Research Article

pISSN: 2345-5357
APPLIED FOOD BIOTECHNOLOGY , 2022, 9 (3):205-216 eISSN: 2423-4214
Journal homepage: www.journals.sbmu.ac.ir/afb

Effects of Spirulina platensis Addition on Growth of Lactobacillus

plantarum Dad 13 and Streptococcus thermophilus Dad 11 in Fermented
Milk and Physicochemical Characteristics of the Product
Joshua Christmas Natanael Luwidharto1 , Endang S. Rahayu1,2,3 , Dian Anggraini Suroto1,2 , Rachma
Wikandari1,2 , Ardhika Ulfah1 , Tyas Utami 1,2,3*
1-Department of Food and Agricultural Product Technology, Faculty of Agricultural Technology, Universitas Gadjah Mada, Yogyakarta,
Indonesia
2- Center for Food and Nutrition Studies, Universitas Gadjah Mada, Yogyakarta, Indonesia
3- University Center of Excellence for Research and Application on Integrated Probiotic Industry, Universitas Gadjah Mada, Yogyakarta,
Indonesia

Abstract Article Information

Article history:
Background and Objective: Spirulina platensis includes excellent bioactive - Received 8 Dec 2021
compounds, which provide health-promoting effects. However, use of Spirulina - Revised 29 Jan 2022
platensis in foods includes limitations due to its unpleasant flavor and taste for some - Accepted 5 April 2022
people. Fermented milk products include typical taste and flavor of fresh acid and can
be combined with Spirulina platensis. Probiotics of Lactobacillus plantarum Dad 13 Keywords:
▪ Bioactive compounds
and Streptococcus thermophilus Dad 11 isolated from traditional buffalo milks can be ▪ Cyanobacteria
used as starter culture strains for the fermented milks. This study investigated lactic ▪ Fermentation
acid bacterial growth, acid production and physicochemical characteristics of the ▪ Lactic acid bacteria
fermented milks with Spirulina platensis. ▪ Probiotics
Material and Methods: Milk fermentation was carried out using addition of various
*Corresponding author:
concentrations (0.15, 0.3, 0.45 and 0.6%) of Spirulina platensis powder and then
microbial cell growth, acid production and antioxidant activity were investigated. Tyas Utami*
Fermented milks with selected concentrations of Spirulina platensis were assessed Department of Food and
within 24 h of fermentation at 37 °C using single and mixed cultures to study various Agricultural Product Technology,
aspects of cell growth, acid production, viscosity, water holding capacity and color. Faculty of Agricultural Technology,
Universitas Gadjah Mada, Yogya-
Results and Conclusion: Increases of Spirulina platensis concentrations in fermented karta, Indonesia
milks increased the microbial cell growth, acid production and antioxidant activity. Center for Food and Nutrition
Studies, Universitas Gadjah Mada,
During milk fermentation by adding 0.3% of Spirulina platensis, cells propagated and Yogyakarta, Indonesia
total lactic acid bacteria and probiotic cell counts reached to respectively 8.73-9.19 and University Center of Excellence for
8.92 log CFU ml-1 after 24 h. The titratable acidity reached to 1.08% and pH decreased Research and Application on Integ-
to 4.41. Viscosity increased significantly after 12 h of fermentation, compared to the rated Probiotic Industry, Unive-
rsitas Gadjah Mada, Yogyakarta,
controls. Fermented milks with Spirulina platensis addition by Lactobacillus Indonesia
plantarum Dad 13 and Streptococcus thermophilus Dad 11 cultures can be developed
Tel: 00905446963446
as alternative functional fermented milk products. Fax: +6274589797
Conflict of interest: The authors declare no conflict of interest. E-mail:
tyas_utami@ugm.ac.id
How to cite this article
Natanael Luwidharto JC, Rahayu E, Suroto DA, Wikandari R, Ulfah A, Utami T. Effects of Spirulina platensis Addition on Growth of
Lactobacillus plantarum Dad 13 and Streptococcus thermophilus Dad 11 in Fermented Milk and Physicochemical
Characteristics of the Product. Appl Food Biotechnol. 2022; 9 (3): 205-216. http://dx.doi.org/10.22037/afb.v9i3.37013

1. Introduction
Spirulina (S.) platensis is a cyanobacteria species, which isoleucine are present at highest values [1]. Furthermore, that
includes compounds of excellent nutritional values. It has microorganism includes bioactive compounds such as
been investigated for high protein contents (~74%) and pigments (phycocyanin, chlorophyll and carotenoid) [2],
essential amino acids (EAA); of which, leucine, valine and vitamins A, B, E and K, Fe, Ca, Mn, Zn and unsaturated fatty

