RU2326939C2 - Nutrient milk medium for preparation of bacteria-probiotics biomass - Google Patents

Abstract

FIELD: technological processes.

SUBSTANCE: invention is related to preparation of nutrient mediums for cultivation of bifido and lactobacteria with the purpose of preparation of microorganisms biomass and its further utilisation in production of ferments for medicinal and prophylactic products, and also for production of dietary supplements (DS). Milk nutrient medium contains the basis in the form of non-fat milk with content of solid residual 13-14%, muriatic cysteine or ascorbic acid, lactopeptone, purified yeast extract with index of amine nitrogen 2.1-2.8%, total protein of at least 3-4%, sodium chloride, manganese sulphate heptahydrate and manganese sulphate tetrahydrate.

EFFECT: provides for the possibility of bifido and lactobacteria cultivation with longer shelf life and with higher titres of bacteria biomass in the process of storage.

2 cl, 4 tbl, 5 ex

Description

The invention relates to biotechnology and can be used in the microbiological, food industry and for the preparation of nutrient media for the cultivation of bifidobacteria and lactobacilli in order to obtain biomass of microorganisms and its further use in the production of starter cultures for therapeutic and prophylactic products, as well as for the production of biologically active additives ( Dietary supplement).

Currently, products and biologically active additives with probiotic microorganisms occupy a leading place in the package of measures aimed at the prevention and correction of microflora disorders. The importance of probiotic preparations and products necessitates the development of new effective inexpensive production nutrient media for the production of microbial mass. In the production of biologically active additives with probiotics, as well as therapeutic and prophylactic products and starter cultures, it is important for them to develop a nutritional basis for probiotic microorganisms. Such a framework should include all the basic components that meet the needs of bacteria. Cultivation in such an environment should contribute, at a minimum, to the preservation of the probiotic properties of microorganisms. A production nutrient medium should provide a high reproduction rate: for lactobacilli up to 5-7 hours and for bifidobacteria up to 18 hours, a high concentration of viable microbial cells per unit volume of the medium - at least 1 · 10 ¹⁰ CFU / ml, high safety of viable microbial cells - not less than 70-90 days.

The nutritional needs of lactic acid bacteria are very diverse and are associated with the biochemical activity of microorganisms. As the medium is enriched with protein, the ability of bacteria to ferment lactose increases, the addition of peptones and peptides (products of protein hydrolysis) enhances the growth of lactic acid bacteria to a much greater extent than amino acids. It should be noted that the amount of amino acids required by various lactic acid bacteria is variable and depends on the needs of the strains. Bifidobacteria most require amino acids: proline, serine, alanine, aspartic and glutamic acids. Possessing some proteolytic activity, bifidobacteria can, to a small extent, additionally provide their needs for nitrogenous substances.

All nutrients in the medium must be in easily digestible form, proteins, in particular, in the form of hydrolysates, autolysates. In some cases, an imperfect technology for the production of protein hydrolysates and autolysates can lead to the appearance in their composition of a significant amount of peptides of high molecular weight, ammonia, as well as colored impurities formed due to the destruction of labile amino acids and interaction with aldehydes. The presence of these components in the environment significantly reduces nutritional effectiveness. The quality of protein hydrolysates is largely determined by the selected method of hydrolysis, providing the required depth of hydrolysis, and the degree of subsequent purification of the resulting hydrolyzate.

Nutrient media of various compositions are used to build biomass. The composition of the nutrient medium for the growth of bifidobacteria and lactobacilli is known, which is a milk hydrolyzate, in which peptone, sodium chloride, agar-agar, lactose, hydrochloric acid cysteine are added as additives in the following ratio of components (Instructions for the preparation of sour-milk bifidumbacterin in dairy kitchens. - M .: MZ RSFSR, 1987, - 11 p.), G / l:

Peptone 2.0 Sodium chloride 5.00 Agar agar 0.75 Lactose 10.00 Hydrochloric cysteine 0.10 Milk hydrolyzate rest.

This composition is used in practice for the growth of bifidobacteria and lactobacilli.

However, along with an expensive base - milk hydrolyzate, it also contains expensive additives, such as peptone, hydrochloride cysteine. In addition, this composition is not an effective nutrient medium for growing bifidobacteria and lactobacilli.

