RU2388814C2 - Nutrient medium for growth of microorganisms - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: nutrient medium contains pantolysate from antler cake produced by extraction of antler cake in the ratio of 1:4-1:5 accordingly at the temperature of 98-99°C at the pressure of 1.5 atm. for 3-4 hours, peptone, sodium chloride and agar-agar with the specified ratio of components.

EFFECT: reduced time required to cultivate microorganisms and improved selective properties of nutrient medium.

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Description

The invention relates to the production of culture media and can be used in medicine and veterinary medicine in the microbiological diagnosis of bacterial diseases of animals and humans.

Known liquid nutrient media based on raw materials of animal origin, in particular based on meat water (meat and peptone broth, meat and peptone liver broth, etc.). Meat water as a semi-finished product for the production of nutrient media is prepared on the basis of minced meat (1 kg of minced meat is mixed with 2 l of distilled water, boiled for 1 hour, left to stand, filtered, added with water to its original volume, stirred in a container and sterilized for 30-40 minutes at 120 ° FROM). For the manufacture of meat and peptone broth, 1% peptone, 0.5% sodium chloride are added to meat water and boiled for 30-40 minutes, the required pH level is set and boiled again for 30 minutes, settled, filtered, packaged and sterilized at 1 atm. within 30 minutes. Dense and semi-liquid nutrient media are prepared by introducing into the broth agar-agar in an amount of 2-3% (solid media) or 0.15-0.4% (semi-liquid medium) (see Microbiological diagnosis of bacterial diseases of animals. Reference. M., 2005, p. 33-41 - prototype).

However, the use of meat for the production of nutrient media is not economically feasible, especially when using meat suitable for nutrition. In addition, the medium has a low spectrum of detection of various types of microorganisms (i.e., information content).

It is necessary to develop an economically feasible effective nutrient medium that excludes the use of expensive meat as raw materials, which has increased information content and with short periods of cultivation of microorganisms during primary sowing.

The specified technical result is achieved by the fact that as a liquid fraction, which is the basis for the manufacture of culture media for the cultivation of microorganisms, previously obtained by heat-treated in an aqueous medium raw materials of animal origin, followed by the introduction of 1% peptone, 0.5% sodium chloride at pH 7, 6 and 1% agar-agar, pantolizate is used based on antler meal, which is a by-product in the production of pantocrine. Pantolizate is prepared by extraction of a mixture of meal and water in the ratio of meal: water 1: 4-1: 5, process temperature 98-99 ° C, under a pressure of 1.5 atm, for 3-4 hours.

Antler meal, from which pantocrine is prepared by alcohol extraction, contains 31% amino acids by weight of the original antler (glycine, aspartic acid, proline, lysine, arginine, alanine, leucine, series, histidine, threonine, valine, tyrosine, phenylalanine, isoleucine, taurine , sarcosine, glucosylin). The mineral composition of antler meal is represented by calcium, magnesium, iron, silicon, phosphorus, sodium, aluminum - the basis of the cinder's ash elements. In addition, nickel, copper, tellurium, manganese, lead, barium, and cobalt, tungsten, strontium, molybdenum, and boron are contained in small amounts in antler flour. The class of lipids is represented by phospholipids, sterols, fatty acids, triglycerides. Available and vitamins of group B.

A general chemical analysis of antler meal shows that from 100 g of antler meal (ground pant) 3% of dry matter passes into pantocrine, and 90% of the total amino acid content and 30% of lipids remain in the meal. The prerequisite for studying the possibility of using an aqueous extract of antler meal as a basis for growing microorganisms was the following.

To build the body of a microbial cell, as well as an energy source, nitrogen, carbon, hydrogen and oxygen are required, while the supply of hydrogen and oxygen is provided by water, carbon is an integral part of organic compounds, including proteins and amino acids, and proteins are the source of nitrogen animal origin. All microbial cells consist of a complex complex of not only organic, but also mineral compounds involved in the formation of coenzymes, through which complex substances such as proteins, fats and polysaccharides are hydrolyzed to simpler and water-soluble compounds that are easily assimilated by microbes. The presence of such minerals as phosphorus, sodium, potassium and magnesium is necessary to create the physicochemical conditions of the nutrient medium required for life of microbial cells and build the cytoplasm. The lipid component of antler meal is also an important factor in the life of microorganisms.

Studies of the physico-chemical properties of antler meal are given in table 1.

