Method to produce probiotic preparation lacto-amylovorin

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.

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The invention relates to the field of biotechnology and concerns a method for obtaining probiotic lactoamilovorin on the basis of Lactobacillus amylovorus BT-24/88 (deposited in Russian national collection of industrial microorganisms under N-6253).

Probiotics are widely used in veterinary medicine. On the basis of probiotics, including lactoamilovorin create effective drugs and biologically active food additives that are used to combat goiter, prevention and treatment of various diseases of the digestive tract, improving and strengthening the body's defenses.

To date, there are a large number of probiotics, obtained using different species of enterobacteria (B.bifidum, Str.faecium, E. coli and others), Bacillus subtilis, Rumenococcus albus, and lactobacilli (Lactobacillus plantarum, fermentum, amylovorus).

Describes a method for the preparation Subtilis, with the content of the dispute and live vegetative cells of bacteria Bacillus subtilis BKM-D by aerobic cultivation at pH in the range of 6.8 to 7.2 with recharge environment 40% glucose (EN 2184774, 2002).

Also known is a method of obtaining dry probiotic preparation, including the cultivation in a liquid nutrient medium on the basis of enzymatic hydrolysate of casein strains of enterobacteria, such as, Bifidobacterium globosum BF-4, or Streptococcus falcium VGNKI-27 for 12-15 hours with subsequent cooling is receiving native culture fluid and its concentration in 10-15 times. The resulting concentrate is washed biomass of 2.5-3.5%solution of sucrose or lactose and dehydrated at a temperature of minus 25-35°C. this concentration of native culture fluid is conducted by the method of ultrafiltration hollow fibers, or by microfiltration on a flat membranes, or separation (EN 2065305, 1996).

A known method of producing Lactobacterin, including obtaining pure cultures of Lactobacillus fermenti and Lactobacillus plantarum, depositing them on the prepared nutrient medium, growing 8-10 h at 37°C and freeze drying of the drug (RU 2200566).

In the early nineties there were reports on the allocation of a new species chromalveolata lactobacilli Lactobacillus amylovorus (L.K. Nakamura Lactobacillus amylovorus, a new Starchhydrolyzing species from cattle wastecom fermentations. Int. I. Syst. Bacteriol., 1981, 31, N 1, p.56-63), J.Bacteriol, 2011, 3, No. 5, p.1, Microbial Drag Resistance 2000, V/12(4), p.284-288, however, information about the possibility of their use in industry is extremely small.

In 1992 he created the probiotic actonelbuy, inhibiting the digestive tract hemolytic bacteria and stimulating microorganisms, gidrolizuemye complex polysaccharides that increase enzyme activity in the small intestine, providing preventive and therapeutic effect of diarrhea. The drug is now widely used in veterinary medicine (Applied Microbiology 58,3355-3359, 1992). For produced by the production lactoamilovorin using Lactobacillus amylovorus L-471.

Described conditions bacteriocin - peptide with antibacterial activity against Pseudomonas aeruginosa produced by Lactobacillus amylovorus DCE 471, and is used as a preservative in foods and feed (Applied Microbiology, 66(2),606-613, 2000. Environ Let, 286, 199-206, 2008).

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

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

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

The strain is grown on liquid milk medium at 37°C for 24 hours. Schematic description of the technology of production of probiotic makes no assumptions about the quality of the target product, stability of results, which is important for its industrial use.

The purpose of this invention is to provide a method of producing lactoamilovorin on the basis of pure cultures of Lactobacillus amylovorus BT-24/88 to consistently obtain the target product with high quality is their values titer of anti-microbial bodies not only in the culture fluid, but in all forms of drug release (liquid and dry powder).

To achieve this goal were created liquid artificial environments on the basis of fermentolizate skimmed milk powder, allowing maximum use of the potential of Lactobacillus amylovorus BT-24/88 (table 1). It was also shown that the treatment of the culture liquid containing actonelbuy order to obtain it in the form of a dry powder, there is a significant reduction in the titer of anti-microbial bodies, which can be avoided by using protective environments with the addition of certain quantities of stabilizers, chosen by us experimentally. In addition, the release of the drug in liquid form also showed that in the process of storage is significantly lower titers of anti-microbial phone Us were selected stabilizers, added at the end of the process of biosynthesis of probiotic allows you to maintain a stable high titer of anti-microbial bodies in the production of a probiotic in the form of liquid (table 2).

Actonelbuy get using Lactobacillus amylovorus BT-24/88 with a broad spectrum of antagonistic activity against pathogenic and pathogenic microorganisms that are resistant to extreme environmental conditions of the digestive tract and antibiotics. The non-pathogenic strain, toxic the x and toxigenic properties does not show (U.S. Pat. EN 2054478, 1996).

