CN118077697A - Liquid composite microbial pesticide and preparation method thereof - Google Patents

The invention belongs to the technical field of microbial pesticides, and particularly relates to a liquid composite microbial pesticide and a preparation method thereof. The liquid composite microbial pesticide provided by the invention comprises composite strain fermentation liquor and ethyl 2-methylbutyrate, wherein the composite strain fermentation liquor consists of the following raw materials in percentage by weight: 70-80% of 5-ALA fermentation liquor and 20-30% of lactobacillus plantarum fermentation liquor. The invention provides a liquid compound microbial pesticide, which is prepared by carrying out compound mixed fermentation on a plurality of strains selected specifically, ensures that each strain realizes synergistic effect in the mixed fermentation process, effectively improves the yield of 5-ALA, can effectively prevent and treat mollusks such as slugs, snails and the like, is beneficial to reducing crop loss, reduces the use of chemical pesticides and provides a brand-new biological solution for preventing and treating agricultural pests such as slugs, snails and the like.

Liquid composite microbial pesticide and preparation method thereof

Technical Field

The invention belongs to the technical field of microbial pesticides, and particularly relates to a liquid composite microbial pesticide and a preparation method thereof.

Background

Mollusks such as slugs and snails are pests with special adaptability and are easy to cause damage to crops and horticultural plants in warm and humid seasons or areas. Slugs and snails feed on young leaves, shoots, flowers, fruits and roots of plants, especially on young seedlings and new tissues. The feeding behavior of the plant growth points is damaged, photosynthesis and nutrition accumulation are affected, and normal development of the plant is further hindered, and the wounds caused by feeding the plant growth points are extremely easy to induce diseases such as soft rot, fusarium wilt and the like, so that the leaves or seedlings are rotted and dead. After the slug and snail destroy the crops in large area, the yield of the crops can be reduced by more than 40 percent, and even the crops are lost in serious cases.

Conventional pesticides are typically designed for the nervous system or metabolic processes of insects, whereas slugs, snails, whose nervous system and metabolic pathways are significantly different from insects, and snails have a coat protection, which, although not completely blocking penetration of chemicals, is somewhat less effective on their internal tissues. While slugs, while not having a housing, may also have a mucilage layer that affects the contact and absorption of the agent. Due to the physiological structure and ecological habit of slugs and snails, the traditional pesticide has poor control effect.

5-Aminolevulinic acid (5-Aminolevulinic acid, hereinafter abbreviated as 5-ALA) is a natural non-protein amino acid, widely exists in animal, plant and microbial cells, and is an essential precursor substance for biosynthesis of tetrahydropyrrole compounds such as chlorophyll, heme and vitamin B ₁₂. 5-ALA is taken as an endogenous substance of organisms, plays an important role in the life process, and has wide application scenes and great market potential.

The existing research generally considers that the function of 5-ALA applied to agricultural production has two sides: can be used as plant growth regulator at low concentration (less than 100 mg/L), and has effects of improving photosynthetic efficiency, enhancing stress resistance, and promoting yield and quality; and at concentrations above 5 mmol/L (corresponding to about 800 mg/L), as biopesticides, bactericides, and herbicides. At present, no report on using 5-ALA or derivatives thereof (such as 5-aminolevulinate) as a medicament for controlling snails, slugs and the like exists in the domestic and foreign literature.

However, 5-ALA has been synthesized mainly by chemistry for a long time, and has high cost, and its application is basically limited to high value fields such as medicine; in recent years, due to the rapid development of genetic engineering and synthetic biology technology, the cost is greatly reduced by constructing genetic engineering bacteria for producing 5-ALA by high-efficiency fermentation, but genetic engineering bacteria for producing 5-ALA in the industry are not probiotics, after fermentation, sterilization treatment is needed to be carried out, and the materials are further used as metagen raw materials for formula application, so that the process and the technical complexity are increased to a certain extent, and certain limitations and challenges still exist in actual large-scale production.

The patent application CN 109929774B of the patent of the application discloses a Bacillus sp.PK9 strain and application thereof in preparing 5-aminolevulinic acid, wherein the strain is a natural strain and is also a Bacillus probiotic, not only can 5-ALA be synthesized by taking glucose as a carbon source, but also can be used for synthesizing 5-ALA by taking lignocellulose or potato residue fermentable sugar liquid and the like which are rich in resources and low in cost as the carbon source, and meanwhile, the strain can be directly fermented to produce a functional agricultural viable bacteria preparation. In addition, the inventor discovers that the fermentation liquor of the strain has strong killing activity on mollusks such as slugs, snails and the like in subsequent research work. Therefore, the strain of the application is utilized to develop an agricultural microbial agent for killing molluscs, which is environment-friendly and has the function of regulating plant growth, and is an innovation worthy of deep exploration.

However, the Bacillus sp.PK9 has higher cost when adopting glucose fermentation, and hydrolysis pretreatment is needed when lignocellulose (corncob, bagasse or switchgrass) or potato residues are utilized, so that the process is complicated and the process is complex; and the yield of 5-ALA produced by fermentation is low, which is about 23-26 mg/L.

Therefore, how to improve the fermentation process of Bacillus sp.PK9, improve the yield of 5-ALA, reduce the cost and develop a green and environment-friendly liquid compound microbial pesticide for controlling mollusks such as slugs, snails and the like is a technical problem to be solved urgently.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a liquid compound microbial pesticide and a preparation method thereof. The invention provides a liquid compound microbial pesticide, which is prepared by carrying out compound mixed fermentation on a plurality of strains selected specifically, ensures that the synergy is realized in the mixed fermentation process of each strain, effectively improves the yield of 5-ALA, can effectively prevent and treat mollusks such as slugs, snails and the like, is beneficial to reducing crop loss, reduces the use of chemical pesticides and provides a brand-new biological solution for preventing and treating agricultural pests such as slugs, snails and the like.

The technical scheme of the invention is as follows:

the liquid composite microbial pesticide comprises composite strain fermentation liquor and ethyl 2-methylbutyrate, wherein the composite strain fermentation liquor consists of the following raw materials in percentage by weight: 70-80% of 5-ALA fermentation liquor and 20-30% of lactobacillus plantarum fermentation liquor.