Known nutrient medium for increasing the biomass of bifidobacteria and lactobacilli based on pea hydrolyzate in the following ratio of components (RF Patent No. 2061037, IPC С12N 1/20, publ. 27.05.96 g), wt.%:

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 amine nitrogen content 130-150 mg% the rest is up to 100%.

The above composition has a low content of low molecular weight peptides and amino acids (amine nitrogen 130-150 mg%), therefore, it does not provide the production of biomass of high concentration - not more than 10 ⁸ CFU / ml. At the same time, the time of building up the microbial mass (at least 24 hours), titratable acidity and shelf life do not meet the requirements.

Also known is a nutrient medium for the accumulation of biomass of bacteria based on whey in the following ratio of components (RF Patent No. 2087532, IPC С12N 1/20, publ. 20.08.97 g), wt.%:

Clarified whey 7.5-10.0 Lactose 0.3-0.5 Yeast autolysate 2.0-3.0 D, L-cysteine 0.05-0.1 NaH ₂ PO ₄ · 2H ₂ O 0.7-0.8 NH ₄ Cl 0.1-0.15 MgSO ₄ · 7H ₂ O 0.015-0.020 Agar agar 0.75-0.080 Water rest pH 7.2-7.4

A medium of this composition, although it gives a high growth rate, does not have enough nutrients (proteins, peptides, amino acids) for the long-term preservation of microbial cells, which is very important for drugs and dietary supplements-probiotics. An additional effect of high growth indicators is a significant increase in titratable acidity, which negatively affects the organoleptic properties of the final product. The presence of expensive components (agar-agar, cysteine) in the production of drugs and dietary supplements-probiotics leads to an increase in the cost of the final product.

The corn-lactose medium (CLS) for the cultivation of bifidobacteria and lactic acid bacteria is widely known (TU 10-02-02-789-192-95):

Agar agar 2.5 g Peptone 10 g Corn extract 40 ml Sodium Citrate 6.6 g Magnesium sulfate 0.12 g Potassium Disubstituted Potassium Phosphate 2.0 Lactose 10 g Hydrochloric cystine 0.15 g

However, CLS does not contain hydrolyzed protein components of milk or yeast cells, which does not ensure the cultivation of long shelf life of the final product. The presence of a non-standard component in the composition of the medium — corn extract — determines the non-standard nature of the medium itself, as well as the unsatisfactory organoleptic properties of the obtained dietary supplements and starter cultures.

The closest analogue (prototype) to the claimed one is a milk nutrient medium for the production of a liquid concentrate of bifidobacteria, containing hydrochloric acid cysteine (L-cystine), D (-) - lactose, milk protein hydrolyzate and yeast autolysate and sodium chloride. The pancreatic hydrolyzate of milk dairy is used as a hydrolyzate of milk proteins, enzymatic autolysate of yeast is used as a yeast autolysate, and milk return normalized by dry residue is used with a total content of amine nitrogen in the nutrient medium of 260-300 mg% in the following ratio of its components, wt.% (RF patent No. 2169763, IPC C12N 1/20, A61K 35/74, A23C 9/12, publ. 06/27/2001):

Pancreatic milk hydrolyzate 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 Milk return (base), normalized by dry residue The rest is 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%. In addition, soy isolate is additionally added to the nutrient medium in an amount by weight of 0.2-0.3%. To activate soy isolate, sodium citrate is additionally introduced into the nutrient medium in an amount by weight of 0.1-0.2%.

However, pancreatic hydrolyzate of milk protein proteins contains, along with high-molecular and low-molecular-weight peptides, partially hydrolyzed casein and other components that are poorly absorbed by bacteria, and enzymatic autolysate of yeast, along with vitamins and stimulating bacterial growth factors, contains toxic substances - products of enzymatic autolysis that do not favor growth conditions bacteria (T. M. Ervolder, A. V. Gudkov and L. P. Semenova. The effect of enzymatic hydrolysates on the accumulation of biomass bifidobacteria. Journal of Dairy Industry, N 12, 1980, p.15-17), resulting in reduced time and titer of biomass of bifidobacteria and lactobacilli during storage.

The technical result of the claimed invention is the creation of such a nutrient medium that would enable the cultivation of both bifidobacteria and lactobacilli with longer shelf life and higher titers of bacterial biomass during storage.

