KR20080070168A - A method for formulating stable microbial agent of bacillus subtilis ah18 against temperature fluctuation - Google Patents

Abstract

A method for formulating a microbial agent is provided to produce a Bacillus product stable over temperature fluctuation at efficient cost by generating heat-resistant spores and mixing the spores with an inorganic fine porous carrier, thereby being effective for distributing the microbial agent without a special treatment process. A method for formulating a microbial agent of Bacillus subtilis AH18 showing stability against temperature fluctuation comprises the steps of: (a) culturing selected Bacillus subtilis AH18 strain with increasing temperature; (b) applying heat stress of a fatal temperature of 57 deg.C to formed spores to generate heat-induction resistant spores; (c) removing remaining growth cells from a mixture of the spores to generate a culture material including only the spores; and (d) adding the Bacillus subtilis AH18 thermostable spore culture material to a pre-sterilized and dried meso and fine porous inorganic carrier to prepare a resistant spore-carrier mixture formulation, wherein the inorganic carrier is selected from the group consisting of a synthetic zeolite NaY(SiO2:Al2O3:Na2O=46:20:11), synthetic zeolite NaA(SiO2:Al2O3:Na2O=39:34:20), natural zeolite(clinoptilolite) or kaolin clay mineral.

Description

A method for formulating stable microbial agent of Bacillus subtilis AH18 against temperature fluctuation

1 is a graph showing the survival rate of growth cells at 80 ℃.

2 is a graph showing the thermal stability of Bacillus subtilis AH18 (Accession No .: KACC 91205P) endospores produced under different conditions.

3 is a graph showing the thermal stability of Bacillus subtilis AH18 endospore-carrier formulation.

4 is a graph showing the effect of the stability of the Bacillus resistant spores of the inorganic carrier against the temperature change of room temperature.

The present invention relates to a method for formulating a Bacillus microbial formulation that is stable to temperature changes, and more particularly, by generating heat resistant spores during formulation of the microbial formulation and incorporating an inorganic microporous carrier in the final step, A method of making a microbial preparation for cost effective and keeping the product dormant against high and low temperature variations.

In agriculture, microbial agents are being explored which are effective to replace conventional chemical pesticides in an environmentally friendly manner to suppress disease of crops.

The microbial agent is a direct use of the function of the living microorganisms, and when applied to soil, etc., it can be said to have an effect according to the use by the activity of the specific containing useful microorganisms indicated, and finally shows an effect that helps plant cultivation.

The microbial agent is composed of living microorganisms and, in some cases, carriers that adsorb the inoculation bacteria, organic nutrients for activating the inoculation bacteria, and other auxiliaries. It is due to the function of the microorganisms contained.

Unlike general products, which are finished products, microbial products are progressive products that must maintain their vitality until the time they are consumed. They must be approached from a new angle in all aspects of production and consumption. In other words, producers should not end with production but maintain living functions until consumption, and consumers should be aware of the problems that can be derived from production in advance so that they can be supplied with vital products. In addition, producers and consumers should be free from the simple stance of using microbial agents and have an active understanding and interest in ecosystem variations that can occur after use as members of the natural environment.

Bacteria multiply due to division, and when the growth conditions are poor, most of them go to sleep or die, but bacteria such as Bacillus genus can form spores to resist stress and can survive for a long time. Most of the filamentous fungi and actinomycetes grow in the mycelial state and form spores to prepare for the next generation. As such microorganisms survive and adapt to the natural ecosystem according to their growth and preservation methods, the microorganisms are greatly affected by the habitat environment.

Soils have high buffering capacity and differ in physicochemical properties from micro-parts, resulting in high microbial survival under some adverse conditions, but generally low in artificially produced media. For this reason, microorganisms are resistant to such spores as Bacillus , yeast, Trichoderma , and Mucor . Degradation is common with In order to reduce the reduction rate, various kinds of substances are added, but after a certain period of time, the number of microorganisms is lower than the survival rate necessary for the expression of the effect or is killed. This product is meaningless to microbial agents and is merely inorganic.

Several strains have been identified and characterized by several research groups that can inhibit major plant pathogens, but few examples have been commercially formulated. Such commercial formulations are rare because they lack the cost-effective formulation methods to deliver the beneficial properties of the microbial preparation and provide easy handling procedures in the distribution process.

