RU2432392C1 - METHOD TO PRODUCE PROBIOTIC PREPARATION BASED ON SPOROGENOUS STRAINS Bac subtillis AND Bac licheniformis - Google Patents

Abstract

FIELD: agriculture. ^ SUBSTANCE: method to produce a probiotic preparation based on sporogenous strains Bac.subtilis and Bac.licheniformis includes their cultivation, mixing of bacterial cells biomasses with protector of microbial cells and subsequent sterile dehydration. At the same time the method uses the strain Bac. subtilis VKPM V-10172 and the strain licheniformis VPKM V-10135. Biomasses of strains are mixed at the ratio of 1:1. A protector of microbial cells is a gelatine-saccharose protective medium in amount that provides for cell titre in the finished product after drying of at least 2X1012 CFU/g. ^ EFFECT: method makes it possible to produce a preparation with high efficiency of probiotic component, which ensures higher digestibility of fodders and increases weight gain of farm animals and birds. ^ 3 tbl, 1 ex

Description

The invention relates to biotechnology, in particular to methods for the production of probiotic preparations based on the bacterial strains Bac.subtilis and Bac.licheniformis. Such preparations can be used as a feed additive for the preparation of feed rations, designed to increase the digestibility of feed of farm animals and poultry.

A known 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, then the native culture is cooled to a temperature of 15-20 ° C and concentrated 10-15 times, the resulting concentrated biomass is washed with 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 in a semi-periodic mode of operation (see RF patent No. 2065305, publ. 08.20.1996).

However, this method has several significant disadvantages. Firstly, the need to use casein hydrolyzate significantly increases the cost of the drug. Secondly, the use of ultrafiltration on hollow fibers or microfiltration on flat membranes as a method of concentration makes this method expensive and requires a lot of investment and highly trained personnel. Also, the use of cryogenic dehydration technology significantly complicates the process. In addition, the use of only two types of bacteria as a mixed probiotic culture makes this mixed population unstable, which reduces the effectiveness of its use.

A known method of obtaining a dry probiotic preparation, which includes separate cultivation under conditions of deep cultivation of microorganisms Lactobacillus acidophilus and Rhuminococcus albus to a predetermined achievement of the titer of each microorganism, mixing the obtained uterine culture with sunflower meal in a predetermined ratio, with Bacillus subtilis being additionally used as microorganisms B-8130, from Lactobacillus acidophilus, Lactobacillus acidophilus Suav is used. VKPM B-4625, from Rhuminococcus albus - Rhuminococcus albus KR, while the grown uterine cultures are mixed in a 1: 1 ratio, autoclaved sunflower meal is seeded with it in the following ratio of components, wt.%:

autoclaved sunflower meal 45-55

uterine culture of Lactobacillus

acidophilus suav.

VKPM 4625,

Rhuminococcus albus kr 20-30 uterine culture of Bacillus subtilis VKPM B-8130 20-30

the mixture is incubated at 30-45 ° C for 4-8 days to obtain at least 1 × 10 ⁸ cells / g of the preparation in a semi-moist preparation, and the resulting preparation is mixed with sunflower meal in a ratio of 1: 10-1: 12 to obtain the preparation with a residual moisture content of 8-10%.

The closest way to the production of a probiotic additive is to mix the biomass of bacteria Bacillus subtilis B-2250 and / or Bacillus licheniformis B-2252 and excipients of the sorbent carrier and a moisture-sensitive filler. As a sorbent carrier, it contains aerosil of hydrophilic grade A and hydrophobic grade AM, a moisture-intensive filler — ion-exchange cation exchange resins of the KB-4P-2 and KU-2-8chs grades in a given ratio of components by weight of dry matter. A dry microencapsulated form of a probiotic additive is obtained by the method of capillary-sorption drying of a mixture of components to a moisture content in the finished product of 8-25%. The result is a microencapsulated form of a probiotic supplement containing a probiotic, convenient to use and use, capable of providing stability of properties at all stages of preparation (RU 2252956 C2, 2005).

