Method of obtaining bacterial concentrate of bifidobacteria in liquid form
IPC classes for russian patent Method of obtaining bacterial concentrate of bifidobacteria in liquid form (RU 2540022): C12N1/20 - Bacteria; Culture media therefor A61K35/74 - Bacteria A23C9/12 - Fermented milk preparations; Treatment using micro-organisms or enzymes (whey preparations A23C0021000000) Another patents in same IPC classes: Barley-and-milk starter preparation method / 2540015 Invention relates to a starter preparation method. 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Claimed is strain of Lactobacillus delbrueckii subspecies lactis CNCM I-3741, reducing content of cholesterol in blood. Strain is applied for obtaining fermented dairy products.	FIELD: chemistry. SUBSTANCE: invention relates to biotechnology and medical industry and can be used in production of bacterial concentrates, biologically active food additives, fermented food products. Method of obtaining bacterial concentrate of bifidobacteria in liquid form includes preparation of nutritional medium with addition of growth components based on clarified cottage cheese whey or on water base with addition of up to 1.5% of glucose, or on soybean whey with addition of lactose in amount 1%. Strain of bifidobacteria B. bifidum 83, activated with β-halactosidase, is introduced into prepared nutritional medium in amount 3-5%, biomass is grown, cooled, poured in containers. EFFECT: invention makes it possible to increase manufacturability and intensity of the process, increase biochemical activity of bifidobacteria and consumer properties of obtained product. 7 tbl, 5 dwg, 4 ex   The present invention relates to biotechnology, food processing and medical industry and can be used in the production of bacterial concentrates, biologically active additives to food, fermented food products. A known method of producing a liquid concentrate of bifidobacteria, providing for the preparation of the nutrient medium, the introduction of seed, cultivation of biomass of bifidobacteria, cooling and packing of liquid product (see EN NO. 2326940, C12N 1/20, AK 35/74, A23C 9/12, 12.10.2007). The disadvantage of this method is the difficulty of preparation of the nutrient medium, multiple transplants to obtain production of the leaven and the low activity of the fermentation of milk in obtaining milk product. The closest in technical essence of the present invention is a method of obtaining a bacterial concentrate bifidobacteria in liquid form, providing for the preparation of the nutrient medium, the introduction of inoculum, increase biomass, bottling, capping (see EN No. 2373277, CL. A23C 9/12, 2006). The disadvantage of this method is low biochemical activity of the inoculum and the associated multiple subcultures to obtain production of the leaven, which complicates the process. In addition, the high content of NaCl in the nutrient environment etc which leads to deterioration of consumer properties of the finished product. The technical result of the invention is to improve the biochemical activity of bifidobacteria and consumer properties of the finished product. This technical result in the implementation of the invention is achieved in that in the method of obtaining a bacterial concentrate bifidobacteria in liquid form, providing for the preparation of the nutrient medium with the addition of growth components, the introduction of inoculum, increase biomass, cooling, bottling, according to the invention a nutrient medium is prepared on the basis of the clarified cheese whey or water-based with the addition of glucose to 1.5%, or soy whey with the addition of lactose in the amount of 1%, and as the inoculum used activated β-galactosidase strain of Bifidobacterium Bifidobacterium bifidum 83in the amount of 3-5%. Distinctive features of the claimed invention is new culturing conditions, using inoculum of bacteria with high biochemical activity and optimization of nutrient media on the basis of cheese whey, soy whey and water framework using glucose and lactose as a source of fermentation. To implement the proposed method of obtaining concentrate experimental studies were performed on the selection and optimization of nourishing the environment and to study the effect of carbohydrate composition on the qualitative characteristics of the concentrate. When creating concentrates bifidobacteria it is necessary to select conditions for the cultivation of bifidobacteria, which provide inoculum with high biochemical activity. It is known that an important role in the cultivation of microorganisms is the activity of a seed. Previously we have developed an efficient biotechnological method of activation of bifidobacteria for obtaining an active inoculum (see EN NO. 997643, A23C 9/12, 23.02.1983). However, our studies showed that not all species and strains of bifidobacteria are activated by our method. To activate the cultures of bifidobacteria used pasteurized skim milk, made the drug yeast β-galactosidase (Maxilact L 2000) at 2 u/ml and kept in the incubator for 2 hours for reaction of transglycosylase. The milk is then at 120°C with a holding time of 10-15 min, made dry preparations of different strains of bifidobacteria obtained from the FSUE "State Diagenetic", and left to ferment. About biotech activity of bifidobacteria judged by titrated and active acidity, the number of viable one. The research results are summarized in table 1. Table 1 - Influence of processing milk β-galactosidase on the biochemical activity of bifidobacteria.
Name strain	Types of generations	Duration the fermenting, h	Titratable acidity, T°	Active acidity, pH	The number of viable cells CFU/cm3
1	2	3	4	5	6
Bifidobacterium. Bifidum 83	I	40±1	4	5,11±0,02	2*108
II	19±1	44	to 5.21±0,01	8*109
III	16±1	54	5,06±0,03	2*109
Bifidobacteriu m adolescentis DSM 20083			60
I	21±1	51	the 5.25±0,01	1*109
II	16±1	54	to 5.21±0,01	3*109
III	12±1	60	5,06±0,03	6*109
Bifidobacterium adolescentis Goa	I	-
Bifidobacterium adolescentis 415	I	-

