RU2524435C1 - Method of production of bacterial concentrate and its application as biologically active food supplement or inoculum for direct application for kurunga - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method comprises growing symbiotic inoculum of kefir fungal inoculum and thermophilic lactic acid bacteria Lactobacillus bulgaricus, Lactobacillus acidophilus and Lactobacillus helveticus (1:0.5:0.5:1) on the nutrient medium based on cottage cheese whey, rye flour and growth components at 30±2°C for 8-10 hours. After receiving the inoculum the biomass is isolated to obtain the liquid bacterial concentrate. In another embodiment for preparation of the inoculum of direct application the resulting liquid bacterial concentrate is mixed with the protective medium and frozen at a temperature not higher than (-20°C). The use of the resulting concentrates is proposed as a biologically active food supplement or the inoculum of direct application for kurunga.

EFFECT: inventions enable to increase the biomass yield and to maintain stability of microflora of the bacterial concentrate during prolonged storage to obtain the biologically active food supplement or the inoculum of direct application.

4 cl, 8 tbl, 3 dwg, 6 ex

Description

The present invention relates to biotechnology and the dairy industry and can be used for the preparation of bacterial concentrates used as biologically active food additives, and for direct starter culture in the production of a heterofermentative product - kurung.

There is a method of producing starter culture for preparing a fermented milk drink of the Kurung type, which provides for the fermentation of skim milk with kefir fungal yeast and bread crumbs in a ratio of 1.5: 0.5 for 8 hours until a clot with an acidity of 120 ° T in a wooden container (see RU No. 2059380, A23C 9/12, publ. 05/10/96, bull. No. 13).

However, the acidity of the product obtained by the above method is low and does not ensure compliance with the organoleptic properties of karung, which should have an acidity of 180-200 ° T. It should be noted that breadcrumbs are sources of Saccharomyces cere-visiae yeast that are not characteristic of kurunga microflora. The data on the preferred cultivation of the starter culture in a wooden container indicate the instability of the symbiosis of the microflora of kefir fungal yeast and bread crumbs. The proposed duration of cultivation of the starter culture is 8 hours, insufficient to achieve the most active stage of development of lactobacilli and yeast producing antibiotic substances, vitamins, etc.

In connection with these reasons, the known method does not allow to obtain a starter culture with characteristic quality indicators for karung. In addition, the above method does not provide resistance to starter cultures for long-term storage.

A known method of preparing a bacterial concentrate, comprising the preparation of a nutrient medium based on whey, inoculum, cultivation, deoxidation of the nutrient medium, separation of the obtained biomass (see SU 1082371 A, A23C 9/12; C12N 1/20, 30.03.84)

When using the above method, a culture of acetic acid bacteria is introduced into the nutrient medium, after which they are inoculated with lactic streptococci, which does not provide the required acidity, alcohol content and volatile organic compounds in the finished product.

The closest in technical essence to the present invention is a method for producing a symbiotic bacterial concentrate, which involves preparing a nutrient medium based on cottage cheese whey and potato broth, introducing a symbiotic starter culture obtained by cultivating kefir fungal starter culture and thermophilic lactobacilli (Bulgarian and acidophilus sticks) at a temperature of (30 ± 2) ° С for 2-3 days, biomass accumulation, deoxidation of the nutrient medium, separation of the obtained biomass, cm Shivani with a protective environment, bottling, freezing. Kefir fungal starter and thermophilic lactobacilli can be used in the following ratios 1: 0.5: 0.5 or 3: 0.5: 0.5 (see RU 2287939 C2, A23C 9/12, 12/23/2004).

For reasons that impede the achievement of the following technical result when using the above method adopted as a prototype, the known method for producing a bacterial concentrate requires long-term cultivation of symbiotic starter culture for 2-3 days to obtain the optimal pH of the medium, as well as a small biomass yield .

Thus, in the production of symbiotic bacterial concentrate, the main task is to improve the conditions for obtaining symbiotic starter culture and the composition of the nutrient medium to increase the efficiency of the technology.

The technical result of the invention is to reduce the duration of the production of symbiotic starter culture, increase the biomass yield and maintain the stability of the microflora of the bacterial concentrate during long-term storage.