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acids (UFA) [3]. Compounds of S. platensis have been 2. Materials and Methods
shown to provide health-promoting effects such as
antioxidant [4], anti-cancer, anti-inflammatory, anti-viral 2.1 Materials
and immune system booster effects [5]. Introduction of S. S. platensis powder (Spirulina TN57, Indonesia) and fresh
platensis in food products includes public resistance due to milk (PT Sukanda Djaya, Indonesia) were purchased from
the unpleasant flavor and taste of S. platensis for some the local supermarkets. The L. plantarum Dad 13 and S.
people, caused by the oxidation of UFAs and minerals that thermophilus Dad 11 were provided by the Food and
produces metallic off-flavors [6]. Fermented milk products Nutrition Culture Collection (FNCC), Center for Food and
include typical taste and flavor of fresh acids, resulted from Nutrition Studies, Universitas Gadjah Mada, Yogyakarta,
carbonyl compounds [7]. Thus, it may be combined with S. Indonesia. De Mann Rogosa Sharpe (MRS) (Merck,
platensis. Furthermore, fermented milk is the most popular Germany), bacteriological agar (Oxoid, USA) and calcium
functional food with high acceptance and is regularly carbonate (CaCO3) (Merck, Germany) were used for MRS
consumed by people on long-term diets [3]. Several studies media. Phenolphthalein (PP), 2,2’-diphenyl-1-1-picrylh-
have assessed benefits of S. platensis in dairy products. ydrazyl (DPPH) and molybdate reagent were purchased from
Beheshtipour et al. [6] reported that the addition of 1% of Sigma-Aldrich, USA. Sucrose (PT, Sugar Group Company,
Arthrospira (A.) platensis powder into yogurts increased cell Indonesia) and skim milk (PT, Mirota KSM, Indonesia) were
growth of the probiotic mixture of Lactobacillus (L.) purchased from the local supermarkets.
acidophilus LA-5 and Bifidobacterium lactis BB-12
2.2 Bacterial Strain and Growth Condition
(reaching up to 8.42 log CFU ml-1). Pan-utai et al. [8] showed
that yogurt fermentation by 0.5% of A. platensis IFRPD 1182 Culture stock was stored at -20 °C as 1:1 mixture with
and mixed cultures of L. bulgaricus YC-X11 and 20% of sterile aqueous sucrose solution and 10% of skim
Streptococcus (S.) thermophilus YC-X11 produced a higher milk. Culture was inoculated in MRS broth and incubated at
acidity than that the control yogurt did. Moreover, addition 37 °C for 24 h. Then, culture was inoculated using MRS agar
of S. platensis into the fermented milk can improve its deep tube at similar conditions and rejuvenated every two
physical characteristics such as increasing viscosity and weeks as working cultures. For starter culture preparation,
water holding capacity (WHC) [9-11] and chemical cultures from working cultures were inoculated in MRS
characteristics such as contents of minerals and antioxidants broth media and incubated at 37 °C for 24 h. Then, cultures
[12]. However, ability of lactic acid bacteria (LAB) to utilize were reinoculated in skim media and incubated at 37 °C for
S. platensis biomass varies within various microbial strains 24 h. The LAB cell number in starter culture was calculated
and fermentation conditions. before inoculation into milk and expressed as colony-
Usually, LAB are widely used in Indonesian traditional forming unit (CFU) ml-1.
fermented foods. Several studies have isolated indigenous 2.3 Milk Fermentation
LAB from Indonesian fermented foods [13-16]. In fact, L.
The first stage included milk fermentation with the
plantarum Dad 13 and S. thermophilus Dad 11 are
addition of various concentrations (0.15, 0.3, 0.45 and 0.6%)
indigenous LAB isolated from dadih, an Indonesian
of S. platensis powder. Fermented milk was prepared based
fermented buffalo milk. The authors’ recent studies reported
on the protocol by Barkallah et al. [12] with some
that L. plantarum Dad 13 potentially served as a probiotic
modifications. Fresh milk, skimmed milk (2%), sucrose (8%)
strain because it could survive in volunteers’ digestive tract,
and S. platensis powder were mixed, pasteurized at 80 ºC for
increase populations of Lactobacilli and L. plantarum in
30 min, cooled down and inoculated with the starter cultures
faeces of healthy people and prevent growth of pathogenic
(1% v v-1). Fermentation proccess was carried out at 37 °C
Escherichia coli [14,17]. Safety aspects of this probiotic
for 24 h using a single culture (either L. plantarum Dad 13 or
strain have been studied [15]. It can be inoculated as a single
S. thermophilus Dad 11) and mixtures of the two cultures.
starter or mixed culture to produce probiotic fermented milks
Cell count, titratable acidity (TA), pH and antioxidant
[18,19]. Mixed cultures of L. plantarum Dad 13 and S.
activity of the milk were assessed before and after the
thermophilus Dad 11 have been used as starter cultures in
fermentation (24 h). The selected concentration of S.
fermented milks, including sensory acceptance similar to that
platensis powder was further studied for its fermentation
of commercial yogurts [20]. These cultures show their
patterns (second stage). The cell counts, TA, pH, viscosity
antioxidant activity and ability to ferment black soymilks
and color were assessed every 6 h during the fermentation.
[13]. Effects of S. platensis addition into the fermented milks
The WHC, minerals and phycocyanin contents were
with L. plantarum Dad 13 and S. thermophilus Dad 11 are
analyzed after 24 h of fermentation. For the milk
still unknown. Therefore, the objective of the current study
fermentation using mixed cultures, numbers of LAB and L.
was to investigate physicochemical characteristics and
plantarum were enumerated using MRS and LPSM agar
viabilities of L. plantarum Dad 13 and S. thermophilus Dad
media, respectively.
11 in fermented milks with S. platensis.

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2.4 Enumeration of the Bacterial Population [19,21] 2.8 Color Analysis of the Fermented Milks [8]
Number of LAB was calculated using serial dilution and Color of the samples were assessed using colorimeter
pour plate methods and MRS agar containing 0.5% CaCO3. (Konica Minolta, Chroma Meter, CR400, Japan). The
Colonies with clear zones were counted after incubation at instrument was standardized using standard white plates.
37 °C for 48 h and expressed in colony-forming units (log Color analysis was expressed from three values of lightness
CFU ml-1). Enumeration of probiotic L. plantarum in mixed (L*) as 0−100 (black to white), a* as green (-) to red (+) and
cultures was carried out using spread plate method on L. b* as blue (-) to yellow (+).
plantarum selective media (LPSM) agar. The LPSM agar
2.9 Mineral Content Analysis of the Fermented Milks
was prepared based on a method by Bujalance et al. [21].
[24,25]
Colonies of L. plantarum were counted and expressed as
colony-forming units (log CFU ml-1) after incubation at 37 Calcium (Ca), phosphorus (P) and iron (Fe) in the
°C for 48 h. fermented milk products were analyzed. A sample of 5 ml
was incinerated using muffle furnace at 550 °C for 6 h. Then,
2.5 Assessment of Acid Production [19] 50 ml of HNO3 (1:3) were added to the ash, which was
Acid production was assessed using titratable acidity (TA) crushed until dissolved. Solution was filtered and the
and pH measurements. The TA was measured via sample supernatant was collected. The Ca content was assessed
titration using 0.1 N NaOH with 1% of phenolphthalein as using titrimetric method. Moreover, P and Fe contents were
indicator. Moreover, pH measurement was carried out using assessed using spectrophotometric methods.
pH meter (Eutech pH, USA). Calibration with standard
2.10 Phycocyanin Content Analysis of the Fermented
buffer (pH 4 and 7) was carried out before the analysis.
Milks [8]
2.6 2,2'-diphenyl-1-picrylhydrazyl Radical Scavenging Sample (1 ml) was centrifuged (Centrifuge 5810 R,
Assay [22] Eppendorf AG, Hamburg, Germany) at 5000 rpm for 10 min.
Sample preparation began with addition of 1 ml of sample Then, the supernatant was mixed with 1 ml of distilled water
into 5 ml of 70% methanol using shaker water bath (Sibata and sonicated (Eyela Sonicator Cleaner, Singapore) for 30
WS-240, Japan) at 120 rpm for 1 h at room temperature. min. Supernatant solution was centrifuged (Centrifuge 5810
Then, sample was macerated at 4 °C for 24 h in dark. This R, Eppendorf AG, Hamburg, Germany) at 10000 rpm for 5
was centrifuged (Centrifuge 5810 R, Eppendorf AG, min and the supernatant was collected and assessed using
Hamburg, Germany) at 3000 g for 15 min. The crude extract spectrophotometer (Shimadzu UV-1601, Japan) at 615 and
was collected and stored at -20 °C until the antioxidant 652 nm. Phycocyanin content was calculated using the
activity analysis was carried out. The antioxidant activity following equation:
was assessed based on the extract ability to scavenge 2,2’- (𝜆 615 𝑛𝑚 − 0.474.𝜆 652 𝑛𝑚)
𝑃ℎ𝑦𝑐𝑜𝑐𝑦𝑎𝑛𝑖𝑛 = Eq. (3)
5.34
diphenyl-1-picrylhydrazyl (DPPH). Sample extract (1 ml)
was added to 3 ml of 0.1 mM DPPH solution and incubated 2.11 Statistical Analysis
for 30 min in dark. Methanol was used as control. The Results were provided as means ±SD (standard deviation).
absorbance was measured using spectrophotometer Analysis was carried out using one-way ANOVA and
(Shimadzu UV-1601, Japan) at 517 nm. The radical Duncan's multiple comparison test if significant differences
scavenging activity was calculated using the following were detected (p < 0.05). Furthermore, statistical analysis
equation: was carried out using IBM SPSS Statistics Software v.26
𝑠𝑎𝑚𝑝𝑙𝑒𝑠 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒
𝑅𝑆𝐴 (%) = (1 − ) × 100% Eq. (1) (IBM, USA) (n = 4).
𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒

2.7 Viscosity and Water Holding Capacity Assessments 3. Results and Discussion
[23]
3.1 Growth and Acid Production of LAB in Fermented
Viscosity of the samples were assessed using Brookfield
Milk with Various Addition of Spirulina platensis
Viscometer (Model DV2T, Brookfield Engineering
Laboratories, USA) after stirring the product for 30 s at 4 °C. The initial LAB count was approximately 6.8 log CFU ml-
1
Samples were assessed using spindle no. 4 at 60 rpm. For the and reached to 8.49 log CFU ml-1 after 24 h of milk
assessment of WHC, 5 g of the samples (Y) were transferred fermentation at 37 C for single and mixed cultures (Table
into a 15-ml tube and centrifuged at 4500 g for 10 min at 4 1). The higher the addition of S. platensis, the greater the
◦
C (Centrifuge 5810 R, Eppendorf AG, Hamburg, Germany). number of LAB in the final products. Addition of S. platensis
The pellet was weighed (W) and WHC of the samples was significantly increased (p<0.05) the cell growth.
calculated using the following equation:
𝑊𝐻𝐶 (%) = (1 − 𝑊 𝑌
) 𝑥 100 Eq. (2)

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Table 1. Cell count, titratable acidity, pH and antioxidant activity of the fermented milks with various quantities of Spirulina
platensis biomasses, added before the fermentation (37 °C, 24 h)

Cell Counts Titratable Radical Scavenging Activity (%)

Starter Cultures Spirulina pH-value
(log CFU ml −1) Acidity (%) Initial Final
0% 8.33±0.13a 0.88±0.02a 4.96±0.01a 16.02±0.99a 34.05±1.81d
Lactobacillus 0.15% 8.65±0.10b 1.02±0.03b 4.85±0.04b 17.97±1.14 ab 36.76±1.50e
plantarum Dad 13 0.3% 8.77±0.11bc 1.04±0.06b 4.77±0.04c 19.43±0.50bc 41.75±1.66f
Total LAB 0.45% 8.81±0.09c 1.19±0.02c 4.55±0.02d 20.86±0.89 c 44.03±1.76g
0.6% 9.10±0.06d 1.28±0.03d 4.53±0.01d 20.90±0.97c 44.69±2.08g
0% 8.50±0.12a 0.78±0.08a 4.78±0.07a 16.02±0.99a 30.57±0.76d
Streptococcus
0.15% 8.79±0.08b 0.94±0.03b 4.52±0.07b 17.97±1.14 ab 33.32±1.25ef
thermophilus Dad
0.3% 8.86±0.07bc 1.05±0.03c 4.33±0.02c 19.43±0.50bc 35.22±2.14f
11
0.45% 9.00±0.07cd 1.23±0.02d 3.89±0.04d 20.86±0.89c 40.58±2.44g
Total LAB
0.6% 9.08±0.12d 1.27±0.01d 3.81±0.01e 20.90±0.97c 42.12±0.63g
8.63±0.05a 0.85±0.02a 4.61±0.06a 16.02±0.99a 31.09±2.77d
0%
(8.21±0.11a)
8.79±0.04b 1.10±0.06b 4.33±0.02b 17.97±1.14ab 35.01±2.18e
0.15%
Mixed Cultures (8.38±0.21b)
Total LAB 8.92±0.07c 1.26±0.02c 4.27±0.03c 19.43±0.50bc 37.10±1.92e
0.30%
(Lactobacillus (8.66±0.07c)
plantarum) 9.07±0.09d 1.33±0.03d 3.75±0.03d 20.86±0.89c 42.23±1.79f
0.45%
(8.76±0.10c)
9.16±0.04e 1.34±0.03d 3.66±0.04e 20.90±0.97c 42.63±1.25f
0.60%
(8.80±0.05d)
* Results are provided as mean ± SD. Results for each strain with different superscripts were significantly different (p<0.05) from Duncan's multiple range
test. Cell counts for single culture were measured as total LAB using MRS medium, while the mixed cultures were measured as total LAB using MRS
medium and Lactobacillus plantarum using LPSM medium. The values in parentheses showed the cells count of Lactobacillus plantarum in fermented milk
with Spirulina platensis addition.

