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QUANTITATIVE METHODS AND
ANALYTICAL TECHNIQUES IN
FOOD MICROBIOLOGY
Challenges and Health Implications
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QUANTITATIVE METHODS AND

ANALYTICAL TECHNIQUES IN
FOOD MICROBIOLOGY
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Challenges and Health Implications
Edited by
Leonardo Sepúlveda Torre, PhD
Cristóbal Noé Aguilar, PhD
Porteen Kannan, PhD
A. K. Haghi, PhD
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First edition published 2022
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Library and Archives Canada Cataloguing in Publication
Title: Quantitative methods and analytical techniques in food microbiology : challenges and health implications / edited
by Leonardo Sepúlveda Torre, PhD, Cristóbal Noé Aguilar, PhD, Porteen Kannan, PhD, A. K. Haghi, PhD.
Names: Sepúlveda Torre, Leonardo, editor. | Aguilar, Cristóbal Noé, editor. | Kannan, Porteen, editor. | Haghi, A. K., editor.
Description: First edition. | Includes bibliographical references and index.
Identifiers: Canadiana (print) 2021039336X | Canadiana (ebook) 20210393394 | ISBN 9781774637265 (hardcover) |
ISBN 9781774637425 (softcover) | ISBN 9781003277453 (ebook)
Subjects: LCSH: Food—Microbiology. | LCSH: Foodborne diseases—Microbiology. | LCSH: Food—Safety measures.
Classification: LCC QR115 .Q83 2022 | DDC 664.001/579—dc23
Library of Congress Cataloging‑in‑Publication Data
CIP data on file with US Library of C

ongress
ISBN: 978-1-77463-726-5 (hbk)

ISBN: 978-1-77463-742-5 (pbk)
ISBN: 978-1-00327-745-3 (ebk)
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About the Editors
Leonardo Sepúlveda Torre, PhD

Full Professor, Bioprocesses and Microbial Biochemistry Group,
School of Chemistry, Autonomous University of Coahuila, Saltillo,
Coahuila, México
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Leonardo Sepúlveda Torre, PhD, is a chemist specializing in Broma-
tology at the School of Chemistry of the Autonomous University of
Coahuila (UAdeC). His postgraduate studies were in related topics on
Food Biotechnology in the Food Research Department of the UAdeC.
In 2011 he made a research stay at the “Institute of Biotechnology and
Bioengineering at the University of Minho (Uminho), Braga, Portugal.
He worked as a collaborator at the Center of Biological Engineering”
in Uminho, Braga, Portugal, with the link project “Biotechnologies for
regional food biodiversity in Latin America.” At the same institution, he
carried out his postdoctoral stay in 2015–2016 on “Assisted extraction by
fermentation of polyphenols from agro-industrial waste.” He is currently
a professor-researcher in the School of Chemistry of the UAdeC, respon-
sible for the group Bioprocesses and Microbial Biochemistry. He teaches
classes in algebra, differential calculus, differential equations, accounting,
and financial administration, general microbiology, food microbiology in
chemical engineering, chemical, and QFB programs.
Cristóbal Noé Aguilar, PhD

Full Professor, Associate Editor of Heliyon (Microbiology) and Frontiers
in Sustainable Food Systems (Food Processing), Bioprocesses and
Bioproducts Research Group, Food Research Department, School of
Chemistry, Autonomous University of Coahuila, Saltillo, Mexico
Cristóbal Noé Aguilar, PhD, is a Director of Research and Postgraduate
Programs at the Universidad Autonoma de Coahuila, Mexico. Dr. Aguilar
has published more than 160 papers in indexed journals, more than 40
articles in Mexican journals, and 250 contributions in scientific meetings.
He has also published many book chapters, several Mexican books, four
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vi About the Authors
editions of international books, and more. He has been awarded several

prizes and awards, the most important of which are the National Prize of
Research (2010) from the Mexican Academy of Sciences; the Prize “Carlos
Casas Campillo (2008)” from the Mexican Society of Biotechnology and

Bioengineering; National Prize AgroBio-2005; and the Mexican Prize in
Food Science and Technology. Dr. Aguilar is a member of the Mexican
Academy of Science, the International Bioprocessing Association, the
Mexican Academy of Sciences, the Mexican Society for Biotechnology
and Bioengineering, and the Mexican Association for Food Science
and Biotechnology. He has developed more than 21 research projects,
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including six international exchange projects.
Porteen Kannan, PhD

Assistant Professor, Department of Veterinary Public Health,
Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences
University, India
Porteen Kannan, PhD, is an Assistant Professor in the Department of
Veterinary Public Health at Madras Veterinary College, Tamil Nadu Veter-
inary and Animal Sciences University, India. The research activities of Dr.
Kannan include food safety and anti-microbial resistance. He performed
his postdoctoral studies at the US Department of Agriculture, Maryland,
USA, with a specialization in foodborne pathogens. He has published his
work in both national and international journals. He is actively involved in
mentoring both MVSc and PhD students.
A. K. Haghi, PhD
Professor Emeritus of Engineering Sciences, Former Editor-in-Chief,
International Journal of Chemoinformatics and Chemical Engineering
and Polymers Research Journal; Member, Canadian Research and
Development Center of Sciences and Culture
A. K. Haghi, PhD, is the author and editor of 200 books, as well as over
1,000 published papers in various journals and conference proceedings.
Dr. Haghi has received several grants, consulted for a number of major
corporations, and is a frequent speaker to national and international audi-
ences. Since 1983, he has served as a professor at several universities. He is
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About the Editors vii
former Editor-in-Chief of the International Journal of Chemoinformatics

and Chemical Engineering and Polymers Research Journal, and is on the
editorial boards of many international journals. He is also a member of
the Canadian Research and Development Center of Sciences and Cultures