The specified technical result is achieved in that in a milk nutrient medium for obtaining biomass of probiotic bacteria containing a base in the form of a milk sample normalized by the dry residue, hydrochloric acid cysteine (L-cystine) or ascorbic acid, milk protein hydrolyzate and growth factors from yeast cells, sodium chloride, according to the invention, the medium further contains manganese sulfate MnSO ₄ · 4H ₂ O, and magnesium sulfate MgSO ₄ · 7H ₂ O, as the milk protein hydrolyzate is used enzymatic hydrolyzate syvo otochnyh milk proteins as growth factors from yeast cells using purified yeast extract at the following ratio of components, wt.%:

serum enzymatic hydrolyzate milk proteins 5.0-7.0 hydrochloric cysteine (Ĺ-cystine) or ascorbic acid 0.001-0.002 sodium chloride 0.03-0.05 Manganese sulfate MnSO ₄ · 4H ₂ O 0.001-0.005 magnesium sulfate MgSO ₄ · 7H ₂ O 0.01-0.02 purified yeast extract 1.5-2.0 Milk return (base), normalized by dry residue The rest is up to 100%

Milk culture medium contains purified yeast extract with an amine nitrogen index of 2.1-2.8%, a total protein of at least 3-4%, an enzymatic hydrolyzate of milk whey proteins (lactopeptone) with a total protein of at least 11%, amine nitrogen of at least 4%, and milk return contains lactose in an amount of not more than 15% and is normalized by dry solids to 13.0-14.0%.

The total amine nitrogen in the nutrient medium is contained in an amount of not less than 300 mg.%.

Introduction to the basis of the medium of the enzymatic hydrolyzate (lactopeptone) of whey proteins of milk (lactalbumin and lactoglobulin) enriches it with a mixture of high molecular weight and low molecular weight peptides, which are more effective growth promoters of bifidobacteria and lactobacilli than, for example, pancreatic protein hydrolyzate (the main component is milk back. Whey proteins of milk in amino acid composition are similar to proteins of human milk. Compared to casein, they are more easily hydrolyzed, contain essential sulfur-containing acids in an amount that meets human needs, 4 times more cysteine and 19 times more tryptophan and their use is more preferable in the production of probiotics for children.

The purified yeast extract contains B vitamins and stimulating growth factors of bifidobacteria, as well as the optimal content of amine nitrogen, which provides the most favorable growth conditions for bifidobacteria and lactobacilli, and does not contain toxic yeast metabolism products that inhibit bacterial growth in comparison with the enzymatic yeast autolysate.

A tangible increase in the quality of the yeast extract is achieved by improving the technology for its production - the soluble state is converted to 30-50% yeast protein, the digestibility of the cellular protein is increased by 8-10% due to loosening of all layers of the cell wall.

Yeast extract contains:

- essential amino acids - lysine, methionine, glycine, alanine, histidine, aspartic acid, threonine, glutamic acid, valine, etc .;

- vitamins - riboflavin (B ₂ ), thiamine (B ₁ ), pyridoxine (B ₆ ), cyancobalamin (B ₁₂ ), niacin (PP);

- protective peptides.

The components providing the reducing properties of the claimed medium are mainly cysteine or ascorbic acid, as well as the amino acids glutamine and asparagine, which are rich in purified yeast extract, which is part of the medium.

To ensure growth and development, lactic acid bacteria need inorganic compounds, especially manganese ions and magnesium ions. Manganese prevents autolysis of cells and is necessary for normal processes of fat metabolism. Mn ⁺⁺ and Mg ⁺⁺ remove the toxic effect of Zn ⁺⁺ if it is present in the medium.

The invention allows to obtain a high initial titer and increase the shelf life without inhibiting growth and increasing the active acidity of probiotic preparations obtained using the proposed nutrient medium.

