RU2372782C1 - Method for production of frozen concentrated leaven based on symbiosis of probiotical bacteria - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: production method of frozen concentrated leaven based on symbiosis of probiotical bacteria provides for preparation of nutrient medium based on enlightened curdy whey with addition of cobalt chloride in quantity (0.1-0.2) mg/ml. Combined culture starter in quantity (3-5)% consisting of bifidobacteria and propionate bacteria taken in ratio 1:1 separately activated β-galactosidase is added to cooled ((34±1)°C ) medium. After that cell growing is performed, bacterial mass is separated from cultural medium, mixed with protecting medium, poured and frozen.

EFFECT: this provides increased probiotical properties of starting culture, increased quantity of bifidobacteria and propionate bacteria cells and high activity of milk fermentation.

4 cl, 4 dwg, 5 tbl, 3 ex

Description

The present invention relates to the microbiological industry, biotechnology and can be used for the preparation of dairy products, as well as biologically active food additives based on biomass of probiotic microorganisms.

It is known that obtaining biological products is one of the difficult areas in biotechnology. The most promising in technological and economic terms is the use of concentrated preparations obtained on nutrient media, which make it possible to obtain crops with desired properties. The use of frozen concentrated starter cultures allows you to increase the volume of production, simplify the process, reduce its duration and improve the sanitary and hygienic indicators of the finished product.

A known method of producing a bacterial concentrate of bifidobacteria for the production of fermented milk products, providing for the preparation of a nutrient medium based on clarified whey and growth components, the introduction of a bifidobacteria inoculum, cultivation, separation of the obtained biomass, mixing with a protective environment, freezing [SU No. 1686718 A1, MKI A23C 9 / 12, publ. October 20, 1993].

The disadvantage of this method is the long-term cultivation of bifidobacteria, which is (32-40) hours, while the number of microorganisms in the finished sourdough reaches only 10 ^8-9 CFU / cm ³ .

The closest way to the claimed invention in terms of features is a method for producing a bacterial concentrate of propionic acid bacteria, which involves the preparation of a nutrient medium based on clarified whey and growth components, with the addition of cobalt chloride to increase the vitamin-forming ability of propionic acid bacteria, the introduction of activated β-galactosidase cultures of propionic acid bacteria Propionibacterium shermanii MSU strain, cell extension, separation of the obtained bio aces, mixing with protective medium, freezing [RU №2309982 C2 S12N 1/20, 9/12 A23S, S12R 1/15, publ. November 10, 2007 Byul. No. 31].

However, the use of monocultures of probiotic microorganisms reduces the biotechnological and probiotic properties of the concentrate.

The objective of the present invention is to provide a concentrated starter culture based on the symbiosis of bifidobacteria and propionic acid bacteria with pronounced biotechnological and probiotic properties. Concentrated starter culture can be used to develop new probiotic preparations recommended for the effective prevention of violations of the microbiocenosis of the gastrointestinal tract and the prevention of dysbiosis of various etiologies.

The creation of a symbiosis of probiotic microorganisms is primarily associated with a number of its undoubted advantages in comparison with monocultures. Symbiosis of cultures has a wider range of transformable substances and is more technologically advanced in practical use.

The technical result provided by the implementation of the invention is to increase the probiotic properties of concentrated starter culture, increase the number of bifidobacteria and propionic acid bacteria cells and ensure high activity of milk fermentation.

The specified technical result in the implementation of the invention is achieved by the fact that in the method of preparing concentrated starter culture, including the preparation of a nutrient medium, the introduction of the inoculum in an amount of (3-5)%, cell growth, separation of the bacterial mass from the culture medium, mixing it with a protective medium, filling and freezing, according to the invention, as a inoculum for fermentation of a nutrient medium, a combined sourdough is used consisting of separately activated cultures of bifidobacteria and propiono acid bacteria, while cobalt chloride is introduced in an amount of (0.1-0.2) mg / ml.

Distinctive features of the proposed method are the new conditions for inoculum cultivation, namely the use as a inoculum of a combined starter culture consisting of separately activated β-galactosidase cultures of bifidobacteria and propionic acid bacteria, which allows to realize to the maximum extent the physiological, biochemical and technological potential of microorganisms.

To implement the proposed method for producing concentrated starter culture, experimental studies were conducted, during which the optimal temperature conditions for the balanced development of microflora of combined starter culture were selected, the ratio of cultures was selected, and conditions for the accumulation of biomass with a high titer of probiotic bacteria were determined.

