RU2384613C2 - Nutritional medium for cultivating bacteria - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: nutritional medium contains glucose, yeast extract, sodium chloride and for protein basis solution of milt broth is used in the ratio of 1:2 (liquid milt broth : distilled water) or 4.45 g of dry milt broth per 1 l of distilled water in the assigned ratio.

EFFECT: invention enables to increase stability of the nutritional medium and accelerate bacteria cultivation terms.

1 tbl, 6 ex

Description

The invention relates to general and medical microbiology and can be used in scientific research and practical work for the bacteriological diagnosis of a number of opportunistic microorganisms.

Currently, there are a sufficient number of nutrient media for the cultivation of microorganisms: meat-peptone broth, liquid Hottinger medium, with different contents of amine nitrogen, cardiac cerebrum, etc. Recently, casein-soya mediums, tryptone-soya and trypcase-soya broths, have become widespread. As a rule, the nutrient medium consists of a protein and carbohydrate base, as well as additional components that enhance the growth of microorganisms: vitamins, amino acids, mineral components. For the preparation of a protein base, meat, fish broths or protein hydrolysates are most often used. The main source of raw materials for the production of such broths is meat, fish, slaughter blood, heart, etc. (Protein hydrolysates in the production of culture media. - M.: All-Russian Research Institute of Control Systems, 1990, 50 p.).

A known nutrient medium for the cultivation of conditionally pathogenic bacteria of the genus Listeria. It consists of a protein base, which is used meat-peptone broth, a solution of sodium chloride and water (French patent No. 2671561, CL C12Q 1/04).

The disadvantage of the above nutrient media is the lack of amino acid composition, which leads to an uneven growth of such microorganisms as bacteria belonging to the genus Listeria. In addition, these environments are expensive and, as a rule, imported, therefore, they are not available to a wide network of practical laboratories involved in the diagnosis of conditionally pathogenic microorganisms. In this regard, the development of simple, inexpensive, nutrient media for the cultivation of bacteria, in particular Listeria, Yersinia and the like, is very relevant.

The closest technical solution to the claimed invention, in terms of features, is a nutrient medium based on trypticase-soy broth with yeast extract ("HiMedia LabLtd", India (http://www.himedialabs.ru/ml417/), including a protein base from casein hydrolyzate (17.0 g / l) and papain broth of soy flour (3.0 g / l), a carbohydrate base in the form of glucose (2.5 g / l), as well as sodium chloride (5.0 g / l ), potassium hydrogen phosphate (2.5 g / l), yeast extract (6.0 g / l). The price of this nutrient medium is about $ 30.

The reasons that impede the achievement of the specified technical result when using the known medium adopted for the prototype is that its protein base contains an amino acid composition insufficient for stable cultivation of bacteria. Therefore, to prepare this nutrient medium, one has to use such expensive ingredients as a hydrolyzate of casein and papain broth of soy flour, as a result, this medium is multicomponent, difficult to prepare, expensive and inaccessible to practical laboratories.

The objective of the invention is to increase the stability and growth rate of bacteria by providing a constant amino acid composition in the nutrient medium, reducing its cost, expanding the raw material base for preparing the protein base of the nutrient medium through the use of fish waste, and availability in practical laboratories.

The specified technical result in the implementation of the invention is achieved due to the fact that in the preparation of a nutrient medium for the cultivation of bacteria as a protein base, use a solution of decoction from fish milk in a ratio of 1: 2 (liquid decoction from fish milk: distilled water) or 4.45 g of dry decoction of fish milk per 1 liter of distilled water, with the following ratio of components, g / l:

yeast extract 10.0 glucose 1,0 sodium chloride 5,0 broth solution from fish milk up to 1 l

The pH of the medium is 7.2-7.4.

The use of a decoction of fish milk in the proposed nutrient medium as the protein base determines the rapid and stable growth of microorganisms due to the content of a sufficient amount of amino acids and trace elements in it. Fish milk is an affordable, inexpensive source of raw materials. They contain nucleoproteins, consisting of nucleic acids and simple proteins - protamines and histones. Protamines and histones are built from amino acids, among which arginine, lysine and histidine predominate; protamines include tyrosine, tryptophan, serine, valine and proline, and histones include serine, valine, alanine, phenylalanine and tyrosine. The milk of a number of fish contains such water-soluble vitamins: B ₁₂ in amounts from 7 to 50 μg%; B ₁ (thiamine) - 50-185 mcg%; In ₂ (riboflavin) - 40-600 mcg%; In _s (folic acid) - 125-1300 mcg%; RR (nicotinic acid) - 1.1-5.7 mg%, etc. (V.P. Zaitsev, I.V. Kizevetter, L.L. Lagunov and others. "Technology of fish products. M: Food industry, 1965, p. 73-74). The chemical composition of chum milk of the 6th stage of maturity is reflected in table No. 1 (Yu.I. Kasyanenko, T. N. Pivnenko. “Comparative physicochemical characteristics of low molecular weight deoxyribonucleic acid (DNA) from marine hydrobionts” - “Izvestiya TINRO”, 1999, vol. 125, p. 152-158).

