RU2169763C1 - Milk nutrient medium for bifidobacterium liquid concentrate preparing - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: invention relates to method of preparing nutrient media used for bifidobacterium culturing and can be used in microbiological and food industry. Nutrient medium has a base, L-cysteine hydrochloride, D -(-)- -lactose, milk protein hydrolyzate, yeast autolyzate and sodium chloride. Skimmed milk pancreatic hydrolyzate is used as milk proteins hydrolyzate; yeast enzymatic autolyzate is used as yeast autolyzate; skimmed milk is used as a base normalized by dry residue in total content of amine nitrogen in nutrient medium 260-300 mg% and in the following quantitative ratio of its components, wt.-%: skimmed milk pancreatic hydrolyzate, 5.0-7.0; L-cysteine hydrochloride, 0.001-0.003; D-(-)-lactose, 0.03-0.09; sodium chloride, 0.01-0.05; yeast enzymatic autolyzate, 1.0-2.0; skimmed milk (base) normalized by dry residue, the balance up to 100%. Autolyzate with index of amine nitrogen 350-400 mg% is used as yeast enzymatic autolyzate. Skimmed milk is normalized by dry residue to value 13.0-14% and amount of added lactose is decreased by 1.5-2 times as compared with analogue. Method involves additional addition of soybean isolate in the amount 0.2-0.3% by mass to nutrient medium. Sodium citrate is added additionally to nutrient medium in the amount 0.1-0.2% by mass for activation of soybean isolate. EFFECT: increased storage period of bifidum-containing preparation by using proposed nutrient medium. 5 cl, 2 tbl

Description

 The invention relates to biotechnology and can be used in the microbiological, food industry and for the preparation of nutrient media used for the cultivation of bifidobacteria in order to obtain their biomass with a high content of amino acids, vitamins and other biologically active substances, which is very important in the formulation of medical and dietary products and biologically active additives (BAA). Milk culture medium can be used in the production of therapeutic and prophylactic dairy products, eubiotics and dietary supplements.

 The selection of an effective and inexpensive nutrient medium that meets the needs of bifidobacteria is one of the main tasks in the production of medical and dietary products, dietary supplements, as well as eubiotics that normalize the biocenosis of the intestinal mucosa. Given that in addition to bifidobacteria, biologically active substances - vitamins, amino acids, unsaturated fatty acids, play an active role in the treatment and prevention of dysbacteriosis and related diseases, one of the main tasks is to create a nutrient medium rich in these substances.

 Bifidobacteria have biosynthetic deficiency in some substances. The constant habitat of bifidobacteria is the intestine, in which organic substances are in an easily digestible form, so bifidobacteria have lost a number of functions of the breakdown and synthesis of some organic substances. The cultivation of these microorganisms requires many growth factors, such as, for example, biotin, pantothenic acid, cysteine, riboflavin, purine and pyrimidine bases, amino sugar, oligosaccharides, unsaturated fatty acids.

 The specific needs of bifidobacteria in nitrogen-containing substances. The strains that live in the intestines of infants are most demanding on organic nitrogen, some strains can use ammonium salts as their sole source of nitrogen. Some strains of bifidobacteria grow in the presence of nitrogen-containing oligosaccharides (N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine).

 Of the amino acids, bifidobacteria most often require proline, serine, alanine, aspartic and glutamic acids. The needs for minerals are still poorly understood, but it is known that sodium plays a special role in the life of bifidobacteria, accelerating their development and increasing the yield of biomass. Milk proteins are characterized by high biological value and an original set of amino acids, which allows to improve the overall balance of amino acids of the entire final product. Carbohydrates of milk are represented by a kind of sugar - lactose, which is one of the main growth factors for bifidobacteria. The intake of lactose in the intestines of humans, especially fermented, has a normalizing effect on the composition of beneficial microflora.

Milk has a large assortment of trace elements. Of particular importance is the calcium of milk, which can be considered as easily digestible. A favorable balanced complex of vitamins is presented in milk, especially vitamins A, B ₂ , vitamin D, carotene, thiamine and ascorbic acid.

