RU2450051C1 - METHOD OF PRODUCING Escherichia coli VL-613 BASED SYMBIOTIC PREPARATION - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method of producing an Escherichia coli VL-613 based symbiotic preparation involves sowing, culturing bacteria in a liquid medium based on Hottinger broth, concentrating the bacterial suspension and then mixing with a stabiliser, packing and lyophilising the preparation. During the process of culturing Escherichia coli VL-613, redox potential of the bacterial culture is reduced to minus (80-100) mV right after sowing. Before the end of the culturing process, pO₂ is kept at 20±5% of saturation of air with oxygen and pH is kept in the 7.0-7.4 range. Glucose is fed in doses until achieving concentration of 0.1-0.2% while limiting growth of Escherichia coli VL-613 with glucose. The culturing process is 4-6 hours long. The preparation contains 80-85% living cells and enables to completely replace synthetic lysine in complete diets when growing broilers.

EFFECT: high quality and stability of the Escherichia coli VL-613 based symbiotic preparation.

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Description

The invention relates to biotechnology and zootechnology and can be used in the industrial production of a symbiotic preparation from a strain of Escherichia coli VL-613.

The symbiosis of birds and microorganisms plays an important role in the normal functioning of the bird organism and the realization of their genetic productivity potential.

The role of the microflora of the gastrointestinal tract in bird nutrition (synthesis of amino acids, vitamins, enzymes and other biologically active substances) is especially pronounced. Probiotics are one of the most fruitful ways of using beneficial forms of microorganisms in poultry farming. The effectiveness of the use of probiotics is not inferior to antibiotics (feed and veterinary use), but at the same time they do not have side effects on the bird's body and intestinal microflora, i.e. are environmentally friendly. Their use allows to obtain poultry products that do not contain residues of chemotherapeutic action and antibiotic preparations.

With intensive poultry farming under conditions of industrial technology for poultry keeping, biologically complete feeding is a decisive factor in obtaining high productivity.

At the same time, it is envisaged to provide the bird with not only high-quality protein and energy components, but also with limiting amino acids and other vital substances.

It is known that the lack of limiting amino acids cannot be compensated for by animal feed, the share of which in combined feeds for poultry is also decreasing, and the price for them is growing. To increase the usefulness of plant feeds, additives of synthetic amino acids are widely used.

Among the essential amino acids, lysine occupies a special place. It is part of the structural tissue proteins and protein enzymes, helps to improve digestion, plays an important role in the formation of the skeleton, increase productivity, has a beneficial effect on the reproductive functions of birds.

Currently, in many countries, when feeding productive animals and poultry, they use feed additives of bacteria that produce amino acids, including lysine. Preparations - producers of this amino acid, depending on the production technology, differ significantly from each other, primarily in terms of the concentration of the active substance. This circumstance becomes the main reason for developing the production technology of such drugs, which, when introduced into the gastrointestinal tract of animals and birds, produce one of the essential amino acids - lysine.

Symbiotics are products of biotechnological production that contain live microorganisms that produce amino acids (including essential ones), enzymes, vitamins in the gastrointestinal tract of animals and birds, and thus increase productivity.

Lyophilically dried E. coil baculture is a symbiotic preparation, and the same baculture applied to carriers (zeolite, glauconite and others) and dried by convection is a feed additive. The symbiotic preparation is applied by the method of evaporation, and not by adding to the feed.

Known methods for the cultivation of bacteria of the genus Escherichia, which are carried out on solid (1) and liquid (2) nutrient media. However, the method of cultivating Escherichia in dense nutrient media is not technological and unproductive - the culture of Escherichia is grown for 24 hours in Petri dishes on solid nutrient medium. Cultivation in liquid nutrient media is carried out without regulating the main technological parameters - the redox potential, the amount of oxygen dissolved in the culture fluid and lasting for 24-48 hours, due to which these methods are not suitable for the industrial preparation of the drug.

There is also a method of producing a feed additive containing a culture of a strain of Escherichia coli - producer of lysine (3). However, in this case, the cultivation of Escherichia is carried out in an uncontrolled mode - without regulating the redox potential, the amount of oxygen dissolved in the culture fluid, pH, for 6-12 hours, which does not allow to obtain a culture stable in biological properties.

The aim of the invention is to reduce the time of growing the culture, reducing the cost of the drug, as well as improving the quality and stability of obtaining a symbiotic drug based on Escherichia coli VL-613.

