Fermentation Conditions of Lactobacilli for the Production of Lactose-Free Starter Culture

: This research aims to determine the fermentation conditions and viability in a medium simulating the gastrointestinal tract and to examine the antagonistic potential of Lactobacillus acidophilus ( Lb. acidophilus) and Lactobacillus rhamnosus ( Lb. rhamnosus) in producing a lactose-free starter. Our study examined Lactic Acid Bacteria (LAB). The sources of Lb. acidophilus strains are camel milk (shubat), Lb. rhamnosus mare milk (koumiss), traditional homemade dairy foods in Kazakhstan. Lb. acidophilus and Lb. rhamnosus are resistant to gastric juice acidity at a pH above 3, therefore they will be able to pass through the stomach intact. The cell populations remained ≥10 6 CFU mL. When Lb. acidophilus was treated at pH 3.0-5.0, the cell population was significantly higher (p≤0.05). Simulated gastrointestinal transit was studied by assessing NaCl resistance. The cell counts in the medium with 5% NaCl were significantly higher (p≤0.05) than those in the medium with 2% NaCL. Lb. acidophilus and Lb. rhamnosus showed growth corresponding to ~7 log CFU at 5-7% NaCl. Lb. acidophilus and Lb. rhamnosus were tested for their antibacterial activity by agar-well diffusion test. Three common pathogens, E. coli, S. marcescens, and S. Typhimurium were used for the investigation of antagonistic activity. Lb. acidophilus and Lb. rhamnosus showed significantly high inhibition zone ranges (p≤0.05) from 10-12 mm against E. coli and S. marcescens . A medium zone of inhibition 5.0-6.0 mm against S. typhimurium. For co-fermentation, pasteurized milk inoculated with Lb. acidophilus and Lb. rhamnosus cells were fractionated into 25 mL portions and placed in 50 mL conical tubes. It was spread on a nutrient medium of Petri dishes and incubated for 24 h at 37°С. Lb. acidophilus and Lb. rhamnosus starter culture reached a significant cell population of about ~8 logs Colony Forming Unit (CFU/mL) during co-culture fermentation, pH in Pasteurized Milk (PM) rapidly decreased to 3.0.