 Cow's milk is not a natural habitat for bifidobacteria; therefore, they develop slowly in milk. No significant proteolytic activity was detected in bifidobacteria, that is, they can absorb milk proteins only after partial hydrolysis; does not promote the growth of bifidobacteria and oxygen dissolved in milk.

A known nutrient medium based on pea hydrolyzate in the following ratio of components (RF Patent RU 2061037, IPC C 12 N 1/20, publ. 20.10.96):
Peptone - 0.15 - 0.25
Sodium Chloride - 0.2 - 0.4
Agar-agar - 0.075 - 0.1
Lactose - 0.5 - 2.0
Pea hydrolyzate, diluted with water to an amine nitrogen content of 130 - 150 mg% - the rest is up to 100%
However, this composition of the medium has a low content of assimilable forms of nitrogen and does not give the expected effect when increasing the biomass of bifidobacteria (the number of viable bifidobacteria is not more than 10 ⁸ CFU / ml). Lactic acid microorganisms, devoid of specific components of milk during cultivation, develop poorly in such an environment, give a vague picture of growth, and the morphology of bacteria is heterogeneous.

Also known is a nutrient medium for the cultivation of bifidobacteria used in the production of bifidumbacterin (RF Patent RU 2080376, IPC C 12 N 1/20, publ. 1995):
Iron sulfate - 0.002 - 0.1
Sodium chloride - 4.0 - 6.0
Glucose - 8.0 - 12.0
Agar-agar - 0.75 - 1.0
Ascorbic acid - 0.8 - 1.0
Hydrolyzate of colostrum casein-whey mass - The rest is up to 100%
However, the amine nitrogen of such a medium is very low 70–110 mg%; therefore, the medium has weak growth properties, which results in biomass production with an insufficiently high level of viable cells (2 • 10 ⁹ CFU / ml). In addition, this medium is intended for the cultivation of only bifidobacteria. In view of the inaccessibility and high cost of raw materials (colostrum casein-whey mass), this nutrient medium for large-scale cultivation of bifidobacteria and propionic acid bacteria is unpromising.

 It is known that the base medium for anaerobic bacteria is meat and peptone broth, rich in free amino acids with the addition of agarizing substances such as dextrose or agar agar. This allows you to create high reducing properties in the environment (its depletion of oxygen), necessary for the growth of anaerobes. To enhance the reducing properties, sodium thioglycolate or thioglycolic acid is also introduced into the meat and peptone broth (Bittner J. - In: Biology and Pathology of Anaerobic Bacteria, Bucharest, 1975, p. 61-69).

 However, the meat-peptone broth based medium is not only expensive and non-standard, but also unsuitable for producing fermented propionic acid bacteria in the food industry due to the presence of thioglycolates in it.

Also known for the cultivation of bifidobacteria and lactic acid bacteria is a corn-lactose medium:
Agar Agar - 2.5 g
Peptone - 10 g
Corn Extract - 40 ml
Sodium citrate - 6.6 g
Magnesium sulfate - 0.12 g
Disubstituted potassium phosphate - 2.0 g
Lactose - 10 g
Hydrochloric cystine - 0.15 g
The disadvantages of this environment are:
- non-standard corn extract, which often has a high contamination with spore forms of bacteria;
- products prepared on its basis have insufficient preservation of bacteria during storage;
- unsatisfactory organoleptic properties of the resulting products associated with the characteristic flavor of the corn extract;
- does not contain protein components of milk.

Known nutrient medium for the cultivation of bifidobacteria of the following composition (GOST 25102-82 or SanPIN 42-123-49-40-88 p. 5.2. And 7.2. Hydrolyzed milk environment):
Peptone - 2.0 - 2.5
Sodium chloride - 2.0 - 3.0
Cysteine (L-cystine) hydrochloride - 0.1 - 0.15
Lactose - 8.0 - 10.0
Agar-agar - 0.7 - 0.8
Hydrolyzed milk - Up to 1 L
pH - 7.2 - 7.4
An environment of this composition gives good results in the cultivation of bifidobacteria and for the quantification of viable bacteria. However, only milk hydrolyzate is used as the basis of this medium, which determines the high cost of the medium and low organoleptic properties, expressed in the bitter taste of the final product. Therefore, this medium is unsuitable for the preparation of dietary supplements or medical and dietary products on its basis.