The authors investigated the effect of different temperature parameters of fermentation on the growth rate of bifidobacteria and propionic acid bacteria. The results of the experiment are presented in figure 1. Analysis of the data of figure 1 showed that a change in the optimal temperature of the cultivation of bacteria leads to a decrease in their growth rate. Whereas at an intermediate temperature of 34 ° C there is a uniform development of microorganisms, the values of the average specific growth rate of bifidobacteria and propionic acid bacteria are approaching at this temperature. In this regard, for the joint cultivation of probiotic microorganisms, the temperature regime of fermentation (34 ± 1) ° С was chosen.

The selection of the optimal ratio of microorganisms in the combined starter culture was carried out taking into account the biotechnological properties of the studied cultures and their associations. The data obtained are shown in table 1.

As can be seen from table 1, all the studied associations of microorganisms have good organoleptic and biotechnological properties. With the joint cultivation of propionic acid and bifidobacteria, the number of viable cells is an order of magnitude higher than that of individual cultures, the content of VFA and vitamin B ₁₂ increases, which indicates a symbiotic relationship between microorganisms. As a result of the experiment, it was also established that the sample with a crop ratio of 1: 2 has the most viscous consistency and the greatest water-holding ability, but at the same time, a more acidic taste is observed, which is explained by a high content of volatile fatty acids, a more intense clot formation of 6 hours.

Variants of the ratio of cultures of bifidobacteria and propionic acid bacteria 2: 1, 3: 1 in the combined sourdough have good organoleptic characteristics, contain a slightly lower amount of VFA, CO ₂ and a longer duration of milk fermentation (7.0-7.5) hours. This is due to the fact that the culture of bifidobacteria predominates in the starter cultures, which is characterized by a longer duration of milk fermentation compared with propionic acid bacteria at a cultivation temperature of 34 ° C.

Data on the study of the accumulation of vitamin B _{12 by the} microflora of starter cultures indicate that propionic acid bacteria synthesize a significant amount of vitamin B ₁₂ and its content in the starter culture is 38 μg / ml. It was found that in the combined starter cultures, the amount of vitamin B ₁₂ increases. This can be explained by the beneficial effect of metabolites secreted by bifidobacteria in the logarithmic phase of growth - amino acids, vitamins, peptides on the synthesis of vitamin B ₁₂ . It is known that amino acids, especially arginine and histidine, have the most stimulating effect on the synthesis of cobalamin.

The greatest amount of vitamin B _{12 was} found in the combined starter culture with a ratio of 1: 2 bifido and propionic acid bacteria, which is 53 μg / ml.

Reducing the dose of propionic acid bacteria in combination starter cultures contributes to a proportional decrease in vitamin B ₁₂ .

Thus, the optimal combination in the combined starter culture of bifidobacteria and propionic acid bacteria 1: 1 was selected taking into account the balanced content of viable cells of these cultures and the organoleptic properties of the resulting combined starter culture. This sourdough tastes better, it also has a moderate acidity of 68 ° T, an acceptable duration of fermentation of milk 6.5 hours, a high content of volatile fatty acids, vitamin B ₁₂ and good rheological indicators.

The next stage of research is devoted to the study of the probiotic properties of the resulting combined starter culture.

The mechanism of action of propionic acid and bifidobacteria is multifactorial. The metabolites of bacterial cells have a positive effect on the macroorganism by the complex of biologically active substances formed by them. The most important of these are antimutagenic and antimicrobial substances.

The data in table 2 indicate that the synthesis of antimicrobial substances and antimutagenic substances by probiotic bacteria is enhanced in symbiotic relationships.

From literary sources it is known that exopolysaccharides (EPS) not only improve the rheological properties of dairy products, but also act as factors contributing to the adhesion of beneficial microorganisms on the intestinal walls. Therefore, in further studies, the biosynthesis of EPS by propionic acid, bifidobacteria and their association was studied. The results are presented in figure 2.

It should be noted a higher synthesis of EPS in propionic acid bacteria, but when co-cultured with bifidobacteria, the number of exopolysaccharides decreases. It is known that an excess of a carbon source in the medium has a stimulating effect on the yield of EPS. During fermentation, the basic metabolic functions of cells undergo a complex regulatory system that controls the digestibility of lactose, the main source of carbon and energy for bacteria. The differences in the physiology of propionic acid and bifidobacteria, manifested during separate and joint cultivation during lactose metabolism, can explain the different yield of exopolysaccharides. In addition, from literature it is known that exopolysaccharides have bifidogenic properties. It is likely that EPS synthesized by propionic acid bacteria stimulate the growth of bifidobacteria, as evidenced by the high number of viable bifidobacteria cells in the combined starter culture.