Further, the preparation method of the 5-ALA fermentation liquor in the composite strain fermentation liquor comprises the following steps:

s1, inoculating a second-level seed solution of saccharomyces cerevisiae into a mixed fermentation tank filled with a mixed fermentation medium for aerobic fermentation, and when the concentration of ethanol in the fermentation liquid reaches 2 g/L, ending the fermentation at the stage to obtain a saccharomyces cerevisiae fermentation liquid;

S2, inoculating a secondary seed solution of corynebacterium glutamicum into the saccharomyces cerevisiae fermentation liquid obtained in the step S1, adjusting the pH value to 7.8, performing aerobic fermentation, and when the glutamic acid content in the fermentation liquid reaches 4 g/L, ending the fermentation at the stage to obtain a saccharomyces cerevisiae and corynebacterium glutamicum mixed fermentation liquid;

S3, inoculating Bacillus sp.PK9 secondary seed liquid into the saccharomyces cerevisiae and corynebacterium glutamicum mixed fermentation liquid obtained in the step S2, adjusting the pH value to 7.2, performing aerobic fermentation, and ending the fermentation at the stage when the 5-ALA content in the fermentation liquid is more than or equal to 300mg/L to obtain the 5-ALA fermentation liquid.

Further, the components of the mixed fermentation medium in the step S1 are as follows:

The solvent is deionized water, and the solute and the concentration are respectively as follows: 70-80 g/L of molasses, 0.5-1.0 g/L of soybean peptone, 2.0-5.0 g/L of urea, 10-20 g/L of solid malt extract, 3.0-5.0 g/L of monopotassium phosphate, 1.0-2.0 g/L of magnesium sulfate, 0.01 g/L of manganese sulfate, 200 mu g/L of thiamine hydrochloride, 1.0-1.5 g/L of polysiloxane defoamer SXP-101 and pH of 6.8-7.0.

Further, the inoculation amount of the second-level seed liquid of the saccharomyces cerevisiae in the step S1 is 1-5%, and the liquid loading amount of the mixed fermentation medium in the mixed fermentation tank is 60-70% of the tank body; the temperature of the aerobic fermentation is 30-32 ℃, the fermentation time is 1-3 h, the rotating speed is 150-200 r/min, the ventilation is carried out according to the ratio of the ventilation volume to the volume of the fermentation tank of 1:1, and the tank pressure is 0.06-0.07 Mpa.

Furthermore, the inoculation amount of the second-level seed liquid of the saccharomyces cerevisiae in the step S1 is 5%, and the liquid loading amount of the mixed fermentation medium in the mixed fermentation tank is 65% of the tank body; the temperature of the aerobic fermentation is 31 ℃, the fermentation time is 3 h, the rotating speed is 150 r/min, the ventilation rate is equal to the ratio of the volume of the fermentation tank to 1:1, and the tank pressure is 0.06 Mpa.

Further, the inoculation amount of the secondary seed liquid of the corynebacterium glutamicum in the step S2 is 5-10%; the temperature of the aerobic fermentation is 31-33 ℃, the time is 36-48 h, the rotating speed is 800-1000 r/min, the ventilation is carried out according to the ratio of the ventilation volume to the volume of the fermentation tank of 1:1, and the tank pressure is 0.06-0.07 Mpa.

Further, the inoculation amount of the secondary seed liquid of the corynebacterium glutamicum in the step S2 is 7%; the temperature of the aerobic fermentation is 31.5 ℃, the time is 38 h, the rotating speed is 1000 r/min, the ventilation rate is in a ratio of 1:1 with the volume of the fermentation tank, and the tank pressure is 0.06 Mpa.

Further, the access amount of the Bacillus sp.PK9 secondary seed solution in the step S3 is 5-10%; the temperature of the aerobic fermentation is 32-37 ℃, the time is 24-36 h, the rotating speed is 400 r/min, the ventilation is carried out according to the ratio of the ventilation volume to the fermentation volume of 1:1, and the tank pressure is 0.06-0.07 Mpa.

Further, the access amount of the Bacillus sp.PK9 secondary seed solution in the step S3 is 10%; the temperature of the aerobic fermentation is 35 ℃, the time is 32 h, the rotating speed is 400 r/min, the ventilation rate is equal to the ratio of the ventilation rate to the fermentation volume of 1:1, and the tank pressure is 0.06 Mpa.

Further, the preparation method of the lactobacillus plantarum fermentation broth comprises the following steps:

And (3) inoculating the lactobacillus plantarum secondary seed solution into a fermentation tank according to an inoculation amount of 1-5%, wherein the liquid loading amount of a fermentation medium in the fermentation tank is 80-90% of that of the tank body, the fermentation temperature is 32-37 ℃, and standing anaerobic fermentation is carried out for 24-48 hours to obtain lactobacillus plantarum fermentation liquid.

Furthermore, the secondary seed liquid of the lactobacillus plantarum is inoculated into a fermentation tank according to the inoculation amount of 5 percent, the liquid loading amount of a fermentation medium in the fermentation tank is 90 percent of the tank body, the fermentation temperature is 36 ℃, and the standing anaerobic fermentation is adopted for 32 h to obtain the lactobacillus plantarum fermentation liquid.

Further, the formula of the fermentation medium adopted in the preparation process of the lactobacillus plantarum fermentation liquid is as follows:

The solvent is deionized water, and the solute and the concentration are respectively as follows: 70-80 g/L of molasses, 0.5-1.0 g/L of soybean peptone, 2.0-5.0 g/L of urea, 3.0-5.0 g/L of potassium dihydrogen phosphate and pH of 6.8-7.0.

In addition, the invention also provides a preparation method of the liquid composite microbial pesticide, which comprises the following steps:

Mixing 5-ALA fermentation liquor and lactobacillus plantarum fermentation liquor according to a certain proportion, filtering by a biological fermentation liquor filter to remove thalli and fermented insoluble substances, obtaining clarified composite strain fermentation liquor containing 5-ALA, adding ethyl 2-methylbutyrate into the fermentation liquor, and uniformly mixing to obtain the final product.

Furthermore, the addition amount of the ethyl 2-methylbutyrate is 0.01-0.02% of the weight of the composite strain fermentation liquor.

The adopted ethyl 2-methylbutyrate is a plant essential oil component, and aims to enhance the snail attracting effect of the pesticide.