Known nutrient medium for the accumulation of biomass of bifidobacteria, containing a nutrient base, a carbon source is lactose, a reducing substance is D, L-cysteine, agar-agar and water. As a nutrient base, it contains whey clarified by sequential processing of skim milk with acid and alkali, and dry or liquid yeast autolysate, and additionally sodium phosphate monosubstituted two-water, ammonium chloride and magnesium sulphate heptahydrate (RF patent N 2087532, IPC C 12 N 1 / 20, publ. 20.08.97), in the following ratio of components, wt.%:
Clarified whey - 7.5 - 10.0
Dry or liquid yeast autolysate - 0.2 - 3.0
Lactose - 0.3 - 0.5
D, L-cysteine - 0.05 - 0.1
Sodium phosphate monosubstituted, two-water - 0.7 - 0.8
Ammonium chloride - 0.1 - 0.15
Magnesium sulphate heptahydrate - 0.015 - 0.02
Agar-agar - 0.075 - 0.08
Water - The rest is up to 100%
The medium has a pH of 7.2 - 7.4 and fairly high growth qualities with a significant reduction in the duration of growth of bifidobacteria and obtaining its mass with a titer of up to 10 ⁹ CFU / ml.

However, as a rule, with a decrease in the duration of growth of bifidobacteria (cultivation time), a significant increase in titratable acidity ( ^o T) of the final product occurs, which in turn reduces its organoleptic qualities and the preservation of viable bacteria.

The closest analogue (prototype) is the nutrient medium used to obtain the bifidobacteria concentrate and containing the basis of the nutrient medium, hydrochloric acid cystine, baker's yeast autolysate, D (-) - lactose, ascorbic acid, milk hydrolyzate (RF Patent N 2104706, IPC ⁶ A 61 K 35/74, C 12 N 1/20, A 23 C 9/127, publ. 1997). As the basis of the nutrient medium use sterile milk with a dry milk content (SOM) of 11-14% in the following ratio of components of the nutrient medium, wt.%:
Milk hydrolyzate - 7.0 - 9.0
D (-) - lactose - 0.05 - 0.15
Hydrochloric cystine - 0.001 - 0.007
Ascorbic acid - 0.003 - 0.009
Baker's yeast autolysate - 0.5 - 2.0
Sterile milk (base) - Up to 100%
Such an environment has high growth properties and organoleptic characteristics of the finished product.

 However, the prototype environment does not provide a sufficiently long shelf life (more than 2 months) of bacterial concentrate.

 The objective of the invention is to obtain a nutrient medium while maintaining high growth properties for both bifidobacteria and other useful anaerobes, for example propionic acid bacteria, providing longer shelf life of the bacterial concentrate without significantly changing their titer and organoleptic properties of the finished product.

This task is achieved in that in a nutrient medium for obtaining concentrated biomass of bifidobacteria in the production of probiotic preparations containing a base, hydrochloric acid cysteine (L-cystine), D (-) - lactose, milk protein hydrolyzate and yeast autolysate, according to the invention, the medium further comprises sodium chloride, a pancreatic hydrolyzate of milk dairy is used as a hydrolyzate of milk proteins, an enzymatic autolysate of yeast is used as a yeast autolysate, and nor alizovanny dry residue skim milk at a total content of amine nitrogen in a medium of 260-300 mg% and the quantitative ratio of components (wt.%):
Pancreatic hydrolyzate milk back - 5.0 - 7.0
Salt cysteine (L-cystine) - 0.001 - 0.003
D (-) - lactose - 0.03-0.09
Sodium chloride - 0.01 - 0.05
Enzymatic yeast autolysate - 1.0 - 2.0
Milk return normalized by solids (base) - Remaining up to 100%
As an enzymatic autolysate of yeast, an autolysate with an amine nitrogen index of 350-400 mg% is used. The milk return is normalized by dry solids to a value of 13.0 - 14%, and the amount of lactose introduced is reduced by a factor of 1.5 - 2 compared to the analogue.