The specified technical result is achieved by the fact that in the method for producing a bacterial concentrate, which provides the preparation of a nutrient medium based on cottage cheese whey, the introduction of a symbiotic starter culture obtained by cultivating kefir fungal starter culture and thermophilic lactobacilli Lactobacillus bulgaricus, Lactobacillus acidophilus at a temperature of (30 ± 2) ° C, accumulation of biomass, deoxidation of the nutrient medium, separation of the resulting biomass and bottling, according to the invention, rye flour is added to the nutrient medium in an amount of (2 ± 0.5)% of the growth of the nutrient medium, Lactobacillus helveticus is additionally introduced into the symbiotic sourdough, while the ratio of kefir fungal sourdough, Lactobacillus bulgaricus, Lactobacillus acidophilus and Lactobacillus helveticus is 1: 0.5: 0.5: 1, respectively, the cultivation of the symbiotic sourdough is carried out for 8-10 hours

In addition, a feature of the method is that a liquid bacterial concentrate is used as a biologically active food supplement.

In addition, a feature of the method lies in the fact that the frozen bacterial concentrate is used as a direct starter culture.

Distinctive features of the claimed invention are the quantitative and qualitative composition of the symbiotic starter culture, namely, the additional introduction of Lactobacillus helveticus culture into it, the optimal ratio of kefir fungal starter culture Lactobacillus bulgaricus, Lactobacillus acidophilus and Lactobacillus helveticus is 1: 0.5: 0.5: 1, respectively, which allows to achieve optimal active acidity in 8-10 hours of cultivation, as well as introducing into the nutrient medium (2 ± 0.5)% rye flour to activate the growth of yeast.

In addition, a distinctive feature of the claimed invention is the use of a liquid bacterial concentrate obtained by the proposed method as a biologically active food supplement, and a frozen bacterial concentrate as a direct starter culture.

For the implementation of the proposed method, experimental studies were conducted.

At the first stage of the research, the authors studied the possibility of reducing the duration of the preparation of symbiotic starter culture. For this, during the formation of a microbial consortium, the active acidifier L.helveticus was added to the combination of kefir fungal starter culture and thermophilic lactobacilli L.bulgaricus, L.acidophilus, taken in a ratio of 1: 0.5: 0.5, respectively.

It is known that L.helveticus is found in naturally formed populations of fermented milk products of heteroenzymatic fermentation and is in symbiosis with other representatives of their microflora. The culture is characterized by high proteolytic activity and milk coagulation rate.

The research results are presented in table 1.

Table 1 Selection of the optimal ratio of kefir fungal yeast, L. acidophilus, L. bulgaricus and L. helveticus The name of indicators The ratio of kefir fungal yeast: L.acidophilus: L.bulgaricus: L.helveticus 1: 0.5: 0.5: 0 (control) 1: 0.5: 0.5: 0.5 1: 0.5: 0.5: 1 1: 0.5: 0.5: 1.5 Duration of cultivation, h 8-10 8-10 8-10 8-10 Titratable acidity, ° T 70 one hundred 120 140 Active acidity, units 4.8 4.3 3.9 3,5 The number of viable cells, KOU / cm ³ : - yeast, fermenting lactose; 3 · 10 ⁴ 9 · 10 ⁵ 8 · 10 ⁶ 2 · 10 ⁴ - yeast that does not ferment lactose; 4 · 10 ³ 6 · 10 ³ 4 · 10 ⁴ 5 · 10 ³ - mesophilic lactobacilli; 7 · 10 ⁸ 1 · 10 ⁸ 4 · 10 ⁷ 1 · 10 ⁶ - thermophilic lactobacilli 2 · 10 ⁷ 8 · 10 ⁸ 1 · 10 ⁹ 9 · 10 ⁹ Mass fraction of alcohol,% vol. 0.2 0.53 0.76 0.5 Mass fraction of lactic acid, mg / cm ³ 634 675 752 845 Proteolytic activity, μg / ml 155 158 165 185

From table 1 it can be seen that the dynamics of titratable and active acidity is proportional to the increase in the dose of L.helveticus in the combined starter culture. So, in experiments 1: 0.5: 0.5: 1 and 1: 0.5: 0.5: 1.5 the titratable acidity reached 120-140 ° T in 8-10 hours, in the control this indicator was 70 ° T.

