International Journal of Advanced Science and Technology

Vol. 29, No. 5, (2020), pp. 6792 - 6801

Immunomodulatory Effects of Probiotics Bacteria Lactobacillus

acidophilus and Streptococcus thermophilusas Fermenter in
Soygurt

Sayu Putu Yuni Paryati1*, Siska Telly Pratiwi1, Evi Apriani Sitorus1, Dianti
Nursafitri Sundarti1, Susanti Ratunanda2, Indarti Trimurtini3 and Eka Noneng
Nawangsih1
1
Department of Microbiology, Faculty of Medicine, Universitas Jenderal Achmad
Yani, Cimahi, West Java, Indonesia
2
Department of Clinical Pathology, Faculty of Medicine, Universitas Jenderal
Achmad Yani, Cimahi, West Java, Indonesia
3
Department of Anatomy, Faculty of Medicine, Universitas Jenderal Achmad Yani,
Cimahi, West Java, Indonesia
*[email protected]

Abstract
Probiotics commonly are produced from Lactic Acid Bacteria (LAB). Lactobacillus
acidophilus and Streptococcus thermophilus are known to grow well in soy milk media.
Probiotics conferred many benefits, one of them is as an immunomodulator. Many studies
have shown that probiotics can stimulate the immune system, either non-specific or
specific immune responses, such as stimulating increased activity of macrophage
phagocytosis and antibody titer. This study aimed to examine the effects of
immunomodulatory L. acidophillus and S. thermophillus as bacteria fermenter in soygurt.
Twenty four of mice were divided in four groups: control negative, mice that treated the
soygurt L. acidophillus, mice that treated the soygurt S. thermophillus, and mice that
treated the combination soygurt. Mice were given the soygurt in 10 days and induced E.
coli on days 9. The mice immune response was measured by observing the activity of
phagocytosis and antibody titer. The study did not show activity of phagocytosis. The
result of study showed that of monocyte numbers increased in peritoneum liquid. It
indicated the enhancement of immune response in mice. There was significant difference
between control group and treated group (p≤0,05) but no significant difference (p≥0,05)
among the treated groups. There are significant differences in antibody titres by treating
soygurt L. acidophilus (p = 0.030) and soygurt combination (p = 0.031) compared to the
control group. The mice grouptreated S. thermophilus yield an increase in antibody titer
but not significant (p≥0,05) compared with the control group.

Keywords: Lactobacillus acidophilus, macrophage phagocytosis activity, soygurt,

probiotic, Streptococcus thermophilus.

1. Introduction
Probiotics are live microorganisms that can provide beneficial effects for the health of
the host body when consumed in sufficient quantities by improving the balance of
intestinalmicroflora [1], [2]. Generally, probiotics derived from lactic acid bacteria (LAB),
for example, Lactobacillus acidophilus and Streptococcus thermophilus [3], [4]. The
bacteria L. acidophilus able to survive in the acidic conditions of the gastrointestinal tract,
resistant to bile salts, stick to the epithelium of the gastrointestinal tract and produce
compounds antimicrobial [5]. S. thermophilus bacteria is often used as a starter in the
production of fermented milk and play a role in the early stages of the fermentation
process. Lactic acid bacteria stimulate the growth of other bacteria on fermented

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milkmedia. S. thermophilus role in the fermentation process in making the atmosphere of

digestive tract becomes more acidic so that the pathogenic bacteria are not resistant to the
acid environment and not able to grow in the digestive tract [6], [7].
Fermented beverages consumed by people which are generally made from milk, such
as yoghurt. Yoghurt with cow dairy ingredients can’t be consumed by people with lactose
intolerance, and high in saturated fatty acids on yoghurt can increase total blood
cholesterol when consumed in large quantities [8], [9]. One alternative for individuals
who do not like dairy cows and have lactose intolerance is consume soy milk.
Composition of soy milk is similar to cow's milk, but soy milk is less favored by people
because it has an unpleasant taste (beany flavor). L. acidophilus and S. thermophilus can
grow well in soybean milk.
Consumption of probiotics in sufficient quantities (106-108 cfu/mL) can provide health
benefits to the host [1]. One of the health benefits of probiotics consumption is as an
immunomodulator in enhancing the phagocytic activity of macrophages.
Immunomulatory effects of probiotics begins with the interaction of probiotics with
intestinal epithelial cells that regulate the immune response of the gastrointestinal tract. L.
acidophilus and S. thermophilus in mice increased phagocytic activity of peritoneal
macrophages [10]. Research conducted in 2000, showed the enhancement of phagocytic
activity of leukocytes and peritoneal macrophages of mice given Lactobacillus rhamnosus
(HN001), Lactobacillus acidophilus (HN017) or Bifidobacteriumlactis (HN019) for 10 or
30 days. Phagocytic index mice treated kefir with Lactobacillus paracasei and
Lactobacillus brevis increased compared to control mice [11].
This study aims to determine the immunomodulatory effects of soygurt fermented by
Lactobacillus acidophilus and Streptococcus thermophilus especially to know their
phagocytic activity and antibodies titer.