 Moreover, soy isolate is additionally added to the nutrient medium in an amount by weight of 0.2 - 0.3%, and to activate the soy isolate, sodium citrate is additionally added to the nutrient medium in an amount by weight of 0.1 - 0.2%.

 In the context of the mass production of probiotic biological products, it is very important to improve the organoleptic properties of products obtained on the basis of the nutrient medium, as well as increase their biological value by increasing the content of free amino acids, vitamins and unsaturated fatty acids.

 Introduction to the basis of the medium of pancreatic hydrolysis of milk protein proteins enriches it with a mixture of high molecular weight and low molecular weight peptides, which are effective stimulators of the growth of bifidobacteria (T. M. Ervolder, A. V. Gudkov and L. P. Semenova. The effect of enzymatic hydrolysates on the accumulation of biomass of bifidobacteria Zh. "Dairy industry", N 12, 1980, - p.15-17).

 The enzymatic autolysate of yeast contains vitamins and stimulating growth factors of bifidobacteria Bifidobacterium bifidum, as well as the optimal content of amine nitrogen, which provides the most favorable growth conditions for bifidobacteria.

 D - (-) - lactose is a moderate stress factor for bifidobacteria cells and provides the initiation of polymorphism of bifidobacteria cells (rod-shaped, coccoid and transitional forms between them). The polymorphism of cells in a population gives it additional resistance to adverse environmental factors, i.e. provides longer shelf life of the drug bifidobacteria in liquid form without loss of activity (Lazareva GI Abstract of dissertation of the candidate of biological sciences “Study of the physiological properties and structural and functional organization of bifidobacteria.” -Minsk, 1991, 25 p.) .

 In order to achieve the required depth of hydrolysis of the skim milk, some technological parameters were optimized - the amount of added pancreatin, the temperature of hydrolysis, the method of filtering the precipitate, and work was carried out on the selection for hydrolysis of the optimal quantitative value of the dry residue of skim milk - COM. As a result, a modified hydrolyzate-milk medium was obtained, which differs from the known GM medium and the prototype medium with an increased content of amine nitrogen - 410 ± 9 mg% and a pH of 6.8.

 In the inventive medium, as a bifidostimulating additive, an enzymatic autolysate of yeast was used, which increases the content of B vitamins in the medium. An autolysate was prepared according to a known scheme (SanPiN). Pancreatin supplements in various amounts from 0.1 to 0.5% were used to seed autolysis, to increase the yield of free amino acids that increase the value of the final product, to accelerate the process of preparing the autolysate and the process of fermentation of the final product. In the prototype used autolysate prepared without additives. The best option was with 0.1% pancreatin, which gave a significantly higher content of amine nitrogen (350-400 mg%) and acceptable pH values. In the variants with a different amount of pancreatin, despite the high content of amine nitrogen, there was no noticeable increase in growth properties, low pH values were obtained. In addition, these options are uneconomical in the production environment.

 The components providing the reducing properties of the claimed medium are mainly cysteine, as well as the amino acids glutamine and asparagine, which are rich in yeast enzymatic autolysate, which is part of the medium (Ibragimova S.I., Neronova N.M., Rabotnova I.L. , J. Microbiology, t.40, N 5, 1971, S. 833-834).

 During the development of technological regimes for producing a dairy base with a high content of COM, it was shown that in the process of sterilization of the dairy base, casein is partially coagulated with the formation of microcoagulates, which then during storage of the bifidobacteria concentrate lead to a noticeable stratification of the product - whey sediment appears, reaching more than 10%, which is unacceptable. Therefore, in order to stabilize casein and increase the protein content in milk, studies were carried out on the introduction of soy proteins in the milk environment.