This creates the optimal activity of the medium (pH), which promotes the growth of yeast, so in the experiment 1: 0.5: 0.5: 1, the number of yeast fermenting lactose reached 8 · 10 ⁶ cfu / cm ³ , not fermenting lactose - 4 · 10 ⁴ cfu / cm ³ . A further increase in the dose of L.helveticus led to a decrease in the number of yeast.

Also, in all combinations, a slight decrease in the content of mesophilic lactobacilli was observed, associated with a decrease in the number of acid-unstable mesophilic streptococci.

The prospects for improving the formed microbial consortium are the study of extracellular metabolites synthesized by microorganisms. In the experiment with the ratio of kefir fungal yeast, L. acidophilus, L. bulgaricus and L. helveticus, taken in a ratio of 1: 0.5: 0.5: 1, respectively, the most active development of alcoholic fermentation is observed. The balanced course of lactic acid and alcoholic fermentation in the microbial consortium contributes to the formation of organoleptic properties characteristic of the heteroenzymatic Kurung product.

Proteolytic activity may vary depending on combination with other cultures. Therefore, when selecting strains of lactic acid bacteria to create bacterial concentrates, it is especially important to consider proteolytic activity in addition to other indicators. The products of proteolytic decomposition of proteins, primarily amino acids, when accumulated during the fermentation of milk, have a significant effect on the formation of consistency, taste and smell, as well as biological value.

According to table 1, there is a direct correlation between the dose of L.helveticus, the rate of milk coagulation and the proteolytic activity of microbial consortia. The highest tyrosine concentrations of 165 and 185 μg / ml were detected in experiments 1: 0.5: 0.5: 1 and 1: 0.5: 0.5: 1.5, respectively, and the lowest in experiments 1: 0.5: 0.5 (prototype) - 155 μg / ml. An increase in the proteolytic activity of microbial consortia promotes the formation of a fine clot characteristic of karung, in the control the clot had a denser consistency.

Thus, the most optimal ratio of kefir fungal yeast, L.bulgaricus, L. acidophilus, L. helveticus is 1: 0.5: 0.5: 1, respectively. The selected ratio contributes to an increase in the number of yeast fermenting lactose, to the parallel development of lactic and alcoholic fermentation, characteristic of kurung, and ensures the achievement of an optimal pH of the medium in 8-10 hours, which reduces the duration of the production of symbiotic yeast.

Next, studies were conducted to develop a liquid bacterial concentrate based on the resulting symbiotic sourdough.

To prepare the nutrient medium, curd whey was used, which is the most balanced and affordable source of carbon and nitrogen nutrition for the development of lactic acid bacteria and yeast.

To stimulate the growth of yeast, not fermenting lactose, rye flour was introduced into the nutrient medium to increase the biomass of the symbiotic yeast.

Rye flour contains all the nutrients (carbohydrates, protein, minerals, vitamins) necessary for the development of microflora of symbiotic starter culture. Carbohydrates of rye flour are composed of fiber, pentosans, hemicelluloses, starch, dextrins, maltose, glucose and other sugars. A significant part of polysaccharides and protein substances are water soluble.

By amino acid composition, rye flour proteins are characterized by a high content of essential amino acids - lysine, arginine and threonine.

For control, we used a nutrient medium with the addition of 7-10% of potato broth (prototype). In the experiment, we used a symbiotic starter culture consisting of kefir fungal starter culture, L. acidophilus, L. bulgaricus and L. helveticus, taken in the ratio 1: 0.5: 0.5: 1, respectively. The results of the study of the quantitative accounting of microorganisms of biomass obtained on nutrient media with different doses of rye flour are presented in figa and 1b.

Quantitative accounting of yeast biomass of symbiotic starter culture indicates (figa) that the introduction of rye flour stimulates the growth of yeast microflora. Thus, the introduction of (2 ± 0.5)% rye flour activates the growth of yeast that do not ferment lactose, and increases the number of cells to 1 · 10 ¹⁰ cfu / cm ³ . Active growth of thermophilic and mesophilic lactobacilli was also observed (Fig. 1b). This is probably due to the fact that some lactobacilli that are part of the formed symbiotic sourdough L.acidophilus, L.helveticus, L.casei, L.plantarum and others have maltase activity.

A further increase in the dose of rye flour to 3% has a somewhat depressing effect on the development of microorganisms of symbiotic sourdough.

The study of the morphological features of the colonies of microorganisms in experiments showed that the colloidal properties of pentosans and rye flour proteins increase the viscosity of the system and promote cohesion of microorganism cells (figure 2).

