Bacillus amyloliquefaciens capable of efficiently degrading pyrethroid pesticides, bacterial preparation and application of bacillus amyloliquefaciens and bacterial preparation
By isolating and applying bacterial preparations of Bacillus amyloid MR17, the problem of difficult degradation of pyrethroid pesticides is solved, and efficient degradation of a variety of pyrethroid pesticides is achieved, and environmental and food safety is improved.
Patent Information
- Application Number
- CN202411611439.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-12
- Publication Date
- 2025-02-11
AI Technical Summary
The prior art is difficult to effectively degrade pyrethroid pesticides, especially β-cyanthrin, which poses a threat to the environment and food safety.
Bacillus amyloliquefaciens MR17, which efficiently degrades pyrethroid pesticides, was screened and isolated, and its bacterial preparations were developed to degrade these pesticides through fermentation broth or solid preparations.
It has achieved efficient degradation of a variety of pyrethroid pesticides including β-cyanothrin, enriched the resource library of degraded strains, and provided new solutions for environmental and food safety.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microbial degradation, and relates to a Bacillus amyloliquefaciens strain capable of efficiently degrading pyrethroid pesticides, a bacterial preparation and application thereof. Background Art
[0002] Pyrethroids are synthetic analogs of natural pyrethroids. They are a class of highly effective, broad-spectrum, low-toxic and biodegradable synthetic insecticides. Due to their wide insecticidal spectrum, good effects, low residues and non-accumulative effects, they have been widely used in agricultural pest control in the past 30 years, and have also achieved good results in pest control of vegetables and fruit trees. However, due to the large-scale use and unreasonable treatment of pyrethroid pesticides, residual pesticides have spread into the soil and various water environments, causing various environmental and food safety problems, directly or indirectly threatening human health. For example, some beneficial insects, including silkworms, honey bees and trichogrammatids, have died in clusters due to the poisoning of residual pyrethroid pesticides. Pyrethroid pesticides are also highly toxic to aquatic organisms such as fish, clams and swamp shrimp. When the concentration of pyrethroid pesticides reaches 10ng / L, it is enough to kill all invertebrates in the water. In addition, some pyrethroid pesticides also have neurotoxicity, cumulative toxicity and reproductive toxicity, and even have the risk of carcinogenicity, teratogenicity and mutagenicity. Therefore, the research on bioremediation of pesticide-contaminated environment and the solution to the pollution of environment and food by various pesticides including pyrethroid pesticides have received more and more attention.
[0003] β-Cypermethrin is a broad-spectrum insecticide in the pyrethroid pesticide class. It has high insecticidal activity against many types of pests. It can be used for pest control of fruit trees, vegetables, grains, cotton, oil, tea and other crops, as well as a variety of forest trees. It is a pesticide with a large amount of use in my country. With the increase in the use of β-Cypermethrin, the residues of this pesticide in the environment continue to accumulate, causing pollution to the ecological environment and directly affecting the quality of agricultural products and food safety. Therefore, it is very important to explore methods for the efficient degradation of pyrethroid pesticides including β-Cypermethrin.
[0004] Microbial degradation is a safe, efficient, low-cost remediation method that does not cause secondary pollution. It is widely considered to be one of the effective means to control pesticide environmental pollution and reduce pesticide residues in agricultural products. At present, researchers at home and abroad have isolated a variety of pyrethroid pesticide degradation strains from the natural environment, including Pseudomonas, Bacillus licheniformis, Rhodococcus pyridinus, Bacillus cereus, Bacillus lysinicus, etc. However, there are limited reports on the use of probiotic strains of Bacillus amyloliquefaciens to degrade pyrethroid pesticides, and there are even fewer reports on the application of microbial preparations in agricultural products and the environment.