Further, the preparation method of the secondary seed liquid of acid-resistant saccharomyces cerevisiae, corynebacterium glutamicum, bacillus sp.PK9 and lactobacillus plantarum can adopt a conventional culture process, and specifically, the preparation method adopted in the invention comprises the following steps:

The preparation method of the acid-resistant saccharomyces cerevisiae secondary seed liquid in the step S1 comprises the following steps:

(1) Transferring the acid-resistant saccharomyces cerevisiae strain stored at the inclined plane at the temperature of 4 ℃ into a seed culture medium (250 mL triangular flask), and culturing for 14-24 hours at the constant temperature of 32-37 ℃ under the condition of 150-200 r/min to obtain acid-resistant saccharomyces cerevisiae primary seed liquid;

(2) And (3) inoculating the acid-resistant saccharomyces cerevisiae primary seed liquid into a seed tank according to an inoculation amount of 1-5%, wherein the liquid loading amount is 80-90% of the tank body, the fermentation temperature is 32-37 ℃, the aerobic fermentation is carried out for 12-24 hours (200-400 r/min, the ventilation rate is ventilation according to a ratio of 1:1 of the volume of the fermentation tank, and the tank pressure is 0.06-0.07 Mpa), so as to obtain the acid-resistant saccharomyces cerevisiae secondary seed liquid.

The formula of the primary and secondary seed culture media of the saccharomyces cerevisiae is as follows: the solvent is deionized water, and the solute and the concentration are respectively as follows: sucrose 100g/L, soybean peptone 0.5-1.0 g/L, urea 2.0-5.0 g/L, potassium dihydrogen phosphate 3.0-5.0 g/L, magnesium sulfate 1.0-2.0 g/L, and pH 6.8-7.0.

The preparation method of the corynebacterium glutamicum secondary seed solution in the step S2 comprises the following steps:

Transferring the Corynebacterium glutamicum strain stored in the inclined plane at 4 ℃ into a seed culture medium (250 mL triangular flask), and culturing for 14-24 hours at constant temperature under the conditions of 32-37 ℃ and rotating speed of 150-200 r/min to obtain primary seed liquid of the Corynebacterium glutamicum;

And (3) inoculating the first-stage seed liquid of the corynebacterium glutamicum into a seed tank according to an inoculation amount of 1-5%, wherein the liquid loading amount is 80-90% of the tank body, and the fermentation temperature is 32-34 ℃. Aerobic fermentation is carried out for 12-24 h (200-400 r/min, ventilation is carried out according to the ratio of the ventilation amount to the volume of a fermentation tank of 1:1, and the tank pressure is 0.06-0.07 Mpa), so as to obtain the corynebacterium glutamicum secondary seed liquid.

The formula of the primary and secondary seed culture media of corynebacterium glutamicum is as follows: the solvent is deionized water, and the solute and the concentration are respectively as follows: molasses 50-60 g/L, ethanol 2 g/L, soybean peptone 0.5-1.0 g/L, urea 2.0-5.0 g/L, potassium dihydrogen phosphate 3.0-5.0 g/L, magnesium sulfate 1.0-2.0 g/L, manganese sulfate 0.01-g/L, thiamine hydrochloride 200 [ mu ] g/L and pH 6.8-7.0.

The preparation method of the Bacillus sp.PK9 secondary seed liquid in the step S3 comprises the following steps:

(1) Inoculating Bacillus sp.PK9 strain preserved at 4 ℃ in a conventional LB culture medium plate, and performing activation culture for 24-48 h at 32-37 ℃;

(2) Selecting single colony with fixed form on a flat plate, culturing in a seed culture medium (250 mL triangular flask) containing 50-100 mL at constant temperature of 32-37 ℃ and rotating speed of 150-200 r/min for 14-24 h to obtain primary seed liquid;

(3) And (3) inoculating the primary seed liquid into a seed tank according to an inoculum size of 1-5% for fermentation for 12-24 hours, wherein the fermentation temperature is 32-37 ℃, the rotation speed is 400 r/min, the ventilation rate is in a ratio of 1:1 with the volume of the fermentation tank, and the tank pressure is 0.06-0.07 Mpa, so as to obtain the secondary seed liquid.

The formula of the Bacillus sp.PK9 primary and secondary seed culture medium is as follows: the solvent is deionized water, and the solute and the concentration are respectively as follows: 2-4 g/L of glucose, 5-10 g/L of tryptone, 10-15 g/L of yeast powder, 3 g/L of monopotassium phosphate and an initial pH value of 7.3-7.5.

The preparation method of the lactobacillus plantarum secondary seed solution in the step S4 comprises the following steps:

Inoculating lactobacillus plantarum strains stored in an inclined plane at 4 ℃ into a seed culture medium (250 mL triangular flask and sealed bottle mouth), and standing and culturing for 36-48 hours at 32-37 ℃ to obtain lactobacillus plantarum primary seed liquid;

And (3) inoculating the lactobacillus plantarum primary seed solution into a seed tank according to an inoculum size of 1-5%, wherein the liquid loading amount is 80-90% of the tank body, the fermentation temperature is 32-37 ℃, and standing and anaerobic fermentation are carried out for 24-48 hours to obtain the secondary seed solution.

The formula of the lactobacillus plantarum primary and secondary seed culture medium is as follows: 10.0 g/L of peptone, 5.5 g/L of beef extract, 5.0 g/L of yeast extract, 10.0 g/L of glucose, 5.0 g/L of lactose, 5.0 g/L of sodium chloride and pH of 6.8-7.0.

The application patent application CN109929774B discloses Bacillus sp.PK9 and application thereof in preparing 5-aminolevulinic acid, the preservation number of the Bacillus sp.PK9 is CGMCC 16179, and the inventor surprisingly finds that the 5-aminolevulinic acid (5-ALA) prepared by fermenting the Bacillus sp.PK9 has a certain killing effect on mollusk snails, slugs and the like in the subsequent research and development processes of the strain. Therefore, in order to better realize the industrialized application of Bacillus sp.PK9, the application combines the specific strains by screening, adopts acid-resistant saccharomyces cerevisiae, corynebacterium glutamicum and Bacillus sp.PK9 for combined synergistic effect, has no antagonism among three strains and good co-existence, and can fully utilize the metabolic complementarity among different strains, promote the synthesis of target products and improve the overall fermentation efficiency and the product quality by the fermentation of different stages.

