RU2267968C2 - Method for production of bioactive supplement - Google Patents

Abstract

FIELD: biotechnology, in particular production of bioactive supplements useful in treatment and prophylaxis of various gastrointestinal diseases in any kind of agriculture animals and poultry.

SUBSTANCE: claimed method includes introducing of lactobacterium cultures Lactobacillus acidophilus and Lactobacillus delbrueskii subsp. Bulgaricus in equal ratio in broth containing lightened milky whey, yeast extract, manganese sulfate, and potassium phosphate. Lactobacterium biomass is grown by deep cultivation process at 44±1°C. During cultivation medium pH is adjusted and double neutralization is carried out. After 2-3 h form cultivation starting Lactobacillus lactic subsp. lactic III Defatted milk, 40 % glucose solution are introduced into broth followed by further cultivation. Before cultivation finishing sodium citrate is introduced and mixture is cooled. Cooled biomass is centrifuged ad separated. Concentrated biomass is blended with sucrose/gelatin protective medium and potassium iodide, pre-packed in penicillin flacks, and freeze dehydrated.

EFFECT: inexpensive harmless and convenient preparation having high antagonistic properties against pathogenic microflora and increase concentration of healthy cells.

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Description

The invention relates to biotechnology, in particular to methods for producing a biologically active additive for the treatment and prevention of gastrointestinal diseases of various etiologies for all types of farm animals and poultry.

A known method of obtaining a probiotic, which consists in introducing strains of lactic acid bacteria in a nutrient medium, cultivating, concentrating the bacterial mass, followed by drying (see RU 2063755 C1, 07.20.1996). The disadvantage of this method is the complexity of the process, high costs.

Patent RU 2035184 C1, 05/20/1995, which describes a method for producing a bacterial preparation for the production of a feed additive, comprising preparing a nutrient medium, introducing a culture of lactobacilli, bifidobacteria and mesophilic lactic streptococcus, culturing, separating the bacterial mass by centrifugation, mixing the obtained bacterial mass with a protective environment, followed by freeze-drying.

The objective of the invention is to develop a new method for producing biologically active additives to stimulate natural resistance and increase the productivity of farm animals, economical and highly effective when used in farms. The technical result consists in obtaining a cheap preparation, convenient for storage, transportation, use, with high antagonistic properties in relation to pathogenic microflora, with a high concentration of viable cells, harmless, without age restrictions.

The technical result is achieved due to the fact that to obtain a biologically active additive as a culture of lactobacilli, lyophilized dried strains of lactobacilli Lactobacillus acidophilus G-1 (VKPM B-3488), Lactobacillus delbrueckii subsp are used. bulgaricus L 20/2 (VKPM B-3141), Lactococcus lactis subsp. lactis III (VKPM B-3961), deposited in the All-Russian Collection of Industrial Microorganisms of the State Research Institute of Genetics and Breeding of Industrial Microorganisms. First, strains of lactobacilli Lactobacillus acidophilus G-1 (VKPM B-3488) and Lactobacillus delbrueckii subsp are cultured. bulgaricus L 20/2 (VKPM B-3141) in an equal ratio of 3-4 wt.%, which are added to the nutrient medium, which is used as clarified milk whey with the addition of 1.0 wt.% yeast extract, manganese sulfate - 160 mg / l, potassium phosphate - 0.1 wt.%. Increase the biomass of lactobacilli by the method of deep cultivation for 12-13 hours at a temperature of 44 ± 1 ° C. During cultivation, the pH of the medium is adjusted with gaseous ammonia. The neutralization is carried out twice until a pH of 5.9-6.2 is reached. Then, after 2-3 hours from the start of cultivation, a strain of Lactococcus lactis subsp is introduced into the medium. lactis III (VKPM B-3961) and 1 wt.% of the volume of the nutrient medium — skim milk and 40% glucose solution and continue cultivation for 12–13 hours. Before cultivation, 2 wt.% lemon acid sodium and begin to cool the medium to a temperature of 15-18 ° C. The cooled biomass is centrifuged at 16,000 rpm. The concentrated bacterial mass is mixed with a sucrose-gelatin protective medium (10 wt.% Sucrose and 1 wt.% Gelatin) with the addition of 0.1-0.5 wt.% Potassium iodide in a ratio of 1: 8 and packaged in 5 ml penicillin vials . Freeze-drying is carried out with preliminary freezing for 4-5 hours to a temperature of minus 40-45 ° C at a vacuum of at least 7 mlV and an evaporator temperature of minus 45-60 ° C, and the shelves are heated from plus 15-20 ° C. And from the moment the product reaches positive temperature, drying is continued for 10-12 hours at a temperature of no more than 40 ° C, the total drying time is 24-28 hours with a residual moisture content of the target product of not more than 3 wt.%.