Formulation strategies that allow microorganisms to survive long-term survival with temperature variations because products formulated with microbial formulations are often placed at temperatures above 50 ° C. during transport and storage in summer and are also exposed to various temperatures during the entire shelf life. This is one of the main considerations.

In addition, the formulation of the microbial agent should inhibit the activity of the microorganisms when the product is in a temperature range that promotes the growth of bacteria, otherwise over-proliferation may cause the product to explode on or before opening. Therefore, the purpose of the formulation is to keep the strain in a “dormancy”, which will determine the final quality of the microbial product.

For this reason, gram positive strain Bacillus is mainly formulated as a microbial preparation using endospores, which are preferred for gram negative and provide strain stability at high temperatures. Studies on sporulation using B. megaterium and Clostridium perfringens can increase the thermal resistance of subsequent spores by applying thermal stress. It shows that it is.

Likewise, B. subtilis cells pre-exposed to mild heat stress are also known to produce more heat stable spores. However, by sampling the Bacillus products on the market and examining the concentrations of bacteria indicated on the product label, many products do not meet their claim of bacteria, either because stable spores have not been adequately produced during production or This suggests that the formulation process cannot maintain a "spore dormancy".

It has been taught that the inert state of various microorganisms and survivors depends on the growth medium, water content and inert components of the carrier. However, for endogenous spore forming bacterial species, the moisture content is known to have little effect on the dormant state.

On the other hand, temperature variations in the environment may be a significant environmental factor in determining the endogenous spore safety in the formulation of Bacillus products as mentioned above, and the spore quality and different inorganic carriers in the final formulation may be hot and cold. Finding out whether or not it affects the dormant state of the endogenous spores under water is an important task. In addition, if the survival of endogenous spores and bacterial strains is closely related to the growth conditions and the ingredients mixed during formulation, the formulation cost should be low to save the commercial market.

The development of microbial preparations consists in the selection of microorganisms, determination of optimum conditions for mass production and formulation. In order to exhibit the expected effectiveness of the microbial preparation, beneficial organisms must be supplied to the consumer alive and alive through various environmental conditions during the distribution process. Therefore, the formulation method to provide strain stability is a step in determining the quality and commercial success of the final product.

Accordingly, it is an object of the present invention to establish a method for formulating a microbial agent that can withstand temperature changes in the storage and distribution environment in formulating the microbial agent.

This object of the present invention is to produce a heat-stable endogenous spores of Bacillus subtilis AH18, and to formulate a stable Bacillus subtilis AH18 microbial formulation by using an inorganic carrier to maintain the spore stability even at high and low temperature variations of the environment Achieved by the method.

The present invention comprises the steps of screening Bacillus subtilis AH18 (Accession No .: KACC 91205P) strain and culturing at elevated temperature; Applying heat stress to the formed spores to generate heat induced endospores; Removing residual growth cells from the spore mixture to produce a culture in which only spores are present; And adding the Bacillus subtilis AH18 culture to a sterilized and dried inorganic carrier to prepare an endospore-carrier mixture formulation.

The present invention also provides an inorganic carrier which is mixed with Bacillus subtilis AH18 culture in the above method.

The present invention also provides a microbial preparation prepared by the above method.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, the present invention is not limited to these examples.

< Example >

Strain Selection and Culture

Bacillus subtilis AH18 was preselected according to plant growth promoting properties and cultured using Luria-Bertani (LB) medium at 37 ° C. This strain had a broad growth temperature range of 15-55 ° C. in LB medium with an optimal growth temperature of 37 ° C. Solid medium was LB or 0.1 × LB prepared using 1.5% agar.

0.5 mL of overnight starter culture was injected into 50 mL of LB medium and incubated at 37 ° C. and 200 rpm on a rotary shaker. The cultured cells were grown for 4 hours until the culture reached the late exponential growth phase (10 ⁸ CFU / mL).

The culture was further incubated for 2 hours at growth temperature to form spores due to food depleted. To produce spores with thermal stability, cells were grown at 42 ° C. for 4 hours until late exponential growth and heat stress was applied for 2 hours at a lethal temperature of 57 ° C.

