Mamayeva L.A., Zh.M.Suleimenova, G.E.Zhumaliyeva

Biotechnological bases of production of fermented milk products for medical and prophylactic purposes

ALMATY – 2018

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UDC 637.146.34BBKMamaeva L.A., Zh.M.Suleimenova, G.E.Zhumaliyeva

ISBNSUMMARY

The theoretical basis of this monograph is the analysis of numerous works of domestic and foreign authors who develop and improve the biotechnology of the production of products for therapeutic and preventive nutrition. In the present work we present information from publications of domestic and foreign authors on the problem under study.

This monograph presents the results of our own research on the use of highly active strains in the production of fermented milk products for functional nutrition.

It is recommended for bachelors, students, masters, graduate students and specialists engaged in the field of biotechnology and food industry.

Velyamov M.T.  -  Head of Laboratory Biotechnology and Food Quality and Safety, Doctor of Biological Sciences, Professor, Academician of the Academy of Agricultural Sciences of the Republic of Kazakhstan 

Lesova Zh.T.  - Candidate of Biological Sciences, Associate Professor of Almaty Technological University 

Serikbaeva A.D.  - Doctor of biological sciences, professor of Kazakh National Agrarian University

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CONTENTINTRODUCTION 41. Functional nutrition, meaning and prospects for their use 61.1 Current trends in the use of probiotics, prebiotics and synbiotics 71.2 Lactic acid bacteria, the significance and prospects of their use 161.3 Microflora of starter cultures used in the production of sour-milk products for dietary nutrition

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2. Research and selection of highly active lactic acid microorganisms 352.1 Morphological and Cultural Properties of Isolated Lactic Acid Bacteria 352.2 Antagonistic properties of isolated cultures of lactic acid bacteria 392.3 Study of the sensitivity of lactic acid bacteria to antibiotics 402.4 Investigation of phage resistance of lactic acid bacteria 422.5 Determination of physico-chemical and technological indicators of lactic acid bacteria

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2.6 Determination of the aroma-forming, proteolytic and lipolytic activity of lactic acid bacteria

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3. Selection of a consortium of lactic acid bacteria with probiotic properties 494. Investigation of the influence of stimulating factors of nutrition of lactic acid microorganisms

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4.1 Investigation of the chemical composition of grain processing products 594.2 Investigation of the effect of germinated wheat on lactic acid microorganisms

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5. Investigation of microbiological processes and physicochemical properties when storing curd whey

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5.1 Study of the influence of whey proteins on the vital activity of lactic acid microorganisms

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6.Development of a composition for a dairy-cereal product based on the use of probiotic microorganisms

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CONCLUSION 84LIST OF USED LITERATURE 88

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INTRODUCTION

Nutrition, as the fundamental process underlying the vital activity of all living organisms, without exception, is of great interest from a wide variety of points of view.

Now in Kazakhstan, as in all countries of the developed world, there is a significant change in the attitude of people, and especially socially active layers of the population, to one's own health.

Health is the greatest value of human life. Everything that makes our life full and happy depends on the state of health: the quality of life, its duration, physical activity, etc. There is a well-founded scientific opinion that with rational nutrition, the duration of human life can reach 120-150 years. Food provides the body with the energy necessary for movement and work, serves as a source of "plastic" substances, proteins, fats and carbohydrates, as well as vitamins and mineral salts, through which the cells and tissues are renewed. The production of hormones, enzymes and other regulators of metabolic processes in the body also occurs due to food products. The nature and usefulness of nutrition depends on the metabolism in the body, the functioning of organs and systems, tissues and cells. With proper nutrition, the internal environment of the human body is ensured, which is the key to health, physical activity and longevity. It provides a full functioning of the immune system, increases the resistance of the body, its ability to withstand diseases. To maintain the normal flow of energy, plastic and catalytic processes, the food should be full. The nutrition of a healthy person should correspond to his physiological needs, depending on sex, region of residence, the nature of work and other factors. Food should be varied.

The diet should include all the groups of products necessary to fill the energy costs and the functioning of all organs and systems of the body. Improper diet leads to disruption of metabolic processes in the body, weakening of immunity, the emergence of chronic diseases, premature aging. Excess nutrition is a frequent cause of diseases of the circulatory system, obesity, atherosclerosis, diabetes mellitus, gout, polyosteochondrosis. Lack of food due to protein deficiency of food causes severe illness in children: slowing growth, mental development, bone formation, the appearance of changes in the pancreas and liver.

In Kazakhstan, according to medical experts, from 75 to 90% of citizens are more or less subject to dysbiosis - a violation of normal intestinal microflora [1]. And also there is a deficiency of vitamins of group "B", including vitamin B12 in the diet of the population.

To effectively solve urgent problems in the field of ecology, nutrition and health of the inhabitants of the Republic of Kazakhstan, a promising direction is the development of technology for sour-milk products using polystyrene bacterial starter cultures with probiotic properties for dietary daily nutrition of people living in areas with unfavorable ecological conditions.

To the most important category of functional nutrition are currently considered probiotics - biological preparations containing living strains of normal human

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microflora. The strains of bifidobacteria, lactobacilli, propionic acid microorganisms have been successfully used for decades in probiotic pharmacopoeial preparations of the first generation and various fermented milk products of a functional purpose. Of great interest is the use of bacteriocins produced by a group of lactic acid bacteria in the food industry [8]. The producers of these bacteriocins are used as a starter culture in various food industries. The resulting bacteriocin ensures the dominance of the desired microflora and the suppression of extraneous microflora, which ensures the safe flow of microbiological processes.

The creation of bio-products of functional nutrition based on symbiosis of probiotic microorganisms and food additives with pronounced prebiotic properties is considered as a strategic direction of alternative medicine, which contributes to the maintenance and restoration of human health.

The analysis of the published fundamental works testifies to the high degree of effectiveness of the use of microbial consortia on the basis of symbiosis of cultures with pronounced probiotic properties, as well as bacteriocin producing microorganisms showing their positive orientation to the organism through regulation of intestinal microflora, suppression of the number of undesirable microorganisms, changes in microbial metabolism, stimulation of body immunity.

The basis for writing this monograph was a steady increase in the interest of specialists in the use of probiotics and bacteriocins in the biotechnology of fermented milk products for functional nutrition.

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1. FUNCTIONAL NUTRITION, IMPORTANCE, AND PROSPECTS OF THEIR USE

The concept of "functional nutrition" originates in the East. The concept of positive (functional) nutrition was first formulated in Japan in the early 80-ies of the last century. Japanese researchers define three basic qualities of functional products: the necessary nutritional value, pleasant taste, positive physiological effect. Healthy food products are not medicines and can not be cured, but they help prevent diseases and aging of the organism in the current ecological situation [48, 64, 70].

The production of functional products will expand and strengthen the base of the dairy industry, increase the production of dairy products with dietary and medicinal properties.

With the deterioration of the quality of milk and the increasing incidence of diseases in humans, especially digestion, technologists in the field of improving its quality, processing milk and creating fundamentally new food products face big challenges.

In the modern structure of nutrition, functional foods occupy an intermediate place between products of mass consumption and dietary products, in particular, products provided for special medical purposes [31, 37].

According to GOST R 52349 [39], a functional food is a food product intended for the systematic use in the composition of food rations by all age groups of a healthy population, reducing the risk of development of diseases associated with nutrition, preserving and improving health due to the presence in its composition of physiologically active functional ingredients.

This group combines substances (and their complexes) of animal, vegetable and mineral origin, as well as living microorganisms, which have the ability to exert a beneficial effect on one and / or several metabolic reactions of the human body when administered systematically in amounts comparable to the daily intake physiological need for them [39, 82, 24].

As part of a daily diet, functional foods can participate in regulating or improving protective biological mechanisms, helping to prevent specific diseases, or simply slowing down the physical process of aging, increasing endurance and improving the state of mind of a person [33, 10].

The categories of functional nutrition include probiotics, prebiotics, dietary fiber, oligosaccharides, vitamins, minerals, polyunsaturated fatty acids, sugar alcohols, cholines, amino acids, proteins, peptides, alcohols, organic acids, isoprenoids, antioxidants [65].

1.1 Current trends in the use of probiotics, prebiotics and synbioticsFrom a functional point of view, different types of microorganisms are divided

into 3 main groups:- Pathogenic microorganisms causing various diseases;

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- latent (conditionally pathogenic) microorganisms which, under normal protective forces of the organism, do not harm it, but when the immune system is broken they become pathogenic microbes;

- useful microorganisms, the most important for human health - are some representatives of lactic acid bacteria, on the content of which the immune response and protection against pathogenic microorganisms depend, that is, human health.

The term "probiotic" is relatively new and it is believed that it was first used by Ferdinand Vergin in 1954 [6]. In his article, he compares the harmful effects of antibiotics and the beneficial effects of beneficial bacteria, calling them "probiotic." The word "probiotic" is of Greek origin: pro - for, "bios" - life, that is, "for life" or "creating life." Verzhdin defines probiotics as a mixed culture of bacteria. They favorably affect the body, which, taking them, improves its microflora.

Later Lilly and Stillwell Term in 1965, in contrast to antibiotics, probiotics described as microbial factors that stimulate the growth of other microorganisms. Richard Parker in 1974 proposed the term "probiotics" to denote living microorganisms and their fermentation products, which have antagonistic activity against the pathogenic microflora. In 1989, Roy Fuller stressed the need for the viability of probiotics and put forward the idea of heir positive effect on patients [7].

Later in 2002, an official consensus was reached on the definition of probiotics. The definition adopted by the International Organization of Health (WHO) reads: "Probiotics (also called bacteria, or cultures) are living microorganisms that, when taken in sufficient numbers, have a positive effect on human health" [7].

The most complete modern designation of probiotics is given in the Russian Industry Standard "Protocol of Patient Management. Disbacteriosis of the intestine, "according to which probiotics are preparations from living microorganisms and substances of microbial origin, which, with the natural method of administration, have positive effects on the physiological, biochemical and immune responses of the host organism through the optimization of its microbial ecological system [7].

Probiotics can contain representatives of one type of bacteria (monoprobiotics), as well as the association of several species of microorganisms (associated probiotics). They can be assigned to a wide range of living organisms (humans, animals, birds, fish, etc.) regardless of the species of the host from which the strains of probiotic bacteria were originally isolated.

However, most often probiotics are assigned to representatives of the animal species from which the corresponding strains were isolated from the biomaterial. It should be noted that autoprobiotics begin to be introduced into practice, the active principle of which are strains of normal microflora isolated from a particular individual and intended for correction of its microecology [7, 11].

Common measures include prescribing antibiotics and using probiotics. The latter are preserved and tolerated by normal flora and prevent more than half of all cases of diarrhea, in particular, dangerous forms and pseudomembraneous colitis.

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Antibiotics used in conjunction with probiotics, in 80% of cases, complications do not. Probiotics are used in cases involving high risk in the elderly, seriously ill or long-term hospitalized persons.

To the means of bacterial therapy, in addition to probiotics, prebiotics include non-digestible substances or dietary ingredients that selectively stimulate the growth and biological activity of intestinal microorganisms, or alter the metabolic activity of its normal microflora, improving the composition of the microbiocenosis.

The mechanism of probiotic action is based on the fact that the human microflora, as mentioned above, is represented in the intestine by lactic acid microorganisms, and they produce enzymes such as hydrolases. These enzymes break down prebiotics, and the energy thus obtained is used by lactic acid microorganisms for growth and reproduction. In addition, in this process, organic acids are formed. They, by lowering the acidity prevent the development of pathogenic microorganisms that do not have enzymes for the processing of prebiotics. The latter stimulate and activate metabolic reactions of useful representatives of human microflora [9, 12].

By chemical characteristics prebiotics can be classified into the following groups: monosaccharides and alcohols (xylose, xylobiose, raffinose, sorbitol, etc.); oligosaccharides (lactulose, fructooligosaccharide, galacto-oligosaccharide, xylo-oligosaccharide, etc.); polysaccharides (pectins, dextrin, inulin, etc.); enzymes (β-galactosidase of microbial origin, proteases of saccharomycetes, etc.); peptides (soy and milk); antioxidants (vitamins B, vitamin E, ascorbic acid), as well as other dietary supplements - amino acids, plant extracts, organic acids, etc. [6]. The general characteristics of probiotics and prebiotics are presented in Table 1.

Table 1 - General characteristics of probiotics and prebioticsProbiotics Prebiotics

To colonize the intestine with a foreign microflora

To stimulate the growth of its own microflora

Preparations: Linex, Bifidumbacterin, Lactobacterin, Acipol, Probiophore and so on.

Preparations: Bon-Sante, Lactusan, Prelax, Lactofiltrum, Inulin, Bran, and so on.

Composition: Probiotics contain living cells of the normal flora of the intestine: bifidobacteria, lactobacilli and so on.

Composition: Prebiotics contain substances that are nutraceuticals (food) for beneficial intestinal microflora

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Treatment strategy: probiotics infect (colonize) the intestine with exogenous (foreign) microflora

The treatment strategy: prebiotics stimulate the growth of the intestinal (own) microflora of the intestine

Penetration: Only 5-10 percent of live bacteria contained in probiotics reach the colon

Prohodimost: prebiotics are not digested in the upper sections of the gastrointestinal tract and in unchanged form reach the colon

Storage: Probiotics should be stored in a dark, cool place: the number of living bacteria in probiotics depends on the conditions and shelf life

Storage: Prebiotics are carbohydrates (sugars), the conditions of the storage of which have little effect on their bifidogenic properties

Table 2 - Prebiotics and synbioticsPrebiotics Synbiotics

Lactulose (Dufalac, Lactusene) Biovestin-lacto (B. bifidum, B. adolescentis, L. plantarum and bifidogenic factors)

PAMBA (para-amino-methyl-benzoic acid)

Maltodophylus (B. bifidum, L. acidiphilus, L. bulgaricus and maltodextrin)

Lysozyme Bifido-tank (a complex of lacto- and bifidobacteria and complex fructooligosaccharides from Jerusalem artichoke)

Calcium pentothenate Laminolact (Enterococcusfaecium L-3, amino acids, pectins, sea kale)

A large group of prebiotics of natural or artificial origin is oligosugars with a carbohydrate chain of 2-10 carbohydrate residues. Oligosugars are not digested and not absorbed in the small intestine. in the brush border there are no enzymes for their cleavage. Unchanged, oligosugars enter the large intestine, where they undergo bacterial fermentation [6]. This group of prebiotics includes dufalac (lactulose), a nonabsorbable and non-digestible in the small intestine, a synthetic disaccharide consisting of fructose and galactose. Lactulose realizes its effect only in the large intestine, where, according to some researchers, it serves as a source of

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energy and nutrient substrate, mainly for bifido- and lactobacilli. Thus, lactulose is a bifido- and lactogenic prebiotic, normalizing the disturbed intestinal microbiocenosis.

The development of these bacteria against the background of optimum pH of the contents of the colon leads to an increase in their biomass and, accordingly, the volume of intestinal contents. The final products of lactulose metabolism are lactic, formic and volatile fatty acids (acetic, oily, propionic). The latter, among other biological effects (hypocholesterolemic, hypolipidemic, antiproliferative action) have an osmotic effect and a corresponding laxative effect. Thus, lactulose combines the properties of a prebiotic and a mild osmotic laxative. These unique features can be successfully used for constipation of various genesis, accompanied by impaired intestinal microflora. The use of lactulose and its analogues can be combined with the use of antibiotics (for other diseases), and in this case, the drug serves as a means of preventing dysbacteriosis [3, 4].

Calcium pantothenate is involved in the processes of acetylation and oxidation in cells, carbohydrate and fat metabolism, the synthesis of acetylcholine, stimulates the formation of corticosteroids in the adrenal cortex, is utilized by bifidobacteria and promotes an increase in their biomass. PAMBA - para-amino-methyl-benzoic acid (analogous to ambene), the inhibitory effect of proteolytic enzymes of opportunistic bacteria and fungi, stimulating the growth and reproduction of bifido- and lactoflora and full-bodied colibacillus.

Lysozyme contributes to the normalization of impaired microflora. The most active against gram-positive pathogenic and opportunistic bacteria. Lysozyme has a bifidogenic, immunomodulating, anti-inflammatory effect, stimulates metabolic and reparative processes and erythropoiesis, improves digestion, improves anti-infectious and antitoxic resistance of the organism, has antibacterial action and shows synergy with many antibiotics [5-13].

To date, a large number of experimental data and clinical observations on the pronounced prophylactic and therapeutic efficacy of probiotics based on lactobacilli have been published [32-37].

The fundamental principle of probiotics is the development of drugs based on microorganisms - representatives of normal human microflora: bifido- and lactobacilli. Some authors argue that probiotics should be made only on the basis of microorganisms from among the representatives of resistant (endogenous) normoflora, which requires the selection of natural or selection of highly active strains [38]. The most known microorganisms that use as a basis for these drugs are the bacteria of the genus Lactobacillus. In total, the genus includes more than 50 species of lactobacilli [38-40]. They have been attracting the attention of researchers for more than a century, since they are widely represented in the microflora of various parts of the digestive tract and the vagina. Due to the pronounced antagonistic properties, cultures of lactic acid bacteria have found wide application in medical practice, food industry, animal husbandry and veterinary medicine [41-46]. Also the most commonly used strains of lactic acid bacteria, such as Bifidobacterium and Streptococcus, because they are perceived as

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the most desirable components of the intestinal microflora. These bacteria are traditionally used in the industry that produces fermented lactic acid products, and have the status of GRAS (usually considered reliable) [47, 48].

In medical practice, lactose-containing biopreparations are prescribed for children and adults in the treatment and prevention of dysbacteriosis of various etiologies, in the treatment of general intestinal infection (GII), chronic diseases of the gastrointestinal tract (GIT) with severe dysbiotic phenomena, especially in the case of deficiency of lactoflora, etc. In connection with the resistance of production strains of lactobacilli to many antibiotics, these drugs are used against chemo- and antibiotic therapy [49].

L. bulgaricus - a special kind of bacteria that differs significantly from other probiotic bacteria. L. bulgaricus is found on green plants, in milk and dairy products. In natural conditions, the bacterium can be found on the roots and oak bark (Quecus), in the flowers of the Bulgarian rose (Rosa damascena), corneline (Cornus mas), keep the tree (Paliurus), etc.

Getting with food into the human colon, bacteria multiply rapidly and within a few hours reach hundreds of millions in 1 gram of weight. Unlike other probiotic bacteria, as well as L. acidophillus, Bifidobacterium, etc. L. bulgaricus are not permanent inhabitants of the human colon, if they are not introduced with food, then after 10-15 days they disappear from the body.

L. bulgaricus differ from other bacteria in that they live independently in nature and only with lactic fermentation and in the human body do they cooperate with certain types of probiotic bacteria. In this symbiosis, they help each other, which many times enhance their useful properties. This symbiosis makes it possible to maximize immunostimulating, anti-cancer, antibacterial, detoxifying, antiatherosclerotic and other useful qualities of L. bulgaricus.

Today, the assortment of drugs, the active principle of which is living lactobacilli, is quite diverse. One of the first monocomponent probiotic drugs is Lactobacterin, which contains the microbial mass L. rlantarum, its production was developed and established in 1980. Lactobacterin is used to treat children from 3 years of age and adults suffering from chronic colitis of various etiologies, as well the drug is used in the complex treatment of patients with ulcerative colitis, somatic diseases complicated by dysbiosis (DB) [29].

The mechanism of action of probiotics is still unknown, although there are several hypotheses. There are numerous evidences confirming that the action of probiotics is associated with the stimulation of the human immune system [49]. Lactobacilli is referred to as "soft" immunostimulants. The spectrum of the immunomodulatory effect of the drug includes the effect on the response of cellular and humoral immunity; Blasten exhibits antiradiation and antitumour properties [50].

The mechanism of action of probiotics is directed to compulsory colonization of the intestine with competitive strains of bacteria - probionontons, carrying out nonspecific control over the number of opportunistic pathogenic microflora,

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displacing it from the intestinal population and inhibiting the increase in pathogenicity factors in its representatives.

The Nurmi concept is a compelling example of effective protection against infection caused by living microorganisms. So one-day chickens get protection from salmonella infection, using a complex of intestinal flora of adult chickens. J. Reid, A.W. Bruce and others [51], considering the importance of microbial adhesion for the ecology of the urogenital and gastrointestinal tract, and the role of host and microbial factors in bacterial interference have shown that lactobacilli are an agent for the treatment and prevention of diseases.

The most important requirement for probiotic bacteria is their ability to survive when passing through the upper sections of the gastrointestinal tract. There they are under the influence of various factors such as acidity of gastric juice, the state of gastric acids, chemicals in gastrointestinal juices, etc. Probiotic bacteria and strains should have qualities that allow them to cross this barrier and reach the living and in sufficient quantity of the department large intestine, where they are populated and multiply.