As a result of the studies, it was found that with the joint cultivation of microorganisms, the antimutagenic properties increase and the antimicrobial activity of the combined starter culture is enhanced. The exopolysaccharides formed by bacteria enhance the probiotic effect of the combined starter culture.

Further, the authors' studies were aimed at creating a concentrated starter culture of bifidobacteria and propionic acid bacteria for direct introduction into milk.

An analysis of the technology for producing concentrated starter cultures showed that the most important stage is the accumulation of biomass of microorganisms, the effectiveness of which depends more on the cultivation conditions and the activity of the seed.

In this regard, at the first stage, the biochemical activity of the combined starter culture of propionic acid and bifidobacteria was studied. The results are presented in table 3.

Table 3 Qualitative characteristics of the inoculum Indicators Characteristic Fermentation activity, h 14-16 Acidity: titratable, ° T 73 ± 2 active pH 4.63-4.58 The number of viable cells, CFU / cm ³ bifidobacteria 4 · 10 ¹⁰ propionic acid bacteria 5 · 10 ¹⁰ The volume of the product (cm ³ ) in which it is not allowed: BGKP (coliforms) 10 S. aureus 10 Pathogens (including salmonella) one hundred Yeast, CFU / cm ³ , no more 5 Mold, CFU / cm ³ , not more than 5

As can be seen from the data obtained, the combined starter culture has a sufficiently high enzymatic activity, moderate acidity and a high content of viable cells of starter cultures.

Propionic acid bacteria are active producers of vitamin B ₁₂ . It is known that cobalt increases the vitamin synthesizing ability of propionic acid bacteria, however, its content in natural media is minimal, therefore, cobalt chloride was included in the composition of the nutrient medium.

The authors studied the effect of cobalt ions on the yield of biomass and on the synthesis of vitamin B ₁₂ . The fermentation process was controlled by the optical density of the biomass and the number of viable cells counted at the end of the fermentation process. According to these indicators, the average specific growth rate of bacteria was found. Cobalt chloride was added in an amount of 0.05; 0.1; 0.2; 0.3 mg / l. As a control, a medium that does not contain cobalt ions. The research results are presented in figure 3 and 4 and in table 4.

From figure 3 it is seen that cobalt increases the biosynthesis of vitamin B ₁₂ . With an increase in the concentration of cobalt ions, there is a greater accumulation of vitamin. It should be noted that in the proposed method it was experimentally established that with the joint development of bifidobacteria and propionic acid bacteria, a certain amount of cobalt not only controls the formation of cobalamin, but also serves as a growth factor compared to the prototype (figure 4). In the prototype, the introduction of cobalt ions into the nutrient medium increases the synthesis of vitamin B _{12 by} propionic acid bacteria, but the cell growth rate decreases.

Table 4 The amount of viable microflora depending on the content of cobalt ions in the nutrient medium Concentration of CoCl ₂ , mg / ml The number of viable cells, KOU / ml B. longum B379M R. shermanii KM 186 Combined Ferment B. longum B379M R. shermanii KM186 0 7 · 10 ¹¹ 6 · 10 ¹¹ 3 · 10 ¹² 4 · 10 ¹² 0.05 3 · 10 ¹¹ 5 · 10 ¹¹ 3 · 10 ¹² 5 · 10 ¹² 0.1 6 · 10 ¹⁰ 2 · 10 ¹¹ 6 · 10 ¹² 8 · 10 ¹² 0.2 1 · 10 ¹⁰ 8 · 10 ¹⁰ 5 · 10 ¹¹ 7 · 10 ¹¹ 0.3 7 · 10 ⁹ 2 · 10 ¹⁰ 5 · 10 ¹⁰ 8 · 10 ¹⁰

As a result of studies, the optimal dose of cobalt chloride was selected in the range of (0.1-0.2) mg / l, which made it possible to obtain biomass not only with a high content of vitamin B ₁₂ , but also with a sufficient number of viable cells of probiotic bacteria (10 ¹¹ - 10 ¹² ) cfu / ml, which is (1-2) order higher compared to the prototype (table 4.).

