be a healthy
alternative for vegans and vegetarians while positively affecting
consumers’ health and improving the taste of soy cheese.
Soy-based cream cheese is a cream cheese analog known for its
creaminess and spreadability. Chemically, it can be defined as a
microgel with a structure formed of protein-covered soy fat glo-
bules [4, 17], resulting from the homogenization of tofu, fat, and
stabilizers. Therefore, its processing begins with the acidic coagu-
lation of soymilk to obtain tofu. However, there are still few studies
[18–20], on this cream cheese analog, particularly with the addition
of probiotics. Nevertheless, advances in processing, such as
Probiotic Plant-Based Cheese 95
membrane technology, enzymatic protein modification, and high

hydrostatic pressure and ultrasound treatments, are promising to
contribute to this product’s sensory characteristics, texture, and
cost efficiency [21].
The fermentation of these substrates to obtain a probiotic
plant-based cheese is an alternative that can improve the products’
nutritional, sensory, and shelf life since starter cultures can be
selected that, in addition to promoting acidification, can produce
extracellular polysaccharides that collaborate with the firmness of
the product. This chapter describes the processes for obtaining
probiotic pea cheese (see Subheading 2), probiotic tofu (see Sub-
heading 3), probiotic soy-based cream cheese (see Subheading 4),
and probiotic chickpea petit suisse cheese (see Subheading 5).
2 Pea Cheese Probiotic
Pea cheese is plant-based, produced by solubilizing pea protein in

water and blending it with oil to create a plant-based emulsion.
Coagulation of pea protein for gel formation can occur through the
action of heat, high pressure, acidification, and fermentation. There
is still a little exploration of fermentation-induced pea gel forma-
tion in this last one. Concerning the production of probiotic pea
cheese, there are still few studies that explore this process. The
primary materials and processing steps for the pea cheese probiotic
are described below:
2.1 Preparation of 1. Pea protein isolate (PPI).

Probiotic Pea Cheese 2. Extra-virgin olive oil.
2.1.1 Materials 3. Sucrose.
4. Glucose.
5. Salt.
6. Starter culture (Streptococcus thermophilus and Lactobacillus
delbrueckii ssp. bulgaricus).
7. Probiotic culture (e.g., Lactobacillus acidophilus, Lacticaseiba-
cillus paracasei, and Bifidobacterium).
2.1.2 Methods 1. Suspend a pea protein isolate (PPI) in distilled water until a
final protein concentration of 10% (w/w) (see Note 1).
2. Stir the mixture at 9500 rpm for 3 min with Ultra-Turrax, to
ensure protein hydration.
3. Add 1% glucose (w/w) and 1% (w/w) sucrose during the
stirring step to ensure that there is a substrate for the bacteria
to grow and acidify the matrix, given the low sugar content of
the PPI.
4. Emulsify the mixture using extra-virgin olive oil (around 10%

olive oil, w/w) with Ultra-Turrax at 13,500 rpm for 3 min (see
Note 2).
5. Homogenize the sample under continuous operation and high
pressure at two stages (150 and 50 bars) in one pass to decrease
oil droplet size and avoid phase separation (see Note 3).
6. Pasteurize (see Note 4) the matrix at 95 C for 5 min in a water
bath and cool down to 43 C before microbial inoculation. For
starter culture inoculation utilize 0.02% inoculum. Add probi-
otic culture and mixture. Containers used for preparation must
be previously dry autoclaved at 121 C for 20 min.
7. Ferment at 43 C until reaching pH 4.5 (see Note 5).
9. Mold the product.
10. Store at refrigeration temperature.
2.1.3 Notes 1. It is known that 10% protein is an optimal protein concentra-

tion for a stable liquid matrix prepared with the PPI. This
concentration keeps the matrix density low, which allows easy
pre-processing and homogeneous inoculation in a liquid
media.
The use of PPI is a functional starting raw material for
fermentation-induced pea protein gels.
In the case of using another source of raw material for the
production of probiotic pea cheese, such as pea protein con-
centrate (78% protein), it is recommended to adjust the pH of
the suspension, as different pH values during pre-treatment of
pea protein led to different ratios between soluble and insolu-
ble protein aggregates in the protein slurry before fermenta-
tion. Therefore, these are determining factors during the
formation of the gel, directly influencing the rheological prop-
erties. For example, the solubility of pea protein concentrate is
highest at pH 8.0 and lowest at pH 6.0.
2. Olive oil or other oil types provide the matrix with the fat
needed for the cheese and help to stabilize the pea proteins in
suspension, which will likely sediment over time if only sus-
pended in water. It is recommended that the initial pH of the
PPI emulsions be 7.0. In pea protein emulsions, this protein
acts as a surfactant due to its excellent emulsifying properties,
which contribute to the stability of the emulsion. Thus, avoid-
ing the need to use surfactants.
3. Hydration and homogenization of proteins with two pressure
stages are important operations in producing a PPI matrix that
guarantees stability and avoids phase separation during
fermentation.
4. Pasteurization of the matrix before fermentation is necessary to

ensure the safety and growth exclusivity of the inoculated
starter culture. Matrices with high starch contents will form a
gel before fermentation, and it will be impossible to inoculate
bacteria into a liquid medium. Therefore, less purified pea
protein ingredients can present challenges during matrix pro-
cessing. Thus, it is necessary to use purer protein fractions, such
as protein isolates (80–95% protein).
5. The time required to reach pH 4.5 can vary between 5.5 and
7 h. The final pH affects rheology and meltability. Higher pH
values (6.0–7.0) provide the cheese analog with more viscous
properties and lower pH (4.5–5.5) with higher elasticity.
3 Probiotic Tofu
Tofu is a soy-based product that is precipitated with coagulants in

the form of curds. The curds’ size and the pressing time length
determine the style of tofu, which can be soft, regular, firm, or extra
firm. The probiotic culture can be added before or after the coagu-
lant. However, there are still few studies that have investigated the
incorporation of probiotics in tofu. The primary materials and
processing steps are described below:
3.1 Preparation of 1. Food-grade whole soybeans (see Note 1), full-fat soy flakes, or
Probiotic Tofu full-fat soybean flour.
3.1.1 Materials 2. Drinking water.
3. Coagulant (see Note 2).
4. Probiotic cultures.
9. Incubator for fermentation.
3.1.2 Methods 1. Wash dry whole soybeans and soak them in water overnight.
The volume of water is usually 2 to 3 times the volume of
the bean.
2. Drain the soaked beans and rinse with fresh water 2–3 times.
3. Ground the wet, clean soybeans in a mill with fresh water. The
water:bean ratio is usually in the range of 6:1 to 10:1.
4. Filter the soy milk through a sieve, cloth, or press bag. Remove
residue and wash once or twice with water (cold or hot),
stirring and pressing again to maximize milk production.
5. Heat raw milk at a boiling temperature and keep it at this

temperature for 5–10 min (see Note 3).
6. Once the milk is heated, transfer it to another container. At the
same time, mix the powdered coagulant with a small amount of
hot water to make a coagulant suspension. Then add the coag-
ulant to the hot soy milk at 70–85 C; depending on the type of
coagulant used, let the mixture sit for about 10 min so that the
proteins can coagulate (see Note 4).
7. After noting the beginning of coagulation, add the probiotic
culture and mix.
8. Transfer the mixture to a sterile press cloth and place it into the
mold. Discharge excess liquid by pressing (see Note 5).
(usually 37 C) until the desired final pH, for about 20 h.
3.1.3 Notes 1. Preferably beans with large seed sizes and light hilum to pro-
duce tofu of whiter color, higher yield, and better overall
quality.
2. Coagulants can be enzymes, salts, or acids. The widely used
ones for tofu making are calcium sulfate, magnesium chloride,
or a mixture of both, nigari, and glucono-6-lactone (GDL,
known as lactone).
3. To avoid burning the milk at the bottom of the cooking vessel,
slow heating with frequent stirring is necessary. Alternatively,
soy slurry may be heated before filtering into soymilk. Heating
soy milk is important to denature the proteins so that they
coagulate into curds in the presence of the coagulant. How-
ever, prolonged heat treatment should be avoided as it can
destroy nutrients such as essential amino acids and vitamins,
Maillard browning, and the development of cooked flavors,
leading to lower yields and poor-quality tofu.
4. Factors affecting coagulation include the temperature at which
a coagulant is added, the type and concentration of coagulants,
the mode of adding coagulants, and the duration of coagula-
tion [14]:
• The suitable amount of coagulant: coagulates all soy milk
and generates a clear whey with an amber or pale yellow
color and sweet taste.
• Excessive coagulant: the whey has a slightly bitter taste and
a yellowish color, and the curd has a coarse, hard texture.
• Insufficient coagulant: the resulting whey is cloudy, with
some remains of uncoagulated soy milk.
• High temperature: rapid coagulation resulting in tofu with

low water-holding capacity, hard and coarse texture, and
thus a low bulk yield.
• Temperature too low: coagulation becomes incomplete,
making tofu too soft to retain its shape.
• Time too short: coagulation is incomplete.
• Time too long: the system’s temperature decreases to such
an extent that the subsequent shaping step becomes com-
plex. For silken tofu, the dwell time should be about
30 min; for regular tofu, 20–25 min; and for firm tofu,
10–15 min.
The suggested additional temperature and concentra-
tion for the main coagulants are: calcium sulfate (CaSO4)
0.5% (w:v) at 80–85 C, magnesium chloride (MgCl2) 5%
(w:v) at 70–72 C; Glucono-δ-lactone (GDL) 0.020 mM at
80 C. The incubation time for coagulation is 10 min.
5. The curd is pressed to form tofu. The size of the curd and the
length of the pressing time determine the style of tofu pro-
duced, which can be soft, regular, firm, or extra firm tofu—the
softer the tofu, the lower the protein and fat levels and the
higher the water content.
4 Soy-Based Cream Cheese Probiotic
This product is mainly obtained from tofu, which results from the
acidic coagulation of soymilk. However, there are still few studies
that have investigated the incorporation of probiotics in soy-based
cream cheese. The main materials and processing steps are
described below:
4.1 Preparation of 1. Soft or firm tofu (see Subheading 3).

Probiotic Soy Cream 2. Palm oil.
Cheese
3. Carrageenan gum.
4.1.1 Materials 4. Pectin.
5. Maltodextrin.
6. Salt.
7. Stainless steel semi-industrial blender or high-pressure
homogenizer.
8. Probiotic cultures.
4.1.2 Methods 1. Homogenize (see Note 1) the tofu with palm oil (5–10%,
w/w), carrageenan gum (4%, w/w), pectin (1%, w/w), malto-
dextrin (2–4%, w/w), and salt (1–3%, w/w). These ingredients
are responsible for the stability and texture of probiotic soy

cream cheese (see Note 2).
2. Add the probiotic culture (see Note 3) to the soy cream cheese
using sanitized or sterilized utensils.
3. Aseptically fill in appropriate packaging and store under
refrigeration.
4.1.3 Notes 1. Homogenization time in stainless steel semi-industrial blenders

varies from 2 to 5 min to produce a homogeneous emulsion.
On an industrial scale, the mixture is homogenized in a high-
pressure homogenizer with pressure ranging from 10 to
25 MPa. It is noteworthy that high shear rates make formula-
tions more spreadable.
2. In addition to maltodextrin, soy protein concentrate or isolate
can be added to the mixture to obtain a smooth soy cream
cheese. Carrageenan gum and pectin increase the viscoelasticity
of the cream and reduce product syneresis during cold storage.
Furthermore, salt concentrations above 6 g/kg and high-fat
content (>280 g/kg) can also reduce syneresis [17].
3. It is recommended that the probiotic concentration be at least
8 log CFU/g because there is a reduction in this value during
storage and after ingestion of the product. Therefore, viability
tests of probiotics are necessary to ensure a concentration
above 6 log CFU/g of product at the time of consumption
and in the distal part of the gastrointestinal tract.
5 Chickpea Probiotic Petit Suisse Cheese
Chickpea probiotic petit suisse cheese is the food produced from

the fermentation of chickpea extract, followed by obtaining quark
cheese. This product can be flavored and added with emulsifiers and
stabilizers. There are still few studies that investigate the use of
chickpeas for the preparation of petit suisse. The main materials
and processing steps are described below:
5.1 Preparation of 1. Food-grade whole chickpea.

Chickpea Probiotic 2. Drinking water.
Petit Suisse Cheese
3. Container or tank for soaking chickpeas.
5.1.1 Materials 4. Stainless steel semi-industrial blender or grinder.
5. Water bath.
6. Probiotic cultures (see Notes 1 and 2).

5.1.2 Methods The preparation of chickpea probiotic petit suisse cheese starts with
the preparation of the chickpea extract, and can be obtained by the
following steps:
1. Select and wash whole or hulled chickpeas to remove dirt and
unwanted material.
2. Soak the chickpea in drinking water at a ratio of 1:3 chickpea:
water (w/v) at room temperature for 12 h.
3. Drain the soaked chickpea and rinse with fresh water 2–3 times.
4. Grind the chickpea with water for up to 10 min. The water:
chickpea ratio is usually in the range of 6:1 (w/v). Food indus-
tries generally do not perform the soaking step due to the long
time required. In this case, chickpeas can be ground with hot
water at 80–90 C for up to 5–10 min for adequate extraction
of compounds.
5. Cool the slurry to 30 C and filter through a sieve, cloth, or
pressing bag. The slurry also can be filtered on one or more
scraped filters in batch or semicontinuous mode or by continu-
ous centrifugation filtration.
6. Add sugar (5–15%, w/w) to the chickpea extract and give heat
treatment. Before inoculating the probiotic culture, this chick-
pea extract must be pasteurized in a water bath, discontinuous
or continuous heat exchanger (see Note 3).
7. Cool the aqueous extract rapidly to fermentation temperature
(usually between 37 C and 43 C), according to the manu-
facturer’s recommendations.
8. Add the inoculum, homogenizing with the help of sanitized or
sterile utensils.
(usually between 37 C and 43 C) until the desired final pH
(see Note 4).
10. Desorb for up to 12 h at 4–8 C in a synthetic filter or sieve to
get quark cheese (see Note 5).
11. Add guar gum (0.3–0.7%, w/w) and xanthan gum (0.3–0.7%,
w/w), and mix with quark mass (see Note 6).
12. Add, if you prefer, fruit pulp (10 to 30%, w/w) previously heat-
treated and other additives such as flavorings (see Note 7).
13. Aseptically fill in appropriate packaging and, preferably, store
under refrigeration.
6 Notes
1. Check the manufacturer’s instructions for use in less than the

recommended volume in the microbial culture envelope. More
instructions are given in Chap. 5, in the Notes to Subheading
3.
2. Exopolysaccharide-producing probiotic culture is recom-
mended to obtain better texture properties.
3. The chickpea extract must have a typical taste and odor, an
absence of spoiling microorganisms, pathogens, and an
absence of fermentation-inhibiting substances. The heat treat-
ment must be carried out in such a way as to guarantee the
safety of the product. If the heating is done in an open tank, at a
lower temperature and longer time, an increase in the solids
content of the aqueous extract will occur.
4. Generally, aqueous extracts are fermented to a pH of about 4.3.
The fermentation time depends on the characteristics of the
culture and temperature employed and typically ranges from
4 to 30 h.
5. Additional hygienic care must be taken in the draining step to
avoid microbiological risk. It is recommended that this step be
carried out under refrigeration. The whey released to obtain
quark cheese has a high concentration of water-soluble com-
ponents, such as carbohydrates, salts, and proteins, so it can be
used to manufacture other food products.
6. Other thickening agents can be used to ensure the texture
properties of the product, such as carrageenan gum (2–5%,
w/w) and pectin (0.5–2%, w/w).
7. The acidity of fruit pulp can affect the viability of probiotic
microorganisms during product storage. Therefore, perform
feasibility tests to verify that the probiotic count is as desired.
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Chapter 7
Probiotic Fruit Juices

Oscar O. Romero-Chapol, Abigail Varela-Pérez, Ana G. Castillo-Olmos,
Hugo S. Garcı́a, and Cynthia Cano-Sarmiento
Abstract
Regular consumption of probiotic bacteria is done mainly by ingestion of dairy or fermented foods;
however, some health issues, changing lifestyles, and feeding habits deprive part of the population of
their benefits. Therefore, consumption alternatives, such as fruit juices, represent a challenge attributed
to the presence of intrinsic conditions that affect the viability of bacteria. In this chapter, we describe a
method incorporating probiotic bacteria encapsulated in an alginate matrix using an emulsification process
as a pretreatment into fruit juices. We also provide the reader with the techniques for morphological analysis
by scanning electron microscopy, as well as the characterization of the juice and the evaluation of cell
viability against simulated gastric conditions.
Key words Probiotics, Juice, Encapsulation, Viability, Scanning electron microscopy
1 Introduction
Currently, the demand for functional products formulated with

probiotics has gained more importance due to their beneficial
effects on several diseases and the advantages they provide by
modulating immune responses and gut microbiota through several
mechanisms [1–3]. Probiotics are “live microorganisms which,
when administered in adequate amounts, confer a health benefit
on the host” [4]. Different foods have been fermented or inocu-
lated with probiotics to be examined as potential carriers of these
microorganisms; however, it must be considered that the beneficial
effect on the host might be modified according to the probiotic
strain or mixture of them that is used [5–7]. Some Lactobacillus and
Bifidobacterium species are probiotics and the most commonly
employed strains in several food products [6]. Traditionally, pro-
biotics are added to fermented milk or other dairy products. How-
ever, it is also evaluated by adding it to other matrices such as juices,
as some consumers are vegans, lactose intolerant, or have protein
105
106 Oscar O. Romero-Chapol et al.
allergies. For this reason, probiotic fruit juices represent an ade-

quate alternative to the preparation of non-dairy probiotic foods, in
addition to supplying nutrients such as minerals, vitamins, fiber,
and antioxidants. One of their main advantages is the pleasant taste
for all age groups, resulting in a functional food that can be
accepted by the public [8]. Fruit juices (100% fruit content), nec-
tars (25–99% fruit), and juice drinks (up to 25% fruit content) have
become crucial sectors in the food industry. Nevertheless, in the
case of probiotic liquid matrices, fruit juices are preferentially
employed since, unlike nectar and juice drinks, they do not contain
food additives such as preservatives or sweeteners that allow this
type of product to achieve a low price in the market but also carry a
negative effect based on the excessive consumption of these sweet-
eners that can have on the health of consumers, leading to dysbiosis
in gut microbiota and increased obesity risk [9, 10]. Fruit juices
contain nutrients naturally beneficial to health, like calcium, retinol,
vitamins, nicotinic acid, riboflavin, pantothenic acid, β-carotene,
biotin, dietary fiber, anthocyanins, and antioxidants. Some of
these compounds have demonstrated harmful effects against cer-
tain pathogenic microorganisms and, in contrast, promote the
growth of bacteria with beneficial properties. Also, another attri-
bute of juices is that they are readily digested in the stomach
compared with a dairy matrix; thus, probiotic bacteria remain in
the stomach’s acid environment for a shorter time [1, 6, 11,
12]. Studies employing fruit juice to prepare probiotic foods have
used the juices of pineapple, cranberry, strawberry, sweet lemon,
mango, apple, peach, pomegranate, grape, orange, blackcurrant,
banana, papaya, and pear, among others [11–13]. Specific bacteria
from the genus Lactobacillus, such as L. plantarum, L. acidophilus,
and L. casei, have proven greater acid tolerance for survival in
strawberry, orange, and cranberry juices [14, 15].
There are bacteria that, when added to juices, can carry out a
fermentation process, resulting in a reduction in sugar in the food
and a high rate of bacterial survival because of their adaptation to
the media. In addition, metabolites are produced during fermenta-
tion that help increase the product’s attributes, including bacter-
iocins which impede contamination during storage. However, even
though fruits are an adequate matrix for probiotic growth, the
viability of the probiotics in the juice is more complicated in com-
parison to a dairy matrix because of the requirement for lactic acid
bacteria to shelter from the acid environment of fruits, along with
not containing enough peptides and free amino acids present for
the metabolism of a probiotic culture [1, 13]. Elements that influ-
ence probiotic viability include intrinsic matrix factors such as
titratable acidity, pH value, molecular oxygen, water activity, artifi-
cial flavors and colorings, chemical or microbial agents, and pre-
servatives like hydrogen peroxide and bacteriocins. On the other
hand, the type of probiotic strain, compatibility of different strains,
Probiotic Fruit Juices 107
percentage of inoculum, and the stress bacteria suffer throughout

the gastrointestinal tract. In general, fruits have a low pH and high
quantities of organic acids, which increase in concentration in
non-dissociated form; hence the survival of probiotic bacteria is
defined by the highly acidic environment and the intrinsic antimi-
crobial activity of the organic acids that accumulate. For example,
Lactobacilli resist and survive better than Bifidobacteria in fruit
juices, with a pH value of 3.7 to 4.3. Moreover, other parameters
also affect probiotic survival during production, processing, and
manipulation: a thermal sterilization process, pasteurization,
cooling rate, volume, packaging material, and storage temperature
[11–13].
An essential factor in the manufacturing of fruit juices is the
method of sterilization, which can be divided into thermal and
non-thermal; non-thermal methods of sterilization employ pulsed
electric fields, high-pressure processes, ultrasound, and ionizing
radiation, all of which achieve the inactivation of microorganisms
at temperatures between 40 and 55 °C [16]. On the other hand,
thermal methods of pasteurization are applied at temperatures
between 60 and 90 °C from a few seconds to some minutes [17].
Pasteurization is a heat process that increases a food’s shelf life,
inactivating certain enzymes and microorganisms (yeasts, molds,
and bacteria) while producing a loss of flavor and changes in com-
position. As a result, primary properties like food taste are fre-
quently modified. However, this technique has been refined, so
fewer properties are modified in the final product; da Costa et al.
[18] prepared orange juice pasteurized at 80 °C for 20 min, cooled
to 37 °C, and added oligofructose before fermentation with Lacto-
bacillus paracasei ssp. paracasei, the juice was stored at 4 °C for
28 days, and its physicochemical and sensory properties were eval-
uated. The authors reported that it was possible to develop symbi-
otic cultures in orange juice added with oligofructose (a prebiotic)
and probiotics without changing the physicochemical characteris-
tics and acceptance of pure juice, despite using pasteurization.
On the other hand, the main objective of high-pressure sterili-
zation is to maintain the viability of probiotic bacteria while elim-
inating possible pathogens present in the food matrix. However,
this process induces alterations in the cell membrane and morphol-
ogy, inhibits the synthesis of proteins and enzymes, and affects the
interruption of translation and transcription, in addition to the
functions of reproduction and survival in the bacteria of interest
[16]. The effectiveness of high-pressure homogenization treat-
ments depends on the pressure, time, number of cycles, and tem-
perature. However, food contains carbohydrates, vitamins, and
minerals that can be susceptible to high pressure. Hence, the design
and the development of probiotic juice turns into a comprehensive
task that needs to consider technical and microbial aspects without

affecting or having the most negligible possible effect on the qual-
ity, nutrient composition, and functionality of the final food prod-
uct. Accordingly, Oliveira et al. [19] developed a juice mix from
mango and carrot, which was processed both thermally and with
high hydrostatic pressure and supplemented with L. plantarum.
Bacteria caused a reduction of pH and increased acidity in juices but
did not modify their antioxidant capacity or the α- and β-carotene
content. Specifically, the high-energy process maintained microbio-
logical quality, unlike pasteurized juices with high psychrotrophic
counts after 35 days of storage. Processing juice at high pressures
proved to be more effective than the thermal process, with fewer
modifications in composition, while maintaining the excellent via-
bility of L. plantarum [19].
Finally, microorganisms can be inactivated by sterilizing with
electric pulses by applying high-voltage pulses to liquid or semi-
liquid foods placed between two electrodes with treatments from
micro to milliseconds. This leads to the formation of pores in the
microbial cell membrane that entails a rupture of the cell, which
causes the release of cell contents and intrusion of surrounding
media [16].
In light of a critical risk of juice viability being lost due to
previously described variables, bacteria can be protected by encap-
sulation and subsequently added to the food matrix. With this,
survival rates can be conserved during all the processes involved in
encapsulation and throughout its shelf life [20]. However, supple-
mentation of probiotic bacteria inside a liquid food matrix, as in the
case of fruit juices, represents a technological challenge where both
factors need to be favored by the mixture. On one side, it is
necessary to protect probiotic bacteria against the intrinsic factors
of juice that affect cell survival and, consequently, their beneficial
effects in the host, while on the side of the food matrix, the growth
of the bacterial population might cause damage due to CO2 pro-
duction and alterations in sensory factors like taste, texture, and
color [21]. Depending on the technique used for encapsulation,
viability varies, with reported percentages ranging between 80 and
95% [22]. Additionally, fewer modifications to juice composition
can be achieved through encapsulation.
Table 1 shows a compilation of some techniques, materials, and
bacteria used to develop probiotic juices and the results of applying
these technologies.
The ionic gelation process that uses alginate as an encapsulant
matrix is one of the techniques mainly employed for the develop-
ment of probiotic juices because of its compatibility with the com-
ponents present in the mixture, high viability rate during storage,
and, finally, for preserving the sensory properties of juice [2, 25].
Table 1
Juice with encapsulated and non-encapsulated probiotics
Encapsulation
technique and
Juice Bacteria used wall material Results References
Pineapple, L. casei DSM 20011 Vibration Preservation of viability of [15]
orange, technology; encapsulated bacteria at
raspberry sodium 28 days of storage in
alginate raspberry and orange juice
compared with free cells;
~ 57% viability for
encapsulated systems
and < 15% in free systems
Apple, L. rhamnosus, B. longum, Emulsification Viability in the juice after [23]
orange L. salivarius, and ionic 6 weeks of storage of ~60
L. plantarum, gelation; and 64% for encapsulated
L. acidophilus, sodium systems; no viability for
L. paracasei, B. lactis type alginate free cells. Encapsulated
Bi-04, B. lactis Bi-07 groups present less
diminution of pH
Apple L. rhamnosus GG Emulsification Viability of encapsulated [2]
and ionic probiotic during storage
gelation; of ~100 and ~ 80% in case
sodium of free cell administration
alginate
Grape L. acidophilus PTCC 1643, Emulsification Sensory aspects like acidity, [24]
B. bifidum PTCC 1644 and ionic color, turbidity, and
gelation; soluble solids are
sodium preserved better in
alginate encapsulated systems.
Viability after storage of
~88% in encapsulated
systems and ~ 78% in free
cells
Apple L. rhamnosus GG Extrusion and Viability of encapsulated [25]
ionic gelation; bacteria during storage of
sodium ~80 and ~ 20% in case of
alginate and free cells. More excellent
alginate–inulin score in sensory
evaluation (~ 1 point) of
juices with encapsulated
bacteria
2 Case Study
2.1 Materials/ The use of distilled water sterilized by autoclave at a pressure of

Reagents 10 psi and temperature of 115 °C is recommended, as well as the
use of reagent-grade components for the preparation of all solutio/
ns. Reagents must be prepared at room temperature (~ 25 °C);
temperature control is not necessary for most solutions. All material
inoculated with bacteria must be sterilized before its disposal. In
the case of culture media (MRS broth, MRS agar, and Gomori
buffer), previous sterilization is mandatory to avoid contamination.
2.2 Cell Culture Freeze-dried or frozen cells of probiotic strains are reactivated at
least three times before use. Probiotic bacteria of the genus Lacto-
bacillus must be inoculated at 1% v/v in MRS broth at 37 °C and
pH 5.9 for 24 h at 150 rpm [3]. In the case of probiotic bacteria of
the genus Bifidobacterium, supplement media with 0.05% w/v
L-cysteine is recommended (see Note 1) [26].
2.3 Encapsulant A bacterial suspension is obtained by centrifugation at 10,000 × g

Mixture and 4 °C for 10 min. Two washes with 0.9% w/v saline solution
(NaCl) must be conducted before resuspension at the same vol-
ume. A mix of 25 mL of 10 log CFU/mL bacterial suspension with
100 mL of 3% w/v sodium alginate, 100 mL of canola vegetable
oil, and 1 mL of Tween® 80 has to be prepared. The mixture must
be homogenized at 4000 rpm for 7.5 min with an Ultra-turrax®
model T-25 rotor-stator type homogenizer (IKA, Staufen,
Germany).
2.4 Cell Viability A Gomori buffer (phosphate buffer) is prepared for the liberation
(Buffers) and quantification of encapsulated bacteria. For example, to pre-
pare 0.1 M K2HPO4 and KH2PO4 stock solutions at pH 7 and 25 °
C, it is necessary to mix 61.5 mL of K2HPO4 and 38.5 mL of
KH2PO4. The buffer is left to rest for 24 h at 4 °C before use.
2.5 Gastrointestinal Enzyme concentrations are 75 U/mL α-amylase, 2000 U/mL

Solutions pepsin, 60 U/mL gastric lipase, 100 U/mL pancreatin, and
10 mM bile solution. Solutions employed to modify pH through-
out the simulation are HCl and NaOH at 1 M; in each stage,
different volumes of 0.3 M CaCl2 are added. At every stage of the
simulation, one phase is used; saliva solution must have concentra-
tions of 15.1 mM KCl, 3.7 mM KH2PO4, 13.6 mM NaHCO3,
0.15 mM MgCl2(H2O)6, 0.06 mM (NH4)2CO3, 1.1 mM HCl,
and 1.5 mM CaCl2(H2O)2; gastric solution needs to have concen-
trations of 6.9 mM KCl, 0.9 mM KH2PO4, 25 mM NaHCO3,
47.2 mM NaCl, 0.12 mM MgCl2(H2O)6, 0.5 mM (NH4)2CO3,
15.6 mM HCl, and 0.15 mM CaCl2(H2O)2; finally, the small
intestine solution must have concentrations of 6.8 mM KCl,
0.8 mM KH2PO4, 85 mM NaHCO3, 38.4 mM NaCl, 0.33 mM
MgCl2(H2O)6, 8.4 mM HCl, and 0.6 mM CaCl2(H2O)2.
3 Methods
3.1 Encapsulation Encapsulant mixture is dispersed in 0.1 M CaCl2 solution by drip-

Process ping it with a 21 G needle placed 15 cm above the solution at
2.5 mL/min, employing a peristaltic pump coupled with a head
and a silicone hose; once capsules are formed, they are stored in
0.1 M CaCl2 solution for 12 h at 4 °C to promote greater reticula-
tion (see Note 2).
3.1.1 Viability Assay Viability and encapsulation efficiency is evaluated by dissolving 1 g

of capsules in Gomori buffer for 5 min with stirring at 2000 rpm
with a vortex; from this solution, 100 μL are used as inoculum,
serial decimal dilutions are done, and then the mixture is cultured in
MRS agar for subsequent counting (see Note 3). In the case of free
cells, buffer and agitation are omitted.
3.2 Inoculation and Juice is inoculated with 10% w/v probiotic capsules (see Notes 4
Characterization of and 5) [27].
Fruit Juices with
Encapsulated Probiotic
3.2.1 Characterization of Each week the CIELab scale parameters L*, a*, and b* are deter-
Probiotic Juice mined (see Note 6) with a colorimeter to determine the total
change of color (see Note 7). The pH and amount of soluble solids
are monitored weekly (see Note 6) using a pH meter and a
refractometer.
Interpretation of the color space might help to illustrate in
visual form the change of color over time (see Fig. 1).
4 In Vitro Evaluation
Evaluation of in vitro gastrointestinal simulation is carried out

employing the COST INFOGEST methodology proposed for
in vitro digestion [28, 29], where three stages of digestion are
simulated: oral, gastric, and intestinal.
Capsules (5 g) and/or free cells (5 mL) are mixed with 4 mL of
salivary solution, 0.75 mL of α-amylase solution, and 25 μL of
CaCl2; the pH is adjusted to 7 if required, and the mixture made
up to 10 mL with ultrapure type I water. The mixture is incubated
for 2 min at 37 °C. Once the incubation period is over, the reaction
is finished by placing the mixture in an ice bath to slow down
enzyme activity.
To the resultant salivary phase mixture, 8 mL of gastric solu-
tion, 0.667 mL of pepsin solution, 0.48 mL of gastric lipase, and
5 μL of CaCl2 are added to form the gastric phase; after adding this,
the pH is adjusted to 3.0 ± 0.2 and the solution made up to 20 mL
Fig. 1 CIELab space color changes in probiotic pomegranate juice

Evaluation of color of pomegranate juice during storage. “Control” sample is simple pomegranate juice,
“Free” contains pomegranate juice with Lacticaseibacillus rhamnosus GG (LGG), and “Encapsulated” is
pomegranate juice with capsules charged with LGG, which were elaborated by high-energy processes
with ultrapure type I water. Once the gastric phase is ready, it is

incubated at 37 °C for 2 h. Following incubation, samples are
cooled in the same way as the salivary phase in an ice bath.
In brief, to obtain the intestinal phase, 8 mL of small intestine
solution, 5 mL of pancreatin, 40 μL of CaCl2, and 3 mL of bile
solution are mixed along with solution derived from the gastric
phase; for intestinal phase simulation, the pH is adjusted to
7.0 ± 0.2 and the solution made up to 40 mL with ultrapure type
I water. Then, the intestinal phase is incubated for 2 h at 37 °C with
agitation at 95 rpm.
At the end of each stage of gastrointestinal simulation, aliquots
are taken to conduct morphological analysis by microscopy
(see Fig. 2) and viability through culture in Petri dishes.
5 Morphological Analysis
Scanning electron microscopy (SEM) is used to visualize morpho-

logical changes that the capsules may present initially or when
subjected to various external factors, allowing these changes to be
appreciated in greater detail. The same microscopy technique can
be used to observe bacteria. In addition to SEM, the morphology
of the capsules can be monitored through different morphometric
parameters.
Fig. 2 SEM visualization of microcapsules during in vitro digestion

Capsules elaborated with high-energy techniques; (a) oral phase, (b) gastric phase, and (c) intestinal phase
5.1 Sampling, Morphological analysis is applied to capsules once they are

Preparation, and SEM prepared, during shelf life, and at the end of each gastrointestinal
Visualization digestion stage. In all cases, one or two capsules are dried for 1 h in
a laminar flow cabinet at 25 °C; the same drying method is applied
for free bacteria, with the difference that the sample is taken directly
from a colony in a Petri dish. Capsules and cells are dried once they
are deposited on a stub with carbon tape. The conditions usually
employed for SEM analysis are 10 and 8 kV voltages, a low vacuum,
a secondary electron detector, and a work distance of 10 mm (see
Fig. 2) (see Note 8).
5.2 Preparation and Morphometric parameters like Feret diameter, area, and circularity
Visualization of are obtained through photographs in JPG format taken with a
Capsules with professional camera at 15 cm from the objective with a resolution
Morphometric of 24.5 megapixels; for that, a medium containing 100 capsules is
Parameters taken and placed in a Petri dish with millimetric paper (see Fig. 3),
and they are analyzed through the ImageJ software (see Note 9).
6 Notes
1. The optimum growth temperature for most probiotic strains

(predominantly Lactobacillus) is between 30 and 40 °C; how-
ever, 37 °C is recommended along with media with a pH range
between 5.5 and 6.2. To promote an adequate mass exchange,
the use of an incubator with agitation is recommended. In the
case of Bifidobacterium, the range of pH might vary between
6.5 and 7.0 because of its lower acid tolerance [30].
2. It is possible to carry out an alternative method of dosage to
obtain a smaller particle size by employing atomization
through a SUJ1A configuration nozzle with a flux of CO2 at
10 SCFH (standard cubic feet per hour) and 50 psi of pressure,
Fig. 3 Preparation of microcapsules for SEM visualization and digital image analysis
Preparation of capsules for determination of morphometric parameters via (a) digital camera and (b) scanning
electron microscopy. Capsules obtained by high-energy techniques
15 cm work distance, and a CaCl2 solution in gentle stirring.