(CRDCSC), Montreal, Quebec, Canada. He holds a BSc in urban and
environmental engineering from the University of North Carolina (USA),
an MSc in mechanical engineering from North Carolina A&T State
University (USA), a DEA in applied mechanics, acoustics, and materials
from the Université de Technologie de Compiègne (France), and a PhD in
engineering sciences from Université de Franche-Comté (France).
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Contents
Contributors.......................................................................................................... xi
Abbreviations........................................................................................................xv
Preface............................................................................................................... xvii
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1. Classification of Microorganisms and Food Microbiology
Generalities................................................................................................... 1
Alfredo Ivanoe García-Galindo, Leonardo Sepúlveda, and Cristóbal Noé Aguilar
2. Risk and Safety in Microbiology.............................................................. 11

Josefina Rodríguez and Cristobal N. Aguilar
3. Food-Microorganism Interaction............................................................. 25
Nathiely Ramirez-Guzman, Leonardo Sepúlveda, and Cristóbal Noé Aguilar
4. Food Preservation...................................................................................... 39
Cristian Torres-León and Cristóbal Noé Aguilar
5. Food and Diseases: What to Know in the Fight to

Ensure Food Safety.................................................................................... 57
Cristian Torres-León, Leonardo Sepúlveda, and Cristóbal Noé Aguilar
6. Microbial Products of Importance in the Food Industry....................... 75

José L. Martínez-Hernández, José Sandoval-Cortes, and Cristóbal Noé Aguilar
7. Hygiene, Control, and Inspection in Foods............................................. 97

José Sandoval-Cortes, José L. Martínez-Hernández, and Cristóbal Noé Aguilar
8. Molecular Methods for Microorganism Detection.................................111

Adriana C. Flores-Gallegos, Raúl Rodríguez-Herrera, and Cristóbal Noé Aguilar
9. New Molecular Methods for the Detection of Microorganisms........... 133

Raúl Rodríguez-Herrera and Cristóbal Noé Aguilar
10. Fungal Production and Function of Phytase......................................... 145

Alberto A. Neira-Vielma, Cristóbal Noé Aguilar, Anna Ilyina,
Georgina Michelena-Álvarez, and José L. Martínez-Hernández
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x Contents
11. Advances in the Biotechnological Process for

Obtaining Ellagic Acid from Rambutan................................................ 165
Nadia D. Cerda-Cejudo, Jose J. Buenrostro-Figueroa, Leonardo Sepúlveda,
Cristian Torres-León, Mónica L. Chávez-González, Cristóbal Noé Aguilar, and

J. A. Ascacio-Valdés
12. Novel Methods of Food Preservation..................................................... 189

C. Guillermo Valdivia-Nájar and Lorena Moreno-Vilet
13. Strategies During Citrus Waste Utilization: Fermentative Route for

Single-Cell Protein Production............................................................... 213
Andrea Guadalupe Flores-Valdés, Gloria A. Martínez-Medina,
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José Luis Martínez-Hernández, Anna Iliná, Cristóbal Noé Aguilar,
Nathiely Ramírez Guzmán, and Mónica L. Chávez-González
14. Impact of Functional Ingredients from Plant Food Byproducts on

Human Gut Microbiota........................................................................... 237
Ricardo Gómez-García, Débora A. Campos, Ana R. Madureira, and Manuela Pintado
15. Trends, Analytical Approaches, and Applications of the VITEK

System for Identification and Classification of Bacteria and Yeasts...... 255
Alaa Kareem Niamah, Shayma Thyab Gddoa Al-Sahlany, Deepak Kumar Verma,
Mamta Thakur, Balaram Mohapatra, Smita Singh, Mónica L. Chávez-González,
Cristóbal Noé Aguilar, Ami R. Patel, and Kolawole Banwo
Index.................................................................................................................. 273
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Contributors
Cristóbal Noé Aguilar

Bioprocesses and Bioproducts Research Group, Food Research Department, School of Chemistry,
Autonomous University of Coahuila, Saltillo Unit – 25280, Coahuila, México,
E-mail: cristobal.aguilar@uadec.edu.mx
Shayma Thyab Gddoa Al-Sahlany
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Department of Food Science, College of Agriculture, University of Basrah, Basra City, Iraq
J. A. Ascacio-Valdés
Autonomous University of Coahuila, Saltillo – 25280, Coahuila, México,
E-mail: alberto_ascaciovaldes@uadec.edu.mx
Kolawole Banwo
Food Microbiology and Biotechnology Unit, Department of Microbiology, University of Ibadan,
Oyo State, Nigeria
Jose J. Buenrostro-Figueroa
Research Center for Food and Development A.C., Cd. Delicias – 33088, Chihuahua, México
Débora A. Campos
Universidade Católica Portuguesa, CBQF-Centro de Biotecnologia e Química Fina-Laboratório
Associado, Escola Superior de Biotecnologia, Rua Diogo Botelho 1327, Porto – 4169-005, Portugal
Nadia D. Cerda-Cejudo
Autonomous University of Coahuila, Saltillo – 25280, Coahuila, México
Mónica L. Chávez-González
Autonomous University of Coahuila, Saltillo – 25280, Coahuila, México,
E-mail: monicachavez@uadec.edu.mx
Adriana C. Flores-Gallegos
Bioprocesses and Bioproducts Research Group, Food Research Department,
Autonomous University of Coahuila, Saltillo, México, E-mail: carolinaflores@uadec.edu.mx
Andrea Guadalupe Flores-Valdés