The cohesion of microbial cells is most pronounced in a nutrient medium with (2 ± 0.5)% rye flour. Figure 2c shows large accumulations of yeast and lactobacillus cells, indicating cell cohesion in the microbial consortium. It should be noted less yeast aggregation in the control (prototype) and experience with 1% rye flour (figa and fig.2b).

In a nutrient medium with 3% rye flour (Fig. 2d), small yeast cells were detected. Obviously, an increase in the dose of rye flour above 2.5% negatively affects their development, although it contributes to the aggregation of microbial consortium cells. This is probably due to the high concentration of solids in the nutrient medium and the colloidal properties of starch. The vast majority of bacteria and yeast are known to be hydrophytes.

From the data of figure 3 it is seen that the introduction of (2 ± 0.5)% rye flour provides a maximum in comparison with other experiments, the increase in optical density of 0.98 units, exceeding 0.55 units. the value of optical density in the control experiment. The yield of inoculum biomass after 22-24 hours, cultivated in a nutrient medium with (2 ± 0.5)% rye flour, was 27 g / l, in the control - 16.6 g / l.

Thus, the optimal amount of rye flour introduced into the nutrient medium is (2 ± 0.5)%, providing a high biomass yield and active growth of yeast and lactobacilli.

A microbial consortium gives certain advantages to a bacterial cell, for example, an increase in biochemical activity, and adaptation to environmental factors is ensured by mechanisms that guarantee the stability of the microbial consortium. Such mechanisms include intercellular cohesion and bacterial adhesion.

Table 2 presents the results of the study of the adhesive properties of the biomass of a microbial consortium consisting of kefir fungal yeast and thermophilic lactobacilli L.acidophilus, L.bulgaricus and L.helveticus, taken in a ratio of 1: 0.5: 0.5: 1, respectively, obtained on various nutrient media: with the addition of (2 ± 0.5)% rye flour and 7-10% of potato broth.

table 2 Influence of biomass accumulation conditions on its adhesive properties Name of experience SPA KUE,% I AM Adhesive properties On a nutrient medium with 7-10% of potato broth (prototype) 3.2 79 4.0 ± 1.5 Medium adhesive On a nutrient medium with (2 ± 0.5)% rye flour (invention) 4.6 85 5.4 ± 1.1 Highly adhesive

The data presented in table 2 indicate that the introduction of rye flour in a nutrient medium to increase the biomass of the microbial consortium increases its adhesive properties, as evidenced by the adhesion index (IAM) of 5.4, as well as the average adhesion index (SPA) of 4.6 and the participation coefficient erythrocytes in the adhesive process (CUE) 85%.

Based on these data, we can assume that the change in adhesiveness is associated with an increase in the concentration of solids of the nutrient medium and the processes of formation of the microbial consortium. The composition of the microbial consortium includes microorganisms having different temperature optima and resistance to environmental activity (pH). Despite the lack of optimal conditions, the populations of these microorganisms are actively developing in the microbial consortium being created. It is obvious that microorganisms in response to extreme cultivation conditions include evolutionarily developed mechanisms of adaptation to environmental changes that go beyond tolerance for this species.

The presence of solid particles of fiber in a nutrient medium with rye flour promotes the immobilization of microorganisms on their surfaces. As part of the aggregates, microorganisms are in close proximity and immersed in a matrix consisting of surface cell structures, exometa-bolites and the adsorbed colloidal components of rye flour. The matrix not only spatially holds microorganisms, but also binds them together, protecting against adverse physicochemical factors, providing a generally stable balanced composition of the microbial consortium.

The antibiotic activity of the liquid bacterial concentrate in relation to pathogenic and opportunistic microorganisms was determined by the method of successive dilutions by the size of the zones of growth inhibition of test cultures. Escherichia coli I ₅₃ and Shigella sonnei 2848 were used as test cultures. For the control experiment, the initial test cultures were used in hydrolyzed milk without the addition of a liquid bacterial concentrate. The research results are presented in table 3.

