SU969715A1 - Process for producing activator for autolysis of microorganisms - Google Patents

Description

(St) METHOD FOR OBTAINING AN AUTOLYSIS ACTIVATOR The invention relates to microbiological and medical industry, in particular, to methods for producing an autolysis activator of microorganisms. Closest to the proposed technical essence and the achieved effect is a method of obtaining an activator of autolysis of microorganisms, involving the hydrolysis of the raw material with trypsin. According to a known method, autolysis regulators are isolated from the fermentolysate of the biomass of microorganisms by extraction with 5 to 10% trichloroacetic acid, followed by precipitation from the extract with volumes of organic solvents. Enzymatic lysis is subjected to suspension of biomass with a concentration of dry substances. For lysis, the first stage is used (the lithium action procedure, in the second stage, the enzyme of proteolytic action, for example trypsin. MICROORGANISMS with an enzyme ratio; substrate 1: 500 C1. The disadvantages of this method are that the autolysis controller can be used as activation and inhibition of autolysis. The nature of the effect of the regulators depends on the substrate (autolytic bacteriaJ). Therefore, to select a regulator of a given nature of action on this microorganism, it is necessary to check the and regulators, which complicates the use of autolysis regulators in practice.In addition, the disadvantages of this method are also the complexity of the procedure for isolating regulators and the need for special equipment that is resistant to trichloroacetic acid. the effectiveness of the action of autolysis activator. 396 The goal is achieved by the fact that according to the method of obtaining the activator of autolysis of microorganisms, involving the hydrolysis of the feedstock thrips otherwise, casein or sodium caseinate is subjected to hydrolysis, and hydrolysis is carried out for 0, h with an enzyme: substrate ratio of 1: 1000 - 1: 5000. The method is carried out as follows. Casein or sodium caseinate is dissolved in water, the protein concentration is from 1 to 5%. If necessary, to improve the dissolution of the protein suspension is heated to 60-8 () - C. At the end of the dissolution process, the temperature of the JBOpa plant is reduced to and adjusted to pH 8-9 with NaOH solution. Then the enzyme trypsin is introduced into the solution, the ratio of the enzyme to the substrate is from 1: 1000 and 1: 50.00. The enzymatic hydrolysis is carried out for a period of 0.5-2 hours, preferably 2 hours. After the end of the hydrolysis, the temperature of the hydrolyzate is raised to 90-100 ° C and kept at this temperature for 1530 minutes to inactivate the enzyme. The hydrolyzate is then cooled and freeze-dried or spray-dried. In this case, as fillers, salts such as table salt, ammonium sulfate, etc. can be used (depending on the intended purpose of the preparation), the amount of 50-150 salts added to the dry weight of hydrolyzed protein when drying. As a hydrolyzable substrate, not only casein or caseinate can be used, but also other proteins, such as milk albumin, bovine serum albumin, gelatin, but the resulting autolysis activator has a less pronounced effect. The universality of the activating effect on the autolysis of various microorganisms is preserved. The most active fraction of the activator, derived from casein or sodium caseinate, contains peptides with a molecular weight of 800-1000 daltons. In order to increase the effectiveness of the activator, this fraction can be isolated from the hydrolyzate by gel filtration on sephadex or acrylex. Example 1.50 g of sodium caseinate is dissolved in 1 l of water while the solution is cooled to a pH of 8.6 with 1% NaOH solution and 0.01 g of trypsin is added as an aqueous solution in a volume of 0.01 l (ratio: enzyme to substrate 1: soon). The hydrolysis is carried out at constant stirring for 2 hours. Then the hydrolyzate is heated to cyPenia and kept in a boiling water bath for 15 minutes, then cooled and lyophilized. The resulting preparation is used to activate the autolysis of microorganisms. To determine the degree of autolysis activation, homogeneous aqueous suspensions of biomass of various microorganisms are prepared with a standard optical density of 0.951.05 when measured at FEK-56M at a wavelength of 5tO nm in a 5 mm thick cuvette. Then the reaction mixtures are made up of equal volumes of a standard suspension and an aqueous solution of an activator with a concentration of t mg / ml. In the control, equal to the first volume of water is added to the standard suspension. The initial on-tus density of the reaction mixture DO and the optical density after incubation for one hour at 37 ° C are measured. Calculate the intensity of autolysis 3 3, 100. The results are shown in the table, options 1-10 (. With a hydrolysis duration of 2 h and the ratio of enzyme: substrate 1: 5000). From the table it is seen that in variants the autolysis intensity of microorganisms will increase to 2, 9-15 times in comparison with the control, and in variants 1 and 2, the intensity of autolysis is high in microorganisms that have not been lysed in the absence of an activator. This can be explained by the transition of the latent form of autolytic enzymes to the active one in the presence of an autolysis activator. Example 2. 100 g of casein is dissolved in 2 l of water at 75 ° C, the solution is cooled to pH 9.0 with 1% NaOH solution and 0.10 g of trypsin as an aqueous solution in 0.10 l is added water (enzyme: substrate ratio 1: 1000} Hydrolysis is carried out for 0.5 h. Then the hydrolysis is kept in a boiling water bath for 30 min, then cooled and lyophilized. The resulting preparation is used to activate the autolysis of microorganisms. Determination of the degree activation is carried out as described in example 1.

