SU1339130A1 - Method of determining respiratory activity of microorganisms - Google Patents

Abstract

This invention relates to microbiology, in particular to biotechnological processes for the production of various bacterium preparations and products of microbiological synthesis. The aim of the invention is to increase the detection sensitivity. The method for determining the respiratory activity of microorganism cultures is that an indicator is added to the microbial cells to be examined, which use a suspension of Saccharomyces sequial baker's yeast, obtained by cultivating on the medium before accumulation of zincoporphyrins in cells, providing luminescence for 10 -10 s and inactivation. - at 20-25 minutes, followed by washing to a concentration of 4-5 g / l. The time is measured from the moment the microorganism culture is introduced into the test sample until the luminescence intensity of the indicator culture is fully restored to a stationary value, the magnitude of which measures the respiratory activity of the culture. 5 ill., 1 tab. § (L co 00 co co

Description

This invention relates to microbiology, namely to biotechnological processes for the production of various bacterial preparations and products of microbiological synthesis.

The purpose of the invention is to increase the detection sensitivity.

The method for determining the respiratory activity of microorganism cultures is as follows.

A sample of the culture suspension is placed in a quartz cuvette. A sample of oxygen is introduced into the sample in the form of a certain volume of physiological solution, to which a definite volume of indicator solution is prepared, prepared by special treatment from a suspension of microorganism cells expressed under certain conditions that ensure the accumulation of significant amounts of intracellular substances of zinc porphyrin nature. .

The time from the moment the microorganism culture of the indicated solution was introduced into the sample under study was measured until the luminescence intensity of the indicator culture was completely restored to its essential value. The concentration of residual oxygen in the suspension in this case is less than 10 M

Saccharomyces cerevisial baker's yeast, previously grown under conditions ensuring accumulation in cells of high pools of zinc-porphyrin nature, is used as an indicator solution of a substance that has the ability to delayed fluorescence, which is quenched with dissolved oxygen.

The method of preparation of the indicator culture is as follows.

The culture of industrial schtamma Sac charomyces cerevisial is grown on a standard synthetic nutrient medium of the following composition, g: 12} 200.5

0.5

NaCl5,0

MgS04, 138

 12H .., 01, 35

1.8

Ammonium citrate4, 5

Sucrose 50,0

ZnS () 4-, 0029

(the concentration is matched by experiment and is optimal for the accumulation of long-lasting luminescent compounds).

The cultivation is carried out with no forced aeration in flasks for 72 hours.

At the end of the cultivation process, a control measurement of the level of the intensity of the delayed fluorescence is carried out in order to estimate the concentration of the synthesized zinc-porphyrin compounds. The specific concentration of fluorophore per unit biomass is calculated.

The culture suspension of yeast cells is inactivated by heat treatment at 80 ° C for 20-25 minutes. The repeated control of the characteristics of the delayed fluorescence of the yeast culture is carried out. The glow should be completely absent. This is due to the fact that, under these heat treatment conditions, endogenous cellular respiration is inactivated and oxygen in the medium dissolves retards the fluorescence of cellular zinc porphyrins. The obtained inactivated culture is centrifuged at 1500 rpm for 15 min and resuspended in physiological solution to a final concentration of 100 g / l.

The indicator culture is stored in a tightly closed container at a low temperature (+ 4 ° C) for six months under conditions that do not require sterility.

Before measuring the respiratory acti-TtffiHocTH of the microorganism culture under investigation, the indicator suspension was diluted with physiological saline to a concentration of 5 g / l. The temperature of the indicator solution is adjusted to i

The cultures of the culture sample under study. When measuring, it is necessary that the concentration of the indicator fluorophore in the mixture remains constant. As shown by preliminary studies, usually, in one dimension, 1 ml of the analyzed culture and 1 ml of indicator solution are required.