Note: I and II generation - milk treated with β-galactosidase,

II generation with milk or without enzyme.

Analysis of the data table. 1 shows that the most actively developing Bifidobacterium adolescentis DSM 20083, as evidenced by the lowest duration of fermentation of milk. Interesting is the fact that other strains of Bifidobacterium adolescentis not activated enzyme preparation of β-galactosidase, because during prolonged cultivation in milk is not the way the tsya clot.

It should be noted that activated culture of bifidobacteria Century bifidum 8₃in the second generation is characterized by high biochemical activity, and in the third generation is able to actively ferment the milk without the addition of β-galactosidase.

Thus, activation of bifidobacteria enzyme preparation of β-galactosidase depends on the species and strain toiletries.

Teoreticheskoi justification mechanism stimulating action of β-galactosidase on the growth of bifidobacteria are presented in the monograph (Hamagaeva I. S. Scientific basis of biotechnology of dairy products for children and diet. The monograph.- Ulan-Ude, in SGTU, 2005, 279 S.).

In further studies we used activated culture of Bifidobacterium Bifidobacterium bifidum 8₃as it is typical for the gastro - intestinal tract of people of different age groups, children and adults, whereas Bifidobacterium adolescentis mainly prevalent in the elderly (see Shenderov B. A. Medical microbial ecology and functional nutrition. So III: Probiotics and functional food, M., Publishing house "Grant", 2001, 288 S.).

Description inoculum of bifidobacteria fat-free milk are presented in table. 2.

As can be seen from the table.2, the inoculum of B. bifidum Bifidobacterium 8₃has a high bio is himicheskoi activity and contains 10 ⁹SOME 1 cm³.

Table 2. - Characteristics of inoculum bifidobacteria.

Further studies explored the influence of the dose of the inoculum on the growth of biomass of bifidobacteria (table.3).

From the data presented in table.3, it is seen that with increasing dose inoculum is more intensive growth of bifidobacteria. It should be noted that the optimal dose of inoculum is 3-5%, while the number of viable cells by 24 hours of cultivation is 10¹¹CFU/cm³that provides high activity of the concentrate. Increase the dose of inoculum to 7% impractical from an economic point of view, as a slight increase of viable cells requires undue cost for the preparation of inoculum.

It should be noted that for the cultivation of bifidobacteria and the floor is possible drugs are used to different environments, this environment Blaurock (see Goncharov, I. methods of cultivation of bifidobacteria. Laboratory business. 1969), casein-yeast and has received widespread hydrolysate-dairy environment (see, for example, RU # 2052253, A23C 9/12, 20.01.1996). But despite the complexity of making the environment and the duration of the passages in the environment, accumulation of liquid concentrate contains bifidobacteria 10⁹CFU/ml and has a low biochemical activity, not fermented milk food and environment. It must be emphasized that the concentrates include preparations containing the cells is not less than 10¹⁰SOME cm³.

Thus, in the production of bacterial concentrates the main task is the selection of the optimal culture conditions for the growth of microbial mass and receiving the finished products with high number of viable cells of Bifidobacterium and ensure the activity of the fermentation of milk and food environments.