 Protein soy isolates along with the enrichment of milk with amino acids also perform a stabilizing function in the production of fermented milk drinks, which is widely used in the dairy industry. It is known that soy protein isolates have a large water-holding ability (1 g of isolate binds up to 7 g of water). This allows you to use them to structure complex fatty milk mixtures. Soy proteins also have buffering properties and inhibit the increase in acidity in fermented milk products. Soy proteins contain a relatively high content of essential amino acids, which makes it possible to increase the biological value of products by soy additives. Isolated and purified soy proteins have certain advantages compared to soy milk, which is characterized by a large non-standardity in basic milk indicators due to the imperfection of domestic technologies for its production. In addition, the advantage of isolated soy proteins over soy milk is that in isolated soy proteins there is no raffinose sugar, which can cause flatulence. It is important to note that in addition to stabilizing the milk clot and reducing the acidity of the sour milk clot, the introduction of a soy protein isolate, in particular SUPRO-760, allowed to increase the nutritional and biological value of the finished product, since SUPRO-760 is a complete digestible protein, reduces the level of serum cholesterol and muscle fatigue. The protein is rich in important essential amino acids - isoleucine - 4.3%, leucine - 7.2%, valine - 4.4%, arginine - 6.7%, glutamic acid - 16.8%. Thus, the isolated soy protein SUPRO-760 made it possible to optimize the amino acid composition of the nutrient medium and, therefore, the liquid concentrate of bifidobacteria prepared on its basis, bringing it closer to the "balanced nutrition formula" developed by A.A. Pokrovsky.

 It is known that in milk the content of essential amino acids is about 41-42%, while the content of free amino acids does not exceed 4-5%. Compared to milk, the content of free amino acids in the liquid concentrate of bifidobacteria reaches 14.7%, of which the proportion of essential amino acids is 50.1%.

 A rich set of essential amino acids in bifidobacteria liquid concentrate obtained using soy protein isolate, such as: valine - 8.3%, leucine - 13.4%, lysine - 7.7%, and methionine-3.1%, It has important physiological significance, since a lack of valine leads to a disorder in coordination of movements, a lack of leucine causes changes in the kidneys and thyroid gland, and a lack of lysine leads to disruption of blood formation processes. In a liquid concentrate of bifidobacteria, the arginine content was increased to 7.7% against 2% in the initial version of the product. An increase in arginine is important because this amino acid stimulates the body's immune system.

 Despite the fact that the introduction of soy protein isolate increases the cost of the nutrient medium, the biological activity of a liquid concentrate of bifidobacteria made on this basis is enhanced. In this case, the scope of ZhKB is expanding to the field of medicine related to the treatment of common dystrophies, dystrophies of individual organs or to improve the nutrition of all body systems. In this case, cholelithiasis can be not only a starter for the dairy industry or dietary supplement, but also a medicine in a certain dose in the treatment of various pathologies of organs and systems of non-epidemiological etiology.

 The following are examples of the compositions of the claimed nutrient medium.

Example 1. Nutrient medium for obtaining concentrated biomass of bacteria with a minimum content of components, wt.%:
Pancreatic milk hydrolyzate - 5.0
Hydrochloric cysteine (L-cystine) - 0.001
D (-) - lactose - 0.03
Sodium chloride - 0.01
Enzymatic yeast autolysis - 1.0
Milk return (base), normalized by solids to 13% - The rest to 100%
The content of amine nitrogen in the medium is 260 mg%.

Example 2. Nutrient medium for obtaining concentrated biomass of bacteria with an average content of components, wt.%:
Pancreatic hydrolyzate milk back - 6.0
Hydrochloric cysteine (L-cystine) - 0.002
D (-) - lactose - 0.06
Sodium chloride - 0.03
Enzymatic yeast autolysate - 1.5
Milk return (base), normalized by solids to 13% - The rest to 100%
The content of amine nitrogen in the medium is 280 mg%.

Example 3. Nutrient medium for obtaining concentrated biomass of bacteria with a maximum content of components, wt.%:
Pancreatic milk hydrolyzate - 7.0
Hydrochloric cysteine (L-cystine) - 0.003
D (-) - lactose - 0.09
Sodium chloride - 0.05
Enzymatic yeast autolysate - 2.0
Milk return (base), normalized by solids to 14% - The rest to 100%
The content of amine nitrogen in the medium is 300 mg%.

