RU2235770C2 - Method for preparing peptone "kaspiy" - Google Patents

Abstract

FIELD: biotechnology, clinical microbiology.

SUBSTANCE: invention can be used in culturing broad spectrum of microorganisms. Fish or piscine flour used as the protein raw is mixed with water, heated to temperature 70-80^oC, kept for 30-40 min, diluted with water, acidified with hydrochloric acid to pH = 1.6-2.0 and fermented using pepsin at 48-50^oC for 18-20 h. Then products of enzymatic lysis are alkalinized to pH =3.8-4.2, boiled for 10-15 min, filtrate is alkalinized to pH = 7.8-8.2, boiled for 10-15 min and filtered again. Filtrate is concentrated to the content of dry matters 18-55%. Invention expands the raw base for preparing peptones, promotes to the more complete purification of peptones from inert substances and provides the best conditions for growth of microorganisms as compared with the known method.

EFFECT: improved preparing method.

1 tbl, 2 ex

Description

The invention relates to medicine, namely to medical microbiology, and can be used for the cultivation of a wide range of microorganisms.

Peptones, products of incomplete protein breakdown, are widely used in the preparation of simple and complex nutrient media as sources of nitrogen and carbon (1, 2, 3, 4).

A known method of producing peptone according to Marten by self-digestion of fresh pork stomachs (5).

The disadvantages of this method are the need for thorough thorough cleaning of pig stomachs from fat and bile, which is a rather time-consuming process, non-standardity of pig stomachs - pepsin sources and the duration of the peptone production process.

There is also a method for producing peptone by enzymatic hydrolysis of proteinaceous raw materials, first with pepsin and then with trypsin (7).

The disadvantage of this method is the high cost due to the use of two enzyme preparations and a large number of technological operations, which makes the process of obtaining peptone more expensive.

Closest to the proposed method is a method for producing peptone according to Ramon by enzymatic hydrolysis of cooked meat with pepsin of pork stomachs in the presence of hydrochloric acid for 24 hours at 56 ° C, followed by sterilization and purification by standing for at least 5 days and decontamination (6 )

The disadvantages of this method are the use of only meat as a raw material, which limits the raw material base for producing peptones, the non-standard source of pepsin - pig stomachs, the duration of the process and the low degree of purification of peptones from non-hydrolyzed protein, leading to turbidity and the formation of sediment after sterilization, which virtually eliminates the possibility the use of this peptone in the composition of culture media without additional purification.

The present invention solves the problem of expanding the raw material base to obtain peptone, reducing the duration of the process and increasing the degree of purification of the target product.

To achieve the technical result in the proposed method, proteinaceous raw materials - fish (for example, sprat, pollock, anchovy) or fishmeal are mixed with water, heated to a temperature of 70-80 ° C, kept at this temperature for 30-40 minutes, then diluted water, the mixture is acidified with hydrochloric acid to a pH of 1.6-2.0 and fermented with pepsin for 18-20 hours at 48-50 ° C, after which the fermentolizate is made alkaline to pH 3.8-4.2, boiled for 10- 15 minutes and filtered, the filtrate was alkalinized to 7.8-8.2, boiled for 10-15 minutes and filtered. The filtrate was concentrated and dried.

Distinctive features of the proposed method from the above known, closest to it, are the use of fish or fishmeal as a raw material, which expands the raw material base for peptones and partially reduces the cost of the process, as well as the fact that peptone is purified at different values pH - first at pH 3.8-4.2, and then at pH 7.8-8.2. This treatment contributes to the complete removal of residual proteins, providing high transparency and the absence of sediment after sterilization of peptone, which is one of the important indicators characterizing the quality of peptone. Processing peptones at various pH values allows not only to increase the degree of peptone purification, but also significantly reduces the cleaning time - from 5 days according to the known method to 8-12 hours by the proposed method.

The method is as follows.

