RU2027754C1 - Nutrient medium for microorganism growing and a method of preparing of casein pancreatic hydrolysate - Google Patents

Abstract

FIELD: microbiology, veterinary, medicine. SUBSTANCE: nutrient medium for growing of bifidobacteria, lactobacteria and streptococci has carbon source - lactose, baker yeast extract, nitrogen source - pancreatic hydrolysate of casein, cystine, tap water and, additionally, manganese and magnesium sulfate salts and ascorbic acid at the following ratio of components, g/l: casein pancreatic hydrolysate 0.14-0.16; baker yeast extract 4.5-5.5; lactose 19-21; cystine 0.15-0.25; MgSO₄·7H₂O 0.15-0.25; MnSO₄·4H₂O 0.04-0.06; ascorbic acid 0.4-0.6, and tap water up to 1 l. For preparing of casein pancreatic hydrolysate casein solution is prepared, heated at 79-81 C, at pH 8.0-8.2 for 2h, cooled its, and hydrolysis is carried out with pancreatin at 49-51 C for 2 h at pH=8.0-8.2, acidified up to pH 4, heated up to 100 C for 5-10 min, cooled up to 75-85 C and subjected for ultrafiltration on diaphragm with pore size 50 mcm. Medium is used for medicinal bacterial preparations production and veterinary probiotics. EFFECT: improved method of nutrient medium preparing. 3 cl, 1 tbl

Description

 The invention relates to the microbiological, medical, veterinary industry and can be used in the manufacture of therapeutic drugs, as well as veterinary probiotics.

 Known industrial hepatic nutrient medium Blaurock containing liver broth, peptone, lactose and cystine.

 However, this environment is expensive, consists of scarce components and is unstable in growth properties.

 A nutrient medium based on pancreatic casein hydrolyzate with the addition of vitamins, amino acids and an RNA hydrolyzate, with a carbon source, lactose, contains a number of expensive and scarce components, the technology for its preparation is rather laborious and time-consuming, which complicates its industrial use.

 Closest to the proposed is a medium based on casein hydrolyzate and yeast autolysate.

 However, instability of its growth characteristics is noted, the yield of cells when grown on this medium does not exceed 2-3 billion / ml. This medium is applicable only for the cultivation of bifidumbacteria.

 The purpose of the invention is the expansion of technological capabilities of the environment and increase the yield of biomass of microorganisms.

For this, the nutrient medium for growing microorganisms contains a carbon source - lactose, a source of vitamins - baker's yeast extract, as a nitrogen source - pancreatic hydrolyzate of casein, cystine and additionally manganese and magnesium salts, ascorbic acid in the following ratio of components, g / l: Pancreatic hydrolyzate casein (N _am ) 0.14-0.16 Baker's yeast extract 4.5-5.5 Lactose 19.0-21.0 Cystine 0.15-0.25 MgSO ₄ ˙ 7H ₂ O 0.15- 0.25 MnSO ₄ ˙ 4H ₂ O 0.04-0.06 Ascorbic acid 0.4-0.6 Tap water up to 1 l
The content of casein hydrolyzate, as is customary, is given in terms of amine nitrogen.

 Known technology for the preparation of pancreatic hydrolyzate of casein.

 However, it is quite laborious and lengthy - up to 2 weeks. The variability of the characteristics of the resulting hydrolysates is also noted. In this regard, it seems relevant to improve the technological process of preparing pancreatic hydrolyzate of casein with high reproducibility of its basic characteristics and a significant reduction in the duration of preparation of the hydrolyzate.

Preparation of the casein hydrolyzate it is carried out with preliminary heating at 79-81 ^{° C} for 2 hours, hydrolized casein pancreatin (pancreatin and casein ratio 20: 1). At 49-51 ^{° C} for 2 hours at a pH of 8.0-8.2 . Then acidified to pH 4.0-4.5 hydrolyzate is heated ^to 100 ° C for 5-10 minutes and cooled to 75-85 ^C was subjected to ultrafiltration on a membrane with a pore size of 50 microns. The clarified filtrate was re-heated ^to 100 ° C, held for 5-10 minutes, cooled to 85-95 ^{° C} and filtered, then introduced chloroform. The content of amino nitrogen should be at least 450 mg.%, And the total - at least 1500 mg.%, Dry residue - at least 18%.