The results are shown in table 5, options 1T-13 (with a duration of hydrolysis of 0.5 m and the ratio of enzyme: substrate 1: 1000). Under the action of the activator, the intensity of the autolysis of methanoic acid bacteria, to culture 170, increases by A, 2 times, E. coPH 17 by 6, P times, and in actinomycete Streptomyces albogrlseotus, from O to 52.8%.

Example 3. 50 g of sodium caseinate are dissolved in 2 l of water at., The solution is cooled to k2 ° C, set to pti 8.6 and 0.02 g of trypsin is added as an aqueous solution in volume

Effect of activator on autolysis intensity

0.02 l. Enzyme ratio; the substrate is 1: 2500. The hydrolysis is carried out at 37 ° (for an hour. Then the hydrolysis is heated to boiling and kept in this state for 20 minutes. Upon cooling, the hydrolyzate is lylated.

The test of the activating drug on the autotolysis of microorganisms was carried out in the same way as in example 1. The results are shown in the table, ariants (with a duration of hydrolysis of Lh and an enzyme: substrate ratio of 1: 2500). The degree of activation of autolysis is for methanol acids of bacteria, cultures 122 and 170, respectively 2.7 and 4.3 times, for E. sY I MRF.-BLO - I, 5 times, for 8. nesentericus 1, the intensity of autolysis increases from 0 to 18.7-5. microorganisms

Streptonyces a1bogriseolus

V. Mesentericus 1

Bif fdobacteruin E.coli MRE-600

P .. coP M17

Methanooxide acid bacteria culture culture 122

Methanooxide acid bacteria culture 170

Lactobact.plantsrun

Candida tropical is VSB-909

Rhdotorula aurant iaca

16.2 2.9

i,

27.3 20.9 3.0 14.1 2.9 25.0 3.0

11Metho-oxidizing bacteria, culture 1706.2

12E, comp M17 5.1

13Streptornyc s altjogrlseolusО

Imeta nose kysl yucci

bacteria culture 1706.1

: .Sh 15Met anoki with yu | di e

Claims (1)

bacteria, the cultivation of the activator of autolysis of microorganisms provides a highly efficient activator of autolysis, which, in comparison with the known method, allows to activate the autolysis of representatives of all groups of microorganisms. In addition, simplification is achieved by eliminating the trichloroacetic acid treatment step. The invention The method of obtaining an activator of autolysis of microorganisms, provides 969715 .8 Continuation of the table .2 6.0 26,2 i, 3 NOM, in order to simplify the process and increase the efficiency of the activator of autolysis, casein or sodium caseinate is hydrolyzed, and hydrolysis is carried out for 0, 5-2 h when the ratio of enzyme: substrate is 1: 1, 110: 1: 50PO. Sources of information taken into account pr1 examination 1. USSR author's certificate Vf 759526, cl. C 07 R 7 / 0,2, 1978.