Example 1. To 1 kg of minced fish add 1 liter of water, the mixture is stirred, heated to 70-80 ° C to denature the proteins, kept at this temperature for 30-40 minutes, then 1 liter of water is added, after which the suspension is acidified with hydrochloric acid to a pH of 1.6-2.0, 10 g of pepsin with an activity of 100,000 units are added and hydrolysis is carried out at 48-50 ° C for 18-20 hours. After the hydrolysis is completed, the suspension is alkalinized with a 30% sodium hydroxide solution to pH 3.8-4 , 2, boil for 15 minutes and filter, the filtrate is again basified to pH 7.8-8.2, boil for 15 minutes, filtered, the filtrate is concentrated to 18-55% dry weight ETS and dried by known methods, such as spraying, penosushkoy or a fluid bed.

Example 2. It differs from example 1 in that 3 l of water is added to 1 kg of fishmeal GOST 2116-82, heated to 70-80 ° C, maintained at this temperature for 30-40 minutes, then 3 l of water is added, then according to example 1.

Peptone is characterized by a high content of true peptone of at least 75%, nitrogen of amino acids is 2.0-2.5%, there is no free protein, indole and heavy metals. After sterilization, the peptone solution remains transparent, there is no precipitate.

For biological quality control, dry peptone in an amount of 15-20 g is dissolved in 1 l of distilled water, 5 g of sodium chloride and 1 g of agar are added. To cultivate the strain Str.Dick 1 add 5 g of glucose. The mixture is heated until the agar is completely melted, boiled for 1-2 minutes, cooled to 45-50 ° C, poured into sterile Petri dishes, dried in an oven at 37 ± 1 ° C for 45 minutes. Peptone quality control is carried out by seeding test strains of E. coli, Sh.flexneri, S.typhi H 901, S.Paratyphi A and B, Kl.pneumoniae, St.aureus, Str.faecalis, Str.Dick 1, Ps. aeruginosa, Str. epidermidis, E.aerogenes, from dilutions of 10 ^-6 , which corresponds to 100 microbial cells.

The results are taken into account after 18-20 hours of incubation of crops at 37 ° C. The table shows a comparative characteristic of the growth of test strains of microorganisms on peptones obtained by the proposed and known methods.

The table shows that the peptone obtained by the proposed method, in size and number of grown colonies exceeds the known peptone, which indicates its higher growth properties compared to peptone obtained in a known manner.

In addition, the proposed method can significantly reduce the time of the process of obtaining peptone and solves the issue of replacing animal meat with cheaper raw materials - fish, fish meal.

Peptone obtained by the proposed method can be used to grow microorganisms of various taxonomic groups and study their cultural and morphological properties, as well as as a nutrient base for the production of agar and non-agar media for various purposes (sugar and bile broth, serum and blood agar, etc. e).

The method is simple to perform, does not require additional costs and can be easily reproduced in enterprises producing nutrient media.

BIBLIOGRAPHIC DATA

1. Handbook of microbiological and virological research methods. Under. ed. M.O. Birger. M .: “Medicine”. 1982, p. 46-47.

2. The Oxoid Manual, FIFTH Edition, 1982. S. 237-244.

3. Microbiological dictionary - reference book A. P. Krasilnikov. Minsk, “Belarus”, 1986, p.228.

4. Microbiology Manual Merck, 1990, p. 258-261.

5. Guide to microbiology, clinic and epidemiology of infectious diseases. Under. ed. N.N. Zhukova-Verezhnikova. M .: Medgiz, 1962, volume 1, p. 353.

6. Ibid., P. 354 (prototype).

7. Physico-chemical methods of control of ingredients, nutrient media and biological products. Moscow, 1970, p. 63-64.

Claims (1)

A method for producing peptone, which involves mixing fish raw materials with water in a predetermined ratio, enzymatic hydrolysis followed by purification by boiling and filtering, concentrating and drying, characterized in that fish meal is additionally used as fish raw materials, and fish raw materials mixed with water are kept at a temperature of 70- 80 ° C for 30-40 minutes, the resulting suspension is diluted with water, enzymatic hydrolysis is carried out with pepsin at a pH of 1.6-2.0 for 18-20 hours, peptone is purified by first adjusting the pH to 3.8-4.2, behind it to 7.8-8.2, and refluxing is performed at each of the indicated pH for 10-15 min and concentrated to a solids content of 18-55%.