The proposed method for measuring the respiratory activity of microorganisms is based on the fact that the analyzed culture is active and utilizes the introduced portion of oxygen. By reducing the dissolved oxygen concentration in the mixture to a level of 10 M and below, the effect is eliminated (Oxygen quenching of delayed fluorescence of zinc porphyrins of the indicator culture. The method allows monitoring the rate of oxygen consumption of the analyzed culture based on measuring the dynamics of recovery of the intensity of delayed fluorescence of the indicator culture, activity of the cultures microorganisms that do not have their own delayed fluorescence, for example, to measure the rate of oxygen consumption to lturoy Esche- richia coli M-17 in the manufacture of a therapeutic and prophylactic drug Ko libakterina, culture Baccillus sub- tilis (producing riboflavin), culture Brevibacterium flavum (producing homoserine).

Example 1. Culture of Escheri- chia coli M-17. The culture medium is M9. The source of carbon and energy is 0.3% glucose. Periodic cultivation in a fermenter with a working volume of 2 l. The aeration rate of 1 l / 1 medium per minute. The rotation speed of the stirrer is 900 rpm.

In the process of growing culture samples are taken; liquids (0.1–1.0 ml depending on the respiratory activity of the population) and placed in a quartz cuvette. About 1 ml of the indicator suspension was added thereto and a thin layer of technical liquid oil was applied. The suspension is irradiated with visible light with a halogen lamp. The radiation receiver is a photomultiplier. The fluorescent signal is amplified with a microvoltmeter and is recorded on a recorder tape. Long luminescence is recorded with a lifetime of 2-10. By the presence of fluorescence

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the suppressive signal is judged on the absence of dissolved oxygen in the sample, and the analysis is started after the appearance of the signal. Portions of saline were introduced into the sample (0.1-1.0 ml depending on the respiratory activity of microbial cells). The time from the moment of introduction into the sample of the portion of oxygen dissolved in the physiological solution to the time the steady-state luminescence signal of the indicator cells reaches the stationary level is recorded.

Fig. 1 shows the results of measuring the concentration values of biomass (1), the rate of consumption of dissolved oxygen by the E. coli M-17 culture during cultivation using the proposed method (2). For comparison, the results of measuring the respiratory activity of a population using a gas analyzer (3) are given. The minimum sensitivity of the gas analyzer (0.5%) does not allow measuring the rate of oxygen consumption less than 0.2 g l h (Fig. 2) with the aeration and displacement mode used. At the same time, the proposed method allows, under these conditions, to register the indicated value up to values of 0.001 gl.

During the entire 10-hour culture process, no prolonged luminescence was observed in the living E. coli M-17 cells themselves, which does not allow the limestone method to be used for testing the respiratory activity of this population.

Example 2. Culture E. coli M-17. The culture medium is m - peptone broth. The source of carbon and energy is glucose (1%). Cultivation in a fermenter with a working volume of 30 l in order to produce the therapeutic and prophylactic preparation colibacterin. The conditions of aeration and mixing, as well as the measurement procedure for the respiratory activity of the bacterial suspension, are similar to those described in Example 1.

Table 1 presents the results of measuring the rate of oxygen consumption by the population of E. coli M-17 in the cultivation process by the proposed and known methods.

The accumulation of long-lasting luminescent compounds in the living cells of Escherichia coli during the study

cultivation regulations are noted only at the end of the process (6-7 hours). Therefore, using the known method of measuring the respiratory activity of microorganisms during the majority of the cultivation process of E. coli M-17 is impossible. The application of the proposed method allows controlling the rate of oxygen consumption by a given microbial population throughout the entire growing process.

Example 3. Bacillus culture

subtilis 304-23, genetically modified by 15 lcii in the process of periodic kulne strain - riboflavin producing. The culture medium is a selective medium. Fermenter volume of 2 liters. The conditions of aeration and mixing, as well as the measurement procedure for the respiratory activity of the microorganism, are similar to Example 1.