The acid-resistant saccharomyces cerevisiae used in the invention is low-temperature acid-resistant saccharomyces cerevisiae Saccharomyces cerevisiae, the strain number is CGMCC 2.786, glutamic acid can be generated in cells in the fermentation process, and the acid-resistant saccharomyces cerevisiae is used as a nutrition promoter of the subsequent Bacillus sp.PK9 to improve the fermentation efficiency, on the other hand, the acid-resistant saccharomyces cerevisiae can also be used for producing ethanol by utilizing molasses in fermentation raw materials, so that the necessary nutrition raw materials can be provided for the subsequent corynebacterium glutamicum fermentation stage; meanwhile, the acid-resistant saccharomyces cerevisiae can ferment solid wort in the mixed fermentation medium to generate malt fragrance, and can attract snails to eat and play a role in trapping.

The adopted strain number of the corynebacterium glutamicum Corynebacterium glutamicum is CGMCC 1.688, molasses in fermentation raw materials and ethanol produced by acid-resistant saccharomyces cerevisiae fermentation can be used as raw materials to further produce glutamic acid, and the glutamic acid is used as a necessary raw material for producing 5-ALA by the subsequent Bacillus sp.PK9 fermentation, so that the Bacillus sp.PK9 can be effectively promoted to produce 5-ALA by utilizing molasses, the content of 5-ALA in the finally prepared fermentation liquid is obviously improved, and the method is directly applied to pesticides without purification.

Furthermore, the lactobacillus plantarum fermentation liquid and the 5-ALA fermentation liquid are combined according to a certain proportion to prepare the liquid compound microbial pesticide with the pH value of about 3.5-4.0, on one hand, the strain number of the lactobacillus plantarum adopted by the invention is CGMCC 1.2469, the acid production is strong, the 5-ALA is used as a functional non-protein amino acid, the stability is enhanced under the pH environment, the insecticidal effect is improved, the using amount is less, and on the other hand, the lactobacillus plantarum adopted by the invention can generate bacteriocin in the fermentation process to inhibit the growth of harmful bacteria, so that the product quality and the insecticidal effect of the prepared pesticide are improved.

In addition, the invention also aims at optimizing fermentation processes of different strains, and firstly, the mixed fermentation medium adopted in the process of preparing the 5-ALA fermentation liquor is rich in various nutrient elements, is very suitable for the synergistic growth of acid-resistant saccharomyces cerevisiae, corynebacterium glutamicum and Bacillus sp.PK9 in the invention, and can ensure that each strain can be rapidly and stably proliferated and efficiently secrete 5-ALA and other insecticidal active substances in the fermentation process.

Meanwhile, the invention can stimulate the biosynthesis potential of the strain to the greatest extent and improve the yield of the 5-ALA by carefully regulating and controlling the fermentation conditions such as temperature, pH value, fermentation time, dissolved oxygen level, stirring rate and the like. In addition, the metabolic substances in the fermentation process can be dynamically adjusted by adopting a staged fermentation mode, and the accumulation of 5-ALA and other active substances with insecticidal action is promoted, so that the yield of products and the insecticidal effect are obviously improved.

The inventor of the application unexpectedly discovers that when the concentration of 5-ALA in the pesticide prepared by the application is in the range of 15-30 mg/L, the pesticide can have good killing effect on mollusks such as slugs and snails, and the concentration of the pesticide is more than 20 times lower than that of the 5-ALA recorded in literature reports. And at this concentration, it is also the concentration at which 5-ALA is used that has a growth regulator function for most crops.

Therefore, the liquid composite microbial pesticide provided by the invention can be effectively applied to prevention and control of common soft pests slug and snail in agricultural production, and has the plant growth regulating function.

The invention also provides a use method of the liquid composite microbial pesticide, which comprises the following steps: after diluting the liquid compound microorganism pesticide of the invention by 10-20 times, spraying the liquid compound microorganism pesticide onto leaf surfaces and grass, wherein the application amount is as follows: 1000-2000 mL/mu.

Compared with the prior art, the liquid composite microbial pesticide and the preparation method thereof provided by the invention have the following advantages:

(1) The liquid compound microbial pesticide provided by the invention aims to realize the synergy among strains in the mixed fermentation process by determining the optimal strain combination and proportion and optimizing the fermentation process, and the control effect on mollusks (such as slugs and snails) is strengthened jointly by interaction mechanisms such as metabolism synergy (such as sharing metabolites, complementary nutrition requirements and the like) and biological antagonism (such as inhibiting harmful microorganisms and optimizing microecological environments).

(2) The invention adopts multi-strain synergistic fermentation to obviously improve the final metabolite 5-ALA of Bacillus sp.PK9, and the molluscicide prepared by mixing the 5-ALA fermentation liquor prepared by the invention with lactobacillus plantarum fermentation liquor has the advantages of simplified production process, no need of further purifying the 5-ALA and effective saving of production cost.

(3) The liquid composite microbial pesticide provided by the invention has remarkable ecological friendliness, has small influence on the environment, and accords with the green agriculture concept. Besides the insecticidal function, the pesticide also has the plant growth regulating function, can promote the healthy growth of plants, strengthen the resistance of the pesticide to the attack of molluscs, and realize the double effects of pest control and crop growth regulation.

Drawings

FIG. 1 is a flow chart of a process for preparing a liquid composite microbial pesticide according to the present invention;

FIG. 2 shows the content of 5-ALA in the fermentation broth of 5-ALA prepared according to the invention at various fermentation times.

Detailed Description

The invention is further illustrated by the following description of specific embodiments, which are not intended to be limiting, and various modifications or improvements can be made by those skilled in the art in light of the basic idea of the invention, but are within the scope of the invention as long as they do not depart from the basic idea of the invention.

In the following examples and comparative examples, the reagents not specifically described were conventional reagents, which were purchased at conventional reagent manufacturing and sales companies, and the methods used, if not specifically described, were all prior art, and some of the raw material manufacturers were as follows:

The strain number of the corynebacterium glutamicum Corynebacterium glutamicum is CGMCC 1.688; the low-temperature acid-resistant saccharomyces cerevisiae Saccharomyces cerevisiae has the strain number of: CGMCC2.786; the lactobacillus plantarum Lactobacillus plantarum has the strain number of: CGMCC 1.2469, all purchased from China general microbiological culture Collection center (CGMCC).