[0005] Therefore, screening probiotic strains that can effectively degrade pyrethroid pesticides including β-cypermethrin, and developing microbial preparations based on them, and applying them to the degradation of residual pyrethroid pesticides in the environment can not only enrich the resources of pyrethroid pesticide degradation bacteria, but also provide a theoretical basis for the application of probiotic preparations in pesticide degradation. Summary of the invention
[0006] One of the purposes of the present invention is to provide a Bacillus amyloliquefaciens strain and a bacterial preparation that can efficiently degrade pyrethroid pesticides. The second purpose of the present invention is to provide the use of the Bacillus amyloliquefaciens strain and the bacterial preparation in degrading pyrethroid pesticides, so as to enrich the strain resource library that can efficiently degrade pyrethroid pesticides, and provide a new idea for solving the problems of excessive pyrethroid pesticide residues in agricultural products and pyrethroid pesticide pollution in the environment.
[0007] In order to achieve the above-mentioned invention object, the technical solution provided by the present invention is as follows:
[0008] A strain of Bacillus amyloliquefaciens MR17 that efficiently degrades pyrethroid pesticides. The Bacillus amyloliquefaciens MR17 was deposited in the General Microbiology Center of the China Culture Collection Administration on July 8, 2024, with the deposit number CGMCCNO.31203.
[0009] The above-mentioned Bacillus amyloliquefaciens MR17 is isolated from Daqu and is obtained by adding β-cypermethrin to the culture medium for targeted enrichment, domestication and purification.
[0010] A microbial preparation, comprising the above-mentioned Bacillus amyloliquefaciens MR17.
[0011] In the technical solution of the above-mentioned microbial preparation, the Bacillus amyloliquefaciens MR17 is the only active ingredient or the main active ingredient of the microbial preparation.
[0012] In the technical scheme of the above-mentioned microbial preparation, the microbial preparation can be the fermentation broth of Bacillus amyloliquefaciens MR17; the microbial preparation can also be a solid preparation directly prepared from the bacterial body of Bacillus amyloliquefaciens MR17, such as a suspension, a powder, etc.; the microbial preparation can also be a preparation prepared by combining the Bacillus amyloliquefaciens MR17 as the sole active ingredient or the main active ingredient with the commonly used auxiliary ingredients in the prior art. The microbial preparation can be prepared by referring to the existing method. For example, the microbial preparation can be a dry powder preparation prepared by mixing the Bacillus amyloliquefaciens MR17 bacterial body with the protective agent and other auxiliary ingredients in the prior art, and freeze-drying or spray-drying. The protective agent can be sucrose, glucose and corn starch, etc.
[0013] The present invention verifies through experiments that: (1) the fermentation broth of the Bacillus amyloliquefaciens MR17 of the present invention has a good degradation effect on a variety of pyrethroid pesticides including β-cypermethrin, deltamethrin, bifenthrin, cypermethrin, fenvalerate and flucythrinate; and (2) the bacterial cells of the Bacillus amyloliquefaciens MR17 of the present invention are prepared together with auxiliary components into a freeze-dried powder, and the freeze-dried powder also has a good degradation effect on β-cypermethrin after rehydration.
[0014] Based on the above experimental results, the present invention also provides the use of the above-mentioned Bacillus amyloliquefaciens MR17 or the above-mentioned microbial preparation in degrading pyrethroid pesticides.
[0015] In the technical solution of the above application, the above Bacillus amyloliquefaciens MR17 is inoculated into the sample to be degraded, or the above microbial preparation is added into the sample to be degraded, so as to degrade the pyrethroid pesticides in the sample to be degraded.
[0016] In the above-mentioned technical solution, the pyrethroid pesticide includes at least one of β-cypermethrin, deltamethrin, bifenthrin, cypermethrin, fenvalerate and cyfluthrin.
[0017] In the above-mentioned technical solution, when degrading pyrethroid pesticides, it is preferred to control the degradation temperature to 30-37° C. and the degradation pH value to 6-7.
[0018] In the above-mentioned technical solution, when degrading pyrethroid pesticides, the degradation time is preferably controlled to be 48 to 72 hours.
[0019] In the technical solution of the above application, when degrading pyrethroid pesticides, it is preferred to control the concentration of pyrethroid pesticides in the sample to be degraded to not exceed 100 mg / L and not be 0.