Introduction
The aim of this study was to determine the viability of lactobacilli in a gastrointestinal tract simulated environment and to investigate the antagonistic potential of probiotic starter bacteria Lb. acidophilus and Lb.rhamnosus.To determine a suitable fermentation period for mixed cultures of Lb. acidophilus and Lb.rhamnosus to achieve low pH values in pasteurized milk.
The original contribution of the study is the use of a probiotic product containing domestic microorganisms will correct dysbiosis processes that have arisen against the background of the underlying disease or milk sugar deficiency.The use of autological lactobacilli preparations is comparable in effectiveness to the use of preparations of commercial lactobacilli strains.
Low-lactose and lactose-free dairy products are classified as functional foods, which provide adequate nutrition for people with lactase deficiency.Their functionality is also enhanced by the probiotic potential of microbial cultures.Strains of LAB isolated from traditional homemade dairy foods in Kazakhstan; from solid fermented dairy products (Kurt, irimshik), liquid cow's milk (ayran, homemade butter), mare's milk (koumiss), and camel's milk (shubat).
The obtained research data on the study of microorganism cultures with probiotic potential are scientifically substantiated and expand the understanding of biological and technological properties of lactic acid bacteria cultures, allowing for the optimization of methodological approaches in the design of starter.On the basis of a comparative evaluation of biological and technological properties of microorganisms bacterial compositions based on Lb. acidophilus and Lb.rhamnosus were produced and a starter was developed.
The significance of probiotic cultures and their beneficial effects in dairy products as mass consumption products, the use of which aims to best fulfill the needs of special dietary products.Fermentation of yogurt starter based on probiotic cultures, better metabolized: Complete utilization of lactose and efficient degradation of galactose.Also, today the prescription of infant formulae supplemented with probiotic strains has become more frequent (Zhang et al., 2021).Ice cream is the most favorable for the inclusion of probiotic strains since the viability of probiotics during production and storage is higher compared to other dairy products (Mohammadi et al., 2011).However, the study shows that fermentation of ice cream blended with probiotics is recommended as a preferred method over including probiotics in the ice cream mix without further fermentation (Gao et al., 2021).The use of probiotic strains is not limited to fermented milk products, yogurt, and cheese, but there are a number of other products that contain probiotics.For example, butter.There is some data showing that high levels of saturated fatty acids in butter are linked to many cardiovascular diseases and diabetes.Probiotic bacteria decrease cholesterol levels (Pimpin et al., 2016).
The experiments conducted by us in the framework of our project, aimed at the implementation of the program "strategy Kazakhstan 2050" from 1.09.23 of the message of the president of the Republic of Kazakhstan, will allow the agrarian sector of the Republic of Kazakhstan to achieve food and environmental sustainability.The laboratory of microbiology and biotechnology of the Astana branch of "Kazakh Research Institute of Processing and Food Industry" LLP has now created a laboratorian starter for manufacturing lactose-free lactic acid products.The distinction from foreign analogs is that the preparation is based on microorganisms isolated in the territory of the Republic of Kazakhstan.This fact has a certain economic effect, as it allows to save costs on the import and transportation of expensive inoculants.After completing all the research experiments in the implemented project, the economic efficiency of the use of a domestic starter will be calculated.
Scientists from higher educational universities of the Republic of Kazakhstan and research institutes are actively studying and using starter cultures to produce preventive and medicinal dairy products.
In the presented study we identified the predominant species of lactic acid bacteria isolated from Kazakh national dairy foods were strains of the genus Lactobacillus acidophilus, rhamnosus, plantarum, fermentum, paracasei, and bulgaricum.Similar bacterial strains were identified in the teamwork of scientists of Buketov Karaganda University, in co-operation with scientists of the Medical University of Karaganda and Nazarbayev University of Astana.They also applied methods to study the biological and probiotic properties of isolated bacterial strains (adhesion to erythrocytes, antibacterial activity against indicator microorganisms, and analysis of morpho-cultural properties of isolates) (Amirkhanova et al., 2021).
Moreover, one of the works of Kazakhstani scientists in the field of microbiological diversity of shubat and ayran was conducted with the support of the JSC "center for international programs" and international scholarship "Bolashak" of the ministry of education and science of the Republic of Kazakhstan.As a result of research work, acid resistance, tolerance to bile acid salts, and antibiotic resistance of isolated strains of microorganisms were analyzed (Zhadyra et al., 2021).
A recent study published by Kazakhstani scientists on "Effects of low lactose mare's milk yogurt consumption on gut microbiota function" was funded under the scientific program of the ministry of education and science of the Republic of Kazakhstan "development of technology using new strains of beneficial microorganisms, enzymes, nutrients, and other complexes in the production of special dietary foods" INN BR10764998.National scientists studied the effect of low-lactose yogurt on the intestinal microbiota of 15 male and female mongrel rats suffering from lactose intolerance.The laboratory rats had a normal diet for 7 days, but for the following 4 weeks, probiotic yogurt (low lactose yogurt) was included in the diet.Fecal samples of the animals before and after the diet (first collection) were examined.A control collection of stool samples was examined on day 28.The microbial community was then determined and quantified using the 16S rRNA method which allowed to carry out genetic identification of microorganisms.The resulting starter is based on lactobacillus, Bifidobacterium, and Streptococcus strains.Sour milk yogurt made from a mixture of mare and cow milk with a low lactose content of 0.002% was used as substrate.This product was fermented for 8 h, at 38°C.As a result, the low lactose sour milk product had a characteristic sour milk flavor and a clot of 1100 T. So eventually consumption of low lactose fermented milk products resulted in a change in microbial biodiversity where an increase in the microbial population of Heliobacteraceae, Eubacterium, Muribaculaceae, Lactobacillaceae and others was observed.Lactose-free yogurt contributed to the decrease in the population of Ruminococcaceae, Peptostreptococcaceae.Thus, the developed starter was aimed at correcting intestinal dysbacteriosis in laboratory rats suffering from lactase deficiency (Samat et al., 2022).
Based on the literature analysis, our group of scientists studied the fermentation conditions of lactobacilli isolated from domestic raw materials to produce lactose-free starters under laboratory conditions.The research we conducted does not require ethical approval as the research has not been conducted on animals or humans.At this stage, the members of the research group have developed a laboratory starter.We are working on writing an application to the ministry of education and science of the Republic of Kazakhstan for financial assistance to continue the current study.
Lactose malabsorption is a common condition worldwide characterized by decreased lactase activity.Lactose is a milk sugar that is hydrolyzed by the enzyme lactase into the monosaccharide's glucose and galactose (Di Stefano et al., 2002;Suchy et al., 2010;Scrimshaw and Murray, 1988).
Symptoms of lactose intolerance are a chronic gastrointestinal disorder characterized by pain, diarrhea, abdominal bloating, weight loss, flatulence, and abdominal cramps (Krieger-Grübel et al., 2020;Ratajczak et al., 2021).Lactose intolerance affects about 75% of the world's population and can be associated with various genetic factors.The global prevalence of lactose intolerance is 2-5% in Northern Europe (Scandinavia, Germany, UK), 17% in Finland and Northern France, and about 50% in South America and Africa.In North American adults (79% of Native Americans, 75% of African Americans, 51% of Hispanics and 21% of Caucasians).90-100% in Southeast Asia, including the Republic of Kazakhstan (Heine et al., 2017).In addition, many studies have shown that avoiding milk and dairy products can increase the risk of bone fractures, osteoporosis, and nutrient deficiencies.However, dairy products are one of the main sources of calcium, potassium, vitamin D, B vitamins, and high-quality protein (Leis et al., 2020).Shokryazdan et al. (2017) state that bacteria, yeasts, and fungi can be used as probiotics.In particular, IBCs can be selected as probiotic cultures.According to other researchers, starter bacteria (Lb. acidophilus, Lb. plantarum, Lb. rhamnosus, Bifidobacterium breve, B. lactis, B. longum, and Streptococcus thermophiles) induce B-galactosidase activity which naturally reduces lactose content, making the product suitable for people with lactose intolerance (Hemarajata and Versalovic, 2013;Ahmed et al., 2013).Probiotic products should contain live microorganisms.A combination of different populations of microorganisms related to lactobacilli species have traditionally been used as probiotics in fermented milk products (De Vrese and Schrezenmeir, 2008).Innocente et al. (2016) reported that probiotic fermented foods should contain at least 10 7 CFU/g of live bacteria.Probiotics have now been shown to improve lactose metabolism.The criteria for selecting probiotics are to reduce pH, and lactose content and increase lactic acid concentration.They should have biological activity against opportunistic microorganisms and high viability to colonize the gastrointestinal tract (Lim et al., 2015).
In the 1970s the idea of probiotics was first proposed by Russian Nobel Prize winner Ilya Mechnikov, known for his book life extension.The term "probiotic" was first coined by W. Collat as "a microbial population favorable to the intestinal microflora".Fuller then listed criteria specific to probiotics.Later, a fuller proposed a definition of probiotics as "a preparation of live microorganisms consumed by humans that induces beneficial effects by qualitative or quantitative effects on the gut microflora".Floch MH, Madsen KK, and Jenkins DJ then expanded the definition to "Probiotics are human-associated microorganisms consumed either with food or as a supplement to improve health".The definition of probiotics was last updated in 2018 by the US National Institute of Health: The national centre for complementary and Integrative Health.The title was reworded as follows: "Probiotics are live microorganisms that provide health benefits when consumed, usually by improving gut flora."Products marketed as probiotics include dairy products and products that are not consumed orally, such as skin creams" (Shokryazdan et al., 2017;De Vrese and Schrezenmeir, 2008).
The present study describes the potential of the newly developed probiotic fermented sour milk starter.Our results are based on sourdough which was fermented with two lactic acid bacteria and isolated from Kazakhstan's homemade sour milk products kurt, irimshik, cow, mare, and camel milk.The study subjects were identified by 16rRNA genetic analysis and the construction of a phylogenetic tree.Two isolates of Lactobacillus acidophilus and Lactobacillus rhamnosus can be practically used as probiotic cultures in sourdough production because of their beneficial acidifying capacity.The cultures studied had significant resistance to low pH and 5-7% NaCl.The studies conducted and results obtained demonstrate the ability of Lactobacillus acidophilus and Lactobacillus rhamnosus to remain viable in gastric acid and bile salt tolerance (ph and NaCL treatment), to possess antagonistic activity against opportunistic microorganisms.The antagonistic activity of Lactobacillus acidophilus and Lactobacillus rhamnosus demonstrated a high range of inhibition zones of 5-6 mm against all indicator bacteria: E. coli, S. marcescens, S. Typhimurium.
It is well recognized that live, active probiotics should be present in a ratio of 10 8 -10 9 СFU/mL.However, it is currently a great challenge to maintain the viability of probiotic cultures.Despite this, Lactobacillus acidophilus and Lactobacillus rhamnoses showed high viability when co-cultured and the starter had an adequate pH.