Further research to obtain the biomass of probiotic microorganisms previously developed a nutrient medium on the basis of whey (see EN 2129794 C1. Hamagaeva I. C. a method of obtaining a dry product for the production of dairy products, 10.05.1999).

The growth of biomass and the number of viable cells of Bifidobacterium studied in nutrient medium on the basis of the whey when making 5 inoculum activated Century culture bifidum 8₃(Fig. 1, 2).

It is evident from Fig. 1 shows that when you make 5% inoculum in a nutrient medium, an increasing biomass of bifidobacteria Century bifidum 8_3,as evidenced by data on the change in optical density and biomass yield.

The same behaviour is observed for the number of viable cells (Fig.2). It should be noted thatafter 20-24 hours of cultivation, the number of viable cells of Bifidobacterium reaches 10¹⁰-10¹¹SOME 1 cm³(Fig.2).

The obtained results show that the use of inactivated cultures of bifidobacteria as inoculum intensifies the growth of biomass and provides a high number of viable cells in the concentrate. Characteristics of the concentrate are presented in table 4.

Organoleptic characteristics of the concentrate of bifidobacteria must meet the requirements specified in table 4.

By now accumulated a significant number is the number of data about the existence of the category of children and adults with disorders of carbohydrate metabolism, manifested in lactose intolerance - lactose, caused by the absence or reduced activity of lactase in the intestinal mucosal. Lactase deficiency is also associated with dysbiosis, preventing the formation of normal biocenosis in the gut. This demonstrates the feasibility of creating new, more efficient and versatile dairy products for children and adults.

We have investigated the possibility of application of the nutrient medium prepared on the basis of water with the addition of glucose and all other components in the production of the bacterial concentrate bifidobacteria.

In the preparation of the nutrient medium used distilled water. Glucose in varying amounts was added to the prepared nutrient medium, i.e. in the environment, prepared with water with the addition of all components, as in the production of concentrate based on acid whey. The quantity of glucose was 1%, 1.5% and 2%. Control served bacterial concentrate bifidobacteria based on acid whey. To assess the effect of glucose on the growth of bifidobacteria compared the average specific growth rate of the cultures (µ_cf), calculated on a segment of the exponential phase of bacterial growth at a temperature of cultivation at 37°C. the Results are presented in Fig.3.

With the according to the data obtained Fig.3 the maximum growth rate was achieved by adding to the culture medium of glucose in the amount of 1.5%.

About the activity of biochemical processes were tried and the number of viable cells of Bifidobacterium, calculated at the end of the fermentation process. The results are shown in Fig.4.

As can be seen from Fig.4, adding glucose to the culture medium in an amount up to 1.5% of the cell growth averaged 4·10¹²that is one order higher than in the medium with added glucose 1%, and approximately the same environment with the addition of 2% glucose.

As a result of experiments it was found that when introduced into the nutrient medium glucose 1.5% of intensified growth of bifidobacteria, and observed the dynamic growth of the number of viable cells (up to 10¹²). Further increase in the dose of glucose does not result in a significant increase in viable cells of Bifidobacterium.

Qualitative characterization of the developed bacterial concentrate bifidobacteria water-based are presented in table 5.

Received data which allow us to conclude about the possible use in the production of concentrate of bifidobacteria environment water-based with the addition of glucose.

Epidemiological studies conducted in different countries have shown that good nutrition, a balanced basic nutrients, vitamins, micronutrients, and also includes products with antioxidant effect, can greatly reduce the content of cholesterol in blood and mortality from cardiovascular disease. One of such products which are of great interest, is soy.

Russia is one of the last places in the world for the cultivation of soybeans, which, undoubtedly, is currently becoming a serious problem, and on the other unfavorable structure of the protein nutrition of the population indicates the need to increase the use of soy protein products directly in human nutrition, and food industries.

Wide applicability of soy products in the treatment of diseases of the digestive system associated primarily with dietary restrictions, as well as numerous favorable effects of soy products on the digestive system. Clinical studies have proven therapeutic effectiveness of soy food additives at various diseases of the digestive system. Consumption of soy products allows you to adjust the protein component of the diet, increase in stravitelne processes, normalizes the motor activity of the gastrointestinal tract, has a positive effect on the immune system.

We have studied the possibility of using soy whey in the preparation of nutrient medium for bacterial production of concentrate of bifidobacteria.