Probiotic bacteria have a positive effect on the human body, colonizing the intestinal wall, they begin to produce important enzymes and cell bioproducts for life. Probiotic bacteria are not permanent inhabitants of the human microbial flora, so they should be taken with food regularly and for a long time to feel their positive impact on health.

It is advisable to prescribe probiotic preparations in accordance with the principle of microecological adequacy taking into account microbiological disorders, phase and stage of intestinal dysbacteriosis. Some drugs in adults are advisable to use for preventive purposes, others are prescribed for treatment. Important is the nature of the underlying disease, in which the intestinal dysbacteriosis develops. Therefore, for viral diseases it is more appropriate to use lactose-containing drugs, with bacterial infections - bifido- and lacto-containing. Spore probiotics are recommended for the treatment of dysbacteriosis occurring with the predominance of Protea and fungi of the genus Candida. Treatment, as a rule, is complex with the appointment of specific bacteriophages, vitamins, sorbents, desensitizing agents, in some cases intestinal antiseptics and antibacterial drugs (in these cases with the subsequent administration of probiotics). In addition to the described medicines, foreign companies produce numerous preparations with a probiotic function of a similar effect related to the class of biologically active food additives [52, 53].

The clinician should provide for the possibility of associating a particular disease with the development of microecological abnormalities of the gastrointestinal tract, assessed by the results of a study of the small and large intestines, including bacteriological analysis of faeces. The results of correction of impaired microflora conclusively prove that in most cases the use of drugs with probiotic function is most physiologically and clinically significant for the patient. The use of probiotic drugs showed their clear clinical and microbiological advantage and pronounced therapeutic effect.

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The most promising at this stage are the development and production of new multivalent and combined preparations with immobilized probiotic bacteria of various taxonomic groups and their biologically active metabolites, necessary for medical practice [54-58].

The drug "Acilact", which includes lactobacilli L. species of acidophilus, is developed. Unlike lactobacilli of the L. plantarum species, the acidophilic lactobacilli used in the preparation of Acilactum belong to the category of microorganisms obligate for humans, they have the ability to live well in the intestines, and therefore they are one of the most important components of preparations that correct the microflora. A polycomponent Acylacte, a preparation containing three strains of L. acidophilus (100ASH, NK1, K3SH24) was developed. One dose includes at least 108 CFU / g of live lactobacilli [59].

Among lactobacilli species L. acidophilus belongs to the leading role as a component of leaven for numerous therapeutic and dietary fermented milk products. The first information about the use of acidophilic bacteria for the prevention and treatment of human diseases refers to the year 1910, when acidophilic milk appeared on the market. At present, in various countries of the world, acidophilic lactobacilli is introduced into monoculture or in combination with various kinds of bifidobacteria in the composition of biologically active preparations, food additives and fermented milk products (both mono-species and complex composition). Much attention to lactobacilli is because representatives of this genus do not participate in the occurrence of any pathological processes in the human body, but, on the contrary, have a positive effect on human health.

Acidophilic lactobacilli are widely used for the prevention and treatment of patients with various types of acute and chronic diseases of the digestive tract, inflammatory processes of the respiratory tract, bacterial infections of the urogenital system.

Representatives of L. acidophilus are used in the same way as antioxidants and drugs that reduce lipid peroxidase and stimulate the growth of other lactobacilli and bifidobacteria. These microorganisms have antitumor activity and stimulate various links of immunity.

In medical practice, the drug "Narine", prepared on the basis of the strain L. Acidophilus 317/402, developed by Armenian scientists, proved to be very useful. They also obtained a new therapeutic and dietary dairy product "Narine", recommended for wide use in the clinic for the complex treatment of patients with intestinal infections caused by opportunistic enterobacteria, in the treatment of allergic skin and mucous membrane damage, inflammatory diseases of the respiratory system, genitourinary system [34].

The combined preparation "Acipol" has found wide application as a probiotic preparation of wide action, consisting of a mixture of living antagonistically active bacteria L. acidophilus and polysaccharide of kefir fungi. According to the mechanism of action, "Acipol" is a multifactorial therapeutic agent, has inhibitory activity against pathogenic and opportunistic microorganisms, which causes a corrective effect on the intestinal microflora, enhances the digestion and

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metabolism [60]. The warmed kefir fungi are an immunomodulator that stimulates the protective properties of the body.

Bondarenko V.M. The preparation Biobakton, which contains a lyophilized acidophilus culture (L. acidophilus), mixed with carrot powder, was developed [33].

Currently, other polycomponent preparations from the associations of various lactic acid bacteria have been well recommended. For example, "Maltidophilus" is a dried at low temperatures L. acidophilus, L. bulgaricus, B. bifidum based on maltodextrin. The biologically active supplement "Probionic" includes lyophilized strains of B. infantis, B. longum, L. acidophilus, as well as sorbitol, mannitol, fructose, fructo-oligosaccharide and natural strawberry filler. The drug "Primadofilus" contains a mixture of microorganisms dried at low temperatures (L. acidophilus, L. rhamnosus, B. infantis, B. adolescentis) based on maltodextrin. The drug "Travis" contains L. acidophilus, L. bulgaricus, Str. thermophilus. Known to a wide range of consumers, the drug "Acidophilus" contains dried L. acidophilus, L. bulgaricus, Str. thermophilus and as a basis - goat's milk or carrot juice [61].

In the world pharmaceutical market, there are lactose-containing preparations such as Normoflora (Bulgaria), Violetalin, Enterol (France), Lactinex (USA), Bioflor (Israel), Lactulose-Dufalac (Netherlands) , they include: L. acidophilus, L. plantarum, L. bifidus, L. casei, L. lactis, L. brevis, L. salivarius, L. rhamnous.

In Russia, also developed the drug "Floradofilus" - a mixture of lyophilized dried microorganisms B. bifidum, B. longum, L. acidophilus, L. bulgaricus, Str. thermophilus.

Among import lactose-containing preparations, "Lineks" has well proved itself. It includes live lyophilized bacteria L. acidophilus, B. infantis, Str. faecium, representing the biomass of the symbiotic association of normal human microflora. Linex contributes to the creation of an acidic environment in various biotopes of the gastrointestinal tract, normalizes peristalsis of the intestine, has a pronounced immunogenic effect, and performs two leading roles in correcting dysbiosis in children-decontamination and the settlement of the biotope by normal symbionts [61, 62].

A drug under the trademark "Lactone" (manufacturer of LLP "Denovo Impex", Republic of Kazakhstan) is developed and manufactured, which contains dry biomass of lactobacilli, cyanocobalamin and folic acid. The intensive growth of market demand and the available imported lactose-containing preparations make domestic producers look for technological solutions for the creation of competitive medicinal forms of domestic probiotics. So, in Kazakhstan, preparations "Plantafermin" and "Polylactobac", developed by the employees of the Institute of Microbiology and Virology of the National Academy of Sciences of Kazakhstan, "Kazakhstan Academy of Nutrition" CJSC and "Leovit" LLP.

Gavrilova N.N. The preparation "Polylactobac" is widely used, which consists of co-grown lactic acid bacteria (L. cellobiosus20, L. fermentum127, L.casei 139, 173a) and propionic acid bacteria (Propioni bacterium shermanii) [63].

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The biopreparation "Plantafermin", proposed by the group of Kazakhstan scientists, is a lyophilized microbial mass of living cells, associations of antagonistically active strains of L. fermentum27, L. plantarum22, L. acidophilus27, B. longum7W, dried in a hydrolyzate-milk medium with the addition of a sucrose-gelatin protective medium, as well as lyophilized Jerusalem artichoke powder [64].

Studies have shown that a high level of normal microflora does not serve as an indicator that it fully manifests its useful properties. Under the influence of unfavorable factors, not only the species composition of bifidobacteria and lactobacilli, but also their enzymatic activity, can change. Therefore, simultaneous use of pro- and prebiotics is advisable. A special place is occupied by probiotic products that contribute to the restoration of natural factors of protection of the human body [65, 66].

The steadily developing direction in this area leads to the improvement of the production of such products. The production of probiotic drugs is based on the improvement of biotechnological processes and, along with the use of traditional strains of lactic acid bacteria for the dairy industry, also provides for the use of new types of microorganisms that have probiotic properties. This group of products is the most advanced in the international market, and the attention of practitioners to expanding its range is not weakening [67]. The composition of sour-milk bioproducts is gradually becoming more complicated, they become multicomponent and have specified functional properties, which is very important for improving the population [68].

Thus, the analysis of literature sources of scientific research has shown that the development of complex probiotic preparations based on lactobacilli and bifiobacteria is a promising direction of biotechnology. Bifidobacteria create conditions for the metabolic activity of lactobacilli, and lactobacilli promote the reproduction of bifidobacteria. The latter colonize mainly in the large intestine, and lactobacilli - and in the remaining parts of the digestive tract. Combined products containing both a plant component that can stimulate the growth of lactic acid bacteria and milk, preserved with lactic fermentation, are used in some foreign countries as products that protect the human body from the diseases of civilization.

1.2 Lactic acid bacteria, the significance and prospects of their use

Among microorganisms that have a wide range of applied nature, a special position is occupied by lactic acid bacteria. A number of scientists of our country and abroad, investigating the beneficial properties of lactic acid bacteria, use them for the manufacture of traditional fermented milk products: koumiss, shubat, which not only have a beneficial effect on the body, but also contribute to strengthening the immune system. With the development of microbiology, knowledge of the morpho-cultural and physiological-biochemical properties of lactic acid bacteria and the scope of their use have expanded [4-11].

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Lactic acid bacteria are a group of microaerophilic gram-positive microorganisms fermenting carbohydrates with the formation of lactic acid as one of the main products. Lactic acid fermentation became known to people at the dawn of civilization. Since then, it is used at home and in the food industry for processing and preserving food and beverages.

Research E.I. Kvasnikova, devoted to lactic acid bacteria, showed that these bacteria are an extremely interesting and practically important group of microorganisms [12, 18]. They are widely distributed in the biosphere, and under the influence of anthropogenic and technogenic factors, their total quantity in the biosphere is continuously increasing. In nature, lactic acid bacteria are found on the surface of plants (for example, on leaves, fruits, vegetables, grains), in milk, external and internal epithelial covers of humans, animals, birds, fish (for example, in the intestine, vagina, skin, mouth, nose and eyes) [12-18].

Due to the formation of large amounts of lactic acid, to which they themselves are largely tolerant, lactic acid bacteria can multiply quite rapidly under suitable conditions, displacing other microorganisms. The natural storage media of these lactic bacteria are milk and dairy products, sour dough, sauerkraut and the like. Many forms of lactic acid bacteria grow very slowly and replaced by a concomitant microflora [19].

In natural and industrial conditions, other microorganisms, usually gram-negative rod-shaped bacteria, cocci forms, mold fungi, yeast, etc., often prevail. This causes certain difficulties in their investigation and requires a wide variety of media and methods that ensure their nutritional needs and growth conditions and depressing growth of extraneous microflora. The latter is achieved by the use of various substances, for example, sodium azide, thallium acetate, sorbic and acetic acids, ethyl alcohol, as well as a significant decrease in the pH of the medium (up to 5 and below) [20, 21]. The isolation and, especially, the quantitative accounting of lactic acid bacteria in various natural and production substrates are associated with a number of problems. This is due to the fact that representatives of this group are very demanding on power supplies and do not grow on simple environments. Most people need a number of vitamins (thiamine, pantothenic, nicotinic and folic acid, biotin) and amino acids, as well as in purines and pyrimidines. To grow them, you need complex media containing herbal infusions, meat and yeast extracts, whey, protein hydrolysates and the like. Surprisingly, some lactobacilli grow on media containing blood and form cytochromes and may even be able to carry out phosphorylation in the respiratory chain [22].

To isolate lactic acid bacteria from various sources, a number of media are recommended. These are liquid and agar media, which include ingredients rich in amino acids and vitamins, tomato juice, yeast extract and autolysate, protein hydrolysates [23, 24]. The ability to form lactic acid as the main product of fermentation is the main property of lactic acid bacteria, according to which they are combined into a separate large group of microorganisms. The fermentation of carbohydrates by the type of lactic fermentation, as a rule, correlates with a number of other signs. Lactic acid bacteria are immobile, do not form a spore, are catalase

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negative, positively stained Gram, do not form a pigment, do not restore nitrates to nitrites [12]. But it should be noted that some researchers have described strains that differ from typical properties in some properties. They are mobile, restore nitrates to nitrites, form spores, catalase positive [25, 26].

According to the cell form, lactic acid bacteria are divided into coccoid and rod-shaped bacteria. The diameter of the coccoid forms is from 0.5-0.6 to 1 μm; they are located singly, in pairs or in the form of chains of different lengths. Rod-shaped bacteria are diverse in form - from short coccoids to long filiformes of various lengths (from 0.7-1.1 to 3.0-8.0 μm), located singly or in chains. The composition of the medium and the conditions of cultivation greatly influence the shape of the cells. The formation of elongated rod-shaped cells is observed when developing on media containing ethyl alcohol with a high active acidity, in media with a deficiency of vitamin B12, under the influence of ionizing radiation [27].

High and medium doses of gamma-ray irradiation (of the order of 100-300 thousand X-rays) of two-day cultures Lb. ptantarum and Lb. Brevis more strongly inhibit the division of cells than their growth. Therefore, in cultures immediately after exposure to irradiation, as a rule, long rod-like and filamentous, as well as degenerate, swollen cell forms are formed. Changed MCD cells also appear when exposed to lysozyme, when substances that reduce surface tension are added to the medium, as well as antibiotics [30, 31].

Elongation of the cell form, their thinning and granulation was observed in the cultivation of individual lactic acid bacteria (MCB) on media with a high active acidity (pH 3.7), with a significant content of table salt (up to 6%), at temperatures much higher than optimal (45 ° C instead of 18 ° C). The same long, thin, granulated rods are often excreted from wandering vegetables, pickles, wines, wort, tomato products.

Some lactic bacteria develop elongated rod-shaped cells and develop them in media with a high content of ethyl alcohol, especially if the medium does not provide enough nutrition for these microorganisms [30]. In general, alcohol suppresses the function of cell multiplication, while the function of growth is suppressed weaker.

The length of cells in different cultures of the same species of lactic acid bacteria depends to a certain extent on the composition of the medium, the presence of oxygen, the way of incubation. Thus, streptococci (Lc. Lactisssp. Cremoris, Str, bovis) can stretch out and resemble rod-shaped forms, and rod-shaped bacteria form streptococcus like chains (for example, Lactobacillus caseissp. EaselLb. Plantarum, Lb. brevis). As McDonald points out [27] in cultures Str. cremoris (Lc.lactisssp. cremoris) and Str. lactis (Lc. lactisssp. lactis) grown in a chemostate on medium with 2% milk powder (as well as other media), long filamentous cells are found. This is obviously due to a decrease in the activity of bacterial autolytic enzymes involved in the process of divergence of cells formed during cell division.

Lactic acid bacteria multiply by dividing cells, sometimes by lacing. The process of reproduction of some lactic acid bacteria with the help of gonidia is described, in which granules (gonidia) are formed at the tips of the rods, increasing

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in size, stretching and turning into rods, and the formation of filterable forms in lactic acid bacteria. Japanese researchers have proven the presence of lactic acid bacteria in the process of spore formation [26].

If the cells divide in the same plane, this leads to the formation of chains; when the division proceeds in two planes, so-called tetrads are formed, which are characteristic of bacteria of the genus Pediococcus.

A number of researchers describe the surface colonies of various types of lactic acid bacteria:

- thermophilic lactobacilli form rough colonies;- mesophilic smooth, and the form of colonies correlates with other signs of

bacteria - the ability to grow at a certain temperature and form long, often threadlike or short sticks [30].

It can often be observed that in culture, along with rough colonies, there are also smooth ones, although the latter turn into rough in the course of time. Such a phenomenon is observed, for example, in L. acidophillus, and smooth colonies of this species are often mistakenly identified with smooth colonies of Lb. caseissp. сasei [31]. In bacteria of the genus Leukonostok, S-, O- and R-types of colonies are found, several types can be established among the R-types.

Researchers made many attempts in the direction of finding a connection between the ability of bacteria to form certain types of colonies, their morphological features and physiological activity. A review of these works is given by E.I. Kvasnikov [12]. The study of the shape of the colonies of lactic acid bacteria was given great attention during the heyday of dissociation studies of microorganisms. Many authors considered the transition of some forms of colonies to others as the dissociation of microorganisms, which is based on mutations. Other scientists have linked the transformation of one type of colonies to others with the conditions of existence of culture, especially with nutrition [28].

The source of energy for lactic acid bacteria is lactic fermentation, in which ATP is formed during the anaerobic oxidation of organic substrates during substrate phosphorylation.

Lactobacilli are microphilic Gram-positive bacteria that do not form spores and do not produce catalase. Based on the production of carbonic acid from glucose, the demand for thiamine, the fermentation of fructose to mannitol and the production of fructose-diphosphate-dolase, lactobacilli are divided into two groups: homo- and heterofermentative [32]:

- homofermentative bacteria during fermentation of sugars form mainly lactic acid and insignificant amounts of fumaric and succinic, volatile acids, ethyl alcohol and carbon dioxide;

- heteroenzymatic - along with lactic acid form significantly larger amounts of acetic acid, ethyl alcohol, carbon dioxide and other products, using 50% of sugars.

Some lactic bacteria, for example, Str. faecalis, fermentation of carbohydrates proceeds according to the Entner-Dudorov scheme.

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However, a significant number of studies have been carried out, indicating a change in the character of the final fermentation products of lactic acid bacteria, depending on a number of final factors (pH, the presence of carbon dioxide and oxygen, the physiological state of the cells), indicating that it is impossible to differentiate sharply the groups of hetero- and homofermentative ICDs [33] .

Lactic acid bacteria belong to the genera Lactobacillus, Leuconostoc, Streptococcus, Lactococcus and Pediococcus [34-36].

The genus Lactobacillus unites rod-shaped bacteria, among which there are homo- and heterofermentative species.

The genus Leuconostoc combines heterofermentative coccoid bacteria with oval or ovoid forms.

The genus Pedococcus combines homofermentative coccoid bacteria, the division of cells of which occurs in two planes, and as a result, tetrads or clusters are most often formed.

The genus Streptococcus united previously homofermentative coccoid bacteria of spherical or oval form, dividing in one plane and arranged in pairs or chains. They were divided into fecal, dairy, oral and pyogenic. Recently, all lactic streptococci, according to the determinant of Berg (IX ed.), Are isolated into the genus Lactococcus [37].

The most common group of bacteria of the genus Lactococcus are round or slightly oval cells with a diameter of 0.5-0.6 to 1 μm, located singly, in pairs or in chains of various lengths. Bacteria of the genus Leuconostoc are elongated and ovate, 0.5 to 0.8 μm in diameter and 1.6 μm long, sometimes they are rod-shaped up to 1-3 μm in length, arranged in chains, without forming clumps of cells.

Types of Leuconostoc are found on plants and morphologically similar to streptococci. They ferment the sugars heterofermentatively, that is, along with a small amount of lactic acid also form acetic acid and aromatic substances (acetoin and diacetyl).

Formation of acetoin and diacetyl from salts of citric acid. However, the aroma appears only at a pH below 5. Since Leuconostoc does not ferment milk to a small extent, it is necessary to use the culture of S. lactis for the appearance of the aroma accompanying the milk ripening process.

Leuconostoc (formerly called betacocci), which are aroma-forming components of starter cultures of lactic acid bacteria for cooking oil. They are also involved in the formation of the flavor of chunky cheeses. Their most important types: Leuconostoc cilrovorum and Leuconostoc dextranicum.

In the dairy farm, lactobacilli are gram-positive lactobacilli. They are of different length and thickness, do not form a spore and oxidize milk much faster than streptococci at pH 3.2-3.5. Lactobacilli achieve optimal development at low pH and reduced oxygen content. According to M. Berga's classification in the dairy farm, all species of Tribus Lactobacilleae are divided into three genera: thermobacteria, streptobacteria and betabacteria.

Lactic acid bacteria have a certain proteolytic activity, due to the action of proteases and peptidases. During the proteolysis of milk proteins, especially at the

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beginning of the cultivation of strains in milk, there is an accumulation of amino acids (aspartic acid, glycine, serine, glutamic acid, threonine, tyrosine, valine, phenylalanine, isoleucine), as well as peptides. Lactobacillus bacillus bacteria have a greater proteolytic activity than coccoid forms. Thus, Lb. bulgaricus, Lb. caseissp.casei can translate up to 25-30% of casein into a soluble form, whereas Lc. lactisssp. lactis and Lc. lactisssp. cremoris - 15-17% [39, 40].