Extraction and Counting
In June 2020, twelve samples were collected from Zhibek Zholy village located in the Arshali district of Akmola oblast.Around 100 mL of each sample was collected and placed in a sterile glass bottle.The samples were then taken to our laboratory and further isolated and microbiologically analyzed for LAB.Sixteen strains of LAB were isolated from solid fermented dairy products (Kurt, Irimshik), cow's milk (ayran, homemade butter), mare's milk (koumiss), and camel's milk (shubat).
Microorganisms were isolated in a Lamsystem laminar box.We prepared MRS agar HiMedia, nutrient medium MRS agar is sterilized at t 120°C under pressure 1A in autoclave "Steam Sterilizer VK-75-01".LAB colonies were cultured in the thermostat TS-200 SPU, produced by JSC "Smolensk SPU SKTB" at 37°C for 48 h.Cooled to room temperature 37°C, then distributed in sterile Petri dishes 15-20 mL each in three repetitions.In this method, the number of bacteria per unit volume of the sample is reduced by serial dilution before the sample is spread over the surface of the agar cup.Be sure to mix the tubes with nutrient broth before each serial transfer.Transfer 0.1 mL of the last three dilutions (10-5, 10-6, 10-7) into each of the three nutrient agar cups and label the cups.
Using a sterile dispenser "Dispenser lenpipet light 1000 mL" (manufacturer Thermo Fisher Scientific, Finland), drop the test suspension into the Petri dish, distributing the cell suspension over the surface of the petri dish.Evenly spread 0.1 mL samples of cow's milk (ayran, homemade butter), mare's milk (koumiss), and camel's milk (shubat) and crushed irimshik and kurt in a porcelain mortar over the entire surface of one of the nutrient media.Agar dishes until the medium no longer looks wet.Sowings were done with different pipettes (tips).The pipette is held strictly vertically.Droplets are not rubbed but can be slightly enlarged by slightly rocking the petri dish.The cultures are incubated and then the number of colonies grown is determined.After sowing the petri dishes are placed in the thermostat with lids downwards.Colonies of lactic acid bacteria are cultured in the thermostat TS-200 SPU produced by Smolensk JSC "SKTB SPU" at a temperature of 37°C for 48 h.Then we count the obtained lab cells.Solid objects like "Kurt" and "irimshik" were rubbed with a porcelain mortar and pestle.The process of rubbing "Kurt" and "irimshik" with porcelain mortar and pestle was as follows.The hardness and rough surface of the porcelain mortar and pestle provide a perfect grinding surface.It is ideal for grinding hard objects such as irimshik and Kurt.The pestle and mortar should be rinsed with distilled water after each use.Grind the irimshik and Kurt to a fine powder and repeat this process until the powder is completely dissolved.We recommend that the pestle and mortar be thoroughly cleaned before first use.Wetted with physiological NaCL solution.Cycloheximide at a concentration of 0.01 was added to MRS to inhibit fungal growth.
Bacterial isolation was carried out according to Abosereh et al. (2016) with some modifications: Successively diluted samples (one milliliter from each sample starting from 10 7 , 10 8 , 10 9 ) were examined on MRS agar.The plates were incubated under anaerobic conditions at 37°C for 48 h.Lactobacillus colonies were randomly selected and numbered.This process was repeated three times to obtain well-separated purified colonies.The strains were then phenotypically examined by morphology and Gram staining.