The spore mixture was incubated for 10 minutes at 80 ° C. and residual trophoblast cells were removed before analyzing the thermal stability of the spores at 90 ° C., resulting in a culture with only spores.

Thermal Stability Analysis of Endogenous Spores

The thermal resistance of the spores was analyzed by a similar procedure to the Mohehedi and Waites method. The methodology was changed as follows.

Spore-only cultures were incubated in a 90 ° C. water bath and samples were collected at 20 minute intervals for 60 minutes. Viable cells were counted by dilution plating on 0.1 × LB plates incubated at 37 ° C. for 16 hours. Plot logNo / N vs. time to generate a survival curve, where No represents the initial cell number and N represents the number of viable cells at a given time point. SigmaPlot ver. 8.02 software was used. Error bars were calculated at P ≦ 0.05.

Carrier MIC ( minimum inhibitory concentration , Minimum inhibitory concentrations)

The inorganic carrier was a synthetic zeolite manufactured by the Korea Institute of Ceramic Engineering and Technology (KICET; Seoul, Korea) or a zeolite processed by natural light by various manufacturers. Synthetic carriers were provided by KICET, and natural carriers were commercially purchased from designated sources.

The carrier was sterilized and dried at 80 ° C. for 2 days before use. Exponentially grown Bacillus subtilis AH18 cells were loaded with 0.04% -10% inorganic carriers to a final concentration of 10 ⁴ CFU / mL in LB. Incubation was carried out at 37 ° C. for 20 hours with uniform shaking at 200 rpm using a 24-well micro-titer dish.

Dilution smears were performed to determine the number of viable cells in each well, and the concentration at which the carrier concentration allowed viable numbers below 10 was defined as MIC (minimum inhibitory concentration).

Endogenous spores at room temperature carrier  Formulation stability analysis

Carriers that did not show growth inhibition at the highest concentration of 10% were selected. These were finally synthetic zeolite NaY (1), synthetic zeolite NaA (4), natural zeolite (7), kaolin clay mineral (12), pearlite (14, 15) (each number corresponds to that listed in Table 1). .

An endospore-carrier mixture was prepared by mixing 1 mL of the heat-induced spore preparation with 10 g of an inorganic carrier to give a final moisture concentration of 10% (v / w). Despite the moisture, the formulations still retained the flaky appearance of the original carrier and showed no signs of aggregation or caking, which is believed to be due to the porous nature of the carrier.

One gram of each endospore-carrier preparation was sealed in heat sealable vacuum bags to prevent dehydration, but not in a vacuum. A total of 10 bags of 1 g sample were prepared for each formulation. The formulated ones were stored at room temperature in the laboratory from October until May of the next year when the temperature change was in the range of 15-25 ° C. The viable cell number of each 1 g preparation was measured monthly for a total of 6 months by dilution plating on 0.1 × LB at 37 ° C. A total of four independent experiments were performed for each formulation. Curves showing changes in mean viable cell numbers for 6 months were obtained from sigma plot ver. Created using 8.0. Error bars are omitted for clarity.

weapon Carrier  Physical properties

Physical properties representing the porous properties of the inorganic carriers were obtained and listed in Table 1.

TABLE 1

Porosity of Inorganic Carrier

¹ Typical Chemical Composition: 1, Na ₁₂ Si ₁₂ Al ₁₂ O _{48 ?} 216H ₂ O; 4, Na _x Si _{12 - x} Al _x O _{48 ?} nH ₂ O (x = 3-4, n = 220-260). The composition of the ferrite is indicated in the ferrite samples I and II, respectively.

^{2 The} average particle size was determined by electrophoretic light scattering for particles less than 1 micron on average and by laser diffusion for micro-sized particles.

The average particle size was measured by the electrophoretic light scattering method (ELS-8000, Photal) for particles smaller than microns, and the laser diffraction method for particles of micron size (Mastersizer 2000, Malvern). Was measured.

Mercury intrusion and nitrogen gas adsorption were used to calculate the porosity, surface area, average pore diameter, and pore size distribution. The mercury porosimeter was used to determine the pore size using the mercury porosimeter. The distribution was measured. BET surface area and average pore diameter values using nitrogen gas adsorption were measured by ASAP2010.