Table 3 Antibiotic activity of liquid bacterial concentrate for test cultures of E. coli I ₅₃ and S.sonnei 2848 The control Dilution of bacteria E. coli I ₅₃ S.sonnei 2848 E. coli I ₅₃ S.sonnei 2848 Abundant growth Abundant growth Lack of growth Growth inhibition Lack of growth Growth inhibition 1: 8 1:64 1:16 1: 128

As can be seen from the data in Table 3, the microflora of the liquid bacterial concentrate has bactericidal and bacteriostatic effects in relation to E. coli I ₅₃ and S.sonnei 2848. The bactericidal action in relation to E. coli was manifested in a 1: 8 dilution, and to S.sonnei - 1:16. The bacteriostatic effect of the concentrate was noted at dilutions of 1:64 and 1: 128, respectively.

To study the antibiotic activity of the bacterial concentrate against tubercle bacillus, Mycobacterium tuberculosis was used as a test culture with inoculation of sputum in patients with active tuberculosis on Levenshtein-Jensen medium. As control was used sowing the initial test culture for hydrolyzed milk, without adding a liquid bacterial concentrate. After exposure to the test culture, together with dilutions of the starter culture on hydrolyzed milk for 7 days, the microflora was reseeded on Wednesday for quantitative accounting of Mycobacterium tuberculosis - Levenshtein-Jensen. The results of studies of the antibiotic activity of a liquid bacterial concentrate are presented in table 4.

Table 4 Antibiotic activity of liquid bacterial concentrate to M. tuberculosis Breeding Leaven Control: (test culture) 1: 1 1: 2 1: 4 1: 8 1:16 1:32 1:64 1: 128 Abundant growth Complete lack of growth Traces Traces Traces Traces Traces The presence of test culture growth The presence of test culture growth

From the data in table 4 it is seen that when diluting a liquid bacterial concentrate and hydrolyzed milk in a 1: 1 ratio, a bactericidal effect on tuberculosis mycobacteria was observed, i.e. complete lack of growth, which was also confirmed by microcopying the drug with Tsil-Nielson staining. Before breeding 1:32, inclusive, a bacteriostatic effect is observed, then there is no significant suppression of growth.

The high adhesiveness and antibiotic activity of the liquid bacterial concentrate makes it promising for use as a probiotic product. Qualitative indicators of liquid bacterial concentrate are presented in table 5.

Table 5 Qualitative indicators of liquid bacterial concentrate The name of indicators Characteristic indicators Consistency Homogeneous, separation of serum is allowed Color Light yellow to cream with dark brown inclusions of rye flour Taste and smell Pure, sour and yeast, with a touch of rye flour Active acidity, units 6.0-6.2 The activity of fermenting milk to an acidity of the clot 120 ° T, h 10-12 Temperature at the outlet from the enterprise, ° С, no more +6 Duration of storage, months 3 The number of microorganisms, cfu / cm ³ , not less than: thermophilic lactobacilli 8 · 10 ¹¹ mesophilic lactobacilli 6 · 10 ¹⁰ lactose-free yeast 9 · 10 ¹⁰ lactose fermenting yeast 7 · 10 ⁹ Antibiotic activity, (test culture E.coli I ₅₃ ) before dilution 1:64 (S.sonnei 2848 test) before breeding 1: 128 (test culture M. tuberculosis) before breeding 1:32 Adhesive properties,% SPA 4.6 KUE 85 I AM 5.4 ± 1.1 Micro picture
Thin sticks, rounded yeast cells, solitary and / or clusters in clusters The volume of the product (cm ³ ) in which it is not allowed: BGKP (coliforms) 10 S.aureus 10 Pathogens (including salmonella) fifty Yeast, cfu / cm ³ , no more 10 Mold, cfu / cm ³ , no more 10

Summarizing the data presented in table 5, we can conclude that the liquid bacterial concentrate obtained by the claimed method contains a high number of viable cells, has antibiotic and adhesive activity, which makes it possible to use it as a biologically active food supplement.

Further studies were devoted to the study of the shelf life of a liquid symbiotic bacterial concentrate (Table 6).