Table 1 The chemical composition of milk of chum salmon of the 6th stage of maturity,% Name of substance Content Deoxyribonucleic Acid (DNA) 12.0 Ribonucleic Acid (RNA) 0.3 Acid soluble products 0.1 Protein 8.7 Water 78.5 Other 0.4

When preparing a protein base for a nutrient medium, you can use a decoction of fish milk in both liquid and dry form. At the same time, the qualitative and quantitative composition of the nutrient medium should be considered essential features of the proposal, as the values of the content of components in the claimed nutrient medium are optimal and determine the relative balance of the cost of the medium with its growth characteristics. Going beyond the declared values of the components leads either to an overexpenditure of raw materials, or to a decrease in its growth properties.

Preparation of a nutrient medium: a decoction of fish milk is prepared in advance. To do this, take 500 g of milk of various types of fish (salmon, cod, herring, etc.), which are pre-crushed, add distilled water in a ratio of 1: 2. Boil for 1 hour. Then it is filtered and sterilized with a pressure of 1 atm for 30 minutes. The broth can be prepared for future use, stored in a household refrigerator at a temperature of + (4-8) ° C, up to six months, or dried, and then used as necessary. Dry broth from fish milk can be stored for a long time in a household refrigerator or at room temperature, it can be transported to other regions, while its quality is preserved.

The resulting broth contains a wide range of amino acids that play an important role in the metabolism of bacteria.

The nutrient medium can be prepared both on the basis of a liquid decoction of fish milk, and on the basis of a dry decoction of fish milk.

Preparation of a nutrient medium based on a liquid decoction of fish milk.

The protein base for the nutrient medium is prepared by diluting a liquid decoction of fish milk with distilled water in a ratio of 1: 2. Then, to 10 g of yeast extract, 1 g of glucose, 5 g of sodium chloride, add up to 1 l of the protein base of the nutrient medium. The mixture is stirred. The resulting culture medium is poured into containers, a well-known method. Using 20% NaOH, the pH of the nutrient medium in each tank was adjusted to 7.2-7.4, and then sterilized at 0.5 atm for 20 minutes.

The resulting culture medium is a clear amber-colored liquid, odorless.

To determine the growth properties of the claimed nutrient medium prepared on the basis of a liquid decoction of the milk of various fish species, the following bacterial test strains were used: Listeria monocytogenes NCTC 10527 (4b serovariant); L. monocytogenes CLIP 75936 (1/2 serovariant); L. ivanovii NCTC 11846; L. innocua SLCC 3379; L. seeligeri SLCC 5921; L. grayi; Yersinia pseudotuberculosis 2781; Y. enterocolitica 164; Escherihia coli.

The research results are presented in table.2.

The number of bacteria cultured in the claimed nutrient medium and in the prototype medium was determined by the Koch plate method after daily cultivation of bacteria at a temperature of 37 ° C. As can be seen from the table, the growth properties of bacterial test strains when cultured in a nutrient medium based on trypticase-soy broth (prototype) and in the inventive nutrient medium prepared on the basis of a decoction of milk of various fish species are stable. During the day of cultivation, the number of bacteria increases by 9 orders of magnitude. Thus, the growth properties of the claimed environment are not inferior to the prototype.

Preparation of a nutrient medium based on a dry broth of fish milk.

The mass of dry broth from fish milk was determined experimentally based on the required amount of amino acids in the nutrient medium. To obtain 1 l of the protein basis of the nutrient medium, 4.45 g of a dry broth of fish milk is taken, dissolved in 1 l of distilled water. Then, to 10 g of yeast extract, 1 g of glucose, 5 g of sodium chloride add up to 1 l of a solution of the protein base of the nutrient medium. The mixture is stirred. The resulting culture medium is poured into containers, a well-known method. Using 20% NaOH, the pH of the nutrient medium in each tank was adjusted to 7.2-7.4, and then sterilized at 0.5 atm for 20 minutes.

The resulting culture medium is an odorless, clear, amber-colored liquid.

Comparative data on the dynamics of the number of test strains of bacteria in the claimed nutrient medium, prepared on the basis of a liquid or dry broth from salmon milk, are presented in table 3.