Cell count of probiotic L. plantarum Dad 13 was slightly Production of acid decreased pH. Results showed that the
lower than the total count of LAB. Beheshtipour et al. [6] higher the S. platensis addition was, the lower pH of the
reported that cell growth of the probiotic mixtures of L. fermented milk was. Moreover, addition of more than 0.3%
acidophilus LA-5 and B. lactis BB-12 in yogurts with A. S. platensis resulted in pH lower than 4.0. Pan-utai et al. [8]
platensis addition (up to 1%) was significantly greater than reported that yogurt fermentation by mixed cultures of L.
that in the control, which reached to 8.42 log CFU ml-1 after bulgaricus YC-X11 and S. thermophilus YC-X11 with 0.5%
fermentation at 40 °C. Results reported by Yamaguchi et al. A. platensis IFRPD 1182 addition at 40 °C for 4 h produced
[26] showed mild increases in bacterial population of the 0.78% TA and pH 4.21. Furthermore, TA and pH of control
mixed cultures of L. acidophilus LA-5, B. lactis BB-12 and samples were 0.55% and 4.39, respectively. Bchir et al. [10]
S. thermophilus in freeze-dried yogurts by addition of S. reported similar results as TA and pH of yogurts produced
platensis (3.4 × 107 CFU g-1), compared to control yogurt using mixed cultures L. bulgaricus and S. thermophilus PAL
(2.2 × 107 CFU g-1). This study showed a higher population YOG 3-30 D and added with 0.5% of S. platensis were 0.8%
of LAB in fermented milk with addition of S. platensis. and 4.7, respectively. Higher TA values were reported by
Fermentation of probiotic L. plantarum Dad 13 was optimum Agustini et al. [11] as TA and pH of yogurts with 1% of S.
at 37 °C and hence called mesophilic LAB [19]. Celekli et platensis addition using mixed cultures of L. bulgaricus, S.
al. [27] reported that growth of S. thermophilus in ayran thermophilus and L. acidophilus were 1.56% and 4.71,
added with various concentrations of S. platensis was not respectively after 6 h of fermentation at 43 °C. Positive
significantly different from that without addition of S. effects of S. platensis on cell growth and metabolic activities
platensis, which was 8.78–8.9 log CFU ml−1 after might be due to the nutritional compounds of S. platensis.
fermentation at 42 °C. It can be assumed that the ability of The S. platensis biomass includes amino acids (AA) [1], B-
LAB to utilize S. platensis biomass for their growth varies group vitamins and various minerals such as Mg, Fe and Mn
within various strains and fermentation conditions. [3], which include potentials as growth factors for L.
Natueally, LAB utilize lactose and other sugars for their plantarum Dad 13 and S. thermophilus Dad 11. Moreover, S.
growth and metabolic activities, producing acids. The initial platensis biomass contains exopolysaccharides, hypoxa-
TA was nearly 0.2%. After the milk fermentation without S. nthine and adenine. These elements can stimulate growth and
platensis addition, TA increased to 0.77–0.88%. Higher activity of LAB [3].
additions of S. platensis (0.15, 0.3, 0.45 and 0.6%) resulted
in greater acid productions. The TA reached to 1.2 and 1.33%
for single and mixed cultures, respectively (Table 1).

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3.2 Antioxidant Activity of the Fermented Milks with 3.3 Profile of Milk Fermentation with Addition of 0.3%
Various Concentrations of Spirulina platensis of S. platensis: Cell Growth and Acid Production
Results showed that higher S. platensis addition led to a Cell growth patterns of a single culture of L. plantarum
higher antioxidant activity of the milk (Table 1). Milk Dad 13 and S. thermophilus Dad 11 in fermented milks with
fermentation by single or mixed cultures increased 0.3% S. platensis biomass were similar to those without
antioxidant activity of nearly two folds after 24 h of addition of S. Platensis; however, higher cell populations
fermentation at 37 °C. However, increases in antioxidant were achieved (Figure 1).
activity during the fermentation was similar within various 9.50

Cell Counts (log CFU ml-1)

(A) 0% 0,3%
concentrations of S. platensis and starter cultures. It is 9.00
noteworthy that antioxidant activity of S. platensis did not
8.50
change during fermentation. These results were in line with
8.00
phycocyanin contents of this study. The phycocyanin
contents were relatively constant during milk fermentation in 7.50
single and mixed cultures. Liu et al. [4] showed that 7.00
antioxidant activity of the fermented milks containing 0.6% 6.50
of S. platensis powder using mixed cultures of L. 0 6 12 18 24
acidophilus, B. bifidum, L. casei, B. infantis, B. longum and Fermentation Time (h)
L. lactis was ±33% after 48 h of fermentation. The current
results showed higher antioxidant activities at shorter times 9.50
0% 0,3%
Cell Counts (log CFU ml-1)
(B)
of fermentation (24 h). Higher antioxidant activities were 9.00
also reported by Barkallah et al. [12], who stated that yogurts
8.50
with S. platensis addition of 0.25% using mixed cultures of
8.00
L. bulgaricus YC-X11 and S. thermophilus YC-X11
included an antioxidant activity of 52.41% after 4 h of 7.50
fermentation at 43 °C. 7.00
During the fermentation, antioxidant activity of the 6.50
fermented milks without S. platensis addition significantly 0 6 12 18 24
increased from 16.02 to 30.57–34.05%. It could occure Fermentation Time (h)
because metabolic activity of the LAB produced bioactive
compounds that acted as antioxidants. Fermentation of black Figure 1. Cell growth patterns during milk fermentation
soymilks using L. plantarum Dad 13 and S. thermophilus with 0.3% of Spirulina platensis biomass using single
Dad 11 increased their antioxidant activities due to the free cultures of (A) Lactobacillus plantarum Dad 13 and (B)
Streptococcus thermophilus Dad 11 (37 °C, 24 h)
aglycone isoflavone, produced from isoflavone
glycoside hydrolysis by -glucosidase [13]. Taha et al. [28] Moreover, mildly different growth patterns were seen in
reported that fermentation of buffalo milk yogurts by mixed milk fermentation with 0.3% S. platensis cultivated by the
cultures of yogurt starters, L. acidophilus 20552 ATCC and mixed culture (Figure 2). The LAB in milk fermentation with
L. helveticus CH 5 produced peptides with high antioxidant 0.3% S. platensis faster propagated and reached to the
and antibacterial activities. It occured because these starter stationary phase, compared to the control sample which
cultures produced enzymes that hydrolyzed milk proteins propagated rapidly until Hour 12 of the fermentation, while
into bioactive peptides. Further studies are needed to the control sample propagated until hour 18 of fermentation.
investigate proteolytic activity and formation of the bioactive Similar acid productions were achieved in fermented milks
peptides in fermented milks using L. plantarum Dad 13 and inoculated with single and mixed cultures. Acid production
S. thermophilus Dad 11. The present results showed that showed similar profiles of fermentation without and with S.
fermented milks containing various quantities of S. platensis platensis addition; however, higher TA and lower pH were
biomass increased cell growth, acid production and achieved with addition of Spirulina biomass (Table 2). These
antioxidant activity of samples after 24 h of fermentation. results revealed that addition of S. platensis supported cell
However, fermented milks containing more than 0.3% S. growth and metabolic activities. This was seen possibly
platensis biomass included a final pH of less than 4.0, which because the cyanobacteria provided AAs and minerals that
was too acidic and could affect physical and sensory were essentials for these cultures.
characteristics of the product. Rani et al. [29] demonstrated
that pH less than 4.0 could lead to body and texture defects
such as gel shrinkage and syneresis. Therefore, addition of
0.3% S. platensis into milks prior to fermentation was
selected for further studies.
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9.50 5.00
Cell Counts (log CFU ml-1)