Fig. 3 shows the results of measuring the respiratory activity of the population of Vas. subtil is in the process of periodic cultivation of the proposed method. It has been found that throughout the whole investigated process of the cell You. subtilis do not contain long-luminescent compounds of zinc-porphyrin nature, which makes it impossible to use a known method. The application of the proposed method allows to produce; assessment of the rate of consumption of dissolved oxygen by the population of you. subtilis throughout the whole process: growing.

Example 4. Culture Micrococ CUS glutamicus 95, a genetically modified strain producing lysine. The composition of the culture medium (the content of the components of the medium in 1 l) 2,: ammonium phosphorus acid 2; ammonium sulphate 2; magnesium sulfate 0.5; manganese sulfate 0.04; molasses 30; corn extract 5.

The procedure for growing the strain and measuring the respiratory activity of the cells is similar to Example 1.

Figure 4 shows the results of measuring the rate of consumption of dissolved oxygen by microbial cells by the proposed method in the process of growing M. glutamicus 95. The results of the studies showed the need to use a known method to control the respiratory activity of the population.

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due to the absence in the cells of the microorganism under study during the whole process of growing slow-fluorescent compounds of cincorphyrin nature.

Example 5. Brevibacterium flavum 2T culture, a genetically modified strain producing homoserine. 1Q The culture conditions and measurements of the respiratory activity of the cells are similar to Example 4.

Figure 5 shows the dynamics of the respiratory activity of the ass examined.

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tivirovaniya. The measurements were carried out by the proposed method. The accumulation of intrinsic slow-fluorescent substances of zincoporphyrin nature in the cells of the studied microorganism was not observed. Therefore, using a known method to measure the rate of oxygen consumption by microbial cells in the process under study is impossible.

Thus, the method of controlling the respiratory activity of microorganisms provides measurements of the rate of consumption of dissolved oxygen by microbial moduli whose cells do not contain their own slow-moving fluorescent compounds that change their luminescence parameters depending on the content of dissolved oxygen in the culture fluid.

The dynamic range of the oxygen consumption rates measured by the proposed method ensures reliable monitoring of the respiratory activity of microbial populations throughout the entire process of growing the listed microbial species.

Claims (2)

Invention Formula The method for determining the respiratory activity of microorganisms by growing them on a nutrient medium, taking a suspension sample in question, irradiating it with light, then registering a long-term luminescence and introducing into the sample a portion of oxygen dissolved in physiological saline, measuring the time from the end of aeration until the luminescence recovers evaluate the respiratory activity of the culture, and the rate of consumption of the solution n 13391 oxygen by the cells of the microorganism under study is determined by measuring the time from ending aeration before recovering s luminescence of long-term luminescent compounds, characterized in that, in order to increase the sensitivity of the determination, in the sampled culture liquid, Sposov Values of speed, g / lh, ratlichnis | 1 eat “nanny ktgrvaly h) from the beginning of the cultivation  ZlllllirilZ Known ---. ,, 112,95gO, 11 ; i ::: Eii :: i; i; iii ::: j::; z: Prevdlagaye0 .0710,01O, .0Гil.luiO.lO, 03 2. P10, Ov 2.91: 0,103,3110,123, OOlO, P eight indicator, which is used as a suspension of Baker's yeast Saccharomyces cerevisial, obtained by cultivation, to accumulate in the cells of zincoporphyrins, providing luminescence of 10 -10 - s and inactivated for 20-25 minutes, followed by washing to a concentration of 4-5 g / l. tbl N c and I 7.5 Ki, 2 / .v UO..G.  b 12 16 th 2 zs zg, " Phi1.3 eight 7624-32 FIG. 0 8 f.v / , -f- -I1-: 1 - gb g zg yv ss s fig. five Editor A.Shishkin Compiled by L. Borisova Tehred M. Dndyk Order 4183/17 Circulation 499 Subscription VNIIPI USSR State Committee for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab., 4/5 Arbitrary and polygraphic enterprise, Uzhgorod, st. Project, 4 / to- N  ss s 71 / V Proof-reader I.Erdeyi