Bacillus Subtilis AH18  Production of endogenous spores

When exponentially growing Bacillus subtilis AH18 cells 10 ⁶ CFU were warmed to a temperature of 80 ° C., only 100 cells or less survived after 10 minutes as measured by dilution smears (see FIG. 1). In addition, growth cells of Bacillus subtilis AH18 could not survive at 80 ° C. for more than 10 minutes, while certain cells (measured by microscopic examination) had exhibited heat-resistance for at least 20 minutes. These cells, which survived high temperatures, then survived another 20 minutes.

Condensation cell size and resistant Gram staining properties observed under the microscope suggested that these heat-resistant cells were endogenous spores produced at high temperatures. This experiment concluded that growth cells of Bacillus subtilis AH18 could not survive at 80 ° C. for more than 10 minutes.

To determine whether spores produced under heat stress are more thermally stable than those produced by depletion, the late exponential culture of Bacillus subtilis AH18 produced at elevated temperature of 42 ° C. was performed at 57 ° C. for 2 hours. The culture produced heat-stress endospores. For comparison, the same late exponential culture was produced at a normal incubation temperature of 37 ° C. and further incubated at 37 ° C. for 2 hours to produce spores by nutrient depletion. Each cell culture was allowed to stand at 80 ° C. for 10 minutes to remove any viable trophoblast cells, blocking flock that could cause a rapid decrease in thermal stability analysis at 90 ° C. After this step, the remaining surviving spores were 1-2 × 10 ⁵ CFU / mL when produced by nutrient depletion and 1-7 × 10 ⁴ CFU / mL when produced under heat stress (results not shown). ).

At 90 ℃ Bacillus Subtilus AH18 Thermal stability

Spore-only cultures were incubated at 90 ° C. to analyze the thermal stability of different spore preparations.

As shown in FIG. 2, spores produced by nutrient depletion decreased about 1,000-fold over 60 minutes, while those produced under heat stress showed only about 10-fold reduction during the same period.

These results suggested that Bacillus subtilis AH18 endospores produced under thermal stress may be 100 times more thermally stable than those produced by nutrient depletion when compared at 90 ° C. for 1 hour.

On the other hand, through further experiments, Bacillus subtilis AH18 endospores produced at a lethal temperature of 57 ° C. when analyzed for 1 hour at 90 ° C. were 100 times more heat than those produced at 37 ° C. depletion. Resistance was shown.

Bacillus Subtilus AH18 Weapons for survival Carrier  effect

To select formulated carriers for Bacillus subtilis AH18, their effects on the survival of Bacillus subtilis AH18 were tested by measuring the minimum inhibitory concentrations for various synthetic and natural salt inorganic carriers (results not shown). .

Bacillus subtilis AH18 in control wells increased 10 ⁵ fold after 20 h incubation from the starting inoculum of 10 ⁴ CFU, whereas the number of cells in the wells containing the inorganic carriers varied with the concentration of the carrier and the carrier showing growth inhibition. MIC of was 0.01-4% due to the different chemical composition or physical structure of the carriers.

However, some carriers did not inhibit cell growth even at the maximum use concentration of 10%, and Bacillus subtilis AH18 cells grew as densely as the control. Inorganic carriers that showed no inhibitory effect at 10% were synthetic zeolite NaY, synthetic zeolite NaA, natural zeolites, kaolin clay minerals and pearlite from two different sources.

Bacillus Subtilus AH18  Inorganic against thermal stability of endogenous spores Carrier  effect

To prepare endogenous spore-carrier formulations, synthetic zeolite carriers NaY (1) and NaA (4), and natural zeolites (clinoptilolite) (7), natural kaolin clay minerals (12) and different industries Two Perlite I and II (14 and 15, respectively) produced by the jar were mixed with heat-stress induced endospores culture at 10% (v / w).

These preparations were used to analyze the effect of inorganic carriers on the thermal stability of Bacillus subtilis AH18 endospores.

