Probiotic yogurt

1. MONGOLOAN STATE UNIVERSITY OF LIFE SCIENCES
SCHOOL OF ANIMAL SCIENCE AND BIOTECHNOLOGY
BORKHUU NOMIN-ERDENE
FEASIBILITY STUDY FOR THE PRODUCTION OF
PROBIOTIC YOGURT
Supervisor’s /Du Ming/
/E.Uugantsetseg/
2020

2. CONTENTS
INTRODUCTION
Rationale for the research
The purpose of the research
Objectives of the research
Novelty of research work
Structure and scope of research work
CHAPTER ONE: THEORETICAL PART
1.1 Mongolian sour milk products
• Kumis
• Hormog
1.2 Classification and morphology of lactic acid bacteria
1.3 Studies of lactic acid bacteria
1.4 Probiotic lactic acid bacteria
1.5 Probiotic dairy products
CHAPTER 2: RESEARCH MATERIALS AND METHODS
2 .1 Methodology for the isolation of pure cultures of lactic acid bacteria
2.2 Identify gram-positive and negative bacteria
2.3 Detection of catalase activity
2.4 Method for determination of general acidity (MNS 0400: 1983)
2.5 Acid resistance identification methods
2.6 Determination of gastric juice tolerance
2 .7 Method for determination of bile acid tolerance
2.8 Paper disk method for determination of antibiotic resistance
2.9 Method for determination of acidity
CHAPTER 3: FINAL RESULTS OF WORK
3.1 Isolation of pure lactic acid bacteria from probiotic yogurt
3.2 Determination of lactic acid bacterial morphology
3.3.1 Determination of acid resistance of lactic acid bacteria
3.3.2 Determination of bile resistance of lactic acid bacteria
3.4 Determination of antibiotic resistance of lactic acid bacteria
3.5 Determination of lactic acid bacterial milk production activity
3.6 Results of sensory analysis in yogurt
DISCUSSION
CONCLUSION
LIST OF REFERENCE
APPENDIX

3. INTRODUCTION
Rationale for the research
Today, the world's food consumption is growing, and it is important to choose new,
innovative and useful products for the human body. Therefore, those who want to eat
healthy food are more interested in milk and dairy products, and the market and
consumption of this type of product is increasing. Mongolian milk and dairy products are
dominated by sour milk products and have high probiotic activity. Recently, the effects of
probiotics on improving the immune system and preventing cancer have been intensively
studied. Researchers have found that probiotics are effective in the prevention and
treatment of chronic intestinal inflammation and diarrhea, as well as in the treatment of
diseases such as urogenital infections and allergies. Some milk and dairy products
produced in Mongolia are imported from abroad. Therefore, there is a real need to
determine the activity of probiotic bacteria and to produce probiotic milk and dairy
products under industrial conditions.
The purpose of the research
The purpose of this study was to study the probiotic properties of lactic acid bacteria
isolated from traditional Mongolian sour milk products and their potential for use in the
production of dairy products.
Objectives of the research
• To study the acid and bile tolerance of probiotic properties of pure cultures of lactic acid
bacteria isolated from camel's milk
• Distinguish pure cultures of lactic acid bacteria from PROBIOTIC yogurt, which is widely
sold in our market.
• To study the acid and bile tolerance of cultures isolated from probiotic yogurt
• Comparison of acid and bile tolerance of pure probiotic lactic acid bacteria isolated from
yeast with cultures isolated from probiotic yogurt.
Sensory evaluation of coated yogurt

4. Novelty of research work
In our country, all the dairy products used in the production process are imported
from abroad. The novelty of this work is that we started this research in search of
microorganisms that could replace these imported assets, and achieved certain results.
Structure and scope of research work
This research work is divided into 3 chapters, including the theoretical basis, materials and
methodology, research results, discussions, conclusions, list of used works and appendices.
The results of the theory and research are presented in 10 tables, 16 figures and 5 graphs,
using a total of 24 publications and 37 pages.

5. CHAPTER ONE : THEORETICAL PART
1.1 Mongolian sour milk products - Kumis and Hormog
• Kumis
The product of yogurt fermentation (yogurt) is a combination of alcohol and lactic acid
oxidation, called whole milk products or fermented milk. [1] Fermented milk has
nutritional value and is of medical and sanatorium importance. This type of dairy contains
the most optimal amount and proportion of nutrients and is easily absorbed by the human
body. This is primarily due to the fact that fermented protein compounds are at the half-life,
and lactic acid, alcohol and carbon dioxide stimulate the secretion of gastric juice and
stimulate the digestive system.
Most fermented milk plays an important role in the treatment and treatment of
gastroenteritis. They contain a relatively large number of vitamins and antibiotics.
Fermented milk is a source of lactic acid bacteria and has the ability to slow down the
process of putrefaction in the human body, kill a variety of acid-sensitive and disease-
causing bacteria. The mare's milk is fermented with yeast and lactic acid bacteria, and in
Europe it is called kumis, and in Inner Mongolia it is called chigger. This product is
important for the population of Central Asian countries such as Mongolia, Kyrgyzstan,
Kazakhstan, Yakut and Uzbekistan.
In the Mongolian context, fermented milk is fermented at home and the fermentation is
kept constant. A temperature of 20-30 ° C is required to avoid the formation of large
amounts of acid and ethanol. As a result of the fermentation process, the acidic pH is not
less than 4 and the amount of ethyl alcohol is not more than 2%. In addition to lactic acid
bacteria and yeast, fermented milk is rich in microelements, antibiotics, vitamins (A, B1, B2,
B12, C, D, E), ethyl alcohol, lactic acid and carbon dioxide. The micro flora of sour kumis is
dominated by Lactobacillus bulgaricus, Lactobacillus rhamnosus, the soft kumis is
Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus casein, while the soft
kumis is dominated by Streptococcus thermophiles and Streptococcus cremoris. In
addition to lactic acid bacteria, Kluyveromyces marxianus var, which breaks down lactose?
Yeast such as lactic and yeast Kluyveromyces marxianus var. marxianus, Kluyveromyces
marxianus var, lactic species are predominant, and Saccharomyces cerevisiae are yeasts.
[10]
The quality of fermented milk depends on the characteristics of the raw material. (For
example, fermented milk has a therapeutic value). This is because mare's milk is richer in
biologically active substances than other animal's milk. In particular, mare's milk is rich in
immune globulin protein, which promotes the body's resistance to disease. Therefore,
mare's milk is used in many countries around the world for the treatment of tuberculosis,
measles and mumps. [5]