Food Research Department, School of Chemistry, Autonomous University of Coahuila,
Saltillo – 25280, Coahuila, México
Alfredo Ivanoe García-Galindo

Center for Interdisciplinary Studies and Research, Autonomous University of Coahuila,
Saltillo, México
Ricardo Gómez-García
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xii Contributors
Nathiely Ramírez Guzmán

Center for Interdisciplinary Studies and Research, Autonomous University of Coahuila,
Anna Iliná

Anna Ilyina
Autonomous University of Coahuila, C.P. – 25280, Saltillo, Coahuila, México
Ana R. Madureira
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José Luis Martínez-Hernández
Bioprocesses and Bioproducts Group, Food Research Department, Autonomous University of
Coahuila, Saltillo, México; Nanobioscience Group, School of Chemistry, Autonomous University of
Coahuila, C.P. – 25280, Saltillo, Coahuila, México, E-mail: jose-martinez@uadec.edu.mx
Gloria A. Martínez-Medina
Georgina Michelena-Álvarez
Cuban Institute for Research on Sugarcane Derivates, Postal Zone 10, San Miguel del Padrón,
La Habana City, Cuba
Balaram Mohapatra
Department of Bioscience and Bioengineering, Indian Institute of Technology Bombay,
Bombay – 400076, Maharashtra, India
Lorena Moreno-Vilet
Department of Food Technology, CONACYT-Department of Food Technology, Centre of Research
and Assistance in Technology and Design of the State of Jalisco, A.C. Zapopan, Jalisco, México
Alberto A. Neira-Vielma
Nanobioscience Group, School of Chemistry, Autonomous University of Coahuila, C.P. – 25280,
Saltillo, Coahuila, México, E-mail: aneiravielma@uadec.edu.mx
Alaa Kareem Niamah

Department of Food Science, College of Agriculture, University of Basrah, Basra City, Iraq,
E-mails: alaakareem2002@hotmail.com; alaa.niamah@uobasrah.edu.iq
Ami R. Patel
Division of Dairy and Food Microbiology, Mansinhbhai Institute of Dairy and Food Technology-
MIDFT, Dudhsagar Dairy Campus, Mehsana – 384-002, Gujarat, India
Manuela Pintado
Associado, Escola Superior de Biotecnologia, Rua Diogo Botelho 1327, Porto – 4169-005, Portugal,
E-mail: mpintado@porto.ucp.pt
Nathiely Ramirez-Guzman
Center for Interdisciplinary Studies and Research, Autonomous University of Coahuila, Saltillo,
México, E-mail: nathiely.ramirez@uadec.edu.mx
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Contributors xiii
Josefina Rodríguez
Center for Interdisciplinary Studies and Research, Autonomous University of Coahuila, Saltillo,
México
Raúl Rodríguez-Herrera
Bioprocesses and Bioproducts Research Group, Food Research Department,

Universidad Autonoma de Coahuila, Saltillo, Mexico, E-mail: raul.rodriguez@uadec.edu.mx
José Sandoval-Cortes
Analytical Chemistry Department, School of Chemistry, Autonomous University of Coahuila ,
Saltillo, México, E-mail: josesandoval@uadec.edu.mx
Leonardo Sepúlveda
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Smita Singh
Department of Life Sciences (Food Technology), Graphic Era (Deemed to be) University,
Dehradun, Uttarakhand – 248002, India
Mamta Thakur
Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and
Technology, Longowal – 148106, Punjab, India
Cristian Torres-León
Research Center and Ethnobiological Garden, Autonomous University of Coahuila,
Viesca – 27480, Coahuila, México, E-mail: ctorresleon@uadec.edu.mx
C. Guillermo Valdivia-Nájar
Centre of Research and Assistance in Technology and Design of the State of Jalisco,
A.C. Zapopan, Jalisco, México, E-mail: gvaldivia@ciatej.mx
Deepak Kumar Verma
Department of Agricultural and Food Engineering, Indian Institute of Technology Kharagpur,
Kharagpur – 721-302, West Bengal, India; Bioprocesses and Bioproducts Research Group,
Saltillo Unit – 25280, Coahuila, México, E-mail: rajadkv@rediffmail.com
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Abbreviations
AP active packaging
ArGa arabinogalactan
ATCC American type culture collection
ATP adenosine triphosphate
AXOS arabinoxylo-oligosaccharides
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BIA biospecific interaction analysis
BOD biochemical oxygen demand
CBHI cellobiohydrolases I
CE capillary electrophoresis
CP cold plasma
DAF DNA amplification fingerprinting
DF dietary fiber
DGGE denaturing gradient gel electrophoresis
FAO Food and Agriculture Organization
FDA Food and Drug Administration
FIR far IR radiation
FISH fluorescence in situ hybridization
FOS fructooligosaccharides
FRET fluorescent resonance energy transfer
GM genetically modified
GMOs genetically modified organisms
GOS galactooligosaccharides
GRAS generally recognized as safe
HAV hepatitis A virus
HHDP hexahydroxydiphenoyl
HMF 5-hydroxy methyl furfural
HPP high pressure processing
HTST high temperature short time
IBD inflammatory bowel disease
IDF insoluble dietary fiber
IOR ionizing radiation
IR infrared
ISM industrial, scientific, and medical
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xvi Abbreviations
ITS internal transcribed spacer