For the preparation of bacterial concentrate of soy whey was prepared soy milk, i.e. on 1 l of water was added to 90 g of soy flour and boiled for 10 minutes. In soy milk was added magnesium chloride and separating the serum from the clot. Next was prepared 3 prototype of a nutrient medium with different content of soy whey. In the first sample, the ratio of soy whey and water was 30%:70%; in the second sample - 50%:50% and in the third sample the content of the soy whey was 100%.

In each experimental sample was made growth components as the production of a concentrate of bifidobacteria based on acid whey. Control served bacterial concentrate bifidobacteria based on acid whey. To assess the impact of soy on serum growth of bifidobacteria compared the average specific growth rate of the cultures (µ_cf.), calculated on a segment of the exponential phase of bacterial growth at a temperature of cultivation at 37°C. the Results are presented in Fig.5.

According to the data obtained maximum soon the th growth was achieved in the preparation of the concentrate on the environment, containing 100% soy whey.

About the activity of biochemical processes were tried and the number of viable cells of Bifidobacterium, calculated at the end of the fermentation process. The results are shown in Fig.6.

As can be seen in Fig.6, in the preparation of the concentrate in a medium containing 100% soy serum, cell growth is 2·10¹²that is one order higher than in the medium with soy whey lower concentrations and is about the same with the environment based on acid whey.

Next were selected optimal concentration of the components contained in the environment. Selection of the optimal dose of lactose is shown in Fig.7.

According to the data obtained maximum growth rate was achieved in the preparation of the concentrate in a medium containing 1% lactose. The number of viable cells of Bifidobacterium, calculated at the end of fermentation, also in this sample reached the maximum number (3·10¹²).

The data obtained allow us to conclude about the possible use in the production of concentrate of bifidobacteria environment of soy whey with added lactose.

Qualitative characterization of the developed bacterial concentrate bifidobacteria soy-based

presented in table 6.

The data obtained allow us to conclude about the possible use in the production of concentrate of bifidobacteria environment soy-based with the addition of lactose.

Summarizing the obtained results, we can conclude that the use of a fundamentally new biotechnological approaches to obtaining the inoculum, consisting in the preliminary activation of the strain of bifidobacteria Century bifidum 8₃the enzyme preparation of the yeast β-galactosidase, provides a high biochemical activity and improves consumer properties of bacterial concentrates.

The use of active inoculum of bacteria allows to obtain bacterial concentrates on nutrient media on the basis of the clarified cheese whey, water-based with the addition of glucose, soy whey with the addition of lactose to achieve the above technical result.

Thus, the developed bacterial concentrates on different nutrient media have a high activity of the fermentation of milk and food environments contain 10¹⁰-10 ¹²CFU/cm³viable cells of Bifidobacterium and provide high consumer properties of the finished product.

Comparative analysis of the present invention with the prototype are presented in table 7.

The main advantages of the proposed method.

1. High biochemical activity of bifidobacteria caused by the use of inoculum activated strain Century bifidum 8₃that provides 10¹⁰-10¹²SOME 1 cm³and increase the usefulness and probiotic properties of dietary SUPPLEMENTS.

2. Liquid concentrated starter culture of bacteria can be used as starter cultures for direct Deposit, as they actively fermented milk (3 UNITS activity sours 300 liters of milk).

3. The use of cheaper nutrient medium, where the basis is whey, water, soy whey, whereas in the prototype is used gidrolizirovanny milk.

4. Technology and the intensification of the production process (absence of numerous passages in a nutrient medium in the preparation of the BAA).

The inventive method is as follows.

When receiving a liquid concentrate of bifidobacteria environment to increase bifidobacteria is clarified cheese whey, or nutrient medium is water-based with the addition of up to 1.5% glucose, or serum with the addition of lactose in the amount of 1%.

The basis of the nutrient medium contribute the growth of the new components, the pH of the medium is set in the range of 7.0±0,1). Prepared medium is sterilized at t=121°C for 30 min, then cooled to a temperature (37±1)°C and contribute inoculate - activated β-galactosidase culture of Bifidobacterium Bifidobacterium bifidum 8₃in the amount of 3-5% and increasing biomass periodic culturing for (24±2) h at a single neutralization after 12 hours of intense sterile solution of sodium carbonate (Na₂CO₃). After completion of the cultivation process separating the upper layer of the culture fluid, the bacterial suspension is cooled to (4±2)°C, poured into aseptic bottles with a capacity of 10-12 ml, corked and labelled.