 The difference of the proposed nutrient medium is the use of pancreatic hydrolyzate of casein, prepared according to the original method.

PRI me R 1. Preparation of pancreatic hydrolyzate of casein (PGA). Take 15 kg of casein and dissolve in 75 kg of drinking water. Heated to 79 ^about C and adjusted to pH 8.0 with 40% NaOH solution with constant stirring. The mixture was allowed to stand for 2 hours. (Constantly stirred) at 79 ^{° C,} then cooled to 49 ° ^C Contribute pancreatin weighed (0.75 kg) dissolved in 2.0 dm of water at 40 ^C. The hydrolysis is carried out for 2 hours at a temperature of 49 ^about C and a pH of 8.0 with constant stirring. After completing the hydrolyzate is acidified to pH 4.0 (with concentrated HCl), heated ^to 100 ° C, held for 5 minutes, cooled to 75 ^{° C} and filtered on an ultrafiltration unit with a pore size of 50 microns. The filtrate clarified in the acidic zone was adjusted to 100 ^° C, held for 5 minutes, cooled to 75 ^° C and filtered through cardboard. For storage of PGC make 10 ml / l of chloroform. Stored at 2 ^{° C} to 1 g of amine nitrogen content should be> 450 mg%, and the total> 1500 mg% -.., Dry residue - at least 18%.

 Preparation of growth nutrient medium based on Freight One.

Prepare a medium of the following composition, g / l: Casein pancreatic hydrolyzate (N _am ) 0.14 Bakery yeast extract 4.5 Lactose 19.0 Cystine 0.15 MgSO ₄ ˙ 7H ₂ O 0.15 MnSO ₄ ˙ 4H ₂ O 0.04 ascorbic kis- lot 0.4 Tap water to 1 liter each component is sterilized separately at ¹¹⁰ ° C for 30 minutes, and ascorbic acid - at ¹⁰⁰ ° C for 5 minutes.

The resulting nutrient medium is used for growing bifidumbacteria (strains N 1 GISK and BF - 4 VGNKI VP). The process is carried out in a fermenter. The culture temperature ^of 37 C, at pH = 6.8 for 15 hours. The seed dose given at the rate of 5% by volume of the medium. The cell yield of both studied strains is higher than on the control medium of the prototype. The results are presented in the table.

PRI me R 2. Take 15 kg of a portion of casein and dissolved in 75 kg of drinking water, heated to 81 ^about C and adjust the pH to 8.5 with 40% NaOH with constant stirring. The mixture was incubated for 2 hours (constant stirring) at 81 ^{° C} and then cooled to 51 ° ^C. Contribute pancreatin weighed (0.75 kg) dissolved in 2.0 dm of water at 55 ^C. The hydrolysis is carried out for 2 hours at a temperature of 51 ^about C and a pH of 8.2 with constant stirring. After completing the hydrolyzate is acidified to pH 4.5 (with concentrated HCl), warmed to 100 ^C. After 10 min, cooled ^to 85 ° C and filtered on an ultrafiltration unit with a pore size of 50 microns. The filtrate clarified in the acidic zone was brought to 100 ^° C, held for 10 minutes, cooled to 95 ^° C and filtered through cardboard. For storage of NGL make 10 ml / l of chloroform and stored at 8 ^{° C} to 1 g of amine nitrogen content should be> 450 mg%, and the total> 1500 mg% dry residue -.. At least 18%.

The nutrient medium is prepared as in example 1. The resulting medium of the following composition, g / l: Pancreatic casein hydrolyzate (N _am ) 0.16 Extract of baker's yeast 5.5 Lactose 21.0 Cystine 0.25 MgSO ₄ ˙ 7H ₂ O 0,25 MnSO ₄ ˙4H ₂ O 0,06 Ascorbic acid 0.6 Tap water to 1 liter is used for growing Lactobacillus plantarum strain-8 PA-3 in a fermenter at pH 6.8 and ³⁷ ° C for 15 h with aeration of 0.2 volume of air per volume of medium per minute and speed of rotation of the mixer 200-500 rpm The inoculum dose is given at the rate of 5% of the medium volume. The cell yield is higher than on the control medium of the prototype. The results are presented in the table.