Lactic acid bacteria possess both endogenous and exogenous proteolytic enzymes [41, 42]. The extracellular proteinase enzyme produced by Lc. lactisssp. lactis, by some properties (the optimum of action, thermal stability) is similar to trypsin. The greatest proteolytic activity is manifested by the reaction of a medium close to a neutral medium (pH 7.0). Probably, therefore, the neutralization of milk with chalk increases proteolytic activity during the entire incubation period of Lactic Acid Bacteria.

In addition to extracellular and intracellular proteolytic enzymes, protease associated with the cell wall was found in lactic acid bacteria. They perform the initial action on milk proteins and play a significant role in ensuring the normal growth of bacteria in milk. Studies have shown that the proteolytic activity of Lc. lactisssp. lactis, Lb. helveticum and Lb. delbrueckiissp. lactis [43-45], was detected during the first hours of fermentation and during the active multiplication of the cells. This fact indicates that the process of decomposition of casein is driven by enzymes associated with growing bacteria, rather than enzymes released during autolysis of cells [44].

A number of authors show a direct relationship between the degree of maturation of cheese, its taste, aroma and the content of free amino acids in it, as well as the products of the conversion of these amino acids accumulated by proteolytically active lactic acid bacteria [46].

Lactic acid bacteria either do not have lipolytic activity, or exhibit it to a certain extent when exposed to some substrates [47].

It should be noted that lipolysis in cheese refers to the most complex multifactorial processes, since the lipolytic system of the product is heterogeneous and is capable of catalytic transformation with the formation of a large number of different compounds [48, 49]. This circumstance to an even greater extent determines the need to establish the basic regularities of lipolytic processes in cheese, which is an indispensable condition for the management of enzymatic reactions in the production of a product, and, correspondingly, the intensity of its maturation process and the formation of organoleptic parameters.

The presence of phospholipase activity in lactic acid bacteria has been established, and significant inter-species and intraspecific differences have been identified on this basis. The quality of many fermented dairy products depends on the ability of the microorganisms that make up the ferment to produce flavor and aroma components. One such component is diacetyl, which is responsible for the "oily" flavor of dairy products, and the ability to produce it as a result of the assimilation of the citrate contained in milk is characteristic of bacteria from p. Lactococcus and various types of Leuconostoc [50, 51].

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During the last decade, many studies have been devoted to elucidating the mechanism of diacetyl formation [52, 53]. All strains of MKB forming diacetyl form acetoin. But not all strains producing acetoin form diacetyl.

The ability of bacteria to assimilate citrate and the formation of diacetyl is unstable, and, with a fairly high frequency, variants appear that do not form diacetyl. Studies have shown that instability is associated with the localization of genes that determine aroma formation (as well as fermentation of lactose, proteinase activity, phage resistance, production of nisin, etc.) on plasmids [54, 55]. Loss of cells of a plasmid leads to a loss of culture of a property.

In general, many of the technological properties of starter strains of lactic acid bacteria are unstable. These include the ability to ferment lactose, proteinase activity, phage resistance, the formation of nisin. Due to intensive research it was shown that instability is associated with the localization of genes that determine all these properties on plasmids. Carrying plasmids is a widespread phenomenon in the world of microbes, their presence gives the cell certain advantages. Plasmids play an important role in the evolution of bacteria, participating in the process of natural selection. Despite the variety of controlled cell functions, plasmids have a number of common properties: they consist of DNA having a circular closed structure, located outside the chromosome and capable of self-replication. The common property for all plasmids is that they give the cell additional, not required functions, but most often beneficial. Loss of cells of a particular plasmid leads to loss of culture of a particular technological property. Plasmids can be lost by the bacterium or eliminated, but the loss of the plasmid does not affect the basic properties of the cell. They can undergo mutations, and also recombine among themselves and the chromosome [50, 51].

A method is known for obtaining a phage-resistant starting culture for fermenting milk, which involves the transformation of the food lactobacillus with a phenotype deficient in fermentation, resistant to lacto- and streptococcal phages. Transformation is carried out by introducing a genetic element carrying the genetic locus of lactose fermentation. The result is a transformed lactobacillus that replicates this genetic element, which is capable of fermenting lactose and does not contain a marker of resistance to antibiotics [55].

Plasmids that determine the production of nisin, as a rule, are linked to the genes of assimilation of sucrose, and the dimensions of plasmids in different strains vary. The same can be said about plasmids that determine phage resistance or proteinase activity, and their size. The production of diacetyl is also controlled by the genes of plasmid DNA, and to date all the plasmids detected are more or less the same mass.

It is known that many production properties of lactococci are determined by plasmids, such as the consumption of lactose, proteolytic activity, aroma formation, antagonism against the bacteria of other systematic groups, resistance to bacteriophages, etc. New plasmids appear to have an endogenous origin, i.e. the genetic material of these plasmids may not be present on the chromosome of the parent strain. It is possible that the observed changes in plasmid profiles in other

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strains occur as a result of genetic interaction between the plasmids and the chromosome.

The antagonistic activity of the lactic acid culture play an important role in the complex of colonization resistance mechanisms, its ability to colonize the gastrointestinal mucosa, which consists of the adhesion of microorganisms to the epithelial cells of the intestine, competition for binding receptors and blockade of adhesion and colonization of mucous membranes with factors of both immunoglobulin nature and factors protection of the host organism. The role of adhesins in different microorganisms is performed by surface-located fimbriae, antigens of protein or polysaccharide nature, lipoteichoic acids, phospholipids, etc. The adhesion process allows microbes to remain in a certain niche, which allows effective colonization of the gastrointestinal mucosa by symbionts.

Therefore, one of the ways to prevent colonization of the intestine by a pathogen is to suppress its attachment by saturating the epithelial receptors with ligands of symbiont adhesion. It is known that pretreatment of intestinal enterocytes with the fimbial antigen of the adhesion of a non-pathogenic strain, for example, E. coli, creates resistance to infection by pathogenic strains possessing the same adhesin. As studies have shown, the process of adhesion of lacto- and bifidobacteria occurs due to the fimbri-like structures and components of the cell wall, apparently, the proteins of the outer membrane and lipoteichoic acid. The products of the vital activity of lactic acid bacteria were probably one of the first antiseptics of the microbial origin of folk medicine. Sour-milk products were used to treat burns and wounds [12].

I. Mechnikov advanced the theory that lactic acid bacteria contribute to better health and longevity. He suggested that "intestinal auto-toxicity" and those arising from its substance can be sold by modifying intestinal bacteria and replacing proteolytic microbes such as clostridium producing toxic substances (including phenols, indoles and ammonia after protein digestion), and beneficial microorganisms. He developed a diet with the addition of milk, fermented by a bacterium, which he called a "Bulgarian stick." The theory of healing of the intestine has not lost its significance even at the present time [56-60].

The antagonistic properties of lactic acid bacteria began to be applied spontaneously in practice already in antiquity. The preservative effect of lactic acid fermentation was used when fermenting vegetables, fruits, in ensilage of fodder, salting fish, storing meat.

The bactericidal activity of lactic acid products and their beneficial effect on the human and animal organism was explained by many researchers by the effect of the lactic and other acids contained in them (acetic, formic, etc.), alcohols, peroxides and other metabolites accumulated by lactic acid microorganisms during their growth and development . The action of such metabolites is based on a decrease in pH and inhibition of the growth of bacteria causing food damage [61, 62]. Further studies of the phenomenon of antagonism of lactobacilli have shown that they are producers of antibiotic substances. So, Lc. lactisssp. lactis, for example, isolate nisin, Lc. lactisssp. cremoris-diplokokcin, Lb. acidophilus -

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acidophilus and lactocidin, Lb. Plantarum-lactolin, Lb. Brevis Breveen, Lb. delbrueckiissp. bulgaric and - bulgarian, Lb. gasseri - gassericin A [61-72] and a number of purified but unnamed substances [73, 74].

It is established that the antibiotic substances of lactobacilli are characterized by a certain spectrum of action. For example, nisin bacteriostatically acts on propionic acid bacteria, staphylococci, sarcin and some lactic acid bacteria, but does not suppress the growth of yeasts, molds and gram-negative bacteria. The mechanism of action on cells is associated with its ability to react with sulfhydryl groups and, thus, turn off the action of a number of important coenzymes (glutathione, acetyl-coenzyme A) [75, 76]. The antibiotic-acidophilus secreted by the acephilic rod inhibits the development of putrefactive bacteria, streptococci and staphylococci, typhoid, paratyphoid A and B, dysentery, tuberculosis and diphtheria. Studies have been published on the mechanism of action of nisin and pediocin on the cytoplasmic membrane of cells; influence of membrane potential, pH, composition of membrane proteins and lipids on the specificity and efficiency of bacteriocins, the role of specific amino acids and structural domains of bacteriocins in their action [77]. Lactocidin is active against acid-fast bacteria, as well as mold fungi. Lactolin retards the growth of Clostridiumtyrobuturum, Saccharomycescarlsbergensis, Candidaalbicans.L. caseissp. Casei has antagonistic properties in relation to intestinal and hay bacillus, pyogenic micrococcus, as well as bacteria of typhoid and dysentery [78].

A method for obtaining bacteriocin Lc is known. lactisssp. lactisNRRL-B-18535, suppressing the growth of Staphylococcusaureus, S. epidermidis, S. rhamosus, Pediococcuspentosaceus, P. acidilactici, Lb. delbrueckiissp. bulgaricus, Lc.lactisssp. cremoris, Lc. lactisssp. lactis, Leuconostocmesenteroides, Lb. plantarum, Lb. fermentum and Listeriamonocytogenes [76].

The antagonistic activity of lactobacilli is associated with the production in large quantities of organic acids (mainly dairy), antibiotic-like substances of different chemical composition, spectrum and mechanism of action (lactocin), hydrogen peroxide. The fact of the expressed influence of L. acidophilus on the immune system of the body through stimulation of monocyte migration, activation of phagocytic activity was established.

The antagonistic properties of lactic acid bacteria and, in particular, lactobacilli, are used in the dairy industry to reduce the pasteurization time of milk, to improve the quality of cheeses, and to extend the shelf life of dairy products. The activity of lactic acid bacteria, isolated from flour and grain cereals, has been proved in relation to the causative agent of potato disease - B. subtilis.

Antibiotics have found application in medicine [79, 80], veterinary medicine [81], the food industry in the canning of vegetables, fruits, soups, sauces, soybeans, meat products, etc. [82-84], in agriculture with ensilage [ 85].

Prolonged use of chemotherapeutic and antibiotic drugs often has an undesirable effect on the body, while the use of antimicrobial substances of lactic acid bacteria is harmless and promising [76, 77,78].

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Fundamental researches of biological and medical science allowed developing and introducing into practice preparations based on different types of living microbial cultures, called probiotics [78, 79]. The ancient Thracians, who live in the territory of present-day Bulgaria in 6000-7000 BC, knew the probiotic effect of lactic acid bacteria. They used bacteria and caused fermentation of fresh sheep milk to produce yoghurt, cheese, cheese, etc. - the first probiotic products in the world. For the population that lived on the Bulgarian land, these were and continue to be traditional products that support the human body, protecting it from a number of diseases.

The Thracians were also familiar with the medicinal qualities of bacteria. They made special mixtures of fermented milk, an extract of medicinal herbs and honey. They treated sick and wounded people, they impregnated them, and then dried linen cloths for dressings, for the treatment of wounds and other skin diseases, or given to soldiers before the battles.

Studies of probiotics have expanded and covered more and more areas. So, if in the beginning probiotics were considered as microorganisms or substances that contribute to achieving an optimal balance between representatives of the intestinal microflora, they were later identified as containing live microbes food additives that favorably affect the host organism by normalizing the intestinal microbiocenosis. Then this definition was expanded and began to include microorganisms that improve the resistant microflora not only of the intestines but also of other organs [80-83].

Currently, probiotics consider all drugs containing live microbes in an amount sufficient to change microflora or are able to colonize a certain area of the host, beneficially influencing health [84, 85].

The significant displacement of bacterial pathogens is not only the effect of bacterial competition caused by oral use of microorganisms. The antagonistic substances of lactic acid bacteria also prevent the introduction of pathogenic microorganisms into epithelial intestinal cells. As a result, the gastrointestinal tract is not colonized by pathogens, the liver is significantly released from the products of bacterial protein metabolism. In general, adsorption of the gastrointestinal tract and other energy-producing components increases, and poorly soluble salts are converted into soluble compounds. This effect is comparable with the action of small doses of antibiotics and is achieved by the addition of probiotics [86].

Thus, lactic acid microorganisms that stimulate the development of normal human microflora (bifido- and lactobacilli) are an important component of functional products. They conduct large-scale work in the human body. This was first established by the Russian scientist I.I. Mechnikov [86].

Useful microorganisms activate the immune system, protect us from the expansion of pathogenic and opportunistic bacteria, neutralize toxins, remove heavy metals, radionuclides from the body, synthesize vitamins, and normalize mineral metabolism.

Recently, many different products have appeared on the basis of such microorganisms - from drugs to food; For example, well known to all lactic acid

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products. The modern conditions of human life in the big city gave birth to the "megacity syndrome", which manifests itself in the disturbance of metabolism, weakening of immunity, changes in the microbial ecology of the digestive tract. One of its components is a decrease in the level of bifido- and lactoflora and a corresponding increase in the number of conditionally pathogenic microorganisms in humans. Currently, more attention is paid to the development of lactic acid products with the introduction of probiotics, which include several microorganisms belonging to different genera and species. In this case, first of all, the interaction of microbes in the natural habitat and, first of all, the symbiosis of individual strains are taken into account.

Thus, the isolation of highly active strains of lactic acid cultures and the selection of a consortium are of decisive importance in the production of fermented milk products. Isolation and selection of strains of lactic acid bacteria make it possible to select strains that can meet all the requirements for a particular product. The complexity of constructing biopreparations of the multi-species composition consists not only in the purposeful selection of strains with certain properties, but also in the study of their compatibility when creating consortia of microorganisms.

1.3 Microflora of starter cultures used in the production of sour-milk products for dietary nutrition

Sour-milk products include various types of yogurt (yogurt, schnikovik, varenets, fermented baked milk, yoghurt, etc.), kefir, kumis, acidophilic drinks [69]. In addition, produce dairy products from buttermilk and whey. Studies in recent years suggest the use of dietary dairy dessert products of lactic acid bacteria. The most interesting in the sphere of consumption are combined desserts containing vegetable ingredients and a fermented milk component [70].

Milk whole and skim milk, cream, condensed and powdered milk, sodium caseinate, buttermilk and other milk raw materials, as well as cereals, fruit and berry and vegetable fillers, food flavors, dyes, sweeteners, stabilizers of the structure are used to produce sour milk desserts.

An acidic product is a product produced by fermentation of milk or cream with kefir fungi and / or pure cultures of lactic acid, propionic acid, acetic acid microorganisms and / or yeast and / or their mixtures, the total content of lactic acid microorganisms in the finished product at the end of the shelf life of at least 106 CFU in 1 g of product [71-73].

In the process of fermentation, complex microbiological and physicochemical processes take place, as a result of which a specific taste, odor, consistency is formed.

It is proved that lactic bacteria entering the intestine create an acidic environment, which prevents the development of putrefactive bacteria that cause the breakdown of food proteins before the formation of indole, skatole and other substances that are poisons. These substances, absorbed into the blood, disrupt the vital functions of the organism [74].

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Fermented milk products contain nutrients essential for the body in easily digestible form. These products are well digested, have dietary and medicinal properties. Dietary properties are due to the presence of lactic acid, carbon dioxide, alcohol, B vitamins produced by certain bacteria.

The presence of lactose in milk and its capacity for fermentation make it possible to organize the production of various types of fermented milk products, depending on the composition of the pure bacterial cultures used and the preparation technology [75-77]. The production of fermented milk products is based on the knowledge of biotechnology, which is based on microbiological processes.

With regard to sour-milk products technology is currently developing in the following areas:

- perfection of classical technologies of fermented milk products using strains of lactic acid bacteria created with the help of new breeding methods;

- development of a new generation of fermented milk products using various microorganisms, as well as probiotic microorganisms that produce biologically active substances [75-77].

At present, there has been a steady trend to enrich sour-milk products with a wide spectrum of special fermented microflora, since the living cells of these starter cultures, as well as the products of their metabolism, play a very important role in the prevention of various diseases. The consumption of such sour-milk products promotes protection against gastrointestinal infections, improving the functioning of the immune system, reducing the cholesterol in the blood and protecting against cancer, preventing the appearance of atherosclerosis, the restoration of nitrates to nitrites. With the use of dairy products for special purposes, there is an improvement in metabolic processes in the body, forces are restored, and fatigue is reduced. It is of interest to use these products even in psychotherapy [77, 78].

Lactic acid cultures are used in the production of dietary products, they are diverse and have different properties. Scientific research suggests the use of lactic acid bacteria in the production of fermented dairy products: B. longum, L. bulgaricus, L. yogurti, L. acidophilus.

In the production of most dairy products and preparations made using acidophilus bacillus and bifidobacteria, S. lactis, S. thermophilus cultures are added to accelerate clotting, to obtain clots with a more dense consistency and to improve taste. 79

In many countries, bifid products are very popular among consumers. Thus, in North America and Europe retail sales of bio-milk and bio-yogurt are rapidly increasing. Dairy products, fermented with bifidobacteria, have good organoleptic characteristics and have pronounced therapeutic properties. The assortment of products with bifidobacteria is very wide: yoghurt, kefir, yogurt, cottage cheese, quick-ripening cheese, sour cream, butter, ice cream, curd desserts, children's dry mixtures, etc. The richest assortment of such products exists in Japan, Germany,

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France, Great Britain, Czechia. In some countries (USA, etc.), bifid-containing dairy products are widely used in clinics [80, 81].

The ability of sour-milk products, especially prepared on combined ferments, is established to strengthen the activity of the digestive glands; This is partly explained by the accumulation of protein decay products in sour-milk preparations. Acidophilic bacillus is a variety of Bulgarian, but it has not lost its ability to enter it into the intestine there for a long time. Many races of the acidophilic microbe have the property of synthesizing various units of the vitamin B group, as well as substances similar to antibiotics; this contributed to the wider introduction of acidophilic lactic acid products in the practice of nutrition for both the adult population and children of various ages. Most of the acidophilus rod has a tendency for pronounced mucus formation, and therefore the product is sometimes obtained with pronounced viscousness [82].

A sour milk product has been developed to accelerate the process of mowing milk and giving it dietary properties based on starter from mesophilic and thermophilic microorganisms. To this end, cultures of L. bulgaricus 176, S. thermophilus 108, S. diacetilactis 13 C 65-64 and cultivated in a ratio of 1: 3: 3: 3 are introduced into monocultures of thermophilic streptococcus and S. diacetilactis [82, 83].

The use of lactic acid bacteria and bifidobacteria in functional foods at the beginning of the 21st century will replace the existing market of chemical medicines by half and, thereby, will make it possible to solve the problem of healthy microbial ecology of man.

Among the various representatives of normal human microflora, bifidobacteria and lactobacilli occupy a special place, since it is they who play the leading role in maintaining and normalizing the intestinal microbiocenosis, the resistance of the organism to intestinal infections, the improvement of protein and mineral metabolism, etc. All this makes it possible to treat lacto- and bifidobacteria as the basis for creating new food products - products with probiotic properties.

Recently, in the technology of dairy fermented products, bifidobacteria are widely used, which refer to microorganisms that constantly inhabit the human intestine. This type of lactic acid bacteria makes up 80-90% of the normal intestinal flora of children and young animals of farm animals during the period of breastfeeding. They actively suppress the development of putrefactive and pathogenic microbes, control the intestinal environment, reproduce the B vitamins and produce natural antibiotics, improve the quality of skin and respiration.

Joint cultivation of bifidobacteria with lactic acid bacteria in milk has a number of advantages. Lactic acid bacteria bind dissolved oxygen in the milk and, thus, create anaerobic conditions favorable for the growth of bifidobacteria; proteolytically active strains of lactobacilli break down casein with the formation of bifidogenic factors: peptides and amino acids. In addition, due to the production of a large amount of acetic acid, bifidobacteria impart a somewhat uncharacteristic taste and odor to the product, which are not characteristic of traditional lactic acid products. On the contrary, the joint cultivation of lacto- and bifidobacteria

27

stimulates the accumulation of metabolic products: organic and volatile acids, acetaldehyde, diacetyl, which contributes to the improvement of the organoleptic characteristics of the product. A faster decrease in pH with mixed fermentation reduces the risk of contamination of the product with an extraneous microflora [84].

