RU2541454C1 - Nutrient culture medium for swine erysipelas strain erysipelothrix rhuisipathie - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: nutrient culture medium for swine erysipelas strain Erysipelothrix rhuisipathie refers to general biotechnology and veterinary microbiology. As a nitrogenous nutrition source, the nutrient medium contains a mixture of fish autolysate and alkaline mussel hydrolysate in the following proportions: alkaline mussel hydrolysate 20-50%; fermentation peptone 1%; potassium phosphate 0.3%; sodium phosphate 1.8%; fish autolysate - the rest.

EFFECT: method can be used for preparing the microbiological nutrient media for erysipelas strain upstream.

Description

The invention relates to general biotechnology and veterinary microbiology and can be used to prepare microbiological culture media for increasing the biomass of the strain of the pathogen of pig erysipelas.

The causative agent of swine erysipelas Erysipelothrix rhuisipathie is a demanding crop. When the strain is activated after long-term storage, the rate of biomass growth is of great importance. As a rule, traditional nutrient media based on meat raw materials are used for scientific and commercial purposes. The meat and peptone broth MPB and ΜΠΑ from the Hottinger pass provide good growth of microorganisms without changing their cultural and biological properties. Sprat and fish meal hydrolysates are also widely used in the production of dry culture media for the cultivation of microorganisms, as proteins of fish in their structure and amino acid composition are most similar to meat proteins and are able to meet the needs of microorganisms in organic nitrogen compounds. Nevertheless, the search for high-quality and inexpensive nutrient media is an urgent task.

Known Nutrient medium for the cultivation of microorganisms (see patent No. 2089609, C1, C12N1 / 20, RU), containing a source of nitrogen nutrition, sodium chloride, agar. As nitrogen nutrition, pancreatic hydrolyzate of fish meal with amine nitrogen content of 4.4 ± 0.2%, total nitrogen of 12, ± 0.5% is used. A significant drawback is that the hydrolyzate has a high cost, due to the need for import from abroad. The closest in combination of features to the claimed invention is “Lively medium“ Aquamed ”for cultivating microorganisms” (div. Pat. No. 34711 U, C12N1 / 20, UA), where the nutrient medium is a mixture of an alkaline hydrolyzate from mollusks with an acid hydrolyzate from fish raw materials in a ratio of 3: 1 - 1: 3. The final concentration of amine nitrogen was 60-120 mg%. The composition of the medium also includes glucose or maltose, peptone and agar. A disadvantage of the known medium is insufficient for the cultivation of the strain of the pathogen of pig erysipelas, the final concentration of amine nitrogen.

Signs that are common with the essential features of the invention. A nutrient medium for cultivating a strain of the causative agent of swine erysipelas Erysipelothrix rhuisipathie, is that the composition of the medium includes enzyme peptone, as well as alkaline hydrolyzate from marine raw materials.

The basis of the invention A nutrient medium for the cultivation of a strain of the pathogen of pigs Erysipelothrix rhuisipathie tasked with the use of marine products to construct a nutrient medium that will ensure the optimal growth of the strain of the pathogen of pigs Erysipelothrix rhuisipathie for the preparation of the vaccine.

This object is achieved in that for the cultivation of a vaccine strain of the causative agent of swine erysipelas Erysipelothrix rhuisipathie prepare a nutrient medium consisting of a fish autolysate, which is additionally injected with 20-50% alkaline mussel hydrolyzate.

Sprat mussel hydrolyzate and autolysate are obtained using known technologies (see Krutchensky G.V., Kulyasova V.E. On the issue of obtaining protein hydrolysates from mollusk ices // Izvestiya TINRO.- 1976.- 99.- P. 102- 104); Likhvar N.A. Dry culture media from fresh and frozen waste from the fishing industry. Scientific notes DagNIVI for the production of nutrient media. Makhachkala, 1956, 2.).

The authors investigated the level of biomass formation of the industrial strain of the pathogen of pig erysipelas Erysipelothrix rhuisipathie No. 286 on various media (Table 1).

As can be seen from the table. 1 culture showed on all mediums, except the reference one, a low growth rate, which indicates the exactingness of the nutritional conditions. In some media, polymorphism of swine erysipelas pathogens was also noted: elongated chains of E.rhuisipathie appeared, which is a sign of unfavorable culturing conditions, in particular due to unbalanced nutrition.

