RU2522811C2 - METHOD OF PREPARING SYMBIOTIC BACTERIA OF GENUS Xenorhabdus, ISOLATED FROM NEMATODES OF GENUS Steinernema feltiae protense, FOR STORAGE - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method comprises growing in the nutrient medium of symbiotic bacteria of the genus Xenorhabdus, isolated from nematodes of the genus Steinernema feltiae protense. The culture of these symbiotic bacteria of the genus Xenorhabdus is mixed with the protective medium containing lactose, polyglucin, thiourea, ascorbic acid, dextrin and water in a ratio of 1:1-2. Freezing is carried out at -40°C followed by freeze-drying to a residual humidity of 3-5%.

EFFECT: invention enables to increase the shelf life of pure cultures of symbiotic bacteria.

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Description

The invention relates to the microbiological industry, and in particular to methods for producing symbiotic bacteria of entomopathogenic nematodes of the Steinemematidae family for storage and accumulation in the industrial production of biomass of these parasites used as biological plant protection agents against pests. When cultivating entomopathogenic nematodes in a nutrient medium, the preparation of a pure culture of symbiotic bacteria, which occur in two forms: primary and secondary, is of great importance. Nematodes multiply in the presence of the primary form, while secondary forms of bacteria can stop the development of nematodes. Therefore, the term "symbiotic bacteria" refers to the primary form.

There is a method of obtaining pure colonies of primary forms of symbiotic bacteria, while the culture of symbiotic bacteria is prepared and stored on agarized jambs in test tubes (4). Pure cultures of the primary forms of symbiotic bacteria are obtained by the method of G. Poinar (Poinar GO The presence of Achromobacter nematophilus in the infective stage of a Neoaplectana sp. // Nematologica. 1966. Vol.12, No. 1. P.31-35 [9] ) by infection with nematodes of caterpillars of the large wax moth (Galleria melonella) grown according to the method of Datka et al. (Dutky SR, et al. A technique for the mass propogation oe the DD-136 nematode // J. Insect Pathol. - 1964. - Vol.6, No. 4. - P.417-422 [6]), followed by selection of bacterial cells from insect hemolymph and plating them on McConkie nutrient agar containing dyes - bromothymol blue (0.004%) and triphenyltetrazolium chloride (0.025%) .

 Three days after the growth of bacteria at 25 ° C, the colonies of the primary forms are selected according to the morphological characteristics of the colonies and the nature of their color. The identification of primary forms of symbiotic bacteria is carried out according to the method of Akurst (4). To obtain a large number of cultures of symbiotic bacteria, it is necessary to repeat the whole process constantly and repeatedly, since the culture is stored for 10-15 days.

The disadvantages of this method are the short shelf life of the obtained bacterial culture (10-15 days) and the inability to accumulate a large mass of culture, the complexity of the process associated with the need to repeat the selection step again

a pure bacterial culture from a natural population of invasive nematode larvae. In this regard, time costs reduce the stability of the industrial production of nematode drugs in general.

The closest known methods of preparing for storage of symbiotic bacteria, in which a culture of symbiotic bacteria is grown in Erlenmeyr flasks for three days on a shaker at a temperature of 25-28 ^° C. Then bacterial cells are frozen by adding 17% to the nutrient broth with bacteria glycerol followed by rapid cooling to -18 ^° C. For rapid thawing of bacteria, cultures are placed for reactivation in a water bath at 56 ° C for reactivation (Akhurst RJ Morphological and functional dimorphism in Xenorhabdus spp. bacte ria symbiotically associated with the insect pathogenic nematodes Neoaplectana and Heterorhabditis // J. Gen. Microbial. 1980. Vol. 121, No. 2. P.303-309 [4]).

The disadvantages of this method of preparation is that in a frozen state a pure culture of bacteria is also stored for a short period (15-20 days) and with a significant decrease in biological activity. To accumulate biomass, it is necessary to repeat the stage of freezing and reactivation of the bacterial culture. The process of industrial production of a biological product, which involves the constant presence of a large number of pure cultures of the primary form of symbiotic bacteria. For the long-term storage and accumulation of large biomass of a pure bacterial culture on an industrial scale, this preparation method is also not suitable.

The objective of the invention is to obtain a culture of symbiotic bacteria with high survival and biological activity and the possibility of long-term storage for accumulation when using biomass of the primary form in the industrial production of biological products based on entomopathogenic nematodes.

The problem is solved in the following way: in a culture of symbiotic bacteria (genus Xenorhabdus) of entomopathogenic nematodes (family Steinemematidae) received in a ratio of 1: 1-2 protective medium. As the medium used: 20% lactose, 10% polyglucin, 3% thiourea, 3% ascorbic acid, 3% dicstrin and water - the rest is up to 100%. Then the resulting culture is frozen at -40 ^° C, followed by freeze drying to a residual moisture content of 3-5%.