LAB lactic acid bacteria
L-Araf L-Arabinofuranosyl
LED light-emitting diodes

LiP lignin-peroxidase
MIR medium IR radiation
MnP manganese peroxidase
MW microwaves
NIR near IR radiation
NoV norovirus
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NTT nonthermal technologies
OH ohmic heating
PCR polymerase chain reaction
PEF pulsed electric fields
PFBs plant food by-products
PL and UV pulsed light and UV processing
PL pulsed light
PNAs peptide nucleic acids
PP passive packaging
PPO polyphenol oxidase
qPCR quantitative PCR
RF radiofrequency
RFLP restriction fragment length polymorphism
RT-PCR real-time PCR
SCAR sequence characterized by amplified region
SCFA short chain fatty acids
SCP single cell protein
SDF soluble dietary fiber
SMF submerged fermentation
SSF solid-state fermentation
TGGE temperature gradient gel electrophoresis
T-RFLP terminal restriction fragment length polymorphism
US ultrasound
UTH ultra-high temperature
UV ultraviolet
Vis visible
WA water activity
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Preface
Since ancient times, human society has been closely related to the ability
to acquire enough food, so that not only the basic needs of survival were
met, but also the preservation of food place allowed humans to be able to
devote time to the arts, crafts, and sciences.
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The development of one of the oldest activities of human beings,
agriculture, is closely linked to the ability to preserve food, first by tech-
niques developed over the centuries by trial-and-error observation, and
more recently by the increasing application of science and engineering.
The basis of these advances is our knowledge of food microbiology. Long
before, Antoine van Leeuwenhoek described his living animalcules, many
of the conditions that controlled microbial deterioration had been empiri-
cally identified. However, it was the emergence of microbiology science
that promoted the preservation of food from an art to a science, allowing
food to be processed, distributed, and marketed with a high degree of
confidence in terms of product quality and safety. Thus, food microbiology
has been an important part of the discipline since its early days.
The scope of food microbiology is highly inclusive, as it interacts
with all subdisciplines of microbiology (e.g., public health microbiology,
microbial genetics, fermentation technologies, microbial physiology, and
biochemistry). In addition, food microbiologists have been at the forefront
of many microbiological concepts and advances. For example, the devel-
opment of biofilms and the ability to detect low numbers of metabolically
stressed microbes from highly complex matrices are two areas where
food microbiologists are providing critical insights into the behavior
of microbiological systems. In addition, new research topics have been
raised as a result of the unique challenge given to food microbiologists,
such as predictive microbiology, probiotics, microbial risk assessments,
and natural antimicrobials.
This book has been prepared to provide up-to-date and detailed scien-
tific information on food microbiology. The book is organized into 15
chapters, five of which focus on the two fundamental aspects of the matter,
food, and microorganisms. Each section consists of detailed information,
from the generalities to the particular aspects of each topic of relevance
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xviii Preface
to be addressed, including basic microbiology, safety, food, pathogenic

microorganisms, food conservation, sanitization, hygiene procedures, etc.
The microbial diversity found in food is described from the classifica-
tion by kingdoms and the main groups of microorganisms present in them.

Although the main issue is about microbial food pathogens, the book
also covers another important aspect of food microbiology, such as food
systems and measures to prevent and control food, foodborne diseases,
etc. Uncontrolled and unwanted microbial growth destroys large quantities
of food age, causing significant losses both economically and relative to
nutrient content. In addition, the consumption of foods contaminated with
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particular microorganisms or microbial products can also cause serious
illnesses, such as food-mediated infections and food poisoning. Every
minute, there are about 50,000 cases of gastrointestinal diseases, and
many individuals, especially children, die from these infections. The most
important preventive measures are for the development and continuous
implementation of effective interventions to improve overall food safety.
—Editors
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CHAPTER 4
Food Preservation
CRISTIAN TORRES-LEÓN1 and CRISTÓBAL NOÉ AGUILAR2
1
Research Center and Ethnobiological Garden, Autonomous University
of Coahuila, Viesca – 27480, Coahuila, México,
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E-mail: ctorresleon@uadec.edu.mx
Bioprocesses and Bioproducts Research Group, Food Research
2
Department, Autonomous University of Coahuila, Saltillo, México
4.1 INTRODUCTION
The main objective of food preservation has been on controlling microbial

populations, with a specific emphasis on pathogenic microorganisms.
Food preservation implies inhibiting the growth of microorganisms
[1]. Hostile environments for microorganisms are an adequate food
preservation strategy The application of heat treatments, reduction of
storage temperatures, application of good manufacturing practices and
the addition of additives define the food shelf-life and safety [2]. The
most important hurdles are water activity (aw), temperature (high or low),
acidity (pH), dehydration, preservatives, and non-thermal technologies
(non-conventional).
4.2 FOOD GROUPS
4.2.1 VEGETABLES
Cereals, sugar, fungi (grown as food), fruits, and vegetables are suscep-
tible to microbial and physical-chemical deterioration after harvest.
This generates the need to use preservation techniques. The postharvest
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40 Quantitative Methods and Analytical Techniques in Food Microbiology
preservation foods as of fruits and vegetables is a challenge owing to their