Identification and Phylogenetic Analysis
Strains were identified using the 16S rRNA gene amplified by PCR using the direct primer 8f 5'-AgAgTTTgATCCTggCTCAg-3 and 806R-5' ggACTA CCAgggTATCTAAT.PCR amplification included prolonged denaturation at 92°C for 3 min; 32 cycles: 95°C for 3 sec, 55°C for 40 sec, 72°C for 60 sec; final elongation for 10 min at 72°C.The primers we used in this study are specific to all bacteria.They are universal and designed to classify related groups of lactic acid bacteria based on sequencing results.
The PCR program was performed using a gene Amp PCR system 9700 thermal cycler (applied biosystems).PCR products were purified from unbound primers by enzymatic method using exonuclease I (ferments) and shrimp alkaline phosphatase (ferments).The sequencing reaction was performed using the big dye terminator v3.1 cycle sequencing kit (applied biosystems) according to the manufacturer's instructions, followed by fragment separation on a 3730×l DNA Analyzer (applied biosystems).The sequences obtained were identified in the gene bank using the BLAST algorithm.Mega software version 6.0 was used to generate phylogenetic trees using the Neiighbor-Joining (NJ) algorithm (Wang et al., 2016).This method allowed the identification of bacteria isolated from Kazakhstan home-made dairy products.Further identification of bacteria allowed the prediction of phylogenetic relationships.

