RU2475535C1 - Method to produce probiotic preparation lacto-amylovorin - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: probiotic lacto-amylovorin is produced by means of depth growth of the strain Lactobacillus amylovorus RNCIM B-6253 on the liquid nutrient medium, which contains the following: corn extract - 10 ± 0.01 ml, lactose - 20 ± 0.005 g, chemically deposited chalk - 0.5 ± 0.01 g, triple-substituted citric-acid sodium - 7.5 ± 0.05 g, 3-water acetous sodium - 2 ± 0.01 g, iron sulfate - 0.1± 0.001 g, 5-water manganese sulfate - 0.16 ± 0.002 g, hydrolysate of dry nonfat milk - up to l. Subsequent extraction of the target product in liquid condition or in the form of a dry powder is carried out with preliminary addition of a stabiliser to a cultural liquid or to a protective medium used when producing the probiotic in the form of the dry powder. The stabiliser is selected from: sodium metabisulfite, sodium hydrosulfite and ascorbic acid.

EFFECT: invention provides for stable production of a probiotic with high values of a titre of antimicrobial bodies not only in a cultural liquid, but also after its treatment required for production of different pharmaceutical forms of a preparation.

3 cl, 2 tbl, 3 ex

Description

The invention relates to the field of biotechnology and relates to a method for producing a probiotic of lactoamilovorin based on a strain of Lactobacillus amylovorus BT-24/88 (deposited in the All-Russian collection of industrial microorganisms under N В-6253).

Probiotics are widely used in veterinary medicine. On the basis of probiotics, including lactoamilovorin, they create effective medicines and biologically active food supplements that are used to combat dysbiosis, prevent and treat various diseases of the digestive tract, increase and strengthen the body's defenses.

To date, a large number of probiotics are known that are obtained using various types of enterobacteria (B. bifidum, Str.faecium, E. coli, etc.), Bacillus subtilis, Rumenococcus albus, as well as lactobacilli (Lactobacillus plantarum, fermentum, amylovorus).

A method is described for producing the preparation Subtilis with the content of spores and live vegetative cells of bacteria Bacillus subtilis BKM B-2250D by aerobic cultivation at pH of the medium in the range of 6.8-7.2 with feeding of the medium with 40% glucose (RU 2184774, 2002).

There is also known a method of obtaining a dry probiotic preparation, including growing in a liquid nutrient medium based on enzymatic hydrolyzate of casein strains of enterobacteria, for example, Bifidobacterium globosum BF-4 or Streptococcus falcium VGNKI-27 for 12-15 hours, followed by cooling of the native culture fluid and its concentration by 10-15 times. The resulting biomass concentrate is washed with a 2.5-3.5% solution of sucrose or lactose and dehydrated at a temperature of minus 25-35 ° C. In this case, the concentration of the native culture fluid is carried out by ultrafiltration on hollow fibers, or by microfiltration on flat membranes, or by separation (RU 2065305, 1996).

A known method of producing lactobacterin, including obtaining pure cultures of Lactobacillus fermenti and Lactobacillus plantarum, applying them to the prepared nutrient medium, growing for 8-10 hours at a temperature of 37 ° C and freeze drying of the drug (RU 2200566).

In the early nineties, there were reports of the isolation of a new type of starch-hydrolyzing lactobacilli Lactobacillus amylovorus (Nakamura LK Lactobacillus amylovorus, a new Starchhydrolyzing species from cattle wastecommentation. Int. I. Syst. Bacteriol., 1981, 31, N 1, p.56, p. 56, ), J. Bacteriol, 2011, 3, No. 5, p. 1, Microbial Drag Resistance 2000, V / 12 (4), p. 284-288, however, there is very little information about the possibility of their use in industry.

In 1992, the probiotic lactoamilovorin was created, which inhibits hemolytic bacteria in the digestive tract and stimulates microorganisms that hydrolyze complex polysaccharides, increase the enzymatic activity in the small intestine, and have a prophylactic and therapeutic effect in case of diarrhea. The drug is currently widely used in veterinary medicine (Applied Microbiology 58.3355-3359, 1992). For the production of lactoamilovorin, a strain of Lactobacillus amylovorus L-471 is used.

The conditions for the preparation of bacteriocin, a peptide with antibacterial activity against Pseudomonas aeruginosa produced by the Lactobacillus amylovorus DCE 471 strain and used as a food and feed preservative, are described (Applied Microbiology, 66 (2), 606-613, 2000. Microbiol Let, 286 , 199-206, 2008).

The main disadvantage of these methods is the significant length of the process of obtaining various forms of release of probiotics, leading to the loss of their activity.