Solid wort in the mixed fermentation medium, purchased from Shanghai Ind food and beverage Co., ltd;

Polysiloxane defoamer SXP-101 in the mixed fermentation medium is purchased from Huarun chemical Co., ltd.

Example 1a liquid complex microbial pesticide and method of making same:

the liquid composite microbial pesticide consists of composite strain fermentation liquor and ethyl 2-methylbutyrate;

the composite strain fermentation liquor consists of the following raw materials in percentage by weight: 75% of 5-ALA fermentation broth and 25% of lactobacillus plantarum fermentation broth.

The preparation method of the liquid composite microbial pesticide comprises the following steps:

S1, inoculating acid-resistant saccharomyces cerevisiae secondary seed liquid into a mixed fermentation tank according to an inoculum size of 5%, wherein the liquid loading amount of a mixed fermentation medium in the mixed fermentation tank is 65% of that of a tank body, the fermentation temperature is 31 ℃, the aerobic fermentation is 3 h (150 r/min, the aeration rate is aerated according to a ratio of 1:1 of the volume of the fermentation tank, and the tank pressure is 0.06 Mpa), and when the ethanol concentration in the fermentation liquid reaches 2 g/L, the fermentation at the stage is finished, so that saccharomyces cerevisiae fermentation liquid is obtained;

S2, inoculating a secondary seed solution of corynebacterium glutamicum into the saccharomyces cerevisiae fermentation liquid obtained in the step S1 according to the inoculation amount of 7%, adjusting the pH value to 7.8, performing aerobic fermentation at the fermentation temperature of 31.5 ℃ at 38 h (1000 r/min, performing aeration according to the ratio of the aeration amount to the volume of a fermentation tank of 1:1, and performing tank pressure of 0.06 Mpa), and when the glutamic acid content in the fermentation liquid reaches 4 g/L, ending fermentation at the stage to obtain a saccharomyces cerevisiae and corynebacterium glutamicum mixed fermentation liquid;

s3, inoculating Bacillus sp.PK 9 secondary seed liquid into the mixed fermentation liquid obtained in the step S2 according to the inoculation amount of 10%, adjusting the pH value to 7.2, performing aerobic fermentation at the fermentation temperature of 35 ℃ to 32 h (400 r/min, performing aeration according to the ratio of the aeration amount to the fermentation volume of 1:1, and performing tank pressure of 0.06 Mpa), and when the 5-ALA content in the fermentation liquid is more than or equal to 300mg/L, ending the fermentation at the stage to obtain 5-ALA fermentation liquid;

S4, inoculating the secondary seed liquid of the lactobacillus plantarum into a fermentation tank according to an inoculum size of 5%, wherein the liquid loading amount of a fermentation medium is 90% of that of the tank body, the fermentation temperature is 36 ℃, and standing anaerobic fermentation is adopted for 32 h, so that lactobacillus plantarum fermentation liquid is obtained;

S5, mixing the 5-ALA fermentation liquor obtained in the step S3 and the lactobacillus plantarum fermentation liquor obtained in the step S4, wherein the 5-ALA fermentation liquor accounts for 75% of the total weight of the two, the lactobacillus plantarum fermentation liquor accounts for 25%, filtering through a biological fermentation liquor filter to remove thalli and fermented insoluble substances, obtaining clarified composite strain fermentation liquor containing 5-ALA, and uniformly mixing the clarified composite strain fermentation liquor with ethyl 2-methylbutyrate accounting for 0.02% of the weight of the composite strain fermentation liquor, thereby obtaining the liquid composite microbial pesticide.

The components of the mixed fermentation medium in the step S1 are as follows:

The solvent is deionized water, and the solute and the concentration are respectively as follows: 80 g/L molasses, 1.0 g/L soybean peptone, 5.0 g/L urea, 20 g/L solid malt extract, 3.0 g/L potassium dihydrogen phosphate, 1.0 g/L magnesium sulfate, 0.01 g/L manganese sulfate, 200 μg/L thiamine hydrochloride, 1.2 g/L polysiloxane defoamer SXP-101, and pH 6.8.

The components of the fermentation medium for fermenting lactobacillus plantarum in the step S4 are as follows:

The solvent is deionized water, and the solute and the concentration are respectively as follows: 70 g/L molasses, 0.5 g/L soybean peptone, 2.0 g/L urea, 3.0 g/L potassium dihydrogen phosphate and pH 6.8.

The preparation method of the acid-resistant saccharomyces cerevisiae secondary seed liquid in the step S1 comprises the following steps:

(1) Transferring the acid-resistant saccharomyces cerevisiae strain stored in the inclined plane at 4 ℃ into a seed culture medium (250 mL triangular flask), and culturing at constant temperature of 34 ℃ and 150 r/min for 20 h to obtain acid-resistant saccharomyces cerevisiae primary seed liquid;

(2) The first-stage seed liquid of the acid-resistant saccharomyces cerevisiae is inoculated into a seed tank according to the inoculation amount of 5 percent, the liquid loading amount is 80 percent of the tank body, the fermentation temperature is 32 ℃, the aerobic fermentation is 20 h (250 r/min, the ventilation amount is aerated according to the ratio of the volume of the fermentation tank to 1:1, and the tank pressure is 0.06 Mpa), so as to obtain the second-stage seed liquid of the acid-resistant saccharomyces cerevisiae.

The formula of the primary and secondary seed culture media of the saccharomyces cerevisiae is as follows: the solvent is deionized water, and the solute and the concentration are respectively as follows: sucrose 100 g/L, soybean peptone 0.5 g/L, urea 2.5 g/L, potassium dihydrogen phosphate 3.5 g/L, magnesium sulfate 1.5 g/L, and pH 6.8.

The preparation method of the corynebacterium glutamicum secondary seed solution in the step S2 comprises the following steps:

Inoculating Corynebacterium glutamicum strain stored in 4 deg.C inclined plane into seed culture medium (250 mL triangular flask), and culturing at constant temperature of 35 deg.C and rotation speed of 150 r/min for 18 h to obtain primary seed liquid of Corynebacterium glutamicum;

the first-level seed liquid of corynebacterium glutamicum is inoculated into a seed tank according to the inoculation amount of 5 percent, the liquid loading amount is 80 percent of the tank body, and the fermentation temperature is 32 ℃. Aerobic fermentation of 18 h (280 r/min, aeration rate according to the ratio of 1:1 of the volume of the fermentation tank, and tank pressure of 0.06 Mpa) to obtain secondary seed liquid of corynebacterium glutamicum.