6.  Hormog
Hormog, one of Mongolia's traditional fermented foods, has been used since ancient
times. Hormog is fermented camel's milk and is used for diseases such as tuberculosis,
edema, and viral infections of the large intestine. Internationally, camel milk and dairy
products have been actively studied for more than a decade and have been shown to have
anti-cancer, anti-diabetic and anti-hypertensive properties [6].
Lactic acid bacteria and yeasts are involved in the composition of sourdough, a sour
drink made from camel's milk, so alcohol is formed along with lactic acid. Because camel's
milk is high in sugar, it creates favorable conditions for the combined oxidation of lactic
acid and alcohol. [11]
1.2 Classification and morphology of lactic acid bacteria
Lactic acid bacteria are more or less common in the environment. However, the number
and size of their cells often depend on the carbohydrate content of the enzyme and other
factors that promote bacterial growth. Milk and dairy products contain lactose, which is an
ideal environment for lactic acid bacteria to live. Lactose, amino acids, vitamins, and other
substances in milk and dairy products cause lactic acid bacteria to multiply rapidly, but the
buffering properties of these products allow millions and billions of lactic acid cells to
accumulate to form a natural "preparation" of lactic acid bacteria. First of all, lactose-
degrading bacteria multiply rapidly. Therefore, it is easy to distinguish the source of pure
culture assets used in the dairy industry from dairy products.
Lactic acid bacteria are not unique morphological characteristics
Almost all of them have specific (incorrect) rods, multiply in gram-positive, aerated
environments, and do not form endospores. They are in the form of bands, branched
filaments or simultaneously in the form of rods, filaments and cocci, and the life of one of
them alternates in the form of rods. [4]
Biochemical properties of lactic acid bacteria
Lactic acid bacteria convert pyruvic acid to lactic acid, and the predominant fermentation
of lactic acid in the final product is called homofermentative lactic acid fermentation, which
is usually carried out by Streptococcus, Lactococcus, and Lactobacillus bacteria. The
breakdown of lactic and acetic acids to form CO2 is called heterofermentative lactic acid
fermentation. Leuconostoc and Lactobacillus species are involved in this process. Oxygen
requirements vary from obligate aerobic facultative anaerobes to microaerophilic obligate
anaerobes. [4]
Classification
Lactic acid bacteria are divided into four main types: Lactobacillus, Leuconostoc,
Pediococus, and Streptococcus. It grows well in anaerobic conditions, but is also able to
survive in an oxygenated environment. [9]

7. 1.3 Lactic acid bacteria studied
Since 1958, R.Indra, P.Khorloo, R.Baldorj, Ts.Namsrai, Ts.Demberel, L. Scientists
Damdinsuren, R.Sukhbaatar, and D.Tsoodol isolated pure cultures of lactic acid
bacteria from sour milk products and determined the physiology, biochemical
properties, and species of the culture.
Since 1959, he has been working at the National University of Mongolia on the
chemical composition of mare's milk, fermentation methods, and the composition of
microorganisms. Baldorj studied.
In 1966, the Bulgarian microbiologist Khor.Chomakov, in his scientific article “Lactic
acid bacteria of Mongolian ferment”, noted the importance of the production of
highly active lactic acid bacteria by acid production.
In 1970, the composition of fermented capital microorganisms was studied and it
was determined that lactose-fermenting and non-fermenting yeasts of the type
Saccharomyces of alcoholic oxidation live in symbiosis with lactic acid bacteria:
Lactobacterium bulgaricum and lactic acid Streptococcus.
In 1974, Prof. Ts.Namsrai's work “Perfection of fermented milk” describes the
general protein of mare's milk and fermented milk, the amount of protein and free
amino acids depends on the fermentation time of fermented milk, solution, protein
groups, fermentation milk protein digestion rate by digestive enzymes, protein
microbial activity. studied the therapeutic properties of fermented milk, such as.
In 1993, many Mongolian scientists studied the microflora of sour milk products
and invested in yogurt. They invested in the Food Industry Research and Design
Institute. had stopped.
Researchers at the Veterinary Research Institute identified a total of 15 baghs, 21
species and 41 species of sour products such as fermented milk, yogurt and sour
cream. Lactic acid bacteria such as Lactobacillus, Lactococcus, and Streptococcus
predominated among these lactic acid bacteria. [3]
In 2014, Prof. B. Батжаргал, Э. Uugantsetseg and others isolated 42 species of lactic
acid bacteria from traditional Mongolian fermented milk products, fermented milk,
and studied their antioxidant activity. [32]
In 2016, Prof. B. Батжаргал, О. Ganzorig, in collaboration with Japanese scientists,
studied the taxonomy of lactic acid bacteria isolated from Mongolian sour milk airag
at the genetic level. [39]
In 2017, Prof. B. Prof. Batjargal B. Ochirkhuyag, E. Uugantsetseg and others isolated
112 pure cultures of lactic acid bacteria from 25 samples of camel's milk prepared
by traditional methods and determined their protein degradation activity. [2]

