Method for production of bacterial concentrate of probiotic microorganisms consortium

FIELD: food industry.

SUBSTANCE: invention relates to biotechnology and may be used as a direct addition starter for probiotic sour cream preparation. The method involves preparation of a nutritional medium where one additionally introduces sodium selenite in an amount of 30-50 mcg/ml, sterilisation and cooling to 35°C, inoculum introduction into the nutritional medium; the inoculum is represented by combined starter based on Bifidobacterium bifidum 8₃, Lactococcus lactis subspecies cremoris 24₄, Propionibacterium freudenreichii subspecies shermanii AC-2503 taken at a ratio of 40:30:30, biomass propagation and separation from culture liquid. One performs mixing with a protective medium, dispensing, closuring and freezing.

EFFECT: invention allows to enhance organoleptic properties of exo-polysaccharide potential and increase storage life of the bacterial concentrate.

2 cl, 5 tbl, 8 dwg, 3 ex

The invention relates to biotechnology and can be used as a starter direct Deposit for preparation of probiotic cream.

A method of producing a bacterial concentrate, comprising preparing culture media, sterilization, cooling, making inoculum, increase biomass, branch biomass from the culture liquid, mixing with a protective environment, bottling, corking, freezing, drying and storage (see THE 9229-001-48774768-02).

The disadvantage of this method is low biochemical activity of the concentrate and the complexity of its preparation in a production environment.

The closest way to the claimed invention on the totality of symptoms is the way the bacterial concentrate, comprising preparing culture media, sterilization, cooling, making inoculum, increase biomass, branch biomass from the culture liquid, mixing with a protective environment, bottling, corking, freezing, storage (see EN NO. 2372782, AS 9/12, 20.11.2009,).

However, the disadvantage of this method is that when using this concentrate is not formed characteristic sour cream organoleptic and structural-mechanical properties and it has a short shelf life.

Object of the invention is higher is their consumer, rheological properties and shelf life of the finished product.

In addition, the creation of a consortium of probiotic microorganisms from specially selected cultures of probiotic microorganisms will increase the biochemical and functional properties of concentrates.

The technical result is ensured in the implementation of the present invention is to improve the organoleptic properties, Exo-polysaccharide potential and the decrease in hydrolytic and oxidative processes during storage of the product.

This technical result in the implementation of the invention is achieved in that in the method of preparation of the bacterial concentrate consortium probiotic microorganisms, including preparation of culture media, sterilization, cooling, making inoculum, increase biomass, branch biomass from the culture liquid, mixing with a protective environment, bottling, corking, freezing, storage, according to the invention in a nutrient medium further added sodium Selenite at doses of 30-50 mg/ml, and the supernatant using a combination of yeast-based Bifidobacterium bifidum 8₃, Lactococcus lactis subspecies cremoris 24₄Propionibacterium freudenreichii subspecies shermanii AC-2503, taken in the ratio of 40:30:30.

Distinctive features of the proposed method is optimizat what I nutrient medium through the introduction of sodium Selenite, as well as the use of specially selected strains of probiotic microorganisms, their choice of the optimal ratio and culturing conditions of probiotic microorganisms, which marked a high sensory, rheological properties, reduction of oxidative processes and the stability of the product during storage.

For the implementation of the proposed method experimental studies were performed, which were selected dose of sodium Selenite, starter cultures of probiotic microorganisms and their optimal value.

Selection of strains for the production of valuable properties is an important aspect of the development of a probiotic product. The basis of starter cultures for traditional sour cream are the lactic acid bacteria Lactococcus lactis subspecies lactis and Lactococcus lactis subspecies Cremoris. Many years of experience in selecting the sourdough microflora shows that the best combination of strains having similar activity of acid generating. Therefore, as the basis of starter culture for probiotic product along with bifidobacteria selected lactic acid bacteria of the species Lactococcus lactis subspecies Cremoris, because this kind of microorganisms that are actively developing in milk, forms a dense cream-shaped clot and being less active kislotoobrazoutei, low postconditional potential.