As can be seen from the table, the growth properties of the test strains of bacteria of the claimed nutrient medium, prepared on the basis of both liquid and dry broth from fish milk, are identical. However, the growth of listeria in the claimed nutrient medium, prepared on the basis of both liquid and dry decoction of salmon milk, begins after 1 hour of cultivation of bacteria, while in the prototype environment after only 8 hours (see drawing).

The claimed technical solution meets the criterion of "inventive step" in view of the fact that for the first time for the cultivation of bacteria, a liquid or dry broth made from fish milk (salmon, cod, herring, etc.) is used as a protein base.

A significant difference of the proposed technical solution is:

- the possibility of using the proposed environment to accelerate the cultivation of opportunistic bacteria;

- replacement of expensive imported nutrient medium with a cheap, easily accessible nutrient medium prepared on the basis of a decoction of fish milk;

- the use of a decoction of fish milk in both liquid and dry form.

The result is:

- an increase in the stability and growth rate of bacteria;

- cheapening of the nutrient medium;

- expansion of the raw material base for its preparation.

The claimed technical solution meets the criterion of "industrial applicability", as the medium is easy to manufacture, it can be prepared both in any microbiological laboratory and on an industrial scale.

Examples of specific production of a nutrient medium.

Example 1

When preparing 1 liter of culture medium for the cultivation of bacteria of the genus Listeria, a liquid broth is prepared from salmon milk. To do this, take 500 g of salmon fish milk (pink salmon), which are pre-crushed, add distilled water in a ratio of 1: 2. Boil for 1 hour. Then it is filtered and sterilized with a pressure of 1 atm for 30 minutes.

A protein base of the nutrient medium is prepared from a liquid broth. For this, the broth of milk is diluted with distilled water in a ratio of 1: 2, respectively.

Then, to 10 g of yeast extract, 1 g of glucose, 5 g of sodium chloride add up to 1 l of a solution of the protein base of the nutrient medium. The mixture is stirred. The resulting culture medium is poured into containers, a well-known method. Using 20% NaOH, the pH of the nutrient medium in each tank was adjusted to 7.2-7.4, and then sterilized at 0.5 atm for 20 minutes.

The resulting culture medium is a clear amber-colored liquid, odorless.

Example 2

The preparation of 1 liter of a nutrient medium based on a dry broth from chum milk is carried out as follows: from 4.45 g of a dry broth from chum milk and 1 liter of distilled water, the protein base of the nutrient medium is prepared. Then, to 10 g of yeast extract, 1 g of glucose, 5 g of sodium chloride, add up to 1 l of a solution of the protein base of the nutrient medium. The mixture is stirred. The resulting culture medium is poured into containers, a well-known method. Using 20% NaOH, the pH of the nutrient medium in each tank was adjusted to 7.2-7.4, and then sterilized at 0.5 atm for 20 minutes.

The resulting nutrient accumulation medium is an amber-clear, odorless liquid.

Example 3

A nutrient medium for the cultivation of bacteria of the genus Yersinia is prepared analogously to example 1.

Only as raw materials for the preparation of broth use herring milk.

The resulting culture medium is a clear amber-colored liquid, odorless.

Example 4

A culture medium for the cultivation of bacteria of the genus Yersinia is prepared analogously to example 2.

Only as raw material for the preparation of broth use cod milk.

The resulting culture medium is a clear amber-colored liquid, odorless.

Example 5

The nutrient medium is prepared analogously to example 1.

Only as raw material for the preparation of broth use Navaga milk.

The resulting culture medium is a clear amber-colored liquid, odorless.

Example 6

A nutrient medium for the cultivation of bacteria of the genus Escherihia coli is prepared analogously to example 2.

Only as raw materials for the preparation of broth use milk of rudd.

The resulting culture medium is a clear amber-colored liquid, odorless.

The nutrient medium is developed on the basis of cheap, domestic raw materials, easy to manufacture, much cheaper than the prototype environment. For its preparation, you can use substandard milk of fish 5-6 degrees of maturity, i.e. flowing that cannot be used for cooking foods. Moreover, its growth properties are not inferior to control environments, and the preparation technology is much simpler, which allows to expand the raw material base and reduce the cost of the nutrient medium for the cultivation of bacteria.

Claims (1)

A nutrient medium for the cultivation of bacteria, including a protein base, glucose, yeast extract and sodium chloride, characterized in that the protein base is a broth solution from fish milk in a ratio of 1: 2 (liquid broth from fish milk: distilled water) or 4.45 g dry broth from fish milk per 1 liter of distilled water with the following ratio of components, g / l:
yeast extract 10.0 glucose 1,0 sodium chloride 5,0 broth solution from fish milk up to 1 l
The pH of the medium is 7.2-7.4.

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