This method needs to filter capsules with a vacuum pump and
11 μm porous size filter paper before viability analysis.
3. Viability is evaluated with the following equation:
N
Viability ð%Þ = × 100
N0
where viability is given as a percentage, N0 is the number of
bacteria (log CFU/mL) before encapsulation, and N is the
number of bacteria (log CFU/mL) released by the
capsules [2].
4. Clarified juices are recommended for product development; to
facilitate analysis of cell viability; the volume of juice used
depends on the beneficial effect sought to generate a functional
food. Chelating agents in juices can destabilize the capsule wall,
causing loss of viability by the interchange of Ca+ with Na+ and
K+ ions in juice [31].
5. Selecting a food matrix (juice) with beneficial properties is
recommended to generate a functional food. The volume of
juice must be based on existing studies that show their effec-
tiveness. To obtain the beneficial effect of specific probiotic
bacteria, it is necessary to have a cell concentration in food of
between 6 and 8 log CFU/g [32].
6. It is recommended that all characterization analysis of the food

matrix is done once viability has been assessed to avoid con-
tamination. In addition, the frequency with which the food
matrix is analyzed should provide kinetic data on viability and
food evolution.
7. The change of color is analyzed with the following equation:
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ΔE ab = ðL 2 - L 1 Þ2 þ ða2 - a 1 Þ2 þ ðb 2 - b 1 Þ2
Where L1 and L2 are the luminosity of samples 1 and 2, a1 and
a2 are the red/green coordinates of samples 1 and 2, and b1 and
b2 are the yellow/blue coordinates of samples 1 and 2. Sample
1 refers to the control parameters of juice at time 0 of storage
[33]. To obtain adequate measures of color, it is recommended
to use a vessel that fits with the colorimeter sensor and has dark
walls and a lid to avoid the passage of external light through the
colorimeter sensor.
8. The conditions used for visualization of bacteria through SEM
as well as the drying period of the sample vary in agreement
with each bacterial strain; if bacteria tend to have large amounts
of mucin, extending the drying time up to 8 h is recom-
mended, besides using another method of drying such as des-
iccator drying.
9. The circularity of capsules is calculated from the following
equation:
4πA
Circularity =
P 2:
Where A is the capsule area and P is the perimeter; if values
of circularity area are equal or close to 1, capsules are consid-
ered to have a morphology geometrically comparable to a
perfect circle [34]. The image analysis software (ImageJ)
directly provides parameters like Feret diameter and area.
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Chapter 8
Probiotic Fermented Vegetables

Hadi Pourjafar, Tatiana Colombo Pimentel, and Tahis R. Baú
Abstract
Fermented foods (as functional foods) are rich in various nutrients that benefit consumers’ health.
Fermented vegetables are a group of fermented foods that have a long history and have been used by
humans in the distant past until today as healthy, delicious, and popular foods, and most importantly, with a
long shelf life. Probiotic fermented vegetables are products that have been fermented and produced by
probiotics; either these microorganisms are naturally and intrinsically present in the vegetables or added
manually. In some cases, despite probiotics in fermented vegetables, the primary fermentation process is
carried out by non-probiotic microorganisms, and probiotics’ role in this process is minor. But in any case, it
acts as a carrier for probiotics to be transferred to the consumer’s body and digestive system. Therefore,
fermented vegetables are an excellent source for isolating and commercializing different and beneficial
probiotic strains. Here we will describe the process of obtaining probiotic vegetable pickles, probiotic
sauerkraut, and probiotic natto.
Key words Fermented vegetables, Health-promoting, Probiotic vegetable pickles, Probiotic sauer-
kraut, Probiotic natto
1 Introduction
Functional food products, especially fermented foods, are products

that have one or more positive effects on health yonder a rudimen-
tary diet [1, 2]. Meanwhile, the health role of fermented probiotic
products is also prominent, and extensive studies have been con-
ducted in this field. As a result, the probiotic product market is
growing very rapidly owing to enlarged customer consciousness
about the impact of these products on health. Currently, probiotic
products, especially probiotic fermented food products, comprise
60–70% of the total functional food market [3–5]. The worldwide
shop for probiotic products was around 24.8 billion € in 2011,
above 31.1 billion € in 2015, and is anticipated to reach around
43 billion € by 2020 [6]. Today, non-dairy fermented probiotic
products have received more attention than dairy products due to
119
120 Hadi Pourjafar et al.
their low fat and more straightforward production process. In

addition, they are used for people with allergies to dairy products.
Therefore, it has more demand from consumers [6–8].
The dietetic and biological potential of vegetables has led to
their alteration into products with multiple characteristics in
upholding the steadiness of microorganisms. Vegetables are rich
in vitamins, minerals, and valuable fibers and usually cause health
effects on the consumer’s body. Many of them are even used to
treat various diseases. Suppose these products are fermented by
probiotic microorganisms, in addition to the presence of probiotics
inside these products, which are transferred to the consumer’s body
either completely or as paraprobiotics and postbiotics, some vege-
table compounds are fermented and converted into healthier com-
ponents for the consumer’s body. These can be referred to as
beneficial organic acids, such as gluconic and glucuronic acids.
Studies have revealed that alkalis, bioflavonoids, potassium salts,
and vitamins in vegetables can positively impact the inhibition and
cure of cardiovascular illness [8–11].
Furthermore, it has been revealed that the valuable properties
of vegetables can be enriched via a natural procedure like lactic
fermentation. Currently, scientists are investigating the lactic fer-
mentation of vegetables as a natural preservation process. Further-
more, various vegetables have natural prebiotic ingredients that
encourage the growth of certain probiotic microorganisms in the
fermented food product and gastrointestinal tract [12].
Many studies have been done concerning probiotic fermented
dairy products, and most of the products available and accessible to
consumers are mostly dairy products. However, many consumers
are looking for a better alternative to dairy products due to some
disadvantages, such as people’s high-fat content and lactose intol-
erance. Hence, the consumption of probiotic fermented vegetable
foods can be a worthy substitute for some people, such as vegetar-
ians and people with allergic reactions to milk proteins [6, 7].
The fermentation process is an old method to increase the shelf
life of vegetable products, which happen to be highly perishable.
Good examples of old and well-known fermented vegetables
include products such as Chinese “PaoCai,” Korean “Kimchi”
(sauerkraut/sour cabbage), Indonesian “Tempeh,” Japanese
“Natto” and “Miso” (fermented soybeans), Chinese “Jiangshui”
(Chinese cabbage, potherbs, radish sprouts, mustard, and fermen-
ted celery), which their health-promoting effects have been proven.
Moreover, various probiotic strains have been isolated from them
[13]. In general, two modes can be considered for probiotic fer-
mented vegetables; one is fermented products that only act as a
carrier for probiotics, and the fermentation process is mainly done
by other microorganisms (non-probiotic microorganisms). The
second case is products that are fermented by probiotic strains.
However, separating these two modes in natural probiotic
Probiotic Fermented Vegetables 121
fermented products is difficult unless one or two specific probiotic

strains are intentionally added to produce commercial probiotic
fermented foods. Probiotic vegetables can be manufactured by
adding probiotics, e.g., into the vegetables/juice or fermentation
with the probiotic microorganism. The second way (fermentation)
is more valued because the probiotic grows into the vegetable
texture/juice into a more adapted probiotic and a low-sugar man-
ufactured good, possibly improving its viability [14, 15]. Further-
more, during fermentation, some metabolites of probiotic
microorganisms, for instance, bacteriocins, exopolysaccharides,
and bacterial cellulose, can increase the quality of the probiotic
fermented product and upsurge their shelf life over the storage
period [6].
A noteworthy point in fermented vegetables is the high acidity
and a significant drop in pH in some of these products compared to
fermented dairy products. As a result, probiotic strains should be
used in these products if they are resistant to acidic conditions
(pH ~ 3.5) [7]. It has been revealed that in fermented vegetables
(pH 3.7–4.3), Lactobacilli can struggle and survive better than
Bifidobacteria [16]. However, some vegetables may have ingredi-
ents that sustain the viability of probiotics, such as saccharides and
organic acids that may be used as a carbon source, ascorbic acid that
drops O/R potential, and cellulose that can protect the probiotics
during the fermentation process and storage time [17]. Cabbage,
soybean, beetroot, carrot, and celery are some instances of vegeta-
bles used as substrates for the transfer of probiotics. Various probi-
otic strains, regular species of Lactobacillus and Bifidobacterium,
such as Lactobacillus acidophilus, L. casei, L. paracasei,
L. rhamnosus, L. fermentum, L. plantarum, and Bifidobacterium
bifidum, B. breve, B. longum have been primarily employed in the
development of various probiotic fermented vegetables [6, 7].
It has been investigated that the suitability of cabbage juice
[18, 19], tomato [20], and beet juice [21] using L. acidophilus,
L. casei, L. plantarum, and L. delbrueckii after 4 weeks of storage at
4 °C and the amount of the active four probiotics in all fermented
products ranged from 105–108 CFU/mL. In another study, Kun
et al. (2008) disclosed that carrot juice can encourage the growth of
B. lactis Bb-12, B. bifidum B7, and B. bifidum B 3.2. Entire
probiotics displayed cell counts of 1010 CFU/mL [22]. In a
study by Zhu et al. (2020), the survivability of L. sanfranciscensis
in tomatoes was examined during 4 weeks of storage at 4 °C, and
results indicated that the survival rate of L. sanfranciscensis in
samples was >106–107 CFU/mL at the end of storing period [23].
One of the most critical fermented plant products with many
fans and good health effects is fermented soybean, from which
several strains of probiotics have been isolated, and some have
even been commercialized. For example, it has been determined
that in the tempeh, traditional Indonesian soybean cake, Rhizopus
microsporus var. oligosporus IFO 8631, a high concentration of

Gamma-Aminobutyric Acid/GABA (GABA is the primary inhibi-
tory neurotransmitter in the central nervous system/CNS. Its pri-
mary role is decreasing neuronal excitability all over the nervous
system. It reduces a nerve cell’s capability to receive, make, or send
chemical messages to other nerve cells. Various homeopathic situa-
tions are related to the alteration levels of GABA. Manifold medi-
cines that target the GABA receptor) were detected [24]. In
addition, some fermented soybeans, such as Japanese Miso
and Malaysian fermented soybean, are a source of probiotic yeast
and lactic acid bacteria/LAB like Zygosaccharomyces sapae I-6 L and
L. plantarum LAB12, respectively, which have anti-inflammatory
properties [25, 26]. Various investigations have shown that many
probiotic strains isolated from fermented soybean products belong
to the Bacillus genus. For example, Bacillus licheniformis 141 as a
probiotic, isolated from Cheonggukjang, a Korean traditional soy-
bean paste, which increases the lifecycle of Caenorhabditis elegans
nematode over serotonin signaling; Bacillus paralichemiformis and
Bacillus sonorensis isolated from fermented soybean paste that ame-
liorated obesity, Nonalcoholic Fatty Liver Disease/ NAFLD and
insulin resistance in a mice model of obesity [13, 27, 28]. Therefore,
various plant products, especially fermented soybean products, can
be an important source of bacterial and yeast probiotics. In Table 1
some probiotic fermented vegetable products are presented. This
chapter describes the processes for obtaining probiotic vegetable
pickles (see Subheading 2), probiotic sauerkraut (see Subheading 3),
and probiotic natto (see Subheading 4).
2 Probiotic Vegetable Pickles
Probiotic vegetable pickles are products obtained by fermenting

vegetables, producing acids in a brine initially composed of water,
sugar, and salt. Although the use of microorganisms claiming pro-
biotics in pickled vegetables is still scarce, this product may be a
promising source of probiotic microorganisms. The main materials
and processing steps for obtaining probiotic vegetable pickles are
described below:
2.1 Preparation of 1. Vegetable raw materials (e.g., cucumber, carrot, pepper, and
Probiotic Vegetable others).
Pickles 2. Drinking water.
2.1.1 Materials 3. Chlorine-based sanitizer (e.g., sodium hypochlorite or sodium
dichloroisocyanurate).
4. Salt.
5. Glucose or sucrose.
Table 1
Some selected probiotic fermented vegetable products
Fermented vegetable Isolated possible

product probiotic strains Health-promoting effects Reference/s
Sauerkraut (Croatian) Lactobacillus brevis SF15 Stimulation and regulation of [29, 30]
L. paraplantarum SF9 the immune system
Sauerkraut (Chinese, PaoCai) L. plantarum LAP6, Prevention of Salmonellosis, [31–33]
L. plantarum S2-5, serum cholesterol
L. plantarum P2, reduction,
L. plantarum S4-1, immunomodulation
Pediococcus pentosaceus (stimulation of
MP12 Th1 type cytokines)
Sauerkraut (Indonesian) L. plantarum sa28k Serum cholesterol [34]
reduction,
Sauerkraut (Kimchi) Lactobacillus Anticancer effects, [13, 27, 35–
acidophilus KFRI342, Serum cholesterol 38]
L. plantarum NR74, reduction, Antioxidant ability
L. plantarum C182,
L. plantarum Ln4,
L. plantarum EM,
L. plantarum LPpnu,
L. plantarum Ln1,
Leuconostoc mesenteroides
F27,
B1,
LMpnu,
Lactococcus lactis KC24,
Kimchi Weissella cibaria JW15 Serum triglycerides [39]
reduction, feces ammonia
emissions reduction
Fermented radish leaves L. gaserri HA4 Strengthening the digestive [40]
(Dua-muoi) system, facilitate the
digestion of food
Fermented green turnip L. fermentum Immunomodulation [41]
(Japanese, Nozawana- Nz8
zuke)
Vegetable pickle (Tuscan L. plantarum HY Antioxidant ability [42, 43]
pickles) L. plantarum JR14
Fermented leek L. plantarum LK8, Antioxidant ability [44]
L. plantarum IMDO 788,
L sakei IMDO 1358,
IMDO 1347,
Weissella confusa LK4
(continued)
Table 1
(continued)

Fermented soybean Rhizopus microsporus var. Creation GABA [24]
(Tempeh) oligosporus IFO 8631
Korean soy sauce Bacillus subtilis MKSK- Antimicrobial activity [25]
E1,
B. subtilis MKSKJ1
Fermented soybean Zygosaccharomyces sapae Anti-inflammatory [26]
(Japanese, Miso) strain I-6 activity
Pickled vegetable (Iranian, Bacillus amyloliquefaciens Antioxidant activity, facilitate [45]
Torshi) 1020G the digestion of food
B. safensis 437F,
B. atrophaeus 1630F
Thai fermented L. plantarum KJ03 Serum cholesterol [46]
stink bean (Sataw-Dong) reduction
Pickled cucumber (Korean, Saccharomyces cerevisiae DNA protection capacity [47]
Jangajji) KU200278, against
Saccharomyces cerevisiae ROS
KU200281
Jiangshui (Chinese fermented Brevebacterium casei, Serum cholesterol [48]
vegetables) Lactococcus raffinolactis reduction
Jiangshui Lactiplantibacillus Ant-pathogenic bacteria [49]
plantarum
Mixed fermented vegetable Lactobacillus plantarum, Probiotic properties [50]
juices (purple cabbage, Saccharomyces cerevisiae
tomato and carrot)
Fermented vegetable Enterococcus hirae Increases immune responses, [51]
immune gene expression,
protection to Aeromonas
hydrophila infection
Pickled vegetables (in the Levilactobacillus Probiotic properties, [52]
Middle Eastern, African, namurensis, resistance against clinically
and Asian sub-continent Lentilactobacillus important antibiotics
regions) buchneri,
Lentilactobacillus
parafarraginis,
Pectobacterium
carotovorum,
Weissella confuse,
Lactiplantibacillus
pentosus,
Leuconostoc carnosum
(continued)
Table 1
(continued)

Fermented vegetable-fruit Saccharomyces cerevisiae, Anti-oxidant and anti-aging [53]
drink, combination Streptococcus thermophiles activities
of vegetable juice (broccoli, TCI125
celery, asparagus, carrot
extracts) and fruit juice
(mulberry, grapes, passion
fruit, blue berry, pineapple,
apple, bayberry, cranberry,
sugar cane, lemon extracts)
in a mass ratio 1:1
Fermented curly kale juice Leuconostoc mesenteroides, Ant-pathogenic bacteria [54]
Lactobacillus plantarum,
L. sakei,
L. coryniformis
6. Probiotic culture (e.g., Lactiplantibacillus plantarum and Lac-

ticaseibacillus casei) and others such as Levilactobacillus brevis
e Leuconostoc mesenteroides (see Note 1).
7. Container or tank for washing and sanitizing vegetables.
8. System for heat treatment of brine and bleaching.
9. Glass packaging.
11. Refrigeration system.
2.1.2 Methods 1. Select vegetable raw materials (see Note 2).

2. Wash the vegetable raw materials with drinking water.
3. Sanitize using chlorine-based products (see Note 3).
4. Rinse with fresh water 3–4 times to remove the sanitizer.
5. Cut the vegetables into cubes, slices, or rounds (see Note 4).
6. Blanch in boiling water for 2–4 min, followed by cooling in
chilled water (see Note 5).
7. Prepare the brine with drinking water, add sucrose or glucose
(1–3%, w/v) for the bacteria to grow, sodium chloride (3–5%,
w/v) (see Note 6), and heat at 95 °C for 5 min in a water bath
and cooled to microbial inoculation (see Note 7).
8. After cooling to 20–30 °C, add the probiotic culture and
homogenize (see Note 8).
9. Pack the vegetables in a glass container and add the brine (see
Note 9).
10. Ferment at the time and temperature (usually 20–30 °C) indi-
cated for the probiotic culture used until it reaches a pH lower
than 3.8 (see Note 10).
2.1.3 Notes 1. Check the manufacturer’s instructions for use in less than the
recommended volume in the microbial culture envelope.
More instructions are given in Chap. 5, in the Notes to
Subheading 3.
2. Raw materials must be at the appropriate degree of maturation
without physiological damage, mechanical injuries, or
deterioration.
3. It is recommended to use sodium hypochlorite or sodium
dichloroisocyanurate (150 ppm for 15 min). However, other
sanitizers approved by a regulatory agency can be used.
4. Some vegetables can be used whole, for example, mini cucum-
bers and baby carrots.
5. The bleaching inactivates enzymes, reduces undesirable micro-
organisms, softens the texture, facilitates packaging, and con-
tributes to preserving the color of vegetables.
6. In high concentrations of NaCl, the growth of Leuconostoc
mesenteroides and other less salt-tolerant species is inhibited.
7. CaCl2 can partially replace NaCl to avoid excessive softening if
the objective is to obtain a crunchy texture during storage.
Pasteurization of the brine before fermentation is necessary to
ensure the safety and growth exclusivity of the inoculated
starter culture.
8. It is recommended that the starter culture probiotic concentra-
tion be at least 7 log CFU/mL in brine to inhibit spoiling
microbiota. In addition, after excessive acid accumulation, a
reduction in the probiotic count can be observed during stor-
age. Therefore, it is recommended to use Lactiplantibacillus
plantarum and Lacticaseibacillus casei LA284 probiotics,
which are acid resistant to pickle processing conditions,
keeping their viability stable during storage [55]. Lactobacillus
acidophilus has also been shown to be suitable for use in pickled
vegetables. In addition, mixed cultures can be used, containing
Levilactobacillus brevis and Leuconostoc mesenteroides ensured
the most preferred end products regarding sensory properties
[56]. The main species responsible for fermentation are homo-
fermentative lactic acid bacteria, such as Lactiplantibacillus
plantarum. During prolonged fermentation, lactic acid can
be metabolized if spoilage microorganisms are present, trigger-
ing the production of spoilage by-products such as propionic
acid, butyric acid, and CO2. The CO2 can cause economic
losses, affecting the texture and bloater damage.
9. On an industrial scale, fermentation is usually carried out in

tanks, and the product is packaged in smaller containers.
10. The fermentation time of pickles from vegetables fermented by
Lactiplantibacillus plantarum is 20–30 °C for 72 h, but this
may vary according to the microorganism used. When lactic
acid bacteria are used, the pH of the product usually decreases
rapidly in the first 24 h of fermentation due to the intense
production of lactic acid by the probiotics.
3 Probiotic Sauerkraut
Sauerkraut is a product made from cabbage by fermentation of a

mixed culture of bacteria, in the presence of sodium chloride and
with a production of lactic acid. As it is a product fermented with
lactic acid bacteria, it is a suitable substrate for the growth of
probiotic bacteria. The main materials and processing steps for
obtaining probiotic sauerkraut are described below:
3.1 Preparation of 1. White cabbage.

Probiotic Sauerkraut 2. Drinking water.
3.1.1 Materials 3. Chlorine-based sanitizer (e.g., sodium hypochlorite or sodium
dichloroisocyanurate).
4. Salt.
5. Leuconostoc mesenteroides and probiotic culture Lactiplantiba-
cillus plantarum (see Note 1).
7. Container or tank for washing and sanitizing vegetables.
8. System for pressing and fermentation.
9. Glass or plastic packaging.
11. Refrigeration system.
3.1.2 Methods 1. Select cabbage heads by removing the outer leaves and central
stalk (see Note 2).
2. Wash the cabbage with drinking water.
3. Sanitize using chlorine-based products (see Note 3).
4. Rinse with fresh water 3–4 times to remove the sanitizer.
5. Cut the cabbage into thin (0.5–2 mm) and even slices.
6. Place the cabbage slices in the fermentation vessel or tank, and
add sodium chloride (1–4%, w/w) (see Note 4).
7. Add a starter culture containing probiotic bacteria (see Note 5).
8. Pressing the product compresses it and facilitates the release of
liquid during fermentation.
9. Keep fermentation vessels closed to promote anaerobic condi-

tions (see Note 6).
10. Ferment at the time and temperature (usually 20–25 °C by
3–10 days) indicated for the probiotic culture used until it
reaches at least 1.5% lactic acid and a pH lower than 3.8.
11. Pack the sauerkraut in previously sterilized glass or plastic jars.
3.1.3 Notes 1. Leuconostoc mesenteroids present rapid adaptation in this sub-

strate. Therefore, its growth is more significant at the begin-
ning of fermentation. At the end of fermentation, the probiotic
Lactiplantibacillus plantarum may predominate, which is
more resistant to acidic conditions [57].
2. White cabbage heads without mechanical injuries or deteriora-
tion should be selected. Before processing, cabbage must be
kept at room temperature for 36–48 h, so its leaves
partially wilt.
3. It is recommended to use sodium hypochlorite or sodium
dichloroisocyanurate (150 ppm for 15 min). However, other
sanitizers approved by a regulatory agency can be used.
4. Salt promotes the removal of water from the plant cell, used by
the fermenting microorganisms. In high concentrations of
NaCl, the growth of Leuconostoc mesenteroides and other less
salt-tolerant species is inhibited.
5. It is recommended that the starter culture probiotic concentra-
tion be at least 7 log CFU/g in product to inhibit spoiling
microbiota.
6. Initially, the fermentation is aerobic, but anaerobic respiration
begins due to the reduction of oxygen by the respiration of the
vegetable and pressing. Therefore, preferably use a fermenta-
tion system that allows the CO2 produced during fermentation
to escape, reducing the internal pressure [57].
4 Probiotic Natto
Natto is a food made from the fermentation of cooked whole

soybeans, popularly consumed by the Asian population. This prod-
uct, which has sticky and viscous filaments, is obtained by fermen-
tation of Bacillus subtilis natto. This microorganism has recently
been investigated for its probiotic potential and was included in the
list of probiotics of the Food and Drug Administration (FDA)
[57]. The materials and processing steps for obtaining probiotic
natto are described below:
4.1 Preparation of 1. Food-grade whole soybeans (see Note 1).

Probiotic Natto 2. Drinking water.
4.1.1 Materials 3. Probiotic culture of Bacillus subtilis natto (see Note 2).
5. Pressure heat treatment system.
6. Polystyrene tray.
7. Room with temperature and humidity control for
fermentation.
4.1.2 Methods 1. Select the soybeans, removing the impurities.

2. Wash dry whole soybeans and soak them in chilled water over-
night. The volume of water is usually 2 to 3 times the volume of
the bean.
3. Cook the soybeans (1.5 kg/cm2) for 20 min (see Note 3).
4. Spray the probiotic culture (3–4 log UFC/g) and mix (see
Note 4).
5. Transfer a portion to a polystyrene tray, in even layers of 4–5
centimeters, covering with a perforated plastic wrap to keep the
moisture.
6. Ferment at 40 °C for 24 h (see Note 5).
7. Mature for 24 h under refrigeration and store at refrigeration
temperature.
5 Notes
1. Preferred grains with tiny seeds and light hilum. Better sensory
characteristics are obtained with soybean cultivars with lower
protein content and higher carbohydrate and lipid content.
2. Commercial cultures can be used for direct vat set (DVS)
inoculation or to prepare the inoculum on a laboratory scale.
For laboratory scale, the strains can be inoculated in nutrient
broth and fermented at 37 °C for 2 days, followed by centrifu-
gation to obtain the pellet, which can be resuspended in sterile
water to obtain the appropriate count of microorganisms [57].
3. Please note that the soybean hulls may come off during
cooking, clogging the pressure release valve. Cooking can
take place without pressure. However, the time will be much
longer, and the beans must be cooked until a very soft texture is
obtained.
4. If inoculation is carried out with vegetative cells, the soybeans
must be previously cooled to 40 °C so as not to reduce the
viability of Bacillus subtilis natto. However, suppose the inocu-
lation is carried out with spores of Bacillus subtilis natto. In that
case, they can be inoculated with still-hot soybeans (80–90 °C)

as these spores are resistant to heat, and thermal shock is
important to activate them [57, 58].
5. Generally, the growth temperature is between 39 and 43 °C,
and the ideal temperature for spore germination is 40 °C
[58]. As the fermentation progresses, higher mucilage viscosity
is observed, in addition to presenting an ammonia aroma.
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Chapter 9
Kombucha Production and Its Bioactive Compounds

Analysis
Chun Zou, Yong-Quan Xu, Yi-Bin Huang, and Jun-Feng Yin
Abstract
Kombucha is a fermented beverage obtained from the fermentation of sweetened tea by a consortium of
yeasts and bacteria. The beverage contains many bioactive compounds from tea extraction and microbial
synthesis. In this chapter, we describe two preparation methods of Kombucha using a symbiotic culture of
bacteria and yeast or a synthetic microbial community as a starter. Moreover, the determination of bioactive
compounds, including organic acids, sugars, and catechins, has been introduced.
Key words Kombucha, Tea, Yeast, Bacteria, Bioactive compounds
1 Introduction
Kombucha is a functional beverage produced by the fermentation

of sugared tea broth with a symbiotic culture of bacteria and yeast
[1, 2]. There are many active compounds found in Kombucha
[3, 4], such as tea polyphenols, vitamins (B1, B2, B6, B12, and
C), organic acids (acetic acid, gluconic acid, glucuronic acid,
among others), and D-saccharic-1,4-lactone acid. These active
compounds provide Kombucha with potential benefits for human
health [4, 5], including antioxidant, antimicrobial, and hepato-
protective effects.
Kombucha is composed mainly of various acetic acid bacteria
and yeasts, sometimes containing a little lactic acid [6]. The tradi-
tional culture method of Kombucha uses a symbiotic culture of
bacteria and yeast (SCOBY) as a starter [7, 8], in which a cellulose
film is formed. Still, it is difficult to control the microorganisms.
Therefore, more and more researchers use a synthetic microbial
community (SMC) as a starter for fine control [9, 10]. In this
chapter, the preparation method of Kombucha using SCOBY or
133
134 Chun Zou et al.
SMC has been introduced in detail. In addition, the determination

of bioactive compounds, including organic acids, sugars, and cate-
chins, has also been described.
2 Materials
2.1 Sugared The composition of the sugared tea broth included the following
Tea Broth materials (g/L) [11]: sucrose, 50–100; tea, 1.5–10.
2.2 Yeast Extract The composition of the YPD broth included the following materi-
Peptone Dextrose als (g/L) [12]: yeast extract, 10; peptone, 20; D-glucose, 20.
(YPD) Broth
2.3 The composition of the HS medium included the following mate-

Hestrin-Schramm (HS) rials (g/L) [13]: glucose, 20; peptone, 5.0; yeast extract, 5.0;
Medium disodium phosphate (anhydrous), 2.7; and citric acid (monohy-
drate), 1.15; and final pH 5.0 0.1.
2.4 De Man Rogosa The composition of the MRS medium included the following
Sharpe (MRS) Medium materials (g/L) [14]: glucose, 20; tryptone peptone, 10; beef
extract, 8; yeast extract, 4; sodium acetate, 5; diammonium hydro-
gen citrate, 2; dipotassium hydrogen phosphate, 2; magnesium
sulfate, 0.2; manganous sulfate, 0.05; Tween 80, 1; and final
pH 6.5 0.1.
3 Methods
3.1 Preparation 1. Preparing the sugared tea broth [15]: Mix 6 g of tea leaves with
Method of Kombucha 100 g of sucrose in 1 L of boiling water and steep for 15 min (see
Note 1). Then, filter the tea leaves and transfer the sugared tea
broth into a sterilized glass jar. Cool the sugared tea broth to
room temperature (see Note 2).
2. Inoculating starter cultures (SCOBY (a) or SMC (b)):
(a) Use a SCOBY (see Notes 3 and 4) as a starter (Fig. 1).
Inoculate 10 g of SCOBY into the glass jar bottle with the
sugared tea broth, and seal with sterile gauze.
(b) Use an SMC as a starter. Inoculate single colonies of yeast,
acetic acid bacteria, and lactic acid bacteria into YPD
broth, HS medium, and MRS medium (see Note 2),
respectively, and culture at 30 C with 160 rpm agitation
for 24 h, at 30 C with 160 rpm agitation for 48 h, and at
37 C with no agitation for 24 h, respectively. Inoculate
combinations of the three strains into the glass jar bottle
with the sugared tea broth, and seal with sterile gauze.
Kombucha Production and Its Bioactive Compounds Analysis 135
Fig. 1 Kombucha production using a SCOBY as starter
3. Culturing Kombucha: place the Kombucha cultures in an incu-

bator (see Note 5) at 28 2 C, and let them stand for
1–2 weeks.
4. Post-fermentation treatment: after the fermentation process,
remove the cellulose film (see Note 4), and collect the fermen-
ted broths (see Note 6).
3.2 Determination of 1. Mobile phase preparation.

Organic Acids (a) Solvent A: Mix 970 mL of 0.1% phosphate solution and
30 mL of methanol in a 1 L glass bottle, then filter the
mixture using a 0.45-μm microporous membrane.
(b) Solvent B: 500 mL of 100% methanol (HPLC-grade).
2. Sample preparation.
Centrifuge the fermented broths at 8000 rpm for 10 min,
and collect the supernatants (see Note 7). Then, filter the
supernatants using a 0.45-μm microporous membrane.
3. Instrument selection.
HPLC equipped with a UV-DAD detector and an Agilent
ZORBAX® SB-C18 column (4.6 150 mm, 5 μm).
4. LC settings [15].
(a) Detection wavelength: 210 nm.
(b) Column temperature: 40 C.
136 Chun Zou et al.
(c) Injection volume: 10 μL.

(d) Flow rate: 1 mL/min.
(e) Gradient program: Maintain solvent A at 100% from 0 to
10 min, linearly ramp to 100% solvent B over 1 min,
maintain solvent B at 100% for 5 min, linearly ramp to
100% solvent A over 1 min, and then maintain solvent A at
100% for 5 min.

Sugars Solvent A: Mix 700 mL of acetonitrile and 300 mL of water
in a 1 L glass bottle, and then filter the mixture using a 0.45-μm
microporous membrane.
and collect the supernatants. Then, filter the supernatants
using a 0.45-μm microporous membrane.
HPLC equipped with an evaporative light-scattering
detector and a Waters XBridge™ Amdie column
(4.6 150 mm, 5 μm).
4. LC settings [15].
(a) Column temperature: 40 C.
(b) Injection volume: 10 μL.
(c) Flow rate: 1 mL/min.
(d) Elution mode: Isocratic mode.
(e) Detector setting: Set drift tube temperature at 80—90 C
and nitrogen pressure at 350 kPa.

