RU2496865C1 - Method to prepare bacterial preparation of acidophilic cultures - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method provides for preparation of a nutrient medium on the basis of a dry nutrient medium MEDIA TW-50 with application of a salt additive into it, such as dibasic sodium phosphate at the specified ratio. The nutrient medium is sterilised, cooled down to the specified temperature and neutralised, with 20% solution of caustic soda, with establishment of the specified pH value of the medium to produce the nutrient medium. The acidophilic culture of the strain 126 and yeast autolysate are introduced at the specified ratio into the produced nutrient medium with subsequent cultivation. At the same time in process of cultivation the acidity of the nutrient medium is maintained with staged deoxidisation by the 20% solution of the caustic soda. The bacterial mass is cooled and separated. The bacterial mass is mixed with the protective medium containing saccharose, gelatine and water, where additionally lactulose syrup is introduced, as a symbiotic protective factor, ascorbic acid, as an anti-enzyme agent, talc powder as a drying and sliding agent at the specified ratio. It is homogenised, poured, frozen and dried with a sublimation method.

EFFECT: invention makes it possible to increase survival rate of acidophilous bacteria under sublimation and storage of a preparation and to reduce hygroscopicity of a finished preparation.

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Description

FIELD OF THE INVENTION

The invention relates to the dairy industry, microbiology, medicine, in particular, to a method for preparing a bacterial preparation of acidophilic culture and can be used to obtain lactic acid microorganisms in the production of biologically active food additives for the prevention of dysbiosis, intestinal dysfunction in children and adults, as well as in farm animals .

State of the art

A known method of producing bacterial starter culture for the production of dairy products by ed. No. 23290, while in order to increase the activity of the obtained bacterial concentrate and speed up the process. Before mixing, sterile skim milk with a solids content of 15-25% is additionally introduced into the protective medium. and mixing the biomass with a protective medium is carried out in a ratio of 1: 4, followed by saturation of the resulting mixture with air to a foamy suspension and freezing.

In the method, the suspension is frozen before drying to (-50) - (-60) ° C, and the drying process is carried out at an initial temperature of (-40) - (-42) ° C and a final temperature of 45-50 ° C, and the drying time is 6 -10 hours (see AS S. SU No. 581701, IPC C12K 3/00, A23C 9/12, publ. 09/25/1979).

The disadvantage of this method is that it does not have high acid-forming properties and has a high cost.

A known method of preparing a bacterial preparation of an acidophilic culture, which provides for the preparation of a nutrient medium based on clarified whey with the addition of salt additives, neutralization of the medium, stage-by-stage deoxidation of the medium during cultivation with acidophilic culture by setting the pH value and feeding with glucose solution, cooling, separation of the bacterial mass mixing it with a protective medium containing sucrose, water and gelatin, freezing and drying, while as a salt howl additives use sodium phosphate disubstituted, neutralization of the medium is carried out with an aqueous solution of ammonia to a pH of 6.2-6.8, moreover, glucose solution of 10% concentration is used for feeding, and freezing and drying are carried out in a two-stage mode (see US Pat. RU 2064269 , IPC A23C 9/12, A23C 21/02, C12N 1/20, publ. 07.27.1996).

The disadvantage of this method and the drug obtained by this method are: not wide range of therapeutic effects, since it is only an effective probiotic, high hygroscopicity of the drug, which allows it to be Packed only in liquid form in bottles and freeze-dried in this container, which significantly reduces consumer qualities of the drug, excludes the possibility of packaging in capsules and other forms of packaging.

The closest in technical essence and the achieved positive effect and adopted by the authors for the prototype is a method of preparing a bacterial preparation of acidophilic culture of strain 12b with reduced hygroscopicity, providing for the preparation of a nutrient medium based on clarified whey with the addition of salt additives, which use sodium phosphate disubstituted, neutralization medium, introduction of acidophilic culture yeast strain 12b, yeast autolysate and gradual deoxidation during cultivation of an acidophilic culture with the establishment of a given pH of the medium, cooling the bacterial mass, mixing with a protective medium containing lactulose syrup as a symbiotic and protective factor, 0.4% ascorbic acid as an anti-enzyme agent, 3% pharmacopoeial powder as drying and sliding agents, homogenization, bottling, freezing and freeze drying. In 1 g of the obtained preparation contains at least 5 × 10 ¹⁰ cells of acidophilic rods of strain 12b (see application RU No. 2009106106 A, publ. 08.27.2010).