The component composition of the protective medium is selected taking into account the permissible parameters of similar media used in microbiology. The use of freeze drying is known for preparing various bacteria for long-term storage (Oparin Yu.G. Damage and protection of biomaterials during freezing and lyophilization. Biotechnology. 1996, 7: 3-11 [2]; Yartsev M.Ya. et al. RF patent № 2053790, A61K 39/02. "A method of manufacturing a vaccine against listeriosis of farm animals" [3]; Manzano M. et al. Simple and Fast PCR Protocol to Detect Listeria monocytogenes from Meat. // J. Sci. Food Agriculture. 1997. - V.74 - n. 1. - p.25-30 [10]; Wang BJC, et al. Fermentation and enzyme technology. NY: John Willey and sons. 1979 180 p. [11]).

However, for our purposes were not developed and not used. By additionally introducing a protective environment, including dextrin and selecting the ratios of its components, as well as the correlation with the biomass of the culture of symbiotic bacteria, it was possible to solve the problem.

Example 1

For lyophilization, a culture fluid with a developed culture of bacteria of the genus Xenorhabdus isolated from nematodes of the species Steinemema feltiae protense fam. Stein-emematidae (the number of bacterial cells in 1 ml of nutrient broth 1.2 · 10 ⁸ ) is mixed with 100 ml of protective medium. The resulting suspension of microorganisms is dispensed in 2 ml in sterile penicillin vials. The bottles are placed on a stainless steel tray and placed in the freezer for freezing. Lyophilized samples should be frozen at -40 ° C for a minimum of three hours. Then the vacuum is turned on and freeze drying is carried out for about 48 hours to a residual moisture content of 3-5% in the samples. Dried samples are sealed sterilely with rubber stoppers and placed in a refrigerator at + 4 ° C for storage.

To prepare 100 ml of the protective medium, the following weights of reagents are taken - 20.0 g of lactose, 10.0 g of polyglucin, 3.0 g of thiourea, 3.0 g of ascorbic acid, 3.0 g of dextrin, the rest is water.

In a 300 ml glass beaker, 80 ml of warm (approximately 60-70 ° C.) distilled water is poured. The glass is placed on a magnetic stirrer with heating turned on and a speed of revolutions of 500-700 revolutions per minute. First, a sample of lactose is poured into the glass, after dissolution of which a sample of polyglucin is fractionally added to the glass. The mixture is mixed until a clear solution is obtained, then weighed portions of thiourea, ascorbic acid, dextrin are added, and again mixed until a true solution is obtained. After that, the magnetic stirrer is turned off, the glass is closed with aluminum foil, and the solution cools to room temperature.

The resulting solution has a strongly acidic reaction, therefore, does not require additional sterilization. Further manipulations with the protective medium must be carried out under sterile conditions. The acidity of the pH of the solution is adjusted on a pH meter by adding a 0.2 M sodium hydroxide solution to 7.0 +/- 0.1 units. The volume of the protective medium is adjusted to 100 ml with sterile distilled water. Then, 100 ml of symbiotic bacteria culture is introduced into the protective medium and mixed in a volume ratio of 1: 1.

Pure cultures of primary forms of symbiotic bacteria for experiments were obtained according to the method of G. Poinar (Poinar GO The presence of Achromobacter nematophilus in the infective stage of a Neoaplectana sp. // Nematologica. 1966. Vol.12, No. 1. P.31-35 [ 9]) by infection with nematodes of the species Steeinemema feltiae protense of Galena mellonella caterpillars grown according to the method of Datka et al. (6), followed by selection of bacterial cells (Xenorhabdus sp.) From insect hemolymph and plating them on McConkie nutrient agar containing bromothymol dyes blue (0.004%) and triphenyltetrazolium chloride (0.025%).

In our experiments, breeding nematodes were used in a nutrient medium with the following component composition: soy flour - 16%, chicken egg - 20%, corn oil - 5%, brewer's yeast 1%, foam rubber crumbs 8% and water - 50%. A similar component composition of IPA with minor changes is recommended by foreign authors for the cultivation of nematodes in a liquid nutrient medium (Buecher EJ, Popiel I. Liquid culture of the entomogenous nematode Steinemema feltiae with its bacterial symbiont // J. Nematol. - 1989. - Vol.21 , No. 2. - P.199-200 [5]; Friedman MJ Commercial production and development // Entomopathogenic nematodes in biological control. - Eds .: R. Gaugler and HKKaya. - Boca Ration, FL: CRC Press, 1990. - P.153-172 [7].