Probiotic is produced by submerged cultivation of Lactobacillus amylovorus BT-24/88 in liquid artificial environment, which is based on hydrolyzed skim milk powder, obtained with the help of enzymes, which then add corn extract, lactose and essential mineral salts (environment No. 1, Wednesday 1-1) at a temperature of 38°C for 24 hours. After the process of biosynthesis to the culture fluid add stabilizer (sodium metabisulfite, hydrosulfite sodium, ascorbic acid), stirred for 10 minutes and produce the product in the liquid state or in the form of a dry powder. The lyophilized product obtained by freeze-drying of the culture fluid in conjunction with a protective environment and our chosen stabilizers.

The claimed method of obtaining a probiotic preparation differs from the prototype:

1. The composition of the liquid nutrient medium (which is based on hydrolyzed skim milk powder, which is the nitrogen source for cultured strain), which allows to obtain a product with high titer anti-microbial phone

2. The use of stabilizers, contributing to the preservation of this level in obtaining various forms of drug release and storage.

These differences allow to draw a conclusion on the conformity of the proposed technical solutions Crete is their "novelty".

The technical result obtained by carrying out the invention, is expressed in the possibility of a stable receiving probiotic with high titer anti-microbial bodies not only in the culture fluid, but after processing required to obtain various forms of drug release, and storing.

The claimed method of obtaining lactoamilovorin is illustrated by the following examples

Example 1.

Receiving the probiotic actonelbuy in liquid form.

Culture of Lactobacillus amylovorus BT-24/88 grown on medium MRS in test tubes at a temperature of 38°C for 24 h Grown culture is transferred into a flask containing 100 ml and forth to zoom in 1000 ml of culture medium (g/l: peptone - 10, enzymatic hydrolysate of casein - 5 KN2RHO46 ammonium citrate - 2, glucose - 10, twin-80 - 1, saline 5 ml, sodium acetate - 1, BCH - 100 ml, cabbage broth - 900 ml. of the composition of the salt solution (g/100 ml): MgSO4×7H2O - 11.5, MnSO4×2H2O - 2.4, FeSO4×7H2O - 0.68, distilled water - 100 ml pH of the medium - 6,8-7,0. Seed grown in flasks for 24 hours at a temperature of 38°C in thermostat and then used for inoculation of the fermenter. Sowing dose of 5-10%.

The cultivation is carried out at a temperature of 38°C under aseptic conditions without aeration, with constant stirring on a nutrient medium No. 2 trace is found of the composition, g/l:

Lactose - 30 g

Corn extract to 5 ml;

Yeast autolysate - 8 ml;

Chalk precipitated - 0.5 g;

Potassium phosphate one-deputizing - 2.0 g;

The sodium acetate 3-water and 3.0 g;

Magnesium sulfate as - 0.6 g;

Iron sulfate, 0.05 g;

Manganese sulphate 5-water - 0.05 g;

Tap water up to 1 L.

The duration of the process 24-26 hours at pH (6,9±0,1). After the end of the process to the culture fluid add sodium metabisulfite based 0.03 g/l and stirred for another 10 minutes. The number of viable cells of Lactobacillus is (3-5)×109CFU/ml

Example 2.

Getting probiotics lactoamilovorin in the form of a dry powder includes the following stages: preparation of inoculum, obtaining inoculum, the cultivation of the producer strain in the fermenter, Department of bacterial mass from the culture fluid, the preparation of a protective environment, mixing the bacterial mass with a protective medium, freezing, freeze drying of a suspension of biomass.

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

The fermentation process is conducted at a temperature of 38°C, pH (6,9±0,1) for 24 hours with continuous mixing without aeration in a nutrient medium No. 1 of the following composition, g/l:

Corn extract - 10;

Lactose - 20;

Chalk chemical is key besieged - 0,5;

Sodium citrate trehzameshchenny - 7,5;

The sodium acetate 3-water - 2;

Iron sulfate - 0.1;

Manganese sulphate 5-water - 0,16;

Hydrolyzed skim milk powder to 1 liter

Skimmed milk powder (30±1) g is dissolved in (250±50) ml of drinking water with a temperature of (43±2)°C, incubated for (45±15) minutes under stirring until complete dissolution. Reconstituted milk filter, add hot water to 1 l, temperature (55±1)°C, pH (6,8±0,1). In the prepared milk contribute enzyme it G3x or GH proteolytic activity of 70 u/g in the amount of (0,23±0,08) g/l, previously activated in a little water. Milk is made by the enzyme maintained at a temperature of (55±1)°C for (2±0,25)PM

800 ml of the obtained hydrolyzed skim milk powder add (7,5±0,05) g sodium citrate translesanas, (0,160±0,002 g of manganese sulfate, (20±0,05) g milk sugar (lactose), (2±0,01 g of sodium acetate, (0,1±0,001 g of iron sulfate, (0,5±0,01 g of PCC, (10±0,01) ml corn extract. Corn extract pre-sterilized at a temperature of (121±2)°C for (30±0,5) minutes, and before you make gidrolizovannogo milk neutralize a 30% solution of sodium hydroxide to pH (6,9±0,1). The mixture is stirred for 10 minutes, then add the guide is olisat skimmed milk powder to 1 liter.