The disadvantage of this method is the low growth and antibiotic activity of bacteria.

Disclosure of invention

The objective of the invention is to develop a method for the preparation of a bacterial preparation of an acidophilic culture with symbiotic factors, high antibiotic and acid-forming activity, a significant decrease in the hygroscopicity of the finished preparation to a level that allows packing it into gelatin capsules, bags of multilayer polymer material and other containers, increasing the survival of acidophilic sticks in the process of sublimation and storage of the drug, which significantly increases consumer achestva drug.

The technical result that can be obtained using the present invention is reduced to high antibiotic and acid-forming activity, reducing the hygroscopicity of the finished product, increasing the survival of acidophilic sticks in the process of sublimation and storage of the drug.

The technical result is achieved using the method of preparing a bacterial preparation of acidophilic cultures, characterized by the preparation of a nutrient medium based on dry MEDIA TW-50 nutrient medium with the addition of a salt additive, which is used as sodium phosphate disubstituted, neutralization of the medium with a 20% sodium hydroxide solution, with the establishment a given value of the pH of the medium, the introduction of the starter culture of acidophilic strain 12b and yeast autolysate and cultivation, while in the process of culturing acidity support by gradual deoxidation with a 20% sodium hydroxide solution, cooling the bacterial mass, mixing with a protective medium containing sucrose 9-10%, gelatin 1-1.5%, water - the rest, and additionally add lactulose syrup 9-10%, as a symbiotic and protective factor, 0.4% ascorbic acid as an anti-enzyme agent, 2.8-3% pharmacopeia talcum powder as a drying and sliding agent, homogenization, filling, freezing and freeze-drying.

Thus, the technical result is achieved due to the fact that in the method of preparing a bacterial preparation of acidophilic cultures, yeast autolysate, lactulose syrup, ascorbic acid, caustic soda, pharmacopeia talcum powder in predetermined quantities are additionally used, which allows to increase the activity of the nutrient medium and obtain in 1 g concentrate 6,5 × ¹⁰ September CFU acidophilus strain rods 12b, thus making yeast autolysate into the culture medium increases the content of amino nitrogen, trace elements, vitamins Ms cultivation bones, which intensifies the reproduction and development of acidophilus rods of strain 12b, additional introduction of lactulose syrup into the protective environment, gives a powerful symbiotic factor, while it does not hydrolyze and is not absorbed in the stomach, but enters the lower intestines, where, being a nutrient medium, useful microflora, for example, lactobacilli, including strain 12b and bifidobacteria, breaks down with the formation of low molecular weight, acetic, lactic and other acids, which leads to inhibition of putrefactive microflora, at et m, intestinal biocenosis is normalized and the general condition of the body, the added ascorbic acid is a strong anti-enzyme agent, which increases the survival of acidophilic sticks during freeze-drying and storage, the introduction of caustic soda to deoxidize the nutrient medium allows the process to be carried out in a gentle manner, as well as increase the survival of acidophilus sticks, the introduction of powdered talc of pharmacopeia facilitates the process of packing the dried preparation, for example, in gelatin capsules, bags of many gosloynogo metal-polymer material, and other containers, and the use for preparing a nutrient medium - medium MEDIA TW-50 (Fig. Certificate of state registration No. 77.99.26.9. U.1071.2.10 of February 25, 2010. Dry nutrient medium MEDIA TW-50 for the cultivation of starter microflora; Products manufactured by Danisco Deutschland GmbH. Busch-Johannsen-Str, 1D-25899 Niebull, Germany, Danisco A / S Edwin Rahrs Vej 38, DK 8220 Bradrand, Denmark (A / S Syntetic, Handvaerkervej 6 DK-6270 Tonder), Denmark; for use in the dairy industry in the production of starter cultures in the production of fermented milk products and cheeses) speeds up and cheapens the process of preparing a bacterial preparation of acidophilic cultures.