 Table 1 shows the comparative characteristics of a liquid concentrate of bifidobacteria (HCB), prepared on the basis of the proposed nutrient medium (examples 1-3), a similar medium and a prototype environment.

 From the analysis of table 1 it can be seen that the cholelithiasis prepared on the basis of the proposed dairy medium has a shorter formation time of clot and acidity of the finished product with a higher level of viability and 100% cell survival during storage compared to similar products prepared on analog media .

Example 4. Nutrient medium with a minimum content of soy isolate to obtain a liquid concentrate of bifidobacteria, wt.%:
Pancreatic milk hydrolyzate - 7.0
Hydrochloric cysteine - 0.003
D (-) - lactose - 0.09
Sodium chloride - 0.05
Enzymatic yeast autolysate - 2.0
Sodium Citrate - 0.1
Protein isolate (SUPRO type) - 0.2
Milk return normalized by dry solids to 14.0% - The rest to 100%
A liquid bifidobacterium concentrate made on such an environment had the following characteristics: titer - 1 • 10 ⁹ - 4 • 10 ⁹ with an acidity of 130 - 160 degrees Turner.

Example 5. A nutrient medium with a maximum content of soy isolate to obtain a liquid concentrate of bifidobacteria, wt.%:
Pancreatic milk hydrolyzate - 7.0
Hydrochloric cysteine (L-cystine) - 0.003
D (-) - lactose - 0.09
Sodium chloride - 0.05
Enzymatic yeast autolysate - 2.0
Sodium Citrate - 0.1
Protein isolate (Supro type) - 0.3
Milk return normalized by dry solids up to 14% - The rest is up to 100%
A liquid bifidobacterium concentrate made on such an environment had the following characteristics: titer - 6 • 10 ¹⁰ - 10 • 10 ¹⁰ with an acidity of 120 - 140 degrees Turner.

 Below are the data (example 2) on the safety of bifidobacteria in a liquid concentrate of bifidobacteria, obtained on the basis of the inventive nutrient medium prepared in accordance with examples N 1, 3 and 5 and the prototype environment.

 Table 2 shows the safety data for bifidobacteria in cholelithiasis.

 The analysis of table 2 shows that the safety of the titer of bifidobacteria obtained using the inventive nutrient medium is two orders of magnitude higher after 3 months of storage and four orders of magnitude higher after 3.5 months of storage of the product compared to the prototype environment.

Claims (4)

1. Milk culture medium for the production of a liquid concentrate of bifidobacteria, containing hydrochloric acid cysteine (L-cystine), D (-) lactose, milk protein hydrolyzate and yeast autolysate, characterized in that the medium additionally contains sodium chloride as a milk protein hydrolyzate use pancreatic hydrolyzate of milk skim, enzymatic autolysate of yeast is used as yeast autolysate, and milk return normalized by dry residue is used as a basis for total amine content nitrogen in a nutrient medium 260-300 mg% and the following ratio of its components, wt.%:
Pancreatic hydrolyzate milk back - 5.0 - 7.0
Hydrochloric cysteine (L-cystine) - 0.001 - 0.003
D (-) - lactose - 0.03 - 0.09
Sodium chloride - 0.01 - 0.05
Enzymatic yeast autolysate - 1.0 - 2.0
Return of milk (base), normalized by solids - The rest is up to 100%
2. The nutrient medium according to claim 1, characterized in that as an enzymatic autolysate of yeast, an autolysate with an amine nitrogen index of 350-400 mg% is used.  3. The nutrient medium according to claim 1, characterized in that the milk return is normalized by solids to a value of 13.0-14%.  4. The nutrient medium according to claim 1, characterized in that the soy isolate is additionally added to the nutrient medium in an amount by weight of 0.2-0.3%.  5. The nutrient medium according to claim 1, characterized in that, to activate the soy isolate, sodium citrate is additionally introduced into the nutrient medium in an amount by weight of 0.1-0.2%.

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