8. 1. 4 Probiotic lactic acid bacteria
One of the great achievements of food biotechnology and medicine in the 20th
century was the formulation of a new concept of probiotic or regulatory food consumption.
According to this concept, food not only provides the body with heat and regenerative
materials, but also promotes human health and reduces the risk of disease by monitoring
or modeling physiological, biochemical and functional reactions. [5]
The origin of the word probiotic is a Latin prefix meaning pro-to, for, and the Greek
symbol βιωτικός (biotic) is a relatively new name derived from the word "bios" meaning
"for life" and is used by humans and animals. represents live bacteria that are beneficial to
health. [15].Probiotics are divided into three types. These include:
• Monoprobiotics - preparations or products that contain only one type of bacteria
• Hamprobiotics - preparations or products that contain a variety of microorganisms
• Synbiotics - preparations or products that contain beneficial microorganisms (probiotics)
and substances that promote their reproduction (prebiotics) [5]
Prebiotics are complementary nutrients for living microorganisms that improve the
microbial balance of the host organism. [19: 567] The main representatives of prebiotics
are carbohydrates, especially fiber. Marcel Roberfroid first described prebiotics in 1995
when he discovered that two fructooligosaccharides, oligofructose and inulin, had prebiotic
properties. In addition, some oligosaccharides in breast milk have prebiotic properties and
are important in supporting the newborn's immune system. [17]
Probiotic active bacteria usually include lactic acid bacteria, including
Bifidobacterium and Lactobacillus species. Probiotics must meet the following
requirements to have a positive effect on the human body.
• The activity of beneficial microorganisms is not lost during food production or they are
viable
• Probiotics are resistant to gastrointestinal acids and alkalis and are inactive
Probiotics need to be attached to the wall of the colon, where they need to be active
to perform protective functions. [5]
Probiotic bacteria are used in the following treatment and spa procedures. These include:
1. Regulate lactose intolerance
2. Prevention of colon cancer
3. Lower cholesterol
4. Lower blood pressure
5. Improve immunity and prevent infection
6. It is useful to reduce inflammation of the colon.

9. Scheme 1. Technology of yogurt production
Milk 40-41T0
Smoothing (7-8 atmospheres, 30 sec)
Separating fat from milk
Heating and Sterilized (90-960
C) 2min
CoolingandCapitalize (430
C,3-5% of the total milkvolume)
Packing
Cooling
Coating (40-450
C)
Storage (0-80
C)

10. 1. 5 Probiotic products
8 types of probiotic products that is beneficial to the human body. We have a
constant metabolism. The function of the internal organs is closely related to the degree of
metabolism. Although the human body contains a certain amount of beneficial
microorganisms that support this process and help it get into the right "stream", these are
not enough and can lead to metabolic disorders. You can get it from the following 8 types of
foods that contain these beneficial bacteria. These are called probiotic products.
1. Natural yogurt - Yogurt with natural and live bacteria that does not contain syrup
or sugar. Hand-coated yogurt is especially useful at home.
Goat's milk fat contains high levels of probiotic bacteria Lactobacillus, Acidophilus
and Bifidus.
2. Miso soup - Japanese miso soup is high in probiotics and protein. This soup is
widely used for digestion and can be used as a sushi bra. Among the probiotic products, it is
the most delicious. When used in combination with tofu and vegetable puree, this low-
calorie soup can help you lose weight. Soups made from rye, peas, rice and miso contain
high levels of Lactobacilli and Bifidus bacteria.
3. Soy Milk - Natural soy milk is high in probiotics. With the latest technology, more
and more nutrients have been added to soy milk. You can find the probiotic product you
are looking for by writing "live and active bacteria" on the label.
4. Kimchi - Recently, the most popular Korean pickle, kimchi, is made from cabbage
fermented with lactic acid bacteria such as Leuconostoc, Lactobacillus and Pediococcus.
Cabbage tastes like sugar and lactic acid secreted by bacteria. In addition to living
organisms, kimchi contains calcium, B-carotene, iron, vitamins A, C, B1 and B2.
5. Milk - Probiotic milk is now sold overseas. Scientists have proven that milk is the
basis for the growth of young organisms and a source of vitamins that help the elderly to
live longer.
6. Dark Chocolate - Dark chocolate is one of the most antioxidant, probiotic products
and important for digestion. High-quality dark chocolate contains four times more
probiotic compounds than dairy products.
7. Pickles - Like kimchi, pickles and other vegetables are a good source of probiotics.
8. Olives - One of the most beneficial probiotic products is the olive fruit. This fruit
can be used in pizza and various salads.