Table 6 Change in the microflora composition of a liquid symbiotic bacterial concentrate during storage Shelf life, days The number of microorganisms KOE / cm ³ thermophilic lactobacilli mesophilic lactobacilli lactose-free yeast lactose fermenting yeast 0 8 · 10 ¹¹ 6 · 10 ¹⁰ 9 · 10 ¹⁰ 7 · 10 ⁹ thirty 8 · 10 ¹¹ 6 · 10 ¹⁰ 9 · 10 ¹⁰ 7 · 10 ⁹ 60 8-10 " 6 · 10 ¹⁰ 9 · 10 ¹⁰ 7 · 10 ⁹ 90 8 · 10 ¹¹ 6 · 10 ¹⁰ 9 · 10 ¹⁰ 7 · 10 ⁹ 120 6 · 10 ¹¹ 3 · 10 ¹⁰ 5 · 10 ¹⁰ 3 · 10 ⁹ 150 4 · 10 ¹¹ 3 · 10 ⁹ 2 · 10 ¹⁰ 4 · 10 ⁸ 180 5 · 10 ¹⁰ 1 · 10 ⁹ 2 · 10 ⁸ 7 · 10 ⁷

From the data of table 6 it is seen that the number of viable cells of the liquid bacterial concentrate stored at a temperature of (6-8) ° C for 120 days (4 months) corresponds to a fresh concentrate. According to the data of table 2 and figure 2, the introduction of rye flour increases the adhesion and cohesion of the microflora of the bacterial concentrate, probably associated with a high survival rate of microorganisms during long-term storage.

Further storage for 150 days leads to a decrease in the content of mesophilic lactobacilli and yeast fermenting lactose, and after 180 days of storage, the number of thermophilic lactobacilli and yeast not fermenting lactose is reduced.

Thus, the results obtained allowed us to determine the optimal shelf life of the liquid bacterial concentrate, which is four months.

The inventive method is as follows:

In clarified curd whey, rye flour is added in an amount of (2 ± 0.5)% of the volume of the nutrient medium, 1 g / l sodium trisodium citrate, 1 g / l monosubstituted potassium phosphate, 0.1 g / l magnesium sulfate, 0.5 g / l sucrose, 0.1 g / l ascorbic acid and sterilized at a temperature of (121 ± 1) ° C. The resulting nutrient medium is cooled to a temperature of (30 ± 2) ° C, 5-7% of the symbiotic starter culture obtained by culturing for 8-10 hours of kefir fungal starter culture, L. bulgaricus, L. acidophilus, L. helveticus, taken in the ratio 1: 0.5: 0.5: 1, respectively, and set the pH of the medium in the range of 6.0-6.5.

The accumulation of biomass of symbiotic sourdough is produced under conditions of periodic cultivation with a double deoxidation of the medium after 12 and 24 hours of cultivation. The cultivation duration is 22-24 hours at a temperature of (30 ± 2) ° C. The biomass is then separated from the culture fluid. The resulting cell suspension is cooled and poured under aseptic conditions into sterile bottles of 5 or 10 cm ³ , sealed, cooled to (6 ± 2) ° C and stored at this temperature for no more than four months.

The resulting liquid symbiotic bacterial concentrate is used as a biologically active food supplement.

To obtain a frozen bacterial concentrate after separation of the biomass from the culture fluid, the biomass is mixed with a protective medium in a ratio of 1: 1 and after capping it is frozen at a temperature of no higher than (-20) ° С. The composition of the protective medium used is given in table 7.

Table 7 The composition of the protective environment Protective Components The content of components, g / l Distilled water 1000 Sucrose one hundred Sodium citrate trisubstituted twenty

Adding sodium citrate to the medium creates a buffer capacity, and the protective effect of sucrose is explained by the ability to hydrate, lower the freezing point of water, and slow down the crystallization rate.

After activation of the frozen concentrate, the number of cells and milk fermentation activity were determined. The results are presented in table 8.

Table 8 Resistance of microorganisms to freezing The number of viable cells, KOU / cm ³ Fermentation activity of 1 liter of milk 0.04 cm ³ , h Indicators Lactose-Free Yeast Lactose Fermenting Yeast Mesophilic Lactobacillus Thermophilic Lactobacillus Before freezing 9 · 10 ¹⁰ 7 · 10 ⁹ 6 · 10 ¹⁰ 8 · 10 ¹¹ 10-12 After freezing 3 · 10 ⁸ 5 · 10 ⁸ 5 · 10 ⁹ 6 · 10 ¹⁰ 10-12

The data in table 8 indicate a high survival rate of cells after freezing, so lactobacilli survived 10 ¹⁰ cfu / cm ³ and yeast 10 ⁸ cfu / cm ³ .