In the control variant (prototype), a pure culture of symbiotic bacteria grown on a nutrient broth was used.

The quality assessment of bacterial cultures was carried out according to three indicators: 1) the output of the bacterial culture after storage; 2) the output of a culture of nematodes that developed in the presence of bacteria after storage; 3) the invasive activity of nematodes after their development in the presence of a bacterial culture obtained by the proposed method in comparison with the prototype.

Cultivation of nematodes on an artificial nutrient medium (IPA) was carried out according to one technique, i.e. using Erlenmeyer flasks with a volume of 500 cm ³ (6).

After completion of the nematode development cycle in IPS, the obtained nematode cultures were evaluated for invasive activity according to the method of Veremchuk and Danilova (Veremchuk G.V., Danilov L.G. Methodological guidelines for determining the invasive activity of nematode cultures of the genus Neoaplectana (Steinemematidae). L., 1978 , 7 p. [1]).

The indicator of invasive activity of nematode-bacterial complexes served as a criterion for the final assessment of the quality of bacterial cultures. The yield of nematode biomass after completion of the nematode development cycle in the IPS is shown in the tables per flask, i.e. 60 g of IPS.

After assessing the quality of bacterial cultures with various methods of their preparation after storage in bacteria isolated from Steinemema feltiae protense nematodes, a four-fold increase in the growth rate of bacterial cells was noted compared with the prototype (Table 1, p. 2).

When assessing the quality of bacterial cells after storage by the intensity of the development of the nematode-bacterial complex on an artificial nutrient medium and its invasive activity against host insects, it was found that the yield of invasive nematode larvae was 10% higher than this in comparison with the prototype (Table 2, p .2).

It was not found that invasive activity was reduced in nematodes that developed in the presence of symbiotic bacteria stored in a lyophilized state (the ratio of culture fluid and protective medium 1: 1) for 7 months (Table 3).

Example 2

The preparation of the bacterial culture was carried out in the same manner as in Example 1, but the ratio of the culture fluid to the protective medium was 1: 2. To prepare a protective environment for lyophilic drying of symbiotic bacteria per 200 ml, 160 ml of warm (about 60-70 ° C) distilled water are poured into a glass beaker with a capacity of 300 ml. Based on 200 ml of the protective medium for freeze drying of symbiotic bacteria, the following weights of reagents are taken - 40.0 g of lactose, 20.0 g of polyglucin, 6.0 g of thiourea, 6.0 g of ascorbic acid, 6.0 g of dextrin, the rest - water. The volume of the protective medium is adjusted to 200 ml with sterile distilled water. After that, 100 ml of symbiotic bacteria culture is introduced into the protective medium and mixed with the protective medium in a volume ratio of 1: 2.

When assessing the quality of bacterial cultures after storage in bacteria isolated from Steinemema feltiae protense nematodes and prepared by the proposed method, a three-fold increase in the growth rate of bacterial cells compared with the prototype was noted (Table 1, p. 3).

When assessing the quality of bacterial cells after storage by the intensity of development of the nematode-bacterial complex on an artificial nutrient medium and its invasive activity against host insects, it was found that the yield of invasive nematode larvae was at the same level as the prototype (Table 2, p .3).

It was also not found a decrease in invasive activity in nematodes that developed in the presence of symbiotic bacteria prepared according to the proposed method and stored in a lyophilized state (the ratio of culture fluid and protective medium 1: 2) for 7 months (Table 3, p. 3).

Thus, the symbiotic bacteria prepared by the proposed method (when the ratio of the culture fluid and the protective environment is 1: 2), after 7 months of storage, according to two indicators correspond to the prototype, and in terms of bacterial growth intensity are 3 times higher than the corresponding parameters of the prototype.

The proposed method for the preparation of pure cultures of symbiotic bacteria of the genus Xenorhabdus isolated from nematodes of the species Steinemema feltiae protense allows to increase the shelf life of the culture up to 7 months with the preservation and even increase of its quality indicators. This allows you to accumulate a seed stock of pure cultures of symbiotic bacteria with high rates of survival, biological activity for a successful and stable mode of functioning of industrial production compared to the prototype.

The proposed method allows for improving the quality of the bacterial culture to ensure its long-term storage and accumulation. To reduce the complexity of the process, eliminating the repeatability of the stage of obtaining a biological product based on nematodes, also to improve their quality. Thus, one of the critical issues in the technologies of mass production of biological preparations made on the basis of entomopathogenic nematodes is removed.