A method for producing a fermented milk product using a consortium of lactic acid cultures of L. acidophilus is known. The AC-1 consortium was obtained by combining 4 strains of L. acidophilus, forming clots of a creamy consistency, having close biochemical and cultural properties. Bacteria formed R-form colonies, the milk was rolled at a temperature of 37-38 ° C. When applying 1% of the starter, the antibiotic activity in relation to the E. coli, Fixer strain, Protein, Salmonella, Staphylococcus in 24-28 hours - was very high. The death of the cells of pathogenic microorganisms was respectively from 99.9% to 100%, the moisture retention capacity of 3-3.5 ml / 100 ml [85].

Strain S. lactis 75-P - has increased activity against the bacteriophage, lactic streptococcus is resistant to the antibiotic and retains the ability to grow at a concentration of streptomycin in broth up to 1000 U / ml, homofermentative, Gram-positive, optimal growth temperature of 28 ° C [43] .

For the preparation of fermented milk products, the selection of lactic cultures on the activity of fermentation, organoleptic parameters of fermented bunches, microscopic preparation. Mesophilic lactic streptococci S. lactis, S. cremoris S. acetoinicus were chosen. The method of preparing the ferment for sour-milk products included skewing milk at 30 ° C for 6-9 hours to an acidity of 78 ° T. The strain forms diacetyl (D) - 29 mg / l, acetaldehyde – 7,2 mg / l, acetoin – 56,7 mg / l, volatile acids -29,5 ml. During the fermentation process, the following free amino acids accumulate in milk: aspartic acid, threonine, series, glutamic acid, proline, glycine, methionine, leucine, isoleucine [85].

To obtain good quality ryazhenka and yogurt used a consortium of lactic cultures S. thermophilus, L. Delbruskii subsptranicus bulgaricus VKPM AC-7. The purpose of the invention was to identify the symbiosis of thermophilic lactate streptococcus and Bulgarian bacillus, which have the ability to ferment milk at 42 ° C for 2.5-4.0 h, form a dense or medium bunch of homogeneous consistency, expressed by sour milk taste, clot syneresis from 32 to 50% , the acidity of the bunch is 85-90 ° T [80].

During the production of the fermented milk product Bifilife, a production starter is introduced on the basis of a consortium of strains of bifidobacteria of B. bifidum, B. longum, B. breve, B. infantis and B. adolescentis in 1: 1: 1: 1: 1 ratio, in a nutrient medium. The selected strains must actively increase the biomass in sufficient quantities to provide a therapeutic effect. Clinical trials of Bifilife have shown that its use contributes to faster clinical improvement in diseases of the gastrointestinal tract, in patients with oncology, liver, genitourinary, wound infection [77].

In some countries, in the production of fermented products, gradual repeated fermentation of milk is used. In the United States, for example, in the manufacture

28

of a number of therapeutic dairy products, milk is first fermented with lactic streptococci, followed by a joint culture of Bifidobacterium and L. acidophilus, the products obtained have a pleasant aroma, have high nutritional value and therapeutic effect. In addition, fermentation of milk with acidophilic and bifidobacteria increases the biological value of the product by increasing the content of available lysine [18]. In Bulgaria, a method of preparing a dietary fermented milk product with bifidobacteria is proposed. As a starter, use a mixture of cultures of L. acidophilus, B. bifidum, Leuconoctoccremoris in a ratio of 1: 1: 1 to 2: 3: 1 [83]. In Poland, a method for obtaining fermented milk products, mainly beverages such as yoghurt and soft curds, involves fermenting the prepared mixture with a ferment consisting of 80 to 90% bifidobacteria and 10-25% mesophilic streptococci or thermophilic bacteria, ripening at 41-44 ° C [83].

In the Czech Republic, in the manufacture of certain types of fermented milk products, separate cultivation of bifidobacteria is used in conjunction with lactic streptococci and acidophilus rod, and then mixing the fermented products in different proportions. In the same country, a method has been developed for the production of hard cheeses with bifidobacteria. In the composition of the microflora of the starter species of bifidobacteria B. longum and B are introduced. bifidum [80].

Bacterial starter cultures and concentrates are produced by leading companies in Denmark, Germany, France and other countries for the manufacture of starter cultures, which are also directly introduced into milk for ripening. Culture DVS (DirectVatSet) - direct erection in the tank, produces the company Chr. Hansen (Denmark). DVS cultures for fermented milk products contain strains of S. thermophilus, L. delbruckii, which give them varying degrees of viscosity, pure sour-milk flavor and aroma.

Constancy of composition, ease of handling, high activity, and absence of risk of bacteriophage contamination are the main advantages of concentrated DVS culture. One of the main advantages of DVS is the production of high-quality fermented products with long shelf life. The shelf life of DVS crops at 18 ° C is 12 months, at a temperature of 5-6 ° C - 6 months. Cultures are packed in aluminum foil bags, and then packaged in cardboard boxes [19].

In Russia and the CIS countries, for all types of dairy products, direct starter cultures are produced by the Texel concern, the Crhodia group (France). Their production began in 1902. Currently, the collection has more than 100,500 strains of lactic acid bacteria.

Scientists of the All-Union Scientific Research Institute have selected strains of S. lactis, S. cremoris, S. acetoinicus and a method for preparing sourdough for sour-milk products has been developed. The selection of mesophilic lactobacilli streptococci S. lactis, S. cremoris, S. acetoinicus on the activity of milk fermentation, organoleptic indices, phage ratio, growth in litmus milk, the compatibility of cultures with co-cultivation in the selected ratio occurs on sterile skim milk at 25-26 ° C . In order to obtain ferments that are resistant to seasonal

29

changes in the quality of milk, cultures are checked by the structure of the population, proteolytic activity, antagonism to a heat-resistant stick [3].

The possibility of using a combined starter of bifidobacteria, Bulgarian bacillus, kefir fungi taken in a ratio of 1: 0.5: 0.5 was investigated. The milling is carried out at a temperature of 35 ° C. Leaven has antibiotic properties in relation to technical harmful and pathogenic microflora [1].

For the normalization of the intestinal microflora, medical preparations of bifidobacteria are used in medical practice. In the East Siberian Technological Institute, lyophilized starter strains of bifidobacteria were developed. Employees of the department developed a new technique for preparing bifidobacteria for drying, a protective environment was selected, drying regimes and storage times for a dry preparation were specified [5].

In recent years, much attention is paid to starter cultures, improving the consistency of products (yoghurt, and others). These are strains capable of producing exopolysaccharides. It has been established that their use prevents senesis, gel breakage, increases the viscosity of products.

For 4 years, the research center in Switzerland, together with various universities, worked to create new improved starter cultures for the preparation of fermented milk products. As a result, a large collection containing more than 3500 microorganisms was formed. The L. acidophilus strain was selected. This strain is resistant to the effects of gastric juice, well combined with the usual microflora, activates immune cells that produce antibodies [76].

In recent years, widespread use of dairy products with the use of microorganisms, which are representatives of the normal microflora of the human gastrointestinal tract. Their use in nutrition causes a significant improvement in the body's activity, contributes to its recovery and, thus, in some cases helps to avoid the use of medicines.

It has been proved that such sour-milk products should be used not only for prevention, but also for the treatment of almost all diseases, including diseases of the gastrointestinal tract. The biological value of therapeutic fermented milk products is due not only to the component composition of the raw materials used, but also to the composition of the useful microflora used.

Often used in practice as probiotic drugs are microorganisms: lactobacilli - L. acidophilus, L. bulgaricus, L. casei, L. rhamnosus, L. brevis, L. cellobelosus, L. fermentum, L. plantarum; bifidobacteria - B. bifidum, B. infantis, B. breve, B. adolescentis, B. longum, B. animals, B. thermophilum.

Gram-positive cocci - Str. salivarius, Str. thermophilus, Str. diacetylactis, Enterococcusfaecium, Lactococcuslactissp. cremoris. Yeast - Saccharomycesboulardii, S. cerevisiae. [67-69].

The mechanism of stimulation of normal microflora growth by a consortium of microorganisms containing probiotics is to inhibit the growth of pathogenic microflora by the production of nutrients; activation of immunocompetent cells, stimulation of endogenous microflora growth as a result of production of vitamins and other growth-stimulating factors; normalization of pH; neutralization of toxins,

30

changes in microbial metabolism, manifested in an increase or decrease in the activity of enzymes. Many strains produce bacteriocins - antibacterial substances that also inhibit the growth of other microbes. This ability is highly possessed by enterococci and lactobacilli. Probiotics have a direct antitoxic effect, they are able to neutralize cyto- and enterotoxins of viruses and bacteria: enteropathogenic and enterotoxigenic Escherichia, clostridia, cholera. So, in the study of Alvarez-OlmosM.I. [20] showed a decrease in the secretion of sodium and water in the intestines of patients with acute infectious diarrhea against the background of S. boulardii. Resta-Lenert S., Barrett K.E. [21, 22] showed that L. acidophilus and St. thermophilus does not act on intestinal secretion on their own, but reduces it after stimulation with enterotoxin. This explains the rapid elimination of physiological dysfunction of the intestine against the background of the appointment of probiotics in infectious diarrhea. The strongest direct antimicrobial and antitoxic action was shown in competitive probiotics of S. boulardii and B. subtilis. The antitoxic effect was demonstrated in L. acidophilus (in relation to rotaviruses, Cl. Difficile, E coli), in L. rhamnosus GG (to rotaviruses, Cl. Difficile, E. coli), Ent. faecium SF-68 (to Cl. difficile; E. coli); thermophilus (to E. coli), in L. plantarum (to E. coli). Bifidobacteria have a preventive effect against intestinal infections and necrotizing enterocolitis of newborns [23], a curative effect has been proved for some strains, in particular B. longum BB536 [24].

Research was carried out at the State University "Semey" at the Department of "Technology for the production of dairy products" to select the optimal composition of the ferment microflora for fermented milk products. As a result of the experiments, the theoretical assumption was confirmed that the starter cultures from three types of cultures containing bifidobacteria and lactobacilli are promising. On the basis of the research, a technology has been developed for the production of a number of fermented dairy products, a gel-like sour-milk product based on skim milk using the association B. longum, S. thermophilus, L. Delbruckii subsptranicus bulgaricus; liquid fermented milk products using the association B. longum, S. thermophilus, L. helveticus; pasty dairy product based on a composition with a high solids content using B. longum, S. thermophilus and kefiric fungi [25].

Many of the strains of bifidobacteria and lactobacilli included in Polybacterin have been successfully used for decades in probiotic pharmacopoeial preparations of the first generation («Bifidumbacterin dry», «Bifilong» and «Acilact») and various fermented dairy products of functional purpose [15]. "Acidopectin" (Acidophilus) has gained special popularity on the market - its main functions are that it helps to restore and maintain normal intestinal microflora; normalizes the work of the gastrointestinal tract; supports the natural protection of the body from infections; prevents the emergence of dysbiosis. With various diseases of the gastrointestinal tract, administration of antibiotics, malnutrition and decreased immunity, the number of acidophilic bacteria in the intestine decreases, which in turn can lead to the development of more serious diseases.

Russian scientists have studied that acidopectin serves as an excellent supplier of live biologically active acidophilic lactobacilli, helps prevent the development

31

of dysbiosis in children and restore normal intestinal microflora. The composition of the drug also includes apple pectin, which creates a favorable environment for reproduction of beneficial bacteria. In addition, pectin contributes to the normalization of the gastrointestinal tract, takes part in the metabolism of carbohydrates, enhances the antioxidant defense of the body and helps to remove toxic substances from the intestine [27].

Thus, the use of lactic acid cultures in the production of dietary products demonstrates the advisability of introducing associations of lactic acid bacteria. Lactic acid bacteria should have proteolytic, antibiotic activity, the ability to form acidic clots in a short time, and be phage-resistant. In the production of fermented milk products, bifidobacteria are used, as well as associations of bifidobacteria with other lactic cultures. Bifidobacteria and acidophilic bacteria have the ability to synthesize antibiotics, suppressing the development of pathogenic microflora, participate in the synthesis of vitamins and amino acids, have a positive effect on the adsorptive capacity of the intestine. The use of drugs containing L. acidophilus and bifidobacteria in the production of dairy dessert products is relevant and promising.

The use of lactic acid cultures to create probiotic drugs is possible in the form of dried cells or fermented food. Milk desserts are one of the directions for creating dietary dishes. As a useful microflora use different types of microorganisms - this acidophilus rod, thermophilic streptococcus, bifidobacteria, Bulgarian rod and many other cultures.

1. RESEARCH AND SELECTION OF HIGH-ACTIVE DAIRY-ACID MICROORGANISMS

1.1 Morphological and Cultural Properties of Isolated Lactic Acid Bacteria

To isolate lactic acid bacteria from various sources, a number of media are recommended. These are liquid and agar media, they contain ingredients rich in amino acids and vitamins, tomato juice, yeast extract and autolysate, protein hydrolysates [16]. Isolation of highly active strains of microorganisms from raw milk, and fermented milk products produced in the South Kazakhstan region, will yield a starter with the necessary properties. Highly active strains of microorganisms will be used in combination to create an industrial starter, and therefore, it is necessary to verify their symbiotic combination.

In the world practice, mainly lactic acid bacteria are isolated from various natural sources and comprehensively studied them, since knowledge of various properties of culture makes it possible to obtain products with specified parameters and to manage the production process on a scientific basis. The process of isolation of lactic acid bacteria included several stages: sampling from local milk raw materials containing lactic acid bacteria; seeding of the enriched culture on a dense nutrient medium and thermostating of crops; in the future, the isolation of colonies into sterile skim milk and the study of isolated strains according to a number of

32

characteristics; the establishment of their belonging to a certain type of lactic acid bacteria; the final stage is the determination of their production value. When selecting the studied cultures, preference was given to those cultures that had a sufficiently high activity of milk fermentation, medium acid-forming capacity, significant antagonistic activity, resistance to antibiotics. The strains of lactic acid bacteria isolated from various sources were microscopically examined and their milk clotting activity and organoleptic parameters were studied. Due to the need to produce viscous, creamy consistency, strains that are capable of forming this kind of bunch were preferred, and the resulting clot should have a sufficiently high moisture retention capacity.

To determine the taxonomic affiliation, their cultural and morphological features were investigated. Description of the colonies is presented in Table 3.

Table 3 - Morphological features of colonies of isolated strains

Strain Column dimensions, mm

Columns Surface

Column profile

Color Edge

1 2 3 4 5 6

SL-12 1-1,2 rough convex white smoothSh-4 0,7-1 smooth flat frosted wavyShA-2 2-2,5 smooth flat frosted smoothCN-1 0,9-1 smooth flat frosted smoothTr-2 0,8-1 smooth convex frosted fimbriatedTr -3 1-1,2 rough flat frosted laconicTr -4 1,5-2 smooth flat white smoothSS-2 0,6-1 smooth flat white smoothSSp-3 1 rough convex frosted smoothSSа-1 0,8-1 smooth flat frosted smoothSPа-1 0,7-1 smooth convex white wavySPм-2 1,5-3 smooth flat frosted fimbriatedSPз-1 1-1,2 rough convex pale gray wavySPз-2 1,5-2 rough convex white wavyК-1 1-2 smooth flat white smoothК-3 0,8-1 smooth flat white smoothКz-2 1-1,2 rough convex Gray smooth

33

Кz-4 0,8-1 smooth convex white wavyTU-5 1,5-2 smooth flat frosted wavyTuz-2 1,5-2,5 smooth flat frosted fimbriated

The resulted morphological and cultural properties of lactic acid bacteria were used for their identification. The ability of lactic acid bacteria to ferment carbohydrates varies greatly with changing cultivation conditions, but this feature can be considered as an auxiliary in identifying them, so the ability to ferment carbohydrates was studied in the isolated microorganisms. The results are shown in Table 4.

Table 4 - The ability of dairy-acid bacteria (DAB) to ferment carbohydrates’

Strain

Ara

bino

se

Mal

tose

Sacc

haro

se

Lact

ose

Glu

cose

Sorb

itol

Raf

finos

e

Xyl

ose

Man

ite

Rib

ose

Star

ch

1 2 3 4 5 6 7 8 9 10 11 12Sl-12 + + - + + - + + - - +Sh-4 + + - + + + - - + - -ShА-2 - + + + + + - - + - -СN-1 + + - + + - + + - - +IT-2 + + + + + - - + - - -Tr-2 - + + + + + - - + - +Тr-3 - - + + + - + - - - +Тr-4 - + + + + - - - - - -SS-2 + + - + + + - - + - -SSp-3 - + + + + + - - + - -SSа-1 + + + + + - - + - - -SPа-1 - + + + + + - - + - -SPм-2 + - - + + - - - - - -SPz-1 + + + + + - - + - - -SPz-2 - + + + + + - - + - +

34

К-1 - - + + + - + - - - +К-3 - + - + + - + + + - +Кz-2 + + + + + - - + - - -Кz-4 - + + + + + - - + - +ТU-5 + + + + + - - + - - -Тuz-2 + + - + + + - - + - -

Further, the main cultural and physiological properties of the isolated strains were studied, the data are presented in Table 5.

In the course of studying the morphological features, we showed that the cells of all cultures under study were immobile, contained no spores, stained positively according to Gram, and were catalase-negative. On these grounds they are typical representatives of the family of lactic acid bacteria.

In accordance with the determinant Bergey, the lactobacillus bacteria were assigned to the genus Streptococcus and Lactobacillus. The studied cultural and morphological features of the cells made it possible to determine the species belonging to the strains studied, of 21 strains of 9 strains Sh-4, SN-1, Tr-4, CC-2, CCa-1, SPz-1, K-1, TU-5 , Ace-2 are representatives of S. lactis; strains SL-12, Cpp-3, K-3 have the species belonging S. cremoris; strains of IT-2, Tr-3, SPa-1, Kz-4 - S. thermophilus; to L. acidophilus are 2 representatives of ShA-2, Tr-2; strain SPm-2 is a representative of L. bulgaricus and isolated strains of wheat grains SPz-2 and Ks-2 are L. plantarum.

Table 5- Morphological features of cells of isolated strains of lactic acid bacteria

Strain of DAB Morphological signs of cells

1 2S. cremoris SL-12 Oval cocci single, diplococci, short chains of 3-5

segments, cell size 0.8 -1.0 μmS. lactis Sh-4 Diplococci, chains of 3-5 segments, cell size 0.8-0.9

microns, 0.8-0.9 micronsL. аcidophilus ShА-2 Single and in the form of chains of rods 10-15 microns

long, 0.8-1.0 microns thick, 0.8-1.0 microns thickS. lactis SN-1 Kokki single, diplokokki, 0,8-1,0 micronS. thermophilus IT-2 Chains of 5-8 segments, cell size 1.2-1.4 μmL. аcidophilus Тr-2 In the form of chains of 3-5 segments of a rod length, 15-

25 microns, a thickness of 1.2-1.5 micronsS. thermophilus Тr-3 Chains of 5-8 segments, cell size 1.3-1.6 μmS. lactis Тr-4 Kokki single, 1.0-1.1 μmS. lactis SS-2 Diplococci, chains of 4-6 segments, cell size 0.8-0.9

micronsS. cremoris SSp-3 Kokki single, diplococci, 1.0-1.1 μm

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S. lactis SSa-1 Kokki single, diplococci, 0,8-0,9 micronS. thermophilus SPа-1 Chains of 5-8 segments, cell size 1.2-1.4 μmL. bulgaricus SPм-2 Short chains of 5-10 μm in length, 1-1.5 μm in thicknessS. lactis SPz-1 Single cocci, diplococci, chains of 3-4 segments, cell size

0.8-0.9 micronsL. plantarum SPz-2 In the form of chains of 3-5 segments of a rod length of

20-25 microns, a thickness of 1.2-1.5 micronsS. lactis К-1 Diplococci, chains of 3-5 segments, cell size 0.7-0.8 μmS. cremoris К-3 Short chains of 3-5 segments, the value of the cells is 0.8

-1.0 μmL. plantarum Кz-2 Diplobacilli, chains of 4-6 segments, cell length 10-15

microns, width 1.2-1.5 micronsS. thermophilus Кz-4 Chains of 5-8 segments, cell size 1.3-1.6 μmS. lactis ТU-5 Diplococci, chains of 3-5 segments, cell size 0.9-1.0 μm

S. lactis Тuz-2 Diplococci, chains of 3-4 segments, cell size 1.0-1.1 μm

2.2 Antagonistic properties of isolated cultures of lactic acid bacteriaThe special importance of lactic acid bacteria is determined by their

physiological, antibiotic and probiotic properties. MKB along with lactic acid form specific substances that exert an antibiotic effect on a number of pathogenic and conditionally pathogenic microorganisms. Antagonistic properties of lactic acid bacteria have been used in practice already in antiquity. The preservative effect of lactic acid fermentation is used when fermenting vegetables, fruits, silage forages, salted fish, and meat in sauerkraut.