This goal is achieved due to the fact that periodic deep cultivation of Escherichia coli VL-613 is carried out in a nutrient medium for 4-7 hours in accordance with the developed process control algorithm, the resulting culture is concentrated, the bacterial mass is resuspended in a protective medium and freeze-dried for long-term preservation biological properties of the symbiotic drug.

The method is as follows.

In a sterile fermenter, which is equipped with an automatic control and regulation system for the main technological parameters (temperature, stirrer speed, pH, pО ₂ , eH), a liquid nutrient medium, Hottinger broth (prepared on the basis of the Hottinger digest with a content of 600-800 mg% of amine nitrogen, is loaded ) prepared according to the following recipe, wt.%:

Hottinger Brew 18.0-22.0 Peptone 0.8-1.2 Disubstituted sodium phosphate 0.7-0.9 Sodium chloride 0.7-0.9 Glucose 0.15-0.25 Yeast extract 0.4-0.6 Distilled water Up to 100.0

Ready sterile nutrient medium should contain 160-180 mg% of amine nitrogen and have a pH of 7.4-7.6 units. pH

An 18-24-hour matrix culture of Escherichia (Escherichia coli pcs. VL-613) inoculated in a liquid medium according to the composition similar to the culture medium in a ratio of 5-10% of the volume of the medium in the fermenter is inoculated into the fermenter with the nutrient medium, and cultured at (37 ± 1) ° C for 4-7 hours.

After inoculation of the fermenter, the redox potential (eH) of the culture fluid is reduced to (-100) - (-80) mV by holding the culture without supplying air to the aeration and the mixer turned off, after which until the cultivation process is completed by changing the air flow to aeration and the stirrer rotation speed maintains the partial pressure of dissolved oxygen (pO ₂ ) in the culture fluid at the level of (20 ± 5)% of the oxygen saturation of the air, the pH of the culture fluid is regulated at the level of (7.2-7.4) units of pH with a supply of 10% - N solution aOH, and a fractional supply of glucose is carried out in doses up to a concentration of (0.1-0.2)% when limiting the growth of Escherichia glucose, which is characterized by a sharp increase in pO ₂ with constant air flow and revolutions of the mixer and stopping the decrease in pH of the culture fluid.

The total concentration of Escherichia at the end of cultivation is (16-30) billion cubic meters / cm ³ .

The resulting bacterial culture is concentrated, the precipitate is mixed with a protective drying medium. After thorough mixing, the bacterial suspension mixed with the protective drying medium is packaged under aseptic conditions in sterile vials and lyophilized.

From one cultivation in a 10 L fermenter, a symbiotic preparation is obtained for feeding 45,000 broilers.

Example 1

The method of obtaining the drug with the minimum values of the parameters

eN of the culture fluid is reduced to - 100 mV; PO _{2 is} maintained at 15%; The pH in the culture fluid is adjusted to 7.2 units. pH fractional supply of glucose is carried out in doses up to a concentration of 0.1%.

Cultivation - within 4 hours.

Accumulation - 16.0 billion cubic meters / cm ³ .

Example 2

The method of obtaining the drug with optimal parameter values

eN of the culture fluid is reduced to - 90 mV; PO _{2 is} maintained at 20%; pH in the culture fluid is adjusted at 7.3 units. pH fractional supply of glucose is carried out in doses up to a concentration of 0.15%.

Cultivation - within 5 hours.

Accumulation - 23.0 billion cubic meters / cm ³ .

Example 3

The method of obtaining the drug with the maximum values of the parameters

eN of the culture fluid is reduced to - 80 mV; PO _{2 is} maintained at 25%; The pH in the culture fluid is adjusted to 7.4 units. pH fractional supply of glucose is carried out in doses up to a concentration of 0.2%.

Cultivation - within 6 hours.

Accumulation - 30.0 billion cubic meters / cm ³ .

Example 4

The method of obtaining the drug with parameter values below the minimum

eN of the culture fluid is reduced to -110 mV; PO _{2 is} maintained at 10%; The pH in the culture fluid is adjusted to 7.1 units. pH fractional supply of glucose is carried out in doses up to a concentration of 0.05%.

Cultivation - within 3 hours.

Accumulation - 9.0 billion cubic meters / cm ³ .

Example 5

The method of obtaining the drug with parameter values above the maximum

eN of the culture fluid is reduced to - 70 mV; PO _{2 is} maintained at 30%; The pH in the culture fluid is adjusted at 7.5 units. pH fractional supply of glucose is carried out in doses up to a concentration of 0.25%.

Cultivation - within 7 hours.

Accumulation - 37.0 billion cubic meters / cm ³ .