Catechins (a) Solvent A: Add 90 mL acetonitrile, 20 mL acetic acid, and
2 mL EDTA-2Na solution (10 mg/mL) into a 1 L volu-
metric flask, add water to make a final volume of 1 L, and
then filter the mixture using a 0.45-μm microporous
membrane.
(b) Solvent B: Add 800 mL acetonitrile, 20 mL acetic acid,
and 2 mL EDTA-2Na solution (10 mg/mL) into a 1 L
volumetric flask, add water to make a final volume of 1 L,
and then filter the mixture using a 0.45-μm microporous
membrane.
and collect the supernatants. Then, filter the supernatants
using a 0.45-μm microporous membrane.
Kombucha Production and Its Bioactive Compounds Analysis 137
HPLC equipped with a UV-DAD detector and a Waters
Symmetry C18 column (4.6 250 mm, 5 μm).
4. LC Settings [16].
(a) Detection wavelength: 278 nm.
(b) Column temperature: 35 C.
(c) Injection volume: 10 μL.
(d) Flow rate: 1 mL/min.
(e) Gradient program: Maintain solvent A at 100% from 0 to
10 min, linearly ramp to 68% solvent A and 32% solvent B
over 15 min, maintain solvent A at 68% and solvent B at
32% for 10 min, linearly ramp to 100% solvent A over
1 min, and then maintain solvent A at 100% for 5 min.
4 Notes
1. The tea types used in Kombucha production are commonly

black tea or green tea. Therefore, the time for extracting tea
juice with boiling water should not be too long, which can
avoid the flavor deterioration of tea juice.
2. All the media mentioned in “2 Materials” should be sterilized
and cooled to room temperature before inoculation.
3. Starter cultures, SCOBY in particular, should not be contami-
nated by undesired microbes. If any mildew is found, the
cultures should be discarded.
4. The SCOBY could be stored in a tea base liquid mixture. This
can be kept at room temperature for up to 3 weeks, but for
more extended storage, the SCOBY should be placed in the
refrigerator at 4 C.
5. Because vitamins in Kombucha are easily degraded under light,
Kombucha should be cultivated in clean and dark conditions.
6. When the fermentation time is too long, the acidity of Kom-
bucha might be too high to drink directly. Therefore, it can be
appropriately diluted and mixed with sugar or honey before
drinking.
7. When the organic acids of Kombucha are determined, the
fermented broths should be diluted at a suitable multiple. It
is due to the high concentration of organic acids in Kombucha.
138 Chun Zou et al.
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narrowed review. Food Chem X 14:1–10 bucha for high-level production of glucuronic
5. Morales D (2020) Biological activities of kom- acid. LWT Food Sci Technol 64:1149–1155
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6. Soares MG et al (2021) Technological aspects ter xylinus B2-1 using the shell extract of
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pounds of interest: a literature review. Trends 14. Zhang CC et al (2019) New selective media for
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7. Harrison K, Curtin C (2021) Microbial com- rhamnosus and Streptococcus thermophilus. J
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8. Lee KR et al (2022) Kombucha fermentation dant profile. Food Chem 363:1–8
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gluconic acid with a microbial community
Chapter 10
Probiotic Beer
Fernanda Meybom, Bárbara Mortl, and Alan Ambrosi
Abstract
The demand for functional beverages that provide health benefits to consumers has increased in recent
years. In this sense, several studies investigate the addition of probiotics in beers. However, there are several
challenges to overcome when adding probiotics to beer, such as the presence of alcohol and hop com-
pounds that prevent the maintenance of a higher viable number of microorganisms. Thus, traditional beer
production routes may not be recommended for this kind of product. Here, we provide a guideline on how
to prepare a probiotic beer that can be used for researching new probiotic microorganisms and highlight
essential points to be considered when developing probiotic beers.
Key words Functional beer, Functional beverage, Brewing, Yeast, Levilactobacillus brevis, Saccharo-
myces cerevisiae
1 Introduction
Beer is one of the oldest fermented beverages, dating back to the

Neolithic age, and, nowadays, the most consumed alcoholic bever-
age in the world. Beer was considered food for several years, and
sometimes, the only beverage safe to drink [1]. The beer industry
has grown remarkably as time passed, especially during the Indus-
trial Revolution, with technological improvements in equipment,
ingredients, and implementation of scientific principles. In the last
two decades, craft beers have experienced exponential growth,
driven by consumers who seek unique drinking experiences. The
current demand for health benefits or awareness about the impor-
tance of a healthy diet has driven the beer market to develop health-
oriented beverages, like low/no alcohol beer, low-calorie beer,
gluten-free beer, and functional beers [2].
Before it was known that microorganisms were responsible for
transforming sugars (from grains) into ethanol, carbon dioxide,
and a variety of volatile compounds, early beers were soured to
some degree due to acidification by wild yeast and bacteria during
139
140 Fernanda Meybom et al.
spontaneous fermentation. Therefore, some traditional sour beers,

which are intentionally acidified through wild lactic acid bacteria
(LAB) and/or acetic acid bacteria (AAB), are considered a classic
style of craft beer. Beer styles like Belgian Lambics and Flanders Red
Ales represent some of the oldest commercial sour beers, which
have recently seen a strong revival [3]. To avoid inconsistencies in
aroma, flavor, quality, and long fermentation periods by using wild
yeasts and wild LAB, pure or mixed commercial LAB cultures are
preferred by brewers to control the brewing process, making a fast
and reproducible biological acidification of wort [4].
Drinking unfiltered and unpasteurized beers rich in live pro-
biotics is related to health benefits that regular beers might not
provide. Probiotics are “live microorganisms that, when adminis-
tered in adequate amounts, confer a health benefit on the host”
[5]. However, if the wild LAB and yeast involved in the spontane-
ous fermentation are not isolated and defined, and, if there is no
evidence from well-designed clinical trials that suggests a possible
health benefit, this undefined microbial consortium cannot be
considered a probiotic [6].
Probiotic beer can be defined as a beverage obtained using
probiotic microorganisms during the fermentation process
[7]. The fermentation process can be conducted in one step, fer-
menting with one probiotic microorganism or co-fermenting using
more than one microorganism [8, 9], or in two steps, fermenting
with Saccharomyces cerevisiae, followed by fermentation with probi-
otic microorganisms [10]. However, producing probiotic beer is
challenging. To guarantee high cell counts of live probiotics, the
recommended minimum dosage is 9 Log colony-forming units
(CFUs) per serving of product [5]. It is recommended to use yeasts
as starter cultures to produce ethanol and carbon dioxide or to be
cultured with probiotic microorganisms because probiotic LAB is
incapable of fulfilling this primary purpose [11]. At the same time,
the antimicrobial characteristic of hops, specifically iso-α-acid
(17–55 ppm), can impair the growth and survival of probiotic
LAB in beers [12]. In this case, using other hop derivatives such
as hop essential oil is an alternative for allowing the growth of
probiotic Lactobacillus spp. [11].
The Bifidobacterium and the strains of LAB—Lactobacillus
acidophilus, Lacticaseibacillus rhamnosus, Enterococcus, and
Streptococcus—are the most known probiotic microorganisms. The
only commercial yeast used as a probiotic is S. cerevisiae var. bou-
lardii [7]. Publications about probiotic beer are recent. Table 1
depicts the main probiotics being investigated to produce probiotic
beers, including the beer style.
Table 1
Main probiotic microorganisms that are being investigated to produce probiotic beers
Probiotic Beer style and process characteristics Comments References

L. acidophilus LA-5 and Bifidobacterium Low and alcohol-free beer Starters: Saccharomyces cerevisiae 70,424 or [10]
lactis BB-12 Wort prepared in a commercial brewery Saccharomyces rouxii 2531
Probiotics were inoculated into the freshly
made beer. After storage, cell counts were
reduced, with greater losses in low-alcohol
beer
Saccharomyces cerevisiae var. boulardii T1 No specific style After the process, cell counts were above 5 Log [13]
compared with Saccharomyces cerevisiae Wort prepared with wheat and barley malts CFU/mL
Safbrew (T58) Each wort was fermented, maturated, and
in-bottle refermented
Free and encapsulated L. rhamnosus GG Pale lager Encapsulation boosts the protection of the [14]
(LGG) in alginate or alginate silica Pasteurized Heineken probiotic’s cells during storage
microcarriers
S. cerevisiae var. boulardii in single and mixed No specific style S. cerevisiae var. boulardii (Sb) showed the [15]
cultures with S. cerevisiae Malted wort ability to overcome stresses such as ethanol
17 strains were isolated from natural content, and it was dominant in almost all
matrices, then five were selected mixed cultures
Mixed cultures increased the antioxidant
capacity and total phenolic content
S. cerevisiae var. voulardii Alcohol-free beer Probiotic yeast was able to grow on a synthetic [16]
Synthetic medium containing dextrin, medium (highest specific growth rate on
glucose, maltose, maltotriose, isomerized glucose)
hop extract, and ethanol The effect of iso--acids on growth rate was not
significant until 30 IBU
The specific growth rate decreased at an
ethanol level of 5% ABV
Probiotic Beer
(continued)
141
142
Table 1
(continued)
Probiotic Beer style and process characteristics Comments References

S. cerevisiae var. boulardii (CECT 1474) India Pale Ale Beer prepared with S. cerevisiae var. boulardii [8]
compared with S. cerevisiae (SF-04) Wort was made with a kit containing 100% as a single yeast starter produced higher
hopped malt extract acidification, higher antioxidant activity,
lower alcohol content, similar sensory
attributes, and higher yeast viability after
45 days compared with the beer prepared
Fernanda Meybom et al.
with a commercial S. cerevisiae

L. paracasei L26 in coculture with Sour beer Unhopped wort was used as pre-culture and [11]
S. cerevisiae S-04, and single cultures as Sweet unhopped wort co-fermented with for co-fermentation. Lactic acid production
control both microorganisms and satisfactory growth of L26 were
Late addition of isomerized hop extract reported
Storage in cold temperatures and live yeast
enhanced the survival of L26 in hopped
wort
Lactobacillus delbrueckii pure culture and Pito—sour sorghum The sensory evaluation found no difference [17]
S. cerevisiae A portion of the wort was conducted in between pito brewed with starter cultures
spontaneous lactic acid fermentation and the traditional pito. Application of the
A portion was fermented in pito pure culture commercial starter cultures in fermenting
followed by S. cerevisiae fermentation pito extends shelf-life by 2 days over
spontaneous pito
S. cerevisiae var. boulardii 17 Yeast extract-peptone-dextrose (YPD) agar S. cerevisiae var. boulardii had great resistance [18]
broths with different concentrations of to alcohol and gastrointestinal conditions
ethanol
S. cerevisiae var. boulardii CNCM I-745 Yeast extract-peptone-dextrose (YPD) agar S. cerevisiae var. boulardii CNCM I-745 was [19]
broth not able to grow at a concentration above
Yeast was tested in YPD broth with 0 to 10% 8% vol. at 28 °C and above 5% vol. at 37 °C
vol. of ethanol Mathematical modeling of yeast stress
resistance is a useful tool
Levilactobacillus brevis and S. cerevisiae SafAle Ale beer Free and immobilized cells remained viable at [20]
S-3 Pale malt extract and hopped wort the end of the storage period (24 days)
After 2 weeks of fermenting, probiotic cells Cell counts, around 5 log CFU/mL after
and dextrose were added to the beer simulated gastric and intestinal fluids
Lacticaseibacillus paracasei subsp. paracasei Wheat beer L. paracasei DTA 81 survivability was [9]
DTA 81 (isolated from stools of infants Sour beer compromised when dry hopping method
1 to 3 weeks old), S. boulardii Wheat beer was tested with hopped wort was applied
17, S. cerevisiae S-04 fermented with S. boulardii 17
Sour beer was fermented with L. paracasei
DTA 81 and S. cerevisiae S-04 in an
unhopped wort
43 wild yeast strains belonging to different Pilsner Pilsner with chickpea flour had an unpleasant [7]
genera such as Lachancea, Kluyveromyces, Pilsner with lentil aroma and taste. Pilsner with lentil had an
Torulaspora, Metschnikowia, Kazachstania, Pilsner with chickpea flour effective fermentative character and pleasant
Brettanomyces, Pichia, Candida, aromatic notes. S. cerevisiae, K. unispora,
Hanseniaspora, Rhodotorula, and L. thermotolerans increased the main
Rodosporidobolus and Saccharomyces aromatic compounds in pilsner with lentils.
Commercial S. cerevisiae US-05 and These yeasts have the potential to produce a
S. boulardii were used as control strains beer with high nutritional and functional
characteristics
Saccharomyces cerevisiae var. boulardii and Pilsen According to the study, S. boulardii presented [21]
Commercial S. cerevisiae W34/70 as first Wort from Heineken Brewery satisfactory tolerance to bile acid, pH, and
fermentation ethanol
Sensorial analysis showed good acceptance of
the probiotic beer
Probiotic Beer
143
Since beer is a complex liquid, obtained from a variety of raw

materials and brewing routes, some variability can be expected in
terms of characteristics, even when prepared at a laboratory scale.
Thus, we present a protocol for the preparation of a probiotic beer
based on a clear beer, with a refreshing taste, low alcohol content,
clean lactic acidity, and a high level of carbonation, inspired by the
sour beer styles (characterized by intentionally high acidity in
beer) [22].
2 Materials
Probiotic beer is made from water, fermentable carbohydrates,

hops, yeast, and probiotic microorganisms. The amount of each
ingredient can be calculated by hand [23] or using free online
calculators (for instance, BeerSmith2 and Brewer’s Friend), consid-
ering the volume of beer and the values chosen for each vital
characteristic of the sour beer style. In this protocol, we consider
the preparation of 1 L of beer with the following characteristics:
OG 1038 g/L, FG 1009 g/L, IBU 0, SRM 10, and ABV 3.7%
v/v. The material can be acquired from local commerce
(see Note 1). All the materials can be used at room temperature.
Water
1 L of fresh, filtered, and chlorine-free water (see Note 2). The
recommended profile for this beer style is 50–60 ppm of calcium,
0–40 ppm alkalinity, 0–50 ppm sulfate, and 0–100 ppm chloride
[24] (see Note 3).
Carbohydrate Source
0.3 kg of extra-light dry malt extract (DME) (see Note 4).
Hops
0.1 g of highly concentrated hop oil extracts (see Note 5).
Microorganisms
0.8 g of dry yeast S. cerevisiae for alcoholic fermentation. Keep the
yeast refrigerated until using it (see Note 6).
1 mL of hydrated L. brevis for acid fermentation. This is the
probiotic strain used in beer production (see Note 7). Keep the
LAB refrigerated until using it.
Equipment
The main equipment required are presented in Table 2.
Probiotic Beer 145
Table 2
Equipment required for preparing 1 L of probiotic beer
Item Reason
2 units of 2 L glass autoclavable container/bottle Wort preparation
1 unit of stove or heating plate Wort heating
1 m of ¼″ food grade silicone hose Transfer beer from the fermentation vessel
1 unit of a rubber stopper with an airlock Releases the CO2 from the fermentation bottle
Bowl Fill with cold water to refrigerate the wort
Temperature chamber (0–25 °C) Fermentation and maturation
Refractometer Measure wort and beer densities (OG and FG)
4 units of 330 mL amber glass bottle Packaging
4 units of metallic caps for the glass bottles Packaging
Bottle capper Packaging
Preparation Sterilization Primary

Wort chilling
of wort of wort fermentation
S. cerevisiae
Maturation Secondary
Packaging
fermentation Sedimented cells
Probiotic LAB
Refrigerated
storage
Fig. 1 Flowchart of the steps required to prepare the sour beer
3 Methods
The preparation of the sour beer follows the flowchart presented in

Fig. 1 (see Note 8). Each step is described separately in this
protocol.
3.1 Preparation of 1. Prepare the wort using the 2 L glass container. Slowly, add the
Wort extra-light dry malt extract (DME) to 1.0 L of the previously
prepared water. The mixture can be performed by using a
magnetic stirrer.
3.2 Sterilization of 1. After full solubilization of the DME, close the flask with alumi-
Wort num foil or a cap (see Note 9).
2. Take the glass container to an autoclave. Set the temperature to
121 °C and let sterilize for 20 min [16].
3.3 Wort Chilling 1. Remove the container from the autoclave and let the tempera-
ture decrease to around 10 °C in the air (ambient condition).
2. Place the container in the water-cooling bath and let the tem-
perature decrease to 18 °C (see Note 10). From here, be careful
handling the wort, because it is susceptible to contamination.
3. After reaching the desired temperature, aerate the wort with
8–9 ppm oxygen [25]. The wort oxygenation process in the
laboratory can be done by shaking the wort in the closed
container for 10 min or until the minimum concentration is
reached (see Note 11). Measurement of dissolved oxygen in
wort can be performed by using an oximeter or other analytical
chemical method.
3.4 Primary 1. Remove the yeast from the refrigerator 1 h before using so that
Fermentation the cells are at a temperature close to the fermentation temper-
ature. 0.8 g of the dry yeast S. cerevisiae is used for alcoholic
fermentation (see Note 12).
2. Progressively sprinkle the dry yeast directly in the fermentation
vessel on the surface of the wort, ensuring the yeast covers all
the wort available to avoid clumps. Let the yeast be hydrated by
the wort.
3. Close the container and shake it slowly to homogenize the
yeast with the wort.
4. Remove the cap and attach the rubber stopper containing the
airlock. This airlock relieves positive pressure due to the pro-
duction of CO2 during fermentation.
5. Place the container in the temperature chamber at 18 °C. The
fermentation process continues until the final extract
(FG) reaches 1009 g/L or it doesn’t change in 48 h.
6. Cool the beer to 2 °C and keep it for 24 h to optimize the yeast
sedimentation.
7. Slowly and carefully remove the supernatant liquid using the
silicone hose and transfer it to a second fermentation vessel.
3.5 Secondary 1. Remove the L. brevis from the refrigerator 1 h before using.
Fermentation 2. Prepare the beer for lactic fermentation (see Note 13) by heat-
ing it to 37 °C.
3. Shake the sachet with the microorganisms and inoculate 10 mL
of the L. brevis directly into the fermentation vessel (see
Note 14).
4. Cover the container and shake it to homogenize. Fermentation
continues until a count of 9 Log CFUs is reached.
Probiotic Beer 147
3.6 Maturation 1. After the second fermentation, cool the beer to 2 °C and keep
it for 72 h for the maturation process (see Note 15).
2. To provide the probiotic sour beer with the flavor of hops, add
0.1 g of hop oil extracts directly to the liquid. After the addi-
tion, shake to homogenize the product (see Note 16).
3.7 Packaging 1. Pre-wash the glass bottles with mild soap and water and sanitize
them with peracetic acid for 15 min (see Note 17). The sour
beer should be bottled without going through a filtration and
pasteurization process to ensure the permanence of living cells.
2. Pack the beer at a temperature of 2 °C and immediately cover it
(see Note 18).
3.8 Refrigerated 1. Keep the bottles at 5 °C to avoid the aging process and contain
Storage the fermentation [26]. The storage time should be as short as
possible, a longer time will always have a negative influence on
the quality and cellular viability of the probiotic beer.
4 Notes
1. Since the ingredients are from natural sources, they may have
slight variations in their characteristics depending on the brand
or even on the batch used. It is advisable to ask the supplier
company for the specific analysis data for the batch of
ingredients used.
2. Potable (tap) water can be treated by a series of filters; usually, a
polypropylene filter (nominal pore size from 5–20μm) is fol-
lowed by one or two steps of filtration through activated car-
bon to remove chlorine from water.
3. This water profile is a suggestion, based on the beer style.
However, for research purposes, it is strongly recommended
to use purified deionized water and analytical reagents to adjust
the salt content and the required pH. Calcium sulfate, calcium
chloride, magnesium sulfate, sodium bicarbonate, magnesium
chloride, and sodium chloride can be used to adjust calcium
(Ca+2), magnesium (Mg+2), bicarbonate (HCO3- 1 ), sulfate
(SO4- 2 ), sodium (Na+1), chloride (Cl-1), and sulfate (SO4- 2 )
ions [27].
4. Malted barley is the main carbohydrate source used for prepar-
ing beer, but other carbohydrate sources can also be used. The
grain bill is calculated from the gravity units and color each
carbohydrate source can offer to reach the desired beer OG and
SRM [23].
However, to simplify laboratory studies, we suggest using

the Dry Malt Extract. Using DME eliminates brewing steps
(grain milling, mashing, and filtration).
5. Hops are used in beer production for impairing the aroma,
flavor, and bitterness. In addition, they provide antimicrobial
and antioxidant properties, which can impair probiotic growth
[28]. Thus, replacing the addition of hops in the boiling stage
with the use of concentrated hop extract in the maturation
stage is an alternative to providing the beer with the hop
aroma without harming the development of probiotics. We
recommend using a concentrated hop extract with the aromatic
character of grapefruit and tropical fruits.
6. Prefer using ale yeast with fast fermentation characteristics and
the ability to form a compact sediment at the end of fermenta-
tion, which helps to improve the clarity of the beer.
7. There is a wide variety of probiotic species that can be used,
such as L. acidophilus, L. helveticus, L. delbrueckii subsp. bulgar-
icus, and L. paracasei.
8. This is a proposed procedure, intended to facilitate the prepa-
ration of the sour beer at a laboratory scale and reach the
desired characteristics, essential for research purposes.
9. If using a screw cap, be careful not to close the flask completely
when autoclaving.
10. Ice can be used in the water bath to help decrease the tempera-
ture. The wort temperature should be just right for the specific
yeast strain. For the sour beer style, the first fermentation with
S. cerevisiae S-04 is performed at 18 °C.
11. In breweries, the wort is oxygenated using a medical oxygen
cylinder with a flow meter. This equipment indicates the
volume of air that dissolves in the liquid with scales from
0–15 L/min.
12. The dosage recommended in this protocol is 80 g/hL for
fermenting at a temperature from 18 to 26 °C [29]. If using
another yeast, follow the manufacturer’s recommendation for
dosage and fermentation temperature.
13. For producing probiotic sour beer, the acidification process is
conducted after the primary fermentation (alcoholic fermenta-
tion) rather than the kettle sour process (common for tradi-
tional sour beer). This procedure avoids removing probiotic
bacteria with the spent yeast from primary fermentation. Then,
beer souring occurs after primary fermentation and removal of
yeast [30].
14. The recommended concentration of the probiotic is 200 mL
for 20 L of beer [31]. If using another variety of probiotic
species, follow the manufacturer’s recommendation for dosage
and fermentation temperature.
Probiotic Beer 149
15. During maturation, beer is saturated with carbon dioxide, and

part of the turbidity-forming components of the beer settles
(clarification) [25].
16. We recommend testing a dosage of 10 g/hL (range
5–40 g/hL) [32]. If using another variety of hop oil, follow
the manufacturer’s recommendation for dosage.
17. Follow the manufacturer’s recommendation regarding the
dilution of the peracetic acid.
18. There will probably be an accumulation of carbon dioxide and
an increase in pressure in the bottle due to the production of
carbon dioxide by the heterofermentation of Lactobacillus spp.
[30]. In this case, the glass bottle is the most suitable because it
supports a higher pressure compared to the plastic bottle. A
low temperature of the beer in the bottle increases the solubil-
ity of carbon dioxide, it is recommended to pack at a tempera-
ture of 2 °C [26].
Acknowledgments
The authors are grateful to the Research and Innovation Support

Foundation of Santa Catarina State (FAPESC), to CNPq (National
Council for Scientific and Technological Development), and
CAPES (Coordination of Improvement of Higher Education
Personnel).
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(2021) Modeling the ethanol tolerance of the
Chapter 11
Probiotic Fermented Sausage

Claudio M. E. Malaghini, Ana Paula Zapelini de Melo, and Silvani Verruck
Abstract
Many changes and innovations in fermented meat products have occurred over time, especially when it
comes to health-promoting products. Some studies have been conducted with the aim of developing
probiotic meat products that can improve the functionality of the gut microbiota. However, the techno-
logical challenges faced during the production of fermented meat sausages make it difficult to apply
probiotics in these food matrices. For probiotics to deliver the expected health outcomes for consumers,
they need to grow in the products and at the end the viable cell count must be sufficient for the
microorganisms to reach the consumer’s gut. Therefore, in this chapter we describe a protocol for probiotic
Friolano-type sausage. Furthermore, the possible sources of defects in the production of probiotic salami
and the best alternatives to overcome them are presented.
Key words Functional foods, Health-promoting compounds, Probiotic salami, Probiotic Friolano
salami, Probiotic meat product
1 Introduction
Meat sausages are products elaborated with meat or edible organs,

seasoned and smoked, and can be cured, cooked or dried, wrapped
in tripe, bladder or other animal membrane properly cleaned
[1]. Fermented sausages are products that undergo a rapid initial
fermentation followed by partial dehydration and may or may not
be smoked. They are meat products consisting mainly of pork or
beef, but can be produced with other meat types, in addition to
pork fat, salt, sugar, curing agent, spices and starter cultures. They
do not require refrigeration and have great stability compared to
other meat products [2].
Friolano Salami is a kind of fermented sausage made exclusively
from pork and lard, ground to a medium particle size of 6–9 mm
and with the addition of the other ingredients required. It has an
irregular cylindrical shape (defined by the shape of the natural wrap)
with length ranging from 15 to 130 cm. Its weight ranges from 0.2
to 4.5 kg and presents non-elastic consistency, compact mass,
151
152 Claudio M. E. Malaghini et al.
delicate aroma, sweet and delicate flavor, and ruby red color with-
out spots. It is a cured product, which can go through the smoking
process, being fermented, matured, and dried [2].
In the world of fermented meat sausages many changes and
innovations have taken place over time, especially when it comes to
products that are beneficial to health, since the demand for these
foods has become a priority for many consumers. With this in mind,
studies have been conducted with the addition of probiotics in meat
sausages [3].
Probiotics are live microorganisms that when properly added to
products present benefits to the consumer’s health, with specific
effects and functional properties [4]. In addition, they contribute
to the balance of intestinal microflora, helping the intestinal transit
and facilitating digestion, relieve the symptoms of lactose intoler-
ance, prevent colon cancer, reduce cholesterol and blood pressure,
stimulate the immune system, produce B-complex vitamins, diges-
tive and protective enzymes, protect against pathogenic microor-
ganisms and control inflammatory vessel diseases [4–6].
However, for them to present the expected results they need to
grow in the products, and at the end of the shelf life the viable cell
count should be enough for the microorganisms to reach the
consumer’s intestine, which makes their application in fermented
sausages difficult, due to their high acidity and salt content, and
lower water activity (aw) [4]. Therefore, in some studies probiotics
are added microencapsulated in foods, which ensures the viability of
the probiotic microorganisms during the process and in the final
product [6]. Therefore, this chapter is directed towards the design
of a probiotic Friolano Salami protocol. Furthermore, the possible
sources of defects in meat sausages and the best alternatives to
overcome them are presented.
2 Materials
1. Pork shank meat.

2. Back fat (lard).
3. Sodium chloride.
4. Curing salts (sodium nitrate).
5. Sugar.
6. Garlic powder.
7. Chili powder.
8. Probiotic culture (Table 1).
9. Starter culture (Table 2).
10. Collagen wrap with 50 a 60 mm.
Table 1
Probiotic or potentially probiotic cultures in meat products and the main results found in the studies
Product Probiotic or potential probiotic used Main results References

Fresh pork sausage L. sakei BAS0117 isolated from Brazilian The strains added allowed desirable [7]
fermented meat products (Italian salami, characteristics to the product during storage
Calabrian sausage, ham, and mortadella)
Italian salami sausage L. acidophilus, Bifidobacterium lactis (potentially The addition of probiotic cultures produced [8]
probiotic) sausages with good physical-chemical,
microbiological, and sensory properties
Fermented pork sausage and loin L. rhamnosus LOCK900 (probiotic strain) Many lactic acid-producing bacteria, including [9]
90% L. rhamnosus, were found during all stages
of the meat process
The added probiotics inhibited lipid oxidation in
loins and pork sausage
Sausage and pork neck fermented and Pure cultures of probiotic strains: The three starter strains could be applied to [10]
dry-cured Bifidobacterium animalis subsp. lactis BB-12 smoked meat products. However, the culture
(strain deposit number: DSM15954), L. acidophilus Bauer did not allow lipid
L. rhamnosus LOCK900 (strain deposit oxidation and discoloration of the products
number: CP005484), L. acidophilus Bauer
(potentially probiotic)
Fermented sausage Isolated from human intestinal tracts: The three strains examined could inhibit the [11]
L. acidophilus FERM P-15119, L. rhamnosus growth of S. aureus and enterotoxins during
FERM P-15120, L. paracasei subsp. paracasei the sausage fermentation process at different
FERM P-15121 temperature variations
Commercial starter culture: L. sake (Chr.
Hansen’s)
Fermented sausage Probiotic lactobacilli strain isolated from infants’ It was observed that the L. rhamnosus remained [12]
feces: L. casei/paracasei CTC1677, L. casei/ viable at high levels (108 CFU/g) and survived
paracasei CTC1678, L. rhamnosus CTC1679 the passage in TGI during sausage
(confirmed probiotic lactobacilli) consumption

Commercial probiotic strains: L. plantarum
299v, L. rhamnosus GG, L. casei Shirota
153
(continued)
154
Table 1
(continued)

Fermented sausage LAB isolated from infants qualified as potential L. rhamnosus CTC1679 was the only strain [13]
probiotics: L. gasseri CTC1700, L. gasseri capable of mastering both repetitions
CTC1704, L. fermentum CTC1693 A putative probiotic effect can be achieved by
Potential probiotic strains with proved ability, eating 10 g/day of fuet with CTC1679
isolated from infant feces: L. casei/paracasei
CTC1677, L. casei/paracasei CTC1678,
L. rhamnosus CTC1679
Claudio M. E. Malaghini et al.
Fermented sausage Probiotic strain: E. faecium ATCC 8459 E. faecium was efficient as a starter for producing [14]
fermented sausage with resistance to curing
salts and sodium chloride and maintained its
viability during the ripening process
Fermented sausage E. faecium CRL183 (potential probiotic) It demonstrated a positive influence of sausage [15]
fermented with E. faecium CRL183 on
microbial diversity
Fermented sausage L. rhamnosus CTC1679 (potential probiotic) L. rhamnosus CTC1679 used as a probiotic starter [16]
culture produced safe, nutritionally enhanced
fermented sausages
The strain showed the ability to act as probiotic
starter cultures remaining viable at high levels
(108 CFU/g) in ripened fuets and surviving
the passage through the human GIT during
the consumption of the sausages
Fermented sausage Commercial probiotic strain: L. sakei (potential The strains presented technological [17]
probiotic) characteristics expected for application in
sausage maturation processes as a starter
culture
Raw fermented sausage L. casei LOCK 0900 isolated from feces of Raw fermented sausages with probiotic strain [18]
healthy infants (potential probiotic) L. casei LOCK 0900 showed good
microbiological quality. The environment of
raw fermented sausages is suitable for the
growth and survival of the probiotic strain
L. casei LOCK 0900
Fermented lamb sausage Commercial probiotic strain: L. acidophilus The number of Lactobacillus (107 CFU/g) and [19]
CCDM 476, Bifidobacterium animalis 241a Bifidobacterium (103 CFU/g) in the final
(potential probiotic) product did not alter its technological
properties. Despite this, there were problems
in using Bifidobacterium as a starter because of
its low concentration after fermentation and
absence after 60 days of storage
Norwegian fermented sausage, Swedish Potential probiotic cultures isolated from The strains met all probiotic criteria and proved [20]
fermented sausage, and Norwegian fermented meat: L. sakei MF1295, L. sakei to be rapidly producing lactic acid,
cured ham MF1296, L. farciminis MF1288, demonstrating the successful application of the
L. plantarum/pentosus MF1290, selected strains as starter cultures for
L. plantarum/pentosus MF1299, L. plantarum Scandinavian-type fermented sausages
MF1291, L. plantarum MF1297, L. pentosus
MF1300, L. alimentarius MF1297
Dry fermented sausage L. rhamnosus GG (probiotic strain), L. rhamnosus L. rhamnosus E-97800 showed the fastest growth [21]
E-97800 (potential probiotic), L. rhamnosus and acidification rate. Therefore, L. rhamnosus
LC-705 (potential probiotic) GG and L. rhamnosus E-97800 were
Commercial strains: Pediococcus pentosaceus considered tasty as the sausages fermented by
the control
Dry fermented sausage Commercial probiotic strains documented: The evaluated strains demonstrated different [22]
L. rhamnosus R0011, L. helveticus R0052, technological capacities in the other conditions
L. rhamnosus Lr- 32, L. paracasei Lpc-37, in which the tests were performed
L. casei Shirota, L. reuteri DSM17938, L. rhamnosus Lr-32, L. rhamnosus R0011,
L. reuteri DSM17918, Enterococcus faecium L. paracasei Lpc-37, E. faecium MXVK29, and
MXVK29 L. casei Shirota strains are the primary

candidates to be used as sausages starters
culture
155
(continued)
Table 1
156
(continued)

Iberian dry fermented sausage Probiotic culture: L. fermentum HL57, Inoculation with L. fermentum HL57 increased [23]
Pediococcus acidilactici SP979 the amount of acetic acid and lipid degradation
products, such as malonaldehyde in Iberian dry
fermented sausages, resulting in a negative
impact on relevant sensory parameters related
to color and flavor. On the contrary,
P. acidilactici SP979 did not remarkably
modify the physical–chemical parameters or
sensory quality of Iberian dry-fermented

sausages
Tunisian dry fermented sausage Autochthonous strains isolated from a Tunisian The use of bacterial strains can inhibit the growth [24]
traditional salted meat “kadid”: L. plantarum, of Gram-negative bacteria and may improve
S. xylosus the sensory properties of sausage due to nitrate
(not confirmed probiotic) reductase and protease activity of the S. xylosus
strain and the acidifying activity of the
L. plantarum strain
Harbin dry sausage Potential probiotics: P. pentosaceus R1, L. brevis Except for L. curvatus R5, all strains isolated [25]
R4, L. curvatus R5, L. fermentum R6 from Harbin dry sausages supported passage in
Confirmed probiotics from fermented dairy the gastrointestinal tract (GIT). Different
products (used for comparison with the components of the strains have different
isolated LAB): L. acidophilus AD1, modes of antioxidant action. LAB isolated
L. plantarum KLDS1.0391, L. curvatus from Harbin dry sausages has strong probiotic
KLDS1.0505, L. sake AS1, L. pentosaceus properties and can be used as potential
KLDS1.0412, L. fermentum KLDS1.0709 probiotics for food processing
Sucuk-type dry sausage Twenty-three probiotic L. plantarum strains L. plantarum AA1-2 and L. plantarum AB20- [26]
producing the conjugated linoleic acid (CLA) 961 were identified as potential strains for CLA
were screened: production. The strain of L. plantarum AB20-
L. plantarum LMG 11405 and L. plantarum 961 can be used to produce sucuk by an
LMG 23521 were selected from the catalog of increase in the amount of linoleic acid of the
BCCM/LMG (Belgian Coordinated product, without alteration on the product’s
Collections of Microorganisms/Laboratory of final characteristics
Microbiology) at the University of Ghent Production conditions such as temperature and
Twenty one L. plantarum strains (AA1-2, AA13- pH were probably the most limiting factors for
59, AA17-73, AB6-25, AB7-35, AB16-65, linoleic acid production
AB20-961, AC3-27, AC10-40, AC18-82,
AC21-101, AC21-1031, AC18-88, AC3-10,
AC3-14, AK4-11, AK6-27, AK6-28,
AK8-31B, BC18-81, BK10-48) isolated from
human sources in Suleyman Demirel
University
Salami Commercial probiotics: L. plantarum 299v, The microbiological counts were different [27]
L. plantarum DSM 9843, L. rhamnosus LbGG according to the type of starter strain used.
or ATCC 53103, L. casei Shirota YIT 9029, L. plantarum 299v kept a concentration
L. reuteri DSM 17938, L. casei ATCC 393 higher than 106 CFU g 1, level of probiotic
bacteria recommended at the time of
consumption to exert a beneficial effect in
humans
The experimental salami proved to be safe since
coagulase-positive coliforms and Staphylococci
were not detected in the salami at the moment
of consumption and after more than 1 month
of storage at a cooling temperature
Nostrano salami Bacterial strains: L. lactis ssp. lactis strain These strains were selected for its ability to grow [28]
340, L. lactis ssp. lactis strain 16, L. casei ssp. under low temperatures and modulate the
casei strain 208, E. faecium UBEF-41 aroma by converting amino acids and fatty
acids, making it possible to produce fermented
matured at low temperature without adding
nitrates and nitrites, resulting in a potentially
safer product with no adverse effect on the
quality of Italian salami
(continued)
157
158
Table 1
(continued)

Italian-type salami Probiotic cultures: L. casei LC 01, L. paracasei The addition of these strains to the sausages [29]
ssp. paracasei ATCC 10746/CCT 0566, caused a reduction in the development of
L. rhamnosus ATCC 7469/CCT 6645 S. xylosus. On the other hand, probiotic
cultures did not interfere in the growth of
P. pentosaceus, which, for most evaluation
periods, showed better development when
together with Lacticaseibacillus
Adapted from ref. [3]
Probiotic Fermented Sausage 159
Table 2
Main starter cultures used in meat sausages
Starter culture Strain

Acid lactic bacteria Latilactobacillus sakei
Latilactobacillus curvatus
Lactiplantibacillus plantarum
Lacticaseibacillus rhamnosus
Pediococus Pediococcus acidilactici
Pediococcus pentosaceus
Staphylococcus and Kocuria Staphylococcus xylosus
Staphylococcus carnosus
Staphylococcus equorum
Kocuria varians
Micrococcaceae Micrococcaceae candidus
Micrococcaceae aquatilis
2.1 Equipment 1. Meat grinder with 6–9 mm disc.