The essence of the method of preparation of a bacterial preparation of acidophilic cultures is as follows:

4.5-5.0 kg of MEDIA TW-50 medium and a salt additive, which is used as 650-700 g of sodium phosphate disubstituted, are added to 95 l of water, the volume is brought up to 100 l, the mixture is sterilized at a temperature of 120-122 ° C for 18-20 minutes, cooled to a temperature of 44-45 ° C and set the pH value to 6.2-6.6 by introducing a 20% sodium hydroxide solution, then 2.5-3.0 kg of acidophilic acid is introduced into the prepared nutrient medium cultures of strain 12b, 0.4-0.5 L of yeast autolysate and cultured at a temperature of 44-45 ° C for 11.0-12.0 hours, while ki are maintained during cultivation flow close to a pH value of 6.2 by deoxidation with a 20% sodium hydroxide solution, after cultivation, the medium is cooled to 21-22 ° C and the bacterial mass is separated in an amount of 1 kg, which is then mixed under sterile conditions with 6.8-7.0 l of a protective medium containing sucrose 9-10%, gelatin 1-1.5%, water - the rest, and additionally add lactulose syrup 9-10% as a symbiotic and protective factor, 0.4% ascorbic acid as an anti-enzyme agent, 2.8-3% pharmacopoeial talcum powder as a drying agent and a sliding agent, homogenization, bottling, freezing and drying, the mass is homogenized, poured into sterile trays, frozen and freeze-dried.

The implementation of the invention

Examples of specific performance of the method of preparation of a bacterial preparation of acidophilic cultures.

Example 1. 4.0 kg of MEDIA TW-50 medium and a salt supplement, 600 g of sodium phosphate disubstituted, are added to 95 l of water, the volume is adjusted to 100 l, the mixture is sterilized at a temperature of 115 ° C for 15 min, cooled to a temperature of 42 ° C and establish a pH value of 6.2 - by introducing a 20% sodium hydroxide solution, then 2.0 kg of acidophilic culture of strain 12b, 0.4 l of yeast autolysate are introduced into the prepared nutrient medium and cultivated at a temperature of 44 ° C for 11.0-12.0 hours, while in the process of cultivation maintain acidity b, close to a pH value of 6.2, by deoxidation with a 20% sodium hydroxide solution, after cultivation, the medium is cooled to 21 ° C and the bacterial mass is separated in an amount of 1 kg, which is then mixed under sterile conditions with 6, 8 l of a protective medium containing sucrose 8%, gelatin 0.5%, water - the rest, and additionally add lactulose syrup 8%, as an antioxidant - 0.35% ascorbic acid, as a drying and gliding agent - 1.5 % pharmacopoeial talcum powder, the mass is homogenized, poured into ster silt trays, freeze and freeze-dry, drying time is 38 hours

Result: the obtained preparation in this way has a low antibiotic and acid-forming activity, high hygroscopicity of the finished preparation, low survival of acidophilus sticks during sublimation and storage of the drug, 1 g of the preparation contains 3.5 × 10 ⁹ CFU of acidophilus sticks.

Example 2. Carried out analogously to example 1, but on 95 l of water 4.5 kg of MEDIA TW-50 medium and a salt additive, which is used as 650 g of sodium phosphate disubstituted, are added, the volume is brought up to 100 l, the mixture is sterilized at a temperature of 120 ° C for 18 min, cooled to a temperature of 44 ° C and set the pH value to 6.2 by introducing a 20% sodium hydroxide solution, then 2.5 kg of acidophilic culture of strain 12b, 0.4 l of yeast autolysate are introduced into the prepared culture medium and cultivated at a temperature of 44 ° C for 11.0 hours, while in the process of cultivation They maintain acidity close to a pH value of 6.2 by deoxidation with a 20% sodium hydroxide solution, after cultivation, the medium is cooled to 21 ° C and the bacterial mass is separated in an amount of 1 kg, which is then mixed under sterile conditions with 6.8 l of a protective medium containing 9.0% sucrose, 1% gelatin, water - the rest, and additionally add 9% lactulose syrup, 0.35% ascorbic acid as an antioxidant, 2 as a drying and sliding agent , 8% pharmacopoeial talcum powder, mass g homogenize, pour into sterile trays, freeze and freeze-dry, drying time is 35 hours

Result: the obtained preparation in this way has high antibiotic and acid-forming activity, low hygroscopicity of the finished preparation, high survival of acidophilus sticks during sublimation and storage of the drug, 1 g of the preparation contains at least 6.5 × 10 ⁹ CFU of acidophilic sticks.