However, the probiotic preparation obtained by the above method, although it has stable physicochemical properties, is not effective enough due to the low content of probiotic microorganisms. In addition, its production is energy intensive and difficult.

For this reason, there remains a need for highly effective probiotic preparations used as feed additives to increase the weight gain of agricultural animals and poultry.

The technical result of this invention is to increase the efficiency of the probiotic component of the resulting product, which provides increased digestibility of feed and increase the weight gain of animals.

This result can be realized using the described method for the production of a probiotic preparation based on spore-forming strains of You. subtilis and you. licheniformis, intended for use as a feed additive to the diets of agricultural animals and poultry, aimed at increasing the digestibility of feed, including the cultivation of strains of you. subtilis and you. licheniformis, mixing the biomass of bacterial cells with the protector of microbial cells and subsequent sterile dehydration, using the Bac strain. subtilis VKPM B-10172 and strain Bac. licheniformis VKPM B-10135, the biomass of the strains is mixed in a 1: 1 ratio, gelatin-saccharose protective medium is used as a protector of microbial cells in an amount that ensures the cell titer in the finished product after drying at least 2 × 10 ¹² CFU / g.

The production method uses new strains of Bac bacteria. subtilis and Bac. licheniformis. The strains are deposited with the GNI Genetics collection under registration numbers: Bac. subtilis VKPM B-10172 and Bac. licheniformis VKPM B-10135. Strains isolated from the natural community in the Kirov region. The following are the properties of each strain.

Bac. subtilis VKPM B-10172

Cultural and morphological features

The cells are straight, rod-shaped, 2-3 microns long, motile. Anaerobically form spores of an oval shape, which are located centrally in the cells. Gram stain - gram-positive. The strain grows well on meat-peptone agar (MPA), wort agar, Gromyko medium, potato agar, as well as on these media with the addition of 10 mg / ml kanamycin, forming shiny creased, creamy-colored colonies with uneven edges. On meat-peptone broth (BCH), the culture forms a uniform turbidity.

Physiological and biochemical characteristics

It ferments lactose, glucose, arabinose, xylose with the formation of acid without gas. Reduces nitrates, provides methylene blue. Coagulase, hyaluronidase, lecitase activity does not possess. It has lipase activity.

The culture is maintained on MPA and potato agar with the addition of kanamycin (50 μg / ml) with reseeding at least 1 time per month, stored on MPA and potato agar with the addition of 50 μg / ml kanamycin.

The optimum growth temperature is 37 ° C, pH is 7.0. The composition of the medium: peptone 5 g / l, yeast extract 3 g / l, NaCl 5 g / l, tap water - up to 1 l.

The optimum growth temperature is 37 ° C, pH is 7.0. The composition of the medium: K ₂ HPO ₄ × 3H ₂ O - 18.3 g / l; KH ₂ PO ₄ - 6.0 g / l; (NH ₄ ) ₂ SO ₄ - 2.0 g / l; NA ₃ C ₆ H ₅ O ₇ × 5.5H ₂ O - 1.2 g / L.

Bac. licheniformis VKPM B-10135

Cultural and morphological features

The cells are straight, rod-shaped, 2-3 microns long, motile. Anaerobically form spores of an oval shape, which are located centrally in the cells. Gram stain - gram-positive. The strain grows well on meat-peptone agar (MPA), wort agar, Gromyko medium, potato agar, as well as on these media with the addition of 10 mg / ml kanamycin, forming shiny cremated colonies of a creamy color with uneven edges. On meat-peptone broth (BCH), the culture forms a uniform turbidity.

Physiological and biochemical characteristics

It ferments lactose, glucose, arabinose, xylose with the formation of acid without gas. Reduces nitrates, provides methylene blue. Coagulase, hyaluronidase, lecitase activity does not possess. It has lipase activity.

The culture is maintained on MPA and potato agar with the addition of kanamycin (50 μg / ml) with reseeding at least 1 time per month, stored on MPA and potato agar with the addition of 50 μg / ml kanamycin.