2. Lard cuber.
3. Mixer.
4. Maturation chamber.
5. Drying chamber.
3 Methods
The protocol for making a probiotic Friolano Salami is illustrated

in Fig. 1.
3.1 Probiotic 1. Use direct vat set (DVS) or direct vat inoculation (DVI) cul-
Cultures tures that allow the addition of probiotic strains directly into
the food matrix. See Chapter 13 for the main suppliers of DVS
probiotic strains (Table 1).
3.2 Friolano Salami 1. Grind the raw pork meat (85% of the raw material) with a
Manufacture 6–9 mm disc at a temperature of 4–7 C and then transfer it
to the blender.
2. Cut the bacon (15% of the raw material) in an incubator at 0 C
into cubes of no more than 1 cm. Add the lard to the meat in
the blender.
3. Add 2.5% sodium chloride, 0.3% sugar, 0.03% chili powder,
0.3% garlic powder, and 0.015% sodium nitrate over the meat
in the blender.
Fig. 1 Steps for the elaboration of a probiotic Friolano Salami
4. Mix the ground meat, minced lard, and the other ingredients
and additives for 2 min in the mixer at 4 C.
5. Add the starter culture and the probiotic culture at 108 CFU/
g. Mix for about 2 min (See Notes 1–3).
6. After a resting phase of 24 h at 0–2 C, the mixture must be
filled using a vacuum filler (See Note 4).
7. Clip the salami and spray an aqueous mold solution on the

surface wrap (See Note 5).
8. Hang the salami and transfer it to the fermentation chamber
(See Note 6).
9. In the fermentation chamber, the dripping phase occurs at
20 C for 14–20 h.
10. Keep the salami in the drying room at 20 C, relative humidity
60 to 80% for 96 to 144 h (See Note 7).
11. Finally, after drying, transfer the salami to the ripening room at
12–18 C for 23 days (See Note 8).
12. The salami can be washed and, thus, packaged in packs without
light and oxygen permeability (See Note 9).
4 Notes
1. The starter culture commonly used in salami is Staphylococcus

xylosus, which produces lipolytic and proteolytic activity
enzymes that are fundamental in the formation and color sta-
bility of the final product, and is involved in aroma formation;
Latilactobacillus sakei with fermenting action, producer of lac-
tic acid and antibacterial metabolites, has also protective action;
and Staphylococcus carnosus, which adds flavor, has protective
and fermenting action.
2. At the end of this stage, the temperature of the mixture rises to
about 6 C.
3. The lactic acid bacteria lower the pH and produce aromatic
compounds in the sausage, also masking the bitterness of the
curing salts. In addition, they produce reducing conditions,
helping to not develop oxidized flavors, and improving color,
since they favor the development of meat pigments by stabiliz-
ing Fe2+. The also protect the pigments from oxidation by
blocking the formation of undesirable compounds in the
product.
4. To avoid air residue in the meat paste, it is very important that
no air be trapped in the salami.
5. Optionally smoking can be done, but knowing the bacterio-
static effect it can have on some probiotics, this step is not
recommended for probiotic Salami.
6. Until pH 4.6–5.4 is reached and for color development.
7. The time of the drying step is given by the weight loss function
chosen as the target, which in turn depends on the quality of
the lean meat fraction used. If an initial step aimed at losing
water from fresh meat is performed in ventilation systems
before grinding, with the objective of losing moisture, the next

drying step can be shorter. At the end of the drying stage, the
temperature of the drying environment is usually a third lower
than it was at the beginning.
8. The length of the ripening chamber will depend on the tar-
geted weight loss. A time of 23 days is sufficient to lower the
water activity (aw < 0.9) and achieve the physical-chemical
characteristics of Friolano Salami. The weight loss at the end
of ripening (intended as a complete cycle) is about 38%. This
weight loss value can vary according to the lean to fat ratio,
diameter, salt concentration, etc. If the previous drying phase
has been carried out correctly in terms of weight loss, during
the first days of maturation some mold colonies appear on the
surface of the wrap. However, this step must be carefully con-
sidered since the lower aw can lead to the loss of probiotic
viability.
9. The viability of probiotics over storage depends on the individ-
ual strain. Some examples of viability time in storage can be
seen in Table 1.
Acknowledgments
The authors are grateful to Coordenação de Aperfeiçoamento de

Pessoal de Nı́vel Superior—Brasil (CAPES)—Finance Code 001.
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8717
Chapter 12
Probiotic in Bakery
Ana Paula Zapelini de Melo, Thais de Oliveira,
Pedro Luiz Manique Barreto, and Silvani Verruck
Abstract
The incorporation of probiotic microorganisms (such as the genera Bifidobacterium, Lactobacillus, Lacti-
plantibacillus, Lacticaseibacillus, and Bacillus) into bakery products is an emerging approach to enlarge the
availability of non-dairy probiotic food on the market. However, the viability and stability of the micro-
organisms may be compromised due to the high processing temperatures employed in the development of
these products. This chapter proposes the design of two independent protocols for the delivery of
probiotics through bakery products: (I) a probiotic bread by adding microorganisms directly to the
dough and (II) an edible probiotic film based on sodium caseinate and chia mucilage for application on
bread surface. Furthermore, for both protocols, the function of each reagent/ingredient and the chemical
reactions involved are described in details, indicating the sources of possible issues and the best alternatives
to overcome them.
Key words Bread ingredients, Functional foods, Probiotic bread, Probiotic edible film, Probiotic
cultures, Non-dairy probiotic
1 Introduction
Probiotics are defined as live microorganisms that provide benefits

to the human health when properly administered [1]. For decades,
dairy products such as yogurts and cheeses were the main matrices
used for the delivery of probiotics. However, the increase in lactose
intolerant and allergic individuals, combined with the evidence of
beneficial effects of probiotic microorganisms on human health, has
propelled the development of new non-dairy probiotics
products [2].
An emerging approach to expand the availability of non-dairy
probiotics food is the incorporation of these microorganisms in
bakery products [3–8]. The development of probiotic bakery pro-
ducts is a challenge, due to the high processing temperatures that
they are subjected to (between 160 and 250 C), which may
compromise the viability and stability of the microorganisms used
165
166 Ana Paula Zapelini de Melo et al.
[9]. To ensure the beneficial effects on human health, especially

related to gastrointestinal health, a minimum concentration of live
probiotic cells must reach the intestine. The suggested minimum
concentration ranges from 108 to 109 CFU/dose [1]. However,
for some products, lower concentrations are sufficient to claim
specific health effects, while for others, a substantially higher con-
centration is required, depending on the strain [1, 2].
The main microorganisms with stated probiotic properties
used in bakery products include the genera Bifidobacterium, Lacto-
bacillus, Lactiplantibacillus, and Lacticaseibacillus. Nonetheless, to
overcome the limitations associated with the processing and storage
of the products, as well as to the gastrointestinal conditions, encap-
sulation techniques may be used as an alternative to protect cell
viability under unfavorable environmental conditions (see
Chapter 14 for detailed information regarding the encapsulation
of probiotic microorganisms) [10]. On the other hand, spore-
forming probiotic strains, such as those of the genus Bacillus,
have recently overcome the technological challenges related to
stressful conditions of bakery products processing (such as high
temperature survival) without the need for encapsulation [5, 8].
The development of probiotic bakery products is still in the
early stages. Most of the studies published in the literature are
directed to the development of probiotic breads. In fact, bread is
an interesting matrix, especially due to its large consumption
worldwide [11]. Protocols for the delivery of probiotics through
breads vary considerably in the literature; however, promising stra-
tegies include, but are not limited to, the addition of microorgan-
isms in dough and as edible coating films [12, 13]. Therefore, this
chapter is directed to the design of a probiotic bread protocol
through the addition of probiotic microorganisms directly to the
dough and, in parallel, an edible film protocol of probiotic coating
based on sodium caseinate and chia mucilage for application on
bread surface.
2 Materials
2.1 Probiotic 1. The main microorganisms with claimed probiotic properties

Microorganisms used in bakery products are presented in Table 1 (see Note 1).
2.2 Edible Film 1. Distilled water.

2. Sodium caseinate.
3. Glycerol.
4. Chia mucilage.
5. Probiotic culture.
Probiotic in Bakery 167
Table 1
Main microorganisms with claimed probiotic properties used in bakery products
Genus Species Strains

Bacillus Coagulans subtilis GBI-306086 [5, 8]
MTCC 5856 [5]
Bifidobacterium Bifidum lactis BB-12 [14] and NCDC 236 [15]
NH019 [6]
Lactobacillus Acidophilus LA-5 [5, 14, 16], PTCC 1643 [17], and NCDC 11 [15]
Lactiplantibacillus Plantarum P8 [3]
Lacticaseibacillus Rhamnosus GG [7] and NCDC 17 [15]
2.3 Bread 1. Wheat flour.

2. Water.
3. Sucrose.
4. Sodium chloride.
5. Active dry yeast (Saccharomyces cerevisiae).
6. Fat.
7. Probiotic culture.
2.4 Equipment 1. Magnetic stirrer.

2. Sonicator (such as VC 505, Sonics & Materials Inc., USA).
3. Centrifuge.
4. Cheesecloth.
5. Vacuum degasser.
6. Pastry brush.
7. Kneading-trough (such as A30 Progress, Brazil).
8. Proofing chamber (such as MSV 750, Heidenreich, Germany).
9. Convection oven (such as Vipinho-0448, Perfecta, Brazil).
3 Methods
The designs of two independent protocols for the delivery of

probiotics through bakery products are presented in Fig. 1: (I) a
probiotic bread by adding microorganisms directly to the dough
and (II) an edible probiotic film based on sodium caseinate and chia
mucilage for application on bread surface.
Fig. 1 Steps for the delivery of probiotics through bakery products: (I) a probiotic bread by adding micro-
organisms directly to the dough and (II) an edible probiotic film based on sodium caseinate and chia mucilage
for application on bread surface
3.1 Probiotic 1. Use direct vat set (DVS) or direct vat inoculation (DVI) cul-
Cultures tures that allow the addition of probiotics directly to the food
matrix, without the need for activation or propagation of the
microbial culture in specific fermentation bulk (see in
Chapter 13 the main exporter suppliers of DVS probiotic
strains) [18] (see Notes 2–4).
3.2 Probiotic Bread 1. Mix dry ingredients (except sodium chloride) at 40 rpm for
1 min. Ingredients are calculated based on the amount of flour
(100%):
– Wheat flour (100%) (see Note 5).
– Sucrose (4%) (see Notes 6 and 7).
– Active dry yeast (Saccharomyces cerevisiae) (2%) in warm
water (45–55 C, until it reaches five times its own weight,
for 15–20 min) (see Notes 8 and 9).
2. Add water (60%) between 4 and 13 C and mix at 40 rpm for
1 min and then at 80 rpm for 7 min (see Notes 10–11).
3. Add 109–1011 CFU/g of lyophilized probiotic culture [19]
(see Notes 1–3).
4. Add sodium chloride (2%) (see Note 12).
5. Add the fat source (3%) under 40 rpm speed (see Note 13).
6. When homogeneity is reached, increase the speed of the
kneading-trough to 80 rpm to start the kneading process (see
Notes 14–16).
7. Let the dough rest for 20–40 min (first fermentation) on a
surface lightly oiled and covered with plastic, to prevent the
dough from drying out, and then carry out the dough division
process (see Note 17).
8. Perform the dough rounding and let the breads rest in a proof-
ing chamber (second fermentation) at 26–30 C, relative
humidity 75–80%, for a minimum of 5 and a maximum of
20 min (see Notes 18 and 19).
9. Mould the dough pieces and return them to the chamber for
the last stage of fermentation, 26–30 C, relative humidity
75–80%, for 60 min (see Notes 20 and 21).
10. Bake at 190 10 C for 12 4 min in a convection oven
(see Notes 22 and 23).
3.3 Probiotic Edible 1. Hydrate the chia seeds in 1:30 distilled water (seed:water,
Film w/w) for 2 h at 50 C under magnetic stirring (600 rpm)
(see Note 24).
3.3.1 Chia (Salvia
hispanica L.) Mucilage 2. Sonicate the solution for 2 min at 500 W, 20 kHz and 30%
Extraction amplitude (see Note 25).
3. Centrifuge at 3488-g at 4 C for 20 min (see Note 26).

4. Collect the chia mucilage (middle layer).
5. Filter mucilage through cheesecloth.
3.3.2 Probiotic Edible 1. Heat distilled water at 50 C.

Film Based on Sodium 2. Under magnetic stirring (600 rpm), add sodium caseinate (5%,
caseinate and Chia w/w) until complete dissolution (see Notes 27 and 28).
Mucilage
3. Add glycerol (30%, w/w) and chia mucilage (1%, w/w), and
keep it under magnetic stirring (600 rpm) until dissolution
(see Note 29).
4. Heat the solution at 60 C for 2 h and then at 90 C for 30 min,
both under magnetic stirring (600 rpm).
5. Cool the polymeric solution to room temperature and add
109–1011 CFU/g of lyophilized probiotic culture under
magnetic stirring (600 rpm) [19] (see Notes 1–3 and 30).
6. Degas the solution under vacuum for 20 min (see Note 31).
7. After baking the bread, wait for them to cool down and apply
with brushing using a pastry brush a thin layer of the probiotic
polymeric solution over their crust (see Note 32).
3.3.3 Other Methods for The main conditions for the delivery of probiotics through breads
Delivering Probiotics available in the literature are summarized in Table 2.
Through Bread
Table 2
Main conditions for delivery of probiotics through breads
Probiotic
viability
Inoculation Inoculation Baking Storage after bake
Probiotic strain dose conditions conditions conditions (CFU/g) References
Bacillus coagulans 107 spores/g DVS 200 25 C 104 (after [8]
GBI-30, 6086 C18 min 10 days 10 days)
B. coagulans 107– MYP 180 C 25 C 103–106 [5]
GBI-306086 108 CFU/ 37 C/ 20 min 7 days (after
B. coagulans g 24 h and 7 days)
MTCC 5856 20 C/
B. subtilis 24 h
PXN 21 BC 40 C/
48 h
GYEA
37 C/
72 h
(continued)
Table 2
(continued)
Probiotic
viability
Inoculation Inoculation Baking Storage after bake
Probiotic strain dose conditions conditions conditions (CFU/g) References
Lactiplantibacillus 109 CFU/g MRS 175 C 25 C 106 (after [3]

plantarum P8 37 C/12 h 8 min 5 days 5 days)
and
37 C/24 h
Lactobacillus 109 CFU/g MRS 175 C 25 C 106 (after [16]
acidophilus LA-5 37 C/12 h 6 min 7 days 7 days)
(encapsulated and
with alginate and 37 C
fish gelatin) /24 h.
Lactiplantibacillus 109 CFU/g MRS 100 C – 108 [4]
plantarum P8 37 C/12 h 15 min
(encapsulated with and
reconstituted 37 C/24 h
skim milk)
Lactobacillus 109 CFU/ MRS 180 C35 min – – [17]

acidophilus mL 37 C/24 h
PTCC 1643 and
(encapsulated with 37 C/48 h
alginate and
chitosan)
Bifidobacterium 1011 DVS 180 C – 105 [6]
animalis spp. CFU/g 40 min
lactis NH019
(encapsulated with
stearic acid,
hydroxypropyl
cellulose, and
sodium alginate)
Lacticaseibacillus – MRS 220 C – – [7]

rhamnosus GG 37 C/48 h 20 min
(encapsulated
with sodium
alginate, hi-maize
resistant starch,
and chitosan)
–: Not described, DVS Direct vat set, MRS De Man, Rogosa & Sharpe Agar, MYP Mannitol Egg Yolk Polymyxin Agar, BC
Glucose Yeast Extract Agar, GYEA Glucose Yeast Extract Agar
4 Notes
1. The microorganisms of the genera Bifidobacterium, Lactobacil-

lus, Lactiplantibacillus, and Lacticaseibacillus are gram-positive
microorganisms with thick cell walls, being able, therefore, to
withstand most of the forces generated in bread production
processes. However, high homogenization speeds can result in
cell disruption and reduced cell viability. Furthermore, these
genera are not able to survive extreme temperature conditions,
therefore, use encapsulation techniques for proper delivery
through breads (see Chapter 14). Bacillus genus microorgan-
isms (such as B. coagulans GBI-30, 6086 spores) can withstand
typical bread processing conditions; thus, they do not require
encapsulation [9, 18, 19].
2. The addition of 109–1011 CFU/g of the probiotic culture in
food is recommended to reach the suggested minimum dose
(108–109 CFU/dose of food) to promote human health ben-
efits. This concentration usually represents the addition of 0.1%
of probiotic in the final product [19].
3. When the direct addition of probiotics to the food matrix
results in losses in cell viability, a rehydration step must be
carried out and, subsequently, the rehydrated cell suspension
must be added to the food matrix. Rehydration is usually
performed in skim milk; however, water, saline solution, or
liquid culture medium can also be used depending on the
strain. The suggested rehydration temperature for thermo-
philes is between 30 and 37 C, while for mesophiles it is
22–30 C [18].
4. Store probiotics following the manufacturer’s instructions to
preserve cell viability by protecting them from light and mois-
ture content at a constant temperature [18].
5. The strength of the flour has effects on the duration of the
fermentation time and is related to its protein content, there-
fore, to achieve optimal dough development (larger volume
and soft crumb), use flours with 13 1% of protein content for
adequate gas retention [20].
6. Sucrose, besides conferring flavor and browning to the crust of
breads (Maillard reaction), controls water activity and prolongs
the shelf life of the products [20].
7. Sucrose concentrations below 10% generally do not inhibit
probiotic microorganisms [18].
8. The fermentation is an anaerobic process produced by the
action of the yeast on the sugar present in the bread dough.
This step is responsible for the production of carbon dioxide
and small quantities of ethyl alcohol, along with the
physicochemical transformations that alter the viscoelastic

properties of the dough. The starch present in the flour is
converted into sugars though enzymatic reactions, and the
resulting sugars from this reaction feed the yeast and outcome
in the formation of carbon dioxide [20, 21].
9. Yeasts are inactivated at 55 C or higher temperatures, there-
fore do not exceed this temperature [20].
10. Water must be between 4 and 13 C to prevent the early start of
the fermentation process, intermediate hardness between
50 and 100 mg L 1 of calcium carbonate and pH approxi-
mately 7.0 [21, 22].
11. The speed of 40 rpm is used for the incorporation of the
ingredients, while the speed of 80 rpm is indicated for the
gluten development and the beginning of the kneading
process [16].
12. Sodium chloride, in addition to its contributions to the flavor
of the final product and controlling water activity, affects the
hydration rate of gluten proteins and inhibits yeast fermenta-
tion due to cell dehydration (osmotic pressure). Therefore,
incorporate sodium chloride in the final steps of the mixing
process. The absence of sodium chloride results in an excess of
softening in the dough, rapid fermentation, and reduction of
the volume of the product, while its excess leads to a reduction
in the fermentative action, causing the gluten to harden exces-
sively [20, 23].
13. Lipids delay the retrogradation of starch gels and provide
softness to baked goods, due to interactions between the
lipid micelles with the hydrophilic segments of the starch
[24]. For a homogeneous dispersion of the fat in the dough,
use saturated fats, with a chain length of C16–C18 (tripalmitin
and tristearin) and with a high melting point (55–60 C) [20].
14. During the mixing process, under application of tension and
shear forces, the gluten proteins are hydrated, providing the
necessary energy for the development of the gluten structure.
Gluten proteins leave their ball-like shape and acquire a linear
shape, facilitating hydrophobic interactions and sulfhydryl-
disulfide interchange reactions. This results in the formation
of threadlike polymers, which in turn, through hydrogen
bonds, disulfide cross-links, and hydrophobic associations,
form a film capable of retaining the gas in the dough [22, 24].
15. For proper gluten development, the dough must be between
27 and 29 C at the end of the mixing stage. Excessive heating
of the dough can compromise fermentation, which will be
stimulated in advance, affecting the bread growth and the
gluten viscoelastic properties [20, 22, 23].
16. Excessive kneading leads to gluten breakdown, and the lack of

it leads to absence of elasticity in the dough [20, 23].
17. The doughs that do not undergo the resting process have
considerably smaller volumes of gas in their composition, influ-
encing the subsequent operations of modeling and dough
weight control. Care must be taken when dividing, as any
compression of the dough during this process will cause a
reduction in the regularity of the weight of the product due
to its degassing, that is, leads to variations in the gaseous
volumes retained in the dough, thus causing damage to its
structure [20].
18. Post-rounding fermentation (second fermentation) assists on
the recovery of dough extensibility, lost during the division and
rounding processes. Optimum temperature at this stage varies
between 26 and 30 C, with a relative humidity of 75–80%.
Temperatures higher than these reduce the gas holding capac-
ity, while lower temperatures delay the fermentation. Proofing
chambers with low relative humidity cause the dough to dry
out and the consequent formation of a crust, while higher
humidity increases the stickiness of the dough, making it diffi-
cult to manipulate [20, 21].
19. Insufficient bulk time provides tough, rubbery, and not easily
moldable gluten, and consequently breads of small volume,
firm crumb, and dense cell structure. On the other hand,
long bulk times result in the release of gas present in the
dough, loss of deformation resistance, and increase in extensi-
bility, and consequently irregular breads with large holes
[20, 21].
20. The final stage of fermentation occurs under the same condi-
tions of the second fermentation, for a longer period of time to
allow the dough to regain an adequate size, due to the loss of
gas resulting from the moulding step. The fermentation pro-
cess is finished inside the oven, when the temperature reaches
55 C [21, 22].
21. The fermentation step does not negatively affect the viability of
probiotic microorganisms [8]. The association between probi-
otic microorganisms and yeasts in the dough during fermenta-
tion is steady due to the absence of nutrients competition
between these microbial populations. The presence of ferment-
able carbohydrates throughout the fermentation process allows
a stable noncompetitive association, and consequently, an
increase in the growth rates of probiotic microorganisms, due
to the excretion of essential amino acids by the yeasts. How-
ever, for this to occur, the growth rates of microbial popula-
tions must be similar and there must be no exhaustion of
fermentable carbohydrates during the process [25, 26].
22. Upon reaching approximately 60 C, the denaturation process

of the flour proteins and changes in the structure of the starch
begins, increasing the viscosity of the dough. The rise in tem-
perature promotes an increase in the number of disulfide bonds
and hydrophobic interactions between nonpolar amino acid
residues in flour proteins, providing stability to the bread
structure [24].
23. The bread baking step is the most critical, as high temperatures
can result in reduced viability and stability of probiotic micro-
organisms. In addition to encapsulation techniques (see Note 1
and Chapter 14), paraprobiotics are an emerging strategy (see
Chapter 15). Paraprobiotics are defined as cells (or cell frac-
tions) of nonviable microorganisms that can provide benefits to
human health when administered in adequate amounts
[27]. However, there are no studies available in the literature
using bakery products for the delivery of paraprobiotics,
making it a possible field to be explored.
24. For complete water absorption by chia seeds, hydrate them
until constant weight (around 2 h).
25. Sonication propagates acoustic waves through chia seeds, gen-
erating cavitation bubbles, that through physical and chemical
effects, break the matrix and promote the transfer of the muci-
laginous gel firmly adhered in the seeds to the solvent
(water) [28].
26. In this step, three layers are formed: (I) the lower layer, which
contains the chia seeds; (II) the middle layer to the chia muci-
lage; and (III) the layer above water.
27. Sodium caseinate must be properly dissolved in the solution to
avoid lumps or particles that could affect the structural integ-
rity of the film [29].
28. The aleatory coil structure and interactions with molecules
coming from hydrogen and hydrophobic bonds, and electro-
static force give sodium caseinate film-forming characteristics.
In addition, the high proportion of polar groups in sodium
caseinate provides adequate barrier capacity for oxygen, carbon
dioxide, and aromatic compounds present in the
environment [29].
29. The combination of the plasticizer glycerol and chia mucilage
provides the films with thermal resistance, a barrier to light and
oxygen, and flexibility due to the reduction of intermolecular
attractions between nearby polymer chains. Furthermore, the
hydrophilic nature of chia mucilage, due to heteropolysacchar-
ides with hydrophilic groups, protects microbial cells against
environmental stress, and therefore maintains the viability of
the probiotics [13, 30].
30. Cooling to room temperature ensures sufficient time for

microorganism cells to adapt to the water activity transition,
maintaining the viability of the probiotics [13].
31. Air must be removed to prevent the formation of bubbles that
could compromise the structural integrity of the film [13].
32. The probiotic coating film does not have the function of acting
as packaging. Therefore, pack the bread coated with probiotic
edible films in polyethylene bags at room temperature (approx-
imately 25 C) for 7 days.
Acknowledgments
The authors are grateful to Coordenação de Aperfeiçoamento de

Pessoal de Nı́vel Superior—Brasil (CAPES)—Finance Code 001.
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Chapter 13
Probiotic and Synbiotic Chocolate

Milena Dutra Pierezan, Callebe Camelo-Silva, Alan Ambrosi,
Marco Di Luccio, and Silvani Verruck
Abstract
Chocolate is a mixture of cocoa by-products, sucrose, and milk solids dispersed in the fat phase (mainly
cocoa butter). It consists of an excellent probiotic matrix due to its low water activity and the considerable
presence of fat and antioxidant compounds. The Lactobacillus, Lacticaseibacillus, Limosilactobacillus,
Bifidobacterium, and Streptococcus comprehend the most applied genus in probiotic chocolate. Its proces-
sing involves the same steps as the traditional chocolate (blending, refining, conching, tempering, molding,
cooling, and storage), with the additional step of probiotic inoculation. In most cases, a single Direct Vac
Set probiotic inoculum, which consists of a freeze-dried cell concentrate, can be added and homogenized
directly into the melted chocolate. Inoculation has to take place after the conching process to ensure cell
viability. Currently, there is not a defined general amount of bacteria to be introduced into the food to
ensure the probiotic health benefits. It must be proven for each strain and inoculum tested. But in general,
cell concentrations between 106 and 1013 CFU/g are being used to ensure the probiotic claim. Further-
more, there is a current controversy over functional chocolate such as the probiotic options due to their
high sugar content. This has motivated researchers and food industries to produce sugar-free and reduced-
sugar options. Considering that some sugar substitute ingredients can act as prebiotics, promoting the
growth of probiotic microorganisms, the combination of prebiotic and probiotic functionality originated
the synbiotic chocolate. Its production also involves the probiotic inoculation step besides the traditional
stages of chocolate processing, but instead of sugar, the prebiotic and other sugar-replacer ingredients are
added, usually into the blending step with the other main ingredients. Therefore, considering the growing
literature involving this theme, the objective of this protocol is to provide up-to-date and detailed
information for the production of different types of probiotic and synbiotic chocolate.
Key words Cocoa, Functional chocolate, Low-sugar chocolate, Low-fat chocolate, Probiotic
microencapsulation
1 Introduction
Chocolate can be defined as a cocoa by-product (cocoa mass, cocoa

powder and/or cocoa liquor), sucrose and milk solids dispersed in
the fat phase, which is mainly composed of cocoa butter [1]. Emul-
sifying agents such as soy lecithin and/or polyglycerol polyricinole-
ate (PGPR) are also usually added, as well as ethyl vanillin, a
179
180 Milena Dutra Pierezan et al.
flavoring agent [2, 3]. The presence of some ingredients can differ
among the different types of chocolate, and other specifications can
be defined for the production of chocolate depending on the
regulations of each country, which can change the possibilities of
product formulation. Furthermore, among innovative functional
chocolate products, the probiotic option has been extensively
studied [1].
Probiotics are live microorganisms that benefit the host’s
health, mainly by improving intestinal problems. However, they
also can provide other health benefits, such as lowering cholesterol
and blood pressure levels and improving mineral absorption and
the immune system [4]. In this context, chocolate has been shown
to be an effective matrix for the active and viable delivery of
probiotics to the gut [5]. The low water activity of chocolate
keeps the probiotics in a low metabolic state, increasing their
viability in the chocolate matrix during storage. In addition, some
cocoa high-fat content by-products can decrease oxygen availability
to the probiotic cell, preventing oxidation and protecting cell via-
bility from thermal inactivation during processing [6]. Although
phenols may act as antimicrobials, they did not decrease the survival
of probiotic bacteria into chocolate [4]. Indeed, more cells tend to
remain viable in dark chocolate, which contains more cocoa antiox-
idant compounds, such as flavonoids, when compared to milk and
white chocolate types [7].
In general, the processing of probiotic chocolate involves the
same process steps as traditional chocolate (blending, refining,
conching, tempering, molding, cooling, and storage), with the
additional step of probiotic inoculation. Once the conching step
can reach temperatures of up to 70 C in some cases, probiotic is
usually added after this, in order to ensure cell viability [8]. In
general, Direct Vac Set (DVS) inoculums, which consist of a con-
centrate of freeze-dried cells, are used. This probiotic powder can
be added directly and homogenized in the melted product
[9]. Among all the probiotic genus, the Lactobacillus, Lacticasei-
bacillus, Limosilactobacillus, Bifidobacterium, and Streptococcus are
the most applied in studies involving chocolate production [7]. In
addition, there is not a defined general amount of bacteria to be
introduced into the food to ensure the probiotic health benefits. It
must be proven for each strain and inoculum tested. But in general,
cell concentrations between 106 and 1013 CFU/g are being used to
ensure the probiotic claim [2, 6].
Besides the health benefits of probiotics, there is a controversy
over the use of this functionality into chocolate due to its high
sugar and calorie content [10]. A high sugar intake is strongly
associated with negative implications such as obesity, diabetes,
and oral health. Thus, combining sweeteners like sucralose, stevio-
side, thaumatin, and sugar alcohols with a bulking agent like inulin,
maltodextrin, and polydextrose has been widely used to partially or
Probiotic and Synbiotic Chocolate 181
totally replace sucrose in probiotic chocolate. Interestingly, some of

these sugar substitutes are prebiotic substances that can induce the
selective growth of probiotic microorganisms. Thus, chocolate that
contains a combination of prebiotic substances and probiotic
microorganisms is called synbiotic chocolate [4, 11].
The synbiotic chocolate can be divided into two subsets, called
complementary synbiotic and synergistic synbiotic. A complemen-
tary synbiotic comprehends a product containing both probiotic
and prebiotic, working independently to achieve one or more
health benefits. The combination might not have solid evidence
of synergistic function, but they provide health benefits separately.
On the other hand, a synergistic synbiotic is composed of a probi-
otic microorganism and a specific prebiotic that have evidence of
supporting the growth or activity of that specific microorganism.
Although the substrate might also enrich other beneficial members
of the gastrointestinal microbiota, its main target is to enhance the
health benefits delivered when compared with probiotic and prebi-
otic separate effects due to the synergistic action [11].
The synbiotic chocolate production also involves the probiotic
inoculation step besides the other traditional stages of chocolate
processing, but instead of sugar, the prebiotic and other sugar-
replacer ingredients are added, usually into the blending step with
the other main ingredients. However, it is important to highlight
that sugar replacement can significantly impact the quality of choc-
olate, including particle size, flow behavior, appearance, texture,
melting profile, and moisture content. Thus, the development of
synbiotic sugar-free and reduced-sugar chocolates with desirable
physical and chemical properties for the food industry is currently a
significant opportunity for scientists [4].
Therefore, considering the growing literature involving this
theme, the objective of this protocol is to provide up-to-date and
detailed information for the production of different types of probi-
otic and synbiotic chocolate.
2 Material
2.1 Chocolate Besides the inoculation of probiotic strains described in the topic
Formulation 2.2, milk chocolate can be produced with 10.4% cocoa liquor,
18.9% cocoa butter, 41.5% sucrose powder, 25.4% milk powder
2.1.1 Probiotic Chocolate
(with 25% fat), 0.5% soy lecithin, and 0.06% ethyl vanillin [2]; dark
chocolate can be produced with 35.9% cocoa liquor, 5% cocoa
butter, 58.8% sucrose powder, and 0.5% soy lecithin [12]; and
white chocolate can be produced with 44.5% sucrose powder,
30% cocoa butter, 16% powdered milk, 9% skimmed milk, 0.3%
soy lecithin, and 0.2% PGPR [3].
2.1.2 Synbiotic Synbiotic chocolate comprises basically the sugar-free and reduced-
Chocolate sugar options (see Notes 1). Its formulation follows the same steps
for each type of chocolate mentioned in topic 2.1.1, except that
the sucrose content can be partially or totally replaced (see Note 2).
Thus, besides the inoculation of probiotic strains described in the
topic 2.2, a synbiotic sugar-free white chocolate, for example, can
be produced with 44.5% maltitol, 30% cocoa butter, 16% powdered
milk, 9% skimmed milk, 0.3% soy lecithin, and 0.2% PGPR. A
variety of sweeteners and bulking agents used in chocolate formu-
lation (see Notes 3 and 4) are summarized in Table 1 [3].
2.2 Probiotic Strains Several probiotic strains are available for use as DVS inoculums in
chocolate production (see Notes 5 and 6). The main cultures
already used in probiotic chocolate and cell viability after prolonged
storage are summarized in Table 2, while the main global exporter
suppliers of DVS probiotic strains are provided in Table 3. The cell
amount to be added into the chocolate varies between different
strains in order to ensure the health benefits (see Note 7) [14–16].
2.3 Equipment for The necessary equipment for a pilot-scale probiotic and synbiotic
Chocolate Production chocolate production includes:
• A mixer with heating such as the planetary Vena mixer BM
30/20 (NV Machinery Verhoest, Izegem, Belgium);
• A pilot-scale-roll refiner such as the Exakt 80S 3-roll refiner
(Exakt Apparatebau, Norderstedt, Germany);
• A chocolate conching machine such as the Buhler Elk’Olino
conche (Richard Frisse GmbH, Bad Salzuflen, Germany);
• A chocolate-tempering machine such as the T5 (Pomati,
Codogno, Italy);
• A chocolate vibration table such as the ZDT-02 (Food Machin-
ery Service Co. Ltd., Nanquim, Jiangsu, China);
• A refrigerator (ranging at least from 0 to 20 C);
• Plastic shapes for molding chocolate in the preferable
dimension;
• A chocolate bar wrapping machine such as the Sleek 40 (Valtara,
Schio, Italy) (optional), including the preferable packaging
material (see Note 8) [21–23].
2.4 Chocolate • Protease peptone (10 g/L);