PRI me R 3. The nutrient medium is prepared as in example 1. The resulting medium of the following composition, g / l: Pancreatic hydrolyzate of casein (N _am ) 0.15 Extract of baker's yeast 5.5 Lactose 21.0 Cystine 0.25 MgSO ₄ ˙ 7H ₂ O 0.25 MnSO ₄ ˙ 4H ₂ O 0.06 pH of the medium 7.2 Tap water up to 1 L is used to grow a deep culture of Streptococcus faecium strain C-74 in a fermenter at pH = 6.8 and ³⁷ ° C for 15 hours under aeration of 0.2 volume of air per volume of medium per minute and the agitator speed of 200-500 rev / min. The seed dose is applied at the rate of 5% of the medium volume. The results are presented in the table.

Example 4. The nutrient medium is prepared as in example 1. The resulting medium of the following composition, g / l: Pancreatic hydrolyzate of casein (N _am ) 0.15 Extract of baker's yeast 5.5 Lactose 21.0 Cystine 0.25 MgSO ₄ ˙ 7H ₂ O 0.25 MnSO ₄ ˙ 4H ₂ O 0.06 pH of the medium 7.2 Tap water up to 1 L is used to grow a deep culture of Lactobacillus acidophilus strain 2585 VGNKI VP in the fermenter at pH = 6.6 and a temperature of 39 ^{° C} for 1 hour under aeration of 0.2 volume of air per volume of medium per minute and the agitator speed of 200-400 rev / min. The seed dose is applied at the rate of 5% of the medium volume. The results are presented in the table.

 The results of examples 1- indicate that the proposed nutrient medium allows to increase the yield of cells by 2-3 times.

 PRI me R 5. Get a nutrient medium in which as a source of nitrogen use casein hydrolyzate obtained by the prototype. Prepare the medium according to the recipe as in example 1. Cultivate B. bifidum pcs. 1. The output of cells on the medium using a casein hydrolyzate prepared as in the prototype is 5 billion / ml, which is significantly lower than on the proposed nutrient medium. Therefore, for the growth characteristics of the environment, an essential role is played by casein hydrolyzate obtained by the proposed method.

 Thus, the proposed environment and method of preparing pancreatic hydrolyzate of casein allows to unify the technology for a number of therapeutic and prophylactic preparations containing several types of microorganisms. In addition, the proposed environment allows to increase the yield of cells, as well as to exclude the use of a number of expensive and deficient components - liver, vitamins, amino acids.

Claims (2)

 NUTRIENT ENVIRONMENT FOR GROWING MICROORGANISMS AND METHOD FOR PRODUCING CASEAN'S PANCREATIC HYDROLYSIS. 1. Nutrient medium for growing microorganisms, containing a nitrogen source - pancreatic casein hydrolyzate, carbon source - lactose, a vitamin source - baker's yeast extract, cystine and tap water, characterized in that it additionally contains manganese and magnesium sulfate salts and ascorbic acid in the following ratio of components, g / l:
Casein Pancreatic Hydrolyzate - 0.14 - 0.16
Baker's Yeast Extract - 4.5 - 5.5
Lactose - 19 - 21
Cystine - 0.15 - 0.25
MgSO ₄ · 7H ₂ O - 0.15 - 0.25
MnSO ₄ · 4H ₂ O - 0.04 - 0.06
Ascorbic acid - 0.4 - 0.6
Tap water, l - Up to 1
2. A method of producing a pancreatic hydrolyzate of casein, comprising dissolving casein in water, heat treating the resulting solution, cooling to a hydrolysis temperature, introducing pancreatin and hydrolyzing at a given temperature and pH, followed by filtration, characterized in that the heat treatment of the casein solution is carried out at 79 - 81 ^o C and a pH of 8.0 - 8.2 for 2 hours, hydrolysis is carried out at 49 - 51 ^o C for 2 hours at a pH of 8.0 - 8.2, the resulting hydrolyzate is acidified to pH 4.0 -4 , 5, heated to 100 ^o C for 5 to 10 minutes, cooled to 75 - 85 ^o C and subjected to ultrafiltration on a membrane with a pore size of 50 μm.

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