11. CHAPTER TWO
RESEARCH MATERIALS AND METHOD
Research materials
The study used 14 pure cultures of lactic acid bacteria from 25 samples of sour
cream prepared from Bayankhongor and Dundgovi provinces, as well
as “ Sain ” yoghurt, “ Elgen ” yoghurt and “Actimel” yoghurt, which are widely sold in the
domestic market .
Survey Methodology
2.1 pure cultures of lactic acid bacteria to distinguish
Samples of fermented milk (10%, 5% v / v) are taken and placed in a sterile,
non-skimmed milk tube and incubated at 37 ° C for 24 h. Repeat this 3 times,
assuming that the microflora in the sample has been restored, and use an MRS at 45 °
C sterilized in a culture medium containing 100 μl of certain culture dilutions.
Inoculate in MRS agar medium and incubate at 37 ° C for 24 h. After 24 h of
incubation, the colonies were selected from the bacteriological loop, inoculated into
a separate colon in an MRS agar medium, and incubated at 37 ° C for 24 h. [18]
2.2 Identify gram-positive and negative bacteria
The Gram method is based on the formation of gentian violet and iodine
complexes in the cell wall of gram-positive bacteria. In 1884, Hans Christian
developed the Gram cell staining method, which is widely used in microbiological
research to identify, identify, and classify bacterial cells. Gram staining of bacterial
cells (1) sequential treatment with pink crystalline solution (gentian violet) and (2)
Gram iodine (Lugol's solution), (3) dye washing (ethyl alcohol), (4) additional
strengthening dye (fuchsin), (5) ) has 5 main stages: washing with water and drying.
Pink stains are classified as Gram-positive (+) bacteria, and red stains are classified
as Gram-negative (-) bacteria.
1. Place filter paper on a specially prepared bacterial paste and stain with pink
crystalline solution (gentian violet) for 1-2 minutes. At this point, the bacterial
cells turn pink.
2. The dye is then absorbed from the filter paste and treated with Lugol's solution
for about 1 min.
3. When the preparation is sprayed with ethyl alcohol (95%), the Gram-positive
bacteria remain pink, while the Gram-negative bacteria become colorless.
4. 4. The product is then stained with a solution of fuchsia (1-2 minutes), during
which the Gram-negative bacterial cells turn red.
5. Wash the paste several times with distilled water and dry it thoroughly under a
microscope to see pink (Gram-positive) and red (Gram-negative) cells on a white
background [19]

12. 2.3 Detection of catalase activity
MRS agar surface feeding environment isolated colonies established 24 hour culture
grown microbial loop slide with sterile distilled water suspensions prepared. Prepared sus
pension at 1-3 drops of hydrogen peroxide (3% H 2 O 2 w/v) solution drops. If no gas
is emitted, the catalase is considered inactive. [20]
2.4 Method for determination of general acidity (МNS 0400: 1983)
Measure 10 ml of the sample into a 150-200 ml conical flask, add 20 ml of distilled
water and add 2-3 drops of 0.1% alcohol solution of phenolphthalein. Rinse the mixture
well and titrate with 0.1 N sodium hydroxide solution. When titrated, shake for 30 seconds
to a pale pink color that does not fade. The turnip's milk acidity, expressed in degrees, is
equal to 10 times the amount of 0.1 N sodium hydroxide (ml) used to neutralize 10 ml of
milk [23].
2.5 Determination of acid resistance
The sterile liquid is inoculated from pure culture in MRS medium and placed in a
thermostat at 37 ° C for 24 h. Dilute the pH of the liquid MRS broth medium with HCl to 1.5,
2.0, and 3.0 and sterilize in an autoclave at 121 ° C for 15 min. Inject the pH in a sterile tube
culture medium for 24 h at a rate of 2% of the culture in the liquid MRS culture medium.
During the first 0, 1, 2, 3, and 24 hours after injection and vortexing, the optical density is
recorded by measuring the spectrophotometer at a wavelength of 600 nm compared to the
control [27].
2.6 Determination of gastric juice tolerance
The sterile liquid MRS broth is inoculated from pure culture medium and placed in a
thermostat at 37 ° C for 16 h. The 16 h culture was centrifuged at 5000 rpm for 10 min to
separate the filtrate. Wash the filtrate twice with 0.1 M pH 7 phosphate buffer. Dilute the
cell to its original volume using a buffer and vortex. Filter the gastric juice containing 0.3%
pepsin with 3N HCl to 2.0 and filter through a 0.22 µm filter into a tube. Inject 2% of the
suspension into a tube of prepared gastric juice. During the first 0, 1, 2, and 3 h, the viability
of the culture is diluted in saline, taken from a series of dilutions, and inoculated into a petri
dish containing MRS agar and incubated at 37 ° C for 24 h. After 24 h, the count of the
grown colony is expressed in units (cfu / ml) of the colony-forming agent in 1 ml. [28]
2.7 Determination of bile tolerance
Lactic acid bacterial cultures were taken from the MRS solid medium under a
bacteriological loop and incubated in the MRS liquid medium at 37 ° C for 16 h. To prepare
the MRS bile medium, mix 0.3% bile with the MRS liquid medium and sterilize at 121 ° C for
20 min. The cultured cells are centrifuged at 5000 g for 10 min. Wash the cells twice with
phosphate buffer solution (0.1 M pH 7.0) and buffer to the original volume. Inject 0.5% into