The formula of the primary and secondary seed culture media of corynebacterium glutamicum is as follows: the solvent is deionized water, and the solute and the concentration are respectively as follows: molasses 50 g/L, ethanol 2 g/L, soytone 1.0 g/L, urea 2.0 g/L, potassium dihydrogen phosphate 3.0 g/L, magnesium sulfate 1.0 g/L, manganese sulfate 0.01 g/L, thiamine hydrochloride 200 μg/L, and pH 6.8.

The preparation method of the Bacillus sp.PK9 secondary seed liquid in the step S3 comprises the following steps:

(1) Inoculating Bacillus sp.PK9 strain stored in 4 deg.C inclined plane into conventional LB culture medium plate, and activating at 35deg.C for culturing 36 h;

(2) Selecting single colony with fixed form on the plate, culturing in seed culture medium (250 mL triangular flask) containing 50 mL at constant temperature of 32deg.C and rotation speed of 150 r/min for 24 h to obtain first-stage seed solution;

(3) The first-stage seed liquid is inoculated into a seed tank for fermentation 22h according to the inoculation amount of 5%, the fermentation temperature is 37 ℃, the rotating speed is 400 r/min, the ventilation amount is in a ratio of 1:1 with the volume of the fermentation tank, and the tank pressure is 0.06 Mpa, so that the second-stage seed liquid is obtained.

The formula of the Bacillus sp.PK9 primary and secondary seed culture medium is as follows: the solvent is deionized water, and the solute and the concentration are respectively as follows: glucose 3 g/L, tryptone 8 g/L, yeast powder 12 g/L, monopotassium phosphate 3 g/L and initial pH 7.3.

The preparation method of the lactobacillus plantarum secondary seed solution in the step S4 comprises the following steps:

Inoculating Lactobacillus plantarum strain preserved at 4deg.C inclined plane into seed culture medium (250 mL triangular flask, sealed bottle mouth), and standing at 35deg.C for 36: 36 h to obtain Lactobacillus plantarum primary seed solution;

inoculating the first-stage seed liquid of lactobacillus plantarum into a seed tank according to an inoculum size of 5%, wherein the liquid loading amount is 90% of the tank body, the fermentation temperature is 36 ℃, and standing anaerobic fermentation is carried out for 46 h, so as to obtain a second-stage seed liquid.

The formula of the lactobacillus plantarum primary and secondary seed culture medium is as follows: 10.0 g/L peptone, 5.5 g/L beef extract, 5.0 g/L yeast extract, 10.0 g/L glucose, 5.0 g/L lactose, 5.0 g/L sodium chloride, pH 6.8.

Example 2a liquid complex microbial pesticide and method of making same:

the liquid composite microbial pesticide consists of composite strain fermentation liquor and ethyl 2-methylbutyrate;

the composite strain fermentation liquor consists of the following raw materials in percentage by weight: 70% of 5-ALA fermentation broth and 30% of lactobacillus plantarum fermentation broth.

The preparation method of the liquid composite microbial pesticide comprises the following steps:

S1, inoculating acid-resistant saccharomyces cerevisiae secondary seed liquid into a mixed fermentation tank according to an inoculum size of 2%, wherein the liquid loading amount of a mixed fermentation medium in the mixed fermentation tank is 70% of that of a tank body, the fermentation temperature is 30 ℃, aerobic fermentation is 1 h (160 r/min, the aeration rate is 0.06 Mpa according to a ratio of 1:1 of the volume of the fermentation tank), and when the concentration of ethanol in the fermentation liquid reaches 2 g/L, the fermentation at the stage is finished, so that saccharomyces cerevisiae fermentation liquid is obtained;

S2, inoculating a secondary seed solution of corynebacterium glutamicum into the saccharomyces cerevisiae fermentation liquid obtained in the step S1 according to an inoculation amount of 5%, adjusting the pH value to 7.8, performing aerobic fermentation at a fermentation temperature of 31 ℃ to 36 h (800 r/min, performing aeration with aeration amount according to a ratio of 1:1 with the volume of a fermentation tank, and performing tank pressure of 0.06 Mpa), and when the glutamic acid content in the fermentation liquid reaches 4g/L, ending fermentation at the stage to obtain a saccharomyces cerevisiae and corynebacterium glutamicum mixed fermentation liquid;

S3, inoculating Bacillus sp.PK9 secondary seed liquid into the mixed fermentation liquid obtained in the step S2 according to an inoculum size of 5%, adjusting the pH to 7.2, performing aerobic fermentation at a fermentation temperature of 32 ℃ for 24h (400 r/min, performing aeration with aeration volume according to a ratio of 1:1 to fermentation volume, and performing tank pressure of 0.06 Mpa), and ending fermentation at the stage when the 5-ALA content in the fermentation liquid is more than or equal to 300mg/L to obtain 5-ALA fermentation liquid;

S4, inoculating the secondary seed liquid of the lactobacillus plantarum into a fermentation tank according to the inoculation amount of 2%, wherein the liquid loading amount of a fermentation medium is 80% of that of the tank body, the fermentation temperature is 32 ℃, and standing anaerobic fermentation is adopted for 24 h, so that lactobacillus plantarum fermentation liquid is obtained;

S5, mixing the 5-ALA fermentation liquor obtained in the step S3 and the lactobacillus plantarum fermentation liquor obtained in the step S4, wherein the 5-ALA fermentation liquor accounts for 70% of the total weight of the two, the lactobacillus plantarum fermentation liquor accounts for 30%, filtering through a biological fermentation liquor filter to remove thalli and fermented insoluble substances, obtaining clarified composite strain fermentation liquor containing 5-ALA, and uniformly mixing the clarified composite strain fermentation liquor with ethyl 2-methylbutyrate accounting for 0.01% of the weight of the composite strain fermentation liquor, thereby obtaining the liquid composite microbial pesticide.

The components of the mixed fermentation medium in the step S1 are as follows:

The solvent is deionized water, and the solute and the concentration are respectively as follows: 70 g/L of molasses, 0.5 g/L of soybean peptone, 2.0 g/L of urea, 10 g/L of solid malt extract, 4.0 g/L of monopotassium phosphate, 1.5 g/L of magnesium sulfate, 0.01 g/L of manganese sulfate, 200 mug/L of thiamine hydrochloride, 1.0 g/L of polysiloxane defoamer SXP-101 and pH 6.9.