After consuming products containing antagonist cultures, they can suppress unwanted intestinal microflora by replicating living cells. Therefore, the use of lactic acid bacteria with a pronounced antagonistic ability in the manufacture of products for therapeutic and prophylactic purposes is a practical necessity.

Table 6 shows the antagonistic activity of the ICD.

Table 6- Antagonistic activity of lactic cultures in relation to pathogenic and conditionally pathogenic microorganisms

Strain Diameter of growth inhibition zones of test cultures, mm

S. typhimuriu

m

E. coli P.aeruginosa

S.aureus B.subtilis

1 2 3 4 5 6S. cremoris SL-12 6±0,5 10,7±0,

513,5±0,5 14,6±0,5 6,3±0,5

36

S. lactis Sh-4 9,2±0,5 6±0,5 12,6±0,5 16,6±0,5 7,4±0,5L. аcidophilus ShA-2

13,0±0,5 12,6±0,5

16,3±0,5 18,8±0,5 26,3±0,5

S. lactisSN-1 12,3±0,5 4,7±0,5 8,5±0,5 14,6±0,5 8,6±0,5S. thermophilus IT-2

14,4±0,5 11,6±0,5

14,4±0,5 15,6±0,5 18,4±0,5

L. аcidophilus Тr-2 11,0±0,5 12,6±0,5

7,3±0,5 24,6±0,5 11,5±0,5

S. thermophilusТr-3 18,7±0,5 4,7±0,5 8,5±0,5 14,6±0,5 8,6±0,5S. lactisТr-4 8,4±0,5 11,6±0,

514,4±0,5 8,6±0,5 18,4±0,

5S. lactis SS-2 11,7±0,5 19,8±0,

511,3±0,5 24,6±0,5 11,5±0,

5S. cremoris SSp-3 8,4±0,5 10,7±0,

512,5±0,5 14,6±0,5 6,3±0,5

S. lactis SSа-1 19,2±0,5 17,6±0,5

16,2±0,5 15,1±0,5 17,7±0,5

S. thermophilus SPа-1

9,2±0,5 6±0,5 12,6±0,5 16,6±0,5 7,4±0,5

L. bulgaricus SPм-2

4,0±0,5 11,6±0,5

6,3±0,5 10,8±0,5 6,2±0,5

S. lactis SPz-1 12,3±0,5 4,4±0,5 9,5±0,5 14,7±0,5 8,6±0,5L. plantarum SPz-2 10,5±0,5 13,8±0,

510,4±0,5 8,9±0,5 9,4±0,5

S. lactis К-1 12,7±0,5 8,7±0,5 9,5±0,5 14,6±0,5 8,6±0,5S. cremoris К-3 18,9±0,5 20,2±0,

514,4±0,5 21,6±0,5 13,4±0,

5L. plantarum Кz-2 11,7±0,5 9,8±0,5 11,3±0,5 8,6±0,5 15,5±0,

5S. thermophilus Кz-4

10,5±0,5 10,7±0,5

7,5±0,5 14,4±0,5 6,8±0,5

S. lactis TU-5 15,0±0,5 21,6±0,5

6,8±0,5 10,8±0,5 6,2±0,5

S. lactisТuz-2 10,7±0,5 12,7±0,5

11,5±0,5 14,7±0,5 11,6±0,5

According to the studies on the antagonistic activity of isolated strains, it can be concluded that L. acidophilus SHA-2, S. thermophilus IT-2, S. lactis SS-2, S. lactis SSa-1, S. cremoris K-3 and S. lactis Tuz-2 have pronounced antagonistic properties and their use in the production of a sour-milk product intended for dietary and preventive nutrition is advisable.

2.3 Study of the sensitivity of lactic acid bacteria to antibiotics

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It is known that lactic acid bacteria - an integral part of the microflora of the digestive tract of humans and animals, which has antagonistic and probiotic activity. Therefore, the detection of strains of lactic acid bacteria, resistant to various antibiotics, is important from the point of view of rational selection of starter cultures for therapeutic and prophylactic sour-milk products, their application in the complex therapy of intestinal diseases.

We conducted a study of the stability of isolated strains of lactic acid bacteria to various antibiotics. The data obtained are presented in Table 7.

Antibiotics affect the enzymatic activity and morphology of cells of lactic acid bacteria. Thus, all the isolated strains are sensitive to ampicillin and erythromycin, which violate the synthesis of the cell wall of microorganisms. The maximum growth retardation zone reached 33.6 mm in S. lactis SN-1, the minimum - 21,7 ± 0,5 mm in L. plantarum Kz-2, penicillin inhibits the ability to ferment monosugars, and the enzyme aldolase, streptomycin - the ability to ferment disaccharides in S. lactis. The more sensitive strains are S. lactis III-4, L. acidophilus Tr-2, S. cremoris SSp-3.

Table 7 - Sensitivity of lactic cultures to antibiotics

Strain

Diameter of growth retardation zones of lactic acid cultures, mm

Am

pici

llin

Gen

tam

icin

Kan

amyc

in

Peni

cilli

n

Stre

ptom

ycin

Eryt

hrom

yci

n

S. cremoris SL-12 27±0,5 1,2±0,1 10,5±0,5 30,2±0,5 9,2±0,1 30,5±0,5S. lactis SH-4 22,3±0,5 0,6±0,1 8,4±0,5 31,6±0,5 4,6±0,1 31,6±0,5L. аcidophilus SHA-2

21,5±0,5 0,6±0,1 6,2±0,5 10,2±0,5 5±0,1 14,4±0,5

S. lactis SN-1 33,6±0,5 0,7±0,1 11,5±0,5 30,6±0,5 3,7±0,1 21,0±0,5S.thermophilus IT-2 22,4±0,5 0 12,5±0,5 21,6±0,5 4,7±0,1 27,4±0,5L. аcidophilusТr-2 31,0±0,5 0,2±0,1 7,7±0,5 31,1±0,5 7,2±0,1 28,6±0,5S. thermophilus Тr-3 28,4±0,5 0 12,5±0,5 30,1±0,5 0,7±0,1 31,4±0,5S. lactis Тr-4 28,6±0,5 0,7±0,1 4,8±0,5 28,2±0,5 0 28,4±0,5

Table continuation 7S. lactis SS-2 31,4±0,5 0,5±0,1 11,6±0,5 30,3±0,5 8,3±0,1 30,2±0,5S. cremoris SSp-3 28,4±0,5 0,6±0,1 11,5±0,5 31,1±0,5 6,2±0,1 29,2±0,5S. lactis SSa-1 28,2±0,5 0 3,5±0,5 5,1±0,5 11,4±0,1 2,3±0,5S.thermophilus Spa-1

29,2±0,5 0,4±0,1 11,7±0,5 29,6±0,5 10,4±0,122,3±0,5

L. bulgaricus SPm-2 22,3±0,5 0,3±0,1 10,4±0,5 30,5±0,5 0 31,4±0,5

38

L. bulgaricus SPm-2 22,3±0,5 0,3±0,1 10,4±0,5 30,5±0,5 0 31,4±0,5

L. bulgaricus SPm-2 22,3±0,5 0,3±0,1 10,4±0,5 30,5±0,5 0 31,4±0,5

L. bulgaricus SPm-2 22,3±0,5 0,3±0,1 10,4±0,5 30,5±0,5 0 31,4±0,5

L. bulgaricus SPm-2 22,3±0,5 0,3±0,1 10,4±0,5 30,5±0,5 0 31,4±0,5

L. bulgaricus SPm-2 22,3±0,5 0,3±0,1 10,4±0,5 30,5±0,5 0 31,4±0,5

S. lactis Spz-1 24,3±0,5 0 12,0±0,5 24,7±0,5 4,3±0,1 28,4±0,5

L. plantarum SPz-2 30,5±0,5 0,6±0,1 8,4±0,5 30,6±0,5 1,5±0,1 30,2±0,5

S. lactis К-1 32,7±0,5 0,4±0,1 9,5±0,5 29,6±0,5 0 19,2±0,5

S. cremoris К-3 22,4±0,5 0,6±0,1 2,3±0,5 31,6±0,5 0 32,3±0,5

L. plantarum Kz-2 21,7±0,5

0,4±0,1

10,3±0,5 29,6±0,5 0 22,3±0,5

S. thermophilus Kz-4

22,5±0,5

0,3±0,1

12,5±0,5 30,5±0,5 0,4±0,1

31,4±0,5

S. lactis TU-5 25,4±0,5

0 8,5±0,5 30,1±0,5 0 28,4±0,5

S. lactis Tuz-2 23,4±0,5

0,6±0,1

12,3±0,5 24,7±0,5 11,6±0,5

30,2±0,5

The lactic acid bacteria are relatively stable to the following antibiotics: gentamicin, kanamycin and streptomycin, which violate not only the synthesis of proteins, but also the processes of replication of the genome of a cell of microorganisms. Thus, growth retardation was not observed with gentamicin in the following cultures: S. lactis SSa-1, S. lactis Spz-1, S. cremoris SL-12 is the most sensitive to this antibiotic. To kanamycin, S. thermophilus Tr-3, S. lactis Spz-1, S. thermophilus Kz-4 and S. lactis Tuz-2, the growth inhibition interval from 11 to 12.5 mm are more sensitive. Streptomycin more strongly inhibited the growth of S. cremoris SL-12 9.2 ± 0.1 mm and S. lactis Tuz-2 11.6 ± 0.5.

The conducted experiments showed that the following strains of S. thermophilus IT-2, L. acidophilus SHA-2, S are the most resistant to the above antibiotics. lactis Tr-4, S. lactis SSa-1, L. plantarum Kz-2 and S. cremoris K-3.

2.4 Investigation of phage resistance of lactic acid bacteria

39

The strains introduced into the starter cultures are tested for sensitivity to bacteriophages. For checks, widespread bacteriophages, known for their aggressiveness to numerous strains, are used. The culture medium infected with a bacteriophage is seeded with a test strain of bacteria. At the same time, if lysis of bacteria occurs, on the dense nutrient medium, there are enlightenment zones, the so-called negative colonies, in which there is no growth of bacteria. In liquid media bacteriophage-sensitive strains of bacteria do not develop (no turbidity) or weak glycolysis (slow accumulation of lactic acid) occurs, or it is absent altogether.

The composition of starter cultures includes strains that are not lysed by any strain or are lysed by not many strains of bacteriophages. To prevent the development of a bacteriophage, many strains of starter are used. In this case, if a phage appears in the leaven, it lyses one or two strains, the rest continue to develop, providing a sour-milk process.

Bacteriophages are one of the main causes of loss of finished products in the production of fermented milk products. Using specially selected phage-resistant starter cultures allows reducing losses from phagolysis. An effective measure to combat the bacteriophage of lactic acid bacteria was the selection of phage-resistant strains and their regular replacement. The use of phage-resistant single-component starter cultures is possible only with a high hygienic culture in the workplace.

When using starter cultures consisting of several strains of lactic acid bacteria, until recently a relatively rare development of the bacteriophage was noted. It is necessary to constantly study the phage background of each enterprise, the isolation of bacteriophages and the selection of starter cultures resistant to previously and newly isolated bacteriophages. With the use of individual strains and unsterilized milk in the production starter, agglutination (gluing) of the cells was sometimes observed. In addition, with the further application of these starter cultures in milk after 2 hours of normal reproduction of the microflora, there was a pronounced agglutination of cells, and after 3 hours - complete lysis.

In many milk-processing enterprises, the work on the phage resistance of lactic acid bacteria is not carried out, and in the production of fermented milk products, many strains of starter are used. The composition of such starter cultures includes several genetically different strains of lactic acid bacteria, which, as a rule, cannot be lysed simultaneously. However, in order to obtain a high quality product, the normal development of all components of the ferment used is required. Consequently, it is still advisable to include strains selected from the results of the test for phage resistance in the composition of starter cultures. The use of specially selected phage-resistant starter cultures allows reducing losses from phagolysis [55].

Table 8 - Sensitivity of Lactic Acid Bacteria to Bacteriophages

Strain Sensitivity to bacteriophages

40

V1 V2 CS. cremoris SL-12 - - -S. lactis SN-4 - - -L. аcidophilus SNA-2 - - -S. lactis SN-1 - - -S. thermophilusIT-2 - - -L. аcidophilus Тr-2 - - -

Table continuation 8S. thermophilusTr-3 - - -S. lactisTr-4 + - -S. lactisSS-2 - - -S. cremoris SSp-3 - - -S. lactis SSa-1 - - -S. thermophilus Spa-1 - - -L. bulgaricus SPm-2 - - -S. lactis Spz-1 - - -L. plantarum SPz-2 - - -S. lactis К -1 - - -S. cremorisК-3 - - -L. plantarum Kz-2 + + -S. thermophilus Kz-4 - - +S. lactis TU-5 - - -S. lactis Tuz-2 - - -Note: (+) - positive; (-) - negative

Analyzing the experiments carried out to determine the resistance to a bacteriophage, it was established that a good growth of cultures was observed in the majority of the test samples, which indicates the stability of these strains to the polyvalent bacteriophage. At dairy enterprises, there is no ongoing work to study the phage background, and the production of fermented milk products uses a multitude of strains of starter. Such ferments contain several different strains of lactic acid bacteria, which, as a rule, can not be lysed simultaneously. To obtain a high quality product, the normal development of all components of the ferment used is required. Consequently, it is still advisable to include strains selected from the results of the test for phage resistance in the composition of starter cultures.

2.5 Determination of physico-chemical and technological indicators of lactic acid bacteria

The coagulation activity and the organoleptic properties of the isolated strains are the most important and decisive indicators that determine their suitability for use in production.

 For further research it is necessary to select strains, when used, the products obtained had high organoleptic characteristics and physicochemical parameters corresponding to the finished product. The high rate of fermentation (with

41

application of 5% of starter), the average acidity (within 70-90оТ), the increased viscosity (more than 5 Pa × s × 10-3) and sufficient moisture retention capacity are of great importance. To strains possessing such properties are the following: S. cremoris SL-12, L. acidophilus SHA-2, S. cremoris K-3, S. lactis SSa-1, S. thermophlus Spa-1, the results of the study of the main indicators are given in the table 9.

The ability to accumulate acid is the main property of lactic acid bacteria used in production. The acid-forming activity of the cultures was evaluated by the Turner method, determining the titratable acidity in the medium. The acid-forming ability of the strains was different. The acid accumulation limit is from 110 ° T to 300 ° T (Table 9). The duration of the coagulation of milk is from 3 to 38 hours.

Table 9 - Physicochemical parameters of fermented milk bunches

Strain of dairy-acid bacteria

Clotting time, h

Titrated acidity, pH

Viscosity, ηef Pa × s

× 10-3

Water retention

capacity,%1 2 3 4 5

S. cremoris SL-12 5-6,5 70 5,20 2,3S. lactis SH-4 4,5-5 75 5,45 3,5L. аcidophilus SHA-2 4,5-5 95 6,05 3,2S. lactis SN-1 4,5-5 110 5,20 2,8S. thermophilus IT-2 3,5-4,5 115 5,00 3L. аcidophilus Тr-2 4-5,5 120 5,15 3.3S. thermophilus Tr-3 4-5 105 5,20 3,6S. lactisTr-4 3,5-4 90 4,55 2,6S. lactis SS-2 6-6,5 75 4,78 3,6S. cremoris SSp-3 3,5-4 85 5,05 2,5S. lactis SSa-1 4-5 75 5,20 2,5S. thermophilus Spa-1 4-4,5 90 5,85 2.4L. bulgaricus SPm-2 7-7,5 130 4,20 5,2S. lactis Spz-1 5-5,5 90 4,20 3,2L. plantarum SPz-2 20-21 135 3,20 4,5S. lactis К-1 4,5 100 4,20 3.6S. cremoris К-3 4,5 75 5,40 2,3L. plantarum Kz-2 25-28,5 145 3,20 5,3S. thermophilus Kz-4 5-5,5 110 4,75 3,8S. lactisTU-5 8-8,5 75 5,40 3.4S. lactisTuz-2 4-5 95 3,80 4.1

The temperature points for reproduction and fermentation process in lactic acid bacteria are different. Even within one species, they can fluctuate depending on the conditions of development. It is established that the activity of the fermentation

42

process depends on how much microorganisms overcome the lag phase and the activity of acid formation is an obligatory criterion for determining the activity of lactic acid bacteria.

The following strains of S. cremoris SL-12, L. acidophilus SHA-2, S. cremoris K-3, S. galactis SSa-1, S.thermophilus Spa-1 were used for the analysis of acid formation activity (Fig. 1).

It has been established that S.kremoris K-3 and S.tactilis SSa-1 and L.cidophilus SHA-2 possess the greatest capacity for acid formation. The clot is formed for 10-12 hours of fermentation (when a loop culture is applied to 10 ml of milk). In this case, the clot is characterized by a dense even consistency and a pleasant sour-milk taste and aroma. The number of viable microorganisms is also the highest in these strains (Figure 3).

0 2 4 6 8 10 12 14 16 18 20 22 240

20

40

60

80

100

120

140

S. cremoris К-3S. lactis SSа-1 L. аcidophilus ShА-2 S. thermophilus SPа-1S. cremoris SL-12,

Time of fermentation. h

Aci

dity

, ° T

Figure 1- Determination of the dynamics of change in acidity different strains

43

Figure 2 - Determination of the number of cells of lactic acid bacteria in 1 ml of the clot

2.6 Determination of the aroma-forming, proteolytic and lipolytic activity of lactic acid bacteria

When developing new lactic acid combination products, it is necessary to study the aroma-forming, proteolytic and lipolytic activity of the lactic acid bacteria we isolated, in order to select strains of practical importance for these characteristics.

There is a direct relationship between the organoleptic qualities of lactic acid products, that is, its taste, aroma and content in

free amino acids, the products of conversion of these amino acids, as well as the number of free fatty acids, mono - and diacylglycerols accumulated by proteolytically and lipolytically active lactobacilli [61, 64].

In the presence of lactic acid alone, the milk clots are characterized by an unexpressed taste. Aroma of the bunch is attached to the by-products of lactic acid fermentation (carbon dioxide, acetic acid, ethyl alcohol, propionic acid, diacetyl, acetaldehyde), protein proteolytic products (peptides, amino acids, sulfur compounds - mercaptan, dimethyl disulphite) and lipolysis (fatty acids).

Table 10 shows the results of determining the aroma-forming, proteolytic and lipolytic activity of the strains isolated by us.

Table 10 - Aromatizing, proteolytic and lipolytic activity of lactic acid bacteria

Strain Aroma-forming ability

Proteolytic activity, γ/ml

Lipolytic activity

S. cremorisSL-12 high 330 highS. lactis SH-4 mean 200 meanL. аcidophilus high 320 high

44

107

106

104

102

109

107

106

104

102

S. lactisSN-1 mean 210 meanS. thermophilusIT- high 300 meanL. аcidophilus Тr-2 high 325 highS. thermophilusTr- mean 200 meanS. lactisTr-4 high 320 highS. lactisSS-2 low 200 meanS. cremorisSSp-3 high 315 highS. lactisSSa-1 high 330 highS. mean 215 meanL. bulgaricusSPm- high 320 highS. lactisSpz-1 low 210 meanL. plantarumSPz-2 mean 300 highS. lactisК-1 low 210 meanS. cremorisК-3 high 330 high

Table continuation 10L. plantarumKz-2 mean 225 meanS. thermophilusKz- mean 315 highS. lactisTU-5 high 235 meanS. lactisTuz-2 high 325 high

On the basis of the complex research carried out, the following Strains of microorganisms are recommended as ferments for the production of fermented milk products: S. lactis SSa-1, L. acidophilus SHA-2 and S. cremoris K-3.

General characteristics of Straina S. lactisSSa-1: a form of cells - cocci, located singly or in pairs, form short chains of 2-4 cells. Cell size 0.8-0.9 microns, all cells stained Gram positive, immobile, do not form spores.

When growing on agarized nutrient media, the superficial colonies had a round shape with an even edge, the surface smooth, matte. Deep colonies of lactic acid cocci had a lenticular shape, white or matte. On the surface of the mown agar grew in the form of a gentle translucent stroke. With growth in milk, all Strain formed a tight clot with a pleasant sour-milk smell. Restores and cools litmus milk, does not form acetoin, decomposes arginine to form ammonia. Do not use sorbitol, raffinose, mannitol, starch as a source of carbon. When 3% of the cultures are introduced, the coagulation of milk occurs after 3.5 - 5 hours. Restores and cools litmus milk. It weakly develops in an alkaline medium (at pH 9.0), with a content of 3-4% NaCl in the medium. It exhibits pronounced antagonistic properties to a number of conditionally pathogenic microorganisms, is not sensitive to gentamicin, shows considerable resistance to penicillin and erythromycin, which generally makes it possible to recommend the use of this Strain for therapeutic and prophylactic sour-milk products and their application in the complex therapy of intestinal diseases.