2. Procedure
The materials used in this research are mice, soygurt with L. acidophilus, S.
thermophilus and mixed of L. acidophilus and S. thermophilus fermenter, deMann
Rogosa and Sharpe Agar (MRSA), Eosin Methylene Blue Agar (EMBA), sucrose, water,
soybean varieties bromo, NaCL 0.9%, Polyethylene Glycol (PEG), agarose, aquadest,
Phosphate Buffer Saline (PBS), feed and drink for mice.

2.1. Reidentification of Bacteria

Bacterial reidentification is performed by macroscopic and microscopic examination.
The macroscopic examination was performed by culturing the media and observing the
morphological characteristics of S. thermophilus and L. acidophilus bacteria on MRS-A
media, whereas E. coli bacteria were performed on EMBA media. The growing bacterial
colonies were assessed for size, color, shape, edges, elevation and smell, and then
compared with the literature. Microscopic examination was done by observation on Gram
staining.

2.2. Soygurt Preparation

Pasteurized and sterilized soybean milk was cooled to 43-45oC, and then was added L.
acidophillus and S. thermophilus fermenters with a dose of 107 cfu/mL. In each 9 mL of
soybean milk mixed with 1 mL of fermenter. The fermenter composition in combination
soyghurt is 1:1 (v/v) between L. acidophilus and S. thermophilus. Milk that has been
given the bacteria fermenter then incubated at 37oC for 48 hours and put in the
refrigerator with a temperature of 24oC, in this temperature soyghurt able to survive for
14 days. Calculations of fermenter bacterial colonies in soy milk media were calculated

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using Total Plate Count method. Calculations performed on soygurt that has been through
the process of dilution. The result of soy milk dilution was then inoculated in MRS-A
medium and incubated for 48 hours at 37oC. Colonies formed in MRS-A media were
calculated using the TPC method.

2.3. Treatment of Research

Twenty four of mice were divided into 4 groups: negative control group, the group of
mice given soygurt L. acidophilus, the groups of mice were given soygurt S. thermophilus
and the group of mice were given soygurt combination. Animals adapted for 7 days, then
given soygurt 0.52 mL/20 gram weight of mice conducted over 10 days. Induction mice
were performed with 103 E. coli 0.1mL on day 9 intraperitoneally. On day 10, mice were
euthanized using CO2 gas and intraperitoneal fluids were taken.

2.4. Calculation of monocyte count and antibody titer

Periotneal liquid was centrifuged at 1,200 rpm, 4°C for 10 minutes. Supernatant was
removed, and the pellet was added RPMI-1640 (Gibco). The number of cells was counted
with hemocytometer, then added to complete medium to obtain cells with density
supension 2,5x106/mL. Microcultures suspension cells was grown in 24 wells that
covered with coverslip. Each of the wells filled in 200 microliter (5x105 cells), incubated
in a 5% CO2 incubator for 30 minutes. A complete medium then added for 1mL on each
well for incubated for 2 hours. Cells were washed twice with RPMI 1mL in each of the
wells and incubation in complete medium was continued until 24 jam [12]. Macrophages
have been cultured in a day before was added latex suspension 200μL/wells and incubated
in a 5% CO2 incubator, 37ºC for 60 minutes. Cells were then washed with PBS 3 times
and then dried at room temperature and fixed with absolute methanol for 30 minutes.
Once dry stained with Giemsa staining 20% and allowed to stand 30 minutes and then
rinsed with water and allowed to dry and then observed using a light microscope with a
magnification 400X [13], [14].
Measurement of antibody titers of mice was assessed using Agarose Gel Precipitation
Test (AGPT) methods. AGPT test is one of the serological test with techniques of
precipitation antibodies by antigen-antibody binding. This is a qualitative test that can
determine the presence of antigen-specific antibody. The AGPT medium was prepared
with a composition of 0.8 grams of Agarose, 2.4 grams of Polyethylene Glycol (PEG), 40
ml of aquadest, and 40 ml PBS with pH 7.4. Furthermore, the mixture of these ingredients
is heated to boiling and allowed for 5-10 minutes to have a half-coagulated consistency.
AGPT media that has been formed poured as much as 3 ml in a petri dish with a diameter
of 6cm to be solid, then put in a refrigerator with temperature 8 oC. Using a perforator, the
medium is perforated with 7 holes to form wells with a hole surrounded by 6 holes as
shown in Figure 1.