The lack of finished nitrogenous substances in the claimed nutrient medium is compensated by the addition of lactopeptone (an enzymatic hydrolyzate of whey proteins of milk) to skim milk and a yeast extract. Whey proteins of milk play an important role in increasing the biomass of probiotic bacteria and shaping the quality of the final product, since they have a high content of essential and sulfur-containing amino acids. Milk-based proteins (mainly casein) are characterized by high biological value and a unique set of amino acids, which helps to improve the overall balance of the amino acid composition of the final product. Milk contains a unique disaccharide - lactose, which has a beneficial effect on the composition of the intestinal normoflora. When milk is heated, for example, during its sterilization, lactose turns into lactulose, which is a powerful bifidogenic factor. Milk contains a significant amount of macro- and microelements, vitamins. Especially important is the digestible calcium of milk, vitamins A, B ₁ , B ₂ , D, C, carotene.

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

Example 1. The minimum content of medium components (wt.%):

enzymatic hydrolyzate whey protein milk 5,0 hydrochloric cysteine (Ĺ-cystine) 0.001 sodium chloride 0,03 Manganese sulfate MnSO ₄ · 4H ₂ O 0.001 magnesium sulfate MgSO ₄ · 7H ₂ O 0.01 purified yeast extract 1,5 Milk return (base), normalized by dry residue The rest is up to 100%

Example 2. The average content of medium components (wt.%):

serum enzymatic hydrolyzate milk proteins 6.0 vitamin C 0.0015 sodium chloride 0.04 Manganese sulfate MnSO ₄ · 4H ₂ O 0.003 magnesium sulfate MgSO ₄ · 7H ₂ O 0.015 purified yeast extract 1.7 Milk return (base), normalized by dry residue The rest is up to 100%

Example 3. The maximum content of medium components (wt.%):

serum enzymatic hydrolyzate milk proteins 7.0 hydrochloric cysteine (Ĺ-cystine) 0.002 sodium chloride 0.05 Manganese sulfate MnSO ₄ · 4H ₂ O 0.005 magnesium sulfate MgSO ₄ · 7H ₂ O 0.02 purified yeast extract 2.0 Milk return (base), normalized by dry residue The rest is up to 100%

The following are the data (tables 1 and 2) for the cultivation of bifidobacteria (Bifidobacterium bifidum strain 791 BAG) and lactobacilli (Lactobacillus strain Lactobacillus acidophilus 57S) using the claimed nutrient medium and prototype medium. From these tables it can be seen that the titer of both bifidobacteria and lactobacilli after cultivation using the inventive nutrient medium is several times higher than using the prototype medium, and the cultivation time is correspondingly shorter.

Table 1 The titer of lactobacilli and the activity of acid formation during cultivation in the claimed nutrient medium No. p / p The composition of the medium Initial titer, CFU / ml Acidity, ° T Fermentation time, hour one Example 1 1.2 · 10 ¹⁰ 115 4.0 2 Example 2 2.510 ¹⁰ 120 3,5 3 Example 3 1.5 · 10 ¹⁰ 126 4.0 four Prototype environment 7.0 · 10 ⁹ 107 5,0 table 2 The titer of bifidobacteria and the activity of acid formation during cultivation in the claimed nutrient medium No. p / p The composition of the medium Initial titer, CFU / ml Acidity, ° T Fermentation time, hour one Example 1 4 · 10 ¹⁰ 130 16 2 Example 2 1 · 10 ¹⁰ 145 eighteen 3 Example 3 1 · 10 ¹⁰ 155 > 18 four Prototype environment 2 · 10 ⁹ 124 24-30

Example 4. The study of the duration of storage of a consortium of strains of lactobacilli in a liquid concentrate of lactobacilli

The duration of storage of a consortium of lactobacillus strains in liquid concentrate was studied (table 3). As a seed dose, a consortium of lactobacillus strains Lactobacillus acidophilus 57S (deposited at the All-Russian Research Institute of Genetics at VKPM under No. 5863), Lactobacillus plantarum P-75 (deposited at the All-Russian Research Institute of Genetics at VKPM under No. B 3962) is used; Lactobacillus casei Sat (deposited at the All-Russian Research Institute of Genetics at VKPM under No. B 5724) in a ratio of 2: 0.5: 0.5. Fermentation is carried out at a temperature of (39-40) ° C for 4-5 hours until the acidity of the finished product is not less than 100 ° T.

A significant period (up to 72 days) of storage with a titer of at least 10 CFU / ml is associated with ensuring the initial acidity of the drug of the consortium of lactobacilli not more than (120-140) ° T, obtaining the initial titer of lactobacilli (not lower than 10 ¹⁰ CFU / ml) due to the use of of the claimed milk culture medium with the addition of lactopeptone and yeast extract.