highly perishable nature [3].
4.2.2 ANIMALS
Foods like meat and meat products, birds, and eggs, fish (and other marine
foods), and milk and derivatives are easily altered by microorganisms
unless they undergo some conservation treatment. Most of these foods
must be refrigerated or even frozen immediately after harvesting, in order
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to inhibit microbial growth and loss of quality [1].
4.3 PRINCIPLES OF FOOD PRESERVATION
4.3.1 PREVENTION OR DELAY (SHELF LIFE) OF THE BACTERIAL

COMPOSITION
The keeping food without germs (asepsis), the elimination existing germs
(filtration), then placing an obstacle to microbial growth (low tempera-
tures, dehydration, anaerobic conditions, chemical preservatives) and the
destroying microorganisms are important forms to prevent spoilage in
food caused by bacteria.
4.3.2 PREVENTION OR DELAY OF FOOD BREAKDOWN
The destroying or inactivating their enzymes (scalding) and preventing or

delaying chemical reactions (avoiding oxidation with the use of antioxi-
dants) are some ways to prevent spoilage of food.
4.3.3 PREVENTING INJURIES CAUSED BY INSECTS, HIGHER

ANIMALS, AND MECHANICAL CAUSES
To protect food against damage by microorganisms a maximum of the

latency phase (a-b) and positive acceleration (b-c) of the growth curve must
be prolonged (Figure 4.1). This is possible by ensuring that the smallest
possible number of microorganisms reach the food the smaller the number
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Food Preservation 41
of microorganisms, the greater the latency phase. Also, contamination by

germs of no active growth (in logarithmic phase), present in containers,
machinery, and utensils should be avoided.
Finally, unfavorable environmental conditions for food germs

(humidity, temperature, WA, pH, or redox potential), or the presence of
microbial inhibitors must be ensured in food (preservatives). The higher
the number of unfavorable conditions, the longer it will take to start growth
[1].
FIGURE 4.1 Growth curve of a bacterial population.

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Note: where; a-b: latency; b-c: positive acceleration; c-d: logarithmic phase; d-e: negative
acceleration; e-f: stationary phase; f-g: accelerated destruction; g-h: decline.
4.4 FOOD CONSERVATION METHODS
Preservation methods may be physical (low temperatures: refrigeration,

freezing, high temperatures: pasteurization, sterilization, elimination
of water: concentration, drying, freeze-drying, removal of air: vacuum
packaging, packaging with CO2 or N2) or chemical like the use of food
additives [4].
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4.4.1 ASEPSIS
Asepsis is defined as freedom from pathogenic microorganisms’ insuf-

ficient dose to cause an infection [5]. It is a way to prevent microorgan-

isms from reaching the food. Applied mainly to raw foods. The type of
microorganism (if it is pathogenic) and the number (size of the danger
and the loading treatment) must be identified to know the potential
risk.
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4.4.2 ELIMINATION OF MICROORGANISMS
Microorganisms can be eliminated by filtration (impenetrable for bacteria),

centrifugation, washing, and expulsion.
4.4.3 MAINTAIN ANAEROBIC CONDITIONS
Pathogenic microorganisms can be controlled by using vacuum closed

containers, (for example, canned food). These microorganisms do not
develop in the absence of oxygen, the same applies to temperature.
4.5 CONSERVATION USING HIGH TEMPERATURES
The destruction of microorganisms by heat is due to the coagulation of

their proteins and especially to the inactivation of the enzymes necessary
for their metabolism. Thermal sterilization technologies have been widely
employed in the food industry since they are well developed and require
low investment cost [6]. The heat treatment to choose to destroy vegeta-
tive cells and spores depend:
• Of other conservation methods to be used (to liven up or accentuate
the applied temperature);
• The effects of heat on the food (should not destroy all its protein
content, weaken the food).
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4.5.1 HEAT RESISTANCE OF MICROORGANISMS AND THEIR

SPORES
Heat resistance of microorganisms is usually expressed as a time of thermal

destruction, which is the time necessary to destroy at a given temperature,
a certain number of organisms under specific conditions. Figure 4.2 shows
the resistance of microorganisms to heat treatment, is observed that some
cells have low resistance (A-B), others have medium resistance (B-C),
and other groups have high resistance (C-D), which are probably spores.
Treatment must reach the entire population.
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FIGURE 4.2 Cell resistance to heat treatment.
The factors that affect bacterial inactivation are:

1. Temperature-Time Relationship: The higher the temperature the
less the destruction time.
2. The Concentration of Spores (Cells): The higher the number
of spores, the more intense the heat treatment to destroy
microorganisms.
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3. Preconditions for Bacteria and Spores: The growth conditions

of the bacteria and the production of spores, therefore as a subse-
quent treatment, will influence their resistance to heat:
i. I ncubation Temperature: Growth at optimal temperatures

increases their resistance.
ii. Age: Young bacteria are less resistant than mature bacteria.
iii. Desiccation: Dissected spores are more difficult to destroy.
4. Composition of the substrate in which bacteria and spores are
heated.
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i. Humidity: Moist heat is more potent to destroy (15–30
minutes at 121°C) than dry heat (3–4 hours at 160–180°C).
ii. pH: They are more resistant to neutral pH; this resistance
falls when the pH (acid) is lowered than when it rises
(basic).
iii. Other Constituents of the Substrate: NaCl in low concen-
trations have a protective effect on certain spores.
4.5.2 HEAT RESISTANCE OF YEASTS AND THEIR SPORES
Spores are the most resistant form of microbial cells and are well-equipped
vehicles for colonization of food [7, 8]. The resistance to heat varies
according to the different species and strains. In general, the spores are
destroyed with barely 5–10°C or more of the temperature that would be
needed to destroy vegetative forms. Ascospores are destroyed at 10–15
min/60°C [7, 8]; none resist brief heating at 100°C. Meanwhile vegetative
forms are destroyed at 50–58°C/10–15 min.
4.5.3 THERMO RESISTANCE OF MOLDS AND THEIR SPORES
Spores are relatively stress-resistant structures but show a large

variation in their ability to survive adverse conditions. Most spores are
destroyed at 60°C/10–15 min. Asexual spores are more resistant than
mycelia; 5–10°C or more is needed to destroy them. Fungal spores are
quite resistant to dry heat; 120°C for 30 min is not enough to destroy
them.
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4.5.4 RESISTANCE TO BACTERIA AND THEIR SPORES
Some pathogens are easily destroyed, others need temperatures of

80–90°C for several minutes. Coccus is generally more resistant than

bacilli. The higher the optimum temperature and maximum growth,
the higher the heat resistance. Bacteria that have a capsule or form
granules are more difficult to destroy, also because of their high lipid
content.
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4.5.5 THERMO ENZYME RESISTANCE
Most of the enzymes in food or microorganisms are destroyed at 79.4°C.
14.6 HEAT PENETRATION
The penetration of heat in food can be done by conduction (from molecule

to molecule), convection (by the movement of liquids or gases), or radia-
tion (energy emitted by molecules). The factors that determine the time
required to raise the temperature of the center of the container to steriliza-
tion temperature are the following:
1. Packaging Material: Heating is slower in jars than in cans.
2. Size and Shape of the Container: The volume of the container
should be considered.
3. Initial Food Temperature: Microorganisms must be longer
in a lethal temperature from the beginning; however, it will not
decrease the destruction time.
4. The Temperature of the Autoclave: This is necessary to reach
the lethal temperature of the microorganisms.
5. Consistency of can Contents, Shape, and Size of Food Portions:
Larger pieces are more quickly cooked than smaller ones.
6. Rotation and Agitation: Accelerate the heat penetration into the
food and completely into the fluid.
7. Artificial rapid cooling is recommended because it is easily adjust-
able. Cooling too slowly can lead to overcooking of the food and
allow the growth of thermophilic germs.
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4.6.1 THERMAL DEATH RATE
To determine the heat treatment, one of these three methods is used:

• Graphical method;
• Mathematical formula; and
• Nomogram method.
The basic aspects of the mathematical formulas are presented below,
for more details the book of Albert and Barbosa-Cánovas can be consulted.
The death rate for any microorganism in a determined medium and
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thermally treated at certain ﬁxed temperature follows ﬁrst-order kinetics.
Thus, if N is the number of microorganisms, its variation with time is
expressed according to Eqn. (1):
dN
= −kN (1)
dt
This equation can be integrated, yielding:
=N N 0 exp ( −kt ) (2)
where; N is the number of microorganisms present at a time t; and k is the

death rate constant. The value of the rate constant depends on the type of
microorganism, the medium, and the temperature [9].
Decimal reduction time (D) is the treatment time required to reduce the
number of microorganisms to the tenth (10) part = DT.
2.303  N 
DT = log10  
k  N0  (3)
and since N = 0.1 N0, the decimal reduction time is expressed as a function
of the rate constant of thermal death as:
2.303
DT = (4)
k
and treatment time is expressed according to Eqn. (5):
 N  (5)
t = DT log10  
 N0 
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4.7 THERMAL TREATMENTS EMPLOYED IN FOOD PROCESSING
The temperature and the time of the treatment of food will depend on the
effect of the heat that exerts on the food and on other methods of conserva-
tion that will be used together. Some foods can be heated to a certain limit.
The greater the heat treatment, the greater the number of germs destroyed
until it reaches the temperature that causes the sterility of the product.
The treatment must at least destroy those microorganisms with potential
danger. In canned foods, all microorganisms that may alter the food during
the last stages of manufacture must be destroyed. The different degrees
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of cooking used in food are classified as pasteurization, cooking around
100°C and cooking above 100°C.
4.7.1 PASTEURIZATION
Pasteurization is a heat treatment that destroys the microorganisms present

in a food (generally liquid), this process is carried out at temperatures
below 100°C. The cooking is employing steam, hot water, dry heat, or
electric currents. Always cooled quickly after heating. Pasteurization is
used when:
• Higher heat treatments would damage the qualities of the product
(e.g., milk);
• One of the objectives is the destruction of pathogenic germs (e.g.,
milk);
• The most important alteration agents are not heat resistant (e.g., the
yeasts of fruit juices);
• Surviving microorganisms are controlled with other additional
methods (commercial milk cooling).
Other storage methods such as refrigeration (e.g., milk), packaging,
and the addition of additives are used as a complement to pasteurization.
Pasteurization times and temperatures depend on the method used in
the product to be treated:
• High temperature, short time: 71.1°C 15 sec;
• Short temperature, long time: 62.8°C/30 min.
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4.7.2 HEATING AT APPROXIMATELY 100°C
Heating to temperatures close to 100° C is enough to destroy all microor-