Figure 1. AGPT media with 7 holes. The hole in the center is inserted E. coli, while in
the other 6 holes are filled with serum in each dilution

In this study, serum was diluted 5 times serially using PBS pH 7.4. The diluted serum
is poured into each well that has been numbered. The AGPT medium containing serum

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and E. coli antigens was then given a damp cotton pad and incubated at 35°C for 48 hours
and then observed. The positive results of this examination are illustrated by the
formation of white precipitation lines between the antigen and serum holes.
The data obtained were tested using analysis of Kruskal-Wallis-test non-parametric
aimed to determine whether there is a difference between groups and followed by Post
Hoc Mann-Whitney test to determine in which group the differences were significant.

3. Results and Discussion

3.1. Reidentification of Bacteria
Bacteria L. acidophilus macroscopicaly showed the characteristic of L. acidophilus
colonies are round, 0.5-1.5mm in diameter, smooth edges, convex, grayish-white color
and acid smelled. Macroscopic examination S. thermophilus showed that the morphology
of colonies of bacteria were round, the size of a pin point, convex, smooth-edged and
release the acid smelled, [15] and E. coli were rejuvenated on differential media EMBA
indicate that the colony was observed according to the parameters characteristic of E. coli
colonies are round, pin point, the edges smooth and firm, convex and colored metallic
green [16]. Bacteria L. acidophilus and S. thermophilusare Gram-positive bacteria while
E. coli is a Gram negative rod-shaped [17].

3.2. Soygurt
According to the Indonesian National Standard (SNI), number of starter bacteria
(probiotics) is at least 107cfu/mL, the other criteria of soy milk as fermented beverages or
probiotics, i.e. the appearance, odor and pH were also observed in this study. The soygurt
has a thick texture, colored white, and produces a characteristic odor soygurt acid.
Soygurt L. acidophilus has a pH of 5.71, S. thermophilus and soygurt combination has pH
of 5.61 and 5.34. The number of bacteria starter is at least 10 7 cfu/mL, and in this study,
soygurt L. acidophilus has 48x1018cfu/mL, soygurt S. thermophilus has 3x1017cfu/mL and
the amount of bacteria on soygurt combination starter is 37x1020 cfu/mL. In the
calculation of the number of bacterial fermenter colonies, obtained amount of bacteria in
soy milk media between 1017-1020 cfu/mL. The amount is more than the standard of
probiotics according to WHO, which is 106-108 cfu/mL. This shows that with the
composition, the growth still meets the standard bacteria as probiotic.

3.3. Macrophages Phagocytosis Activity

This study has been conducted to identify macrophage cell characteristics, the nucleus
contains chromatin folding or curved delicate thread-like cytoplasm and usually contain
azurofilik smooth granules [13] as seen in Figure 2.
Phagocytic activity of macrophages in this study cannot be observed, because is
dominated by the carbon particles, possibly due to the number of macrophages too little as
a result of taking the peritoneal fluid after the induction is too premature, i.e. one day
post-induction. It is proved in the course of a study before, that with the induction of mice
during the six days may increase the phagocytic activity of macrophages. In previous
studies, infection of mice with Lysteriamonocytogenes during the day can already be
observed increased activity of ingestion, but in this study the induction of mice with E.
coli in one day can’t be observed an increase in phagocytic activity of macrophages
allegedly because E. coli produces a polysaccharide antigen K-1 in the capsule (antigen's
capsule) that are resistant to the process of destruction by phagocytic cells [18-19].
Macrophages are the development of monocytes into the tissue that begins with the
proliferation of monocytes, the increase in the number of monocytes as an indicator of the
increase in activity of macrophages. Therefore, in this study the calculation of the number
of monocytes was done.