S. lactis is the microorganism most widely used for the preparation of fermented milk products. A very dense, stabbing clot forms under its action [71].

45

The morphology of S. cremoris K-3 cells in contrast to S. lactis SSa-1 has the form of short chains of 3-5 segments, the value of the cells is 0.8 -1.0 μm. The time of fermentation when applying 3% of the ferment is 5-5.5 hours. The optimum development temperature is 22-30 ° C. Do not give growth in an environment with 4-6% NaCl, restore and roll litmus milk. They have pronounced antagonistic properties, are phage resistant, have considerable resistance to the action of a number of antibiotics (gentamicin, streptomycin), and the application of this Strain in the production of a sour-milk product intended for dietary and preventive nutrition is justified.

S. lactis and S. cremoris are typical representatives of lactic fermentation and are found in almost all dairy products. They play an important role in the maturation and formation of fermented milk products. S. cremoris forms clots, reminiscent of the consistency of sour cream. This property allows the use of this strain in the production of dairy products, characterized by a thick consistency.

Acidophilic lactobacilli are widely used for the prevention and treatment of patients with various types of acute and chronic diseases of the digestive tract, inflammatory processes of the respiratory tract, bacterial infections of the urogenital system. Representatives of L. acidophilus are used in the same way as antioxidants and drugs that reduce lipid peroxidase and stimulate the growth of other lactobacilli and bifidobacteria. These microorganisms have antitumor activity and stimulate various links of immunity. Oral bacterial therapy with acidophilic lactobacilli prevents the occurrence of diarrhea associated with the administration of antibiotics in children (for example, amoxicillin).

It is shown that many strains of acidophilic bacteria have a pronounced virosocidal effect, due to the production of highly active hydrogen peroxide. In high concentrations, L. acidophilus has a virucidal effect against the human immunodeficiency virus.

Lactobacilli have a pronounced antagonistic activity and the ability to adhere, which determines the important role of these microorganisms in maintaining colonization resistance. There are well documented facts that lactobacilli (in particular Lactobacillus GG) have a pronounced ability to prevent exacerbation of ulcerative colitis caused by C.difficile, to have a pronounced therapeutic effect in diarrhea of newborns caused by both bacterial and viral pathogens. Included in the biofilm covering the mucous membranes, the microcolonies of lactobacilli are resistant to the action of the animal lysozyme present in the intestinal contents, as well as to the digestive juices, bile and acids. It should be noted that some lactobacilli themselves actively produce a lysozyme-like enzyme (L. casei, L. fermentum, L. acidophilus).

The antagonistic activity of lactobacilli is associated with the production in large quantities of organic acids (mainly dairy), antibiotic-like substances of different chemical composition, spectrum and mechanism of action (lactocin), hydrogen peroxide. The fact of expressed influence of L. acidophilus on the immune system of the body through stimulation of monocyte migration, activation of phagocytic activity was established.

46

The isolated strains of S. lactis, S. cremoris and L. acidophilus from the milk raw material of the South Kazakhstan Oblast have pronounced antagonistic properties, are phage resistant, have considerable resistance to the action of a number of antibiotics (gentamycin, streptomycin), and the use of these Strain in the production of a fermented milk product for dietary and preventive nutrition, is justified.

3. CODE OF THE CONSORTIUM OF MILK-BINARY BACTERIA WITH PROBIOTIC PROPERTIES

When creating a consortium of microorganisms, with the aim of using it as a complex starter, much attention is paid to the quality indicators of fermented milk: taste, odor, consistency, take into account proteolytic, antibiotic activity and total coagulation time [6, 9].

It is proved that between the microorganisms of the ferment is a complex exchange of products of vital activity, which significantly increases the energy of acid formation and can affect the energy of aroma formation

Probiotics are living useful microorganisms that react most sensitively to adverse effects. Coming with food in the required quantities, these microorganisms have a beneficial effect on human health by normalizing the composition and functions of the microflora. Most often - it's bifido- and lactobacilli, capable of manifesting antagonism against pathogenic and conditionally pathogenic microbes. Under certain conditions, probiotics perfectly coexist with lactic cultures of starter cultures, so they can be included in various types of fermented milk products.

Such products are perfection. Being carriers of useful crops, they are tasty, have excellent organoleptic characteristics, delicate consistency, are well combined with various flavoring and flavoring additives and, most importantly, make up for a person's vital needs in essential nutrients and energy. They are included in nutrition as dietary supplements, in the form of lyophilized powders containing bifidobacteria, lactobacilli and their combinations.

To date, several complex probiotic preparations have been developed and are used, consisting of 2-5 different lyophilized dried living microorganisms. They can be used without prescribing a doctor to restore the intestinal microbiocenosis, to maintain good health, so permission to manufacture and use probiotics as dietary supplements from government agencies controlling the creation of medications is not required.

In medical practice, complex preparations of the first generation are known, including 2 or 3 strains of various microorganisms (Bifikol, Atzilact, Bifilong, etc.) [60].

Disadvantages of many foreign biopreparations are the unadaptedness of the strains of microorganisms used in them for the Kazakh population of people, the high cost of such drugs, which limits the mass curative and prophylactic use of them in the current economic conditions of our country [57, 58].

The quality and nutritional value of most sour-milk products, including sour-milk desserts, are predetermined mainly by the number of ferments used, the

47

microflora of which participates in the formation of taste, aroma and consistency of the finished product. Lactic acid bacteria are able to produce antibiotic substances, inhibit the growth of technically harmful and sanitary-indicative microorganisms. The main criteria for selecting starter cultures for the production of dietary fermented milk products is the activity of acid formation, resistance to phages, as well as the ability to suppress pathogenic and technically harmful microflora, the absence of antagonism between lactic bacteria.

Bifidobacteria are microorganisms that live in the human intestines, but pathogenic, that is, unable to cause disease. Moreover, they can be said to cooperate with the body, participating in protein, fat and mineral metabolism, in the production of vitamins and enzymes. Finally, they actively produce acids that are harmful to salmonella, staphylococci, and causative agents of dysentery.

Net cultures of bifidobacteria are characterized by certain properties: they are gram-positive anaerobic fixed sticks. B. longum grow in the absence of air and at low partial pressure.

Strains of bifidobacteria have antibiotic activity in relation to pathogenic microflora, due to which they can be used for the preparation of therapeutic fermented milk products [85].

L. acidophilus - belongs to the group of thermophilic rods, which are widely used in the production of fermented milk products. The main distinctive property of acidophilic bacteria is their antibiotic activity. They are one of the representatives of the normal microflora of the human intestine, they are contained in the feses of children and adults in conjunction with enterococci, E. coli and other bacteria. They are able to suppress the development of a number of microorganisms, including bacteria of the Escherichia coli group, dysentery, paratyphoid and others. The acid formation limit at ripening is 180-300 oT, at the same time, L (+) and DL - lactic acid are formed [11].

Due to the absence in the studied scientific and technical literature of specific recommendations on the use of the above-described cultures in dairy dessert products, it is of scientific and practical interest to conduct studies on the joint activity of isolated cultures and lactic acid organisms for a bacterial preparation.

To study the possible use as a starter of the Strain of S. cremoris K-3, S. lactis SSa-1, L. acidophilus SHA-2 and the ferment of the bacterial preparation "Bifilact-Extra", which includes lactic acid and bifidobacteria B. longum, L. acidophilus, were cultured together, comparing the results with the result of their separate cultivation.

For the experiments, the starter was introduced into sterile skim milk. Pre-select the basis, i.e. the possibility of combining S. cremoris K-3, S. lactis SSa-1, L. aidophilus SHA-2 for this purpose in a large tube with sterile skim milk (20-30 ml), an equal amount of each Strain is applied, the tubes with milk are thermostated until a clot is formed. The bases are twice poured into sterile skim milk and tested for the compatibility of Strain. At the same time, other cultures of the milk are added to the cultures that are part of the ferment. The compatibility of Strain lactic acid bacteria is determined by the duration of the coagulation of milk

48

by a combination of Strain compared to the duration of coagulation with each culture (Strain) included in their composition (with equal organoleptic parameters). By the number of microorganisms (Figure 3) and acidity determine the possibility of their joint cultivation.

0 2 4 6 8 10 12 14 16 18 20 22 240

20406080

100120140

Streptococcus cremoris К-3Streptococcus lactis SSа-1 L. аcidophilus ShА-2

Time of fermentation. h

Aci

dity

, ° T

Figure 3 - Determination of the dynamics of acidity changes by different strains

Recommendations for maintaining the temperature in the cultivation of symbiotic starter substantially diverge. By changing the temperature during cultivation of symbiotic starter cultures, it is possible to regulate the composition of its microflora in the desired direction. When preparing starter cultures from microorganisms with different temperature optimum, it is advisable to select temperatures close to optimal for mesophilic microorganisms, that is, at a temperature of 32 ° C. With separate cultivation of selected strains, it was found that with time the acidity increases in all the test samples (Figure 3).

When using the ferment of the bacterial preparation Bifilact-Extra, which contains lactic acid and bifidobacteria B. longum, L. acidophilus, in spite of the lower values of titrated acidity in the first hours of cultivation, there is a further rapid increase in acidity, which reaches a maximum value for 20-24 hours - 135 oT. However, the formation of a clot when using this starter occurs only after 16 hours.

When the S. cremoris K-3, S. lactis SSa-1 and L. acidophilus SHA-2 were co-cultivated, a more rapid acidity growth was observed, and for 8 hours of their co-cultivation a thick, even consistency with a pure sour-milk taste was formed.

Thus, the joint cultivation of S. cremoris K-3, S. lactis SSa-1 and L. acidophilus SHA-2 allowed to reduce the fermentation time by 2 times, to obtain a clot, of good quality.

In Figure 4, a comparative diagram is presented, the changes in the number of cells of individual strains of microorganisms depending on the duration of cultivation with a joint (multicomponent starter) and separate (single-component starter) application of the selected strain.

49

0 2 4 6 8 10 12 14 16 18 20 22 240

100

200

300

400

500

600

one-part starter

multicomponent starter

Duration of exposure, h

Figure 4 - Comparative diagram of the change in the number of cells of strains of microorganisms, S. lactis SSa-1 in separate cultivation (one-part ferment) and combined with other yeast strains (multicomponent starter)

0 2 4 6 8 10 12 14 16 18 20 22 240

100

200

300

400

500

600

one-part starter multicomponent starter

Duration of exposure, h

Figure 5 - Comparative diagram of the change in the number of cells of strains of microorganisms of S. cremoris K-3 in separate cultivation (one-part ferment)

and combined with other Strain leaven (multicomponent starter)

In the first 12 hours, intensive growth of the strains studied was observed. The highest growth of cells with the use of multicomponent starter was observed in the culture of S. lactis SSa-1, and during the first hours of co-cultivation the cell growth was almost 100 times and the maximum amount (3,4 x 108) of lactic acid bacteria of this Strain is 12-14 hours with a joint cultivation of S. cremoris K-3, S. lactis SSa-1, L. acidophilus SHA-2.

50

CFU

/ m

l

C

FU /

ml

109

107

106

104

102

109

107

106

104

102

0 2 4 6 8 10 12 14 16 18 20 22 240

100

200

300

400

500

600

one-part starter multicomponent starter

Duration of exposure, h

Figure 6 - Comparative diagram of the change in the number of cells of strains of L. acidophilus SHA-2 microorganisms in separate and co-culture with other

stains

The tendency of increasing the number of strains S. cremoris K-3 cells using a multicomponent starter culture is also observed in the analysis of the effect of joint and separate cultivation on this strain. However, the intensity of influence is less pronounced. Co-cultivation of S.cremoris K-3, S. lactis SSa-1 and L. acidophilus SHA-2 significantly increases the number of viable lactic acid microorganisms. Their number increases in direct proportion to the exposure time. A special growth is observed from 4 o'clock, in the future there is a stationary phase of their growth, in fact even on the second day. A similar picture is observed with microscopy. The presence of L. acidophilus SHA-2 in the form of single rods and short chains and a significant number of single cocci in the form of short chains of different lengths - S. cremoris K-3, S. lactis SSa-1. The main indicators characterizing these strains are given in Table 11.

Table 11 - Main indicators characterizing lactic acid microorganisms

Microorganisms Microscopic picture Coagulation activity, h

Organoleptic properties of the resulting clots

S. cremoris K-3 diplococci, short chain

4-5 dense, even, consistency creamy, taste pure sour-milk

S. lactis SSa-1 single cocci, small chains

4-6 dense, even, consistency slightly viscous, taste pure sour-milk

L. acidophilus SHA-2

single sticks and collected in small

4-5 smooth, the consistency is

51

CFU

/ m

l

109

107

106

104

102

109

107

106

104

102

109

107

106

104

102

chains stitching, the taste is pure sour-milk

B. longum, single sticks and collected in small chains, individual cells are curved, slightly branched sticks

6-8 smooth, consistency stitching, with a slight separation of whey, a taste of pure sour-milk

L. Acidophilus sticks single straight, in the form of short chains and abundant number of cocci in the form of short chains

3,5-4 dense, even, consistency creamy, taste pure sour-milk

The conducted experiments confirm the symbiotic relationship of S. cremoris K-3, S. lactis SSa-1 and L. acidophilus SHA-2.

To check the probiotic properties of the starter, it is necessary to establish the ability of the isolated strains and leaven obtained on their basis to suppress pathogenic and conditionally pathogenic microorganisms. Table 12 shows the antagonistic activity of the ICD.

The antagonistic activity of microorganisms is determined by the action of nonspecific and specific factors. The first include the formation of antibiotics of a polypeptide or unidentified nature, bacteriocins, short chain fatty acids. Nonspecific factors include the formation of lactic, acetic acids, the creation of a low oxidation-reduction potential through the utilization of oxygen, competition for nutrients [15, 99].

The antagonistic effect of lactic acid bacteria is due to an increase in the active acidity of the nutrient medium due to the fermentation of carbohydrates and changes in physicochemical conditions [15, 21]. Antagonism manifests itself in putrefactive bacteria, staphylococci, enterococci, proteolytic sticks, pathogenic and enteropathogenic E. coli, salmonella, fungi of the genus Candida [19, 34].

Table 12 - Antagonistic activity of isolated strains and microorganisms of bacterial preparation "Bifilact-Extra"

Strain

Diameter of growth inhibition zones of test cultures, mm

S. typhimurium

E. coli P.aeruginosaS.aureus B.subtilis

L. аcidophilus SHA-2

13,0±0,5 12,6±0,5 16,3±0,5 18,8±0,5 26,3±0,5

S. lactisSSa-1 19,2±0,5 17,6±0,5 16,2±0,5 15,1±0,5 17,7±0,5

52

S. cremorisК-3 18,9±0,5 20,2±0,5 14,4±0,5 21,6±0,5 13,4±0,5B. longum,L. аcidophilus

14,2±0,5 18,6±0,5 16,2±0,5 24,1±0,5 27,7±0,5

Polystyrene leaven 21,0±0,5 22,1±0,5 19,3±0,5 21,9±0,5 26,8±0,5

When comparing the data of antagonistic activity between the microorganisms "Bifilact-Extra" and the experimentally obtained polystyrene starter culture, we found that S.cremoris K-3, S.lactis SSa-1 and L. acidophilus SHA-2 cultures have greater inhibitory activity to the three test - strains of S. typhimurium, E. coli, P. aeruginosa. As for gram-positive bacteria, S. aureus and Bac.subtilis were most sensitive to B. longum, L. acidophilus. These microorganisms produce bacteriocins well, ensuring their high inhibitory activity.

Thus, the antagonistic activity of the studied strains was compared in a comparative aspect with "Bifilact-Extra", which includes lactic acid bacteria B. longum, L. acidophilus. All the lactic acid microorganisms isolated from the local milk raw material showed a high degree of antagonistic activity to S. typhimurium, E. coli, P. aeruginosa, S. aureus, and Bac.subtilis, with some of them exhibiting greater anthropogenic activity than the microorganisms B. longum, L. acidophilus "Bifilact-Extra".

As is known, treatment of various infections with eubiotics is carried out against the background of antibiotic therapy. In connection with this, an important criterion in the use of these bacteria for therapeutic nutrition is their resistance to antibiotics. In studying this issue, we previously determined the sensitivity of the isolates of S. cremoris K-3, S. lactis SSa-1 and L. acidophilus SHA-2 to 6 antibiotics belonging to different groups: ampicillin, erythromycin, streptomycin, penicillin, gentamicin, kanamycin. The choice of antibiotic data was justified by their wide application in clinical practice in the treatment of various infectious diseases of the gastrointestinal tract, urogenital and upper respiratory tract, with purulent-septic complications in the postoperative period.

Table 13 - Sensitivity of the isolated lactic cultures and bacterial preparation "Bifilact-Extra" to antibiotics

Strain

Diameter of growth retardation zones of lactic cultures, mm

Am

pici

llin

Gen

tam

icin

Kan

amyc

in

Peni

cilli

n

Stre

ptom

ycin

Eryt

hrom

ycin

L. аcidophilus SHA-2

21,5±0,5 0,6±0,1 6,2±0,5 10,2±0,5 5±0,1 14,4±0,5

S. lactis SSa-1 28,2±0,5 0 3,5±0,5 5,1±0,5 11,4±0,1 2,3±0,5S. cremoris К-3 22,4±0,5 0,6±0,1 2,3±0,5 31,6±0,5 0 32,3±0,5

53

B. longum,L. аcidophilus

19,5±0,5 7,6±0,1 3,2±0,5 0 3±0,1 20,4±0,5

Polystyrene leaven 22,4±0,5 0 2,3±0,5 31,6±0,5 0 14,3±0,5

The obtained data testify to the inexpediency of using the bacterial preparation in conjunction with "Bifilact-Extra", which consists of lactobacillus B. longum, L. acidophilus, during treatment with ampicillin and erythromycin, however, this drug can be recommended for treatment with penicillin. We can recommend the simultaneous use of isolated strains of S. cremoris K-3, S. lactis SSa-1 and L. acidophilus SHA-2 with streptomycin and gentamycin.

Thus, the poly-stem leaven based on S. cremoris K-3, S. lactis SSa-1 and L. acidophilus SHA-2 is promising for further study of both possible components of probiotic preparations and its use as a starter for the production of a dairy-cereal product .

The main milk raw material for the production of a sour milk base was whole cow's milk. An important component of milk is lactose, which when lactic ferment forms lactic acid.

Lactic acid stimulates the development of lactic acid rods, which suppress the putrefactive microflora, and promotes the absorption of calcium and phosphorus. In milk there is a high content of calcium and phosphorus, which perform important functions in the human body. Calcium and phosphorus are in well-balanced proportions, which causes high digestibility, enzymes, hormones, antibodies, antibiotics and biologically active substances supplement the value. Thus, milk contains all the nutrients necessary for the human body (proteins, fats, carbohydrates, minerals, vitamins) in well-balanced proportions and in an easily digestible form. As a raw material, whole cow normalized milk was used, in which the chemical composition was studied, the results of the study are given in Table 14.

Table 14 - Chemical characteristics of normalized milk

Name of raw

materials

Mass fraction,% Mineral substances, mg Energy value, kcalfat protein carbohy

dratesСа Р К Na Mg

Milk normalized

2,2±0,2 3,6±0,2 5,1±0,2 122 92 148 50 13 52

With the purpose of preparation of the sour-milk base of the dairy-cereal product, the ferment was prepared, first a laboratory, then a production starter, the main indicators were determined (table 15).

54

Table 15 - Basic indices of the production starter

Type of sourdough

Indicators

titratable acidity, 0T

clotting time, h

viscosity, ηef

Pa × s × 10-3

moisture-retardant ability

organoleptic indices

1 2 3 4 5 6S. lactis SSa-1 75 4-5 5,2 2,5 taste pure fermented

milk, consistency thick

S. cremoris К-3 75 4-6 5.4 2,3 taste pure sour-milk, consistency homogeneous, creamy

Table continuation 18L. аcidophilus SHA-2

95 4-5 6,05 3,2 smooth, the consistency is stitching, the taste is pure sour-milk

B. longum,L. acidophilus

100 5-6 4,1 4,2 the taste is pure sour-milk, the consistency is even, slightly piercing. with a small serum compartment

Polystyrene leaven

80 3,5-4 5,1 2,7 the taste is pure fermented milk, the consistency is uniform, creamy.

To prepare a laboratory starter, the whole milk was sterilized at 100 ° C, cooled to a fermentation temperature of 32 ° C, and fermented and fermented. One of the main requirements for lactic cultures used as a starter is the accumulation of lactic acid, the production of dense clots and the formation of active viable cells of microorganisms.