[0020] In the technical solution of the above application, when degrading pyrethroid pesticides, the inoculation amount of the above Bacillus amyloliquefaciens MR17, or the addition amount of the above microbial preparation, can be determined through experiments after the specific sample to be degraded is determined, with the principle of achieving a relatively high degradation rate for pyrethroid pesticides by adjusting the inoculation amount or the addition amount.
[0021] For example, when the fermentation liquid of the above-mentioned Bacillus amyloliquefaciens MR17 is used to degrade pyrethroid pesticides, the above-mentioned Bacillus amyloliquefaciens MR17 is cultured to the late logarithmic growth stage and inoculated into the sample to be degraded. Generally, the above-mentioned Bacillus amyloliquefaciens MR17 can be cultured to the late logarithmic growth stage, and the OD 600 The value is set to 1.0 to form a seed solution, and the seed solution is inoculated into the sample to be degraded at an inoculation amount of 6% (v / v) to 8% (v / v) to degrade the pyrethroid pesticides.
[0022] Compared with the prior art, the technical solution provided by the present invention produces the following beneficial technical effects:
[0023] 1. The present invention provides a strain of Bacillus amyloliquefaciens MR17 that can efficiently degrade pyrethroid pesticides. The Bacillus amyloliquefaciens MR17 is screened from food sources. The present invention discloses for the first time that the Bacillus amyloliquefaciens has a good degradation effect on a variety of pyrethroid pesticides including β-cypermethrin, deltamethrin, bifenthrin, cypermethrin, cypermethrin and flucythrin. Based on this, the present invention also provides the use of the Bacillus amyloliquefaciens MR17 in degrading pyrethroid pesticides. The present invention enriches the bacterial strain resource library that can degrade pyrethroid pesticides, has great application value in using probiotics to repair pyrethroid pesticide residues, and provides a new idea for solving the problems of excessive pyrethroid pesticide residues in agricultural products and pyrethroid pesticide pollution in the environment.
[0024] 2. The present invention has experimentally confirmed that the Bacillus amyloliquefaciens MR17 of the present invention is prepared into a lyophilized powder together with auxiliary components, and the lyophilized powder has a good degradation effect on β-cypermethrin after rehydration. Based on this, the present invention also provides a microbial preparation containing the Bacillus amyloliquefaciens MR17 and the use of the microbial preparation in the degradation of pyrethroid pesticides, which can better meet the needs of application in actual scenarios. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 This is the colony morphology of strain MR17.
[0026] Figure 2 is a scanning electron micrograph of strain MR17.
[0027] Figure 3 This is a phylogenetic tree constructed based on the 16S rRNA gene sequence of strain MR17.
[0028] Figure 4 These are the growth curve of strain MR17 and the degradation curve of β-cypermethrin.
[0029] Figure 5 The effect of inoculum size on the degradation of β-cypermethrin by strain MR17.
[0030] Figure 6 The effect of temperature on the degradation of β-cypermethrin by strain MR17.
[0031] Figure 7 The effect of pH on the degradation of β-cypermethrin by strain MR17.
[0032] Figure 8 It is the degradation ability of strain MR17 on different concentrations of β-cypermethrin.
[0033] Fig. 9 It is the degradation ability of strain MR17 on different pyrethroid pesticides. DETAILED DESCRIPTION
[0034] The following examples further illustrate the Bacillus amyloliquefaciens, bacterial preparation and application thereof for efficiently degrading pyrethroid pesticides provided by the present invention. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art may make some non-essential improvements and adjustments to the present invention based on the above invention content, which still fall within the scope of protection of the present invention.
[0035] Example 1: Isolation, identification and preservation of pyrethroid pesticide-degrading strain MR17
[0036] (1) Screening and isolation of strains
[0037] Daqu samples were taken from a certain Luzhou-flavor winery in Qionglai City, Sichuan Province, and the Daqu was crushed for use. Take 1g of the crushed Daqu sample, add it to a conical flask containing an MSM medium with a β-cypermethrin concentration of 50 mg / L, and culture it at 30°C and 180rpm for 96 hours. Take the culture solution and transfer it to an MSM medium with a β-cypermethrin concentration of 100 mg / L according to an inoculum of 6% (v / v). According to the above operation, the transfer is continued for multiple times, and the concentration of β-cypermethrin in the MSM medium is increased each time until the concentration of β-cypermethrin in the medium is increased to 1600 mg / L, and the β-cypermethrin-degrading strain is enriched and domesticated.