Next, media were studied which, in addition to sprat hydrolysates, contained from O to 50% mussel hydrolyzate (Table 2). The strain was cultured under standard temperature conditions for 48 hours. The optical density was measured on a photoelectrocalorimeter at a wavelength of 545 nm and a cell thickness of 5 mm. The control sample was an inoculated medium of the same composition.

As can be seen from the table. 2, the addition of a mussel hydrolyzate to the nutrient medium in an amount of 20% of the volume ensured the doubling of biomass both in the reference medium (0.163 against 0.081) and in the experiment (0.130 against 0.063), and the addition of 50% of the mussel hydrolyzate to the medium tripled the growth. At the same time, the difference between the experimental and reference medium, despite the absence of glucose in the test medium, gradually decreased with an increase in the content of mussel gyrolyzate.

Due to the high content of amino nitrogen (an indicator of the nutrient content of media for growing microorganisms), seafood hydrolysates are a valuable raw material for biotechnological industries. The autolysate of fish contains 4-10 times more amine nitrogen than is necessary for the productive growth of commercial strains. Mussel hydrolyzate, when its content in the medium is not more than 20%, doubles the bacterial mass. Thus, the combination of fish autolysate and alkaline mussel hydrolyzate allows you to get a balanced nutrient medium for the cultivation of the pathogen of erysipelas of pigs.

An example implementation of the method. Prepared an experimental series of vaccines against swine erysipelas Erysipelothrix rhuisipathie No. 286 in the amount of 3 DM ³ . It was preliminarily determined that the pH of the fish hydrolyzate was 6.0, and the bmict of nitrogen nitrogen was -740 mg%. The mussel hydrolyzate has a pH of 7.7 and a nitrogen amine content of 247 mg%. From these components, liquid and solid nutrient media were prepared according to standard prescriptions for BCH. The composition of the medium for cultivating an industrial strain on the proposed nutrient medium included a mixture of fish autolysate and 20% by volume alkaline mussel hydrolyzate, enzyme peptone - 1% of the total volume, potassium phosphate - 0.3%, sodium phosphate - 1, 8%, pH the medium was 7.3-7.4. The strain was seeded in test tubes with liquid medium, then transferred to vials with broths, and after cultivation under appropriate conditions, transferred to vials with the same medium and cultured under appropriate conditions. At the end of the cultivation, the typical and purity of the culture was determined by microscopy of smears from the media and the concentration of microbial cells was established.

The culture of the industrial strain of the pathogen of pig erysipelas obtained on media from hydrolysates, compared with media obtained from raw meat, did not differ in cultural and morphological properties and met the requirements of the passport data for the strain. The concentration of microbial cells in 1 cm should be at least 50 million. The authors obtained the following data: on seafood nutrient media, the concentration of microbial cells was 50 million, and on media made from Hottinger broth - 70 million.

Inactivation of microbial cultures was carried out using formaldehyde at a concentration of 0.4%. Verification of the completeness of inactivation was carried out by plating the obtained vaccine on nutrient media microscopically stained with Gram smears. The vaccine was tested for harmlessness on 5 white mice weighing 18-20 g, injecting them 0.3 cm3 intraperitoneally. The results of monitoring the animals for 16 days showed that the vaccine is harmless: the experimental animals remained alive, fed normally, and no changes in behavior were noted.

Control of immunogenic activity was carried out on the same white mice after vaccination with the vaccine. Mice were infected with a control strain of the causative agent of erysipelas in a lethal dose. All control mice (10 animals) that were not vaccinated died within 4-6 days, all vaccinated survived for 10 days. Based on the results of these studies, it was concluded that the vaccine manufactured is immunogenic active.

Thus, the claimed nutrient medium using products of marine origin, provides the growth of the strain of the causative agent of swine erysipelas Erysipelothrix rhuisipathie, suitable for the preparation of an effective vaccine.

Claims (1)

Nutrient medium for cultivating a strain of the causative agent of swine erysipelas Erysipelothrix rhuisipathie, which includes enzyme peptone, potassium phosphate, sodium phosphate, and a hydrolyzate from marine raw materials, characterized in that a mixture of fish autolysate and alkaline mussel hydrolyzate is used as a nitrogen source ratio of components:
alkaline mussel hydrolyzate 20-50% peptone enzymatic one% potassium phosphate 0.3% sodium phosphate 1.8% fish autolysate rest.