(A) Total LAB L. plantarum (A)

0% 0.3%

Viscosity (Pa. s)
9.00
4.00
8.50
3.00
8.00
7.50 2.00
7.00 1.00
6.50
0 6 12 18 24 0.00
0 6 12 18 24
Fermenation Time (h) L. plantarum
Fermentation time (h)

9.50 Total LAB L. plantarum

(B ) 5.00
Cell Counts (log CFU ml-1)

9.00 (B) 0% 0.3%

Viscosity (Pa. s)
4.00
8.50
3.00
8.00
2.00
7.50
1.00
7.00
0.00
6.50
0 6 12 18 24
0 6 12 18 24
Fermentation Time (h) Fermentation time (h)
L. plantarum

(C) 5.00 0% 0.3%

Figure 2. Cell growth patterns during milk fermentation (A)
4.00
Viscosity (Pa. s)

without Spirulina platensis and (B) with 0.3% Spirulina

platensis addition using mixed cultures of Lactobacillus 3.00
plantarum Dad 13 and Streptococcus thermophilus Dad 11
(37 °C, 24 h) 2.00
1.00
Biomass of S. platensis includes 17 AAs that can support
0.00
growth of LAB [1]. Culture of L. plantarum needs arginine, 0 6 12 18 24
glutamic acid, leucine, valine and cysteine or methionine,
while S. thermophilus needs histidine and cysteine or Fermentation time (h)
methionine [30]. Furthermore, S. platensis includes several
minerals such as Mg, Fe and Mn [3] needed by LAB for the Figure 3. Viscosity patterns during milk fermentation with
enzymatic reactions [31]. 0.3% Spirulina platensis addition using single cultures of (A)
Lactobacillus plantarum Dad 13 and (B) Streptococcus
3.4 Viscosity during Milk Fermentation with 0.3%
thermophilus Dad 11 and (C) mixed cultures of
Spirulina platensis Addition Lactobacillus plantarum Dad 13 and Streptococcus
Viscosity and WHC are important physical parameters in thermophilus Dad 11 (37 °C, 24 h)
assessing quality of fermented milk products. Viscosity
profiles during milk fermentation showed a similar pattern Bchir et al. [10] reported that addition of 0.3% of dried S.
using either single or mixed cultures (Figure 3). Viscosity platensis into yogurts with L. bulgaricus and S. thermophilus
increased significantly (p < 0.05) after 12 h of fermentation. PAL YOG 3-30 D significantly increased the viscosity to
After this time, viscosity of the fermented milks with S. 0.85 Pa.s, compared to the control (0.70 Pa.s) after 4 h of
platensis addition was almost two folds greater than those fermentation at 42 °C. Atallah et al. [9] stated that addition
without S. platensis addition. Viscosity of the fermented of 1% of S. platensis into yogurts with a mixture culture of
milks with single and mixed cultures increased from 2.04- L. bulgaricus and S. thermophilus respectively increased
2.44 Pa.s (without S. platensis) to 3.73-4.05 Pa.s (with 0.3% viscosity and WHC to 16.3 Pa.s and 56% after fermentation
S. platensis). Moreover, WHC of the fermented milks with at 42 °C. Results of the current study demonstrated higher
0.3% S. platensis (64-67%) was mildly higher after 24 h of WHC values than those results of Pan-utai et al. did [8], who
fermentation, compared to the controls (60-62%) (Figure 4). reported that yogurts with mixed cultures of L. bulgaricus
YC-X11 and S. thermophilus YC-X11 as well as 0.3% dried
S. platensis addition included WHC values of nearly 55%
after 4 h of fermentation at 43 °C.

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Table 2. Acid production during milk fermentation with 0.3% Spirulina platensis addition at 37 °C

Fermentation Titratable Acidity (%) pH

Strains
Time (h) 0% 0.3% 0% 0.3%
0 0.23±0.01a 0.23±0.01a 6.71±0.08a 6.71±0.08a
6 0.39±0.07b 0.45±0.07b 5.83±0.01b 5.77±0.16b
Lactobacillus plantarum c
12 0.53±0.02 0.57±0.04c 5.49±0.09c 5.42±0.06c
Dad 13
18 0.78±0.06d 0.90±0.04e 5.28±0.04d 5.05±0.01e
de
24 0.84±0.01 0.99±0.06f 4.98±0.02f 4.6±0.01g
0 0.23±0.01a 0.23±0.01a 6.71±0.08a 6.71±0.08a
6 0.41±0.04b 0.44±0.03b 5.93±0.06b 5.79±0.17c
Streptococcus c
12 0.53±0.01 0.68±0.05d 5.58±0.05d 5.30±0.20e
thermophilus Dad11
18 0.83±0.06e 0.97±0.02g 5.30±0.10e 4.90±0.14g
f
24 0.91±0.04 1.06±0.03h 4.81±0.01g 4.45±0.08i
0 0.23±0.01a 0.23±0.01a 6.71±0.08a 6.71±0.08a
6 0.35±0.02b 0.50±0.03c 5.94±0.02b 5.88±0.04b
d
Mixed Cultures 12 0.60±0.05 0.71±0.05e 5.56±0.04c 5.33±0.01d
18 0.89±0.02f 0.97±0.03g 5.13±0.07f 4.72±0.06f
g
24 1.00±0.03 1.08±0.02h 4.64±0.01gh 4.41±0.05g
* Mixed cultures are mixture of Lactobacillus plantarum Dad 13 and Streptococcus thermophilus Dad 11. Results are provided as mean ± SD. Results in
each strain with different superscripts were significantly different (p<0.05) from Duncan's Multiple Range Test

b
69.00
a b
67.00
a
65.00
a
WHC (%)