Table 1 Name of indicator Meal from antler deer (after making pantocrine for injection) Meal from antler deer (after making pantocrine for internal use) Moisture (initial),%,% 1,1 3.0 Hygroscopic moisture,%,% 6.5 9.7 Fat,%,% 0.31 0.48 Total nitrogen,%,% 7.72 7.91 Protein nitrogen,%,% 7.31 7.76 Residual nitrogen,%,% 0.24 0.19 Ash,%,% 43.26 40.34 Protein,%,% 47.74 50.09 Absolute dry matter,%,% 91.42 90,20 Organic matter,%,% 48.34 49.91

Thus, the physicochemical composition of antler meal fully meets the requirements for raw materials for the production of a liquid base of nutrient media, provided that such parameters of the process of extraction of antler meal are developed, in which there will be a sufficient amount of substances necessary for growing microorganisms in the pantolizate.

Preliminary studies on a number of technological parameters on the extraction process determined the positive effect of excess pressure, the degree of dilution of meal with water and the time of the extraction process on the output of components. Based on preliminary studies, an experiment was conducted to identify the optimal combination of extraction process parameters, characterized by the highest percentage of dry matter output at a gentle temperature of 98-99 ° C, meal: water ratios 1: 4, 1: 5, 1: 6 for 2, 3 , 4, 5 hours at a pressure of 1, 1.5, 2 atm. The results of the influence of the parameters of the process of extraction of antler meal on the percentage yield of dry matter are shown in table 2.

table 2 Pressure, atm The time of the process, (hour) with the ratio of meal: water 2 3 four 5 1: 4 1: 5 1: 6 1: 4 1: 5 1: 6 1: 4 1: 5 1: 6 1: 4 1: 5 1: 6 one 1.3 1.7 2.0 2.1 2,3 2,3 2.2 2,4 2,4 2,3 2,4 2,4 1,5 1.8 2.2 2,4 3.6 4.2 4.3 3.8 4.6 4.6 3,7 4,5 4,5 2.0 1.8 2,3 2,4 3.8 4.3 4.3 3.9 4.6 4,5 3.9 4.6 4.6

As can be seen from table 2 of the extraction process at atmospheric pressure, it was characterized by a maximum dry matter yield of 2.4%, providing a total nitrogen content of 100 ml of pantolizate at a level of 190 mg. Carrying out the process at excess pressure (in our opinion, due to a decrease in the viscosity of the broth and more intensive dissolution of proteins) - 1.5 atm, the yield of dry matter increased sharply and reached at a dilution of 1: 4 and 1: 5 and the extraction time of 3 and 4 hours 3.6-4.6%, providing a total nitrogen content of 294-375 mg in 100 ml of pantolizate. It is well known that for the intensive growth of most microorganisms, the content of total nitrogen in 100 ml of broth at a level of at least 200-300 mg is necessary.

Thus, the optimal regime of water extraction of antler meal when receiving pantolysate should be considered the regime: temperature 98-99 ° C, for 3-4 hours, the ratio of meal: water 1: 4 and 1: 5 at a pressure of 1.5 atm, providing output total nitrogen at the level of 294-375 mg per 100 ml of pantolizate. Carrying out the process at 2 atm with all other parameters did not provide a significant difference in comparison with 1.5 atm, as well as 5-hour extraction compared to 4-hour.

Pantolysate-based culture medium was tested on 5 representatives of 2 families of microorganisms: Micrococcaceae and Enterobacteriaceae. Table 3 shows comparative tests of the nutrient mixture (the nature of the growth of microorganisms) according to the prototype (meat and peptone broth) and nutrient mixtures prepared on the basis of pantolizate obtained in the following modes:

- the ratio of meal: raw materials 1: 5, extraction for 3 hours at 1.5 atm and 98-99 ° C;

- ratio 1: 4, time 4 hours, 1.5 atm, 98-99 ° C.