ganisms, except bacterial spores. This temperature is common in home-

made preserves.
4.7.3 HEATING ABOVE 100°C
Temperatures above 100°C, are reached in steam autoclaves under pres-

sure. Autoclave temperatures increase with increasing vapor pressures. The
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acronym UTH (ultra-high temperature) means that the food (usual milk)
was heated to a temperature between 135 and 150°C (for 1–10 s) [10] by
heat injection followed by the instantaneous evaporation, condensation,
and fast cooling. Maintaining for enough time, this treatment capable of
sterilizing the foods. These will have a long shelf life (6–9 months) without
refrigeration [10, 11]. UHT treatment eliminates pathogenic bacteria and
deactivates enzymes [10].
4.7.4 CONSERVATION USING LOW TEMPERATURES
The lower the temperature the slower the chemical reactions, enzymatic
reactions, or microbial growth. A sufficiently low temperature will inhibit
the growth of all microorganisms.
4.7.5 GROWTH OF MICROORGANISMS AT LOW TEMPERATURE
Freezing prevents the prolongation of most microorganisms. Refrigeration

slows their growth rate. Temperatures of 5 or 6°C delay the multiplication
of microorganisms producing food poisoning, except for Clostridium botu-
linum type E [12]. Microorganisms have been found growing up to –17°C.
4.7.6 TEMPERATURES USED IN STORAGE AT LOW

TEMPERATURES
Low-temperature preservation is the most adopted method [13]. Low-

temperature storage inhibits respiratory rate, spoilage reactions, and the
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growth of pathogenic microorganisms [3]. The most commercial storage

units are below –18C.
4.7.7 REFRIGERATION
Consumers prefer refrigerated foods for freshness, low processing damage,

and convenience when cooking [13]. Refrigeration is the main method of
preserving food or as a temporary system until another form of conserva-
tion is applied. Cooling temperature, relative humidity, air velocity, and
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composition of the local atmosphere and possible use of UV radiation
should be considered.
4.7.7.1 TEMPERATURE
The freezing temperature must be selected according to food class, time,

and storage conditions. Some foods have optimal storage temperatures
above freezing, for example, a banana should not be stored in the refrig-
erator, as it has a better storage temperature between 13.3–16.7°C; as for
potatoes, they are better stored at 10–12.8°C.
4.7.7.2 RELATIVE HUMIDITY
Low humidity causes water loss and the wrinkling of food surfaces. If
humidity is lower than the relative humidity of equilibrium, there is a water
loss from the food to the exterior, leading to dehydration of the product
[14]. High relative humidity favors the development of microorganisms
causing alterations.
4.7.7.3 VENTILATION OR AIRSPEED CONTROL
Maintain a uniform relative humidity is important to eliminate odors and

maintain food quality.
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4.7.7.4 COMPOSITION OF THE STORAGE ATMOSPHERE
The amount of proportion of the gases in the atmosphere greatly influ-

ences. In the presence of optimal concentrations of CO2 or O3, the food

remains unchanged for longer. Furthermore, higher relative humidity can
be maintained, without jeopardizing the conservation and qualities of the
food. This also favors the use of a storage temperature higher than the
cooling temperature can be used.
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4.8 IRRADIATION
The combination of UV irradiation with refrigeration helps to preserve

several foods, allowing the use of humidity and higher temperatures than
can be used in refrigeration. Some cheese and meat storage rooms are
installing UV lamps.
4.9 FREEZING
Many microorganisms cannot grow in freezing [14]. Also, when food

is frozen, part of the water is transformed into ice, thus decreasing the
food’s water activity (aw). Many microorganisms cannot develop in low
WA conditions [14].
4.9.1 ADVANTAGES THAT FAST FREEZING PRESENTS OVER SLOW

FREEZING
Rapid freezing forms smaller ice crystals the mechanical so the destruction
of food cells is scarce. Also, with a higher freezing speed, there is greater
microbial and enzymatic inhibition. Fast freezing slows the chemical and
enzymatic reactions of food by stopping microbial growth, the same effect
produces intense or slow freezing.
4.10 DEHYDRATION
Drying is an excellent method of food preservation [15]. Fresh foods are

very perishable because of their high moisture content. In food materials,
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water exists in two ways: both free water (intercellular spaces) and bound
water (intracellular space). Free water is the solvent for microbial growth
[15, 16]. Some examples are hot air drying, sun drying, vacuum drying,
freezing drying [15, 16]. The factors that control dehydration are tempera-
ture, humidity, air velocity, and dehydration time. Dehydrated foods
should be packed immediately after dehydration in suitable packaging.
New research is being developed to develop new drying methods (more
efficient and with less damage to food). Microwave drying [18], infrared
(IR) drying, vacuum impregnation, ultrasound (US) assisted, lyophiliza-
tion process [19], and osmotic dehydration are some of the new foods
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drying technologies [16].
4.11 NON-THERMAL (NON-CONVENTIONAL) PRESERVATION