In addition, the biomass of the combined starter culture grows more intensively, the cultivation process is reduced by 3-4 hours compared with the known method and is (19-21) hours. This indicates that the composition of the nutrient medium contains all the components necessary for the growth of bifidobacteria and propionic acid. -acid bacteria that make up the combined starter culture, and the symbiotic relationships of these cultures during co-cultivation.

To use concentrated starter culture in the dairy industry, it is necessary to create conditions for extending the shelf life of microflora, which is determined by the economic conditions of production. One way to maintain cell viability is by freezing. Therefore, at the next stage of the work, the obtained biomass of combined starter culture was frozen and the effect of cryoanabiosis on cell survival was studied. For this, the suspension of cells of the combined starter culture obtained after centrifugation was mixed with a protective medium in a ratio of 1: 1 and frozen at a temperature not exceeding minus 20 ° С. The results are shown in table 5.

Table 5
Resistance of microorganisms to freezing Indicators The number of bifidobacteria cells, CFU / cm ³ The number of cells of propionic acid bacteria, CFU / cm ³ Fermentation activity of 1 liter of milk 0.01 cm ³ , h Before freezing 7 · 10 ¹² 9 · 10 ¹² 12-14 After freezing 2 · 10 ¹² 5 · 10 ¹² 12-14

As the data obtained by us show, the number of cells after freezing remained at the same level, which is explained not only by the use of a protective environment, but also by the formation of natural protectors - exopolysaccharides.

In addition, according to the proposed method, the number of cells remained at the same high level of 10 ¹² cfu / cm ³ for 7 months of storage. In the known method for producing a bacterial concentrate of propionic acid bacteria, the number of viable cells during storage decreases by an order of magnitude and amounts to 10 ¹⁰ cfu / cm ³ .

Thus, as a result of the studies, it was found that the combined starter culture has a more pronounced antimutagenic, antimicrobial activity and vitamin-synthesizing properties compared with the individual cultures of microorganisms that make up its composition. High resistance of combined microflora to low temperatures is noted. Obtained by the claimed method, the frozen concentrated sourdough has high biochemical properties and contains a high number of viable cells.

Namely, the identified set of distinguishing features of the invention, which consists in using combined starter culture of bifidobacteria and propionic acid bacteria as an inoculum, affects the achievement of a technical result, which is expressed in an increase in the probiotic properties of concentrated starter culture, an increase in the number of bifidobacteria and propionic acid bacteria cells and in ensuring high fermentation activity milk.

The proposed method for producing frozen concentrated yeast based on the symbiosis of probiotic bacteria is as follows.

As the basis of the nutrient medium in the claimed method, clarified curd whey is selected, which is a relatively cheap secondary raw material in the production of cottage cheese, to which medium components are added: buffer salts, ascorbic acid, peptone, agar and cobalt chloride in an amount of (0.1-0, 2) mg / ml. Then set the pH of the medium within 7.0. The nutrient medium is sterilized at (121 ± 1) ° C with a holding time of (23 ± 2) min, cooled to a temperature of (34 ± 1) ° С. As an inoculum for fermentation of a nutrient medium, a combined starter culture is used, consisting of separately activated cultures of β-galactosidase propionic acid and bifidobacteria, in an amount of 3-5%. Cell growth is carried out for (19-21) hours under conditions of periodic cultivation with a single neutralization of the medium after (9-11) hours with a saturated sterile solution of sodium carbonate (Na ₂ CO ₃ ). The resulting bacterial mass is cooled to a temperature of (18-20) ° C, separated from the culture medium and at this temperature is mixed with a protective medium, poured into 2 ml vials and frozen at minus (20-30) ° C. Bottles with concentrated sourdough are closed with sterile plugs and rolled up with metal caps. The frozen preparation is stored for up to 7 months at a temperature of minus (20-30) ° С.

Example 1

A nutrient medium is prepared on the basis of clarified curd whey, to which medium components are added: buffer salts, ascorbic acid, peptone, agar and cobalt chloride in an amount of 0.1 mg / ml. Then set the pH of the medium within 7.0. The prepared medium is sterilized at 121 ° C with a holding time of 23 minutes and cooled to 34 ° C. Then, 5% combined sourdough, consisting of separately activated β-galactosidase cultures of bifidobacteria strain Bifidobacterium longum B379M and propionic acid bacteria bacteria strain Propionibacterium shermanii KM 186 in a ratio of 1: 1, is added to the medium. Cells are grown for 19 hours under conditions of periodic cultivation with a single neutralization of the medium after 9 hours with a saturated sterile solution of sodium carbonate (Na ₂ CO ₃ ). At the end of the process, the bacterial mass is cooled to 18 ° C and the cells are separated from the culture medium by centrifugation. The resulting bacterial mass at a temperature of 18 ° C is mixed with a protective medium in a ratio of 1: 1. Then pour into bottles of 2 ml, frozen in a refrigerator at minus 30 ° C. Bottles with concentrated sourdough are closed with sterile stoppers and rolled up with metal caps. The frozen preparation is stored for up to 7 months at a temperature of minus 30 ° C.