The selected strains have not a high acid-forming activity, a significant antagonistic ability and pronounced resistance to the polyvalent bacteriophage. In general, this makes it possible to use this consortium of microorganisms in the

55

production of a fermented milk product. The resulting sour-milk clot possesses a pure sour-milk taste, an even consistency and the necessary moisture retaining capacity.

Thus, the isolation and use of highly active lactic acid microorganisms made it possible to obtain a production starter that meets the requirements of the production process of a sour milk base for a combined dessert product.

4. INVESTIGATION OF THE INFLUENCE OF THE STIMULATING FACTORS OF NUTRITION OF MILK-ACID MICROORGANISMS

4.1 Investigation of the chemical composition of grain processing productsWheat processing products are of great interest as suppliers of substances

necessary for the body, such as easily digestible sugars and fiber. Wheat is mainly used in the milling industry to produce different varieties of flour. A by-product of the milling industry is wheat bran, embryos, which are very rich in biologically active substances, but they are usually not used in the production of basic types of food products.

Wheat germ seeds exert a strengthening effect on the body, are antioxidants, control and normalize the level of cholesterol in the blood. Scientific evidence supports: wheat germs are rich in B vitamins, they contain proteins, fiber, iron and other minerals. They are used to reduce the risk of cardiovascular diseases. Wheat germ oil is especially rich in vitamin E and essential fatty acids, which the human body can not produce on its own. Wheat germ oil is a part of additives "Tritinat", "Viardo" and others.

Table 16 - Chemical composition of anatomical parts of wheat grain (in% on dry matter)

Grain and its parts Contents in grain

Prot

ein

Star

ch

Suga

r

cellu

lar

tissu

e

Pent

oza

ns Ash

Fat

Whole grain 100,0 16,06 63,07 4,32 4,03 8,10 2,18 2,24The aleuron layer 6,54 53,16 0 6,82 6,41 15,44 10,03 8,16Shell 8,62 10,56 0 2,59 23,73 51,43 1,6 1,47Embryo with scute 3,24 37,63 0 26,36 2,46 9,74 7,55 16,2

7

As can be seen from Table 16, the balance of wheat chemical substances in the main grinding products (according to NS Berkutova) shows that most of the substances not digested by humans remain in the bran [28].

A comparative analysis of the chemical composition of bran and sprouted wheat (Table 17) showed the advisability of using sprouted wheat in the production of combined fermented milk products.

56

Table 17 - Chemical composition of wheat processing productsIndicators Mass fraction of substances,%

in bran in the germinated пшеницеMoisture 7,50+0,30 10,50±0,15

Fat 4,71+0,19 6,4±0,6Protein 17,23±0,44 27,4±0,6Ash 5,36+0,24 5,10±0,20Carbohydrates, including: 65,20+0,98 50,60±0,50Starch 19,10+0,05 41,00±0,50Alimentary fiber 46,10+0,01 9,60±0,50Hemicelluloses 26,3+0,05 5,60 ±0,50Lignin 16,40+0,01 2,40+0,01Pectin 3,40+0,01 1,60+0,01

From Table 17 it can be seen that in germinated wheat the moisture content is 10.5%, the fat content is 8.4%, the protein content is almost 10% higher in wheat germ in comparison with bran, and the carbohydrate content in germinated wheat is less in general on 8%, but thus basically there are assimilated forms of carbohydrates. At the same time, in sprouted wheat and bran, the ash content is close to 4.10% and 5.32% [29]. In germinated wheat, all ingredients are in easily digestible form.

4.2 Investigation of the effect of germinated wheat on lactic acid microorganisms

With the germination of the grain, the caloric content of the cereal crop decreases, and the nutrients become easier to assimilate by humans. Sprouted wheat grains help strengthen immunity, normal brain and heart function. Their consumption reduces the level of cholesterol in the blood. Figures 7, 8, 9 show photos of wheat grains during germination and changes in the internal structure of the grain.

Dietary fibers (cellulose, hemicellulose, pectin substances, lignin), concentrated mainly in the fruit and seed shells of grain, which practically do not undergo chemical changes during germination, swell and partially separate from the aleurone layer (Fig. 7a, b), unchanged Mineral substances are retained. In the sprouted grain, the amount of vitamin E (tocopherol), which retards the aging of the organism and is responsible for the activity of the genital glands of internal secretion, increased 2.3-fold, vitamin C, B1, B2, B6, carotene

Microstructural studies show that the internal elements of the germinated grain are characterized by a pronounced asymmetry inherent in the components of plant origin. On the general background consisting of two types of starch grains (large, globular, with a smooth surface and numerous numerous small ones fixed on

57

them), protein formations of complex configurations are found in the form of islets.

In the grain of various stages of germination, the amount of low-molecular substances soluble in water increases, the content of reducing sugars and protein nitrogenous substances sharply increases, and non-protein nitrogen decreases. At the same time, the content of sucrose in the first hours of germination decreases as a result of increased respiration, and after 24 hours it increases as a result of its synthesis due to starch. With the germination of grain and the development of cereals, dry substances, concentrated mainly in the endosperm in the form of starch, are expended on the synthesis of the anatomical parts of the young plant. This is evident when comparing figures 7, 8, 9 (a, b), on the whole this is accompanied by a decrease in calorie content and the accumulation of biologically active substances.

In the course of this process, the content of bran remains unchanged in absolute value, the correlating content of dietary fiber and fiber increases sharply, and at the output we have a product that concentrates all the functional ingredients of cereals-dietary fibers, vitamins, oligosaccharides, enzymes, protein, polyunsaturated fatty acids , - by reducing the amount of "empty calories" (starch).

а) b)Figure 7- During the first hours of germination and after 12 hours of germination

58

а) b)Figure 8- After 24 hours and after 36 hours of germination

а) b)Figure 9- Changes occurring in the fruit and seed coat during the first hours of

germination and after 36 hours of germination of the grain

In the future, the process is stabilized and a slight decrease in the number of ICDs is observed for 36-40 h, this is due to the fact that the nutrients that feed the grain sprout are reduced (Figure 10).

We have studied the change in the number of lactic acid bacteria in the grain of wheat during its germination. For the study, wheat seeds, which had been germinated for a certain time, were used, which were ground and used as a storage culture. An intensive growth of lactic acid bacteria was established on the media used. Figure 10 shows the quantitative change of lactic acid bacteria in time on the MRS medium.

There is a sharp increase in the amount in the first 10-12 hours; this is due to the intensive moistening of the grains, with the extraction of substances from the grain, which served as nutrients for growth of the milk-acid bacteria.

59

Figure 10- Quantitative change of lactic acid bacteria, in wheat grain during germination

For the following analyzes wheat flour from wheat germ obtained in laboratory conditions was used. The main criterial requirements for milk-grain clot are, decrease in fermentation time, due to stimulation of milk-acid bacteria, increased moisture retention capacity and high organoleptic evaluation.

     The moisture absorption was determined from the formula (1):

Moisture absorbing capacity=(m1/m2) 100%, (1)

where m1 - is the weight of the sample after absorption of moisture;m2 - dry weight

The initial sample of flour from wheat germ contained 10.5% moisture.The initial sample of wheat bran contained 7.5% moisture.

It is established that in the first 30 minutes the flour absorbs 30% of moisture in total, then the intensity increases and after an hour reaches 85%, after 90 minutes - 120%. In the future, the process stabilizes, and the water absorption intensity increases by only about 5% every 30 minutes.

60

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 400

100

200

300

400

500

600

Duration of incubation of grain, h

10 20 30 40 50 60 70 80 90 100

110

120

130

140

150

160

170

180

190

200

210

0

20

40

60

80

100

120

140

160

180

200

Time. mines

Вла

гопо

глащ

ающ

ая с

посо

бнос

ть, %

2

1

1- 1- Moisture absorption capacity of wheat flour2- 2- Moisture absorption capacity of wheat bran.

Figure 11 - Dependence of the moisture-absorbing capacity of grain products on time

The moisture absorbing capacity of wheat bran increases intensively within 1 hour and reaches almost 30%, in the future the water absorption rate decreases. The maximum volume of water absorption is 40 g / 100 g of product.

From the experimental data in Fig. 11 shows that the moisture-absorbing capacity of flour from sprouted wheat is more than 3 times higher than the moisture-absorbing capacity of wheat bran.

Structure of flour starch grains is crystalline, finely dispersed. Starch is characterized by a significant adsorption capacity, so it can bind a large amount of water under normal temperature conditions.

Dextrins are the primary products of starch hydrolysis, colloidal substances that form sticky solutions with water. The molecular weight and properties of dextrins depend on the degree of hydrolysis of the starch. In wheat flour, obtained from sprouted grain, contains about 3 - 5%. Dextrins poorly bind water.

Analyzing the obtained experimental data (Table 18), it was found that when using the consortium of S. cremoris K-3, S. lactis SSa-1 and L. Acidophilus SHA-2 the process of coagulation of milk is accelerated with increasing amount of added flour from germinated wheat, the duration of coagulation is 3.5-4 hours. The maximum titrated acidity of the clot was 80 0T.

61

Table 18 - Physicochemical parameters of lactic acid bunches with germinated wheat

Flour from wheat germ,

%

Clotting time, h

Titrated acidity, ° T

Viscosity, ηef Pa × s ×

10-3

Water-retention ability,%

Sensory evaluation

1 4-4,5 71 5,25 2,4 4,82 4-4,5 72 5,45 2,2 53 3,5-4 72 6,05 2,1 54 3,5-4 73 6,05 2,0 55 3,5-4 73 6,05 1,8 56 3,5-4 75 6,15 1,6 57 3,5-4 76 6,40 1,4 58 3,5-4 77 6,55 1,2 59 3,5-4 78 6,70 1,1 4,910 3,5 80 6,85 1,0 4,8

Control 4,5-5 70 5,00 2,5

The composition of flour from germinated wheat includes dietary fiber - pectin, lignin, cellulose, hemicelluloses, which promote the growth of lactic acid bacteria. In the culture medium of lactic acid bacteria, carbohydrates are additionally introduced, fermentation of carbohydrates and alcohols is an important diagnostic sign of lactic cultures. According to the scientific and technical literature on the composition of wheat germ, they are noted that they are rich in vitamin B, which is necessary for the growth of lactic acid bacteria. Figure 12 shows the stimulating effect of flour from sprouted wheat on lactic acid microorganisms.

62

Figure 12-Dynamics of growth of lactic acid bacteria at various doses of flour from wheat germination

Experimental studies have shown that the total number of bacteria increases with an increase in the dose of flour from sprouted wheat, at the time of clot formation in experiment 3, the number of bacteria is 5.6 × 107, in experiment 5 - 2.3 × 108. Sprouted wheat stimulates the development of lactic cultures with application of 1-10%, the optimal dose of the introduction of germinated wheat provides growth of lactic microflora 3-10%, representing the combination of the ferment of the bacterial preparation S. cremoris, L. acidophilus and S. lactis.

With the increase in the dose of wheat germinated into the milk, the coagulation process is accelerated, the viscosity of the lactic acid clots is also increased, the optimal dose of administration, which ensures the growth of lactic microflora is 3-8%.

When wheat germ is added to the combined dairy product, the end product is enriched with a number of functional ingredients: dietary fibers, oligosaccharides, minerals, unsaturated fatty acids, a number of vitamins actively synthesized by germinating an ingredient that reduces the mass fraction of essential amino acids. The introduction of up to 10% of flour from sprouted grain does not adversely affect the taste qualities of the milk-grain bunch, which has a positive value both for a more economical expenditure of milk, whose production does not yet meet the needs of the population, and when the dairy product is saturated with a grain component in the production of functional appointments.

The generalization of the obtained results gives grounds to believe that sprouted wheat can be used in the production of combined dairy products as

63

0

20

40

60

80

100

120

140

10% germinated wheat8% germinated wheat5% germinated wheat3 % germinated wheat

Ripening time, h

CFU

/ m

l

109

107

105

103

102

additional sources of biologically active substances, proteins, polysaccharides, including structural and mineral substances.

The possibility of using cereals in the production of dairy products provides a balance in the content of proteins, fats and carbohydrates; improvement of structural and mechanical properties; increase the shelf life of the product; cost reduction, etc.

On the basis of complex studies of wheat processing products, it was proposed to use sprouted wheat in raw form together with the shell.

The need for the use of sprouted wheat is due to the following restrictions on the use of other wheat processing products in the production of combined dairy products:

- bran - low digestibility by enzymes of the gastrointestinal tract and deficiency of almost all essential amino acids;

- embryos - ability to rancid in a short time.

5. INVESTIGATION OF MICROBIOLOGICAL PROCESSES AND PHYSICO-CHEMICAL PROPERTIES IN STORAGE OF MILK SERUM

In the process of collecting and storing cheese curd whey before processing, a set of measures are taken that make it difficult to seed an extraneous microflora, as well as inhibit the development of microflora that has entered the serum during the main production process, including microflora of bacterial starter cultures.

The results of microbiological parameters of curd whey are presented in Table 19.

Table 19- Microbiological parameters of curd whey

Serum test Contents in 1 ml of curd wheyTotal number of microorganisms х 103

molds Yeast х 102

colibacillus bacteria х 102

Before deoxidation 78,13±5,40

After deoxidation

161,80±18,10 47,0±5,0 3,30±0,4After separation

393,10±4,87 103,6±9,5 3,30±0,2After storage (24 hours) 432,0±20,65 266,5±34,3 3,88±0,9 20,0

Curd whey should be immediately recycled for processing, as storage leads to rapid accumulation of both lactic acid microorganisms and foreign microflora (Table 20). If this is impossible for any reason, then it must be subjected to special treatment (biological, pasteurization, cooling, canning).

64

Table 20 - Microbiological parameters of curd whey in the technological process

Process stage Contents in 1 ml of curd wheyLactobacilli , cells Mold fungi, cells Bacterium of E. coli,

cells

Syneresis 99 000 - -

Self-pressing 204 000 - 0,03

Pressing 344 000 0,2 0,9

Serum collection 451 200 3,08 1,67

Serum storage (more than 24 hours)

1019000 56,0 41,9

During storage, the composition and properties of the whey are altered. This is facilitated by the action of lactic acid bacteria in the production process and contamination by microflora. Lactose, as the least stable component, undergoes enzymatic hydrolysis.

The enzyme lactase, formed by bacteria, participates in lactose digestion, which leads to an increase in the titratable acidity of curd whey (Figure 13), active serum acidity (Figure 14), turbidity increase (Figure 15) and lactose losses (Figure 16).

0 10 20 30 40 50 60 70 80 900

10

20

30

40

50

60

70

80

90

Duration of storage, h

Titra

ted

acid

ity, °

T

Figure 13 - Effect of storage time on titrated acidity

65

0 10 20 30 40 50 60 70 80 90 1000

1

2

3

4

5

6

7

Duration of storage, h

Act

ive

acid

ity, p

H

Figure 14 - Effect of storage time on active acidity

Figure 15 - Effect of duration of serum storage on turbidity

The pH and turbidity of the curd whey are also altered. In addition, hydrolysis of protein and fat occurs, the taste of whey changes, unwanted and even harmful

66

substances can accumulate. In practice, we can assume that when stored without treatment for 12 h, the whey loses 25% of its energy value. The loss of lactose can be judged by the increase in the titrated acidity of the serum. The effectiveness of the loss of lactose during self-quenching of serum is shown in Figure 16.

1 2 3 4 5 6 7 8 9 100

102030405060708090

Lactose, %

Aci

dity

, ° T

Figure 16 - Lactose loss in milk whey as a function of the increase in its acidity

Taking into account the obtained data, it is necessary to establish the storage capacity of curd whey.

Analysis of literature data shows that [10] for prolonging the shelf life of cheese curd whey is used: heat treatment, preserving using preservatives, thickening, cryoconcentration and other methods. Two methods are investigated in the paper, which do not require the use of additional equipment and raw materials. From the data shown in Figure 17, it follows that low-temperature heat treatment makes it possible to keep the dairy serum up to 2-3 days, further storage is not recommended, as organoleptic indices deteriorate sharply. By the end of the shelf life, the whey gets a sour, musty taste, its color becomes cloudy.

Duration of storage, h

Figure 17 - Dynamics of changes in the acidity of pasteurized whey

67

Also, the second method for increasing the shelf life of curd whey has been investigated - its low-temperature treatment. In the scientific works of A.G. Khramtsova [10] shows the freezing temperature of milk whey - 5 - 80C. A soft mode of serum freezing at a temperature of -4 ° C has been studied.

To study the quality of the blocks from the frozen milk whey curd, an average sample was sampled, heated (unfrozen) by the temperature increase of the sample at 1-20C for 20-30 minutes, which allowed preserving the serum consistency without significant changes.

I stage (storage up to 10 days) - a slight increase in titratable acidity is observed at 4.0 ± 1.0 0T.

II stage (storage up to 20 days) - titrated acidity increases more intensively, by 20 0T.

Stage III (storage up to 30 days) - differs by a sharp increase in the titrated acidity, up to 58 0T, which excludes the possibility of its repeated heat treatment after defrosting. Also, the organoleptic parameters of whey are reduced, by the end of storage it acquires a sour, extraneous taste. Further storage of serum is impractical.

Thus, after studying the storage capacity, it was determined that it is expedient to curd milk whey pasteurized at a temperature regime (72 ± 10С) with an exposure of 15-20 seconds - the traditional mode of heat treatment, after which, immediately sent to the production of milk-grain product. Recommended two modes of storage of whey cheese curd: short-term (up to 2-3 days) at a temperature of 2-40C; long (up to 10-15 days) at a temperature of - 4 - 60C.

5.1 Study of the influence of whey proteins on the vital activity of lactic acid microorganisms

The purpose of the studies was to study the effect of whey proteins on the vital activity of lactic acid microorganisms and, on the basis of this, to identify the optimum amount of raw materials to be introduced, taking into account the organoleptic parameters of the finished product.

In 100 ml of curd whey contains 0.135 mg of nitrogen, about 65% of which is a part of protein nitrogen compounds and about 35% of it is made up of non-proteinaceous compounds. The content of protein nitrogen compounds in milk whey varies from 0.5-0.8% and depends on the method of coagulation of milk proteins taken in the preparation of the main product [22]. The composition of protein nitrogen compounds is given in Table 21. The content of whey proteins in milk whey is stable and on average is 0.74%.

68

Table 21 - Composition of protein nitrous compounds of milk serum

Fractions of proteins Content, % Isoelectric point, рН

The temperature of denaturation, 0С

Lactoalbumine:   β-lactoglobulin A   α-lactoglobulin B   β-lactoglobulin   (A + B)   β-lactoglobulin C   serum albuminLactoglobulin:   euglobulin   pseudoglobulin

   proteosupeptones

0,4-0,5

0,06-0,08

0,06-0,18

5,25,15,35,334,7

6,05,65,3

75-11060-9560-9560-9060-95

75-9075-9070-100

Serum proteins (albumins and globulins) have valuable useful biological properties, contain the optimal set of vital amino acids, and approach the amino acid scale of the "ideal protein" from the point of view of the physiology of nutrition (Table 22). This allows us to refer them to the full proteins used by the body for structural metabolism, mainly for the regeneration of liver proteins, the formation of hemoglobin and blood plasma. The valuable biological properties of whey proteins are due not only to the balance of the amino acid composition, but also to their good digestibility.

Table 22 - The content of essential amino acids in whey proteins and the "ideal protein" in 100 g of protein

Amino acids Whey proteins «Ideal protein»IsoleucineLeucineLysineMethionineCystinePhenylalanineTyrosineThreonineTryptophanValine

6,212,39,12,33,44,43,85,22,25,7

4,07,05,5

3,56,0

4,01,05,0

69

The amino acids of whey include amino acids of protein substances and free amino acids. The total amino acid content of the whey is shown in Table 23.

Table 23 - Total amino acid content in milk whey

Serum Amino acid content, mg / lfree in proteins

Total irreplaceable Total irreplaceableCurd 450 356 5590 2849

The amino acid composition of the main fractions of the curd whey protein is shown in Table 25. Some essential amino acids, such as leucine, isoleucine, methionine, lysine, threonine, tryptophan, are present in milk whey proteins even in larger quantities than in milk proteins.

Table 24 - Amino acid composition of the fractions of whey protein

Amino acids Content,% of total proteincasein albumin globulin

Valine 7,2 4,7 5,8

Leucine 9,2 11,5 15,6

Isoleucine 6,1 6,8 6,1

Proline 11,3 1,5 4,1

Phenylalanine 5,0 4,5 3,5

Cystine 0,34 6,4 2,3

Methionine 2,8 1,0 3,2

Cysteine - - 1,1

Tryptophan 1,7 7,0 1,9

70

ArginineHistidineLysineAspartic acidGlutamic acidSerinThreonineTyrosineGlycine

Alanin

4,13,18,27,122,46,34,96,32,73,0

1,22,911,518,712,94,85,55,43,22,1

2,91,611,411,419,55,05,83,81,47,4

For the growth and development of lactic acid bacteria need compounds of copper, iron, sodium, potassium, phosphorus, iodine, sulfur, magnesium and especially manganese. Data on the content of mineral elements are given in Table 25.