The optimum growth temperature is 37 ° C, pH is 7.0. The composition of the medium: peptone 5 g / l, yeast extract 3 g / l, NaCl 5 g / l, tap water - up to 1 l.

The optimum growth temperature is 37 ° C, pH is 7.0. The composition of the medium: K ₂ HPO ₄ × 3H ₂ O - 18.3 g / l; KH ₂ PO ₄ - 6.0 g / l; (NH ₄ ) ₂ SO ₄ - 2.0 g / l; NA ₃ C ₆ H ₅ O ₇ × 5.5H ₂ O - 1.2 g / L.

The method is as follows. The biomass of B.subtilis and B.licheniformis is obtained by growing under sterile conditions, the biomass is separated in a centrifuge, the paste is mixed with a protective medium (gelatin-sucrose), poured and dried in a freeze dryer. The biomass of B.subtilis and B.licheniformis is obtained by cultivation in rocking flasks with a volume of 750 ml or in fermentation apparatus with a capacity of 500 l to 63 m ³ in a medium with glucose, peptone, corn extract. The culture fluid obtained in the fermentation process is centrifuged. Bacterial biomass and spores (in the form of a concentrated suspension) are mixed with a protective medium containing sucrose and gelatin, poured into penicillin vials of 3 ml and dried in a freeze dryer.

Example

Obtaining an inoculum of Bacillus subtilis and Bacillus licheniformis.

The bacterial culture of B.subtilis and B.licheniformis is stored in a slanted solid medium of MPA or timing No. 1 in test tubes closed with cotton plugs. The optimum storage temperature is 4-6 ° C. To maintain the culture in a viable state, once every 3 months, reseed on fresh nutrient medium MPA or timing is performed and grown at t = 37 ° C until spore formation 16-24 hours.

To obtain inoculum for sowing 100 flasks with a fermentation nutrient medium, from 1 to 5 flasks of the inoculum are prepared. To obtain the inoculum, 2 options are used:

- in a rocking flask with a volume of 750 ml with a liquid nutrient medium with a volume of 100 ml add a piece of 1 × 1 cm ^{2 of the} original culture from the beveled solid MPA agar;

- 0.5-1 ml of spore suspension, washed from two biological tubes with the original culture of 20 ml of physiological saline, is introduced into a 750 ml rocking flask with a 100 ml liquid nutrient medium.

Cultivation is carried out in a thermostatic shaker at N = 180-200 rpm or in fermentation apparatus with a capacity of 500 l to 63 m ³ , at t = 37 ° C for 16-24 hours for a Bacillus subtilis culture and for 40-48 hours for Bacillus licheniformis. The purity of the culture is checked by plating on Petri dishes with agarized MPA medium, followed by temperature control at t = 37 ° C. The absence of growth of extraneous microflora is determined visually by the type of colonies. In addition, the purity of the culture is determined microscopically. In the absence of extraneous microflora, the inoculum is used for inoculation of flasks with a fermentation medium.

Cooking environments

Preparation of a liquid nutrient medium for the inoculum.

The composition of the nutrient medium:

Peptone - 10 g / l

Glucose - 20 g / l

NaCl - 1 g / l

CaCl ₂ - 0.05 g / l

MgSO ₄ - 0.25 g / l

MnSO ₄ - 0.3 g / l

FeSO ₄ - 0.01 g / l

Corn extract - 30 g / l

The pH of the medium is 7.5 ± 0.1, adjusted with a 20% NaOH solution.

Poured into rocking flasks with chippers of 100-150 ml.

Sterilized in a steam sterilizer VK-75 at t = 121 ± 1 ° C for 25 minutes.

TP-2-2 Preparation of a liquid nutrient medium for fermentation.

The composition of the nutrient medium:

Peptone - 10 g / l

Glucose - 20 g / l

NaCl - 1 g / l

CaCl ₂ - 0.05 g / l

MgSO ₄ - 0.25 g / l

MnSO ₄ - 0.3 g / l

FeSO ₄ - 0.01 g / l

Corn extract - 30 g / l

The pH of the medium is 7.5 ± 0.1, adjusted with a 20% NaOH solution.