The components of the fermentation medium for fermenting lactobacillus plantarum in the step S4 are as follows:

the solvent is deionized water, and the solute and the concentration are respectively as follows: 75 g/L molasses, 0.7 g/L soybean peptone, 3.0 g/L urea, 4.0 g/L potassium dihydrogen phosphate and pH 6.9.

The preparation methods of the acid-resistant saccharomyces cerevisiae secondary seed solution in the step S1, the corynebacterium glutamicum secondary seed solution in the step S2, the Bacillus sp.pk9 secondary seed solution in the step S3 and the lactobacillus plantarum secondary seed solution in the step S4 are the same as those of the example 1.

Example 3a liquid complex microbial pesticide and method of making same:

the liquid composite microbial pesticide consists of composite strain fermentation liquor and ethyl 2-methylbutyrate;

the composite strain fermentation liquor consists of the following raw materials in percentage by weight: 80% of 5-ALA fermentation broth and 20% of lactobacillus plantarum fermentation broth.

The preparation method of the liquid composite microbial pesticide comprises the following steps:

s1, inoculating acid-resistant saccharomyces cerevisiae secondary seed liquid into a mixed fermentation tank according to an inoculum size of 3%, wherein the liquid loading amount of a mixed fermentation medium in the mixed fermentation tank is 60% of that of a tank body, the fermentation temperature is 32 ℃, aerobic fermentation is 2h (180 r/min, the aeration rate is aerated according to a ratio of 1:1 of the volume of the fermentation tank, and the tank pressure is 0.07 and Mpa), and when the ethanol concentration in the fermentation liquid reaches 2 g/L, the fermentation at the stage is finished, so that saccharomyces cerevisiae fermentation liquid is obtained;

S2, inoculating a secondary seed solution of corynebacterium glutamicum into the saccharomyces cerevisiae fermentation liquid obtained in the step S1 according to the inoculation amount of 10%, adjusting the pH value to 7.8, performing aerobic fermentation at the fermentation temperature of 33 ℃ and at the aeration rate of 48h (900 r/min, the aeration rate is in a ratio of 1:1 with the volume of a fermentation tank, and the tank pressure is 0.07 and Mpa), and when the glutamic acid content in the fermentation liquid reaches 4 g/L, ending the fermentation at the stage, thus obtaining a saccharomyces cerevisiae and corynebacterium glutamicum mixed fermentation liquid;

S3, inoculating Bacillus sp.PK9 secondary seed liquid into the mixed fermentation liquid obtained in the step S2 according to the inoculation amount of 8%, adjusting the pH value to 7.2, performing aerobic fermentation at 37 ℃ to 36 h (400 r/min, and performing aeration with aeration volume according to the ratio of 1:1 to the fermentation volume, wherein the tank pressure is 0.07 and Mpa), and when the 5-ALA content in the fermentation liquid is more than or equal to 300mg/L, ending the fermentation at the stage to obtain 5-ALA fermentation liquid;

S4, inoculating the lactobacillus plantarum secondary seed solution into a fermentation tank according to the inoculation amount of 3%, wherein the liquid loading amount of a fermentation medium is 85% of that of the tank body, the fermentation temperature is 37 ℃, and standing anaerobic fermentation is adopted for 48 h, so that lactobacillus plantarum fermentation liquid is obtained;

S5, mixing the 5-ALA fermentation liquor obtained in the step S3 and the lactobacillus plantarum fermentation liquor obtained in the step S4, wherein the 5-ALA fermentation liquor accounts for 80% of the total weight of the two, the lactobacillus plantarum fermentation liquor accounts for 20%, filtering through a biological fermentation liquor filter to remove thalli and fermented insoluble substances, obtaining clarified composite strain fermentation liquor containing 5-ALA, and uniformly mixing the clarified composite strain fermentation liquor with ethyl 2-methylbutyrate accounting for 0.02% of the weight of the composite strain fermentation liquor, thereby obtaining the liquid composite microbial pesticide.

The components of the mixed fermentation medium in the step S1 are as follows:

The solvent is deionized water, and the solute and the concentration are respectively as follows: 75 g/L molasses, 0.8 g/L soybean peptone, 3.0 g/L urea, 15 g/L solid malt extract, 4.0 g/L potassium dihydrogen phosphate, 1.5 g/L magnesium sulfate, 0.01 g/L manganese sulfate, 200 μg/L thiamine hydrochloride, 1.5 g/L polysiloxane defoamer SXP-101, and pH 7.0.

The components of the fermentation medium for fermenting lactobacillus plantarum in the step S4 are as follows:

The solvent is deionized water, and the solute and the concentration are respectively as follows: 75 g/L molasses, 1.0 g/L soybean peptone, 5.0g/L urea, 5.0g/L potassium dihydrogen phosphate and pH 6.8.

The preparation methods of the acid-resistant saccharomyces cerevisiae secondary seed solution in the step S1, the corynebacterium glutamicum secondary seed solution in the step S2, the Bacillus sp.pk9 secondary seed solution in the step S3 and the lactobacillus plantarum secondary seed solution in the step S4 are the same as those of the example 1.

Comparative example 1:

In comparison with example 1, comparative example 1 is different in that solid wort is removed from the mixed fermentation medium in the step S1, and ethyl 2-methylbutyrate is not added in the step S5. Other components and preparation methods were the same as in example 1.

Comparative example 2:

In comparison with example 1, comparative example 2 differs in that the fermentation broth of 5-ALA was prepared without fermentation using acid-resistant Saccharomyces cerevisiae and Corynebacterium glutamicum, but only using Bacillus sp.PK9. Other components and preparation methods were the same as in example 1. As a control, the effect of acid-tolerant Saccharomyces cerevisiae and Corynebacterium glutamicum on the yield of 5-ALA was compared. The specific fermentation method comprises the following steps:

Directly inoculating the prepared Bacillus sp.PK9 secondary seed solution into the mixed fermentation medium according to the inoculation amount of 10%, regulating the pH value to 7.2, carrying out tank pressure to 0.06 Mpa, carrying out aeration according to the ratio of the aeration amount to the fermentation volume of 1:1, carrying out constant-temperature fermentation at the rotation speed of 400 r/min and the fermentation speed of 32 h at the temperature of 35 ℃, and detecting the 5-ALA content in the fermentation liquid.