Owls the local cultivation of bifidobacteria with lactic acid bacteria has several advantages. Lactic acid bacteria associated dissolved oxygen in milk and thereby create conditions favorable for the growth of bifidobacteria. Proteoliticeski active strains of lactic acid bacteria break down the casein education bifidogenic factors, peptides and amino acids.

To improve functional properties and improve the consistency of the developed product in the combination starter is advisable to enter the propionic acid bacteria.

Consistency is one of the defining indicators of quality of sour cream. Structural-mechanical properties of fermented milk clots due to the colloidal state of the micelles casinocalifornia complex, denaturation of whey proteins, particle size and physical state of the fat emulsion, the synthesis of natural stabilizers microorganisms ferment.

Accumulated in the process of ripening polysaccharides combine with proteins of the milk, forming the carbohydrate-protein complexes, increasing the hydrophilicity of the bunch, and have a positive impact on the consistency of sour cream. Therefore, when selecting crops for combination starter, you must study the degree of production of polysaccharides studied strains. The results are presented in table 1.

As can be seen from the data presented, the most great capacity for the production of exopolysaccharides found strains of Propionibacterium freudenreichii subsp. shermanii AC-2503, Bifidobacterium bifidum 8₃and Lactococcus lactis subspecies cremoris 24₄.

According to modern concepts the role of natural stabilizers perform exopolysaccharides, density and water-holding capacity of clots, and thus the consistency of the product to a certain extent due to the ability of bacteria ferment to produce exopolysaccharides. Microbial polysaccharides presents a wide range of polymers, the most different in composition, structure, link type.

To obtain finished products with specified quality parameters, you must use a starter culture with a stable set of characteristics.

At the initial stage of studies have examined the biotechnological potential of different strains of Lactococcus lactis subspecies Cremoris. The results are presented in table 2.

From table 2 it is seen that the strains of L. Cremoris have a pronounced biochemical activity. Yeast have good organoleptic characteristics. Revealed the stability of the studied crops to different concentrations of bile, sodium chloride and phenol. The studied strains belong to the high adhesive, the maximum capacity for the adhesive process showed the strain of L. cremoris 24₄who has the highest ecopolitology potential.

In the next series of experiments was studied biotechnological potential of bacteria and propionic acid bacteria. The results are presented in tables 3 and 4.

From table 3 it follows that all strains of propionic acid bacteria have a high biochemical activity, as evidenced by the rapid fermentation of milk and the maximum number of viable cells 10^9-10CFU/cm³. The studied culture showed resistance to high concentrations of bile is, sodium chloride was evolved in an environment with a low pH value, which indicates high survival rate of these crops in the gastrointestinal tract of man. It should be noted that the most high biotechnological potential of P. shermanii AC-2503.

Biotechnological potential of bifidobacteria are presented in table 4.

Analysis of the results showed that the studied strains of bifidobacteria have a high biochemical activity, as evidenced by the rapid fermentation of milk and a significant number of viable cells in cultures of 10⁹CFU/cm³. Yeast-based activated cultures of bifidobacteria have good organoleptic characteristics. It is noted that the strain Century bifidum 8₃when ripening forms a dense clump in comparison with C. longum DK-100.

Based on the results of research to create a consortium of probiotic microorganisms were selected strains of Bifidobacterium bifidum 8₃, Lactococcus lactis subspecies cremoris 24₄and Propionibacterium freudenreichii subsp. shermanii AC-2503 high ecopolitology and biotechnological potential.

When creating a consortium of bifido-, lacto - propionic acid bacteria it is necessary to select the optimal parameters of cultivation, which will be of mutual stimulation, balanced growth and a stable balance of selected cultures and the preservation of the production of the valuable properties of each representative microbial Association.

The difficulty of writing consortium of starter cultures in this case is that cultures that make up the design of the leaven need different temperature Optima and have a different rate of reproduction. Given the different optimum temperature for the development of L. cremoris 24₄- 25-27°C, propionic acid bacteria - 30°C, bifidobacteria - 36-38°C, it was necessary to find conditions for culturing these microorganisms in the symbiotic yeast.