Example 3. Carried out analogously to example 2, but on 95 l of water, 5.0 kg of MEDIA TW-50 medium and a salt additive, which is used as 700 g of sodium phosphate disubstituted, are added, the volume is brought up to 100 l, the mixture is sterilized at a temperature of 122 ° C for 20 min, cooled to a temperature of 45 ° C and set the pH to 6.6 by introducing a 20% sodium hydroxide solution, then 3.0 kg of acidophilic culture of strain 12b, 0.5 L of yeast autolysate are introduced into the prepared culture medium, and cultivated at a temperature of 45 ° C for 12.0 hours, while in the process of cultivation cations maintain acidity close to a pH value of 6.2 by deoxidation with a 20% sodium hydroxide solution, after cultivation, the medium is cooled to 22 ° C and the bacterial mass is separated in an amount of 1 kg, which is then mixed under sterile conditions with 7.0 l of a protective medium containing 10.0% sucrose, gelatin 1.5% and water - the rest, and additionally add 10% lactulose syrup, 0.40% ascorbic acid as an antioxidant, as an absorbent, drying and glidant 4.0% talcum powder pharmaceutical opeynogo, the mass is homogenized, bottled in sterile trays, frozen and freeze-dried, the drying process duration is 35 hours.

Result: the preparation obtained by this method has high antibiotic and acid-forming activity, low hygroscopicity of the finished preparation, high survival of acidophilus sticks during sublimation and storage of the drug, 1 g of the preparation contains at least 6.5 × 10 ⁹ CFU of acidophilic sticks.

Example 4. Carried out analogously to example 3, but on 95 l of water, 5.5 kg of MEDIA TW-50 medium and a salt additive, which use 750 g of sodium phosphate disubstituted are used, bring the volume to 100 l, the mixture is sterilized at a temperature of 122 ° C for 25 min, cooled to a temperature of 45 ° C and set the pH to 6.6 by introducing a 20% sodium hydroxide solution, then 3.5 kg of the acidophilic culture of strain 12b, 0.5 L of yeast autolysate are introduced into the prepared nutrient medium, and cultivated at a temperature of 45 ° C for 12.0 hours, while in the process of cultivation They maintain an acidity close to pH 6.2 by deoxidation with a 20% sodium hydroxide solution, after cultivation, the medium is cooled to 22 ° C and the bacterial mass is separated in an amount of 1 kg, which is then mixed under sterile conditions with 7.0 l of a protective medium containing sucrose 11.0%, gelatin 2%, water - the rest, and additionally add 11% lactulose syrup, 0.40% ascorbic acid as an anti-enzyme, as an absorbent, drying and gliding agent 3.0% Pharmacopowder Talc Powder ynogo, the mass is homogenized, bottled in sterile trays, frozen and freeze-dried, the drying process duration is 40 hours.

Result: in 1 g of the drug obtained by this method, contains at least 5 × 10 ⁹ CFU of acidophilic sticks, as well as the number of introduced components and drying time increased significantly, which led to an increase in cost.

Thus, the most optimal are examples 2 and 3 of the preparation of a bacterial preparation of acidophilic cultures.

The invention in comparison with the prototype and other known technical solutions has the following advantages:

- a bacterial preparation of an acidophilic culture obtained using the proposed method has symbiotic factors;

- high antibiotic and acid-forming activity;

- a significant reduction in the hygroscopicity of the finished product;

- increased survival of acidophilus rods in the process of sublimation and storage of the drug;

- a significant expansion of consumer properties of the drug made using this method.

- use for the prevention of dysbiosis, intestinal dysfunction in children and adults, as well as in farm animals.

- cost reduction.

Claims (1)

A method of preparing a bacterial preparation of acidophilic cultures, characterized in that a nutrient medium is prepared on the basis of MEDIA TW-50 dry nutrient medium with the addition of a salt additive, which is used as sodium phosphate disubstituted, neutralization of the medium with a 20% sodium hydroxide solution with a predetermined value pH of the medium, introduction of acidophilic culture starter culture of strain 12b and yeast autolysate, cultivation, while in the process of culturing the acidity is maintained by phased deoxidation with a 20% sodium hydroxide solution, cool the bacterial mass, mix with a protective medium containing sucrose, gelatin and water, additionally add lactulose syrup as a symbiotic and protective factor of 0.4% ascorbic acid as an anti-enzyme agent, 2.8- Pharmacopeia 3% talcum powder as a drying and sliding agent is homogenized, poured, frozen and freeze-dried.