In 1996, the possibility of obtaining lactoamilovorin using the Lactobacillus amylovorus BT-24/88 strain, deposited in the All-Russian collection of industrial microorganisms under N В-6253 (Pat RU 2054478, 1996) was shown.

This method of obtaining lactoamilovorin based on a strain of Lactobacillus amylovorus BT-24/88 is the closest to the claimed.

The strain is grown in liquid milk at a temperature of 37 ° C for 24 hours. A schematic description of the technology for producing probiotic does not give an idea about the quality indicators of the target product, the stability of the results, which is important for its industrial use.

The aim of this invention is to provide a method for producing lactoamilovorin based on a pure culture of the strain Lactobacillus amylovorus BT-24/88, which allows to stably obtain the target drug with high titer of antimicrobial bodies not only in the culture fluid, but also in all forms of drug release (liquid and dry powder).

To achieve this goal, liquid artificial media were created on the basis of skimmed milk powder fermentolizate, which made it possible to maximize the potential of the Lactobacillus amylovorus BT-24/88 strain (Table 1). It was also shown that when processing a culture fluid containing lactoamilovorin in order to obtain it in the form of a dry powder, there is a significant decrease in the titer of antimicrobial bodies, which can be avoided by using protective media with the addition of certain amounts of stabilizers that we selected experimentally. In addition, the release of the drug in the form of a liquid also showed that during storage there is a significant decrease in the titer of antimicrobial bodies. We selected stabilizers, the addition of which at the end of the probiotic biosynthesis process allows us to stably maintain a high level of antimicrobial body titer when the probiotic is released in the form of a liquid (Table 2).

Lactoamilovorin is obtained using the Lactobacillus amylovorus BT-24/88 strain, which has a wide spectrum of antagonistic activity against opportunistic and pathogenic microorganisms and is resistant to extreme environmental conditions of the digestive tract and antibiotics. The strain is non-pathogenic, toxic and toxigenic properties does not show (Pat. RU 2054478, 1996).

The probiotic is obtained by deep cultivation of the strain Lactobacillus amylovorus BT-24/88 in a liquid artificial medium, the basis of which is a skimmed milk powder hydrolyzate obtained by enzymes, into which corn extract, lactose and necessary mineral salts are then added (medium No. 1, medium 1 -1) at a temperature of 38 ° C for 24 hours. After the biosynthesis process is completed, a stabilizer is added to the culture fluid (sodium metabisulfite, sodium hydrosulfite, ascorbic acid), stirred for 10 minutes and the product is isolated in liquid or dry powder. A dry preparation is obtained by lyophilization of the culture fluid together with a protective medium and the stabilizers we have chosen.

The claimed method for producing a probiotic preparation differs from the prototype:

1. The composition of the liquid nutrient medium (the basis of which is the hydrolyzed skim milk powder, which is a source of nitrogen for the cultured strain), which allows to obtain a drug with a high level of antimicrobial titer.

2. The use of stabilizers that contribute to maintaining this level upon receipt of various forms of release of the drug and during its storage.

These differences allow us to conclude that the claimed technical solutions meet the criterion of "novelty."

The technical result obtained by carrying out the invention is expressed in the possibility of stable production of a probiotic with high titer of antimicrobial bodies not only in the culture fluid, but also after its processing, necessary to obtain various forms of release of the drug, and during storage.

The inventive method for producing lactoamilovorin is illustrated by the following examples

Example 1

Obtaining the probiotic lactoamilovorin in liquid form.

The culture of Lactobacillus amylovorus BT-24/88 is grown on MRS in test tubes at a temperature of 38 ° C for 24 hours. The grown culture is transferred to flasks containing 100 ml and further for scaling of 1000 ml of culture medium (g / l: peptone - 10, casein enzymatic hydrolyzate - 5, KH ₂ PO ₄ - 6, ammonium citrate - 2, glucose - 10, tween-80 - 1, saline solution - 5 ml, sodium acetate - 1, MPB - 100 ml, cabbage broth - 900 ml.Salt composition (g / 100 ml): MgSO ₄ × 7H ₂ O - 11.5, MnSO ₄ × 2H ₂ O - 2.4, FeSO ₄ × 7H ₂ O - 0.68, distilled water - 100 ml, pH of the medium - 6, 8-7.0. Seeds in flasks are grown 24 hours and at a temperature of 38 ° C in a thermostat and further used for inoculation of the fermenter.The sowing dose is 5-10%.

Cultivation is carried out at a temperature of 38 ° C in aseptic conditions without aeration, with constant stirring in a nutrient medium No. 2 of the following composition, g / l:

Lactose - 30 g

Corn extract - 5 ml;

Yeast autolysate - 8 ml;

Chemically precipitated chalk - 0.5 g;

Monosubstituted potassium phosphate - 2.0 g;

Sodium acetic acid 3-aqueous - 3.0 g;

Magnesium sulphate heptahydrate - 0.6 g;

Iron sulfate - 0.05 g;

5-aqueous manganese sulfate - 0.05 g;

Tap water up to 1 liter.