Examples confirming the possibility of carrying out the invention.

Example 1. When receiving a liquid concentrate of bifidobacteria environment to increase bifidobacteria is clarified cheese whey. Clarified cheese whey rascist to pH 6,5-7,0. In the prepared serum make magnesium chloride is 0.4%, the buffer salt is 0.3%, peptone - 1%, agar-agar - 0,75%, pH of the medium is set in the range of 7.0±0,1). Prepared medium is sterilized at t=121°C for 30 min, then cooled to a temperature (37±1)°C and contribute inoculate - activated β-galactosidase of Bifidobacterium bifidum 8₃in the amount of 3% and increasing biomass in conditions of periodic cultivated the I for (24±2) h at a single neutralization after 12 hours of intense sterile solution of sodium carbonate (Na ₂CO₃). After completion of the cultivation process separating the upper layer of the culture fluid, the bacterial suspension is cooled to (4±2)°C, poured into aseptic bottles with a capacity of 10-12 ml, corked and labelled.

Example 2. A nutrient medium is prepared soy-based.

In soy whey make lactose - 1%, MgCl₂to 0.3%, the buffer salt is 1.5%, ascorbic acid and 0.1%, peptone - 5%, agar microbiological and 0.8%, pH of the medium is set in the range of 7.0±0,1). Prepared medium is sterilized at t=121°C for 30 min, then cooled to a temperature (37±1)°C and contribute inoculate - activated β-galactosidase strain of Bifidobacterium Bifidobacterium bifidum 8₃in the amount of 5% and increasing biomass periodic culturing for (24±2) h at a single neutralization after 12 hours of intense sterile solution of sodium carbonate (Na₂CO₃). After completion of the cultivation process separating the upper layer of the culture fluid, the bacterial suspension is cooled to (4±2)°C, poured into aseptic bottles with a capacity of 10-12 ml, corked and labelled.

Example 3. When receiving a liquid concentrate of bifidobacteria environment to increase bifidobacteria is clarified cheese whey. Clarified cheese whey rascist to pH 6,5-7,0. In the prepared serum in osat magnesium chloride - 0,4%, the buffer salt is 0.3%, peptone - 1%, agar-agar - 0,75%, pH of the medium is set in the range of 7.0±0,1). Prepared medium is sterilized at t=121°C for 30 min, then cooled to a temperature (37±1)°C and contribute inoculate - activated β-galactosidase of Bifidobacterium bifidum 8₃in the amount of 5% and increasing biomass periodic culturing for (24±2) h at a single neutralization after 12 hours of intense sterile solution of sodium carbonate (Na₂CO₃). After completion of the cultivation process separating the upper layer of the culture fluid, the bacterial suspension is cooled to (4±2)°C, poured into aseptic bottles with a capacity of 10-12 ml, corked and labelled.

Example 4. A nutrient medium is prepared, water-based.

In the water make glucose and 1.5%, the buffer salt is 0.1% ascorbic acid and 0.1%, peptone - 5%, microbiological agar and 1.5%, pH of the medium is set in the range of 7.0±0,1). Prepared medium is sterilized at t=121°C for 30 min, then cooled to a temperature (37±1)°C and contribute inoculate - activated β-galactosidase strain Bifidobacterium bifidum 8₃in the amount of 5% and increasing biomass periodic culturing for (24±2) h at a single neutralization after 12 hours of intense sterile solution of sodium carbonate (Na₂CO₃). After the process is finished, the cult is growing separating the upper layer of the culture fluid, the bacterial suspension is cooled to (4±2)°C, poured into aseptic bottles with a capacity of 10-12 ml, corked and labelled.

A method of obtaining a bacterial concentrate bifidobacteria in liquid form, providing for the preparation of the nutrient medium with the addition of growth components, the introduction of inoculum, increase biomass, cooling, bottling, wherein the nutrient medium is prepared on the basis of the clarified cheese whey or water-based with the addition of up to 1.5% glucose, or soy whey with the addition of lactose in the amount of 1%, and as the inoculum used activated β-galactosidase strain of Bifidobacterium Bifidobacterium bifidum 8₃in the amount of 3-5%.