Probiotic Viability • HM peptone B – the equivalent of beef extract (10 g/L);
Analysis
• Yeast extract (5 g/L);
2.4.1 MRS Agar • Dextrose (20 g/L);
Formulation (See Note 9)
• Dipotassium phosphate (2 g/L);
Table 1
Sugar replacers and bulking agents in different synbiotic sugar-free types of chocolate and probiotic availability in the final product
Replacement of the Inoculation Temperature Probiotic

total sucrose Type of dose and time of viability after
Substitute content (%) chocolate Probiotic strain (CFU/g) storage storage References
DVS
Maltitol and inulin 100 and 23.2 White L. paracasei Lpc-37 ATCC SD5275 109 13–15 C, 106–108 for [3]
with DP > 23 and and L. acidophilus LA-14 ATCC 90 days both inulin
DP < 10 SD5212 types
Maltitol and inulin 68.7 and 31.3 Milk L. acidophilus and L. paracasei 109 13–15 C, 106 [13]
90 days
Microencapsulation
Polydextrose and 68.1 and 31.3 Dark L. plantarum (299v) and 1013 11 C, ND < 108 [6]
inulin L. acidophilus La3 (DSMZ
17742)
Isomalt and stevia 100 and 0.08 Dark L. plantarum (299v) and 1013 11 C, ND < 108 [6]
L. acidophilus La3 (DSMZ
17742)
Isomalt and stevia 104.3 and 0.09 Milk L. plantarum (299v) and 1013 11 C, ND >107 [7]
L. acidophilus La3 (DSMZ
17742)
DP Degree of polymerization, ND Not described
183
Table 2
184
Probiotic enrichment in different types of chocolate and its cell viability after storage
Inoculation Inoculation
dose temperature Type of Temperature and Probiotic viability after storage
Probiotic strain (CFU/g) ( C) chocolate time of storage (CFU/g) References
DVS
L. acidophilus NCFM® and B. lactis 108 30–32 Milk and 4 and 20 C, 107–108 [17]
HN019 dark 180 days
L. paracasei Lpc-37 ATCC SD5275 109 35 White 13–15 C, 90 days 108–109 [3]
Milena Dutra Pierezan et al.
and L. acidophilus LA-14 ATCC

109 35 Milk 13–15 C, 90 days 106 [13]
SD5212
Lacticaseibacillus rhamnosus, 108–109 40 Dark 18 C, 90 days 108, except L. reuteri (< 106) [18]
L. paracasei F19, Lacticaseibacillus
casei DG and Limosilactobacillus
reuteri DSM 17938
L. acidophilus NCFM, L. rhamnosus 106–107 35 and 40 Milk 20 C, 180 days L. rhamnosus and L. acidophilus [2]
HN001 and, B. lactis HN019 increased to 109 after 90 days.
B. lactis decreased to <106 after
60 days
L. acidophilus NCFM and B. lactis 108–1010 30 Milk and 15 C and fluctuating All strains remained >106 at 15 C [19]
HN019 dark temperature Almost all remained >106 at
(15–30 C), fluctuating temperature
14 months
Microencapsulation
L. acidophilus (LA-5), L. rhamnosus 1012 45 Dark 4 and 25 C, > 107 after 180 days at 4 C > 107 after [8]
(LGG), Lactobacillus 180 days 120 days at 25 C
sanfranciscensis,
Lactiplantibacillus plantarum,
L. casei 431, Bifidobacterium
animalis subspp. lactis (BB-12),
and Streptococcus thermophilus
L. plantarum 564 and L. plantarum 108 40 Dark 20 C, 360 days 106 in 180 days. After that, decreased [20]
299v until 105
Immobilization
L. casei 01 and L. acidophilus (LA-5) 1010 35 White, 4 and 25 C, 60 days All strains remained between 106 and [21]
milk 108 after 60 days at 4 C and
and between 105–106 after 10 days at
dark 25 C
DVS Direct Vac Set inoculum
185
Table 3
Main global exporter suppliers of DVS probiotic strains
DVS Corporate
supplier Probiotic strains availablea Headquarter
Crh L. rhamnosus (GR-1, LGG, DSM33560), L. acidophilus (DDS-1, Horsholm,
Hansen LA-5, UALa-01), L. paracasei (CASEI 431, F-19, UALpc-04), Denmark
L. plantarum (UALp-05), L. casei (UALc-03), L. reuteri (RC-14,
LRC, UALre-16), B. lactis (UAB1a-12), B. animalis subsp. lactis
(BB-12), S. thermophilus (TH-4, UASt-09)
Sacco L. rhamnosus (CRL 1505, IMC 501), L. paracasei IMC 502, Cadorago, Italy
system L. plantarum LPLDL
Danisco L. acidophilus NCFM, L. paracasei Lpc-37 B. lactis (HN019, Bi-07, Copenhage,
Bl-04), L. lactis subsp. lactis, S. thermophilus TA040 Denmark
Synbio L. acidophilus LA1063, L. casei LC122, L. paracasei LPC48, Yangzhou and
tech L. plantarum LP198, L. rhamnosus LRH09, B. animalis subsp. lactis Taiwan, China
Inc. BAL06, S. thermophilua ST37
a
Only the probiotic species already described in the literature as being used in chocolate production were considered
• Sodium acetate (5 g/L);

• Triammonium citrate (2 g/L);
• Manganese sulphate (0.05 g/L);
• Tween 80 (1.08 g/L);
• Agar (15 g/L);
• Magnesium sulphate (0.2 g/L);
• L-Cysteine (0.05 g/L) (only for Bifidobacterium viable cells
count) [24].
2.4.2 M17 Agar • Pancreatic digest of casein (5.3 g/L);

Formulation (See Note 9) • Soy peptone (5.3 g/L);
• Beef Extract (5.3 g/L);
• Yeast Extract (2.6 g/L);
• Ascorbic Acid (0.5 g/L);
• Magnesium Sulfate (0.3 g);
• Disodium-β-glycerophosphate (20 g/L);
• Agar (11.5 g/L);
• Sterile lactose solution (100 g/L) [25].
Reagents, Solvents, and • Acetic acid;

Solutions • Sodium hydroxide;
• Peptone water solution;
• Distilled water [7, 24].
2.4.3 Laboratory • Water bath;

Glassware and Equipment • Microwave oven (optional);
• Glass bottle (volume of 1000 mL);
• Propylene centrifuge tube (volume of 50 mL);
• Sterile petri dishes;
• Pipette (sterile tips of 1 mL);
• Laminar flow cabinet;
• Bacteriological oven/Incubator;
• pHmeter [24, 25].
2.5 Chocolate • A water activity analyzer such as the Aqualab (METER Group,
Physical Analysis Inc., Hopkins, U.S.A);
• A colorimeter such as the Chroma Meter CR-400 (Konica
Minolta, Tokyo, Japan);
• A texture analyzer such as the texture analyzer Model TA.HD.
plus (Texture Technologies, Hamilton, U.S.A);
• A Differential scanning calorimeter;
• A laser scattering particle size distribution analyzer such as the
MasterSizer® (Malvern Instrument, Malvern, U.K);
• A rheometer;
• A crusher equipment;
• An ultrasonic bath.
3 Methods
3.1 Probiotic and All the steps are summarized in the flowchart presented in Fig. 1, as
Synbiotic Chocolate well as the four main equipment used are presented in Fig. 2.
Production
1. Mixing: mix the melted fat (20% of the total cocoa butter
present in the formulation) and the other main ingredients
(cocoa liquor, powdered sugar or sugar substitutes, powdered
milk) during 12–15 min until they become homogeneous at
40 C in a mixer with heating [20];
2. Refining: transfer the chocolate mass to a pilot-scale-roll refiner
to achieve a mean particle size of approximately 20–25 μm (see
Note 10) [3];
3. Conching: transfer the chocolate mass to a chocolate conching
machine to execute two steps (see Note 11):
(a) Dry conching: performed for 45 min at 60 C;
(b) Wet conching: the remaining cocoa butter (80% of the
total), soy lecithin, and flavoring (when present in the
formulation) is added, and then this process is maintained
during 5 h, 25 min at 60 C;
Fig. 1 Flowchart of probiotic and synbiotic chocolate production

Fig. 2 Chocolate processing during mixing, refining, conching and tempering steps. (Adapted from [31, 32])
4. Probiotic inoculation and mixing: transfer the chocolate mass

again to the mixer with heating, where the content of the DVS
is added directly into the melted product (see Note 12) with a
temperature between 30 and 45 C at a minimum number of
rotations for 3 min [17];
5. Tempering: to provide cocoa butter crystallization in a pre-
ferred crystalline form (β-V), transfer the chocolate mass into
a tempering machine to execute three steps:
(a) Heating at 33–35 C (white chocolate) or 45 C (milk and
dark chocolate) until melting [3, 13, 27];
(b) Cooling between 25 and 28 C for 5 min [13];
(c) Heating again at 28–29 C (milk chocolate) or 25 C
(white and dark chocolate) for conversion of any unstable
crystals [3, 13, 27];
6. Molding with vibration: mold the final chocolate into plastic
shapes and maintain it on the vibration table at 27–30 C until
the complete removal of air bubbles;
7. Cooling: cool the molded chocolate at 5 C in a refrigerator for
20 min for complete solidification (see Note 13) [3, 28];
8. Demolding and packaging: demold the chocolate and package
it in a specific equipment or manually [29];
9. Storage: store the packaged chocolate at 20 C or other tem-
peratures, if preferable (see Tables 1 and 2), with a range of
humidity between 10 and 20% and preferable with low oxygen
(see Note 14) [30].
3.2 Quality Analysis 1. Suspend all the components from the culture medium in
Related to Probiotic 1000 mL distilled water in a glass bottle (see Note 15).
and Synbiotic 2. Check the final pH at 25 C, that must be at 6.5 0.2 (see
Chocolate Note 16);
3.2.1 Chocolate Probiotic 3. Heat it in a microwave oven (see Note 17) or a water batch can
Viability Analysis be necessary to dissolve the medium completely;
4. Sterilize by autoclaving at 15lbs pressure (121 C) for 15 min;
Preparation of MRS Agar
5. Cool the MRS agar (45–50 C) before use, and if not used
promptly store in refrigeration (2–8 C) [25].
Preparation of M17 Agar 1. Suspend all the components from culture medium, with the
exception of the lactose in 1000 mL distilled water in a glass
bottle (see Note 15);
2. Heat it in a microwave oven (see Note 17) or a water batch to
dissolve the medium completely;
3. Sterilize by autoclaving at 15lbs pressure (121 C) for 15 min;
4. Cool the bottle until 50 C to add 50 mL sterile lactose
solution and mix;
5. If M17 agar is not used promptly, store it in refrigeration
(2–8 C) (see Note 18) [26].
Viable Cell Count To investigate the probiotic viability in the chocolate formulation
during storage, carry out the analysis described below in the same
day that the chocolate was produced, and repeat it with the desir-
able frequency (for example on 0th, 30th, 60th, and 90th days of
storage) (see Note 19):
1. Take approximately 25 0.2 g of chocolate under aseptic
conditions and mix it with 180 mL peptone water solution;
2. Melt the mixture in a water bath for 15 min at 40 C;
3. Prepare a decimal dilution series;
4. Plate 1 mL of each dilution (triplicate) and add 15–25 mL of
molten selective media (45–50 C) in petri dishes, using the
proper inoculation mode, which will depend on the probiotic
strain included into the formulation (see Note 20);
5. Gently shake the plate to mix the inoculum in the selective
media;
6. Let the media solidify;
7. Incubate petri dishes under adequate conditions, which will
depend on the probiotic strain included into the formulation
(see Note 21);
8. After the incubation period, make the colony count. Register
the results as CFU/g [7].
3.2.2 Chocolate Physical The physical analysis of chocolates is one of the most important
Analysis quality parameters for the food industry. From the results of these
analyses, we can predict, e.g., the shelf life of the product and the
melt in the mouth. Below we describe the procedures of the main
physical analyzes performed on chocolates.
Water Activity 1. Preparing the sample: Grind the chocolate in a crusher.

2. Reading on the device: Transfer the 2 g of chocolate to a water
activity analyzer, and read at 25 C [33].
Color Measurement 1. Preparing the sample: Grind the chocolate in a crusher.

2. Reading on the device: Transfer the 5 g of chocolate to a Petri
plate and place a white sheet under it. Then, the color para-
meters such as L: brightness, a: red-green, and b: yellow-
blue can be measured using a colorimeter. In addition, the
chroma (C*) and whiteness index (WI) parameters can be
calculated using Eqs. 1 and 2. It is advisable to carry out this
analysis if any substance in the chocolate formulation changes
the color of the final product [33].
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
C ¼ a2 þ b 2 ð1Þ
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
WI ¼ 100 ð100 L Þ2 þ ðaÞ2 þ ðb Þ2 ð2Þ
Texture Analysis 1. Preparing the sample: Cut the chocolate into approximately
1 cm2 square.
2. Performing the analysis: As a texture parameter, hardness is the
most important to determine in chocolates. Place the sample in
a texture analyzer and operate using a 500 N load cell with a
pre-test speed of 1 mm s1 and a firing force of 0.1 N. Pre-test,
test, and post-test speeds applied during textural measurement
can be adjusted from 1 mm s1, 1 mm s1, and 10 mm s1,
respectively. The hardness values of each sample must be
measured at least 7 times [3].
Fusion Properties 1. Preparing the sample: Grind the chocolate in a crusher.

2. Performing the analysis: Transfer the 15 mg of chocolate to
aluminum crucibles and analyze them in a differential scanning
calorimeter (DSC) at 0–70 C under a heating rate of 5 -
C min1 and nitrogen flow of 50 mL min1 [34].
Particle-Size Distribution 1. Preparing the sample: Grind the chocolate in a crusher.

2. Performing the analysis: Disperse approximately 0.20 g of
chocolate in vegetable oil (refractive index, RI ¼ 1.45) at
room temperature (20 2 C) until an obscuration of 0.2 is
obtained. Then, keep the sample in an ultrasonic bath for 2 min
to ensure that the particles are freely dispersed. Finally, read on
a laser scattering particle size distribution analyzer [33, 35].
Rheological Measurements 1. Preparing the sample: Melt the chocolate in an oven at 50 C

for 75 min.
2. Performing the analysis: Transfer the chocolate to a rheometer
and shear at a rate of 5.0 s1 for 10 min at 40 C before the
measurement cycles start. Measure the shear stress at 40 C
with increasing shear rate from 0.5 to 60 s1 (ramp up) in 120 s
and then decrease the shear rate from 60 to 0.5 s1 (ramp
down). On each ramp, 50 measurements must be taken. This
measurement cycle must be repeated 30 consecutive times until
thixotropy is eliminated from the samples. The measurement
data can be applied to the Casson Model (Model recom-
mended for chocolates) (Eq. 3) to determine related rheologi-
cal parameters such as yield stress, Casson yield stress, and
Casson viscosity [33, 35, 36].
pffiffiffi pffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffi
τ ¼ τCA þ μ CA γ ð3Þ
Where, τ: yield stress; τCA: Casson yield stress; μ CA: Casson
viscosity, and γ: shear rate.
4 Notes
1. Besides sugar-free and reduced-sugar chocolates, low-fat pro-

ducts have also been produced, using defatted cocoa derivatives
or fat replacers with prebiotic functionality such as inulin,
β-glucan, xanthan gum, and guar gum. However, most studies
have discussed the impact of fat reduction or fat replacement
on the quality attributes of chocolate and do not discuss the
nutritional effect. In addition, there are no studies describing
the production of low-fat chocolate with probiotic or synbiotic
functionality. Thus, investigating the effect of probiotic
fat-reduced chocolate on health is an excellent opportunity
for a study topic [4];
2. Although regulations can change slightly between countries, in
the case of reduced-sugar chocolates, the “light” claim can be
used in the product label when there is approximately 25% of
sugar reduction when compared to the regular version [37];
3. Although polyols can present a prebiotic functionality, high
concentrations can cause abdominal discomforts. Thus, care
must be taken when formulating chocolate with this type of
ingredient [38];
4. It is important to notice that sweeteners and bulk agents can
replace the total content of sucrose by 100% or a little bit more,
which can slightly alter the proportion of other ingredients into
the chocolate formulation;
5. Several strains are already considered probiotic and are cited in
the literature. Besides them, FAO states Guidelines for the
Evaluation of Probiotics in Food [39] when the potential of
some strains still has to be investigated before being added to
the food product;
6. DVS is widely used for probiotic chocolate production. How-
ever, in some cases, other approaches, such as microencapsula-
tion (see Chap. 14), can ensure greater protection of probiotics;
7. To ensure the health benefits provided by probiotic strains into

the chocolate formulation, 101 to 102 CFU/g above the desir-
able amount can be added, considering the potential cell losses
during chocolate processing and storage [7];
8. Most dairy probiotics and other products are stored and sold
on the market in plastic packaging with high oxygen perme-
ability, this poses a serious problem to the growth and survival
of the probiotic. The use of plastic films with high oxygen
barrier properties and active packages with oxygen absorbers
or glass containers can be a solution [28];
9. After opening, the product should be properly stored dry, after
tightly capping the bottle due to the hygroscopic nature of the
product [40];
10. Human taste buds allow for detecting particles larger than
25 μm. Thus, a lower particle size in chocolate products is
desirable. In 3-roller machines, the chocolate mass must be
treated two or three times to obtain this size, while in 5-roll
refiners it can be achieved in a single process. Rotation speed
and temperature can be adjusted to approximately 0.75 m/
s and 30 C for the first roll, 1.25 m/s and 35 C for the
second, 1.80 m/s, 40 C for the third, 2.45 m/s and 45 C,
and 3.70 m/s and 40 C for the fifth [41];
11. The literature describes a range of temperatures employed in
the conching process of different types of chocolate. Overall,
milk and white chocolate conching temperatures range
between 40 and 70 C. Temperatures above 70 C cannot be
exceeded in those cases once it provides the denaturation of
milk proteins into the formulation. But the lack of milk solids
in dark chocolate formulation allows it to be heated between
40 and 80 C. The longer the time and temperature are
applied, the greater the Maillard reaction. However, tempera-
tures above 60 C tend to reduce soy lecithin efficiency. Thus,
it was suggested to establish a temperature of approximately
60 C for all types of chocolate [40, 42];
12. Additives and probiotic powders are generally resuspended in
some liquid ingredients before being added to the product
formulation. However, resuspension of DVS probiotic cells in
UHT milk before adding to chocolate has been shown to cause
significant viability losses for L. rhamnosus GG, L. paracasei
F19, L. paracasei DG, and L. reuteri DSM17938. This fact is
probably justified due to the induction of an early reactivation
of freeze-dried cells when resuspended, becoming more sensi-
tive to stress conditions during the final stages of chocolate
production. Thus, an anabiotic state of cells is preserved when
added directly to chocolate, which has been shown to ensure
greater survival [18];
13. The different chocolate shapes (chocolate bars, bon bons, and
other filling products) are made with the same ingredients and
production steps. But in the case of filled chocolates, the
melted chocolate must be molded into semi-sphere shapes,
an additional step must be included, which consists of filling
the molded chocolate, adding melted chocolate above the
filling or joining a second semi-sphere chocolate to the first.
Then the final product can be cooled [43];
14. Besides a proper packaging material, vacuum storage was
proven to be better than nitrogen or air when it comes to
probiotic viability into chocolate. Also, its survival is inversely
related to storage temperature [30];
15. Currently it is possible to acquire the commercial powder
containing all the necessary ingredients of the selective media.
In those cases, it is necessary only to resuspend the mixture
powder in distilled water, and proceed with sterilization.
16. Occasionally, sterilization may cause the pH to fall outside of
the specified pH limits. In these rare cases, pH adjustment
using acetic acid or sodium hydroxide is recommended [25];
17. Heating in a microwave oven may be uneven across the total
volume of the medium. This can be prevented if you slowly
shake the flask when it starts to boil and reheat further until the
medium is totally dissolved [44];
18. The addition of lactose must be done only in the selective
media that will be plated immediately. The part of the agar
that will be stored for future plating can’t contain lactose,
because it can’t be melted again. Thus, it must be discarded.
19. The inoculation procedure requires skilled labor and must be
conducted aseptically in an adequate laboratory (in a laminar
flow cabinet) and respecting the good laboratory practices to
avoid contamination [45];
20. It is not necessary to use a highly selective media when count-
ing pure cultures added into a food formulation. Thus, for
Lactobacillus, Lacticaseibacillus, and Limosilactobacillus, it is
possible to use a MRS agar in a pour plate technique. For
Bifidobacterium, the MRS agar must be supplemented with
0.05% cysteine and inoculated with pour plate technique. For
Streptococcus, the M17 agar is recommended with a spread
plate procedure [45–47]. For more details about colony count-
ing and selective media, check the Chap. 25 called “probiotics”
present in the “Handbook of Dairy Food Analysis” [48];
21. Lactobacillus, Lacticaseibacillus, Limosilactobacillus, and Bifi-
dobacterium must be incubated at 37 C for 48 h, while the
Streptococcus must be incubated at 42 C for 48 h, all under
anaerobic conditions [7, 49].
Acknowledgments
The authors are grateful to CAPES (Coordination of Improvement

of Higher Education Personnel) for the scholarship (Finance code
001).
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10 Aug 2022
Chapter 14
Microencapsulation of Probiotics
Callebe Camelo-Silva, Lais Leite Figueredo, Vanessa Cortina Zanetti,
Alan Ambrosi, Marco Di Luccio, and Silvani Verruck
Abstract
Probiotics are susceptible to factors such as stomach acid, enzymes, and bile salts. Also, when incorporated
into food matrices, intrinsic or processing factors like low pH, high water activity, or high cooking
temperatures can negatively affect the viability of microorganisms. Encapsulation technology can ensure
the safe delivery of probiotics to the gut and better survival during processing and storage. Several
techniques are used to protect probiotics, for example, emulsion, extrusion, spray-drying, freeze-drying,
liposome, electrospinning, and others. Here, we describe in detail the main methods of encapsulation of
probiotics, including emulsion, extrusion, and spray-drying techniques.
Key words Probiotic, Encapsulation, Emulsion, Extrusion, Spray-drying
1 Introduction
The consumption of probiotic products has increased exponentially

due to the range of benefits these microorganisms can offer to
human health. However, it is still a challenge to ensure the viability
of probiotics to the consumer, as they have a noticeable loss of
viability after passing through the digestive tract. In addition, when
incorporated into commercial products, intrinsic or processing
factors such as low pH, high water activity, or high cooking tem-
peratures can negatively affect the viability of microorganisms [1].
Microencapsulation emerges as an alternative to circumvent
these limitations. This technique is based on trapping probiotics
within an encapsulating matrix, ensuring safe delivery to the intes-
tine at appropriate therapeutic levels to provide human health
benefits [2]. Several microencapsulation techniques can be used
to encapsulate probiotics (Table 1). However, emulsion, extrusion,
and spray-drying techniques occupy a prominent place, considering
199
200 Callebe Camelo-Silva et al.
Table 1
Encapsulation techniques used to microencapsulate probiotics
Encapsulation Encapsulation
technique Probiotic strain Wall materials yield (%) References
Emulsion Lactiplantibacillus Alginate 27–82 [7]
plantarum (MT,
ZH593)
Extrusion Limosilactobacillus reuteri Alginate and (tamarind gum 93–97 [8]
(DSM 20016) or mutamba mucilage or
cassia tora gum or
psyllium mucilage or
konjac gum)
Spray-drying Bifidobacterium animalis Full-fat goat’s milk and/or 94–97 [9]
subsp. lactis BB-12 prebiotics (inulin and/or
oligofructose)
Freeze-drying Lactobacillus acidophilus Microalgae Spirulina 80–92 [10]
(La-05), platensis, Chlorella
Lacticaseibacillus casei vulgaris, Scenedesmus
(Lc-01) quadricauda, and
Lagerheimia longiseta
Supercritical Bifidobacterium animalis Poly-(vinylpyrrolidone)- Not shown [11]
subsp. lactis BB-12, poly-(vinylacetate-co-
Bifidobacterium crotonic acid)
longum BB-46
Liposome Lacticaseibacillus Lecithin and (chitosan or 81–87 [12]
rhamnosus (ATCC gelatin)
10754)
Electrospinning Lacticaseibacillus Pectin and poly (vinyl Not shown [13]
rhamnosus 1.0320 alcohol)
Microfluidics Saccharomyces cerevisiae Alginate Not shown [14]
(PDC1-GFP)
Layer-by-layer Ligilactobacillus Chitosan and alginate Not shown [15]
salivarius Li01 (Li01)
Fluidized bed Lactobacillus acidophilus Xanthan, alginate, chitosan, 35–78 [16]
(PTCC 1643) and gellan
3D printing Bifidobacterium lactis Alginate and gelatin Not shown [17]
(HOWARU®
Bifidous) Lactobacillus
acidophilus
(HOWARU®
Dophilus)
Microencapsulation of Probiotics 201
their low cost, simplicity of handling, and the possibility of produc-

ing large-scale microcapsules. Thus, throughout this chapter, we
will address only these most used techniques.
The encapsulating matrix can be formed using different wall
materials, also known as carriers. Sodium alginate has been widely
used due to its low cost, biocompatibility, food grade, and targeted
delivery of probiotics (soluble in basic medium, for example, in the
intestine) [3]. Wall materials such as chitosan, gelatin, milk pro-
teins, pectin, carrageenan, prebiotics, and different types of starch
have also occupied a prominent place for the microencapsulation of
probiotic strains. The criteria for choosing a suitable encapsulating
agent are mainly based on its physicochemical properties (molecu-
lar mass, solubility, glass transition temperatures, crystallinity, film
formation, and emulsifying properties). A good wall material must
also be easy to handle during the encapsulation process. In addi-
tion, it cannot react or injure the probiotic strain during the encap-
sulation and storage process and, finally, it must meet the solubility
properties of the microcapsule by releasing the probiotics at the site
of action [1]. To describe the methodology of this chapter, we will
consider alginate (ALG) and whey proteins (WPI) as encapsulating
agents and the strain of Lacticaseibacillus rhamnosus GG as active
material. Alginate was chosen because it is necessary to use a
hydrocolloid for the crosslinking process in the emulsion and
extrusion methods. In addition, it is considered GRAS (Generally
Recognized as Safe) and low cost. However, other wall materials
have been widely used [4–6].
2 Material
For the production of microcapsules, the following materials are

needed:
• Freeze-dried probiotic cells;
• De Man Rogosa and Sharpe (MRS) broth;
• Glycerol.
• Bacteriological oven;
• Centrifuge;
• Saline solution;
• Soybean oil;
• Alginate (ALG);
• Whey proteins (WPI);
• Calcium carbonate;
• Acid organic;
• Span 80;
• Calcium chloride;
• Spray-drier.
3 Methods
3.1 Preparation of To obtain the stock solution, freeze-dried probiotic cells can be
Probiotic Suspension rehydrated in sterile skim milk (25 g L 1) or with De Man Rogosa
and Sharpe (MRS) broth added with glycerol (20 g L 1) and stored
in sterile Falcon vials at 20 2 C [18] (see Note 1). Then, the
stock solution is added to sterile MRS broth and incubated
(37 1 C for 48 h) to reach the stationary phase (see Note 2).
After the incubation time, the probiotic cells are harvested by
centrifugation (1000 g) for 10 min at a temperature of
25 1 C and washed twice with saline solution (0.9 g
100 mL 1). Cell pellets should be kept at 4 1 C until encapsu-
lation procedure.
3.2 Encapsulation of The emulsion technique consists of mixing two immiscible phases,
Probiotics by Emulsion called the dispersed or discontinuous phase, and the oily or contin-
uous phase [1]. In this method, ALG gelation can be performed
internally or externally (Fig. 1). In internal gelation, the alginate is
previously solubilized with calcium carbonate, and then an aliquot
of organic acid is added to the mixture after emulsification to
CHEMICAL REACTION
1) CaCO3 + CH3COOH oCO2 + H2O + Ca+2

Sodium Alginate Cell Suspension
+ 2) Ca+2 + 2NaAlg o(Alg)2 + 2Na+
CaCO3 (Internal Gelation)
Extrusion Technique
Emulsion Technique
Drop in calcium
Polymeric Solution Chloride solution
Containing Probioic
Calcium Alginate bead
Another Extrusion System Emulsion in vegetable
Outer core
Solution External Gelation Internal Gelation
CaCl2 Acetic acid CaCl2
Frequency Jet Cutting Electostatic Rotating Co-extrusion Calcium Alginate beads Pregelfied Calcium Alginate
generation Technique Field Disc particles beads
Fig. 1. Extrusion and emulsion technologies [19]. (Adapted from [19])

promote gelation. As the organic acid enters the aqueous phase, it

interacts with calcium carbonate, releasing calcium ions and car-
bonic acid. The calcium ions react with the alginate through com-
plexation with the carboxylic groups of the polymer, forming the
“egg box model” structure [19]. On the other hand, in external
gelation, the complexation reaction of the carboxylic groups of
ALG occurs through contact with a solution of calcium chloride.
1. Preparing the dispersed phase: Mix 5% (w/v) of WPI in
100 mL of sterile distilled water under stirring at 400 rpm.
Then, gently add 1% (w/v) of ALG (see Note 3), and leave
under stirring until the alginate is entirely homogenized.
2. Addition of cell biomass: Aseptically, an aliquot (~ 9 log CFU
mL 1) of the probiotic biomass should be added to the dis-
persed phase and then homogenized at 400 rpm for 5 min. It is
recommended to add a biomass content that reaches a viable
cell count of around 9 to 10 log CFU g 1 (see Note 4).
3. Preparing the continuous phase: Add 300 mL of soybean oil to
a beaker. Add 3% (v/v) of an emulsifying agent (Span 80) in the
same container and leave it under stirring at 400 rpm until
complete homogenization (see Note 5).
4. Mixing the two phases: In a beaker, mix the dispersed and
continuous phases and leave under stirring at 400 rpm for
20 min or until the complete formation of the emulsion (see
Note 6).
5. ALG cross-linking process: While stirring, add an aliquot of a
1.5% (w/v) calcium chloride solution to form the gelled micro-
capsules (see Note 7). Then, turn off the agitation and add
200 mL of sterile distilled water to attract the microcapsules to
the aqueous phase.
6. Collecting the microcapsules: Discard the emulsion superna-
tant, collect the microcapsules by vacuum filtration, and keep
them at 4 C until drying.
7. Drying of microcapsules: Gelled microcapsules can be dried in
a spray-dryer, freeze-dryer, or fluidized bed dryer (see Note 8).
After drying, the microcapsules can be packed in airtight pack-
aging and kept at room temperature until use (see Note 9).
3.3 Encapsulation of The extrusion technique (Fig. 1) involves mixing the cellular bio-
Probiotics by Extrusion mass of the probiotic with the polymeric solution (ALG and WPI)
and then forming droplets by passing the solution through a nozzle
or atomizing nozzle [20].
1. Preparing the dispersed phase: Mix 5% (w/v) f WPI in 100 mL
of sterile distilled water under stirring at 400 rpm. Then, gently
add 1% (w/v) of ALG (see Note 3), and leave under stirring
until the alginate is entirely homogenized.
2. Addition of cell biomass: Aseptically, an aliquot (~ 9 log CFU

mL 1) of the probiotic biomass should be added to the dis-
persed phase and then homogenized at 400 rpm for 5 min. It is
recommended to add an aliquot with a viable cell count of
around 8 to 9 log CFU g 1 (see Note 4). It is worth emphasiz-
ing that the dispersed phase containing the hydrocolloid must
be prepared just before use.
3. Forming the gelled microcapsules: Once the feed solution
(FS) (polymer solution + probiotic) is prepared, the FS is
dripped into a 1.5% (w/v) calcium chloride (see Note 7) gelling
solution under stirring at 200 rpm. The dripping of the FS into
the gelling solution is carried out using an atomizing nozzle. In
this case, the FS is pumped by a peristaltic pump, and the
droplets are quickly transformed into solid particles through
the complexation of ALG with calcium ions. Another simpli-
fied form can be used, for example, using a syringe to perform
the drip (see Note 10). After the dripping step, it is interesting
to leave the microcapsules to rest (~20 to 30 min) in the CaCl2
solution to solidify the microcapsules completely. The forma-
tion of large particles and the low production rate are the main
disadvantages of this technique for use in the food industry.
However, to overcome this, the extrusion process can be com-
bined with ultrasound, jet cutting, electrostatic field, and rotat-
ing disk (Fig. 1).
4. Collecting the gelled microcapsules: Collect the microcapsules
by vacuum filtration and keep them at 4 C until drying.
5. Drying of microcapsules: Gelled microcapsules can be dried in
a spray-dryer, freeze-dryer, or fluidized bed dryer (see Note 8).
After drying, the microcapsules can be packed in airtight pack-
aging and kept at room temperature until use (see Note 9).
3.4 Encapsulation of The spray-drying encapsulation technique (Fig. 2) is well estab-