Poured into rocking flasks with chippers of 100-150 ml.

Sterilized in a steam sterilizer VK-75 at t = 121 ± 1 ° C for 25 minutes.

Preparation of a gelatin-sucrose protective medium for lyophilization of the product.

The composition of the nutrient medium:

Gelatin - 10 g / l

Sugar - 100 g / l

The medium is thoroughly mixed, poured into 100-150 ml vials and sterilized in a VK-75 steam sterilizer at t = 121 ± 1 ° C for 25 minutes.

Cultivation of Bacillus subtilis and Bacillus licheniformis.

In a rocking flask with chippers with a volume of 750 ml with a liquid nutrient medium with a volume of 100-150 ml, the inoculum is inoculated in an amount of 0.5-1 ml. Cultivation is carried out in a thermostatic rocking chair N = 200-220 rpm or in fermentation apparatus with a capacity of 500 l to 63 m ³ at N = 250-270 rpm with an air flow of 1: 1, at t = 37 ° C for 16 -24 hours for a culture of Bacillus subtilis and within 40-48 hours for a Bacillus licheniformis.

The end of the cultivation is determined by reaching a pH of 7.2-7.6 for Bacillus subtilis and a pH of 8.0-8.5 for Bacillus licheniformis by total glucose consumption and copious spore formation. The number of bacteria is determined by ten-fold dilutions with plating on Petri dishes. In 1 cm ^{3 of} culture fluid should be at least 1010 cells.

Processing the resulting culture suspension.

The obtained culture liquids B.subtilis and B.licheniformis are mixed by titer in a ratio of 1: 1. In order to concentrate the culture fluid, centrifugation is carried out. Centrifugation is carried out in a centrifuge at N = 6000-8000 rpm for 25 minutes The supernatant is collected in a neutralizer, disinfected at a temperature of 100 ° C for two hours, and then drained into the sewer. After collecting the paste, the centrifuge is treated with a 4% solution of chloramine, then washed with water.

The obtained biomass of B.subtilis and B.licheniformis cells with a moisture content of 60-80% (bacterial paste) (≈2 g with 100 ml of culture liquid) is discharged into enameled or plastic containers, mixed with a protector (gelatin-sucrose mixture) so that the dry product titer after drying was CFU / g 2 × 10 ¹² . Stirring until complete homogenization is carried out on a mixer at N = 1000 rpm, for 20-25 minutes Before bottling, the suspension is filtered through 4 layers of sterile gauze and poured into 3 ml vials.

Drying

Vials are placed on shelves of a freeze dryer.

Drying mode consists of three segments.

1 segment: freezing from room temperature to t = -40 ° C at a speed of 0.3, holding for 4-5 hours at this temperature.

2 segment: drying under vacuum at a temperature from t = -40 ° C to t = -20 ° C. Exposure at t = -20 ° C for 4-5 hours.

3 segment: drying under vacuum at a temperature from t = -20 ° C to t = + 30 ° C. Exposure at t = + 30 ° C for 0.5 hours.

After the end of 3 segments, the vacuum is released, the bottles are taken out and transferred to the box for capping.

The residual moisture of the drug tablet is not more than 4%.

Packaging of the finished product.

Vials with freeze-dried product are sealed with rubber stoppers and fixed with aluminum caps. Labels are glued on the vials with the drug, placed in cardboard boxes. Cardboard boxes are stacked in corrugated cardboard boxes. A label with the name of the drug and the date of manufacture is put in the box.

Dry biomass of B.subtilis and B.licheniformis is used to prepare a feed additive, which is used to increase the digestibility of feed due to improved digestion of farm animals, birds and fish in conditions of their industrial reproduction.

Examples.