The number of viable cells of Lactobacillus in the culture fluid is (1-5)×1010CFU/ml

Then the obtained culture liquid is used to produce different forms of release of the probiotic.

To obtain probiotic in powder form ready culture liquid is subjected to the separation.

After separation of the precipitate biomass, which is a paste of yellow-brown colour, mixed with a protective environment and a stabilizer (which is used as hydrosulfite sodium), in a ratio of 1 l of solution per 1 kg of biomass. To smooth the mixture is stirred for 20-25 minutes as a protective environment using the solution translesanas 5.5-water sodium citrate (50 g/l) and sucrose (100 g/l), hydrosulfite sodium (3 g/l). The solution is prepared with tap water, sterilize it in an autoclave and cooled to room temperature.

Received a creamy slurry is directed to freeze drying. Residual humidity is obtained the desired product 5%.

The content of viable cells of Lactobacillus amylovorus powder (3-5)×1011CFU/g

Example 3.

Obtaining inoculum, preparation of nutrient medium, cultivation, separation is conducted analogously to example 2, the growing strain spend on the environment, 1-1, and before drying oncentrate culture liquid is mixed with a protective environment and a stabilizer, which is used ascorbic acid at the rate of 5 g/L.

The content of viable cells of Lactobacillus amylovorus freeze-dried concentrate (5-8)×1011CFU/g

The composition of the medium 1-1:

Corn extract - 10;

Lactose - 20;

Chalk precipitated - 0,5;

Sodium citrate trehzameshchenny - 7,5;

The sodium acetate 3-water - 2;

Iron sulfate - 0.1;

Manganese sulphate 5-water - 0,16;

Hydrolyzed skim milk powder to 1 liter

(Hydrolyzed skim milk powder is obtained using proteolytic enzyme Alcalase 2,4L analogously to example 2).

APPLICATION

Table 1
Activity of Lactobacillus amylovorus BT - 24/88 on different environments:
The productivity of the strain CFU/ml
Wednesday 13×1010
Wednesday 25×109
Wednesday 32×109
Wednesday 48×108
Wednesday 1-14 x 10sup> 10

Environment # 2 (composition, g/l):

Lactose - 30 g;

Corn extract to 5 ml;

Yeast autolysate - 8 ml;

Chalk precipitated - 0.5 g;

Potassium phosphate one-deputizing - 2.0 g;

The sodium acetate 3-water and 3.0 g;

Magnesium sulfate as - 0.6 g;

Iron sulfate, 0.05 g;

Manganese sulphate 5-water - 0.05 g;

Tap water up to 1 L.

Environment No. 3 (composition, g/l)

Peptone - 10

Enzymatic hydrolysate of casein - 5;

KN2RHO4- 6;

The ammonium citrate - 2;

Glucose - 10;

Tween-80 - 1;

Sodium acetate - 1;

Saline solution - 5 ml (MgSO4×7H2O - 11.5, MnSO4×2H2O - 2.4, FeSO4×7H2O - 0.68, distilled water - 100 ml);

Cabbage broth - 900 ml.

Wednesday 4:

Skimmed milk powder - 12;

Yeast extract - 10;

Tap water up to 1 L.

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

Packing: Banks polymer.

Dark glass bottles.

Storage conditions. In the dark place at a temperature not higher than 12°C.

Date bookmark deposited. 20.02.2010,

The initial activity of the liquid form of 5×1010CFU/ml

The initial activity of the powder 5-8×1011CFU/g

Table 2
the Liquid formLyophilized powder
The titer of anti-microbial bodies CFU/mlThe titer of anti-microbial bodies, CFU/g
One year storageOne year storage
Without stabilizer5×1073×1075×1073×1072×10101×10102×10101×1010
With sodium metabisulfite6×1095×1095×1096×1095×10107×10107×10105×1010
Ascorbic acid5×1093×109 4×1093×1096×10105×10105×10105×1010
With hydrosulfite sodium3×1094×1093×1095×1097×10106×10105×10107×1010
Non series12341234

1. The method of receiving probiotic lactoamilovorin providing for the cultivation of Lactobacillus amylovorus VKPM B-6253 in a liquid nutrient medium and the preparation of the desired product from the resulting culture fluid in the liquid state or in the form of a dry powder using a stabilizer, characterized in that cultivation of lead in a nutrient medium containing:

10±0.01 ml
corn extract
lactose20±0.005 g
chalk precipitatedof 0.5±0.01 g
sodium citrate trehzameshchenny7,5±0.05 g
the sodium acetate 3-water2±0.01 g
iron sulfate0,1±0.001 g
manganese sulphate 5-water0,16±0,002 g
hydrolyzed skim milk powderto 1 l

2. The method according to claim 1, characterized in that to obtain hydrolyzed skim milk powder is used, it G3x, it GH or alcalase 2,4L.

3. The method according to claim 1, characterized in that the stabilizer is selected from: sodium metabisulfite, hydrosulfite sodium and ascorbic acid.



 

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