TECHNOLOGIES
The development of innovative non-thermal food processing technolo-

gies has received attention due to the increasing consumer demand for
safe foods of high nutritional and functional quality [20]. In non-thermal
preservation technologies, the temperature is not the main factor of the
preservation of food (microorganisms and enzymes) [20, 21]. Some of
the most promising emerging non-thermal technologies to extend food
shelf life are pulsed electric fields (PEF), ultraviolet (UV) irradiation,
high-pressure homogenization, high-pressure processing, IR heating,
microwave heating, pulsed light (PL), ozone processing, and cold plasma
(CP) [22]. Currently, non-thermal emerging technologies have been
widely developed in rich countries. However, in America and Africa very
few countries are investigating these technologies [20].
4.12 CONSERVATION BY ADDITIVES
4.12.1 FOOD ADDITIVES
A food additive is defined by Codex Alimentarius Commission’s (which is

recognized as the international standard) as “any compound not typically
consumed as a food by itself and not normally used as an ingredient in the
food but is intentionally added in the manufacture, processing, treatment,
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preparation, packing, transport, and holding of the food, to perform a tech-

nological function” [23]. The term does not include contaminants added to
food for preserving or enhancing the nutritional potential [23].
4.12.2 ANTIMICROBIAL CONSERVATIVES
The antimicrobial conservatives increase the shelf life of the foodstuffs.

However, the concentrations must be regulated so as not to affect the
organoleptic properties of the food [24]. In general, antimicrobial conser-
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vatives must have a broad spectrum of antimicrobial activity, must not be
toxic to humans and animals, should not affect the original taste of the
food.
4.12.3 NATURAL FOOD ADDITIVES
Currently, there is a trend towards the use of natural additives, these

are accepted by consumers for their perception as safer and healthier.
Compounds derived from the secondary metabolism of most plants as
polyphenols are recognized for having important biological properties
such as antioxidant and antimicrobial activity [25]. Although consumers
are leaning toward natural additives, this offer is still quite limited [26].
4.12.4 ADDITIONAL FOOD PRESERVATIVES
Additives not recognized in some regulations: Natural organic acids

(lactic, malic, citric) and their derivatives; vinegar (acetic acid is a natural
acid, more effective against yeasts and bacteria than against molds), NaCl,
special sugars, their oils, CO2, nitrous, and nitrates. These have been
traditionally used for the preservation of meat products because of the
effective antimicrobial action of nitrite against Clostridium botulinum
[27]. The nitrates added to sausages and canned meats as antimicrobials
can cause headaches [28]. However, the reduction of nitrates to nitrites
(by nitrate-reductase in the bacterial flora) may cause acute toxic effects
and the formation of carcinogenic substances due to the reactions between
nitrogen oxide and amines [29].
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Generally recognized as safe (GRAS): Propionic acid (affects the

permeability of the membrane, used to prevent mold growth and forma-
tion of filamentous elements), caprylic acid, sorbic acid, and sorbates
(inhibit yeasts and molds, lower against bacteria), K, Na, Ca, benzoic acid,
benzoate, and other derivatives of benzoic acid (methyl and propyl esters
of p-hydroxybenzoic acid). Essential oils [30].
KEYWORDS
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• asepsis
• generally recognized as safe
• metabolism
• microbial growth
• microorganisms
• postharvest
• ultra-high temperature
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Chapter · March 2022

Cristian Torres-León Cristobal N Aguilar

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QUANTITATIVE METHODS AND

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© 2022 Apple Academic Press, Inc.

CIP data on file with US Library of C

ISBN: 978-1-77463-726-5 (hbk)

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Leonardo Sepúlveda Torre, PhD

Cristóbal Noé Aguilar, PhD

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editions of international books, and more. He has been awarded several

Casas Campillo (2008)” from the Mexican Society of Biotechnology and

Porteen Kannan, PhD

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former Editor-in-Chief of the International Journal of Chemoinformatics

the Canadian Research and Development Center of Sciences and Cultures

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2. Risk and Safety in Microbiology.............................................................. 11

5. Food and Diseases: What to Know in the Fight to

6. Microbial Products of Importance in the Food Industry....................... 75

7. Hygiene, Control, and Inspection in Foods............................................. 97

8. Molecular Methods for Microorganism Detection.................................111

9. New Molecular Methods for the Detection of Microorganisms........... 133

10. Fungal Production and Function of Phytase......................................... 145

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11. Advances in the Biotechnological Process for

Cristian Torres-León, Mónica L. Chávez-González, Cristóbal Noé Aguilar, and

12. Novel Methods of Food Preservation..................................................... 189

13. Strategies During Citrus Waste Utilization: Fermentative Route for

14. Impact of Functional Ingredients from Plant Food Byproducts on

15. Trends, Analytical Approaches, and Applications of the VITEK

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Cristóbal Noé Aguilar

Andrea Guadalupe Flores-Valdés

Alfredo Ivanoe García-Galindo

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Nathiely Ramírez Guzmán

Food Research Department, School of Chemistry, Autonomous University of Coahuila,

Alaa Kareem Niamah

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Bioprocesses and Bioproducts Research Group, Food Research Department,

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ITS internal transcribed spacer

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to be addressed, including basic microbiology, safety, food, pathogenic

i. I ncubation Temperature: Growth at optimal temperatures