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Figure 2. The morphology of macrophages with Hematoxyline-eosin staining (arrow

sign) characterized by nuclei containing folding chromatin or cytoplasm such as fine
curved threads

In this study, the result showed the increase of the number of monocytes in the
peritoneal fluid. An increasing number of monocytes may indicate the body's immune
response in mice, which monocyte is an early form of macrophages before entering into
the tissue of body [13]. Monocytes have the same function with macrophages in terms of
phagocytosis of foreign microorganisms that enter the body, but monocytes circulating in
the blood and perform its functions in the blood [13], [18].
The increase in the number of monocytes in the peritoneal fluid caused by the
proliferation and migration of monocytes from the blood vessel to the peritoneum as
phagocytic cells that produce the cytokines secrete in the peritoneum such as chemotaxis
factors called chemokines or chemotaxins [17], [20]. The number of monocyte for each
treatment group are described in Table 1.
Table 1 showed that the administration soygurt can increase the number of monocytes
significantly (p<0.05). Giving soygurt in mice proved to boost the immune response. The
immune response was initiated by the attachment of probiotic bacteria to intestinal
mucosa. Homeostatically, epithelial cells are unresponsive to bacterial flagella, whereas
flagellin that has entered the basolateral generates an inflammatory response that depends
on TLR5 [21]. The ingestion of the probiotic however, has been linked to a dampening
down of this response, reducing IL-8 secretion even in the presence of pathogenic
Salmonella dublin [22].

Table. The Number of Monocyte in Each Group

Group n Average and standard p-value
deviation

Control 6 16 ± 12,43

0,003
P1 6 33,83 ± 30,36

P2 6 31,17 ± 21,30

P3 6 31 ± 31,52
Description: P1 = Soygurt Lactobacillus acidophilus, P2 = Soygurt Streptococcus
thermophilus, P3 = combination soygurt
The increase in the number of monocytes peritoneal fluid in the group of soygurt L.
acidophilus because the ability of these bacteria to grow and survive at pH 3.5 to 6.8. L.
acidophilus ability to grow at acid condition makes the bacteria are able to survive in the
stomach acid when passing through the digestive tract, reach the intestine and colonize in

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the intestines. This suggests that L. acidophilus qualify as probiotic bacteria and is very
good to be probiotic [1], [5]. Colonization of L. acidophilus in large numbers in the colon
can increase its activity in terms of providing health benefits to the body, one of which
boost the immune response. Gill et al. in 2000 showed that administration of lactic acid
bacteria can increase some indexes of natural and adaptive immunity including
phagocytic activity of mononuclear cells (monocytes and macrophages) and
polymorphonuclear cells, NK cell activity, production of IFN γ, an antibody response
against oral and systemic antigens [23].
In the combination of soygurt, there are two types lactic acid bacteria, i.e. L.
acidophilus and S. thermophilus with different abilities. Streptococcus thermophilus is not
resistant to acidic conditions, because the optimum pH for nature growth of these bacteria
is 6,5, [7] cause no resistance to acidic conditions of the stomach. Streptococcus
thermophilus plays a role in the early stages of fermentation (as a starter) and the presence
of these bacteria in fermented milk stimulates the growth of L. acidophilus [24]. The
combination of L. acidophilus and S. thermophilus theoretically could increase the
benefits of consumption of probiotics, one of which is to improve the immune response
[8]. This research shows the average number of monocytes in the combination group
lower than soygurt L. acidophilus, it is probably because their nutrient competition
between these two types of bacteria.
The average number of monocytes of P2 group (soygurtS. thermophilus) was 31.17
because this soygurt given after feeding, so that the stomach are not too acidic and
intestinal bacteria can survive and has an impact on the immune system in increasing the
amount of monocytes proliferation and migration into the peritoneal fluid. Statistically,
the Kruskal-Wallis test results showed a significant effect on the provision of soygurt to
the number of monocytes in mice (p≤ 0.05) compared with controls. Giving three types of
soygurt in mice proved to enhance the immune system that begins with the attachment of
probiotic bacteria in the intestinal mucosa. The application of some strains of LAB
produced enhanced activity of peritoneal and pulmonary macrophages and blood
leukocytes, and increased secretion of lysosomal enzymes [25].
The exact mechanism of how LAB stimulate the immune system are not fully
understood. LAB or their products can get access to the gut-associated lymphoid tissue,
especially the Peyer plaques and/or the sistemic immune system's [13], [18]. Peyer
plaques cells produce specific antibodies against the LAB and inhibit the proliferative
response improvement against mitogen B cells and T cells. Translocation of LAB with
mesenteric lymph nodes and Peyer plaques in mice given LAB oraly may support this
possibility. In addition, the intestinal epithelium contains a number of immunoregulatory
cells. Interaction between LAB or their products produced by immunocompetent cells
such as macrophages and T cells secrete (cytokines) that are known have a significant
effect on the functioning of the immune system. The existence of specific receptors of
peptidoglycan, a cell wall component of LAB on macrophages and lymphocytes also
supports this possibility [13], [20], [23], [26].