The duration of the process is 24-26 hours at pH (6.9 ± 0.1). After the process is complete, sodium metabisulfite at the rate of 0.03 g / l is added to the culture fluid and stirred for another 10 minutes. The number of viable cells of lactobacilli is (3-5) × 10 ⁹ CFU / ml.

Example 2

Obtaining a probiotic of lactoamilovorin in the form of a dry powder includes the following steps: preparation of seed, obtaining an inoculum, growing a producer strain in a fermenter, separating the bacterial mass from the culture fluid, preparing a protective medium, mixing the bacterial mass with a protective medium, freezing, freeze-drying a biomass suspension.

Obtaining seed in test tubes and flasks analogously to example 1.

The fermentation process is carried out at a temperature of 38 ° C, pH (6.9 ± 0.1) for 24 hours with continuous stirring without aeration on nutrient medium No. 1 of the following composition, g / l:

Corn extract - 10;

Lactose - 20;

Chemically precipitated chalk - 0.5;

Sodium citrate trisubstituted - 7.5;

Sodium acetic acid 3-aqueous - 2;

Iron sulfate - 0.1;

5-aqueous manganese sulfate - 0.16;

Hydrolyzed skim milk powder - up to 1 liter.

Skimmed milk powder (30 ± 1) g is dissolved in (250 ± 50) ml of drinking water with a temperature of (43 ± 2) ° С, maintained for (45 ± 15) min with stirring until completely dissolved. The reconstituted milk is filtered, hot water is added up to 1 liter, temperature (55 ± 1) ° С, pH (6.8 ± 0.1). The protosubtilin G3x or G10x enzyme with proteolytic activity of 70 u / g in the amount of (0.23 ± 0.08) g / l, previously activated in a small amount of water, is introduced into the prepared milk. The milk with the enzyme added is kept at a temperature of (55 ± 1) ° С for (2 ± 0.25) hours.

In 800 ml of the obtained skim milk powder hydrolyzate, add (7.5 ± 0.05) g of trisubstituted sodium citrate, (0.160 ± 0.002) g of manganese sulfate, (20 ± 0.05) g of milk sugar (lactose), (2 ± 0 , 01) g sodium acetate, (0.1 ± 0.001) g iron sulfate, (0.5 ± 0.01) g chemically precipitated chalk, (10 ± 0.01) ml of corn extract. Corn extract is pre-sterilized at a temperature of (121 ± 2) ° C for (30 ± 0.5) min, and before being added to hydrolyzed milk, it is neutralized with a 30% sodium hydroxide solution to pH (6.9 ± 0.1). The mixture is stirred for 10 minutes, then the hydrolyzed skim milk powder is added to 1 liter.

The number of viable lactobacilli cells in the culture fluid is (1-5) × 10 ¹⁰ CFU / ml.

The resulting culture fluid is then used to produce various forms of probiotic release.

To obtain a probiotic in powder form, the prepared culture fluid is subjected to separation.

At the end of the separation, the resulting biomass sediment, which is a yellow-brown paste, is mixed with a protective medium and a stabilizer (which is used as sodium hydrosulfite), in a ratio of 1 liter of solution per 1 kg of biomass. To obtain a homogeneous mass, the mixture is stirred for 20-25 minutes. As a protective medium, a solution of trisubstituted 5.5-aqueous sodium citrate (50 g / l) and sucrose (100 g / l), sodium hydrosulfite (3 g / l) are used. The solution is prepared in tap water, sterilized in an autoclave and cooled to room temperature.

The resulting creamy suspension is sent for freeze drying. The residual moisture content of the target product 5%.

The content of viable Lactobacillus amylovorus cells in the powder is (3-5) × 10 ¹¹ CFU / g.

Example 3

The preparation of seed, preparation of a nutrient medium, cultivation, separation is carried out analogously to example 2, the strain is grown on medium 1-1, and before drying, the culture fluid concentrate is mixed with a protective medium and a stabilizer, which is used ascorbic acid at a rate of 5 g / l .

The content of viable cells of Lactobacillus amylovorus in freeze-dried concentrate (5-8) × 10 ¹¹ CFU / g

The composition of the medium 1-1:

Corn extract - 10;

Lactose - 20;

Chemically precipitated chalk - 0.5;

Sodium citrate trisubstituted - 7.5;

Sodium acetic acid 3-aqueous - 2;

Iron sulfate - 0.1;

5-aqueous manganese sulfate - 0.16;

Hydrolyzed skim milk powder - up to 1 liter.