The preparation method of the Bacillus sp.PK9 seed solution is the same as that of the example 1.

The composition of the mixed fermentation medium was the same as in example 1.

Comparative example 3:

In comparison with example 1, comparative example 3 was distinguished in that during the preparation of the 5-ALA fermentation broth, acid-resistant Saccharomyces cerevisiae, corynebacterium glutamicum and Bacillus sp.PK9 were simultaneously introduced into a mixed fermentation medium for fermentation, and the other components were the same as in example 1. As a control, the effect of the inoculation sequence of the acid-tolerant Saccharomyces cerevisiae and Corynebacterium glutamicum on the yield of 5-ALA was compared.

The specific fermentation method comprises the following steps:

The acid-resistant saccharomyces cerevisiae secondary seed liquid is respectively inoculated into a mixed fermentation tank according to an inoculation amount of 5%, the liquid loading amount of the mixed fermentation tank is 65% of the tank body, then the lactobacillus glutamicum secondary seed liquid is inoculated according to an inoculation amount of 7%, the prepared Bacillus sp.PK9 secondary seed liquid is inoculated according to an inoculation amount of 10%, the pH value is adjusted to 7.2, the tank pressure is 0.06 Mpa, the ventilation amount is ventilation according to a ratio of the ventilation volume to 1:1, 400 r/min, the constant temperature fermentation is carried out at 35 ℃ for 32h, and the 5-ALA content in the fermentation liquid is detected.

The preparation method of the acid-resistant saccharomyces cerevisiae, corynebacterium glutamicum and Bacillus sp.PK9 secondary seed solution is the same as that of the example 1.

The composition of the mixed fermentation medium was the same as in example 1.

Test example one, 5-ALA fermentation yield detection:

and quantitatively measuring the 5-ALA content in the 5-ALA fermentation broth by adopting a spectrophotometry. The method comprises the following specific steps:

The 5-ALA fermentation broths at different fermentation times in step S3 of example 1, comparative example 2 and comparative example 3 were centrifuged, 400. Mu.L of the supernatant was taken, 200. Mu.L of acetate buffer and 100. Mu.L of acetylacetone were added, and water bath 15 min was boiled. After cooling to room temperature, 700. Mu.L of Ehrlich's reagent (Ehrlich's reagent formulation is as follows: 1 g pair dimethylaminobenzaldehyde was added to 30mL glacial acetic acid, then 8 mL perchloric acid (70%) was added, the volume was set to 50mL with glacial acetic acid, and ready to use) was added, reaction 20min was detected using a spectrophotometer at 554 nm. And simultaneously, 5-aminolevulinic acid is used as a standard substance, and a standard curve is drawn. According to the standard curve, the content of 5-ALA in the 5-aminolevulinic acid fermentation liquid at different fermentation times is calculated.

The 5-ALA yields at different fermentation times in example 1, comparative example 2 and comparative example 3 are shown in FIG. 2.

As can be seen from FIG. 2, the different fermentation modes have a great influence on the 5-ALA production. The yield of 5-ALA using the preparation method of the present invention (example 1) was up to 318 mg/L at 32 h, which is much higher than that of comparative examples 2 and 3. Meanwhile, the yield of 5-ALA of Bacillus sp.PK9 in the invention patent 201910084426.2 is obviously better than that of the Bacillus sp.PK9 (about 23-26 mg/L).

Test example two, pesticide efficacy test:

And selecting a soybean farmland with snail insect pests, and equally dividing the soybean farmland into different treatment groups, wherein the area of each treatment group is 200m ². The dosage of the liquid medicine or the microbial inoculum is calculated according to the use instruction concentration. Spraying in the evening for 1 time.

A blank control group, a positive control group (40% metaldehyde suspending agent), examples 1 to 3 groups, and comparative examples 1 to 3 groups;

The positive control group adopts 40% metaldehyde (common snail prevention and treatment agent) suspending agent, the suspending agent is diluted 400 times, soybean leaves are uniformly sprayed by a sprayer, and the spraying amount is as follows: the use of 200 mL undiluted 40% metaldehyde suspending agent per mu translates into the use of the same.

The treatment methods of the examples 1-3 and the comparative examples 1-3 are as follows: the liquid composite microbial pesticides prepared in examples 1 to 3 and comparative examples 1 to 3 were diluted 20 times and uniformly sprayed on soybean leaves with a sprayer. The spray amount is as follows: the fermentation broth is calculated by using 1500 mL undiluted fermentation broth per mu.

The blank group was sprayed with an equal amount of water uniformly on the soybean leaves using a sprayer.

100 Plants of soybean were randomly selected in the test cell of each treatment group, and the snail amount of the hundred plants of soybean before the application of the drug and the snail residual amount of the hundred plants of soybean 7 days after the application of the drug and 14 days after the application of the drug were examined, respectively. And the rate of reduction of insect population, the correction control effect and the feeding rate were calculated respectively, and the test results are shown in tables 1 and 2. The calculation formula is as follows:

Rate of reduction of insect population (%) = (pre-drug basal number-post-drug number of living insects)/pre-drug basal number x 100%;

Correction control effect (%) = {1- (number of live insects after control in treatment area×number of live insects after control in control area/number of live insects before control area×number of live insects after control in control area) } ×100%;

feeding rate (%) = number of fed leaves in treatment group/number of fed leaves in blank control group x 100%.

Table 1 snail controlling effect (7 days control effect)

Table 2 snail controlling effect (14 days controlling effect)

The test results in tables 1 and 2 show that the effect of the liquid compound microbial pesticide prepared in the embodiments 1-3 is obviously better than the control effect of the traditional chemical reagent metaldehyde in snail control. And the 7 day control and 14 day control of example 1 are both greater than 40% metaldehyde suspending agent. When the mixed fermentation medium of the comparative example 1 is not added with solid wort and is not added with ethyl 2-methylbutyrate, the insecticidal effect is obviously affected, and the control effect is obviously reduced; in comparative examples 2 to 3, when the fermentation strain and the fermentation sequence in the process of preparing the insecticide are changed, the insecticidal effect of the prepared insecticide is greatly reduced.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.