3.4. Effectiveness of Bacteria L. Acidophilus, S. Thermophilus and Combination in

Soygurt Against Total Monocytes
Mann Whitney test results showed there were significant differences increase in the
number of monocytes in all groups given soygurt compared to the negative control group
(p<0.05), but not found significant differences between treatment groups were given
soygurt L. acidophilus when compared with treatment group of soygurt S. thermophilus
(p=0.317). Comparison of L. acidophilus and combination group was not found
significant differences (p=0.317) as well as with S. thermophilus treatment group (p =
0.872). Table 2. shows the value p≥0,05, there were not a significant difference on

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increasing the number of monocytes in mice given soygurt group with bacterial starter L.
acidophilus as a single bacteria compared with the group given soygurt with combination
bacterial starter of L. acidophilus and S. thermophilus. This is probably due to the
competition of nutrients between L. acidophilus and S. thermophilus, so that the average
increase in the number of monocytes soygurt L. acidophilus group and the combination
was not significantly different. Another possibility which cause no significant difference
between groups soygurt L. acidophilus and S. thermophilus combinations are mechanisms
that S. thermophilus play a role in the early stages of fermentation (as a starter) and the
presence of these bacteria in fermented milk stimulates the growth of L. acidophilus.

Table 2. Comparison of the Number of Monocytes Between Groups

Control L. acido- S. ther- Combi-
philus mophilus nation
Control 0,004* 0,006* 0,004*

L. acidophilus 0,317 0,125

S. thermophilus 0,872

Combination

Note: * = p-value <0.05 significantly different

The administration of soygurt with S. thermophilus as a single bacteria had no

significant effect when compared with administration of L. acidophilus and combination.
This occurs because the administration of soygurt in rodents after eating resulted in
stomach conditions are less acidic than before eating, so the number of S. thermophilus
bacteria colonies increases and affects the activity of the immune system.

3.5. Effectiveness of L. Acidophilus and S. Thermophilus as Single Fermenter and

Combination in Soyghurt Against Antibodies Titer
An antibody titer examination was performed on 24 serum samples with 5 series
dilution. The result of AGPT test showed that there was an increase of antibody titer in
the group given soyghurt compared to the control group. Data of antibody titer tested
normality with Shapiro Wilk Test, obtained p <0,05 which means data is not abnormal
distribution, so test continued with non-parametric test Kruskal Wallis Test. The results of
the Walis Kruskal Test can be seen in Table 3.
Statistically showed that there was significant effect on soygurt administration on the
increase of serum antibody titre (p≤0,05). The presence of attachment of lactic acid
bacteria (LAB) in the intestinal mucosa is known to influence the increase of immune
system activity that is cellular and humoral. The presence of lactic acid bacteria
colonization will increase phagocytosis activity by macrophages which will then present
antigenic products to T lymphocytes to be recognized with the help of a presenting
molecule. T lymphocytes are activated to trigger the proliferation and differentiation of
other T lymphocytes in lymphoid tissue. In addition, macrophages also secrete special
activating agents, i.e. interleukin 1, 2 and IFN-γ which are capable of enhancing the
growth and reproduction of T lymphocytes cells. The further formed T cells will be
released in lymphatic fluid and transported into the circulatory system [7]. The largest T
cells found in the body are helper T cells that act as the main regulator for all immune
functions. Activated lymphocyte T cells release lymphokines that work in other cells of
the immune system. Interleukins 4, 5, and 6 are the main interleukins that have a strong

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effect on B lymphocyte activity. These three interleukins cause increased growth and
stimulation of B lymphocytes in the formation of antibodies [8].