When selecting strains accounted for the stability of the technological and functional properties, such as organoleptic properties, the ability to accumulate biomass, the ability to form viscous clots through the synthesis of exopolysaccharides, and compliance with the criteria for probiotic microorganisms.

Cultures of L. cremoris, bacteria and propionic acid bacteria were cultured at 30°C, 35°C, 37°C (see Fig.1).

As can be seen from Fig.1 at an intermediate temperature 35°C the value is of specific growth rates of the studied microorganisms approaching, therefore, at this temperature will be uniform development of selected strains in the Association.

The optimal ratio of microorganisms in the consortium was chosen taking into account balanced growth of crops and organoleptic characteristics of clots. The results obtained are shown in table 5.

The data of table 5 show that the balanced growth of all microorganisms is observed when the ratio of the Century Bifidum 8₃: L. Cremoris 244 : P. Shermanii AC-2503 equal to 40:30:30. If this clot is characterized by a homogeneous moderately viscous consistency and high organoleptic characteristics. Good balance and a strong symbiotic relationship certificate is listbuy high population density cultures, which guarantees the stability of the microbial consortium.

Selected conditions of cultivation of the consortium probiotic microorganisms provide a high biochemical activity and good organoleptic characteristics of the combined starter.

Leaven is a fundamental factor in the production of sour cream. It ferment give the product the most characteristic features of the viscosity, the structure of the clot, consistency, aroma, and also determine the stability of quality indicators during storage.

The structure and texture of fermented milk products largely depend on the ability of starter cultures to produce extracellular polysaccharides.

According to literature data, synthesis and qualitative composition of exometabolites of bacteria depend on the culture conditions. It is known that the cultivation of Bifidobacterium longum VM and Propionibacterium freudenreichii subsp. shermanii strain 186 KM on a nutrient medium with the addition of sodium Selenite increases the viscosity of the culture fluid and increases the concentration of exopolysaccharides (RU # 2333655, A23C 9/12, A23L 1/304, C12N 1/18, 20.09.2008,). As for the lactococcal, such information in the literature is missing. In addition, it was found that the synthesis of biologically active substances depends on the species and strain toiletries.

So on with abuses stage investigated the effects of different doses of sodium Selenite on the biosynthesis of exopolysaccharides (EPS) probiotic microorganisms. The results are shown in Fig.2.

As can be seen from the data shown in Fig.2, the addition of sodium Selenite to the culture medium stimulates the synthesis of exopolysaccharides. The largest increase of exopolysaccharides is observed in the concentration range of sodium Selenite from 30 to 50 mcg/ml Further increase in the concentration of Selenite more than 50 μg/ml did not lead to a significant increase of exopolysaccharides. Interesting is the fact that increasing the concentration of sodium Selenite in the nutrient medium does not lead to stunted growth of microorganisms, and the number of viable cells at the end of cultivation (10¹⁰-10¹¹) CFU/cm³. This indicates the high resistance of microorganisms to sodium Selenite. Resistance to selenium bacteria may be due to the presence associated with cells of polysaccharides, which prevents the penetration of selenium in cells, which are then released into the environment. From literary sources we know about protective biological functions XPS microorganisms, which play a major protective role, because they act much faster in comparison with inducible defense systems.

As a result of the studies selected optimal dose of sodium Selenite 30-50 μg/ml, providing a high number of exopolysaccharides and active the OST of microorganisms.

Further studies have examined the growth of biomass and EPS synthesis by co-cultivation Century Bifidum 8₃, L. Cremoris 24₄and P. Shermanii AC-2503, taken in the ratio of 40:30:30 on a nutrient medium with the addition of sodium Selenite in the amount of 30 µg/ml the Results are shown in Fig.3 and 4.

As can be seen from the data shown in Fig.3, the synthesis of exopolysaccharides cultures combined fermentation begins in the early growth stages and continues throughout the cultivation period. The maximum number of exopolysaccharides noted in the stationary growth phase and is to 39.6 mg/ml

By co-cultivation of crops Century Bifidum 8₃, L. Cremoris 24₄and P. Shermanii AC-2503 on a nutrient medium with sodium Selenite marked balanced growth of microorganisms and the number of viable cells after 20 hours of cultivation is (10¹⁰-10¹¹) CFU/cm³(Fig.4).