The duration of milk fermentation after activating the frozen suspension in milk was 10-12 hours, which indicates the preservation of biochemical activity. This is probably due to the positive influence of the protective environment. The protective effect of sucrose during the freezing of bacteria is explained by their ability to hydrate, lower the freezing temperature of water and slow down its crystallization rate. The addition of sodium citrate into the medium ensures that the reaction of the medium is close to neutral or slightly acidic.

The high survival rate of probiotic bacteria after freezing is also associated with the presence of rye flour in the concentrate, which increases intercellular cohesion and adhesion. Cohesion and adhesion are evolutionarily developed mechanisms for the adaptation of microorganisms to environmental changes, allowing cells to maintain viability in extreme environmental conditions.

Based on the studies, it was found that with cryoanabiosis, the number of starter microflora cells decreases slightly and high biochemical activity remains for six months at a temperature of -18 ° C.

Example 1. Obtaining a liquid bacterial concentrate

In clarified curd whey, rye flour is added in an amount of 1.5% of the volume of the nutrient medium, 1 g / L sodium trisubstituted citric acid, 1 g / L monosubstituted potassium phosphate, 0.1 g / L magnesium sulfate, 0.5 g / L sucrose , 0.1 g / l of ascorbic acid and sterilized at a temperature of 121 ° C for 30 minutes. The resulting nutrient medium is cooled to a temperature of 30 ° C, 5% symbiotic starter culture is introduced (kefir fungal starter culture L.bulgaricus, L.acidophilus, L.helveticus, taken in a ratio of 1: 0.5: 0.5: 1, respectively, time cultivation for 10 hours at a temperature of 30 ° C). Set the pH of the medium within 6.0.

The accumulation of biomass is produced under conditions of periodic cultivation with double deoxidation of the medium after 12 and 24 hours of cultivation. Duration of cultivation 24 hours, temperature 30 ° C.

After the cultivation process, the biomass is separated from the culture fluid. The resulting suspension is cooled and poured under aseptic conditions into sterile bottles of 5 and 10 cm ³ and sealed. The shelf life of the liquid bacterial concentrate at a temperature of (6 ± 2) ° C is four months.

Example 2. Obtaining a liquid bacterial concentrate

In clarified curd whey, rye flour is added in an amount of 2% of the volume of the nutrient medium, 1 g / l sodium trisodium citrate, 1 g / l monosubstituted potassium phosphate, 0.1 g / l magnesium sulfate, 0.5 g / l sucrose, 0 , 1 g / l of ascorbic acid and sterilized at a temperature of 121 ° C for 30 minutes. The resulting nutrient medium is cooled to a temperature of 32 ° C, 7% symbiotic starter culture (kefir fungal starter culture L. cultures, L. bulgaricus, L. acidophilus, L. helveticus taken in a ratio of 1: 0.5: 0.5: 1, respectively, time cultivation for 9 hours at a temperature of 32 ° C) and set the pH of the medium within 6.0.

The accumulation of biomass is produced under conditions of periodic cultivation with double deoxidation of the medium after 12 and 24 hours of cultivation. Duration of cultivation 23 hours, temperature 32 ° C

After the cultivation process, the biomass is separated from the culture fluid. The resulting suspension is cooled and poured under aseptic conditions into sterile bottles of 5 and 10 cm ³ and sealed. The shelf life of the liquid bacterial concentrate at a temperature of (6 ± 2) ° C is four months.

Example 3. In the clarified curd whey make rye flour in an amount of 2.5% of the volume of the nutrient medium, 1 g / l sodium trisodium citrate, 1 g / l potassium phosphate monosubstituted, 0.1 g / l magnesium sulfate, 0.5 g / l sucrose, 0.1 g / l ascorbic acid and sterilized at a temperature of 121 ° C for 30 minutes. The resulting nutrient medium is cooled to a temperature of 30 ° C, 6% symbiotic starter culture is introduced (kefir fungal starter culture, L.bulgaricus, L. acidophilus and L. helveticus taken in the ratio 1: 0.5: 0.5: 1, respectively, the cultivation time 8 hours at a temperature of 30 ° C) and set the pH of the medium within 6.0.

The accumulation of biomass is produced under conditions of periodic cultivation with double deoxidation of the medium after 12 and 24 hours of cultivation. Duration of cultivation 24 hours, temperature 32 ° C.