13. MRS liquid bile medium and MRS bile medium. Incubate in a water bath at 37 ° C and
measure the optical density of the cells at 560 nm of light every hour for the first 8 h. The
control uses a non-bacterial MRS culture medium. [29]
2.8 Paper disk method for determination of antibiotic resistance
Prepare a suspension of antibiotic resistance in 4-5 ml of 0.85% saline compared to
0.5 McFarland (1.5 * 108 cells / ml) and inject it into a solid MRS medium. After 5 to 20
minutes, place 4 to 6 antibiotic discs in a petri dish with sterile forceps. After incubation at
37 ° C for 12 to 24 h, determine the extent of the bacterial antibiotic resistance in the petri
dish in millimeters. The result is represented by the letters R, I, S.
(R-Resistant, I-Moderately Resistant, S-Resistant) [30].
2.9 Method for determination of acidity
Usable equipment and reagents:
-10 ml pepitika
-25 ml burettes
-1% phenolphthalein
-50 mL volumetric flask
- distilled water
-0.1 n NaOH
Procedure: Take 10 ml of yogurt, put it in a 50 ml flask, add 20 ml of distilled water, add 2-3
drops of 1% phenolphthalein solution and mix well. Then titrate with 0.1 N NaOH until pale
pink. The resulting color intensity should not fade for 1 minute.
Record the amount of alkali in the titration. Multiply the alkali used to titrate 10 ml of milk
by 10 to convert it to 100 ml of milk and express the acidity in Turner degrees.
The chemistry of the neutralization method is given by the equation:
H 2 SO 4 + 2NaOH = Na 2 SO 4 + 2H 2 O

14. CHAPTER THREE:
FINAL RESULTS
This study was conducted in February -March 2020 in the Integrated Laboratory of Natural
Sciences, School of Animal Husbandry and Biotechnology, University of Agriculture.
3.1 Isolation of pure lactic acid bacteria from probiotic yogurt
We selected “Sain”, “Elgen” and “Actimel” probiotic yogurts, which are widely sold in our
country, and isolated pure lactic acid culture cultures in order to compare the probiotic
properties of our pure lactic acid bacterial cultures with the traditional method.
In addition to studying the morphology of pure cultures of isolated lactic acid, Gram stain
and catalase enzyme testing were performed. The results are summarized in Table 1.
3.2 Determination of lactic acid bacteria morphological
Morphological properties of pure cultures of lactic acid bacteria isolated from probiotic
yogurt samples
Table1
Name of
the sample
in which
the bacterium
was isolated
Culture
number
Gram
staining
Catalase
activity
Cell
shap
e
Colonial properties
Size (mm) Color Form Market Surface
“Sain” yogurt S + - Stick
s
1-2 White Round Equal Smooth
“Elgen” yogurt E + - Stick
s
1-1.5 White Round Equal Smooth
“Actimel”
yogurt
A + - Stick
s
1-2 White Round Equal Smooth
Isolated cultures were grown on MRS agar solid medium in small, white, flat-edged, round-
shaped colonies with a smooth surface.
Microscopic examination of the cell formulations of the isolated cultures revealed that they
were all rod-shaped, Gram-positive, and catalase-negative.

15. Figure 1. Pure culture of lactic acid bacteria isolated from “Sain” yogurt
Figure 2. Pure culture of lactic acid bacteria isolated from “Elgen” yogurt
Figure 3. Pure culture of lactic acid bacteria isolated from “Actimel ” yogurt

16. 3.3 Pure cultures of lactic acid bacteria probiotic characteristics to results
Probiotic characteristics of 17 cultures selected for the study (14 cultures isolated from
piglets and 3 cultures isolated from probiotic yogurt)
1. Acid resistance
2. Bile tolerance was determined by determination.
In order for probiotic bacteria to attach to the intestinal wall of the host organism and have
a positive effect on their health, they must first survive the stressors of the gastrointestinal
tract. Therefore, we methodologically determined the viability of pure cultures of lactic
acid bacteria in the gastrointestinal tract.
3. 3 .1 Resistance to acid identified
The acid resistance of the pure cultures of the selected lactic acid bacteria was determined
by counting the number of colonies given 24 hours after inoculation in MRS agar medium
for 3 h after inoculation in MRS broth liquid medium with pH = 1.5, pH = 2.0, and pH = 3.0.
Resistance to lactic acid bacterial pure cultures Table 2.
Culture number
The acidic environment of lactic acid bacteria
Endurance , 3 hours
рН = 1.5 рН = 2 рН = 3
2 - - +
217 - + +
X -17 - + +
E- 31 - + +
921 - + +
923 - + +
1 - - +
5 ∕ 3 - + +
2 / 1 - + +
5 - - +
4 - - +
8 ∕ 5 - + +
6 ∕ 2 - + +
325 - + +
E - + +
S - + +
A - + +
Reservations : (-) u did not raining (+) to growth