The essence of the proposed method is the introduction of strains of cultures of lactobacilli Lactobacillus acidophilus G-1 (VKPM B-3488) and Lactobacillus delbrueckii subsp. bulgaricus L 20/2 (VKPM B-3141) in an equal ratio of 3-4 wt.% to a nutrient medium, which is used clarified whey with the addition of 1.0 wt.% yeast extract, manganese sulfate - 160 mg / l potassium phosphate - 0.1 wt.%. Build biomass by the method of deep cultivation in industrial fermenters for 12-13 hours at a temperature of 44 ± 1 ° C. During cultivation, the pH of the medium is adjusted with gaseous ammonia. The neutralization is carried out twice until a pH of 5.9-6.2 is reached. Then, 2-3 hours after the start of cultivation, Lactococcus lactis subsp. lactis III (VKPM B-3961) and 1 wt.% of the volume of the nutrient medium — skim milk and 40% glucose solution. Then, cultivation is continued for 12-13 hours. 2 wt.% Citric acid sodium is added to the nutrient medium and cooled to 15-18 ° C. The cooled biomass is centrifuged at 16,000 rpm. The resulting concentrated bacterial mass is mixed with a sucrose-gelatin protective medium (10 wt.% Sucrose and 1 wt.% Gelatin) with the addition of 0.1-0.5 wt.% Potassium iodide in a ratio of 1: 8 and Packed in penicillin vials. Freeze-drying is carried out according to a mode selected experimentally.

Example 1. Preparing a nutrient medium containing clarified whey, freed from proteins by the thermoacid method (clarified), add yeast extract in an amount of 1.0 wt.%, Manganese sulfate - 160 mg / l, ammonium sulfate - 0.1 wt.% potassium phosphate - 0.1 wt.%. Bring the volume of the nutrient medium with clarified serum to 1 liter. Lactobacillus strains Lactobacillus acidophilus G-1 (VKPM B-3488) and Lactobacillus delbrueckii subsp are introduced into the obtained medium. bulgaricus L 20/2 (VKPM B-3141) in equal proportions in an amount of 3.0 wt.% of the volume of the nutrient medium. Cultivation is carried out by the in-depth method at a temperature of 43 ° C for 12 hours. During cultivation, the pH of the medium is adjusted with a 40% sodium hydroxide solution. Twice neutralization is carried out until a pH of 5.9 is reached. Then, after 2 hours from the start of cultivation, a strain of Lactococcus lactis subsp is introduced into the medium. lactis III (VKPM B-3961) in an amount of 3.0 wt.% and 1 wt.% of the volume of the nutrient medium - skim milk and 40% glucose solution. Then, cultivation is continued for 12 hours, and before the end of cultivation, 2 wt.% Citric acid sodium are added and the medium begins to cool to 15 ° C. The cooled biomass is centrifuged at 16,000 rpm. The obtained concentrated bacterial mass is mixed with a sucrose-gelatin protective medium containing 10 wt.% Sucrose and 1 wt.% Gelatin with the addition of 0.1 wt.% Potassium iodide in a ratio of 1: 8. Packed in penicillin vials and subjected to freeze-drying. The total drying time is 24 hours.

The concentration of viable cells of lactobacilli in the obtained biologically active additive is 13 × 10 ⁹ KOE / g

Example 2. In 1 l of clarified whey add yeast extract in an amount of 1.0 wt.%, Manganese sulfate - 160 mg / l, ammonium sulfate - 0.1 wt.%, Potassium phosphate - 0.1 wt.%. Lactobacillus strains Lactobacillus acidophilus G-1 (VKPM B-3488) and Lactobacillus delbrueckii subsp are introduced into the obtained medium. bulgaricus L 20/2 (VKPM B-3141) in equal proportions in an amount of 3.5 wt.% of the volume of the nutrient medium. Cultivation is carried out by the in-depth method at a temperature of 43 ° C for 13 hours. During cultivation, the pH of the medium is adjusted with gaseous ammonia. Twice neutralization is carried out until a pH of 6.0 is reached. Then, 3 hours after the start of cultivation, a strain of Lactococcus lactis subsp was introduced into the medium. lactis III (VKPM B-3961) in an amount of 3.0 wt.% of the volume of the nutrient medium and 1 wt.% of the volume of the nutrient medium - skim milk and 40% glucose solution. Cultivation is continued for 12 hours. Before the cultivation, 2 wt.% Citric acid sodium are added to the nutrient medium and the medium is cooled to 15 ° C. The cooled biomass is centrifuged at 16,000 rpm. The resulting concentrated bacterial mass is mixed with a sucrose-gelatin protective medium containing 10 wt.% Sucrose and 1 wt.% Gelatin with the addition of 0.1 wt.% Potassium iodide in a ratio of 1: 8. Packed in penicillin vials and subjected to freeze-drying. The total drying time is 27 hours