Probiotics by Spray- lished for large-scale industrial applications and is considered an
Drying economically viable technique. In this technique, the suspension
containing the wall materials and probiotics is atomized in a drying
chamber with concurrent hot air, which instantly removes water
from the atomized solution [21]. Microcapsules are removed from
the drying chamber by a negative pressure cyclone system and can
be collected on the drying chamber bottom or in the collection
flask (see Note 11).
1. Preparing the feed solution: Mix 5% (w/v) of WPI in 100 mL
of sterile distilled water under stirring at 400 rpm. Then, gently
add 1 (w/v) of ALG (see Note 3), and leave under stirring until
the alginate is entirely homogenized (see Note 12).
2. Addition of cell biomass: Aseptically, an aliquot of the probiotic
biomass should be added to the dispersed phase and then
Fig. 2 Spray drying process schematic diagram [22]. (Adapted from [22])
homogenized at 400 rpm for 5 min. It is recommended to add

an aliquot with a viable cell count of around 8 to 9 log CFU
g 1 (see Note 4).
3. Encapsulation process: Turn on the spray-dryer equipment and
operate it with the concurrent flow (see Note 13) with an inlet
temperature of 150 C and an outlet temperature of 50 C (see
Note 14). Program the drying airflow of 35 m3 h 1, and
compressor air pressure of 0.7 MPa [23].
4. Then, turn on the peristaltic pump to pump the FS and pro-
gram supply flow to 20 mL min 1. It was found that slow
drying kinetics leads to significant inactivation of the dehydra-
tion of Lactiplantibacillus plantarum, while a rapid drying rate
could instantly stabilize the cells and thereby prevent this inac-
tivation [24]. In addition, a high drying rate during the first
stage of drying, when facilitated by hydraulic membrane per-
meability, may limit bacterial adaptation because of exposure
for too short a time to the gradual withdrawal of moisture. It is
recommended that the FS be kept under magnetic stirring at
room temperature during the encapsulation process (see Note
15).
5. Before FS entry, sterile distilled water at room temperature
must be pumping until stabilization of the inlet temperature.
6. Collecting the dry microcapsules: After complete evaporation

of the water, collect the microcapsules from the collector
located at the bottom of the equipment, store them in hermetic
packages and keep them at room temperature until use (see
Note 9).
The analysis of microcapsules is an important step in the micro-
encapsulation process. Microcapsules must be characterized before
use to observe their physical, chemical, and biological properties.
Table 2 shows the characteristics of the probiotic microcapsules
obtained by the emulsion, extrusion, and spray-drying techniques
and the main characterization analyses.
4 Notes
1. You can use other cryoprotectants. MRS for LAB only, if strains
from other species (E. coli, Bacillus, Saccharomyces), other
broths should be used.
2. Cells in the stationary phase are more resistant and have a
higher encapsulation yield than cells in the log phase [33].
3. Alginate should be added gently to not form lumps. You can
place it on a foil film and spray it on the solution. Another way
to avoid the formation of lumps is to homogenize them in
warm water (40–50 C).
4. Adding an aliquot of L. rhamnosus GG with a low viable cell
count may compromise delivery to the gut at levels suitable for
promoting human health.
5. Any oil can be used. The emulsifying agent is chosen according
to the lipophilic hydrophilic balance (LHB); generally, the
most used are Tween 80 and Span 80.
6. Using slow agitation rates (400–500 rpm) is recommended.
High agitation rates can damage the probiotics‘cell wall.
7. Other types of salt can be used for ALG crosslinking, such as
calcium citrate. However, it is desirable to use low concentra-
tions. High salt concentrations have a detergent effect, which
dissolves bacterial membranes and even causes cell death.
8. Another drying method can be used. However, those are more
commonly used. The drying of microcapsules is important
both from a microbiological and technological point of view,
as it increases the lifespan of microorganisms. In addition,
drying the microcapsules makes it possible to incorporate pro-
biotics into low-moisture food matrices.
9. These microcapsules can be used in the products described in
other chapters of this edition to improve survivability in pro-
cessing, storage, and TGI.
Table 2
Characteristics of the probiotic microcapsules obtained by the emulsion, extrusion, and spray-drying techniques and the main characterization
analyses
Characteristics of the
probiotic
microcapsules How to determine? Why perform this analysis? Ideal characteristics Reference
Size and morphology Size: Laser diffraction method using The size and morphology of the The microcapsules should not exceed [25]
Mastersizer or dynamic light microcapsules are influenced by the 100 μ in size
scattering (DLS) LUMiSizer wall materials and the encapsulation
technique employed
Morphology: Optical microscopy, Microcapsules’ size can affect their They may have a regular shape (e.g.,
fluorescence microscopy, and performance in protection, spherical, tubular, and oval) or an
scanning electron microscopy probiotic delivery, and the sensory irregular shape
(SEM) quality of the food product
incorporated with the
microcapsules
Chemical structure Fourier transform infrared FTIR identifies the functional groups Not applicable [25]
and surface spectroscopy (FTIR) after chemical modification,
chemistry provides insights into the
interaction between microcapsule
components, confirms the cross-
linking, and probes the degradation
of the polymeric matrix
Raman spectroscopy Confirms immobilization and
encapsulation of probiotic
Density: Bulk density Multi-volume pycnometer and The density of microcapsules is a Not applicable [25, 26]
and true density burette containing toluene significant factor in the processing,
storage, packaging, transportation,

and commercialization of the
product
207
(continued)
208
Table 2
(continued)
Characteristics of the
probiotic
microcapsules How to determine? Why perform this analysis? Ideal characteristics Reference
Porosity The porosity of the microcapsules can Porosity is defined as the void fraction Low porosity is desirable, as the [25, 27]
be calculated via the relationship in the powder sample. It is an presence of pores in the
Callebe Camelo-Silva et al.
between the bulk and true density important property that plays an microcapsules can favor the
of the sample important role in the stability of permeation of substances harmful
SEM and Brunauer-Emmett-teller probiotic powders to probiotics, for example, stomach
(BET) techniques acid
Water activity (aw), aw: Water activity analyzer (e.g., aqua Influences the stability of A high aw, [28, 29]
moisture content, lab, decagon devices) moisture, and hygroscopicity imply
and a faster decline in viability during
hygroscopicity storage
Moisture content: Gravimetrically encapsulated probiotics during aw around 0.3 is considered
with heat storage satisfactory for dried probiotic
microcapsules
Hygroscopicity: Calculated as the High hygroscopicity tends to form
weight of the water absorbed per clusters of microcapsules
mass of the sample (%), and can be
determined by exposing the
microbeads to a NaCl saturated
solution for a period of time
Thermal analysis Differential scanning calorimetry Thermal stability of wall materials Not applicable [30]
(DSC)
Type of order present X-ray diffraction The crystals could damage the cells, Amorphous solids [31]
in powders: which would reduce the viability of
Amorphous or microorganisms, making the
crystalline amorphous structure interesting
Amorphous solids are in general
more soluble, and the
crystallization may entail a
negative impact on the handling
properties
Encapsulation process Encapsulation yield (EY): Measured Determines the effectiveness of Microencapsulation can be considered [25]
based on the ratio of the number of entrapment within microcapsules successful when it yields relatively
viable entrapped bacteria to the and survival of viable cells during higher EY
number of free bacteria the microencapsulation procedure
Simulated Infogest 2.0 Determines the stability of Probiotics should reach the intestine [32]
gastrointestinal encapsulated probiotics in in adequate doses to promote
conditions simulated gastrointestinal human health
conditions such as mouth, stomach,
and intestine
Thermal stability assay Bath with controlled temperature Determines the thermal stability of Probiotics should be resistant to [9, 30]
encapsulated probiotics elevated temperatures
Heat-resistant encapsulated
probiotics favor the probiotication
of thermo-processed foods
Storage stability BOD incubator oven Determines the stability of It is desirable that probiotics remain [30]
encapsulated probiotics during stable for long periods
storage at different temperatures
209
10. The formation of large particles and the slow production rate
are the main disadvantages of this technique for use in the food
industry.
11. For probiotic microcapsules, the ideal in bench spray-dryers or
pilots is to collect only the product from the collector due to
the greater control of the exit temperature of the process.
12. During the process, encapsulated microorganisms can undergo
several stresses, including heat stress and dehydration. Encap-
sulating agents such as gelatin, gum arabic, and cellulose ace-
tate phthalate has been reported as protective agents capable of
forming a physical barrier resistant to hot air [21]. In addition,
disaccharides are encouraged as they can preserve the structure
of probiotic cell proteins and membranes through a connection
at sites that previously interacted with water [34].
13. Spray flow can be applied in three ways (concurrent, counter-
current, or mixed flow). However, the choice of spray flow will
depend on the direction in which air and liquid (e.g., feed
solution) enter the drying chamber. In the first case (concur-
rent), the product is in contact with the colder air, preferable
for drying thermosensitive materials, such as probiotics.
14. The lower the Toutlet, the higher the post-drying viability.
Toutlet is therefore considered to be the principal drying param-
eter that affects the viability of spray-dried LAB, and any lack of
monitoring and control of the latter may be markedly
detrimental [21].
15. Agitation prevents materials in solution from settling.
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Chapter 15
Paraprobiotics Preparation for Use in Food and Beverages

Cássia Pereira Barros, Cynthia Manassi, Silvani Verruck,
Marcia Cristina Silva, Erick A. Esmerino, Monica Q. Freitas,
Abstract
In recent years, the high demand for functional products has driven the food industry to produce healthier
foods. In this context, the incorporation of probiotics has been an excellent strategy to improve the
nutritional value of food products. The current definition of probiotics indicates that microorganisms
need to be alive to provide health benefits to the consumer. However, scientific evidence has indicated that
bacterial viability is not necessary for health promotion. Paraprobiotics are inactivated microbial cells or cell
fractions that, when ingested, confer health benefits. Additionally, they are safer, more economical, and
stable therapeutic options for industrial processes and commercialization. Therefore, this chapter provides
detailed protocols for obtaining potential paraprobiotics for use in food and beverages.
Key words Functional foods, Nonviable microorganisms, Inactivated probiotics, Postbiotics, Inacti-
vation methods
1 Introduction
Probiotics play an important role in promoting health and well-

being through the prophylaxis and therapeutic intervention of
various disorders or diseases. Although the primary function of
probiotic bacteria is the restoration of healthy intestinal microbiota
and, consequently, improvement of gastrointestinal disorders [1],
beneficial systemic (extra-intestinal) effects via mediators of the
immune, endocrine, and nervous systems have been increasingly
explored [2–4].
According to the International Scientific Association of Pro-
biotics and Prebiotics (ISAPP), probiotics are “live microorganisms
that when administered in adequate amounts, confer a health
benefit on the host” [5]. Therefore, according to this definition,
bacterial viability is essential for the beneficial effects associated
with its consumption to be observed. However, certain aspects
213
214 Cássia Pereira Barros et al.
inherent to the use of live bacteria must be considered, such as

restricted application in certain food matrices considered stressful
substrates; high costs with management and control of tempera-
ture, oxygen, and humidity to maintain probiotic viability during
processing, storage, and commercialization [6, 7]; and greater risks
of causing adverse effects in individuals with weakened immune
systems (elderly, transplanted, newly premature babies, etc.) [7–9].
On the other hand, in recent decades, several scientific studies
have shown that dead or inactivated probiotics are also capable of
promoting biological activity in the host through modulation of
the innate and adaptive immune system, in addition to exerting
anti-inflammatory, anti-proliferative, antioxidant, and antagonistic
effects against pathogens [10, 11]. The term “paraprobiotics” was
created to name inactivated microbial cells or cell fractions that,
when ingested, confer health benefits to the consumer [12]. How-
ever, in 2021, ISAPP proposed to update the concept of postbiotics
to “preparation of inanimate microorganisms and/or their compo-
nents that confer a benefit to the health of the host” [13]. Thus, the
concept initially used to refer to the metabolites produced by
microorganisms would also include nonviable microbial cells.
However, paraprobiotics continue to be widely used by the scien-
tific community.
The addition of paraprobiotics, as bioactive ingredients, repre-
sents a potential opportunity for food and pharmaceutical indus-
tries to diversify the niche of functional products, nutraceuticals,
and alternative medicines. When comparing the use of inactivated
probiotic bacteria to their viable counterparts, the following advan-
tages can be highlighted: they have no or minimal interaction with
the components of the food matrix, which makes it possible to
include them in any food without altering the sensory character-
istics and, consequently, extends the commercial validity of the
product; there are no restrictions on their consumption as a food
supplement and they are stable at room temperature, providing
convenience for large-scale production and significant savings for
manufacturers as they do not require a cold chain during storage
and distribution [7, 10].
In recent years, many formulations supplemented with para-
probiotics in dairy matrices (fermented milks and dairy drinks) and
non-dairy matrices (isotonic drinks and pasta) have been success-
fully developed, resulting in several benefits (improvement of intes-
tinal function and microbiota, reduction of chronic stress and
improvement in sleep quality, anxiety relief, postprandial glycemic
control, reduction of glucose and total cholesterol levels, among
others) [14–18]. Therefore, the objective of this chapter is to
provide, in detail, protocols for the preparation of potential para-
probiotics with the aim of conferring functionality to foods and
beverages.
Paraprobiotics Preparation for Use in Food and Beverages 215
Fig. 1 Examples of inactivation processes applied to generate paraprobiotics
1.1 How to Produce The production of paraprobiotics consists of the inactivation of

Paraprobiotics? probiotic microorganisms either by traditional or emerging meth-
odologies, as examples (Fig. 1): application of heat (pasteurization,
sterilization, or tindalization), pH changes (low and high), gamma
irradiation, ultrasound, supercritical CO2 [19], lyophilization [20],
ultraviolet radiation [21], high pressure [22], ohmic heating [23],
and chemical agents. In general, such processes can damage the cell
membrane, break genomic DNA, coagulate cytoplasmic proteins
and/or compromise different physiological functions (inactivation
of key enzymes or deactivation of membrane selectivity) [19],
which renders probiotic bacteria unable to grow in culture media.
It is important to emphasize that each inactivation process has
its own mechanisms of action and, therefore, will affect the bacterial
structural and physiological components in different ways and,
thus, influence the paraprobiotic immunomodulatory activity
[10, 12]. In this sense, it is essential that the methods and opera-
tional conditions chosen, in addition to inactivating, are able to
preserve the metabolic activity and the integrity of the cell mem-
brane, in order to maintain the functionality initially provided by
live probiotics [12, 18, 19].
2 Materials
1. Probiotic strain(s) can be acquired in lyophilized form for

direct addition (Direct Vat Set – DVS) or can be isolated
from fermented products and functional food (as starter cul-
tures for sausages, dairy products, etc.), as well as from the
intestine of humans and healthy animals. We particularly prefer
to use Direct Vat Set (DVS) cultures for practicality.
2. Laboratory tools – Petri dishes (90 mm diameter), spatula,

glass pipet, micro-pipet, laboratory glassware, sterile tubes,
sterile plastic bag and sterile loop;
3. Laboratory equipment – analytical balance, microwave or
stove, autoclave, bacteriological incubator, centrifuge, spectro-
photometer, vortex mixer, thermostatic bath, and sample
homogenizer;
4. Distilled water;
5. Culture media – MRS (De Man Rogosa and Sharpe) broth and
MRS agar. Culture media should be prepared according to the
manufacturer’s instructions provided on the label (see Note 1).
After accurately weighing the dehydrated medium, pour all
ingredients in an Erlenmeyer flask or any volumetric flask and
dilute to 1 L with distilled water. Then mix until complete
dissolution using a glass rod and heat (see Notes 2 and 3).
Afterwards, verify that the pH of the media is adequate and
proceed to sterilization in an autoclave at 121 °C for 15 min.
After removal from the autoclave, identify them correctly with
name and date.
6. Phosphate-buffered saline (PBS, pH 7), (see Note 4);
7. Chemical reagent – 30% hydrogen peroxide (H2O2);
3 Methods
3.1 Probiotics For the isolation of probiotic strains from fermented foods or dairy
Strains and Cell products, the following procedures must be performed:
Suspensions 1. Weigh 25 g of the sample and transfer it quantitatively to a
Preparation sterile plastic bag, add 250 mL of phosphate-buffered saline
(PBS, pH 7) and place in the sample homogenizer for 1 min at
300 rpm (see Note 5).
2. Make tenfold dilutions by adding the 1 mL aliquot of the
sample to 9 mL of phosphate-buffered saline (PBS, pH 7),
homogenize each dilution using a vortex mixer. Transfer
1 mL aliquot of each selected dilution to Petri dishes and add
12–15 mL of MRS agar (pour plate technique), as illustrated in
Fig. 2 (see Note 6). Subsequently, incubate the plates under
anaerobic conditions at 37 °C for 24–48 h (see Note 7).
3. Select colonies with a circular shape, creamy texture, and white
color to be cultivated in MRS broth for 24 h under anaerobic
conditions at 37 °C (Fig. 3). After the incubation period, seed
again on plates containing MRS agar until complete isolation is
achieved.
Fig. 2 Plate counting method by the pour plate technique. (Source: The Figure was partly generated using
Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license
(https://creativecommons.org/licenses/by/3.0/) and using Chemix (https://chemix.org))
Fig. 3 Example of color, texture, and shape of colonies that must be selected for
new cultivation until complete purification is achieved
4. The isolated colonies must be analyzed in relation to their

morphological characteristics by the Gram stain method.
Gram-positive colonies should proceed for the Catalase test.
Catalase test.
Procedure – using a sterile loop, add colony to a glass slide and
add 2 drops of 30% (v/v) hydrogen peroxide (H2O2).
Data interpretation: the formation of gas bubbles indicates the
production of catalase enzyme by the test bacterium, while
no bubble formation is negative for catalase.
5. Purified Gram positive and catalase negative strains are consid-
ered presumptive lactic acid bacteria. They can be kept as
frozen stocks in 20% (v/v) glycerol at -20 °C until time of use.
6. Provide genotypic identification of the isolated strain for
deposit.
On the other hand, if you have obtained Direct Vat Set (DVS)
pouches of commercial lyophilized probiotic culture, simply acti-
vate them as follows:
1. Activate probiotic cultures in MRS broth for 24–48 h at 37 °C
under anaerobic conditions.
2. Harvest cells by centrifugation at 2700 x g, 4 °C for 10 min,
wash twice with phosphate-buffered saline (PBS, pH 7) and
resuspend in the same buffer solution [19].
3. Adjust the cell concentration of bacterial suspensions with
phosphate-buffered saline (PBS, pH 7) to achieve an optical
density at 600 nm (OD 600 nm) of 0.7 to 0.8 (corresponding
to a cell concentration of around 7–8 log CFU/ml) (see Note
8).
3.2 Inactivation As previously mentioned, there are several inactivation processes

Treatment for that can be used to obtain potential paraprobiotics (item 2). The
Paraprobiotic application of heat has been the most used methodology due to its
Production practicality and low cost. The mechanisms of thermal inactivation
may involve damage to cell membrane, loss of nutrients and ions,
ribosome aggregation, rupture of DNA filaments, essential enzyme
inactivation, and protein coagulation. In general, heat treatments
of bacterial suspensions use a temperature range between 70 and
100 °C (see Note 9). In addition, inactivation can also be achieved
through the interspersed use of elevated temperatures followed by
incubation periods with milder temperatures that can be ambient,
cooling, or freezing [24]. Preliminary tests are recommended to
determine the operational conditions (time x temperature bino-
mial) that will result in the inactivation of probiotic cell suspensions
(see Note 10).
Procedures:
1. Adjust the thermostatic bath according to the desired temper-
ature for the treatment.
2. After reaching the target temperature, immerse the tubes con-
taining the cell suspensions (see Note 11).
3. At the end of the treatment, the bacterial suspensions must be
immediately cooled in an ice bath at 4 °C in order to stop
heating, and then evaluate them regarding the effectiveness of
the inactivation treatments under the conditions considered by
the traditional method of plate count.
3.3 Assessment of All bacterial suspensions, treated and untreated (control), should
Probiotic Cell Viability be analyzed for the presence or absence of CFU growth. For the
After Inactivation production of potential paraprobiotics, the absence of CFU in
Treatment plates with pure cell suspensions is necessary, thus indicating that
the treatment and the respective inactivation condition were, in
fact, effective for obtaining paraprobiotics (see Note 12). Other-
wise, the presence of CFU indicates that the inactivation of the
probiotic cells was not achieved in the operational parameters
tested.
Procedures:
1. Make serial dilutions of treated and untreated cell suspensions
in sterile tubes containing phosphate-buffered saline (PBS,
pH 7) in a 1:10 ratio.
2. Inoculate the dilutions in MRS agar using the pour plate
technique, as described in item 2 of topic 3.1.
3. Incubate the plates under anaerobic conditions for a period of
72 h, considering the possibility of growth retardation.
4. After the incubation period, the plates must be analyzed in
order to verify the growth or not of CFU in the tested inacti-
vation conditions.
5. If there is growth of colonies (live probiotics), they must be
analyzed for their morphological characteristics by the GRAM
staining method and catalase test.
4 Notes
1. In order to avoid waste, it is recommended to prepare just

enough medium for each test. After cooling the MRS agar,
distribute approximately 15 mL of the sterile medium in a Petri
dish. Then just store the Petri dishes below 5 °C until
further use.
2. Be extremely careful when heating to prevent the culture
medium to be overheated or burnt.
3. During the preparation of MRS agar, use a glass rod to place a

drop of the medium on a cold surface. If after a few seconds it
solidifies, it indicates that it is ready to be sterilized in the
autoclave.
4. Phosphate-buffered saline (PBS, pH 7) can be replaced with
other saline solutions such as (0.85% NaCl, pH 7) sterile saline
or even 0.1% (w/v, pH 7) peptone water.
5. All bacteriological procedures need to be performed using a
Bunsen burner in a clean and closed working area or inside a
sterile laminar flow hood or similar.
6. Before adding MRS agar, make sure it is not too hot. After
pouring on the plate, homogenize slowly so as not to spread it
on the wall or cover of the plate. Wait for the medium to
solidify completely before taking it to a bacteriological
incubator.
7. An incubation period of 24 h is required for the growth of
microorganisms of the Lactobacillaceae family and 48 h for
microorganisms belonging to the genus Bifidobacterium
spp. [23].
8. If the laboratory does not have a spectrophotometer available,
the adjustment of cell density of probiotic suspensions can be
performed using the McFarland nephelometric scale.
9. Typically, paraprobiotic microorganisms cultivated on an
industrial scale through heat treatments have been able to
retain their main functionalities from viable microbes (probio-
tics) at the intestinal level [24].
10. Although so far few studies have evaluated the impact of differ-
ent inactivation treatments on bacterial structure and compo-
nents [19, 22, 23, 25], there are advanced techniques that can
be complementary to the plate counting method and help in
the choice of methods/more suitable inactivation conditions
in order to maintain the probiotic properties. Flow cytometry
consists of an automated instrument that, associated with fluo-
rescent dyes, allows the simultaneous evaluation of several
cellular parameters, such as alterations in structural and meta-
bolic properties. In this way, it is possible to assess the extent of
cell damage induced by inactivation processes, generating
accurate results in real time [26].
11. During heat treatment in the thermostatic bath, it is recom-
mended to use a thermocouple and multimeter to adjust and
control the temperature more precisely.
12. It is important to highlight that the probiotic inactivation
process produces potential paraprobiotics. However, in vitro,
preclinical or clinical tests are extremely necessary to confirm
whether, in fact, the permanence of the functionality initially
provided by probiotic microorganisms.
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Chapter 16
Postbiotics Preparation for Use in Food and Beverages

Jonas de Toledo Guimarães, Cássia Barros, Houshmand Sharafi,
Mehran Moradi, Erick A. Esmerino, and Adriano Gomes da Cruz
Abstract
Postbiotics are considered novel bioactive compounds, since there is not yet a consensus about its defini-
tion; however, many postbiotic compounds are already used in the food industry to provide food preserva-
tion, in the manufacture of bioactive food packaging, control bacterial biofilm, or provide functional effects
to food products. The postbiotics can be applied to food as a mixture (several compounds, often called cell-
free supernatant) or a separated form; however, there are issues about the production of these postbiotics
and the practical application to different food products. This chapter aims to present the most used
methodologies and the details about its production and application.
Key words Bioactive compounds, Postbiotic metabolites, Probiotics, Cell-free supernatant, Food
preservation
1 Introduction
Probiotics and prebiotics are well-known and defined functional

compounds, but other components derived from microorganisms,
which also provide health benefits but refer only to inanimate
microorganisms and/or their components such as paraprobiotics,
ghostbiotics, heat-inactivated probiotics, and postbiotics, still cause
divergences in the scientific community [1]. The most recent defi-
nition of postbiotics from the International Scientific Association
for Probiotics and Prebiotics (ISAPP), states that postbiotics is “a
preparation of inanimate microorganisms and/or their compo-
nents that confers a health benefit on the host,” therefore, a post-
biotic to be considered effective must contain inactivated microbial
cells or cell components, with or without metabolites, and promote
health effects [2]. However, the above definition also includes
inactivated microorganisms that is commonly known as parapro-
biotics, therefore, other authors still consider the original definition
of postbiotics presented by Tsilingiri, Rescigno [3] as “any factor
223
224 Jonas de Toledo Guimarães et al.
resulting from the metabolic activity of a probiotic or any released

molecule capable of conferring beneficial effects to the host in a
direct or indirect way”.
In this context, due to the lack of a consensus regarding post-
biotics, many different bioactive compounds can be considered
postbiotics, since they did not fit the traditional definitions of
probiotics, prebiotics, and paraprobiotics. According to Moradi
et al. [4], the term postbiotics can be used to characterize the
“bioactive soluble factors (products or metabolic byproducts) pro-
duced by food-grade microorganisms during the growth and fer-
mentation in complex microbiological culture (in this case named
cell-free supernatant [CFS]), food, or gut, which exert some ben-
efits to the food or host.” Furthermore, according to [5], “a post-
biotic must be derived from a well-defined microorganism or
combination of microorganisms for which genomic sequences are
known and prepared using a delineated technological process of
biomass production and inactivation, which can be reliably repro-
duced.” Currently, several postbiotic molecules are being identified
by researchers as a result of extracellular metabolites production or
release of intracellular components from microbial cells. These
components include secreted proteins/peptides, bacteriocins such
as acidophilin, reuterin, and bifidin, cell-free supernatant, organic
acids (e.g. lactic acid and acetic acid), vitamins, short-chain fatty
acids (e.g. butyric acid and propionate), and neurotransmitters,
biosurfactants. [6].
Probiotics are recognized for their diverse effects on consumer
health as long as the supplementation is long enough to colonize
gut microbiota and then produce their beneficial compounds.
Therefore, the bioactive compounds produced by probiotics are
the main factors responsible for their effects, and these compounds,
named postbiotics, can most of the time provide effects similar to
the living microorganisms. The advantage of using postbiotics in
food and beverages instead of probiotics is the absence or reduction
of changes in product’s characteristics, such as physicochemical and
sensory [4]. Despite the known effects of probiotics and their
metabolites, other non-probiotic microorganisms may also pro-
duce metabolites with significant effects on the host or food prod-
uct, being, therefore, considered a postbiotic.
In order to use postbiotics in food and beverages, they must be
properly prepared to maintain their activity, remove the living
microorganisms and reduce the undesired effects (e.g., sensory
changes) on food products. Postbiotics prepared from probiotic
or other known microorganisms, mostly lactic acid bacteria can
present different bioactive compounds such as organic acids,
short-chain fatty acids, carbohydrates, antimicrobial peptides,
enzymes, vitamins, cofactors, immune-signaling compounds, and
complex agents [4]. These postbiotics can be applied with different
Postbiotics Preparation 225
objectives, such as food preservation, in the manufacture of bioac-

tive food packaging, controlling bacterial biofilm, or providing
functional effects to food products [4, 6, 7].
The postbiotic can be applied to food in a mixture (several
compounds, often called cell-free supernatant) or in a separated
form (preparation derivative), which will depend on the objective of
the application or resources available for postbiotic preparation
since the preparation of a specific postbiotic is more expensive and
complex than the preparation of a mixture [4]. Therefore, the
objective of this chapter is to present the main methodologies
used for postbiotics preparation in order to use it in food and
beverages.
2 Materials
2.1 Consumables • Ammonium sulfate

• Ascorbic acid
• Acetic acid
• Catalase
• Dialysis tubing (1 kDa cut-off)
• Erlenmeyers (100, 250 and 1000 mL)
• Glass tubes (10 mL)
• Glass bottles (100 and 1000 mL)
• L-cysteine
• Lithium chloride
• Meta-phosphoric acid
• MRS (De Man, Rogosa and Sharpe) broth (for lactic acid
bacteria)
• NaCl solution 0.9% (v/v)
• NaOH
• Polyethylene bags
• Potassium cyanide solution
• Sterile distilled water and buffer solution (phosphate buffer
saline; PBS, 0.01 M phosphate, pH 7.2)
• Syringe or Buchner filter (0.22 and 0.45-μm pore size)
• Target microorganism (s) in lyophilized form
2.2 Equipment • Automated pipette gun and Micropipette (100–1000 μL and

1000–10,000 μL)
• Autoclave
• Bunsen burner
• Freeze dryer
• Lab vortex mixer
• Laboratory analytical balance
• Laminar flow cabinet
• Magnetic stirrer
• pH meter
• Rotary evaporator
• Refrigerated centrifuge
• Refrigerator (4, -20 and -70 °C)
• Spray dryer
• Standard incubator and CO2 incubator
• UV spectrometer
• Vacuum filtration glassware system
• Vacuum pump
• Water bath
3 Methods
3.1 Cell-Free This form of postbiotic preparation may contain products from
Supernatant (CFS) microbial metabolisms such as metabolites synthesized by the
microorganism on culture/food ingredients or structural sub-
stances produced by them. It is important to highlight that the
microbial products are mainly related to the type of bacterial strain
and culturing medium, which contain only soluble factors such as
products or metabolic by-products that have been secreted into the
medium during bacterial growth [7].
After microbial propagation to produce CFS, the bacterial cells
may be inactivated/ disrupted (e.g., enzymatic treatment, thermal
treatment, sonication, high pressure, Ultraviolet rays, ohmic tech-
nology) in order to release new substances into the postbiotic
mixture such as intracellular metabolites and cell wall-derived mate-
rials. Treatment parameters in every process may vary according to
the type of microorganism and the target postbiotics. Non-thermal
processes were less reported and heat treatment is still the method
of choice in most cases [8]. Then, the postbiotic mixture, indepen-
dently of the post-propagation treatment, is centrifuged and/or
filtered to separate bacterial cells from postbiotic metabolites [9].
Most of the postbiotic CFS preparations are carried out accord-
ing to Fig. 1, however, some modifications may be applied to the
fermentation medium and the microbial strain to obtain different
postbiotics.
Fig. 1 General flowchart of a postbiotic cell-free supernatant preparation
3.1.1 Fermentation Most postbiotic preparations are made with lactic acid bacteria
Medium (LAB); therefore, the MRS broth is commonly used [7]; however,
other media can be used, including some based on dairy ingredients
such as modified cheese whey and milk permeate [10]. Despite the
greater importance of LAB on postbiotics production, other micro-
organisms may also be used such as fungal species [11], but the
fermentation medium will change for fungal species, since their
metabolism are different than LAB.
In case of preservation of the microbial strains, they can be kept
frozen (-80 °C). However, before freezing, the concentrated/
centrifuged probiotic cells must be added of 20% glycerol [12] to
maintain cell integrity during freezing and thawing [13]. Another
microbial preservation method is lyophilization, in which the prod-
uct can be kept in refrigeration or even at room temperature after
the process. In addition, many microorganisms and protective
cultures of certain reputable companies are commercially available
in frozen and freeze-dried concentrated cultures (known as direct
vat set (DVS) or direct-to-vat inoculation (DVI) cultures), and can
simply be used according to provider’s instruction.
The culture characteristics, medium composition, incubation
conditions, and cultivation time can affect metabolites production
by LAB. It was reported that postbiotics from different LAB strains
prepared with low-heat milk revealed significantly higher antifungal
activity than those prepared with milk permeate [10]. The cultiva-
tion of probiotics in MRS media supplemented with glucose, yeast
extract, surfactants, or emulsifiers could increase the production of
postbiotic products such as bacteriocin-inhibitory activity, protein-
aceous and therapeutic substance with antimicrobial activity [7].
Evidences tend to support the hypothesis that a mixture of

multi-strains is more effective than a mono-strain to produce post-
biotics. Furthermore, coculturing can facilitate the expression of
ribosomal synthesized antimicrobial agents (AAs) like bacteriocins
in LAB, also leading to the discovery of novel AAs [7], but in some
cases, a combination of two or three strains does not work [14],
requiring a compatibility test before the experiment. It has also
been shown in laboratory that postbiotics can be produced by
fermentation with proteolytic starter cultures, and if the pH is
maintained close to neutrality during fermentation, it may improve
the release of postbiotic compounds [15].
Procedure
For probiotic bacteria strain cultivation to produce postbiotic com-
pounds some important steps must be carefully performed for safe
production, as briefly presented in Fig. 2. All material and equip-
ment used during the procedure must be sterile or autoclaved
(121 °C for 15 min). The work must be done in aseptic conditions,
preferably using a laminar flow cabinet.
1. Culture media preparation: Currently, the most used probiotic
strains are from Lactobacilli and Bifidobacteria families, being
the MRS media the best option to grow them [9, 10, 16–18]
(see Note 1).
2. Pre-activation: If a microbial strain is lyophilized or kept fro-
zen, a pre-activation is required (see Note 2), incubating the
microorganisms usually in glass tubes containing MRS broth
(10 mL) at 32–37 °C for a specific period of time (mostly 48 h)
under aerobic or anaerobic conditions depending on the type
of strain. Some strains require special nutrients and inhibitors
(see Note 3).
3. Inoculum preparation: The microbial suspension can be cen-
trifuged at 5000 g for 15 min and the obtained pellet is washed
using 0.9% (v/v) NaCl solution or PBS three times. Finally, the
obtained bacterial cells are suspended in PBS to standardize the
microbial concentration before inoculation and propagation
(see Note 4) [19].
3.1.2 Microbial The objective of propagation is to increase the biomass and conse-
Propagation quently the postbiotics yield.
Procedure
1. First Inoculation: The activated cultures are placed in a 100 mL
bottle containing 90 mL MRS broth and, if necessary, other
required nutrients (see Note 5).
2. Incubation: The bottle is incubated for a specific period of time
(see Note 6) at 32–37 °C.
Fig. 2 Schematic procedures of postbiotics (cell-free supernatant) preparation. (Adapted from Moradi et al. [7])
3. Second Inoculation: the grown culture is placed in a 1 L MRS

broth bottle.
4. Incubation: The bottle is incubated for a specific period of time
(see Note 6) at 32–37 ° C.
These steps (1–4) are continuously repeated until enough
probiotic biomass is acquired (see Note 7).
5. Post-propagation treatments: In order to increase the postbio-
tics yield or to extract specific postbiotics, the microorganisms
can be inactivated by the application of post-propagation
conventional treatments (e.g., enzymatic and thermal) or treat-

ments based on emerging technologies (e.g. ultrasound and
high-pressure treatments, pulse electric field, among others),
then, extracting more metabolites from bacterial cells. There-
fore, these treatments can add new intracellular metabolites
providing new functionality to the obtained postbiotic
[7]. Additional steps may be necessary to eliminate intact cells
and recover only the postbiotic fraction such as microfiltration
(see Note 8). Meanwhile, for postbiotics secreted by viable
cells, the methods or technology necessary for their recovery
from supernatants consist basically of eliminating the viable
cells from the medium by centrifugation and/or filtration [9],
as described in the Subheading 3.1.3.
3.1.3 Harvesting The harvesting phase consists in obtaining the desired postbiotics,
and separating them from the living microorganisms. This tech-
nique is usually the same used for the separation and obtainment of
probiotic biomass, but the product will change and additional steps
must be carried out to ensure the presence of only postbiotics.
There are different ways to extract postbiotics. The most used is
the separation of these bioactive extracellular metabolites of the
producing cells by centrifugation and filtration techniques
[20]. The cell-free supernatants containing the active metabolites
can be obtained after the incubation period.
Procedure
1. Centrifugation: To extract the cells from the media a cleaning
procedure is required. First, the media should be centrifuged at
7000 g for 20 min at 4 °C.
2. Supernatant separation: At this moment heavier microorgan-
isms are sent to the bottom of the flasks, then, the supernatant
can be separated from the living microorganisms using an
electric pipette gun and serological pipette tip (see Note 9).
(a) If the purpose is to obtain the microbial cells, the precipi-
tate is washed with 0.9% (v/v) NaCl solution or PBS three
times, repeating the procedure, until no MRS broth
remains.
(b) In the case of obtaining postbiotics, the living microor-
ganisms (the bottom part) will be discarded and the super-
natant, rich in microbial metabolites, obtained for further
procedures.
3. Filtration: Finally, the resulting supernatant is filtered using a
syringe filter (0.22 to 0.45-μm pore size) to ensure sterility and
the absence of microbial cells [21, 22]
3.1.4 Concentration and After centrifugation and filtration, the obtained liquid, rich in
Preservation Methods postbiotics, can be preserved under refrigerated conditions until
its application on the food; however, for a longer conservation
period and to improve usability in the application, the postbiotic
mixture (cell-free supernatant) can be dried by spray drying or
freeze drying methods [7, 23]. It must be taken into account that
some methods may negatively affect postbiotics functionality (see
Note 10).
Freeze Drying
This is the common method of concentration and preservation for
postbiotics used in the literature. Freeze-drying consists of three
steps, as described below.
Procedure
1. Freezing: ice crystals are formed in postbiotics solution at a
temperature – 40 °C under atmospheric pressure within a few
hours.
2. Primary drying: the pressure is reduced below the equilibrium
vapor pressure of ice (pump pressure: 100 mTorr and shelf
temperature: – 60 °C).
3. Secondary drying: unfrozen water was diffused and desorpted
in the postbiotics [23, 24].
4. Storage: The obtained dried postbiotics should be kept at 4 °C
in polyethylene bags for further use (see Note 11) and analysis.
Spray Drying Procedure