63.00
a 0%
61.00 0.3%

59.00

57.00

55.00
Lactobacillus Streptococcus Mixed Cultures
plantarum Dad 13 thermophilus Dad 11

Figure 4. Water holding capacity of the fermented milks with 0.3% Spirulina platensis addition after 24 h of fermentation at
37 °C. Values are expressed as mean ±SD. Values of the strains with different superscripts are significantly different (p<0.05)

The present results were similar to those from yogurts greatly to viscosity, stabilizing and emulsifying
with addition of gelling agent, stabilizer and emulsifier. characteristics of the polysaccharides [34].
Sahan et al. [32] reported that yogurts with the addition of
3.5 Color Changes during Milk Fermentation with 0.3%
hydrocolloid ß-glucan concentrates up to 1% included
Spirulina platensis Addition
viscosity of nearly 4 Pa.s after fermentation at 43 °C. By
adding S. platensis biomass, fermented milk products Color changes were seen during milk fermentation, when
potentially do not need addition of gelling agents, stabilizers S. Platensis was added. Table 3 shows that L*, a* and b*
and emulsifiers to further process. Addition of 0.3% S. values significantly (p<0.05) increased until hour 18 of
platensis increased viscosity and WHC of the fermented fermentation and then was relatively constant until the end of
milks. This might be due to the exopolysaccharides produced fermentation in single and mixed cultures. Color of the
by S. platensis. Sed et al. [33] reported that the addition of fermented milks changed from blue-green to green after 24 h
0.5% of exopolysaccharides from S. platensis to cream of fermentation. Martelli et al. [35] reported that L*, a* and
products could increase their stabilizing and emulsifying b* values increased in skimmed milk media with mixed
characteristics, resulting in better texture characteristics. cultures of S. thermophilus, L. lactis and L. helveticus with
Polysaccharides of S. platensis included rhamnose as the 0.25% S. platensis powder addition after 24 h of fermentation
major component and reached to 53%, which contributed at 37 °C, meaning that color changed to green.

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Table 3. Color Changes during milk fermentation with 0.3% Spirulina platensis addition for 24 h at 37 °C

Fermentation Color
Strains
Time (h) L* a* b*
0 66.61±1.19a -7.63±0.10a 2.94±0.28a
6 66.02±0.80a -7.39±0.09b 2.91±0.16a
Lactobacillus plantarum Dad 13 12 67.27±0.53a -7.35±0.17b 4.03±0.12b
18 74.97±1.01b -6.83±0.15c 6.03±0.05c
24 74.64±0.88b -6.69±0.04c 7.11±0.68d
0 66.61±1.19a -7.63±0.10a 2.94±0.28a
6 66.10±0.65a -7.63±0.02a 3.18±0.50a
Streptococcus thermophilus Dad
12 66.92±0.69a -7.02±0.07b 4.63±0.15b
11
18 74.25±0.97b -6.89±0.07c 6.92±0.10c
24 74.68±0.67b -6.67±0.08d 7.35±0.13d
0 66.61±1.19a -7.63±0.10a 2.94±0.28a
6 66.30±0.58a -7.77±0.07a 3.27±0.37a
Mixed Cultures 12 67.03±0.74a -7.32±0.24b 6.21±0.47b
18 74.74±1.05b -6.75±0.13c 6.93±0.31c
24 74.40±0.55b -6.61±0.05c 7.39±0.06c
* Mixed cultures are mixture of Lactobacillus plantarum Dad 13 and Streptococcus thermophilus Dad 11. Results are provided as mean ± SD. Results in
each strain with different superscripts are significantly different (p<0.05) by Duncan's multiple range test.

Barkallah et al. [12] reported that yogurts containing milks with 0.3% S. platensis addition and as 10, 7.5 and 57%
0.25% of S. platensis biomass included low a* and b* values, for the controls after 24 h of fermentation. Barkallah et al.
showing a light green color. [12] stated that yogurts containing 0.25% of S. platensis and
Color changes of the fermented milks containing 0.3% of a mixed culture of L. bulgaricus and S. thermophilus
S. platensis might be due to chlorophyll degradation caused included higher mineral contents than yogurts without S.
by decreases in pH during fermentation. During Platensis. This was 107.7 mg 100 g-1 for calcium and 9.01
fermentation, pH of single and mixed cultures decreased mg 100 g-1 for iron. Drago and Valencia [37] reported that
from 6.71 to 4.41-4.6. At acidic conditions, hydrogen ions calcium and iron contents respectively increased by nearly
could transform chlorophylls to pheophytins by substituting 7.37 and 36.08% in yogurts produced by a mixed culture of
magnesium ions in the porphyrin ring and releasing a phytol L. bulgaricus and S. thermophilus after 6 h of fermentation
group to pheophorbide. Pheophytin and pheophorbide are at 42 °C. Obadina et al. [38] showed that mineral contents of
products of chlorophyll degradation, producing an olive- calcium and iron respectively increased nearly 13.55 and 21,
green color [36]. 43% after 24 h of fermentation in soymilks fermented by a
mixture of L. acidophilus, S. cremoris, Micrococcus aureus
3.6 Mineral Contents of the Fermented Milks with 0.3%
and S. lactis. Biomass of S. platensis is known to include high
S. platensis Addition
mineral contents such as calcium, phosphorus, potassium,
Selected minerals such as calcium, phosphorus and iron and iron as well as trace minerals such as magnesium, zinc
were assessed in fermented milks with S. platensis addition. and selenium [3]. Therefore, fermented milks containing
Mineral contents of the fermented milks with 0.3% S. 0.3% of S. platensis biomass included higher mineral
platensis addition increased significantly (p < 0.05) after 24 contents than that the controls did. The current results
of h fermentation in single and mixed cultures, compared to showed increases in mineral contents during the fermentation
the controls (Table 4). Calcium, phosphorus and iron process.
respectively increased as 13, 14 and 112% for the fermented

Table 4. Mineral contents of the fermented milks with 0.3% Spirulina platensis addition (37 °C, 24 h)