17. In an acidic environment with pH = 3.0, the whole culture grew evenly, while at pH = 2.0, 4
of the 14 cultures isolated from the hormone did not grow.
Graph 1. Resistance of pure lactic acid bacteria isolated from Mongolian milk and 3 cultures
isolated from probiotic yogurt
The graph shows that lactic acid bacteria isolated from Mongolian lactose have
better acid resistance than pure cultures of lactic acid bacteria isolated from Mongolian
milk and three cultures isolated from probiotic yogurt.
A comparative study of the acid resistance of 14 cultures isolated from yeast and 3
cultures isolated from probiotic yogurt showed that not all of them were able to grow at pH
= 1.5. However, it is tolerant to pH = 2.0
Indicates acid resistance for 10 cultures. In other words, they have been shown to be able
to survive in the acidic environment of the human stomach.
0
10
20
30
40
50
60
70
80
90
100
С Э А 217 2//1 8//3 X-17 5//3 Э-31
THE
NUMBER
OF
COLONIES
GROWN
(PH=2.0)

18. 3. 3.2 Determination of bile resistance of lactic acid bacteria
To determine the bile tolerance of the selected lactic acid bacteria, the cultures were
inoculated at 0.3% bile MRS broth medium at 1% by volume and their optical density was
measured by spectrophotometer at 600 nm for the first 8 h of culture. Their bile tolerance
was calculated by comparing this measurement with the optical density of cultures grown
in a bile-free medium.
Pure cultures of lactic acid bacteria bile resistance Table3
Culture Feeding
environment
Incubation time and time
1 2 3 4 5 6 7 8
Optical density , 600 nm
8 ∕ 5 MRS 0.023 0.036 0.072 0.098 0.228 0.320 0.419 0.541
MRS (0.3% bile ) 0.029 0.043 0.067 0.065 0.154 0.191 0.217 0.312
217 MRS 0.043 0.086 0.392 0.891 1,267 1,550 1,623 1,763
MRS (0.3% bile ) 0.054 0.066 0.258 0.496 0.872 1,036 1,041 1,067
6 ∕ 2 MRS 0.019 0.026 0.056 0.052 0.160 0.361 0.435 0.526
MRS (0.3% bile ) 0.023 0.033 0.049 0.045 0.135 0.286 0.341 0.460
X -17 MRS 0.025 0.060 0.134 0.272 0.682 1,129 1,235 1,365
MRS (0.3% bile ) 0.031 0.056 0.114 0.248 0.431 0.639 0.756 0.804
5 ∕ 3 MRS 0.056 0.149 0.386 0.964 1,447 1,704 1,802 1,869
MRS (0.3% bile ) 0.057 0.134 0.338 0.790 1,248 1,516 1,635 1,725
2 ∕ 1 MRS 0.031 0.066 0.079 0.232 0.492 0.876 0.936 1,108
MRS (0.3% bile ) 0.054 0.057 0.053 0.118 0.268 0.349 0.415 0.506
E
MRS 0.009 0.048 0.052 0.067 0.111 0.287 0.382 0.606
MRS (0.3% bile ) 0.017 0.039 0.05 0.064 0.093 0.181 0.36 0.575
S
MRS 0.011 0.045 0.067 0.089 0.102 0.146 0.352 0.593
MRS (0.3% bile )
0.019 0.038 0.05 0.072 0.098 0.222 0.323 0.589
A
MRS 0.040 0.063 0.091 0.225 0.292 0.397 0.775 0.866
MRS (0.3% bile )
0.025 0.054 0.082 0.189 0.267 0.378 0.791 0.897
Of the 14 cultures isolated from Hormone , 6 cultures numbered 8/5 , 217, 6/2, X-17, 5/3,
and 2/1 were found to be bile tolerant. All three probiotic cultures were bile tolerant.

19. Give ability to withstand the bile of the three cultures of lactic acid bacteria shows
graphically the results point on.
Graph 2. Tolerance of 0.3% bile medium of 6/2 culture
Graph 3. Tolerance of 0.3% bile medium of X-17 culture.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 2 4 6 8
Abs
600
nm
Incubation period ,hour
6-2
MRS цөсгүй
MRS 0.3%-ийн цөстэй
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Abs
600
nm
Incubatation period,hour
X-17
MRS цөсгүй
MRS 0.3%-ийн цөстэй

20. Graph 4. Ability to withstand 0.3% bile medium of 5/3 culture
When cultured in an MRS broth medium with 0.3% bile, the light absorption intensity of all
cultures was increased during 8 h of culture, indicating that these cultures were able to
withstand the bile medium.
Graph 5. Bile resistance of pure lactic acid bacteria isolated from Mongolian milk and
culture isolated from probiotic yogurt
From this graph, it can be seen that the bile tolerance of pure probiotic lactic acid bacterial
cultures isolated from sourdough is higher than that of pure lactic acid bacterial cultures
isolated from probiotic yogurt.
0
0.5
1
1.5
2
0 2 4 6 8
Abs
600
nm
Incubation period, hour
5-3
MRS цөсгүй
MRS 0.3%-ийн цөстэй