Antimicrobial Activity
The antimicrobial activity of Lb. acidophilus and Lb.rhamnosus was assessed by agar diffusion methods (prick method) as described by Reuben et al. (2019).By means of a sterile pipette, a number of small drops of diluted culture medium are dispensed onto a sterile glass substrate.Subsequently, each drop is observed under a microscope.This drop is extracted with a sterile capillary pipette to fresh medium.The individual microorganism present in the drop starts multiplying to yield a pure culture.The indicator strains used in this study were S. Typhimurium, S. marcescens, and E. coli.The inhibitor tests were carried out in three replicates.
Lactobacilli species were inoculated into MRS broth at 37°C for 24 h under anaerobic conditions.One hundred microliters of 10 7 CFU/mL target strains were distributed on MRS agar and left to set in the refrigerator for 4 h to avoid premature growth of the strain.The dishes were incubated at 37°C for 24 h.The antagonistic activity of the probiotic strains was evaluated by the diameter (mm) of the growth inhibition zone (Ratajczak et al., 2021;Heine et al., 2017).The high inhibition zone was 10-5.5 mm, the medium inhibition zone was 5-8.9 mm and the low inhibition zone was 1-4.9 mm.

pH Treatment and Viability
The pH tolerance was tested according to the methodology described by (Lopes et al., 2020).To one milliliter of probiotic strains, 9 mL of sterile distilled water was added to obtain the initial dilution.Serial dilutions were made separately for Lb.acidophilus and Lb.rhamnosus.The initial pH values of the MRS medium were adjusted to 3.0, 4.0, 5.0, 6.4, and 8.5 using 1 mol/L HCl and 1 mol/L NaOH.PH was measured on a Mettler Toledo SevanCompact instrument.The tested probiotic cultures were incubated at 37 °C for 48 h, 72 h and 120 h.All cell concentrations were presented as log CFU/mL.The number of CFU was calculated using Eq.1: where, Eq.1: M = The number of cells in 1 mL A = The average number of colonies that grew from dilution V = The volume of the suspension ml 10 n = The dilution The use of this method makes it possible to assess the resistance of isolated cultures of microorganisms to samples simulating transit through the gastrointestinal tract.Entering the organism, probiotic microorganisms are exposed to various stress factors, so an important selection criterion is their survival and successful passage through the upper digestive tract.Resistance to bile is one of the most important properties of microorganisms introduced in probiotics.In the small intestine, probiotics are exposed to bile acids and pancreatic enzymes.

NaCL Treatment and Viability
The ability of LAB to survive under different salt concentrations was studied in MRS broth and agar.The tolerance to NaCL was determined according to methods by Do Amaral Santos et al. (2014).One milliliter of samples was mixed with 9 mL of distilled water to take an initial dilution.Serial dilutions were made separately for Lb.acidophilus and Lb.rhamnosus.Thus, 1 mL suitable dilution Lb. acidophilus and Lb.rhamnosus were mixed with the MRS broth with different concentrations of NaCl (2, 5, 7%) and inoculated at 37°C for 48, 72, and 120 h.Viable counts of probiotic bacteria were obtained by spreading 1 mL of the dilution mixed with the melted MRS agar.The MRS plates containing 0.1% (w/v) cysteine-HCl allowed differential count and the colonies presented of color white.Plates were incubated under anaerobic conditions at 37°C for 48 h.Then colonies enumeration was expressed as log CFU/mL.
The number of CFU was calculated by the Eq.2: where, Eq.2: M = The number of cells in 1 mL A = The average number of colonies that grew from dilution V = The volume of the suspension mL 10 n = The dilution Strains are resistant to the secretory fluids of the gastrointestinal tract, which is one of the main indicators of probiotic quality, due to the fact that most lactobacilli are inactivated when exposed to bile and low gastric pH.Probiotics must survive passing through the low pH of gastric juice and bile acids in order to reach the intestinal tract, colonize the host epithelium, and exert beneficial effects.The obtained results of resistance of isolates to stress factors indicate the ability of cultures promising as inoculums to survive under unfavorable conditions in the upper gastrointestinal tract.

Statistical Analysis
Significant differences of means (p≤0.05) were compared through independent Student's t-test by using SPSS 23 (IBM Corp., Armonk, New York, USA).Statistical analyses were carried out using Microsoft Excel 2010.Independent Student's t-test was used to test the statistical significance between the mean values of microbial growth during the study of resistance of isolated microbial cultures to samples simulating gastrointestinal transit (pH, NaCl).Cell count data were obtained from three independent experiments.p<0.05 was considered to indicate a significant difference.Independent sampling showed an equal variance of 0.000.The confidence interval showed a plausible difference between the obtained values of microbial growth.
In our study, p<0.05 rejects the null hypothesis which argues that there is a statistically considerable difference in the growth of microorganisms simulating gastrointestinal transit.