[0038] The formula and preparation method of the MSM culture medium are: MgSO4 0.5g / L, NaCl 0.5g / L, KH2PO4 0.5g / L, K2HPO4 1.5g / L, (NH4)2SO4 1.5g / L, pH 7.0, and distilled water; sterilized in an autoclave at 121°C for 20min.
[0039] (2) Purification of strains
[0040] Take the culture fluid obtained by the last enrichment and acclimation in step (1), spread it on an LB solid culture medium plate containing 50 mg / L β-cypermethrin after gradient dilution, and culture it at 30°C for 72 hours. Pick out a single colony for purification until the colony morphology on the LB solid culture medium plate is completely consistent, pick out a single colony with a regular colony morphology, and store it on a seed slant for later use.
[0041] The formula and preparation method of the LB solid culture medium plate are as follows: 5 g / L yeast powder, 10 g / L tryptone, 10 g / L sodium chloride, 2% (w / v) agar powder, pH 7.0, and distilled water; sterilized in a high pressure sterilizer at 121° C. for 20 min.
[0042] (3) Identification of strains
[0043] ①Morphological identification
[0044] The strain MR17 purified in step (2) was diluted and spread on an LB solid culture medium plate, and cultured at 30°C for 24 hours. The colony morphology of the strain MR17 was observed. Figure 1 As shown in Figure 1, the colony morphology of strain MR17 is light yellow, opaque, round, rough, ridged, and irregularly edged. The individual characteristics of strain MR17 were observed using a scanning electron microscope, such as Figure 2 As shown, the cells of strain MR17 were found to be rod-shaped and spore-forming.
[0045] ②Physiological and biochemical identification
[0046] The main biological characteristics of strain MR17 are facultative anaerobic, Gram-positive bacteria. It is positive in catalase test, sucrose fermentation test, and glucose fermentation test, but negative in lactose fermentation, VP test, MR test, starch hydrolysis test, indole test, and nitrate reduction test.
[0047] ③16S rRNA molecular biological identification
[0048] The strain MR17 was added to the LB liquid medium, shaken and cultured at 30°C for 24h, and the bacteria were collected. The formula and preparation method of the LB liquid medium are: 5g / L yeast powder, 10g / L tryptone, 10g / L sodium chloride, pH 7.0, and distilled water; sterilized in an autoclave at 121°C for 20min. The total DNA of the collected bacteria was extracted using a DNA extraction kit from Beijing Tiangen Biochemical Technology Co., Ltd., and the extracted total DNA was used as a template, and the 16S rRNA gene sequence was amplified by PCR using forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1492R (5'-TACGGCTACCTTGTTACGACTT-3').
[0049] The PCR amplification system was as follows: 1 μL DNA template, 0.5 μL forward primer 27F at a concentration of 20 mg / L, 0.5 μL reverse primer 1492R at a concentration of 20 mg / L, 12.5 μC 2×PCR Mix, and 10.5 L ddH2O. The PCR amplification program was as follows: 95°C pre-denaturation for 5 min, 94°C denaturation for 1 min, 50°C annealing for 1 min, 72°C extension for 2 min, 30 cycles, and then extension at 72°C for 10 min.
[0050] After the amplification, an appropriate amount of the amplified PCR product was electrophoresed with 1.0% agarose gel and photographed in a gel imaging system to detect the quality of the extracted genomic DNA. The remaining PCR amplification products were entrusted to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The spliced sequence files were analyzed for homology by the NCBI BlAST program, and the phylogenetic tree was constructed using MEGA 6.0 software to determine the strain species information, such as Figure 3 shown.