As shown in FIG. 3, they did not show a significant negative effect on thermal stability for 1 hour at 90 ° C. irrespective of the carrier irradiated and the survival rate was on par with the spore-only preparation produced as in FIG. 2. Since the spore-only sample was liquid and the endogenous spore-carrier formulation contained 10% water, this could indicate the fact that the thermal stability of the endospores was not significantly affected by moisture content, at least under experimental conditions. have.

6 months at 15-25 ℃ carrier In formulation  Heat-induced Bacillus Subtilus  Stability of AH18 Endospores

To investigate the stability of heat-induced Bacillus subtilis AH18 endospores in inorganic carrier formulations under temperature variations of 15-25 ° C., various cell numbers in 1 g of endospores-carrier preparations of heat-sealed plastic bags were totaled. Measurements were made monthly for six months.

As shown in FIG. 4, preparations with carriers 1 (NaY zeolite), 4 (NaA zeolite), 7 (natural zeolite) and 12 (kaolin clay) were each stable at various cell numbers throughout the investigated period. Resistance to condensed cell morphology and Gram strains suggested that the cells in these preparations still remained spores after 6 months (results not shown).

However, bacterial formulations using perlite showed a gradual increase in cell number, after 6 months, the number of bacteria on carrier 14 increased by 100-fold and that of carrier 15 increased by 10- ⁷ times. The enhanced cell number for sample 15 showed an increase in the same pattern as the formulation of spore only maintained in liquid phase (compare with □ and ◆ in FIG. 4). In each of the Perlite 15 formulations and the spore-only liquid cultures, the cell number increased rapidly after one month, indicating that the endogenous spores' dormant state was broken at the beginning of manufacture. On the other hand, spores in the pearlite sample 14 appeared to remain stable for up to 2 months, but then changed into effector cells, gradually growing over the remaining 4 months.

The difference in proliferation was 10 ⁵ times smaller than using Perlite 14 compared with Sample No. 15 (compared with □ and ■ in FIG. 4). Nevertheless, microscopic analysis of cell morphology and motility suggested that the cells in each of these pearlite preparations were active influenced cells at the end of the irradiation period (results not shown). These results suggested that carriers 1, 4, 7 and 12 were very effective in maintaining dormancy for at least 6 months, whereas pearlite carriers promote germination and thus promote the proliferation of trophoblast cells.

The difference in the degree of spore-stability imparted by the various carriers was investigated by analyzing the physical, mainly porous, properties of these carriers. As shown in Table 1, the difference in chemical composition, structural diversity or average particle size seems to show a slight difference, but suggests that the average pore size of the carrier measured by mercury infiltration is negatively related to the spore dormant state. Synthetic and natural zeolites and kaolin clay minerals with average pore diameters in the 0.63-0.084 μm range (medium and microporous) exhibiting large BET surface areas were able to maintain spore dormancy, whereas the 3.7-8.0 μm range (macroporous) Perlite carriers having a pore size of and showing a small BET surface area could promote spore germination and growth of growth cells.

In comparing the porosity of the meso and microporous and macroporous carriers, porosity of 79% or more was another key factor in promoting spore germination. These results indicate that the synthetic or natural zeolites, or kaolin clay minerals, that exhibit large BET surface areas due to relatively smaller porosity and smaller average pore diameters, are stable for 6 months against Bacillus subtilis AH18 endospores at room temperature from 15 to 25 ° C. Can provide.

In order to maintain viability under high temperature, Bacillus subdilatus are grown at 42 ° C and late exponential phase culture is maintained at a lethal temperature of 57 ° C to artificially induce sporulation. The thermostable endospores of Bacillus subtilis AH18 were produced at the production stage.

Stability of spores quantitatively analyzed at 90 ° C. for 1 hour suggests that spores produced under lethal temperature-stress are 100 times more heat-stable than spores produced under nutrient depletion conditions at normal growth temperatures of 37 ° C. It was. Synthetic and natural inorganic carriers such as zeolites, pearlites, kaolin clay minerals with different chemical compositions, structures and pore diameters to control spore germination and over-proliferation problems that may be in the temperature change range 15-25 ° C. Was introduced into the formulation.