Extraction and Counting
The isolated IBC strains were identified using 16S rRNA.These 16S rRNA sequences were collated to construct a phylogenetic tree and phylogenetic position of the strains studied.The 16S rRNA gene sequence (about 1,400 bp) was determined and examined using the NCBI BLAST software (http://www.ncbi.nlm.nih.gov) for their closest relatives/the reference strains with homology greater than or equal to 99%.The genetic identification process established that the cultures studied belong to a taxonomic group such as Lactobacillus (Fig. 1).

Identification and Phylogenetic Analysis
Their probiotic properties: Antagonistic activity against pathogens, biocompatibility, high viability, and lactose utilization activity were investigated in our previous work (Arynova et al., 2020).Based on the results obtained, we settled on two strains: Lb. acidophilus and Lb.rhamnosus.The ability of Lb. acidophilus and Lb.rhamnosus to ferment lactose was investigated using the Cole ferricyanide method (Cole, 1933).The lactose content in PM with cultured Lb. acidophilus and Lb.rhamnosus was 3.1% (data not shown).In traditional Kazakh domestic dairy products, Lb. acidophilus and Lb.rhamnosus were identified as the dominant species and were present in all samples collected.

Antimicrobial Activity
The antagonistic activity of the sixteen probiotic strains was studied by inoculation on an agar medium.The antagonistic activity of MCB against the three indicator pathogens varies greatly and the results are presented in Table 1.Lactobacillus acidophilus and Lactobacillus rhamnosus had high to medium-range inhibitory activity against all 3 indicator pathogens S. typhimurium, S. marcescens, and E. coli.

pH Treatment and Viability
The effect of pH on the growth properties of the potential probiotic cultures Lb. acidophilus and Lb.rhamnosus was carried out as described under 'materials and methods'.One of the most significant characteristics of a lactose-free starter as a probiotic is its resistance to the acidity of gastric juice.Gastric pH ranges from pH 4.0-6.0.The ability of potential probiotic strains to survive under acidic conditions is a mandatory criterion for the production of probiotic dairy products (Abosereh et al., 2016).
It is assumed that a good probiotic should have the ability to withstand pH in the range of 3.5-4.5,as it is used to assess the acid resistance of the probiotic culture (Kim et al., 2019).
As pH is an important criterion of bacterial function and viability, we studied the effects of different pH values (3.0, 4.0, 5.0, 6.4, 8.5).The evolution of cell populations in Lb. acidophilus and Lb.rhamnosus cultivation is shown in Figs.3-4.
Lb. acidophilus and Lb.rhamnosus shows good growth at pH 3.0.The cell populations remained ≥06 CFU mL during 72 h of incubation.When Lb. acidophilus was treated at pH 3.0, 4.0, and 5.0, the cell population was significantly higher than in the treatment group at pH 6.4, 8.5 (p≤0.05).
Lb. acidophilus and Lb.rhamnosus did not grow at a pH above 5.0.The number of viable cells decreases rapidly in the pH range of 6.4-8.5.At the end of the study, according to the initial counts Lb. acidophilus decreased by~1.4 log CFU, Lb. rhamnosus~1.6log CFU.