Spirulina Calcium (mg 100g−1) Phosphorus (mg 100g−1) Iron (mg 100g−1)
Strains
(%) Initial Final Initial Final Initial Final
Lactobacillus 0% 112.60±3.26a 123.51±1.77b 97.44±0.17a 105.30±1.25c 0.55±0.01a 0.87±0.01c
plantarum
0.3% 119.58±0.69b 135.33±1.60c 102.11±0.24b 116.51±0.81d 0.64±0.03b 1.37±0.03d
Dad 13
Streptococcus 0% 112.60±3.26a 124.35±2.72b 97.44±0.17a 104.74±0.65c 0.55±0.01a 0.86±0.01c
thermophilus
0.3% 119.58±0.69b 135.91±2.82c 102.11±0.24b 116.03±0.21d 0.64±0.03b 1.33±0.02d
Dad 11
Mixed 0% 112.60±3.26a 124.27±1.15b 97.44±0.17a 104.82±0.29c 0.55±0.01a 0.87±0.01c
Cultures 0.3% 119.58±0.69b 136.09±1.33c 102.11±0.24b 116.63±0.77d 0.64±0.03b 1.39±0.02e
* Mixed cultures are mixture of Lactobacillus plantarum Dad 13 and Streptococcus thermophilus Dad 11. Results are provided mean ± SD. Results in
each strain with different superscripts are significantly different (p<0.05) from Duncan's multiple range test

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This could be due to the intracellular quantities of cultures (Figure 5). This finding was similar to the finding by
minerals, which were accumulated by L. plantarum Dad 13 Pan-utai et al. [8], who showed that yogurts with S. platensis
and S. thermophilus Dad 11 during fermentation. Boyaval et addition at a concentration of 0.1–0.3% and a mixed culture
al. [39] reported that the intracellular concentrations of Ca2+ of L. bulgaricus YC-X11 and S. thermophilus YC-X11
increased at the logarithmic phase of S. lactis. In addition, included a phycocyanin content of approximately 0.2 mg g-1
Obadina et al. [38] demonstrated that increases in mineral (db). Barkallah et al. [12] reported that the addition of 0.25%
contents of the fermented soymilks were affected by S. platensis biomass to yogurts using a mixed culture of L.
minerals released from chelated complex compounds bulgaricus YC-X11 and S. thermophilus YC-X11 included a
through the microbial activities. phycocyanin content of 0.297 mg g-1 (db). Wu et al. [40]
demonstrated that phycocyanin included a low stability at
3.7 Phycocyanin Contents of the Fermented Milks with
temperatures ≥ 45 °C and pH 3-4. In this study, fermented
0.3% S. platensis Addition
milks were incubated at 37 °C with pH 4.46-4.6 after 24 h of
Phycocyanin is a natural proteinaceous pigment of S. fermentation. Therefore, phycocyanin contents of the
platensis with bioactive and antioxidant activities. fermented milks with S. platensis were relatively stable
Phycocyanin contents were relatively constant after milk during fermentation.
fermentation [0.220-0.248 mg g-1 (db)] in single and mixed
0.30
a a a
a a
0.25 a
Phycocyanin (mg g db-1)

0.20

0.15 Inital
Final
0.10

0.05

0.00
Lactobacillus Streptococcus Mixed Cultures
plantarum Dad 13 thermophilus Dad 11

Figure 5. Phycocyanin contents of the fermented milks with 0.3% Spirulina platensis addition (37 °C, 24 h). Values are
shown as mean ±SD. Values of the strains with similar superscripts are not significantly different (p < 0.05)

5. Acknowledgements
4. Conclusion
This study was financially supported by the Ministry of
Increased quantities of S. platensis powder increased cell Research and Technology/National Agency for Research and
growth (up to 9 log CFU ml-1) and acid production (titratable Innovation (KEMENRISTEK-BRIN) through the Higher
acid and pH of 1.34% and 3.66, respectively) in milks Education of Research and Development Research Scheme
fermented by L. plantarum Dad 13 and S. thermophilus Dad (PPUPT) with the contract numbers of 8/E1/KPT/2021 and
11. Addition of 0.3% of S. platensis powder significantly 3572/E4/AK.04/2021, and agreement contract numbers of
improved the viscosity to 4.05 Pa.s during milk fermentation. 6/E1/KP.PTNBH/2021, 006/E4/AK.04.PTNBH/2021 and
These probiotic fermented milks with 0.3% of S. platensis 2165/UN1/DITLIT/DIT-LIT/PT/2021 and 6648/UN1-
included 8.9 log CFU ml-1 L. plantarum with 35-40% /DITLIT/DIT-LIT/PT/2021. This study was technically
antioxidant activity. This study can be addressed to develop supported by the Center of Excellence for Research and
functional fermented milks. Further studies are necessary to Application on Integrated Probiotic Industry, Universitas
investigate the cell viability and physicochemical charact- Gadjah Mada, with contract numbers of 006/E4/AK.04.PTN-
ristics during storage. BH/2021 and 6648/UN1/DITLIT/DIT-LIT/PT/2021.
6. Conflict of Interest

The authors report no conflicts of interest.

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syneresis in yoghurt: A review. Indian J Dairy Biosci. 2012; 23. https://doi.org/10.1111/j.1365-2621.2002.tb08870.x
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Holzapfer WH WB, editor. John Wiley and Sons Ltd; 2014: p. https://doi.org/10.1016/S0189-7241(15)30081-3
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 ‫‪Research Article‬‬
‫‪pISSN: 2345-5357‬‬
‫‪eISSN: 2423-4214‬‬
‫‪APPLIED FOOD BIOTECHNOLOGY , 2022, 9 (2):205-216‬‬
‫‪Journal homepage: www.journals.sbmu.ac.ir/afb‬‬

‫اثرات افزودن اسپیرولینا پالتنسیس بر رشد الکتوباسیلوس پالنتاروم ‪ Dad 13‬استرپتوکوکوس‬