21. 3.4 Determination of antibiotic resistance of lactic acid bacteria
Bacteria with probiotic properties must be resistant to antibiotics. This is because
they have a beneficial effect on the human body, as well as resistance to antibiotics, which
allow them to live longer in the gastrointestinal tract..
Lactic acid bacterial resistance to antibiotics Table 4
Culture
notation
K
anamycin
30
mcg
Chloramphenicol
10
mcg
Erythromycin
30
mcg
Ampicillin
30
mcg
Cefazolin
5
mcg
Tetracycline
30
mcg
Sulfidomecin
100
mcg
Amoxicillin
10
mcg
Novobiocin
30
mcg
2/1 9 mm 0 2 mm 15 mm 11 mm 16 mm 0 11 22 mm
5/3 15 mm 0 11 mm 0 mm 5 mm 6 mm 5 mm 0 mm 0 mm
6/2 1 6 mm 0 0 0 0 0 mm 18 mm 0 15 mm
8/5 5 mm 0 0 5 mm 0 9 mm 0 2mm 0 mm
X-17 0 15 mm 0 mm 0 1 6 mm mm 0 0 17 mm
E- 31 5 mm 0 16 mm 0 0 0 0 0 7 mm
325 10 mm 10 mm 10 mm 19 mm 8 mm 15 mm 0 16 mm 20 mm
217 2 mm 0 8 mm 4 mm 5 mm 8 mm 0 5 mm 8 mm
In terms of antibiotic resistance, cultures 6/2, X-17, and 5/3 were found to be resistant to
six commonly used antibiotics: amoxicillin, ampicillin, tetracycline, and penicillin.

22. 3.5 Results of lactic acid bacterial milk formation and acid formation
Yogurt coating
Inject 500 μl of the pre-sterilized MRS broth from the culture medium with an
automatic pipette. In addition, one column of pure bacterial culture grown in solid media is
taken through a bacteriological loop. Then grow in a thermostat at 35C for 24 hours. The
cultured culture is centrifuged for 5 minutes at 10,000 rpm. As a result, two layers are
formed, with the liquid part at the top and the bacterial cells at the bottom. Then pour the
liquid portion and wash the bacterial cell agglomerate twice with 0.9% NaCL solution. Add
another 800 μl of NaCL, shake well, dissolve the precipitate in 20 ml of milk and keep in a
thermostat for 24 h. Then you will prepare the basic yoghurt coating.
The probiotic activity of 6 cultures with probiotic properties was determined at 3%,
5%, and 10% of the volume of skimmed milk, and the cultures of the bacteria were
inoculated into a thermostat at 37 ° C until they formed. At 5 and 10 percent, the cultures
produced milk between 4 and 6 hours.
Pure culture of lactic acid bacteria to form milk (3%) Table 5
Culture number
Milk production activity (3%)
General acidity , T 0 ( after 24 hours )
2 hours 4 hours 6 hours 24 hours
2/1 - - - ± 65
5/3 - - - ± 90
6/2 - - ± + 50
8/5 - - ± + 70
217 - - - ± 65
X-17 - - ± + 75
Reservations: (-) not formed,
(±) weakly formed,
(+) good composition
When 6 cultures that were found to be resistant to acid and bile were valued at 3% of milk
volume, 50% or 3 cultures were poorly milked at 6 o'clock. For the remaining 3 cultures,
milk was formed within 24 hours.

23. Pure culture of lactic acid bacteria to form milk (5%) Table 6.
Culture number
Pursuing less milk (5%) General acidity , T 0
( After 24 hours )
2 hours 4 hours 6 hours 24 hours
2 ∕ 1 - - ± + 80
6 ∕ 2 - - ± + 70
X -17 - - ± + 80
5 ∕ 3 - - ± + 100
217 - - ± + 100
8 ∕ 5 - - ± + 100
Reservations: (-) not formed,
(±) weakly formed,
(+) good composition
When skimmed liquid milk was enriched with 5% of milk volume from pure probiotic lactic
acid bacterial cultures, all cultures began to form little by little at 6 hours, and after 24
hours, all were densely packed. The pure milk culture of lactic acid bacteria was calculated
at 3% by volume of milk and the milking time was slightly shorter than that of fermented
milk. Also, the increase in titer acidity is due to the increase in the amount of capital.
Lactic acid bacterial culture milk production activity (10%) Table 7 .
Culture number Pursuing less milk (10%) General acidity , T 0
(after 24 hours )
2 hours 4 hours 6 hours 24 hours
2 ∕ 1 - ± + + 10 5
6 ∕ 2 - ± + + 100
X -17 - - ± + 100
5 ∕ 3 - ± + + 120
217 - - ± + 115
8 ∕ 5 - - ± + 113
Reservations: (-) not formed
(±) weakly formed
(+) good composition
When the culture was enriched to 10% with skim milk, it was gradually weaned at 4 o'clock,
and after 24 hours it was all weaned.
The formation time was relatively short compared to the 3% and 5% cultures. This is
shortened depending on the number of cells in the culture.
The resulting milk, i.e. yoghurt, was slightly sour in taste, light yellow in color, with a slight
whey secretion, and smelled as soft as yogurt.