NaCL Treatment and Viability
In order to select possible probiotic candidates, we investigate their tolerance to different salt concentrations.
The salt tolerance of probiotic strains is one of the important technological properties in the production of probiotic dairy products.Osmotic stress can cause pronounced inhibition of bacterial growth.High concentrations of NaCL cause a decrease in the adhesion capacity of the functional groups of lactobacilli and increase cell membrane damage (Ma et al., 2020).
In this study, Lb. acidophilus and Lb.rhamnosus were studied and their ability to grow at different concentrations of salts (2, 5, 7% NaCL).The results of cell growth are shown in Figs.4-5.Lb. acidophilus and Lb.rhamnosus showed a slight increase in cell population at 24, 48, and 72 h in 2% NaCL, which were not statistically different (p≥0.05).The cell counts in the medium with 5% NaCL was significantly higher than those in the medium with 2% NaCL (p≤0.05).The tested strains showed the best survival rates at 5 and 7% NaCl.Lb. acidophilus and Lb.rhamnosus showed growth corresponding to ~7 log CFU at 5-7% NaCl for 72 h The quantification of one strain from the other was determined according to the methodology (Schiraldi et al. 2002).Cell growth was followed during experiments by measuring absorbance at 600 nm on a Beckman DU 640 Spectrophotometer (Milan, Italy).Cell counting by direct microscopic analysis was not always reliable, cell density estimate can be affected by an error.However, throughout the experiment, a few samples were extruded through a syringe needle (2-4 times), and diluted, and the number of cells per unit volume was counted in a Burker chamber using an optical microscope.The two Lactobacillus strains were recognized by morphological characteristics.Fermentation was carried out on a Sartorius Biostat® A MO UniVessel® Glass 1L 230V fermenter.The quantities of Lactobacillus acidophilus and Lactobacillus rhamnosus during co-fermentation are shown in Fig. 6.At the beginning of co-fermentation, ~7 log CFU/mL of Lactobacillus acidophilus and Lactobacillus rhamnosus were in 500 mL of Pasteurised Milk (PM).The fermentation process in coculture was carried out for 24 h.The viable cell populations of the test strains increased slowly over 24 h and reached 8 log CFU/mL, showing an increase of 1 log CFU/mL.At 8 h of co-fermentation, their growth was above 7 log CFU/mL.The growth of Lactobacillus rhamnosus ranged from 7.47-7.77log CFU/mL after 20 h.Lactobacillus acidophilus showed a population of 8 log CFU/mL at the end of co-cultured fermentation.
The acidification rate during fermentation of a mixed culture in PM is shown in Fig. 7.After 4 h, the pH values in the PM substrate with the incubated strains drop.Coculture of Lactobacillus acidophilus and Lactobacillus rhamnosus contributed to the pH drop from 6-3 after 24 h.In co-fermentation, Lactobacillus acidophilus and Lactobacillus rhamnosus showed an increase in their population from ~7 log CFU/mL to ~8 log CFU/mL at 12-, 16-, 20-and 24-h fermentation than at 4 h fermentation, these differences were significant (p≤0.05).Thus, MCBs achieved a significant number of viable cell populations by 24 h fermentation compared to 12, 16, and 20 h fermentations.Wang et al. (2020) reported that probiotic bacteria must have the ability to survive and pass through the gastrointestinal tract.Thus, this study was carried out to evaluate several probiotic characteristics of Lb. acidophilus and Lb.rhamnosus.

Discussion
Three common conditionally pathogenic microorganisms, S. typhimurium, S. marcescens, and E. coli, were used to investigate their antagonistic activity, carried out by in vivo diffusion methods.All two strains showed strong antimicrobial activity against E. coli.However, the inhibition was more pronounced in Lb. acidophilus than in Lb. rhamnosus (Fig. 2).Fei et al. (2018) found that L. acidophilus inhibited not only the growth of Gram-negative bacteria but also the growth of Gram-positive bacteria such as L. monocytogenes, S. aureus, and S. hemolyticus.Similar observations have been made by Mpofu et al. (2016) Lb. rhamnosus can inhibit several pathogens.Rajoka et al. (2017) report that Lb. rhamnosus has a higher zone of inhibition against E. coly (8-13 mm diameter).
Lb. acidophilus and Lb.rhamnosus are lactic acidproducing bacteria.Georgieva et al. (2015); and Do Amaral Santos et al. (2014) also identified that the bactericidal activity of Lb. acidophilus with respect to Gram-negative pathogens may be related to the production of lactic acid as a result of carbohydrate glycolysis.Our studies showed that Lb. acidophilus and Lb.rhamnosus has the broadest spectrum of activity and zones of inhibition (with a range of high and medium diameters from 5-12 mm).
These strains were further investigated for resistance to pH and NaCL and their ability to grow under various acidic stresses and salt concentrations.Kailasapathy and Chin (2000) in their work reported that pH values between 3.5 and 4.5 are good environmental conditions for populations of potential probiotic organisms.The survival of Lb. acidophilus and Lb.rhamnosus was based on pH values; low pH increases their survival.Figure 3 shows the survival of Lb. acidophilus and Lb.rhamnosus at 3.0, 4.0, 5.0, 6.4 and 8.5 for 72 h of incubation.At pH 3.0-4.0,survival was higher compared to pH 6.4-8.5 for all strains studied (p≤0.05).The results showed that all strains tested were highly resistant to acidic conditions.Lim et al. (2015); Rathore et al. (2012) studied the survival of Lb. rhamnosus and Lb.acidophilus under acidic conditions showed the highest survival (pH 1.5 -3.0, pH 3.0 -4.0).Zuljan et al. (2014) report in their experiments that an acidic environment plays an important role in bacterial growth.
An acidity tolerance in the pH range of 3-4, suitable for probiotic cultures, is sufficient for successful passage through the stomach and the environment of the human gastrointestinal tract.These results are in agreement with most of the data presented by other authors as well (Dinçer and Kıvanç, 2021;Innocente et al., 2016).
Lb. acidophilus and Lb.rhamnosus were tested for their ability to grow in the presence of NaCl (2, 5, 7%).Two strains were tolerant to NaCl concentrations.Lb. acidophilus and Lb.rhamnosus reached ~6 Log CFU/mL at 48 h.
Other researchers Vinderola et al. (2002); Wang et al. (2020) have also observed a high level of bacterial growth at 7-5% NaCL, so the growth of Lb. acidophilus was increased by 7-5% NaCl among the probiotic strains used in this study Thus, 7-5% NaCl stimulated the growth of strains.Ma et al. (2020) write that NaCL is widely used in food production, but in probiotic products, high salt concentration affects cell viability, metabolism, and physiological functions of isolates.Son et al. (2018) have suggested that probiotic products require a bacterial suspension (1×10 7 CFU/mL) to have an effect on the digestive system.The cofermentation process for the production of fermented probiotic products is shown in Fig. 6.The highest level of Lb. acidophilus and Lb.rhamnosus cell population above 8 logs CFU/mL was observed at the end of cofermentation (Fig. 6).The observed number of probiotic bacteria is in agreement with similar studies carried out by previous authors (Skryplonek et al., 2019).