  Table 25 - Content of mineral elements in serum

Macronutrients Content, g / kg Microelements Content, mg / kg

SodiumPotassiumCalciumMagnesiumPhosphorusSulfur

224,117156,08699,0441,6481,2342,584

кремнийникельжелезоцинк

марганецмедь

алюминийкадмий

1,3220,3730,2600,1580,0090,0282,640

not detected

When studying the influence of whey proteins on the vital activity of lactic acid microorganisms, the positive effect of the introduced raw materials on the quantitative composition of the cultures studied was established, the best result was from 3 to 8% of the application of whey proteins (Figure 18).

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Figure 18 - Dynamics of growth of lactic acid bacteria with various doses of whey proteins

The analysis of the conducted experiments allows to draw a conclusion about the stimulating effect of introduced whey proteins as sources of easily assimilable nitrogen nutrition. Lactic acid bacteria are not able to synthesize organic forms of nitrogen and therefore need to grow in the presence of them in the medium; only some of the lactic acid bacteria use mineral nitrogen compounds to synthesize a number of organic compounds. A number of amino acids are required for satisfactory growth of lactic acid bacteria: arginine, cysteine, glutamic acid, leucine, phenylalanine, tryptophan, tyrosine, valine, the presence of these amino acids in whey and in optimal amounts provides a significant increase in lactic microflora.

6. DEVELOPMENT OF THE COMPOSITION FOR DAIRY-GRAIN PRODUCT BASED ON USE OF PROBIOTIC MICROORGANISMS

Among the priorities of the development of the dairy industry in the Republic of Kazakhstan is the development of scientific bases for the technology of third generation dairy products, as well as dairy products for special purposes, including therapeutic and prophylactic, functional purposes, enriched with vitamins and biologically active additives. A relatively new direction is the production of combined products of functional purpose, intended for dietary and daily food, including nutrition of school-age children.

Perspective and actual directions of development of technologies of new combined products with the corrected composition, in accordance with scientifically justified need, are the following:

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1 2 3 4 5 6 7 8 9 10 11 120

20

40

60

80

100

120

140

8% whey proteins 5% whey proteins3 % whey proteins 1 % whey proteins

Ripening time, h

CFU

/ m

l

109

107

106

104

102

- use of specially selected starter, consisting of probiotic dairy cultures;- use of dairy and vegetable raw materials to stimulate the growth of probiotic

lactic acid microorganisms.Within the framework of this direction, we have studied the reserves of such

valuable protein-carbohydrate raw materials as sprouted wheat and whey proteins containing a significant amount of essential amino acids. The production of a composition for a dairy-cereal product based on the use of probiotic microorganisms consists of the following main steps:

At the first stage, highly active strains of microorganisms from raw milk, from fermented milk products of the South-Kazakhstan Region are isolated. Their identification was carried out, these microorganisms are defined as S. Cremoris К-3, S. lactis SSa-1, L. аcidophilus SHA-2.

In the second stage, the selection of the grain component and whey proteins in the production of the combined fermented milk product was made. The stages of germination and changes in the internal structure of the grain have been studied. The chemical composition of whey proteins was studied. The effect of wheat germ and whey proteins on the growth of lactic acid bacteria was studied, the optimal amount of wheat germ and whey proteins was determined.

In the third stage, the formula for the combined fermented milk product based on sprouted wheat using probiotics was made, and a technology for preparing a combined fermented milk product based on sprouted wheat using probiotics was developed.

Based on the analysis of the results of experimental studies, the formulation of a combined dairy product based on sprouted wheat using probiotics was established.

The main tasks set in this section of research work are:- to determine the main technological parameters of production of a fermented

milk product based on sprouted wheat and whey proteins using probiotics.- to conduct industrial approbation of the technology of the combined

fermented milk product based on sprouted wheat and whey proteins using probiotics.

Scheme 19 shows the stages of research, and the practical results obtained.

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74

Isolation of highly active sour-milk microorganisms

Microbiological basis of production

of a combined fermented milk

product

The number of microorganisms, acid formation, antagonistic activity and phage resistance

Creation and selection of bacterial

starter for the production of milk =

grain product

Selection of combinations of lactobacilli for a multi-stench

starter with probiotic properties

Determination of physico-chemical and technological indicators

The total number of lactic acid bacteria, ripening time, titratable acidity,

symbiotic relationship

Water retention capacity, viscosity of the bunch, time of

fermentation of raw materials in polysthem leaven

Selection of components

stimulating vital activity of lactic acid

microorganisms

Investigation of the chemical composition and biological value of grain raw materials, and flour from germinated wheat

Dynamics of growth of lactic acid bacteria at various doses of flour

from wheat germ

Practical implementation of research results

Investigation of the chemical composition and biological value of whey proteins

Dynamics of growth of lactic acid bacteria at various doses of serum

proteins

Calculation of the formula of the combined fermented milk

product based on sprouted wheat using probiotics

Combined sour-milk product with wheat germ, using a poly-stamping starter

Industrial approbation of milk-grain product

Act of Industrial TestingThe protocol of the extended tasting

of the dairy-grain product

Identification of highly active sour-milk microorganisms

Morphological, cultural properties of lactic acid bacteria

Stages of research Researched factors Controlled parameters

Figure 19 - Scheme of implementation and practical results of work

75

The scheme for producing a fermented milk product based on sprouted wheat using probiotics is shown in Figure 21 and consists of three stages:

1 - preparation of a milky-gelled mixture;2 - preparation of a sour-milk component;3 - mixing of milk-gelled mixture and fermented milk component.The production process must be carried out in compliance with sanitary norms

and rules for dairy enterprises approved in accordance with the established procedure.

The production process of the composition was carried out as follows: a milk-gelled mixture was prepared by mixing gelatin and milk. For this gelatin was pre-soaked in water and left for swelling for 40-50 minutes. After swelling, the solution of gelatin was heated until complete dissolution at a temperature of 500C, and skimmed milk was introduced there, pasteurized at a temperature of 80±20C 8-10 minutes and cooled to 40-420C. Sugar and flavor were then added, mixed and cooled to room temperature.

An acid-milk component with a fat mass fraction of 2.5% was obtained by leavening milk, a poly-stammed starter consisting of strains S. cremoris К-3, S. lactis SSa-1,L. аcidophilus SHA-2 extracted from local milk raw materials in an amount of 5%. Whole milk was normalized, homogenized at a pressure of 15-16 MPa and a temperature of 500C, pasteurized at 800C for 8 minutes, cooled to a fermentation temperature of 320C, and a ferment, wheat flour and whey protein concentrate were added. The quenching was carried out for 3-3.5 hours to an acidity of 70-75 0T. The finished sour-milk component was mixed. The milk-gelled mixture was mixed with the lactic acid component, stirred for 5-10 minutes, cooled to 80C and poured into polystyrene cups.

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Heating the mixture to 500C

Mixing

Sugar, flavoring

Stirring and cooling to room temperature

Bacterial leaven

Cooling up to 320С

Cooling up to 400С

Sprouted wheat

Concentrate of whey proteins

Fermentation for 3-3.5 hours

Mixing

Mixing of the milk-gelled mixture and fermented milk component and cooling to 80C

Packaging

Water

Figure 20 - Diagram of the production of a fermented milk product with wheat germ, based on the use of a polystamm culture with a probiotic effect

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1/3 of the milk

2/3 of the milk

Swelling for 40-50 minutes Pasteurization at 800C for 8 minutes

Milk fat-freeGelatin

One of the determining factors influencing the biological value of the product being developed is the number of viable lactic acid microorganisms in the finished product. Therefore, the ratio of the sour milk base and the gelled part had to be selected taking into account the number of microorganisms (cl / g) and organoleptic parameters. Since the additional components of the developed products are wheat flour (5-10%), whey proteins (3-5%), as well as gelatin, sugar, flavor (in the amount of 5-10%), the amount of milk used should be about 80%. The results of the experiments are shown in Table 26.

Table 26 - Influence of the ratio of sour-milk and gelled components on the number of microorganisms in the final product

Component Number of microorganisms, CFU / g

Organoleptic indicatorsSour milk base,%

Gelled part,%

30 50 8,6х103 consistency excessively dense, rigid, insufficient content of fermented milk ingredient

40 40 7,4х104 consistency slightly dense hard, insufficient content of fermented milk ingredient

50 30 6,1х105 the product obtained has a jelly-like consistency, pure sweet sour-milk taste with a taste of wheat

Examples of the composition for the preparation of a combined fermented milk product with wheat germ, based on the use of poly-stam starter probiotics:

1. To prepare 100 kg of the composition, gelatin is first prepared, 2.45 kg of gelatin is soaked in 20 liters of cold water for 40 minutes, then heated to 50 ° C, then mixed with 30 kg of skim milk containing 8 kg of sugar and cooled to 42 ° C , add flavoring in an amount of 0.05 kg.

To prepare the fermented milk component, 50 kg of skim milk is used, pasteurized at a temperature of 80 ±2 ° C with an exposure time of 10 minutes, cooled to a fermentation temperature of 32 ° C and 2.5 kg of bacterial leaven prepared on the basis of lactic cultures is added: S. cremoris К-3, S. lactis SSa-1 and L. аcidophilus SHA-2, taken in an equal proportion and the amount of 5% of the mass of the mixture. There also bring crushed wheat germ in the amount of 4 kg and 3 kg of whey protein concentrate. After the mixture is fermented for 5 hours. The finished sour-milk component is stirred without cooling.

Then, 59.5 kg of sour-milk component and 40.5 kg of milk-gelled mixture are mixed, cooled to 8 ° C and packed.

The resulting combined fermented milk product with wheat germ, based on the use of probiotic-based polystammal starter, has an excessively sweet taste.

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2. The preparation of a composition for the preparation of a combined fermented milk product with wheat germ, based on the use of a polymer strain of probiotic action, is carried out analogously to Example 1 at the following consumption of components, kg:

skimmed milk - 82Sprouted wheat - 5bacterial culture - 2.41whey protein concentrate - 4gelatin - 1sugar - 5,5flavoring - 0.09The resulting product has an insufficient jelly-like consistency.3. The preparation of the composition for the preparation of a combined

fermented milk product with sprouted wheat based on the use of a polymer strain of probiotic action is carried out analogously to Example 1 at the following consumption of the components, kg.

skimmed milk - 73Sprouted wheat - 12bacterial culture - 2.5whey protein concentrate - 4gelatin - 2.42sugar - 6flavoring - 0.08The obtained sour-milk product with sprouted wheat based on the use of the

probiotic-based poly-stam starter has a non-uniform structure, a grain taste due to a large amount of sprouted wheat and does not allow obtaining the desired result.

The results of the selection of the main ingredients are presented in Table 27.

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Table 27 - Examples of a composition for the preparation of a gelled milk-grain product

№ Used raw materials,%

Sensory evaluationskimmed milk

Sprouted wheat

whey protein concentrate

bacterial culture Gelatin Sugar

1 82 10 3 1 1 3 long fermentation time, the product has insufficient jelly-like consistency, excessive grain taste, insufficient sweetness

2 80 7 4 2 3 4 long fermentation time, excessive grain taste, increased dense consistency, not expressed sweetness

3 80 5 3 2,5 2,5 7 the product has a jelly-like consistency, an excessive sweet sour-milk taste, with a slight taste of wheat

4 80 6 3,5 2,5 2 6 the product has a jelly-like consistency, pure sweet sour-milk taste with a taste of wheat

Average value

80-82

5-5,5 3-4 2,5 2-2,5 5-6 the product meets the requirements: has a jelly-like consistency, pure sweet sour-milk taste with a slight taste of wheat.

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Thus, the preparation of a composition for the preparation of a combined fermented milk product with wheat germ, based on the use of a probiotic-based poly-stam starter, is carried out in the same manner as in Example 1 at the next consumption of the components,%.

skimmed milk - 80-82Sprouted wheat - 3-5,5Whey protein concentrate - 3-5gelatin - 1,95-2,9sugar - 5 -7flavoring - 0,05-0,1bacterial culture - 2-2,5The resulting product has a jelly-like consistency, a pure sweet sour-milk

flavor with a taste of wheat.

Based on the results of the experiments on the preparation of a combined fermented milk product based on sprouted wheat using probiotics, a formulation was established, which is presented in Table 28.

Table 28 - Recipe for a combined fermented milk product based on sprouted wheat using probiotics

Component Amount,% (parts by weight)Milk fat-free 80-82

Sprouted wheat 3-5,5

Concentrate of whey proteins 3-5

Gelatin 1,95-2,9

Sugar 5 -7

Aroma 0,05-0,1

Bacterial leaven 2-2,5

On the basis of the results obtained, a formula for the milk-grain product with sprouted wheat was created based on the use of a poly-stamping quarry with probiotic properties. The obtained combined lactic acid product, for therapeutic and prophylactic purposes, contains the lactic acid bacteria we isolated, which have high probiotic properties.

CONCLUSION81

In recent years, widespread use of dairy products, normalizing the functioning of the human gastrointestinal tract. Their use in nutrition causes a significant improvement in the body's activity, contributes to its recovery and, thus, in some cases helps to avoid the use of medicines. It has been proved that such sour-milk products should be used not only for prevention, but also for the treatment of almost all diseases, including diseases of the gastrointestinal tract.

The special importance of lactic acid bacteria is determined by their physiological-biochemical, antibiotic and probiotic properties [33].

Lactic acid products have a significant bactericidal effect, which determines their beneficial effect on the human body and animals. Many researchers explained this by the effect of the lactic and other acids contained in them (acetic, formic, etc.), alcohols, peroxides and other metabolites accumulated by lactic acid microorganisms during their growth and development. The action of such metabolites is based on the decrease in pH and the inhibition of pathogenic and opportunistic bacteria due to this growth.

As a result of the work done on the isolation of highly active strains, we obtained 21 strains of various lactic acid bacteria. Of these, 9 strains are representatives of S. lactis, 3 strains have a species identity S. cremoris, 4 Strainа – S. thermophilus, to L. аcidophilus there are 2 strains, one strain is representative of L. bulgaricus, and from the wheat grains belonging to L. plantarum, 2 strains were isolated.

Currently, more attention is being paid to the development of lactic acid products based on poly-stem sourdough, which includes several microorganisms belonging to different genera and species. For this, it is necessary to take into account the interactions of microbes in the natural habitat and, first of all, the symbiosis of individual strains. Many of the lactic acid bacteria studied by us have high production-value properties-acid formation, antagonistic activity, and resistance to the action of bacteriophages. The conducted studies to determine the antimicrobial activity of lactic acid bacteria with growth on various media allowed to identify the individual characteristics of each culture, which seems necessary for further recommendation of the use of poly-stamping starter for the production of therapeutic and prophylactic purposes. Knowledge of the antagonistic properties of each culture, the optimal conditions for their maximum manifestation, must be taken into account when composing the formulation of therapeutic and dietary products with specified properties. On the basis of a comprehensive study, the following strains of microorganisms were recommended for starter in the manufacture of a combined fermented milk product S. lactis SSa-1, L. аcidophilus SHA-2и S. cremoris К-3.

Probiotic sour-milk products are produced using microorganisms that are representative of the normal microflora of the human gastrointestinal tract. A special place is occupied by probiotic products, which contribute to the restoration of natural factors of protection of the human body. Their use in nutrition causes a significant improvement in the activity of the organism, contributes to its recovery.

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A steadily developing direction in this area leads to the improvement of the production of such products. Among various representatives of normal human microflora, bifidobacteria and lactobacilli occupy a special place, since they have the leading role in maintaining and normalizing the intestinal microbiocenosis, the resistance of the organism to intestinal infections, the improvement of protein and mineral metabolism and other. All this allows us to consider lacto- and bifidobacteria as a basis for creating new food products - products with probiotic properties.

Bifidobacteria and acidophilic bacteria have the ability to synthesize antibiotics, suppressing the development of pathogenic microflora, participate in the synthesis of vitamins and amino acids, and have a positive effect on the adsorptive capacity of the intestine. Consequently, the use of drugs containing L. acidophilus in the production of dairy products is relevant and promising.

As a production starter, was used culture from strains of S. cremoris K-3, L. acidophilus SHA-2 and S. lactisSSa-1 isolated from local milk raw materials. Strain, used for starter, have sufficiently good acid-forming activity, high antagonistic ability and pronounced resistance to polyvalent bacteriophage. This makes it possible to use this consortium of microorganisms S. cremoris K-3, L. acidophilus SHA-2 and S. lactisSSa-1 in the production of a sour-milk product intended for dietary and preventive nutrition.

Studies have shown that a high level of normal microflora does not always serve as an indicator that it fully manifests its useful properties. Therefore, simultaneous use of pro- and prebiotics is advisable. The introduction into the probiotic products of the components of prebiotic action makes it possible to expand the spectrum of their action. The study of the stimulating effect of prebiotics on the growth, enzymatic and adhesive activity of the own symbiotic microflora, as well as the reduction of its antibiotic susceptibility with the help of pro- and prebiotic preparations, is one of the topical problems of microbiology. Prebiotics include dietary fiber, in a significant amount contained in sprouted grain. We have established that the optimal amount of grain input is 3-5.5% of the main raw material - milk. The microflora of the human colon is capable of producing enzymes that break down the dietary fiber, the products of their metabolism are later used by probiotic lactic acid microorganisms for growth and reproduction. In addition, in this process, organic acids are formed. It is they, reducing acidity, prevent the development of pathogenic microorganisms that do not have enzymes for processing prebiotics. In combination, the actions of probiotics and prebiotics stimulate and activate the metabolic reactions of useful representatives of human microflora.

In conditions of market economy and tough competition, dairy enterprises are trying to develop a new, economically profitable and competitive assortment of dairy products, which is really possible with the full use of milk constituents and non-waste production. Inherent secondary raw materials of the dairy industry in the production of cheeses, cottage cheese and casein is whey.

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The existing shortage of high-tech dairy raw materials contributes to the introduction of its integrated processing. In this regard, whey protein, containing all essential amino acids, it is advisable to consider as an additional source of high-grade protein. The presence of essential amino acids in whey protein when it is added to fermented milk provides a significant increase in lactic microflora. We established that the optimal dose of serum proteins to be introduced is 3-8%.

Investigation of the peculiarities of the effect of sprouted wheat and whey proteins on the biological properties of lactic microflora makes it possible to steadily change the properties of the finished product, develop more effective preparations of natural origin, which in turn will promote active prophylaxis and correct treatment of infectious and inflammatory diseases without the use of antibiotics.

The analysis of scientific data in the field of combined products technology has made it possible to establish the main directions for improving technology. In the range of produced sour-milk additives, along with the traditional set, combined grain products are increasingly developing. The main purpose of grain fillers in these products is to enrich them with vitamins, microelements, dietary fiber, organic acids, and also in stimulating action on lactic acid microorganisms. Combination of vegetable and dairy raw materials is a promising direction in creating qualitatively new food products. The theory and practice of combining dairy raw materials with raw materials of plant origin, including grain components, is one of the actual trends in the food industry and will further develop further in the future.

The obtained results make possible to draw the following conclusions:1. There are 21 strains of different lactic acid bacteria. Of these, 9 strains are representatives of S. lactis; 3 strains have the species belonging to S. cremoris; 4 strains - S. thermophilus; to L. acidophilus are 2 strains; one strain of SPm-2 is representative of L. bulgaricus, and two strains isolated from wheat grains refer to L. plantarum.

2. Based on a comprehensive study of technological parameters (clotting time, moisture retention capacity), biochemical properties (aroma-forming, proteolytic and lipolytic activity), as well as in phage resistance and resistance to a number of antibiotics, the following strains of poly-stamping starter microorganisms: S. cremoris K-3, L. acidophilus SHA-2 and S. lactisSSa-1.

3. The introduction of flour from sprouted wheat speeds up the process of coagulation of milk, stimulates the growth of lactic acid bacteria, increases the viscosity of fermented milk bunches. The optimal dose of flour from wheat germ is 3-5,5%.

4. To stimulate the vital activity of lactic acid bacteria, the introduction of whey proteins rich in essential amino acids is recommended as part of the milk-grain product. The optimal dose of serum proteins to be introduced is 3-5%.

5. The main parameters of the preparation of a poly-stam starter with probiotic properties for the production of a dairy-cereal product of table and therapeutic-

84

prophylactic purposes were determined, and the optimal ratio of its constituent components was selected.

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