The concentration of viable cells of lactobacilli in the obtained biologically active additive is 13 × 10 ⁹ KOE / g

Example 3. In 1 l of clarified whey add yeast extract in an amount of 1.0 wt.%, Manganese sulfate - 160 mg / l, potassium phosphate - 0.1 wt.%. Lactobacillus strains Lactobacillus acidophilus G-1 (VKPM B-3488) and Lactobacillus delbrueckii subsp are introduced into the obtained medium. bulgaricus L 20/2 (VKPM B-3141) in equal proportions in an amount of 4.0 wt.% of the volume of the nutrient medium. The cultivation is carried out by the in-depth method at a temperature of 43 ° C for 13 hours. During cultivation, the pH of the medium is adjusted with gaseous ammonia. Twice neutralization is carried out until a pH of 6.2 is reached. Then, after 2 hours from the start of cultivation, a strain of Lactococcus lactis subsp is introduced into the medium. lactis III (VKPM B-3961) in an amount of 3 wt.% of the volume of the nutrient medium and 1 wt.% of the volume of the nutrient medium - skim milk and 40% glucose solution. Cultivation is continued for 12 hours. Before the cultivation is completed, 2 wt.% Sodium citric acid is added to the nutrient medium and cooled to 15 ° C. The cooled biomass is centrifuged at 16,000 rpm. The resulting concentrated bacterial mass is mixed with a sucrose-gelatin protective medium containing 10 wt.% Sucrose and 1 wt.% Gelatin with the addition of 0.5 wt.% Potassium iodide in a ratio of 1: 8. Packed in penicillin vials and subjected to freeze-drying. The total drying time is 28 hours.

The concentration of viable cells of lactobacilli in the obtained biologically active additive is 6 × 10 ¹⁰ CFU / g

The invention in comparison with the prototype has the following advantages:

- reduced terms of cultivation and freeze-drying, a high concentration of lactobacilli and their preservation unchanged for at least 12 months, low cost and high return on investment, ease of transportation, storage, ease of use. The method allows to obtain a biologically active additive environmentally friendly, the bacteria included in its composition - probiotics are characterized by increased osmotolerance, high colonization activity and the ability to adhere to epithelial cells, carry out the synthesis of amino acids, enzymes, participate in general metabolism, inhibit the development of pathogenic microflora, accelerate the process of digestion and assimilation of feed, contribute to the formation of local local immunity on the intestinal mucosa, eliminate immunodeficiency, develop iysya on the background of dysbacteriosis.

Claims (1)

A method of producing a biologically active additive, including introducing a culture of lactobacilli into a nutrient medium, cultivating, concentrating the bacterial mass by centrifugation, mixing the obtained bacterial mass with a sucrose-gelatin protective medium, followed by freeze-drying, characterized in that Lactococcus lactis subsp is used as a culture of lactobacilli. lactis III, Lactobacillus delbrueckii subsp. bulgaricus L 20/2, Lactobacillus acidophilus G-1, with Lactobacillus acidophilus G-1 and Lactobacillus delbrueckii subsp. bulgaricus L 20/2 in equal proportions to a nutrient medium containing clarified whey, yeast extract in an amount of 1.0 wt.%, manganese sulfate - 160 mg / l, potassium phosphate - 0.1 wt.%, cultivation is carried out at a temperature (44 ± 1) ° С, during cultivation, the pH of the medium is adjusted with gaseous ammonia, neutralization is carried out until pH 5.9-6.2 is reached, and after 2-3 hours from the start of cultivation, Lactococcus lactis subsp. lactis III and 1% of the volume of the nutrient medium skim milk and 40% glucose solution and continue to cultivate for 12-13 hours, an additional 0.1-0.5 wt.% potassium iodide is added to the protective medium, and freeze-drying lead within 24-28 hours to a residual moisture content of not more than 3%.