‫ترموفیلوس ‪ Dad 13‬در شیر تخمیر شده و خصوصیات فیزیکوشیمیایی محصول‬
‫جاشوا کریسمس ناتانائل لوویدارتو‪ ،1‬اندانگ اس راهایو‪ 2 ،1‬و‪ ،3‬دیان آنگراینی سوروتو‪1‬و‪ ،2‬راچما ویکانداری‪1‬و‪ ،2‬آردیکا اولفا‪ ،‬تیاس‬
‫‪ 2 ،1‬و ‪* 3‬‬
‫اوتامی‬
‫گروه تکنولوژی فرآورده های کشاورزی و مواد غذایی‪ ،‬دانشکدهتکنولوژی کشاورزی‪ ،‬دانشگاه گادجاه مادا‪ ،‬یوگیاکارتا‪ ،‬اندونزی‪.‬‬ ‫‪-1‬‬
‫مرکز مطالعات غذا و تغذیه‪ ،‬دانشگاه گادجاه مادا‪ ،‬یوگیاکارتا‪ ،‬اندونزی‪.‬‬ ‫‪-2‬‬
‫مرکز عالی دانشگاه برای تحقیقات و کاربرد در صنعت یکپارچه پروبیوتیک‪ ،‬دانشگاه گادجاه مادا‪ ،‬یوگیاکارتا‪ ،‬اندونزی‪.‬‬ ‫‪-3‬‬
‫تاریخچه مقاله‬
‫چکیده‬
‫دریافت ‪ 8‬دسامبر ‪2221‬‬
‫داوری ‪ 22‬ژانویه‪2222‬‬
‫پذیرش ‪ 5‬آوریل ‪2222‬‬
‫سابقه و هدف‪ :‬اسپیرولینا پالتنسیس حاوی ترکیبات زیستفعال عالی میباشد‪ ،‬که بر ارتقای سالمتی موثرند‪ .‬با این‬
‫حال‪ ،‬استفاده از اسپیرولینا پالتنسیس در غذاها بهدلیل طعم و مزه نامطبوع آن برای برخی افراد‪ ،‬محدودیتهایی دارد‪.‬‬
‫واژگان کلیدی‬ ‫فرآوردههای تخمیری شیر عطر و طعم معمولی اسید تازه را دارند و میتوانند با اسپیرولینا پالتنسیس ترکیب شوند‪.‬‬
‫▪ ترکیبات زیستفعال‬ ‫زیستیارهای‪ 1‬الکتوباسیلوس پالنتاروم ‪ Dad 13‬و استرپتوکوکوس ترموفیلوس ‪ Dad 11‬جدا شده از شیرهای سنتی‬
‫▪ تخمیر‬
‫بوفالو را میتوان به عنوان سویههای آغازگر برای شیرهای تخمیر شده استفاده کرد‪ .‬این مطالعه رشد باکتری الکتیک‬
‫▪ باکتریهای الکتیک اسید‬
‫▪ ریزجلبک‬ ‫اسید‪ ،‬تولید اسید و ویژگیهای فیزیکوشیمیایی شیرهای تخمیر شده با اسپیرولینا پالتنسیس را بررسی کرد‪.‬‬
‫▪ زیستیار‬
‫*نویسنده مسئول‬ ‫مواد و روش ها‪ :‬تخمیر شیر با افزودن غلظتهای گوناگون (‪ 2/55 ،2/3 ،2/15‬و ‪ 2/6‬درصد) پودر اسپیرولینا‬
‫تیاس اوتامی‬ ‫پالتنسیس انجام شد و سپس رشد سلولهای میکروبی‪ ،‬تولید اسید و فعالیت آنتیاکسیدانی مورد بررسی قرار گرفت‪.‬‬
‫گروه تکنولوژی فرآورده های‬ ‫شیرهای تخمیر شده با غلظتهای انتخابی اسپیرولینا پالتنسیس در طی ‪ 25‬ساعت تخمیر در دمای ‪ 33‬درجه سلسیوس‬
‫کشاورزی و مواد غذایی‪ ،‬دانشکده‬ ‫با استفاده از کشتهای منفرد و مخلوط برای مطالعه جنبههای مختلف رشد سلولی‪ ،‬تولید اسید‪ ،‬گرانروی‪ ،‬ظرفیت‬
‫تکنولوژی کشاورزی‪ ،‬دانشگاه گادجاه‬ ‫نگهداری آب و رنگ مورد ارزیابی قرار گرفتند‪.‬‬
‫مادا‪ ،‬یوگیاکارتا‪ ،‬اندونزی‪.‬‬
‫مرکز مطالعات غذا و تغذیه‪ ،‬دانشگاه‬
‫یافتهها و نتیجهگیری‪ :‬با افزایش غلظت اسپیرولینا پالتنسیس در شیرهای تخمیرشده رشد سلولهای میکروبی‪،‬‬
‫گادجاه مادا‪ ،‬یوگیاکارتا‪ ،‬اندونزی‪.‬‬ ‫تولید اسید و فعالیت آنتیاکسیدانی افزایش یافت ‪ .‬در طی تخمیر شیر با افزودن ‪2/3‬درصد از اسپیرولینا پالتنسیس‪،‬‬
‫مرکز عالی دانشگاه برای تحقیقات و‬ ‫سلولها تکثیر یافتند و پس از ‪ 25‬ساعت‪ ،‬تعداد کل باکتریهای الکتیک اسید و سلولهای زیستیار بهترتیب به ‪-2/12‬‬
‫کاربرد در صنعت یکپارچه‬ ‫‪ 8/33‬و ‪ log CFU ml-1 8/22‬رسید‪ .‬اسیدیته قابل تیتراسیون به ‪ 1/28‬درصد رسید و ‪ pH‬به ‪ 5/51‬کاهش یافت‪ .‬پس‬
‫پروبیوتیک‪ ،‬دانشگاه گادجاه مادا‪،‬‬
‫از ‪ 12‬ساعت تخمیر‪ ،‬گرانروی بهطور قابل توجهی در مقایسه با گروه شاهد افزایش یافت‪ .‬شیرهای تخمیر شده با افزودن‬
‫یوگیاکارتا‪ ،‬اندونزی‬
‫تلفن‪+6281328852222 :‬‬ ‫اسپیرولینا پالتنسیس‪ ،‬که به آنها الکتوباسیلوس پالنتاروم ‪ Dad 13‬و استرپتوکوکوس ترموفیلوس ‪ Dad 11‬اضافه شده‬
‫باشد‪ ،‬را میتوان بهعنوان جایگزین فرآورده های تخمیری فراسودمند‪ 2‬شیر توسعه داد‪.‬‬
‫پست الکترونیک‪:‬‬
‫تعارض منافع‪ :‬نویسندگان اعالم میکنند که هیچ نوع تعارض منافعی مرتبط با انتشار این مقاله ندارند‪.‬‬
‫‪tyas_utami@ugm.ac.id‬‬

‫‪1‬‬
‫‪Probiotics‬‬
‫‪2‬‬
‫‪Functional‬‬

‫‪This open-access article distributed under the terms of the Creative Commons Attribution NonCommercial 4.0 License (CC BY-NC‬‬
‫‪4.0). To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.‬‬