24. 3.6 Sensory evaluation of the spread
The probiotic activity of 6 cultures, which were found to have probiotic properties,
was calculated at 3%, 5%, and 10% of the volume of skimmed milk and injected into a
thermostat at 37 ° C until it was formed. At 5 and 10 percent, the cultures produced milk
between 4 and 6 hours. The taste, aroma, color and general acidity of the formed yogurt
were determined.
Sensory analysis of 3% coated yogurt Table 8
Culture
number
Characteristics of the formed yogurt
color smell taste
2/1 Light white Like yogurt Soft taste
5/3 Light beige Like yogurt Soft taste
6/2 Light white Like yogurt Soft taste
8/5 Light beige Like yogurt Soft taste
217 Light white Like yogurt Soft taste
X-17 Light white Like yogurt Soft taste
The resulting milk, i.e. yoghurt, was analyzed for color, odor, and taste, which are
sensory parameters. They all had a slightly sour taste, a light yellow color, and a soft,
yogurt-like smell.
5 % by calculating coated yogurt sensory organs Analysis Table 9.
Culture
number
Characteristics of the formed yogurt
color smell taste
2/1 Light white Like yogurt Slightly sour
5/3 Light white Like yogurt Slightly sour
6/2 Light beige Like yogurt Sour
8/5 Light beige Like yogurt Sour
217 Light white Like yogurt Slightly sour
X-17 Light white Like yogurt Slightly sour
10 % by estimated coated yogurt sensory organs Analysis
2/1 Light beige Like yogurt Soft taste
5/3 Pale yellow ( such
as soy milk discarded )
Like yogurt Slightly sour
6/2 Light white Like yogurt Soft taste
8/5 Pale yellow ( such
as soy milk discarded )
Like yogurt Soft taste
217 Light white Like yogurt Soft taste
X-17 Light beige Like yogurt Soft taste
The resulting milk, i.e. yoghurt, was slightly sour in taste, light yellow in color, with a slight
whey secretion, and smelled as soft as yogurt.

25. DISCUSSION
Probiotics are living microorganisms that retain beneficial health properties in their
host organism. Most commercially available probiotics today are Lactobacillus and
Bifidobacterium. Lactic acid bacteria with probiotic properties are resistant to gastric juice
and bile. As a result, it lives in the gastrointestinal tract, attaches to the intestinal wall and
exhibits therapeutic properties. [32] Therefore, probiotic lactic acid bacteria have been
studied in many countries. The probiotic properties of lactic acid bacteria isolated from
various sources are being studied intensively. [13, 14,19,23]
Researchers who determined the probiotic properties of lactic acid bacteria isolated
from fermented samples [19,21] showed that L. helveticus, L. paracasei sbsp paracasei
microorganisms were able to withstand pH 3.0 acidic environment for 3 hours and 0.3%
bile for 8 hours. Although the results of our study showed that bile acids had the same
growth for 8 hours, our probiotic cultures showed a steady increase in acid resistance at
pH 2 and pH 3 for 3 hours. can be seen to grow better.
In this study, we studied probiotic lactic acid bacteria from traditional sour milk
products and concluded that they could be used as a probiotic product in the future to
make probiotic yogurt.

26. CONCLUSION
In 2016, 14 pure cultures of lactic acid bacteria isolated from autumn dung samples in
Dundgovi and Bayankhongor aimags were selected for the study.
Of the 14 cultures selected for the probiotic study, 10 cultures were able to survive for up
to 3 h in an acidic environment with a pH = 2.0.
Six of the 10 cultures that were acid-resistant in a 0.3% bile medium were able to grow
for up to 8 hours.
In terms of antibiotic resistance, cultures 6-2, X-17, and 5-3 showed resistance to six
commonly used antibiotics: amoxicillin, ampicillin, tetracycline, and penicillin.
Based on the fact that the acid and bile tolerance of these cultures was higher than that of
probiotic cultures A, E, and C isolated from PROBIOTIC yogurt, it is considered that 6-2, X-17,
and 5-3 cultures isolated from HORMOGN can be used as probiotic products in the future.

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29. APPENDIX
Figure 4. Determination of acid resistance of pure cultures of isolated lactic acid bacteria
Figure 5. Growth on MRS agar in an X-17 culture grown for 3 h in an MRS broth medium
with pH = 2.0 and pH = 3.0
Figure 6. Growth on MRS agar of 325 cultures grown for 3 h in an MRS broth medium with
pH = 2.0 and pH = 3.0
Results for antibiotic resistance
Figure 7. Antibiotic resistance range of 6/2 and X-17 cultures

30. Probiotic yogurt isolated pure cultures of lactic acid bacteria probiotic yogurt coated
I. Actimel yogurt
Figure 8. Actimel yogurt pure bacterial cultures to distinguish and yogurt coated
II. Elgen yogurt
Figure 9. “Elgen” yogurt pure bacterial cultures to distinguish and yogurt coated
III. Sain yogurt
Figure 10 . Sain yogurt pure bacterial cultures to distinguish and yogurt coated
First picture- Sample,
Second picture-Separation of pure STB culture from yogurt
Third picture-Yogurt coating using isolated probiotic lactic acid bacterial culture

31. Figure 11 . 217 No. cultures with yoghurt coated
Figure 12 . 5/ 3 cultures with yoghurt coated
Figure 13 . 8 / 5 cultures with yoghurt coated

32. ACKNOWLEDMENT
We would like to express our sincere gratitude to E.Uugantsetseg and Du Ming the supervision,
and the staff of the Agricultural Product Chemical Technology Program for their guidance and
assistance in conducting the research on “Probiotic Yogurt Production Feasibility Study”.