Table 3 Data on the cultivation of a consortium of bifidobacteria using the claimed nutrient medium and the storage of its biomass Production sourdough Consortium-based Lactobacillus Biomass Types of strains of lactobacilli in the consortium 0.5: 0.5: 2 Culture medium Cultivation time, hours Acidity, T ° The titer (CFU / ml) of ZhKB during storage Clot 1 day 72 days 1 day 60 days 72 days B-5724 The inventive environment 4.0. 140 160 Normal B-3962 1 · 10 ¹⁰ 6 · 10 ⁹ 2 · 10 ⁹ B-5863 Also Wednesday - prototype 5,0 170 320 2.510 ⁹ 9.0 · 10 ⁷ 2.510 ⁷ Normal

Example 5. The study of the duration of storage of biomass of a consortium of strains of bifidobacteria in a liquid concentrate of bifidobacteria obtained using the inventive nutrient medium

The duration of storage of a consortium of strains of bifidobacteria in a liquid concentrate of bifidobacteria was studied (table 4). As a seed dose of bifidobacteria, a consortium of strains of bifidobacteria is used: Bifidobacterium bifidum 8-3, Bifidobacterium longum I-3, Bifidobacterium adolescentis B-1 in a ratio of 3: 1: 1.

A seed dose of bifidobacteria is applied in a volume of 5.0-7.0% of the volume of the nutrient medium with a titer of bifidobacteria of at least 10 ⁹ CFU / ml, cultivation is carried out for a time sufficient to obtain a concentrate of bifidobacteria with a biological titer of at least (0.8- 1.0) 10 ¹¹ CFU / ml.

A significant period (up to 72 days) of storage of a liquid concentrate of bifidobacteria with a titer of at least 10 ¹⁰ CFU / ml is associated with ensuring the initial acidity of the preparation of a consortium of bifidobacteria not more than (140-145) ° T, obtaining the initial titer of bifidobacteria (not less than 10 ¹¹ CFU / ml).

Table 4
Data on the cultivation of a consortium of bifidobacteria using the claimed nutrient medium and the storage of its biomass Production sourdough Consortium-Based Bifidobacteria Liquid Concentrate (BAC) The composition of the proposed consortium Strain ratio Cultivation time, hour Acidity, T ° The titer (CFU / ml) of ZhKB during storage Clot 1 day 72 days 1 day 60 days 72 days l) B. bifidum 8-3 60 22 146 150 3 · 10 ¹¹ 1 · 10 ¹⁰ 1 · 10 ¹⁰ Normal 2) B.longum (I-3) twenty 3) B.adolescentis. B-1 twenty Also Wednesday - prototype 29th 137 152 5 · 10 ⁹ 1 · 10 ⁸ 9 · 10 ⁷ Normal

Thus, the technical result of the claimed invention confirms the creation of such a nutrient medium that provides the ability to cultivate both bifidobacteria and lactobacilli with longer storage periods (up to 72 days or more) and higher titers of bacterial biomass during storage.

Claims (2)

1. Milk culture medium for biomass of probiotic bacteria, containing a base in the form of skim milk with a solids content of 13-14%, hydrochloric acid cysteine or ascorbic acid, milk protein hydrolyzate, growth factors from yeast cells and sodium chloride, characterized in that the medium additionally contains manganese sulfuric acid tetrahydrate and magnesium sulfuric acid seven-water, contains lactopeptone as a hydrolyzate of milk proteins, and purified yeast as growth factors from yeast cells extract with an amine nitrogen ratio of 2.1-2.8%, total protein of at least 3-4% in the following ratio of nutrient medium components, wt.%: lactopeptone 5.0-7.0 hydrochloric cysteine or ascorbic acid 0.001-0.002 sodium chloride 0.03-0.05 manganese sulfuric acid, four-water 0.001-0.005 magnesium sulfuric acid heptahydrate 0.01-0.02 purified yeast extract with indicator amine nitrogen 2.1-2.8% total protein not less than 3-4% 1.5-2.0 skim milk with content dry residue 13-14% the rest is up to 100% 2. The milk culture medium according to claim 1, characterized in that skim milk contains lactose in an amount of not more than 15%.