Table 3 Type of microorganism and growth indicators Nutrient Based Media meat water (BCH) pantolysate (in the mode of 1: 5, 3 hours, 1.5 atm, 98-99 ° C) pantolysate (in the mode of 1: 4, 4 hours, 1.5 atm, 98-99 ° C) 1. Staphylococcus St. aureus, St. epidermidis Growth Time 12 hours 9 hours 11 o'clock Growth pattern Intense, uniform turbidity Uniform turbidity Transparent medium Sediment Easily susceptible mucus sediment Easily suspended mucus sediment Easily suspended mucus sediment 2. Streptococcus Str. pneumonic Growth Time No growth 15 hours 21 hours Growth pattern Uniform haze Transparent medium Sediment Sediment rising in the form of a "pigtail" Sediment rising in the form of a "pigtail" 3. Enterobacteriaceae E. coli, Enterobacter aerogenes Time 11 o'clock 4 hours 9 hours the appearance of growth No turbidity Slight turbidity Weak Growth pattern clouding Sediment Sediment rising in the form of a "pigtail" Sediment rising in the form of a "pigtail" Sediment rising in the form of a "pigtail" Growth pattern meat water (MPA) pantolysate (in the mode of 1: 5, 3 hours, 1.5 atm, 98-99 ° C) pantolysate (in the mode of 1: 4, 4 hours, 1.5 atm, 98-99 ° C) 1. Staphylococcus St. aureus, St. epidermidis Growth Time 12 h 9 h 9 h Number of colonies 35, 80 70, 130 47, 115 Colony size 4-5 mm 2-4 mm 2-5 mm Colony shape Round Round Round The edges Equal Equal Equal Surface Smooth, shiny Smooth, shiny Smooth Relief Convex Convex brilliant Transparency Opaque Opaque Convex Consistency Mucous Mucous Opaque Color Gray-white with a yellow tint, gray-white Yellow, gray white Mucous
Yellow, gray white 2. Streptococcus
Str. pneumonic Growth Time no growth 15 h 15 h Number of colonies one hundred 26 Colony size 1 mm 1 mm Colony shape Right Right The edges round round Surface Equal Equal Relief Shiny, smooth Brilliant Transparency Convex smooth Consistency Translucent Convex Color Mucous Translucent White with a bluish tint Mucous
White with a bluish tint 3. Enterobacteriaceae E. coli, Enterobacter aerogenes Growth Time 12-14 h 9-10 h 12 h amount twenty; 40 17, 54 15, 46 colonies 3-4 mm 2-3 mm 2-4 mm Colony size Right round Right Right Colony shape Equal round round The edges Smooth, shiny Equal Equal Surface Convex Smooth, shiny Smooth Relief Translucent Convex brilliant Transparency Pasty Translucent Convex Consistency Cream white Pasty Translucent Color Cream white Paste Creamy White

Analyzing the data of the table, it is seen that when using a nutrient medium with pantolysate, its information content increases when cultivating E.coli, Enterobacter aerogenes, St. aureus, St. epidermidis, while culturing Str. pneumonic there is no need to add 20% of horse serum, without the addition of which this microorganism does not grow on conventional MPB and MPA media. Thus, the new medium with the addition of pantolizate allows not to use meat (to reduce the cost of preparing the medium) in the manufacture of the medium; improves the information content of the environment; reduces the time of appearance of the primary growth and accumulation of the bacterial mass of microorganisms; allows you to do without additional growth factors necessary for the cultivation of Str. pneumonic, which makes this medium preferable for primary sowing, as more microorganisms are cultivated on it.

Example. A nutrient medium for growing microorganisms is prepared as follows: 200 g of antler meal is mixed with 1 liter of distilled water, the mixture is placed in an extractor and extracted at 98 ° C under a pressure of 1.5 atm for 3 hours. The contents of the container were filtered through a cotton-gauze filter, followed by sterilization at 120 ° C for 40 minutes. After sterilization, the extract was filtered a second time. To prepare 500 grams of medium, 487.5 g of extract (pantolizate) is taken and mixed with 5 g of peptone, 2.5 g of sodium chloride and 5 g of agar-agar (for preparing a dense medium), the pH is adjusted to 7.6. After establishing the pH, the medium is boiled for 30 minutes, sedimented, filtered, Packed in test tubes and sterilized at 1 ATM for 30 minutes.

Thus, the use of pantocrine production waste (antler meal) as a raw material in the production of nutrient media for the cultivation of microorganisms (given the technological parameters of the antler meal extraction process that provide a sufficient amount of components extracted from the meal for the growth and development of microorganisms) can reduce the consumption of meat raw materials and ensure high efficiency of the obtained nutrient medium by expanding the spectrum of detection of various types of microorganisms and reducing the timing of their cultivation.

Claims (1)

Nutrient medium for growing microorganisms containing, as a basis, raw materials of animal origin thermally processed in an aqueous medium, peptone, sodium chloride, agar-agar, characterized in that the medium contains pantolizate from antler meal obtained by extraction of antler meal with water in the ratio of 1: 4-1: 5, respectively, at a temperature of 98-99 ° C under a pressure of 1.5 atm for 3-4 hours, with the following ratio of components, wt.%:
peptone 1,0 sodium chloride 0.5 agar agar 1,0 pantolysate rest