Conclusion
Our study examined LAB isolated from traditional homemade dairy foods in Kazakhstan; specifically, up to sixteen strains of lactobacilli were identified via advanced genetic techniques known as "16sRNA sequencing".Up to sixteen strains of Lactobacilli species were identified via advanced genetic techniques known as "16sRNA sequencing".Gene amplified by PCR using the direct primer 8f 5'-AgAgTTTgATCCTggCTCAg-and 806R-5' ggACT ACCAgggTATCTAAT.The PCR program was performed using a GeneAmp PCR System.The sequencing reaction was performed using the BigDye terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).The sequences obtained were identified in the gene bank using the BLAST algorithm.Mega software version 6.0 was used to generate phylogenetic trees using the Neighbor-Joining (NJ) algorithm.The above method enabled us to identify the biodiversity of lactic acid bacteria existing in Kazakhstan home-made dairy products (fermented cow, mare, camel milk, irimshik, kurta, home-made butter).The lactobacilli strain we have identified we are considering for further selection as lactose-free probiotics and want to examine them as valuable resources.
The present study describes the antagonistic activity and tolerance to acid and salt concentrations of Lb. acidophilus and Lb.rhamnosus isolated from traditional Kazakh domestic dairy products.The cultures investigated had significant resistance to low pH and 5-7% NaCl.Based on these results, it can be concluded that Lb. acidophilus and Lb.rhamnosus are potential probiotic cultures and can be used as a starter for dairy products.A cell population of Lb. acidophilus and Lb.rhamnosus of 8 logs CFU/mL and a pH value of 3.0 was achieved within 24 h during the co-culture process.
Development of starters by mixing Lactobacillus acidophilus and Lactobacillus rhamnosus should become domestic fermented dairy products to meet consumer demand and market expansion in Kazakhstan.Based on the results of the research it is planned to conduct pilot testing of starters on the territory of Akmola region, Arshala district, Zhibek Zholy settlement based on the firm "Voskhod-2004" LLP.At the current moment, there is an agreement (contract) and in the future, an act of completed work on testing of starters for lactose-free lactic acid products will be obtained.Next, it is planned to implement this starter for industrial purposes so far only on the territory of the Akmola region, but with the prospect of neighboring regions.
In perspective, the area of our research will be focused on the immobilization of probiotic strains Lactobacillus acidophilus and Lactobacillus rhamnoses.For stability of probiotic viability using spray drying.

Fig. 1 :
Fig. 1: Phylogenetic tree based on 16S rRNA sequences showing the position of Lb. acidophilus and Lb.rhamnosus

Fig. 2 :Fig. 3 :Fig. 4 :Fig. 5 :
Fig. 2: Changes in the number of viable cells (differences between data after 24, 48, and 72 h, in log CFU mL of Lb. acidophilus at different pH values.Columns indicate the standard deviation from the mean value.Indicates a significant difference (p≤0.05) using the student's t-test.Different capitals indicate significant differences between cell populations at different indication times under the same stress conditions

Fig. 6 :Fig. 7 :
Fig. 6: Number of cell populations during co-fermentation in PM.Different uppercase letters indicate significant differences; lowercase letters indicate non-significant differences

Table 1 :
Antagonistic activity of 16 strains of isolated lactic acid bacteria from traditional Kazakh home milk products