It should be noted that the exopolysaccharides play an important role in the formation of symbiotic systems. Thus, the data available in the literature suggest that the recognition and initial contacts symbionts occur through the interaction of cell surface polysaccharides of bacteria, which provide high adaptation, physiological resistance of cells of microorganisms.

While examining the AI morphology of microorganisms under different concentrations of sodium Selenite was noted, that with increase of sodium Selenite there was an accumulation of cells (cohesion). The same dynamics with regard to the cultivation of a consortium of microorganisms (Fig.5).

The data presented in Fig.5, show that by co-cultivation of microorganisms on the environment with the sodium Selenite form micro-colonies, which ensures a high survival rate when exposed to adverse environmental factors. Probably, in terms of intercellular contacts through aggregation of cells sustain their livelihoods.

Thus, as a result of researches it is established that the introduction of the nutrient medium of sodium Selenite helps to increase the synthesis of exopolysaccharides and strengthen the symbiotic relationship between probiotic bacteria, which increases the production of valuable properties of the consortium.

To determine the dose you make frozen bacterial concentrate in the formulation of cream resulted in a series of experiments to study the biochemical activity of the concentrate. It was established that one vial containing 5 ml (5 units of activity) of the leaven of direct application, is able to ferment 200 kg cream (12-14) h at 35°C. the Frozen bacterial concentrate was used for fermentation of cream.

Sour cream is characterized choir is leading organoleptic indicators and contains a high number of viable cells of probiotic bacteria. Sour cream is structured disperse systems. A characteristic feature of sour cream is that its viscous properties undergo significant changes not only when the temperature of the product, but also when changing the gradient of the shear rate, which occurs when different types of mechanical impact on the product.

Research on effective viscosity of the destroyed structure prototype of sour cream were conducted on a rotational viscometer at 20°C. as a control sample used sour cream, fermented bacterial concentrate without the addition of sodium Selenite. The results are presented in Fig.6.

As can be seen from the data of Fig.6, the viscosity of a test sample of sour cream 15% higher than in the control sample. When increasing the rotation speed of the spindle and mechanical effects, the decrease of the effective viscosity. In the prototype sour cream viscosity loss are 60,27, in the control 73,87%.

Thus, the structure of the prototype sour cream is more resistant to mechanical stress, so the consistency of the product will be less susceptible to the influence of technological factors.

The durability of the food product during storage is an indicator of high quality. The preservation of the organoleptic characteristics, biological engineering is the first and nutritional value during a certain period of time provides the functional effectiveness of the probiotic product and its competitiveness. When storing products containing significant amounts of fat, there is a change in the lipids forming substances that reduce the nutritional value and diet quality. This should be considered when determining the shelf life of cream.

Therefore, in the next series of experiments, we have studied changes in quality characteristics of sour cream during storage. Samples of sour cream 15% fat) kept at a temperature (4±2)°C. As is known, the transformation of milk fat occurs mainly as the result of enzymatic hydrolysis and oxidation under the action of atmospheric oxygen. To determine the intensity of these processes in the samples were determined acid value and peroxide value. The results are presented in Fig.7 and 8.

As can be seen from the data of Fig.7, in the sample noted a higher rate of formation of free fatty acids and peroxides. The values of the acid number at the initial stage in the samples differ slightly, because at first there is an accumulation of free fatty acids forming the taste and aroma of sour cream. Increased hydrolysis with further increase in the concentration of free fatty acids leads to the released oxidation of unsaturated fatty acids with oxygen with the formation of ketones and aldehydes that can impair the organoleptic characteristics of the product.

C is achene peroxide value in the prototype below, than in the control (see Fig.8). This can be explained by the fact that in the prototype sour cream is a high number of viable cells of microorganisms ferment that has its own mechanism of antioxidant protection through the synthesis of enzymes and antioxidant properties of the residual amount of selenium. The collective impact of these factors leads to a decrease in the rate of oxidative processes in the prototype of sour cream.