- evaporation (0.5 × 10 ¹² CFU / fl)

- additive in premix (3.3 × 10 ¹¹ CFU / g)

- additive in compound feed (3.3 × 10 ⁹ CFU / g)

Age Norm healthy bird Recovery after antibiotics Vaccination Stress Relief Improving infection up to 5 days 1 bottle per day for 2500 goals, drinking 3-5 days 1 bottle per day for 1250 goals, drinking 3-5 days 1 bottle per day per 1000 heads, drinking 3-5 days 1 vial per day for 825 goals, drinking 3-5 days with 10% glucose solution 5-10 days 300 g per 1 ton of premix 500 g per 1 ton of premix 500 g per 1 ton of premix 750 g per 1 ton of feed 1000 g per 1 ton of feed A 1000 g per 1 ton of feed 10-20 days 175 g per 1 ton of premix 225 g per 1 ton of premix 200 g per 1 ton of premix 500 g per 1 ton of premix 350 g per 1 ton of feed 450 g per 1 ton of feed 400 g per 1 ton of feed 20-30 days 100 g per 1 ton of premix 200 g per 1 ton of premix 175 g per 1 ton of premix 1000 g per 1 ton of feed 3-5 days 200 g per 1 ton of feed 400 g per 1 ton of feed 350 g per 1 ton of feed

Norms of input (g / t) (3.3 × 10 ⁹ CFU / g) into feed and (3.3 × 10 ¹¹ CFU / g) into premix View Compound feed Premix Chickens 500 g / ton 250 g / ton Broilers 1000 g / ton 500 g / ton Furry animals 300 g / ton 150 g / ton Suckling pigs 400 g / ton 200 g / ton Fattening Pigs 300 g / ton 150 g / ton Sows 500 g / ton 250 g / ton

Turkeys 400 g / ton 200 g / ton Calves, foals 500 g / ton 250 g / ton

The rate of daily drinking 1 bottle (0.5 × 10 ¹² CFU) Kind of young Norm Daily Chickens 2500 goals Suckling pigs 10 goals Rabbits 10 goals Puppies of fur animals 15 goals Little kids, lamb 10 kg weight Calves 40 kg weight

The spores of the probiotic together with the feed enter the esophagus, then overcome the viable acidic environment of the stomach and, entering the alkaline environment of the small intestine, grow into vegetative cells, secreting a large number of digestive enzymes, which contribute to a more complete digestion and digestion of the feed.

At the same time, spores that have sprouted compete for nutrient substrates with pathogenic microflora and displace it from the intestine, thereby increasing the animal’s immune status and its protective barrier against infections. In the rectum, vegetative cells that have not completed metabolism sporulate and exit with feces, followed by a sanitizing effect of manure.

The effectiveness of the probiotic is enhanced by the fact that the additive contains aerobic B. subtilis and anaerobic B. licheniformis bacteria, while an aerobic bacterium works on the surface of the feed or mucous membrane in the presence of oxygen, anaerobic in volume of feed and inside the mucosa. However, unlike additives similar in composition, these bacteria are in the composition of the drug in a real ratio of 1: 1, which just guarantees the claimed powerful synergistic effect.

Six qualities of sporogenous probiotic making it a probiotic growth promoter:

- Producer of digestive enzymes (amylases, lipases, proteases).

- Antagonist of pathogens (competition for nutrient substrates).

- Saprotroph (antitoxic effect, lysis of decay products).

- Immunomodulator (macrophage activation, induction of endogenous interferon).

- Tolerance (supplements and / or replaces feed antibiotics, neutral in feed mixes and premixes).

- Stability (withstands high temperatures and granulation pressures in feed mills, unpretentious in storage.

Claims (1)

A method for the production of a probiotic preparation based on spore-forming strains of you. subtilis and you. licheniformis, intended for use as a feed additive to the diets of agricultural animals and poultry, aimed at increasing the digestibility of feed, including the cultivation of strains of you. subtilis and you. licheniformis, mixing biomass of bacterial cells with the protector of microbial cells, characterized in that they use the strain you. subtilis VKPM B-10172 and strain You. licheniformis VKPM B-10135, the biomass of the strains is mixed in a 1: 1 ratio, gelatin-saccharose protective medium is used as a protector of microbial cells in an amount that ensures the cell titer in the finished product after drying at least 2 × 10 ¹² CFU / g, then sterile dehydration is carried out.