The stability of the spores analyzed for all 6 months is effective in maintaining the spores' dormant state with a small average pore diameter and a large BET surface area, and a BET surface area of 11-763m ² / g with a pore diameter of 0.63-0.084㎛. Synthetic and natural zeolites and kaolin clay minerals were able to maintain spore dormancy for 6 months, while the perlite carriers with small BET surface areas of 4.1-0.95 m ² / g had an average pore size of 3.7-8.0 μm, resulting in growth cell growth. It has been shown to promote germination and active growth of vegetative cells. In addition, the spore-dormant carrier showed less than 74% porosity.

Through this, although various inorganic carriers having various chemical compositions and structures do not show a significant difference in maintaining the thermal stability of the endogenous spores under high temperature, to maintain the spore dormant for 6 months at 15 to 25 ℃, There was a significant difference between meso and microporous and macroporous carriers.

In analyzing the different physical properties of the carrier, the average pore diameter of the carrier is considered to be a major factor in maintaining or breaking the spore dormancy (see Table 1), and the average pore size of 0.63 μm or less (medium and microporous) synthesis and Natural zeolites and kaolin clay mineral carriers were effective in maintaining dormancy, while pearlite carriers of 3.7 μm or more (macroporous) were effective in promoting spore germination and growth of Bacillus subtilis growth cells (see Table 1 and FIG. 4). ).

Once the dormant state was broken, more bacterial growth was detected in Perlite II with a large pore diameter (8 μm) in Perlite I with a size of 3.7 μm (Table 1 and FIG. 4, Comparative Samples 14 and 15).

Inorganic carriers with large pore sizes can be introduced when microbial activity is particularly required for survival, but for final microbial products that require constant stability of the strain, it is important to maintain the dormancy of the microorganisms, thus formulating them with large porous materials. It is considered undesirable to do so.

Here, it is unknown why the microporous inorganic carrier is effective to maintain the endogenous dormant state, but when there are small pores that cannot be supported by small microorganisms, the pores absorb moisture and physically isolate from the surface of the pores with spores. It is possible to add some moisture to germination so that it can be extracted. On the contrary, in the case of large pores in which spores can be supported, it is thought that they promote the germination of spores and the growth of vegetative-type cells because they act as a reservoir for moisture by absorbing moisture into the pores. This can be complemented by the fact that when the pore sizes differed, the larger the pores, the more spores awoke in the dormant phase and the proliferation of trophoblasts also occurred (Tables 1 and 4, Comparative Samples 14 and 15). ).

In conclusion, the heat resistant endospores of Bacillus subtilis AH18 can be produced by inducing spore formation by lethal temperature following the elevated temperature in the growth stage, and the heat stable endospores can resist for 1 hour in heat up to 90 ° C. have. The dormant state of the endospores can be maintained at 15-25 ° C. for 6 months when combined with an intermediate or microporous carrier having an average pore diameter of 0.63 μm and having a large BET surface area.

As described above, the method for formulating the microbial preparation according to the present invention generates heat resistant spores during formulation and incorporates an inorganic microporous carrier in the final step, thereby making the Bacillus product cost effective and resistant to high and low temperature variations. It is an effective invention in the microbial preparation industry because it is effective in maintaining a dormant state against the microorganism preparations without special management.

Claims (3)

Selecting Bacillus subtilis AH18 strain and culturing at elevated temperature; Applying heat stress at a lethal temperature of 57 ° C. to the formed spores to generate heat induced endospores; Removing residual growth cells from the spore mixture to produce a culture in which only spores are present; And Adding the Bacillus subtilis AH18 heat stable spore culture to a presterilized and dried intermediate and high strength inorganic carrier to prepare an endosporum-carrier mixture formulation. Formulation method of Bacillus subtilis AH18, characterized in that consisting of. The method of claim 1, wherein the inorganic carrier is a synthetic zeolite NaY (SiO ₂ : Al ₂ O ₃ : Na ₂ O = 46: 20: 11), synthetic zeolite NaA (SiO ₂ : Al ₂ O ₃ : Na ₂ O = 39 : 34: 20), a method of formulating Bacillus subtilis AH18, characterized in that it is selected from the group consisting of natural zeolites (Clinoptilolite) or kaolin clay minerals. Bacillus subtilis AH18 microbial preparation prepared by the method of claim 1.

Images