The combination of the obtained data shows that the cream produced using bacterial concentrate, has good organoleptic properties, rheological performance, high ecopolitology potential and is characterized by increased storage stability.

The inventive method is as follows.

For the preparation of nutrient media whey lighten, make growth components and 30-50 μg/ml of sodium Selenite, sterilized, cooled, add 5% inoculum of a consortium of microorganisms consisting of Bifidobacterium bifidum 8₃, Lactococcus lactis subspecies cremoris 24₄, Propionibacterium freudenreichii subsp. shermanii AC-2503, taken in the ratio of 40:30:30, increasing biomass for 20-22 hours periodic cultivation in a single neutralization environment through 10 hours to maintain the pH at an optimum level solution places the CSOs sodium. Then the bacterial mass is separated from the culture liquid by centrifugation. The obtained cell suspension is mixed with a protective environment at a ratio of 1:1, poured into 5 ml vials, frozen at a temperature of minus 25°C, and then the vials are closed with sterile tubes and rolled metal caps. The frozen concentrate is stored up to 8 months at a temperature of minus 18°C.

Examples confirming the possibility of carrying out the invention.

Example 1. For the preparation of nutrient media whey lighten, make growth components and 30 μg/ml of sodium Selenite, sterilized, cooled to 35°C, contribute 3% inoculum of a consortium of microorganisms, comprising Century bifidum 8₃, L. cremoris 24₄, P. shermanii AC-2503, taken in the ratio of 40:30:30, increasing biomass for 20 hours under conditions of periodic cultivation in a single neutralization environment through 10 hours to maintain the pH at an optimum level with a solution of sodium carbonate. Then the bacterial mass is separated from the culture liquid by centrifugation. The obtained cell suspension is mixed with a protective environment at a ratio of 1:1, poured into 5 ml vials, frozen at a temperature of minus 25°C, and then the vials are closed with sterile tubes and rolled metal caps. Frozen conc is t store up to 8 months at a temperature of minus 18°C.

Example 2. For the preparation of nutrient media whey lighten, make growth components and 50 μg/ml of sodium Selenite, sterilized, cooled to 35°C, add 5% inoculum of a consortium of microorganisms, comprising Century bifidum 8₃, L. cremoris 24₄, P. shermanii AC-2503, taken in the ratio of 40:30:30, increasing biomass for 22 hours under conditions of periodic cultivation in a single neutralization environment through 10 hours to maintain the pH at an optimum level with a solution of sodium carbonate. Then the bacterial mass is separated from the culture liquid by centrifugation. The obtained cell suspension is mixed with a protective environment at a ratio of 1:1, poured into 5 ml vials, frozen at a temperature of minus 25°C, and then the vials are closed with sterile tubes and rolled metal caps. The frozen concentrate is stored up to 8 months at a temperature of minus 18°C.

Example 3. For the preparation of sour cream to normalize the mass fraction of fat of 15%, then pasteurized at a temperature of 95°C with a holding time of 5-10 minutes, homogenized at a pressure of 8.12 MPa, cooled to a temperature of 35°C, fermented frozen bacterial concentrate at the rate of 5% activity (5 cm³200 l cream, stirred for 10-15 min and left for fermentation for the formation of a clot acidity 55-6°T. The duration of fermentation is 10-12 hours. After fermentation, the product is cooled to 15-20°C., mixed, packaged, sent to the cooling and maturation, storage at t=(4±2)°C for not more than 10 days.

1. A method of obtaining a bacterial concentrate consortium probiotic microorganisms, including preparation of culture media, sterilization, cooling, making inoculum, increase biomass, branch biomass from the culture liquid, mixing with a protective environment at a ratio of 1:1, bottling, corking, freezing, characterized in that the culture medium was additionally contribute sodium Selenite at doses of 30-50 mg/ml, and the supernatant using a combination of yeast-based Bifidobacterium bifidum 8₃, Lactococcus lactis subspecies cremoris 24₄, Propionibacterium freudenreichii subspecies shermanii AC-2503, taken in the ratio of 40:30:30.

2. The method according to p. 1, characterized in that the cultivation supernatant is carried out at 35°C.