Table 1 Yield of bacterial cultures of the genus Xenorhabdus sp. on nutrient broth after storage (number of bacterial cells in 1 ml of nutrient broth) Type of nematodes No. Options Cultivation of bacteria in nutrient broth for 48 hours Steinemema feltiae protense one Control - in test tubes on agarized jambs (prototype) after storage 1.2 · 10 ⁸ 2 The method for lyophilization (the ratio of the culture fluid and the protective environment 1: 1) within 7 months after storage 3.8 · 10 ⁸ 3 The method of lyophilization (the ratio of the culture fluid and the protective environment 1: 2) within 7 months after storage 3.610 ⁸

table 2 The yield of nematode culture (family Steinemematidae) in the presence of symbiotic bacteria (Xenorhabdus sp.) After storage Type of nematodes No. Options The yield of a mature culture of nematodes after the development of the nematode-bacterial complex, million Steinemema feltiae protense one Control - in test tubes on agarized jambs (prototype) after storage 15.5 ± 4.2 2 The method for lyophilization (the ratio of the culture fluid and the protective environment 1: 1) within 7 months after storage 16.2 ± 2.5 3 The method of lyophilization (the ratio of the culture fluid and the protective environment 1: 2) within 7 months after storage 14.5 ± 4.4

Table 3 Invasive (entomopathogenic) activity of nematodes against test insects (Galleria mellonella) with bacteria obtained by the proposed method after storage Type of nematodes No. Options The indicator of the pathogenicity of nematodes, LD ₅₀ Steinemema feltiae protense one Control - in test tubes on agarized jambs (prototype) after storage 22.8 ± 2.7 2 The method for lyophilization (the ratio of the culture fluid and the protective environment 1: 1) within 7 months after storage 22.3 ± 3.6 3 The method of lyophilization (the ratio of the culture fluid and the protective environment 1: 2) within 7 months after storage 25.1 ± 6.7

Information sources

1. Veremchuk G.V., Danilov L.G. Guidelines for determining the invasive activity of nematode cultures of the genus Neoaplectana (Steinemematidae). L., 1978, 7 pp.

2. Oparin Yu.G. Damage and protection of biomaterials during freezing and lyophilization. Biotechnology. 1996, 7: 3-11.

3. Yartsev M.Ya., Shishov V.P., Raevsky A.A., Koroteeva L.A., Pavlenko I.V. RF patent No. 2053790, A61K 39/02. "A method of manufacturing a vaccine against listeriosis of farm animals."

4. Akhurst R.J. Morphological and functional dimorphism in Xenorhabdus spp. bacteria symbiotically associated with the insect pathogenic nematodes Neoaplectana and Heterorhabditis // J. Gen. Microbial 1980. Vol. 121, No. 2. P.303-309.

5. Buecher E.J., Popiel I. Liquid culture of the entomogenous nematode Steinemema feltiae with its bacterial symbiont // J. Nematol. - 1989. - Vol.21, No. 2. - P.199-200.

6. Dutky S.R., Thompson J.V., Cantwel G.E. A technique for the mass propogation of the DD-136 nematode // J. Insect Pathol. - 1964. - Vol.6, No. 4. - P. 417-422.

7. Friedman M.J. Commercial production and development // Entomopathogenic nematodes in biological control. - Eds .: R. Gaugler and H.K. Kaya. - Boca Ration, FL: CRC Press, 1990. - P.153-172.

8. Han R., Wouts W.M. Development of Heterorhabditis spp. strains as characteristics of possible Xenorhabdus luminescens subspecies // Rev. Nematol. 1990. Vol.13. P.411-415.

9. Poinar G.O. The presence of Achromobacter nematophilus in the infective stage of a Neoaplectana sp. // Nematologica. 1966. Vol.12, No. 1. P.31-35.

10. Manzano M., Cocolin L., Ferroni P., Cantoni C., Comi G. A Simple and Fast PCR Protocol to Detect Listeria monocytogenes from Meat. // J. Sci. Food Agriculture. 1997. - V.74 - n.1. - p.25-30;

11. Wang B.J.C., Cooney C.L., Demain A.L. Fermentation and enzyme technology. N-Y .: John Willey and sons. 1979 180 p.).

Claims (1)

A method of preparing symbiotic bacteria of the genus Xenorhabdus isolated from nematodes of the species Steinernema feltiae protense for storage, comprising introducing a protective medium into the culture of symbiotic bacteria of the genus Xenorhabdus isolated from nematodes of the species Steinernema feltiae protense, moreover, as a protective medium in the culture fluid at a ratio of 1: 1 -2 enter a medium containing lactose 20 wt.%, Polyglucin 10 wt.%, Thiourea 3 wt.%, Ascorbic acid 3 wt.%, Dextrin 3 wt.% And water - up to 100 wt.%, Freeze at -40 ^° С followed by freeze drying to residual humidity and 3-5%.