After the cultivation process, the biomass is separated from the culture fluid. The resulting suspension is cooled and poured under aseptic conditions into sterile bottles of 5 and 10 cm ³ and sealed. The shelf life of the liquid bacterial concentrate at a temperature of (6 ± 2) ° C is four months.

Example 4. In clarified curd whey make rye flour in an amount of 2.5% of the volume of the nutrient medium, 1 g / l sodium trisodium citrate, 1 g / l monosubstituted potassium phosphate, 0.1 g / l magnesium sulfate, 0.5 g / l sucrose, 0.1 g / l ascorbic acid and sterilized at a temperature of 121 ° C for 30 minutes. The resulting nutrient medium is cooled to a temperature of 32 ° C, 7% symbiotic starter culture is introduced (kefir fungal starter culture, L. bulgaricus, L. acidophilus and L. helveticus taken in the ratio 1: 0.5: 0.5: 1, respectively, the cultivation time 8 hours at a temperature of 32 ° C) and set the pH of the medium within 6.0.

The accumulation of biomass is produced under conditions of periodic cultivation with double deoxidation of the medium after 12 and 24 hours of cultivation. Duration of cultivation 24 hours, temperature 32 ° C.

After the cultivation process, the biomass is separated from the culture fluid by centrifugation. The resulting cell suspension is mixed with a protective medium in a 1: 1 ratio, poured into 2 ml vials (2 doses) and frozen in a freezer at a temperature of minus 20 ° C. Vials with a frozen preparation are closed with rubber stoppers and aluminum caps. The shelf life of the bacterial concentrate at a temperature of minus 18 ° C is six months.

Example 5. The use of liquid bacterial concentrate as a biologically active food supplement.

The obtained liquid bacterial concentrates according to examples 1, 2 and 3 are used as a biologically active food supplement to restore the microflora of the gastrointestinal tract, relieve adverse reactions to anti-TB drugs and as an immunomodulating agent.

It is recommended to take one teaspoon in the diet of children over 5 years old 3 times a day 30 minutes before or after meals, for adults 3 times a day, one tablespoon before or after meals.

Example 6. The use of frozen bacterial concentrate as a direct starter culture.

Frozen bacterial concentrate is used as a direct starter culture for the production of a heterofermentative product - kurung. It is recommended that 4 doses of frozen bacterial concentrate (4 ml) be added to ferment 200 ml of milk. The duration of milk fermentation is 10-12 hours.

Claims (4)

1. A method for producing a bacterial concentrate, which involves preparing a nutrient medium based on cottage cheese whey, introducing a symbiotic starter culture obtained by cultivating kefir fungal starter culture and thermophilic lactobacilli Lactobacillus bulgaricus and Lactobacillus acidophilus at a temperature of (30 ± 2) ° C, accumulation of biomass, nutrient deoxidation separation of the obtained biomass and bottling, characterized in that rye flour is added to the nutrient medium in an amount of 2 ± 0.5% of the volume of the nutrient medium, in an additional symbiotic yeast Lactobacillus helveticus is additionally introduced, while the ratio of kefir fungal starter, Lactobacillus bulgaricus, Lactobacillus acidophilus and Lactobacillus helveticus is 1: 0.5: 0.5: 1, respectively, the cultivation of the symbiotic starter is carried out for 8-10 hours. 2. The use of a liquid bacterial concentrate obtained by the method according to claim 1, as a biologically active food supplement. 3. A method for producing a bacterial concentrate, which involves preparing a nutrient medium based on cottage cheese whey, introducing a symbiotic starter culture obtained by cultivating kefir fungal yeast and thermophilic lactobacilli Lactobacillus bulgaricus and Lactobacillus acidophilus at a temperature of (30 ± 2) ° C, accumulation of biomass, nutrient deoxidation , separation of the obtained biomass, mixing with a protective medium in a ratio of 1: 1, bottling and freezing, characterized in that rye flour is added to the nutrient medium in an amount of 2 0.5% of the volume of the nutrient medium, Lactobacillus helveticus is additionally introduced into the symbiotic starter, while the ratio of kefir fungal starter, Lactobacillus bulgaricus, Lactobacillus acidophilus and Lactobacillus helveticus is 1: 0.5: 0.5: 1, respectively, the cultivation of the symbiotic starter within 8-10 hours 4. The use of frozen bacterial concentrate obtained by the method according to claim 3, as a direct starter culture for the production of fermented milk heterofermented product - kurunga.

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