(The hydrolyzate of skimmed milk powder is obtained using the proteolytic enzyme Alkalase 2,4L as in example 2).

APPENDIX

Table 1 Data on the activity of the strain Lactobacillus amylovorus BT - 24/88 on various media: CFU / ml strain productivity Wednesday 1 3 × 10 ¹⁰ Wednesday 2 5 × 10 ⁹ Wednesday 3 2 × 10 ⁹ Wednesday 4 8 × 10 ⁸ Wednesday 1-1 4 × 10 ¹⁰

Wednesday No. 2 (composition, g / l):

Lactose - 30 g;

Corn extract - 5 ml;

Yeast autolysate - 8 ml;

Chemically precipitated chalk - 0.5 g;

Monosubstituted potassium phosphate - 2.0 g;

Sodium acetic acid 3-aqueous - 3.0 g;

Magnesium sulphate heptahydrate - 0.6 g;

Iron sulfate - 0.05 g;

5-aqueous manganese sulfate - 0.05 g;

Tap water up to 1 liter.

Wednesday No. 3 (composition, g / l)

Peptone - 10

Casein Enzymatic Hydrolyzate - 5;

KH ₂ PO ₄ - 6;

Ammonium citrate - 2;

Glucose - 10;

Twin-80 - 1;

Sodium acetate - 1;

Saline solution - 5 ml (MgSO ₄ × 7H ₂ O - 11.5, MnSO ₄ × 2H ₂ O - 2.4, FeSO ₄ × 7H ₂ O - 0.68, distilled water - 100 ml);

Cabbage broth - 900 ml.

Wednesday 4:

Skimmed milk powder - 12;

Yeast extract - 10;

Tap water up to 1 liter.

Data on the storage of lactoamilovorin in accordance with OST 42-2-72.

Packing: Polymer cans.

Bottles of dark glass.

Storage conditions. In the dark place at a temperature of no higher than 12 ° C.

Bookmark date for storage. 02/20/2010

The initial activity of the liquid form is 5 × 10 ¹⁰ CFU / ml.

The initial activity of the powder is 5-8 × 10 ¹¹ CFU / g.

table 2 Liquid Release Form Lyophilized powder Antimicrobial titer, CFU / ml Antimicrobial titer, CFU / g After a year of storage After a year of storage Without stabilizer 5 × 10 ⁷ 3 × 10 ⁷ 5 × 10 ⁷ 3 × 10 ⁷ 2 × 10 ¹⁰ 1 × 10 ¹⁰ 2 × 10 ¹⁰ 1 × 10 ¹⁰ With sodium metabisulfite 6 × 10 ⁹ 5 × 10 ⁹ 5 × 10 ⁹ 6 × 10 ⁹ 5 × 10 ¹⁰ 7 × 10 ¹⁰ 7 × 10 ¹⁰ 5 × 10 ¹⁰ With ascorbic acid 5 × 10 ⁹ 3 × 10 ⁹ 4 × 10 ⁹ 3 × 10 ⁹ 6 × 10 ¹⁰ 5 × 10 ¹⁰ 5 × 10 ¹⁰ 5 × 10 ¹⁰ With sodium hydrosulfite 3 × 10 ⁹ 4 × 10 ⁹ 3 × 10 ⁹ 5 × 10 ⁹ 7 × 10 ¹⁰ 6 × 10 ¹⁰ 5 × 10 ¹⁰ 7 × 10 ¹⁰ Series Numbers one 2 3 four one 2 3 four

Claims (3)

1. A method of obtaining a probiotic of lactoamilovorin, comprising cultivating a strain of Lactobacillus amylovorus VKPM B-6253 in a liquid nutrient medium and preparing the target product from the obtained culture fluid in a liquid state or in the form of a dry powder using a stabilizer, characterized in that the cultivation is carried out in a nutrient medium, containing:
corn extract 10 ± 0.01 ml lactose 20 ± 0.005 g chemically precipitated chalk 0.5 ± 0.01 g trisubstituted sodium citrate 7.5 ± 0.05 g sodium acetate 3-aqueous 2 ± 0.01 g iron sulfate 0.1 ± 0.001 g 5-manganese sulfate 0.16 ± 0.002 g skimmed milk powder hydrolyzate up to 1 l 2. The method according to claim 1, characterized in that to obtain the hydrolyzate of skimmed milk powder use protosubtilin G3x, protosubtilin G10x or alkalase 2.4 L. 3. The method according to claim 1, characterized in that the stabilizer is selected from: sodium metabisulfite, sodium hydrosulfite and ascorbic acid.