Table 3. Kruskal Wallis Test Between Treatment Groups

Group Median Minimum Maksimum P

Control 0,00 0 1

S. thermophilus 1,00 0 32
0,042*
L. acidophilus 18,00 0 32

Combination 2,00 0 16

*value p≤0,05 there is significant difference between two or more groups

The presence of lactic acid bacteria activity in the gastrointestinal tract due to
consumption of soygurt will result in a continuous pattern of activation, proliferation, and
differentiation of T lymphocytes directly through the activation of T lymphocytes by
LAB, and indirectly by increased levels of interleukin 1, 2 and IFN-γ are secreted by
active macrophages. This causes T cell lymphocyte levels to increase in the circulation of
lymphatic fluid, blood circulation, and lymphoid tissue. In condition of high levels of T
lymphocytes, when pathogenic bacteria enter, these pathogenic bacterial antigens will
activate many T lymphocyte cells, including one of the helper T lymphocytes. Increased
activation of helper T cells, will increase the stimulation of growth and the formation of
antibodies by B lymphocytes so that the levels of antibodies can increase rapidly in the
body [13], [28]. The administration of lactic acid bacteria can increase the activity of
macrophage phagocytosis, interleukin production, interferon formation, activity of NK
cells, and lymphocyte proliferation which will increase antibody formation, so it is
considered capable of acting as an immunomodulator [29].
The increase of serum antibody titre in the group given soygurt with L. acidophilus
fermenter to the control group showed a significant difference (p<0.05). Higher antibody
titer significantly was also found in the combination group of soygurt compared to the
control group (p<0.05), but in the mice group given soygurt with the fermenter S.
thermophilus bacteria as single fermenters, there was no significant difference compared
to the control group (Table 4).

Table 4. Mann Whitney Test Between Treatment Groups

S. thermophilus L. acidophilus Combination

Control 0,283 0,028* 0,029*

S. thermophilus 0,212 0,622

L. acidophilus 0,291
*p-value <0.05 there was a significant difference between the groups
S. thermophilus is known to have an optimum pH 6.5 [30]. The acidic acid pH is
thought to decrease the amount and activity of colonization of S. thermophilus in the
gastrointestinal tract. The low activity of S. thermophilus colonization in the
gastrointestinal tract can cause immunomodulatory effects by probiotic bacteria,
especially in increasing the formation of antibodies is not maximum. In combination

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soygurt, colonization of S. thermophilus is lower, colonization activity by L. acidophilus

may be more dominant, so that although L. acidophilus is given as a combination
fermenter with S. thermophilus, an enhancement in antibody titers found similar with L.
acidophilus treatment as single fermenter.
L. acidophilus is able to grow and live in the pH range of 3.5-6.8, with the ability to
survive at low pH until pH 3, it is probable that these bacteria can pass through the
stomach to reach and colonize in the intestine. This indicates that L. acidophilus is very
good for probiotics because it meets one of the requirements as probiotics, which must be
resistant to acid, especially gastric acid [31], [32]. The highest antibody titer was induced
by L. acidophilus can also be supported based on research done by Parvez et al (2006),
where L. acidophilus is known to increase lymphocyte proliferation by up to 43% which
will increase antibody formation, so it can be used as an immunomodulator [33].
Administration of oral lactic acid bacteria, such as Lactobacillus casei, L. acidophilus,
L. rhamnosus, L. delbrueckii subsp. bulgaricus, L. plantarum, Lactococcus lactis, and S.
thermophilus, increase the number of IgA-producing cells associated with lamina propria
in the mucosa and this effect is dose dependent. It has been reported that most lactic acid
bacteria can induce the inflammatory immune response induced [34]. Probiotics have a
positive influence on the innate immunity, exerting several antiviral properties [35].

4. Conclusion
The results showed that administration of soygurt with L. acidophilus as fermenter
bacteria gave best immunomodulatory effect compared with S. thermophilus fermenter,
and combination of both, but there is no significant difference.

Acknowledgments
Further thanks to the Institute of Research and Community Service of Universitas
Jenderal Achmad Yani for financial support for this study through Unjani's competitive
research grant fund.

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