Spray drying has been proposed as a low-cost alternative to freeze-
drying for the preparation of postbiotics powder.
1. Evaporation: water is partially evaporated from postbiotics
solution by a rotary evaporator (see Note 12).
2. Spray drying: The postbiotic solution is heated to 37 °C and
spray dried under these conditions: inlet air temperature, 119 °
C, feeding by a two-fluid nozzle, outlet air temperature 64 °C,
and flux of feeding, 6 mL/min (see Note 13).
3. Storage: The obtained dried postbiotics should be kept at 4 °C
until use in polyethylene bags for further use and analysis.
3.1.5 Method of After postbiotics preparation, they can be applied to several foods
Application (Fig. 3); however, depending on the food characteristics and how
the postbiotic was extracted and preserved, the method of applica-
tion may also change. The main concerns about postbiotics appli-
cation to food are the physical-chemical changes that may occur
after this addition since some postbiotic mixtures present high
moisture, a brown or yellow color, or different sensory attributes
Fig. 3 Methods of postbiotics application in different kinds of food
[7]. To overcome the color changes, alternative media such as a

dairy- (buttermilk, whey, and permeate solution) or plant-based
solution may help postbiotics to have a more creamy or white
appearance; however, reducing the contact time between postbio-
tics and food may solve the color issue, in some cases. In most cases,
postbiotics solution contains proteolytic and lipolytic enzymes,
boosting gradually food proteolysis and lipolysis and increasing
some food quality index such as thiobarbituric acid (TBA) and
total volatile basic nitrogen (TVB-N) levels after postbiotics addi-
tion. In this case, enzymatic inactivation of postbiotics is recom-
mended to overcome this drawback. Generally, it is clear that a
greater concentration of postbiotics is needed to achieve the same
effect in food than in vitro conditions. The ratio varies from 10 to
100 times higher than in the laboratory condition. It varies accord-
ing to the type of postbiotics, type of food, and method of
application.
Depending on the food type, postbiotics can be used by direct
addition to food formulation, coating, and spray methods. For
example, in fish and meat ground, cheese, bakery, and liquid
food, postbiotics in dry form can be applied in free or encapsulated
forms in food formulation, while in fish and meat fillets, poultry,
fruits, and vegetables spray and coating of postbiotics solutions may
be practical. Moreover, the postbiotics solution may be used as a
preserving solution in certain food such as mozzarella cheese. For

coating and preservation applications, it is recommended to select a
microbial strain with high exopolysaccharides producing feature to
produce viscous postbiotics solutions or add postbiotics to other
viscous polymer solutions (e.g., chitosan, alginate, etc.), which can
improve the rheology and texture of the receiving food. In special
cases, postbiotics can be included in food packaging material in
order to prepare functional packaging materials to extend shelf life
and inhibit surface pathogens.
It is worth mentioning that the chemical composition of each
postbiotics solution or powder should be clarified before food
application. Since there are many compounds with different nat-
ures, there is not a fully clarified method for analyzing the compo-
sition of postbiotics. Several chromatographic (e.g., GC, HPLC)
methods have been used for the identification of postbiotics con-
stituents. For quantification of phenolics, flavonoids, organic acids,
etc., HPLC is the method of choice, while for volatiles and fatty
acids, gas chromatography-mass spectrometry is preferred. Addi-
tionally, for a complete evaluation, the choice of a proper initial
preparation procedure and the use of more than one analytical
instrument/procedure is recommended [7, 25].
3.2 Postbiotic Vitamins are organic molecules that are supplemented in the diet in
Compounds a small amount to facilitate various biological processes in the body.
Identification Most B-complex group vitamins are directly involved as coenzymes
in several energy metabolism reactions. Humans are incapable of
3.2.1 Vitamins biosynthesizing most of the vitamins, and therefore they subse-
quently have to be supplemented exogenously. Most of the vita-
mins have to be supplemented through the diet (vitamin A, D, E,
etc.); however, limited vitamins (folic acid-B9, cobalamin-B12,
Ribofavin-B2) are even synthesized by commensal gut bacteria
and some probiotic bacteria [26]. LAB is capable of producing
other vitamins in a few quantities. Therefore, postbiotics produc-
tion can also be directed to the production of important vitamins.
Separation Procedure
Separation begins with selecting a proper extraction method, which
varies and depends on the vitamin nature. Similar to food, enzyme
extraction from CFS can be done by acid/alkaline hydrolysis, sol-
vent extraction, and solid phase extraction. Folate (vitamin B9) and
riboflavin (B2) have similar separation procedures with minor
differences.
1. Adding extraction buffer:
(a) Folate: extraction buffer [0.1 M phosphate buffer
pH 6.8 + 0.5–1.5% (w/v) ascorbic acid (see Note 14), to
avoid vitamin oxidation and degradation] is added to the
CFS solution.
(b) Vitamin B12: 0.57 M phosphate buffer pH 4.5 + 0.05%

potassium cyanide solution is added to the CFS solution.
2. First Centrifugation: the solution is centrifuged at 5000 g for
10 min.
3. Heating (see Note 15):
(a) Folate: boiling for 5–15 min.
(b) Vitamin B12: heated at 100 °C for 30 min.
4. Second Centrifugation: the heated solution is centrifuged at
1000 g for 10 min.
5. Filtration: the obtained supernatant is filtered with syringe
filter (0.45-μm).
6. Storage: kept at -70 °C for further analysis.
7. Identification and quantification:
(a) Folate: ELISA, enzymatic and microbiological assays (see
Note 16), (ultra) high-performance liquid chromatogra-
phy (U) HPLC, or LC-MS [27–29].
(b) Vitamin B12:: microbiological assay (using L. delbrueckii
subsp. lactis ATCC 7830 as an indicator organism) or
available chromatographic methods [30]. Solid phase
extraction using C18 cartilages may be also used for the
separation of vitamin B12 [31].
3.2.2 Short-Chain Fatty Short-chain fatty acids (SCFAs), comprising 1 to 6 carbon-based

Acids anions can be produced during bacterial fermentation, of which
acetate (C2), propionate (C3), and butyrate (C4) are the most
abundant [32]. SCFAs production tends to participate in preserv-
ing the gut barrier function, contributing to the metabolism of
carbohydrates and lipids, and act in distinct processes in other
tissues, such as adipose tissue remodeling and the immune system.
Due to their involvement in energy and lipids metabolism, these
molecules can contribute to the reduction of inflammatory risk
diseases, obesity, diabetes, and other metabolic failures [33]. Fur-
thermore, they may be responsible for several of the beneficial
effects related to the commensal or probiotic bacteria. Propionate,
acetate, and butyrate are among the most known SCFAs which are
produced by specific microorganisms. The first one is produced
commonly by Lactobacillus spp. While the others are synthesized
by Bifidobacterium spp. [34].
Acetate is the most abundant SCFA produced by most enteric
bacteria as a fermentation product. Pathways for acetate synthesis
are widely distributed among bacterial groups, while pathways for
propionate and butyrate appear to be highly conserved and are
substrate-specific [35]. Therefore, acetate can be formed by hydro-
genotrophic acetogenic bacteria like Acetobacterium woodii, from
formate through the Wood-Ljungdahl pathway. Acetate is used in
part by the bacteria that use them to synthesize some of their
structural components [15].
SCFAs are a potential class of postbiotics, the supply of active

ingredients at the desired location of the intestine is facilitated; for
this reason, diseases located in the intestine have been mostly
studied [32]. It is worth mentioning that for SCFAs production,
an optimum growth condition (e.g., pH, time, temperature, salt,
and SCFA precursors such as glycine, glutamate, threonine, and
aspartate) should be used for bacterial culturing depending on the
type of strain [34, 36].
1. First Centrifugation: the postbiotic solution is centrifuged at
3000 g for 40 min at room temperature (25 °C).
2. Supernatant separation: the supernatant can be separated using
a micropipette and transferred into an appropriate vial.
3. Fatty acids extraction: The obtained CFS (0.75 mL) is mixed
with meta-phosphoric acid (0.3 mL) and the vial is kept at
ambient temperature (25 °C) for 25 min to facilitate the fatty
acids separation.
4. Second centrifugation: The sample is then centrifuged at 5000 g
for 15 min
5. Supernatant separation: the supernatant containing fatty acids
is obtained using a micropipette and the vial stored at -20 °C
for further gas chromatography analysis.
3.2.3 Secreted Proteins/ Enzymes are active proteins that catalyze biochemical reactions
Peptides [37]. Microbial enzymes possess a variety of biochemical, physio-
logical, and regulatory functions. Industrially, enzymes are derived
from a small group of bacterial and fungal strains, mainly Bacillus
subtilis, Bacillus licheniformis, Aspergillus niger, and Aspergillus
oryzae [38]. The main bacteria that produce enzymes of interest
are those of the genus Bacillus. They are important for their high
growth rate that leads to short fermentation times, for their ability
to secrete proteins in the extracellular environment, and for being
recognized as safe. The Bacillus genus is probably the most impor-
tant bacterial source of proteases. It is capable of producing high
yields of neutral and alkaline proteolytic enzymes with remarkable
properties, such as high stability against extreme temperatures, pH,
organic solvents, detergents, and oxidizing compounds [39]. It has
been reported that the maximum production of proteases pro-
duced from Bacillus subtilis is achieved under the following physical
and nutritional aspects: 48 h of incubation time; continuous agita-
tion at 220 rpm, pH 7.5; temperature 45 °C; 2% skim milk; yeast
sludge 300 μL; 0.4% ammonium sulphate; urea 0.2%; and cane
molasses 0.03% [40].
Among postbiotics generated by microorganisms, there are

some peptides with antimicrobial activity, whose mechanism of
action is the formation of pores on a bacterial membrane or inhibit-
ing compounds of bacterial wall synthesis [41]. Those peptides can
be expressed constitutively or induced in response to infection, as
immunomodulatory agents that increase natural innate immunity
and as neutralizing agents for endotoxins [42]. The antimicrobial
peptides are associated with two interrelated characteristics of the
peptides that are their net charge and their propensity to be amphi-
pathic. Both characteristics facilitate their interaction with other
bacterial membrane components such as teichoic acids, involving
electrostatic interactions between positively charged antimicrobial
amino acids and Gram-negative bacterial membrane
lipopolysaccharide [15].
Bacteriocins are one of those peptides with a wide antimicrobial
activity spectrum, which can be produced by both Gram-positive
and Gram-negative bacteria, mainly LAB. Their benefits involve
stability, bioengineering, diversity, production, and safety. Nisin is
one of the first studied bacteriocins, being considered a class I of
antibiotics and reported for many in vitro effects against spoilage
and pathogenic bacteria [15]. Bacteriocins have properties to pre-
vent biofilm formation, due to their unique structure. Pediococcus
acidilactici HW01 releases a pediocin-like bacteriocin (HW01 bac-
teriocin), which is an efficient antagonist of Pseudomonas aerugi-
nosa due to its potential to hinder biofilm progression and
production of virulence factors. HW01 bacteriocin is able to atten-
uate P. aeruginosa KCTC 2004 biofilm formation independently of
cell death [4].
As a postbiotic, bacteriocins have three main mechanisms to
combat biofilm: (a) suppression of twitching motility; this ability of
biofilm is mediated by pili, whereas swimming and swarming are
the results of flagella activity, (b) disturbing quorum sensing (QS);
it alters cell interactions, colonization, and loss of QS signals,
(c) reduction of virulence factors (as pyocyanin, protease, and
rhamnolipid); pyocyanin contributes to biofilm formation and
exposing infection and rhamnolipid from P. aeruginosa is also
responsible for the conservation of biofilm channels [43]. Alterna-
tively, pre-coating surfaces with bacteriocin from Limosilactobacil-
lus fermentum alone is more effective to inhibit P. aeruginosa PAO1
biofilm than its simultaneous use with the living bacteria. In addi-
tion to cell death, L. fermentum bacteriocin causes microenviron-
ment alteration, disorder in cell communication and assembly,
decreased QS signals, and weakened cell membrane integrity by
pore formation [44].
Peptides, bacteriocins, and enzymes have similar separation proce-
dures. Generally, bacteriocins, bacteriocins-like substances (BLS),
and peptides are separated, concentrated, and purified by a combi-
nation of precipitation-centrifugation-dialysis and chro-
matographic and electrophoretic techniques for antimicrobial
tests (see Note 17).
1. Neutralization: to neutralize the effect of organic acids and
hydrogen peroxide, pH is adjusted to 6.2 with NaOH 1 N and
130 U/mL of catalase is added.
2. Precipitation: The process is started by adding ammonium
sulfate (see Note 18) to reach the desired saturation percentage
(70%) (see Note 19)
3. Mixing: the solution is mixed with a magnetic stirrer at 4 °C for
6–8 h.
4. Centrifugation: the mixture is centrifuged (10,000 g for
20 min at 4 °C) to obtain protein precipitant containing BLS.
5. Dialysis: A small volume of 10 mM PBS (or distilled water) at
pH 2.5 is added to the sample and dialyzed for 8 h at 4 °C
against PBS (pH 7) in dialysis tubing or dialysis cassettes (1 kDa
cut-off) (see Note 20) with two changes of buffer (the ratio of
the postbiotics to dialysis solution is 1: 500).
6. Identification and characterization: The obtained solution is
referred to as partially purified bacteriocin and can be used for
antimicrobial tests; however, further steps using chro-
matographic methods (Ion exchange Sephadex G-25 column
and reverse-phase HPLC), sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE), amino acid sequence
and matrix-assisted laser desorption ionization-time of flight
mass spectrometry (MALDI-TOF-MS) can be applied to
purify and characterize a specific bacteriocin from postbiotics
[22, 45–47].
4 Notes
1. Other media could be chosen depending on strain growth

performance, the desired postbiotics functionality, which can
be affected by the type of medium and culture condition. For
example, postbiotics prepared in tryptic soy broth reveal higher
antioxidant capacity than those prepared in MRS [14].
2. Follow one of these procedures for activation depending on the
nature of desired strain:
(a) Transfer 3–5 colonies of a bacterium from agar plate to the
culture broth.
(b) Transfer a frozen solution of the bacterium to a culture

broth (1 to 100 ratio).
(c) Add an appropriate amount of commercial lyophilized
strains according to the company’s instruction
3. In most cases, lithium chloride (0.1% w/v) is added to prevent
the growth of Gram-negative bacteria. To ensure the source of
nitrogen, L-cysteine (0.05% w/v is also added to the MRS
medium [48].
4. To adjust the turbidity of bacterial suspension, use one of these
methods:
(a) McFarland standards: Prepare or buy the desired McFar-
land Equivalence Turbidity Standards (0.5, 1.0, 2.0, 3.0,
4.0)
(b) Use commercial McFarland Densitometer
(c) Spectrophotometric method: Adjust the optical density of
bacterial suspension to a level where the exact bacterial
population in it is already determined. Then to confirm
and ensure the count, prepare different dilutions, culture,
and enumerate according to the common bacterial enu-
meration method. The bacterial suspension should not be
kept in PBS for more than 15 min at room temperature.
However, keeping the bacteria for more than 15 min
causes cell lysis and its inoculation should be avoided.
5. The medium composition should be balanced between precur-
sors and other additives (carbon and nitrogen source, cofac-
tors, or polysorbates), since some precursors may improve the
synthesis of postbiotics by indirect overstimulation of other
biosynthetic routes [34].
6. The incubation time will depend on the strain growth rate and
metabolism.
7. At an industrial level, reactors can be used to produce large
amounts of microbial biomass in less time. It is important to
mention that after each growth phase, a counting analysis
should be done to assess the microbial concentration and
then establish a production process for microbial biomass
obtainment.
8. In general, the loss of viability from living microorganisms
occurs after exposure to factors that alter microbial cell struc-
ture and/or change their physiological functions without dis-
rupting the bacterial membrane and cell wall necessary to
maintain the cell structure intact. The viability may also
decrease by disrupting the bacterial membrane with the appli-
cation of at least two treatments in order to obtain the intracel-
lular metabolites and/or cell wall components in the form of
fragments.
9. Transfer CFS as quickly as possible to a new container to avoid

resuspending bacterial cell in the CFS [22].
10. For example, during spray drying, certain volatile metabolites
are lost or lyophilization removes hydrogen peroxide from
postbiotics solution [7]. Moreover, the antioxidant activity of
lyophilized postbiotics of some LAB is sevenfold lower than
the initial postbiotics solution (Unpublished observations).
11. It is recommended to be used on the day of preparation.
12. As an alternative to increasing the total solids, maltodextrin
(20–40%) needs to be added to postbiotics solution [49].
13. Lactic acid in postbiotics is very hygroscopic and thermoplastic
acid makes the drying process very difficult [49].
14. For riboflavin separation, acetic acid (1% v/v) is commonly
added instead of buffer with ascorbic acid [29].
15. Vitamins must be protected from oxidation by performing the
extraction under dim light and cooling the samples in ice after
heating.
16. In this test, an indicator microorganism (L. casei subsp. rham-
nosus NCIMB 10463 or L. casei NCIM 2364) is used for
quantification of folate based on the AOAC official methods
for folate quantification using available folic acid test kit
according to the manufacturer’s instructions.
17. As an alternative, the organic solvent (ethanol or acetone)
extraction technique can be used for the separation of some
bacteriocins. In this method, postbiotics is exposed to three
volumes of cold acetone at –30 °C [50].
18. This is an effective chemical used commonly for peptides pre-
cipitation without affecting bioactivity [22].
19. Some bacteriocins may precipitate at lower concentrations of
ammonium sulphate, then it is important to determine what
level of salt precipitates the peptide of interest [50]
20. A dialysis bag should be carefully selected as most bacteriocins
have a size smaller than 10,000 Da. For separation of peptides
with a size below 10 KDs, CFS is purified using 10 kDa ultra-
filtration membranes [50].
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Chapter 17
Psychobiotic Carried by Food and Beverage

Cássia Pereira Barros, Erick A. Esmerino, Roberto Laureano Melo,
Abstract
Common mental disorders such as anxiety and depression have increased in prevalence in recent decades,
becoming a global health challenge. In this context, it is necessary to search for therapeutic approaches
aimed at preventing and treating these disorders. The term psychobiotics refers to beneficial bacteria
(probiotics) or support for these bacteria (prebiotics) that influence bacteria–brain relationships. In addi-
tion to improving bowel function, they may also exert anxiolytic and antidepressant effects through
immunological, humoral, neuronal, and metabolic pathways. Therefore, the use of psychobiotics can
help the maintenance and/or restoration of the population’s mental health. This chapter describes in detail
protocols for the elaboration of a food product with psychobiotic potential. In addition, the most used
behavioral tests for preclinical trials that can be applied to confirm the psychobiotic effect are also discussed.
Key words Microbiota, Probiotics, Prebiotics, Mental wellness, Adjunctive therapy, Fermented food
1 Introduction
Common mental disorders such as anxiety and depression have

increased substantially in recent decades. According to the World
Health Organization, depression affects more than 300 million
people, and it is estimated that by 2030 it will be the main cause
of disability in the world [1]. Depression is associated with persis-
tent depressed mood, inability to experience pleasure or interest,
fatigue, psychomotor retardation, poor concentration, and suicidal
thoughts. It is important to emphasize that depressed individuals
may simultaneously suffer from anxiety, a condition represented by
a state of discomfort, fear, and uncertainty about the future
[2, 3]. Therefore, such disorders, in addition to negatively affecting
the quality of life of patients, can also cause great economic losses
due to the drop in productivity at work and the high costs of
treatments for health and well-being.
243
Currently, there is a wide range of therapeutic interventions for

the treatment of mental disorders. However, these interventions
often only relieve symptoms, they take too long to take effect or are
ineffective, which can increase the suicide rate. In addition, most of
the medications used cause undesirable side effects such as mood
swings, sleep disturbances, dependence and addiction, and changes
in the health of other parts of the body [4–6]. In this context, there
is an urgent need for approaches that are faster, safer, and more
effective in restoring and maintaining psychological health.
Among the new therapeutic methodologies, the modification
of the intestinal microbiota has attracted much interest due to the
bidirectional interaction of the microbiome with the nervous sys-
tem via the gut–brain axis. Several studies have shown that con-
sumption of beneficial microorganisms can reduce symptoms of
depression, anxiety, and stress and improve cognition, suggesting
possibilities for a new class of probiotics with psychotropic activity
called “psychobiotics” [7, 8]. The emerging term “psychobiotic”
was created to define the group of probiotic bacteria that, when
ingested in adequate amounts, promote beneficial effects on mental
health [9]. Later, this definition was updated to include any exoge-
nous factor capable of exerting actions in the brain that are
mediated by bacteria [10]. Therefore, the concept of psychobiotics
came to include prebiotics as they contribute to the growth of
beneficial intestinal bacteria.
Psychobiotics can be consumed through a healthy diet, dietary
supplements, and functional fermented foods. Significant improve-
ments in physical and mental health parameters were reported in
clinical trials conducted with petrochemical workers and healthy
students under academic stress after consumption of probiotic
yogurt and fermented milk, respectively, when compared to the
placebo group [11, 12]. Therefore, these findings support that
nutritional therapies with psychobiotics can modulate the psycho-
logical symptoms associated with intestinal dysfunction.
In this context, animal models are important in evaluating the
safety, the efficacy and action mechanism of new drugs [13]. In
addition, these models help to understand the neurobiological
mechanisms inherent to mental health. Currently, preclinical trials
have been considered the backbone of new drug research, having
contributed decisively to the development of current treatments for
depression and anxiety disorders [14].
Given the above, the objective of this chapter is to provide
guidelines for the development of a food supplemented with pro-
biotic bacteria with psychobiotic potential. In addition, the behav-
ioral tests most used for preclinical trials that can be conducted to
confirm the psychobiotic effect of the food product are also
described.
Psychobiotic Carried by Food and Beverage 245
2 General Recommendations Proposed for Application of Psychobiotic Strains in

Foods and Beverages
It is advisable to start with the following criteria:

1. Selection of strain(s) – first, a literature search must be carried
out in order to investigate microorganisms that have already
been identified as probiotics and that, therefore, have been
previously proven in preclinical and clinical trials in humans
which, in addition to providing beneficial effects on the host,
are safe and capable of transiently surviving through the gas-
trointestinal tract, adhering to intestinal epithelial cells and
resisting intestinal acidity, bile, and high-count foods. In addi-
tion, it is possible to choose formulations with single microbial
strains or multi-strain formulations containing two or more
strains. Then, choose those that have already been studied for
the desired psychobiotic effect (Table 1), since not all probio-
tics share the same mechanisms of action and clinical benefits.
Otherwise, it is possible to perform a rapid screening to assess
whether the chosen strain has psychobiotic activity through
in vitro tests. In general, beneficial bacteria with psychobiotic
characteristics are represented by the genera Lactobacillus, Bifi-
dobacterium, and Lactococcus [15]. On the other hand, it
should be noted that recently several species of microorganisms
allocated to the genus Lactobacillus were reclassified after eval-
uating their phenotypic, genotypic, and ecological characteris-
tics in 24 new genera. Among them, Lacticaseibacillus,
Lactiplantibacillus, Limosilactobacillus and Levilactobacillus
have psychobiotic properties.
2. Determination of microorganism concentration, frequency,
and time of consumption – the cell density of the psychobiotic
microorganism normally ranges from 109 to 1011 CFU, with a
consumption frequency of once or twice a day for about 2 to
8 weeks [16, 17].
3. Selection of the food matrix – fermented foods have been used
as psychobiotic delivery vehicles. They are defined as foods and
beverages obtained through enzymatic conversions of food
components mediated by microorganisms. In this context,
they are considered an excellent source of probiotics due to
the large amount of lactic acid bacteria naturally present in the
raw material or intentionally added as starter cultures to pre-
serve and improve the sensory characteristics of the product
[18, 19]. In fact, the psychobiotic effect was first reported after
daily consumption of a dairy drink containing the probiotic
Lactobacillus casei Shirota caused a general improvement in
mood [20]. Subsequently, further research was carried out
with fermented products based on milk (yoghurt, kefir, milk),
Table 1
Examples of bacteria strains with psychobiotic potential
Psychobiotic Effects Gut Microbes Psychobiotic Strains

Anxiolytic activity Lactobacillus spp. L. acidophilus LA5
L. helveticus ROO52
L. helveticus NS8
Lactiplantibacillus spp. L. plantarum DR7
Lacticaseibacillus spp. L. casei Shirota
L. rhamnosus JB-1
Limosilactobacillus spp. L. fermentum NS9
Bifidobacterium spp. B. breve 1205
B. breve CCFM1025
B. longum 1714
B. longum NCC3001
B. longum R0175
Antidepressant activity Lactobacillus spp. L. acidophilus LA5
L. acidophilus W37
L. gasseri OLL2809
L. helveticus NS8
Levilactobacillus spp. L. brevis W63
Lactiplantibacillus spp. L. plantarum 90sk
Lacticaseibacillus spp. L. casei Shirota
L. casei W56
Lactococcus spp. L. lactis W19
L. lactis W58
Bifidobacterium spp. B. bifidum W23
B. breve M2CF22M7
B. breve CCFM1025
B. infantis 35624
B. lactis W52
B. longum R0175
B. longum subsp. infantis E41
Anti-stress activity Lactobacillus spp. L. acidophilus LA5
L. gasseri CP2305
L. helveticus R0052
Lacticasebacillus spp. L. casei Shirota
L. rhamnosus JB-1
Bifidobacterium spp. B. breve CCFM1025
B. logum R0175
soy (milk, paste, sauce, tempeh or natto), rice (bran), algae

(Laminaria japonica, Saccharina japonica), black carrots
(aqueous extract), and green or black teas and sucrose solution
(kombucha) with the aim of investigating any biological activ-
ity they could exert on the central nervous system [21]. How-
ever, it is important to highlight that each fermented food has
specific effects that can change according to the chosen food
matrix, type of fermentation used or microorganisms involved
[19]. Dairy-based matrix are considered ideal for proliferation
of probiotics because they contain a large amount of carbon

and essential amino acids resulting from the hydrolysis of lac-
tose and the proteolytic system involved in the use of casein.
Additionally, the buffering capacity of milk and its fat content
provide a suitable condition for probiotics to better tolerate the
adverse conditions of the gastrointestinal tract [22]. Table 2
includes several studies that show the potential psychobiotic
applications in fermented food products. In general, it was
possible to observe that most of these trials, whether in vivo
murine model or clinical trial, involved the use of fermented
milk as the main carrier matrix of psychobiotics of the genus
Lactobacillus spp. present in concentrations between 106 and
1010 CFU /g/mL in the food consumed daily in varying
dosages for a period between 4 and 12 weeks.
3 General Considerations for the Evaluation of Psychobiotic Activity in the Food

Product by Behavior Tests
Behavioral assessment is an important tool to assess the actions of

psychobiotics. However, some considerations are essential to guar-
antee the reliability and reproducibility of the results obtained. In
principle, the adoption of the principles of use and care of labora-
tory animals are important to avoid or minimize discomfort, dis-
tress, and pain. Thus, animals must be kept in environments where
there is control of temperature and humidity, adequate ventilation
and air quality, as well as light cycle and noise control. All recom-
mendations to ensure an adequate macroenvironment for carrying
out the experimental protocols are present in the federal laboratory
animal regulations and local institutional rules.
In addition to a properly controlled macroenvironment, it is
necessary to pay attention to the animal model to be chosen to
evaluate the action of psychobiotics. Although porcine and zebra-
fish models have become more prevalent, rats and mice are the most
commonly used animals for behavioral assessment. Mice, in partic-
ular, are more useful due of their ability to reproduce and mature
rapidly, and the relative ease to which genetic modification can be
applied through mutational, transgenic, and knockout approaches.
Once the animal model has been chosen, the gender, age and strain
must be standardized. It may seem like an excess of care, but there
are numerous scientific studies that demonstrate that these factors
can interfere with the stress response and, consequently, anxiety-
like behavior. For example, females show less general activity on
tests to assess anxiety, such as the elevated plus maze [42].
Table 2
Psychobiotic strains’ applications in fermented foods products
248
Psychobiotic Strain(s)/
Fermented Food Concentration Model Dose/Treatment duration Outcomes References
Black carrot Lactiplantibacillus plantarum In vivo 2% fermented black carrot Better cognitive function by [23]
SRCM 9 murine with L. plantarum for preventing hippocampal insulin
model 8 weeks resistance associated with lower
amyloid-β deposition in type
2 diabetic rats with dementia
Black soybean milk Levilactobacillus brevis FPA 3709 In vivo 35 mg/kg or 70 mg/kg Antidepressant effect without side [24]
(1 × 106 CFU/mL) murine body weight by oral effects in rat models
model gavage for 28 days
Cássia Pereira Barros et al.
GABA-rich fermented Levilactobacillus brevis DL-11 In vivo 8.83, 16.67 and Relieved anxiety and improved sleep [25]
milk (108 CFU/mL) murine 33.33 mg/kgbody quality, that may be associated with
model weight / day for 4 weeks significant increases of SCFAs in the
intestine and related to changes in
the composition of the gut
microbiota in mice
Milk Lactobacillus casei Shirota (1 × 108 Clinical trial 65 mL for 3 weeks General improved the mood of adults [20]
CFU/mL) whose mood was initially poor/
depressive
Milk Bifidobacterium animalis subsp. Clinical trial 125 g twice daily for Affected the activity of brain regions [26]
lactis I-2494 (1.25 × 1010 CFU/ 4 weeks that control central processing of
dose), Streptococcus thermophilus emotion and sensation in healthy
I-1630, women
Lactobacillus delbrueckii subs.
bulgaricus I-1632 and I-1519
(1.2 × 109 CFU/125 g) and
Lactococcus lactis subsp. lactis
(1.2 × 109 CFU/125 g).
Milk Lactobacillus helveticus IDCC3801 Clinical trial 500, 1000, or 2000 mg of Improved cognitive function in [27]
tablets of skim milk healthy old adults
powder fermented for
12 weeks
Milk Lactobacillus acidophilus, Clinical trial 200 mL/day for 12 weeks Positively affected cognitive function [28]
Lacticaseibacillus casei, and some metabolic statuses in
Bifidobacterium bifidum, and Alzheimer’s disease patients
Limosilactobacillus fermentum (60–95 years old)
(2 × 109 CFU/g for each)
Milk Lacticaseibacillus casei Shirota YIT Clinical trial 100 mL/day for 8 weeks Improved symptoms of stressed [12]
9029 (1 × 109 CFU/mL) and In subjects
vivo
murine
model
Milk Limosilactobacillus fermentum LAB9 In vivo 0.2 mL/day by oral gavage There was a restoration of cholinergic [29]
(109 CFU/0.2 mL) or L. casei murine for 4 weeks neurotransmission and attenuation
LABPC (109 CFU/0.2 mL) model of neuroinflammation in mice
Milk Lactobacillus gasseri CP2305 Clinical trial 190 g/day for 5 weeks Improvement in sleep quality and [30]
(1 × 1010 CFU / 190 g) alleviated stress-associated
symptoms in healthy students
Milk Lactobacillus gasseri CP2305 Clinical trial 2,5 g of powder dissolved Improved physical and mental states [31]
(1 × 1010 CFU/2,5 g) in water/day for 4 weeks in students in the cadaver dissection
course
Milk drink Lactobacillus helveticus CM4 Clinical trial 190 g/day for 8 weeks Improvement in attention and [32]
containing memory in healthy middle-aged
lactononadecapeptide adults
Laminaria japonica Levilactobacillus brevis BJ20 Clinical trial 1.5 g/day for 6 weeks Demonstrated a protective [33]
mechanism against cognitive
impairment associated with
dementia in elderly
Saccharina japonica Levilactobacillus BJ20 Clinical trial 500 mg/twice daily for Changes in memory ability via [34]
algae 4 weeks regulation of SOD antioxidant
system
Psychobiotic Carried by Food and Beverage
Soybean Lactiplantibacillus plantarum C29 Clinical trial 800 mg/day for 12 weeks [35]
(1.25 × 1010 CFU/g)
249
(continued)
Table 2
250
(continued)
Psychobiotic Strain(s)/
Fermented Food Concentration Model Dose/Treatment duration Outcomes References
Improved cognitive function in
individuals with mild cognitive
impairment
Rice bran Saccharomyces cerevisiae IFO 2346 In vivo 1 g/kg/day of a hot water Provided anti-stress and anti-fatigue [36]
murine extract of fermented rice effects in rats and mice
Cássia Pereira Barros et al.
model bran for 2 weeks

Kefir – fermented Milk 4% kefir grains containing Clinical trial 2 mL/kg/day for 90 days Improved memory, visual-spatial/ [37]
Acetobacter aceti, Acetobacter sp., abstraction abilities, and executive/
Lactobacillus delbrueckii, language functions
Limosilactobacillus fermentum,
Fructilactobacillus fructivorans,
enterococcus faecium, Leuconostoc
spp., Lactobacillus kefiranofaciens,
Candida famata, and Candida
krusei
Kefir – fermented Milk Kefir grains dominated by In vivo Daily administration by Increased capacity of GABA [38]
Lactococcus Lactis, Lactobacillus murine oral gavage for 3 weeks production in the gut microbiota of
kefiranofaciens, Bifidobacterium model mouse
breve and Limosilactobacillus
reuteri
Unpasteurized milk and Lactobacillus Clinical trial Free consumption before Decreased stress and anxiety scores in [39]
dairy products and after 12 weeks adults
Yogurt Lactobacillus gasseri SBT2055 Clinical trial 100 g/day for 12 weeks Levels of the stress-induced hormone [40]
(5 × 108 CFU/dose) and adrenocorticotrophic hormone
significantly decreased in adults
Finally, the physical handling of animals is also important,

because it can interfere in the physiology and behavior of subjects.
It is advisable that this handling occurs daily for the animals to
adapt to human physical contact [43]. Moreover, the way in which
this handling is carried out is important. Mice respond better to
passive handling, such as a tube or cupped hands, than to the more
usual method, that is the rapid handling by grasping the base of the
tail [44].
4 Materials
4.1 Probiotic Strain As mentioned before, mono- or multi-species microbial formula-