Example 2

A nutrient medium is prepared on the basis of clarified curd whey, to which the medium components are added: buffer salts, ascorbic acid, peptone and agar; cobalt chloride in an amount of 0.2 mg / ml is also added to the medium. Then set the pH of the medium within 7.0. The finished medium is sterilized at 120 ° C with an exposure of 25 minutes and cooled to 35 ° C. Then, 4% combined yeast is introduced into the medium, which consists of separately activated β-galactosidase cultures of bifidobacteria, strain Bifidobacterium bifidum 8 ₃ and propinoacid bacteria, strain P.fredenreichii subsp.shermanii AC - 2503 in a 1: 1 ratio. Cells are grown for 20 hours under conditions of periodic cultivation with a single neutralization of the medium after 10 hours with a saturated sterile solution of sodium carbonate (Na ₂ CO ₃ ). At the end of the process, the bacterial mass is cooled to 20 ° C and the cells are separated from the culture medium by centrifugation. The resulting bacterial mass at a temperature of 20 ° C is mixed with a protective medium in a ratio of 1: 1. Then it is poured into vials of 2 ml, frozen in a refrigerator at minus 20 ° C. Bottles with concentrated sourdough are closed with sterile stoppers and rolled up with metal caps. The frozen preparation is stored for up to 7 months at a temperature of minus 20 ° C.

Example 3

A nutrient medium is prepared on the basis of clarified curd whey, to which the medium components are added: buffer salts, ascorbic acid, peptone and agar. Contribute to cobalt chloride in an amount of 0.2 mg / ml. Then set the pH of the medium within 7.0. The finished medium is sterilized at 122 ° C with an exposure of 22 minutes and cooled to 33 ° C. Then, 3% combined yeast is introduced into the medium, consisting of separately activated β-galactosidase cultures of bifidobacteria, the Bifidobacterium longum B379M strain and propionic acid bacteria, the Propionibacterium shermanii KM 186 strain, in a 1: 1 ratio. Cells are grown for 21 hours under conditions of periodic cultivation with a single neutralization of the medium after 11 hours with a saturated sterile solution of sodium carbonate (Na ₂ CO ₃ ). At the end of the process, the bacterial mass is cooled to 19 ° C and the cells are separated from the culture medium by centrifugation. The resulting bacterial mass at a temperature of 19 ° C is mixed with a protective medium in a ratio of 1: 1. Then it is poured into vials of 2 ml, frozen in a refrigerator at minus 25 ° C. Bottles with concentrated sourdough are closed with sterile stoppers and rolled up with metal caps. The frozen preparation is stored for up to 7 months at a temperature of minus 25 ° C.

Claims (4)

1. A method of obtaining a frozen concentrated starter culture on the basis of a symbiosis of probiotic bacteria, which involves preparing a nutrient medium containing cobalt chloride, introducing an inoculum in an amount of 3-5%, building cells, separating the bacterial mass from the culture medium, mixing it with a protective medium, filling and freezing , characterized in that as the inoculum use a combined starter culture, consisting of separately activated β-galactosidase cultures of bifidobacteria and propionic acid bacteria ry, wherein cobalt chloride is introduced in an amount of 0.1-0.2 mg / ml. 2. The method according to claim 1, characterized in that the strain of Bifidobacterium longum B379M and propionic acid bacteria strain Propionibacterium shermanii KM 186, taken in a 1: 1 ratio, are used as inoculum using β-galactosidase culture of bifidobacteria. 3. The method according to claim 1, characterized in that the strain of Bifidobacterium bifidum 8 ₃ and propionic acid bacteria strain P.fredenreichii subsp.shermanii AC - 2503 taken in a 1: 1 ratio are used as inoculum using β-galactosidase culture of bifidobacteria. 4. The method according to claim 1, characterized in that the inoculum is introduced at a temperature of (34 ± 1) ° C.

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