The technological parameters of the manufacture of a symbiotic preparation from the strain Escherichia coli VL-613, indicated in the examples, are shown in table 1.

Table 1 The values of the technological parameters of the periodic cultivation of Escherichia Cultivation time, hour eN, mV pH units pO ₂ ,% The amount of glucose,% Accumulation, billion / cm ³ Minimum parameter values 3 -one hundred 7.2 fifteen 0.1 16,0 Optimal parameter values four -90 7.3 twenty 0.15 23.0 Maximum parameter values 5 -80 7.4 25 0.2 30,0 Values below minimum 6 -110 7.1 10 0.05 9.0 Parameter values above maximum 7 -70 7.5 thirty 0.25 37.0

The preparation prepared according to examples 1-3 after freeze-drying contains 80-85% of viable cells and meets the technical requirements, while the preparation prepared according to examples 4 and 5 contains 25-30% of viable cells and is unsuitable for use.

When broilers were grown on complete diets in which synthetic lysine (lysine monohydrochloride) was replaced with a symbiotic preparation from the Escherichia coli strain VL 613, an increase in the productivity of chickens due to improved digestibility and the use of food nutrients was noted.

To determine the effectiveness of replacing crystalline lysine (lysine monohydrochloride) with a lysine producer based on the Escherichia coli strain VL613 in the VNITIP experimental farm, experiment was conducted on broilers of the Cobb-500 cross breed from 7 days old to 5 weeks old according to the scheme shown in table 2 .

For the first seven days, the chickens of all groups received the same granular compound feed with nutrition according to the Cobb-500 cross-country standards; later, loose feed was used according to the experimental design.

table 2 Experience outline Groups Number of goals Feeding characteristics The control 35 Basic diet with synthetic lysine (lysine monohydrochloride) Experienced 35 The main diet - without the addition of synthetic lysine, but with the addition of a symbiotic drug

The research results are presented in table 3.

Table 3 Indicators Groups The control Experienced one 2 3 Live weight, g daily chicken fifty fifty Live weight in the group, g 194.1 + 43.54 204.06 ± 4.02 in 7 days 380.5 ± 7.99 403.86 ± 7.03 in 14 days 754.00 ± 17.63 813.86 ± 17.78 at 21 days 1312.29 ± 28.80 1454.00 ± 29.10 in 28 days 2092.70 ± 45.84 2212.66 ± 43.80 in 38 days 2107.00 ± 46.17 2483.6 ± 44.79 Live weight in 38 days, including cockerels 2024.30 ± 66.19 2299.75 ± 53.19 chickens 1786.28 ± 52.90 2067.50 ± 56.65 The safety of the livestock,% one hundred one hundred Feed consumption per 1 goal., Kg 3,59 3.48 The cost of feed per 1 kg of growth, kg 1.74 1,61 The average daily gain in live weight, g 54.1 59.1

As can be seen from table 3, the live weight of the experimental broilers exceeded the control. Moreover, it was significantly higher than the control over the entire broiler growing cycle. The studied drug allows for the synthesis of lysine in the body of chickens in the most critical period of life, when the digestive system is most vulnerable and immaturity. The use of the drug did not adversely affect the safety of broilers and feed conversion.

Thus, the use of a symbiotic preparation based on Escherichia coli VL-613 when feeding broilers highly productive crosses allows the synthetic lysine (lysine monohydrochloride) to be completely replaced.

Information sources

1. RF patent No. 2175351, С12Р 13/04, C12N 1/20, C12N 15/31, C12N 1/20, C12R 1/185, 01/27/2001.

2. RF patent No. 2189253, A61K 39/108, 04/09/2001.

3. RF patent No. 2347807, C12N 1/20, A33K 1/14, C12R 1/185, 02.27.2009.

Claims (1)

A method of obtaining a symbiotic preparation from Escherichia coli VL-613, including inoculation, cultivation in a liquid nutrient medium based on the Hottinger digest, concentration of the bacterial suspension, followed by mixing with a stabilizer, packaging and lyophilization of the drug; in the process of cultivation of Escherichia coli VL-613 immediately after sowing, the redox potential of the bacterial culture is reduced to minus (80-100) mV, after which, until the end of the cultivation process, pO _{2 is} maintained at the level of (20 ± 5)% of air oxygen saturation , the pH is regulated at the level of (7.0-7.4), and the fractional supply of glucose is carried out in doses to a concentration of (0.1-0.2)% with limitation of the growth of Escherichia coli VL-613 glucose, the duration of the cultivation process is 4-6 hours