(s) tions can be chosen (see Note 1). Probiotic cultures can be acquired
in lyophilized form for direct addition (Direct Vat Set – DVS)
widely used on an industrial scale for its practicality or can be
isolated from fermented foods and dairy products.
4.2 Isolation of To perform the isolation of probiotic strains, the following materi-
Probiotic Strains als are needed:
1. Culturing tools – MRS broth (De Man Rogosa and Sharpe)
and MRS agar.
2. Distilled water.
3. 0.1% (w/v, pH 7) peptone water (see Note 2).
4. Laboratory tools – Petri dishes (90 mm diameter), spatula,
glass pipet, micro-pipet, laboratory glassware, sterile plastic
bag, and sterile loop.
5. Laboratory equipment – analytical balance, microwave, auto-
clave, bacteriological incubator, vortex mixer and sample
homogenizer.
6. Chemical reagent – 30% hydrogen peroxide (H2O2).
7. Culture media should be prepared according to the manufac-
turer’s instructions provided on the label (see Note 3). After
accurately weighing the dehydrated medium, pour all ingredi-
ents in an Erlenmeyer flask or any volumetric flask and dilute to
1 L with distilled water. Then mix until complete dissolution
using a glass rod and heat (see Note 4). In the same way,
prepare the 0.1% (w / v, pH 7) peptone water. For 1 L of
solution, weigh 1.0 g of bacteriological peptone and dilute
with 1000 mL of distilled water. Mix until complete dissolu-
tion. It is not necessary to warm up. Afterwards, verify that the
pH of the media and the peptone water is adequate and pro-
ceed to sterilization in an autoclave at 121 °C for 15 min. After
removal from the autoclave, identify them correctly with name
and date.
4.3 Behavioral Tests To perform the behavioral tests, add a method of taking notes and
collecting data. For this purpose, stopwatches and counters are
important to quantify the different experimental variables. This
data collection can be done in real time, as long as the researchers
are attentive and experienced. However, it is more advisable that
the tests are recorded by some camera recording system and ana-
lyzed later. Some computerized video tracking system (e.g., Noldus
Ethovision, Anymaze) can be used to analyze behavioral tests (see
Note 5). Although these software are helpful, they are very costly
and not essential. Whether the experimenter chooses pen and paper
or a computer-based entry system depends on budget and
convenience.
The apparatus used to carry out the behavioral tests can be
purchased or made by the researchers themselves, in compliance
with the specifications previously described in the protocol. In
general, the apparatus needs to be waterproof and easy to clean,
such as acrylic or sealed wood. Between each test, it is essential to
clean the apparatus with an ethanol solution, since the odor of feces
and urine from the previous animals can interfere with the behavior
of the following animals. Between each test, it is essential to clean
the apparatus with an ethanol solution to remove the odor of feces
and urine.
The specifications of the apparatus used to perform the behav-
ioral tests most used for preclinical trials are listed below. We will
use the specifications for tests used in mice, as they are the most
used animal model for evaluating the psychobiotics effectiveness.
1. Open field test: The apparatus consists of a circular acrylic box
(30 cm in diameter) divided into 12 quadrants distributed in
two different zones (central and peripheral). Some laboratories
also use white acrylic or wood cage (30 cm × 30 cm × 15 cm)
divided into 9 or more quadrants (Fig. 1a).
2. Light-dark box test: acrylic or woodcage (45cm ×27 cm × 27 cm)
unequally divided into two chambers by a black partition con-
taining a small opening (Fig. 1b).
3. Elevated plus maze test: the apparatus consisting of four arms
(30 cm × 5 cm) were placed 50 cm above the ground. Two
opposite arms were delimited by acrylic vertical walls, whereas
the other two opposite arms had unprotected edges (open
arms) (Fig. 1c).
4. Marble burying test: in this protocol, 20 or 25 glass marbles
were evenly spaced in the plastic cage (35 cm × 50 cm × 35 cm)
in the presence of the mouse.
5. Tail suspension test: an apparatus used in which the mice
should be suspended 60 cm above the adhesive tape placed
approximately 1 cm from the tip of the tail.
Fig. 1 Behavioral assays used to measure anxiety-like behavior in rodents: (a) Open field test, (b) Light-dark
box test, and (c) Elevated plus maze test
6. Forced swim test: the apparatus consisted of water-filled poly-

propylene or glass cylinder (radius = 30 cm; depth = 50 cm) at
25 °C. The animals are unable to escape or touch the bottom of
the vessel.
7. Sucrose preference test: For the task, mice are presented with
two bearing sipper tubes. One tube contains tap water, whereas
the second contains a 2–4% sucrose solution. Prior to begin-
ning testing, mice are habituated to the presence of two drink-
ing bottles (one containing 2% sucrose and the other water) in
their home cage.
For conducting clinical trials to assess the efficacy of psycho-
biotics, a simple and quick protocol is the Hospital Anxiety and
Depression Scale (HADS). To perform this test, patients must
answer a simple questionnaire. Very few people have difficulty
completing it, on paper or electronically.
It is important to highlight that although it is not the focus of
this chapter, there are very elegant methods, in addition to behav-
ioral tests, to prove the psychobiotics efficacy in the preclinical and
clinical trials, such as serum glucocorticoid measurement, quantifi-
cation of neurotransmitter and cytokines, as well as brain image
studies.
5 Methods
5.1 Isolation of 1. Weigh 25 g of the sample and transfer it quantitatively to a

Probiotic Strains sterile plastic bag, add 250 mL of 0.1% (w/v, pH 7) peptone
water and place in the sample homogenizer for 1 min at
300 rpm (see Note 6).
2. Make tenfold dilutions by adding the 1 mL aliquot of the
sample to 9 mL of 1% (w/v, pH 7) sterile peptone water, and
homogenize each dilution using a vortex mixer. Transfer 1 mL
aliquot of each selected dilution to Petri dishes and add
12–15 mL of MRS agar (pour plate technique) (see Note 7).
Subsequently, incubate the plates under anaerobic conditions
at 37 °C for 24–48 h (see Note 8).
3. Select colonies with a circular shape, creamy texture, and white

color to be cultivated in MRS broth for 24 h under anaerobic
conditions at 37 °C (Fig. 1). After the incubation period, seed
again on plates containing MRS agar until complete isolation is
achieved.
4. The isolated colonies must be analyzed in relation to their
morphological characteristics by the Gram stain method.
Gram-positive colonies should proceed for the Catalase test.
Catalase test.
Procedure – using a sterile loop, add colony to a glass slide and
add 2 drops of 30% (v/v) hydrogen peroxide (H2O2).
Data interpretation: the formation of gas bubbles indicates the
production of catalase enzyme by the test bacterium, while
no bubble formation is negative for catalase.
5. Purified Gram positive and catalase negative strains are consid-
ered presumptive lactic acid bacteria. They can be kept as
frozen stocks in 20% (v/v) glycerol at -20 °C until time of
use [45].
5.2 Psychobiotic As mentioned earlier, in functional fermented foods, probiotic

Food Processing microorganisms with psychobiotic properties can be part of the
raw material whose fermentation process occurs naturally or inten-
tionally through the incorporation of probiotic cultures into the
food matrix.
In the production of Kefir, for example, fermentation can occur
naturally with lactic acid cultures made with Kefir grains, Lentilac-
tobacillus kefir, species of the Leuconostoc, Lactococcus, and Aceto-
bacter genera, producing lactic acid, ethanol, and carbon dioxide
[46]. The microbiota of grains is composed of lactose-fermenting
yeasts (Kluyveromyces marxianus), non-lactose fermenting yeasts
(Saccharomyces omnisporus, Saccharomyces cerevisiae and Saccharo-
myces exiguus), Streptococcus salivarius subsp. thermophilus, and
different probiotic bacteria of the genus Bifidobaterium spp., Lac-
ticaseibacillus casei, L. heveticus, Lacticaseibacillus rhamnosus,
among others, which vary according to the origin and culture
method applied [47]. It is important to emphasize that the proof
of the beneficial effect is, as a rule, strain-specific and as kefir grains
of different origins contain mixtures of different species of micro-
organisms, for evidentiary purposes, studies in humans must be
carried out with the same mixture in which the alleged effect is
intended to be demonstrated.
The intentional addition of probiotic cultures to foods as func-
tional ingredients can occur at different stages of processing:
1. As a starter culture itself. Example: fermented vegetables. In
the process of making fermented soybeans, the inoculated
probiotic strain is exclusively responsible for the fermentation
of soybeans at 37 °C for 24 h [35].
2. After the fermentation phases. During the processing of probi-

otic yogurt, the probiotic culture can be added after fermenta-
tion to the cooled (41 °C) product before packaging,
simultaneously with the conventional yogurt cultures
(S. thermophilus and Lactobacillus delbrueckii bulgaricus), as
well, they can be incorporated as live probiotic starter
cultures [48].
5.3 Assessment of Probiotics can lose their viability and metabolic activity due to
Probiotic Cell Viability unfavorable environmental conditions during the production
stages and storage period of food products (see Note 9). In general,
the minimum concentration for a given health benefit should be
equal to or greater than 6 log CFU/g/mL of probiotic bacteria at
the time of consumption [49], which is in compliance with the
results reported in Table 2. Therefore, it is necessary to perform the
total count of probiotics during the commercial validity of the
product, according to the protocol described below.
1. Make ten-fold dilutions by adding the 1 mL aliquot of the
sample to 9 mL of 1% (w/v, pH 7) sterile peptone water,
homogenize each dilution using a vortex mixer and sowing
on plates containing MRS agar by the pour plate technique.
Subsequently, incubate the plates under anaerobic conditions
at 37 °C for 72 h.
2. Microbial counts are expressed as log CFU/mL.
3. Analyze the morphological characteristics of the colonies by
the Gram staining method and catalase test.
5.4 Behavioral Tests 1. Procedure: Each mouse is placed individually in the center of
apparatus and allowed to explore the cage for 5 min (see Note
5.4.1 Open Field Test
10).
2. Variables measured: During this time, the number of squares
crossed, number of rearing (standing on hind legs with paws
pressed against the wall of the arena), time of grooming, time
in the center zone, center distance (the distance traveled in the
center of the arena), and center ratio (center distance to total
distance ratio) were assessed. At the end of testing, the number
of fecal pellets was also counted, and the arena was cleaned with
a 10% ethanol solution.
3. Data interpretation: This test is a simple protocol, whose eval-
uation of its components has been widely used to determine
emotionality [50, 51] and spontaneous locomotor activity in
rodents [52]. In addition, this test is also considered an excel-
lent model sensitive to drugs with anxiolytic properties. In this
test, locomotor activity is indicated by the total distance
traveled in the apparatus, while the vertical activity is assigned
by the number of rearing. Concerning defecation, this
parameter appeared, under some circumstances, to represent

an emotional behavior. Lastly, anxiety-like responses were
linked to time in the center zone and center ratio, whereas
grooming time indicates higher stress responsiveness.
5.4.2 Light-Dark Box 1. Procedure: The animals are individually placed in the test. Mice
Test are placed inside the dark side and allowed to freely move
between the two chambers for 5 min (see Note 11).
2. Variables measured: During this time, the time spent on the
light side, number of transitions, and latency to first entry into
the light side were recorded.
3. Data interpretation: This test is based on the rodents’ innate
aversion to brightly lit places and novel environments [53]. In
this test, the latency to first entry into the light side and the
time spent on the light side, number of transitions, and are
associated with anxiety-like behavior. At first, the number of
transitions is associated with spontaneous locomotor activity.
However, when analyzed with the other variables, it may indi-
cate anxiogenesis.
5.4.3 Elevated Plus Maze 1. Procedure: The animals are placed in the center of the maze
Test and allowed to move freely for 5 min. An arm entry was defined
as the entry of four paws into the arm (see Note 12).
2. Variables measured: During this period, the cumulative time
and frequency of entries into open and closed arms were
registered. Then, the percentage of entries and time in open
arms and the time in the central platform were calculated.
During the test, the number of stretch-attend posture (SAP),
which is generally associated with anxiety, occurs when the
rodent elongates its body, and is either standing still or moving
forward very slowly.
3. Data interpretation: The elevated plus maze reflects a conflict
between the rodents’ preference for protected areas and their
natural motivation to explore new environments [54]. Thus,
this test is used to assess anxiety-like behavior [55]. The
anxiety-like behavior is linked to the percentage of entries and
time in open arms. In mice, the SAP behavior occurs when the
mouse is undergoing risk-assessment specifically due to an
internal exploratory-anxiety conflict [56].
5.4.4 Marble Burying 1. Procedure: In this protocol, 25 glass marbles were evenly
Test spaced in the presence of the mouse. After 30 min, marbles
that were up to two-thirds covered were counted (see Note
13).
2. Variables measured: The number of marbles buried is evaluated
during the test.
3. Data interpretation: In both natural and laboratory conditions,

rodents spontaneously use available bedding material to bury
unpleasant sources of discomfort present in their home envi-
ronment. The number of marbles buried is directly related to
anxiety response.
5.4.5 Tail Suspension 1. Procedure: Mice are suspended by the tail with adhesive tape
Test attached ~1 cm from the tip for 6 min.
2. Variables measured: During the test, the time of immobility
and latency to the first immobility episode are evaluated.
3. Data interpretation: This test is based on the observation that
rodents, after the initial execution of oriented escape move-
ments, develop an immobile posture when placed in an
unavoidable stressful situation [57]. In this test, the condition
involves inescapable hemodynamic stress caused by the animals
being suspended by the tail. The immobility assumes a low
resilience, and consequently, a high level of depression-like
behavior (see Note 14).
5.4.6 Forced Swim Test 1. Procedure: The mice are introduced individually into the
water-filled cylinder.
2. Variables measured: During the test, the time of immobility
and latency to the first immobility episode are evaluated (see
Note 15).
3. Data interpretation: The forced swim test is also based on the
development of an immobile posture immediately following a
stressful situation. It is known that immobility time is indicative
of low resilience and highly associated with depression-like
behavior [58].
5.5 Sucrose 1. Procedure: The mice should be placed alone in their home
Preference Test cages in order to accurately measure sucrose solution intake.
After 24 or 48 h of acclimation, the mice have the free choice of
either drinking the 2% sucrose solution or tap water for a
period of 4 days (see Note 16).
2. Variables measured: Water and sucrose solution intake is
measured daily. Sucrose preference is calculated as a percentage
of the volume of sucrose intake over the total volume of fluid
intake and averaged over the 4 days of testing.
3. Data interpretation: The sucrose preference test for rodents is
based on the animal’s natural preference for sweets. A low
preference for sucrose indicates anhedonia and, consequently,
depression-like behavior [59].
5.5.1 Hospital Anxiety 1. Procedure: After patient consent, the researchers should ask
and Depression Scale them to complete the questionnaire. The researchers should be
(HADS) available to hear doubts or problems related to the participants.
2. Variables measured: The HADS is composed of 14 items, 7 of
which is related to the anxiety assessment of (HADS-A) and the
other 7 questions are related to depression (HADS-D). Each of
your items can be scored from 0 to 3, composing a maximum
score of 21 points for each.
3. Data interpretation: The HADS was initially developed to
assess psychological distress in non-psychiatric patients. Sub-
jects scores of less than 7 indicate non-cases of significant
clinical symptoms for anxiety and/or depression, scores
between 8 and 10 indicate mild symptoms, scores between
11 and 14 denote moderate symptoms and scores between
15 and 21 designate severe symptoms of anxiety and/or
depression [60].
6 Notes
1. Formulations with multiple strains must have a proven benefi-

cial effect of probiotic mixture. In the case of formulations
whose strains have already been recognized for their effective-
ness, it is not necessary to carry out a new evaluation
[61]. However, it would be ideal to be able to compare the
psychotropic properties of each strain present within the multi-
strain products versus multi-strain probiotics because the com-
bination can generate synergistic effects on the bioactivity of
the probiotics, as well as, mutual inhibition between the strains
can occur, reducing the effectiveness of the psychobiotic
product [62].
2. 0.1% (w/v, pH 7) peptone water can be replaced with saline
solutions, such as (0,85% NaCl, pH 7) sterile saline or
phosphate-buffered saline solution (PBS, pH 7).
3. It is recommended to prepare just enough medium for each
test. After cooling the MRS agar, distribute approximately
15 mL of the sterile medium in a Petri dish. Then just store
the Petri dishes below 5 °C until further use.
4. Be extremely careful when heating to prevent the culture
medium to be overheated or burnt.
5. If using a video tracking system, it is necessary that the color of
the animal and the apparatus background are different. For
lighter animals, we use a dark background. If the animal is
darker, we use the white background. Lack of contrast can
interfere with the video tracking system performance.
6. All bacteriological procedures need to be performed using a

Bunsen burner in a clean and closed working area or inside a
sterile laminar flow hood or similar.
7. Before adding MRS agar, make sure it is not too hot. After
pouring on the plate, homogenize slowly so as not to spread it
on the wall or cover of the plate. Wait for the medium to
solidify completely before taking it to a bacteriological oven.
8. 24 h for lactobacilli and 48 h for bifidobacteria.
9. Several factors can contribute to a significant reduction in the
viability of probiotic cultures, such as chemical composition of
the food matrix, salt and sugar content, type of probiotic strain
and its interactions with the starter cultures, inoculation rate,
content and availability of nutrients, fermentation temperature
and duration, redox potential and dissolved oxygen, pH and
titratable acidity, concentration of final metabolites, and stor-
age temperature [22].
10. Generally, the animals are placed in the center of the apparatus
to avoid misinterpretations, only counting the central area time
after the animal’s first spontaneous entry into that zone. Often
animals treated with sedative drugs have higher immobility
time in the central area. These animals do not show anxiolysis,
but rather sedation.
11. In the light-dark box test, a very common mistake is the use of
a low brightness environment. In these circumstances, there
will not be much contrast and, consequently, the light side will
be less aversive. Based on this premise, it is essential to use a
luxmeter to measure light intensity. 400-lux illumination in the
white area was sufficiently aversive to significantly reduce the
time spent in that area [53].
12. It is necessary to standardize the way in which rodents are
handled. Make sure that each animal will be placed inside the
apparatus in the same position. Differences are observed when
rodents are placed facing toward an open arm versus toward
the closed arm [54]. In our laboratory, rodents are placed on
the maze facing the same closed arm.
13. In the marble burying test, substrates of a denser nature (i.e.,
river sand) should be used, since this prevents incidental
settling of marbles during the test. In addition, video recording
of the test is recommended in order to accurately differentiate
marble-directed behavior, as opposed to an accidental covering
of marbles caused by the normal exploration or by an increased
locomotor activity of the animals [63].
14. Any manipulation that increases the general motor activity of

the animals is capable of reducing the immobility time, which
can lead to spurious conclusions. For this reason, it is essential
to evaluate mice locomotor activity previously through the
open field test [64].
15. Immobility is defined as floating vertically in water, making
only those minimal movements necessary to keep the head
above water [65].
16. The positions of the two bottles or tubes in each cage or
chamber should be changed each day. In addition, make sure
the bottles used in the protocol are not leaking [59].
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INDEX
A Cell-free supernatant (CFS) ............................... 224–232,

234, 235, 239
Acetic acid............................... 48, 69, 89, 133, 134, 136, Cell inactivation ................................................................ 5
140, 156, 194, 224, 225, 239 Cheddar cheese .....................................37, 44, 46, 47, 49
Acid coagulation .......................................................38, 39
Chocolates ............................................... 61, 68, 179–194
Acidifying..........................................................35, 94, 156 Churning ......................................................69–71, 73–77
Additives ......................................7, 9, 13, 15, 17, 18, 20, Chymosin............................................................ 38, 40–42
24, 62, 101, 106, 160, 193, 238
Coagulant enzymes ......................................................... 35
Allergenicity..................................................................... 81 Coconut oil ..................................................................... 94
Almond ......................................................................24, 94 Color measurement ...................................................... 191
Antagonistic effect ................................................. 15, 214
Conching ....................................180, 182, 187, 189, 193
Antibiotics ........................................................9, 124, 236 Conjugated linoleic acid (CLA) ............................. 45, 68,
Antimicrobial components ............................................. 15 69, 75, 156
Anxiety .......................................214, 243, 244, 247, 248,
Contaminants ............................................ 8, 9, 16–18, 28
250, 253, 256–258 Cooling ............................................ 9, 13, 17, 41, 55, 71,
Aromatic compounds............................69, 143, 160, 175 75–77, 84, 88–90, 107, 123, 157, 176, 180, 189,
218, 219, 239, 258
B
Corn zein......................................................................... 94
Bacteriocins ............................................27, 69, 106, 121, Cottage cheese ................................................................ 40
224, 228, 236, 237, 239 Cream maturation .....................................................70, 76
Bakery .............................. 55, 68, 86, 165–168, 175, 232 Crescenza......................................................................... 41
Beer ....................................................................... 139–149 Critical control point (CCP) ...................... 14, 16, 18, 23
Behavior test......................................................... 247–251 Curd............................. 17, 28, 35–37, 39–44, 47, 97–99
Bifidobacterium ......................................3, 20, 26, 36, 42,
46, 53, 57–59, 72, 73, 83, 95, 105, 110, 113, 121, D
140, 141, 153, 155, 166, 167, 171, 172, 180, Dark chocolate ....................................180, 181, 189, 193
184, 186, 194, 200, 220, 234, 245, 246, 248–250
Deaeration ......................................................................... 9
Bioactivities.....................................................69, 239, 258 Demineralization............................................................. 35
Biofilms ................................................................. 225, 236 Depression ..................................243, 244, 250, 253, 258
Biological activity ................................................. 214, 246
Determination of catechins ........................... vii, 136–137
Biosurfactants ................................................................ 224 Determination of sugars .........................................vii, 136
Blending .........................................................95, 180, 181 Direct vat ..............................................40, 56, 62, 73, 83,
Bovine breeds .................................................................. 68
129, 159, 169, 171, 215, 218, 227, 251
Bread .................................................... 166–170, 172–176
Buffering capacity................................................... 36, 247 E
Bulking agents............................................. 180, 182, 183
Butter ...................... 61, 68–78, 179, 181, 182, 187, 189 Edible films........................................................... 166, 176
Elevated plus maze test................................................. 256
C Emulsion...............................................47, 61, 68, 71, 82,
95, 96, 100, 199–203, 206, 207
Canestrato Pugliese cheese ............................................. 44 Encapsulation ............................................... 8, 24, 45, 63,
Carrier.................................... 36, 37, 105, 120, 201, 247 108, 109, 111, 114, 141, 166, 172, 175,
Cashew nuts .................................................................... 94
200–205, 207, 209
Cattle breeds...................................................................... 7 Enzymatic hydrolysis ...................................................... 37
Cell cultivation ................................................................ 56
Adriano Gomes de Cruz et al. (eds.), Probiotic Foods and Beverages: Technologies and Protocols,
Methods and Protocols in Food Science, https://doi.org/10.1007/978-1-0716-3187-4,
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer
Nature 2023
263
PROBIOTIC FOODS AND BEVERAGES: TECHNOLOGIES AND PROTOCOLS
264 Index
Equipment......................................... 8, 9, 48, 55, 60, 70, 137, 156, 159, 169, 171, 180–184, 189, 190,
74, 76, 77, 139, 144, 145, 148, 159, 167, 182, 194, 227–229, 238, 259
187, 189, 205, 206, 216, 228, 251 Inulin ............................................. 13, 20, 24, 25, 54, 59,
Ethanol ...............6, 9, 69, 139–143, 239, 252, 254, 255 109, 180, 183, 192, 200
Exopolysaccharides ................................. 11, 69, 121, 233
Extrusion .................. 8, 47, 72, 109, 199–204, 206, 207 J
Juice ............................................... 36, 89, 105–109, 111,
F
112, 114, 115, 121, 124, 125, 137
Feeding material................................................................ 8
Fermentation ......................................2, 8, 11, 13–17, 19, K
21, 23, 24, 37, 40, 47, 62, 68, 69, 71, 73, 81–84,
Kombucha ...........................................133–135, 137, 246
86–89, 94–97, 100–102, 106, 107, 120, 121,
125–130, 133, 135, 140, 142–148, 151, 153, L
155, 161, 169, 172–174, 224, 226–228, 234,
235, 246, 254, 255, 259 Laboratory scale ............................. 44, 48, 129, 144, 148
Fermented cream ............................................... 40, 68, 75 Lactation season ................................................................ 8
Fermented milk .............................................1, 2, 5–9, 11, Lactic acid .......................................... 2, 8, 11, 13, 15, 21,
13–28, 36, 62, 105, 244, 247, 248, 250 25, 27, 35, 39, 40, 62, 69, 81, 94, 106, 122,
Fermented vegetables ..................................120–125, 254 126–128, 133, 134, 140, 142, 155, 160, 218,
Festivo cheese .................................................................. 42 224, 225, 227, 239, 245, 254
Feta cheese.................................................................43, 46 Lacticaseibacillus .................................... 3, 19–22, 25, 36,
Filtration ........................................ 9, 14, 84–87, 97, 100, 37, 40, 46, 47, 54, 57–59, 72, 73, 83, 95, 112,
101, 147, 148, 203, 204, 226, 229–231, 234 125, 126, 140, 143, 158, 159, 166, 167, 171,
Flavor ............................................... 8, 15, 17, 18, 23, 27, 172, 180, 184, 194, 200, 201, 245, 246, 249, 254
28, 35, 38, 39, 44, 48, 61, 62, 69, 82–84, 89, 93, Lactobacillus ......................................... 3, 6, 8, 15, 18–21,
94, 98, 106, 107, 137, 140, 147, 148, 152, 156, 25, 36, 46, 47, 53, 57–59, 69, 72, 73, 83, 95,
160, 172, 173 105–107, 110, 113, 121, 123–126, 140, 142,
Forced swim test ........................................................... 257 149, 155, 166, 167, 171, 172, 180, 184, 194,
Free fatty acids (FFAs) .................................................... 45 200, 234, 245–250, 255
Freeze-drying ....................................................... 200, 231 Lactose ....................................................8, 27, 35, 53, 61,
Fresh cheese................................................. 36, 40, 41, 46 68, 82, 105, 165, 186, 190, 194, 247
Frozen desserts................................................................ 53 Lactose intolerance .......................... 36, 81, 93, 120, 152
Fruits................................................ 6, 13, 14, 16, 18, 26, Large-scale production .......................................... 44, 214
55, 88, 89, 101, 102, 106–108, 125, 148, 232 Light-dark box test ..................................... 253, 256, 259
Functional products ............................................. 105, 214 Limosilactobacillus ........................................ 4, 41, 46, 83,
Fusion properties .......................................................... 191 180, 184, 194, 200, 236, 245, 246, 249, 250
Live microorganisms .................................. 1, 2, 5, 23, 27,
G 36, 45, 69, 105, 140, 152, 165, 180, 213
Gelation ...........................37, 39, 72, 108, 109, 202, 203 M

H Marble burying test ...................................................... 259
Maturation............................................36, 39, 45, 56, 60,
Hard cheese ........................................... 38, 43–46, 48, 49
62, 70, 75, 76, 126, 145, 147–149, 154, 159, 162
Hardening .......................................................... 56, 60, 63 Mental disorders................................................... 243, 244
Harvesting ..................................................................... 229 Microbial propagation .................................................. 226
Homogenization ........................................ 56, 60, 61, 82,
Microbial strains ............................. 3, 226–228, 233, 245
94, 96, 100, 107, 172, 203 Microencapsulated ...................................... 8, 54, 74, 152
Hospital ................................................................ 253, 258 Milk chocolate...................................................... 181, 189
Minas frescal cheese ........................................................ 40
I
Mold .....................................................41, 42, 44, 96, 98,
Ice creams .....................................................53–58, 60–63 107, 161, 162, 189
Inactivation processes ................................. 215, 218, 220 Molding .................................................39, 180, 182, 189
Inoculation ........................................... 13–15, 37, 40, 42, Mucilage ............................................... 24, 130, 166–168,
53, 56–58, 71, 72, 83, 87, 88, 96, 111, 123, 129, 170, 175, 200
Index 265
N Semi-hard cheese............................................................. 44
Semi-soft cheese .............................................................. 42
Neurotransmitters ....................................... 122, 224, 253 Sensory acceptance.......................................................... 15
Sensory properties.............................................17, 26, 48,
O
86, 107, 108, 126, 153, 156
Oat extract .................................................................85, 86 Separation procedures.......................................... 232, 237
Oil ................................... 61, 68, 77, 85, 94–96, 99, 110, Shelf life ...................................................... 36, 42, 54, 69,
140, 144, 147, 149, 191, 201, 203, 205 95, 107, 108, 113, 120, 142, 152, 172, 190, 233
Sodium caseinate.......................... 24, 166–168, 170, 175
P Soft cheese .................................................................40–42
Soybeans ........................................ 81, 82, 84, 85, 94, 97,
Packaging............................................ 5, 7, 13–15, 17–19,
120–122, 124, 128–130, 201, 203, 248, 249, 254
21–23, 27, 56, 76, 89, 90, 99, 101, 107, 125–127,
Soy extract .......................................................... 81, 84, 85
147, 176, 182, 189, 193, 194, 203, 204, 207,
Spontaneous ferments..................................................... 83
225, 233, 255
Spray dryer...........................................203–205, 210, 226
Palm fruit oil ................................................................... 94
Stabilizers............................... 20, 55, 56, 59, 61, 94, 100
Paracaseinate.................................................................... 39
Standards .................... 2, 6, 9, 20, 43, 68, 113, 226, 238
Paraprobiotics..............69, 214, 215, 218–220, 223, 224
Starch ....................................................13, 20, 25, 44, 82,
Particle size distribution ...................................... 187, 191
86, 87, 94, 97, 171, 173, 175, 201
Peas ............................................................................94–97
Starter cultures ................................................7–9, 11, 12,
Penicillium camemberti .................................................. 39
14–17, 20, 25–28, 35–37, 40, 42, 44, 83, 89,
Physical analysis ............................................................. 190
95–97, 126–128, 134, 137, 140, 142, 151–155,
Pickles ..................................................122, 123, 126, 127
159, 160, 215, 228, 245, 254, 255, 259
Plant-based beverages ........................................ 81–83, 88
Storage .................................................. 5, 7, 9, 12–14, 18,
Plant-based cheese ....................................................93–95
19, 21, 23, 25–27, 36, 40–45, 47, 53, 54, 58, 60,
Prebiotics .............................................. 13, 20, 24–27, 54,
63, 71, 72, 76, 78, 83, 100, 102, 106–109, 112,
82, 86, 107, 120, 181, 192, 200, 201, 213, 223,
115, 121, 126, 137, 141–143, 147, 153, 155,
224, 244
157, 162, 166, 171, 180, 182–184, 189, 190,
Pre-pasteurization ............................................................. 9
193, 194, 201, 205, 207–209, 214, 231, 234,
Probiotics................................................ 1–5, 7–9, 11, 13,
255, 259
15, 16, 18–28, 35–37, 40–49, 53–60, 62, 63, 68,
Streptococcus ..........................................3, 6, 8, 15, 20, 26,
69, 71–76, 81–83, 86–90, 93–102, 105–114,
39, 41, 95, 125, 140, 180, 185, 194, 248, 254
119–129, 140–145, 147, 148, 152–160, 162,
Sucrose preference test ........................................ 257–258
165–172, 174–176, 180–184, 186–190,
Sugar replacements ....................................................... 181
192–194, 199–210, 213–216, 218–220, 223,
Suspensions ................................................ 72, 78, 87, 96,
224, 227–229, 232, 234, 244, 245, 247, 251,
98, 110, 172, 204, 218–220, 228, 238
253–255, 258, 259
Sweet cream........................................................ 68, 75, 76
Propagation method ....................................................... 56
Sweeteners .................................................... 7, 13, 14, 18,
Proximate composition..................................................... 5
20, 25–27, 55, 61, 106, 180, 182, 192
Psychobiotics ..................... 244–248, 250, 252–254, 258
Symbiotic culture of bacteria and yeast
Purification .................................................. 9, 12, 56, 217
(SCOBY)...........................................133–135, 137
R Synbiotics......................... 13, 53, 54, 181–183, 188, 192
Syneresis..................................................... 25, 35, 39, 100
Raw milk ....................................... 7–9, 14, 23, 28, 77, 98 Synergistic..................................... 8, 11, 15, 25, 181, 258
Refining ....................................................... 180, 187, 189
Rennet ................................................... 40, 41, 43–45, 94 T
Rheological measurements .................................. 191–192
Tail suspension test ....................................................... 257
Rice extracts..................................................82, 83, 86–88
Tempering ............................................................ 180, 189
S Texture analysis ............................................................. 191
Texturization ................................................................... 43
Salt resistance .................................................................. 47 Thermiziation.................................................................. 43
Sanitation....................................................................... 8, 9 Thickeners ......................... 13–15, 18, 20, 24, 25, 88, 89
Sausages ...................................... 151–156, 158–160, 215 Tindalization ................................................................. 215
266 Index
V W
Veganism.......................................................................... 81 Washing ..........................................................87, 125, 127
Vegetable proteins........................................................... 94 Water activity ................................................ 38, 106, 152,
Viability........................................ 2, 8, 11, 13, 19, 24–26, 162, 172, 173, 176, 180, 187, 190, 199, 208
36–38, 40–42, 44, 45, 47–49, 53, 54, 63, 69, 71, White cheese..............................................................42, 43
90, 100, 102, 106–109, 111, 112, 114, 115, 121, White chocolate...................................180–182, 189, 193
126, 129, 142, 147, 154, 162, 165, 166, 171,
172, 174–176, 180, 182–184, 190, 193, 194,
199, 208–210, 213, 214, 238, 255, 259
Viable cell count.................................... 15, 152, 203–205
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Preface to the Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

1 Probiotic Fermented Milk. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

14 Microencapsulation of Probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

ALAN AMBROSI • Graduate Program in Food Engineering, Federal University of Santa

SILVANI VERRUCK • Graduate Program in Food Science, Federal University of Santa

Probiotic Fermented Milk

Probiotic fermented milk is a product derived from traditional

microorganisms [2]. However, the recent research regarding

consensuses to determine which strain/species or groups can be

Ymer (Denmark Skyr (Iceland

Many fermented milk could contain probiotics, such as yogurt,

Codex Alimentarius China USA Canada Australia

However, novel tetra packaging was developed to comply with the

Materials used for probiotic fermented milk production can be

sanitation of the cows’ udder (or other dairy animals) is necessary as

SCC was influenced by parities, calving season, and lactation

and it can be stored for about 7 days [11]. Pre-pasteurization

LAB pure Activity

Reinoculation Mother Inoculation & Bulk starter

Mother starter culture

Substrate Storage & Bulk starter culture

The production method of probiotic fermented milk is similar to

Standardization1 Homogenization Heat treatment2 Cooling Pure culture

Cooling Fermentation6 Packaging5 Inoculation4 Bulk culture3

Stirring & Fermentation6

3.1 Pre-Treatment Pre-treatment includes raw milk standardization, homogenization,

starter culture and possess desired texture of the product. Detailed

should be determined previously during the pilot plant test before

Besides traditional packaging, the novel product pushed the

of packaging are also available for this particular product, such as

1. Several probiotics can be applied as starter cultures, where the

includes (1) probiotic species and strain identification with

Filling, capping Bottle

Bottle production line Main manufacture line Extra addition step

GG MRS broth for 24 h at 37 C. both strains were