[0051] The 16S rRNA gene sequence of strain MR17 is as follows:
[0052]
[0053] The strain MR17 isolated and purified by the present invention is identified as Bacillus amyloliquefaciens through morphological characteristics, physiological and biochemical, and molecular biological identification. The strain is named Bacillus amyloliquefaciens MR17. The strain MR17 has been deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on July 8, 2024, with a deposit number of CGMCC No.31203.
[0054] Example 2: Analysis of the degradation characteristics of β-cypermethrin by strain MR17
[0055] (1) Seed liquid preparation: A single colony was selected from the seed slant of strain MR17 preserved in Example 1 and inoculated into 25 mL of LB liquid medium with an initial pH of 7.0. The culture was cultured at 30° C. and 180 rpm for 24 h. The OD was adjusted with sterile saline. 600 When the dilution reaches 1.0, you get the seed solution.
[0056] (2) Determination of growth and degradation kinetics: The seed solution was inoculated at an inoculum size of 6% (v / v) into 25 mL of LB liquid medium (pH 7.0) containing 50 mg / L β-cypermethrin as the experimental group, and the LB liquid medium without the inoculated strain was used as the blank control. Each group of experiments was repeated three times, and the culture was carried out at 30°C and 180 rpm for 120 h. Samples were taken every 12 h to determine the concentration of β-cypermethrin to understand the degradation of β-cypermethrin by strain MR17. At the same time, the OD value was measured at a wavelength of 600 nm using a spectrophotometer. 600 To understand the growth of strain MR17.
[0057] (3) Detection of β-cypermethrin and establishment of its standard curve equation
[0058] ①Sample pretreatment
[0059] After each group of samples was shaken and evenly mixed, 1.0 mL of culture medium was taken into a 2.0 mL EP tube, and 1.0 mL of acetonitrile was added. Ultrasonic (40 kHz, 300 W)-assisted extraction was performed for 30 min. After mixing, the samples were centrifuged at 13,000 rpm for 10 min. The supernatant was filtered with a 0.22 μm organic phase filter membrane, and the filtrate was collected and detected by high performance liquid chromatography.
[0060] ② Establishment of detection conditions and standard curve equation
[0061] High performance liquid chromatography (HPLC) detection conditions: the chromatographic column is ZORBAX eclipse plus C18 (4.60mm×150mm, 5.0μm), the flow rate is 1.0mL / min, the mobile phase is acetonitrile-ultrapure water (containing 0.1% formic acid), the volume ratio of acetonitrile to ultrapure water (containing 0.1% formic acid) is 85:15, the column temperature is 30°C, the ultraviolet detection wavelength is 210nm, and the injection volume is 10μL.
[0062] Establishment of the standard curve equation: Take a certain volume of 10g / L β-cypermethrin and dissolve it in acetonitrile, prepare standard solutions with a mass concentration gradient of β-cypermethrin (5, 10, 20, 30, 40, 50mg / L), filter through a 0.22μm organic phase filter, and detect according to the above HPLC detection conditions. Quantify the β-cypermethrin in the standard solution by retention time and peak area, draw a standard curve, and obtain the standard curve equation. Calculate the concentration of β-cypermethrin in the sample based on the standard curve equation and the peak area of the sample, and calculate the concentration of the remaining β-cypermethrin in the culture medium.
[0063] Degradation curve of β-cypermethrin (the concentration of β-cypermethrin remaining in the culture medium changes with culture time) and OD of strain MR17 600 The curve of changes with culture time is as follows Figure 4 As shown in the figure, β-cypermethrin control represents the concentration of β-cypermethrin in the culture medium of the blank control group, and β-cypermethrin residue represents the concentration of β-cypermethrin in the culture medium of the experimental group. Figure 4 It can be seen that in the initial culture period of 0 to 6 hours, strain MR17 is in a short growth lag period, and then strain MR17 enters the logarithmic growth period (culture time 6 to 48 hours). During this period, the degradation rate of β-cypermethrin by strain MR17 is the fastest. After 48 hours of culture, the growth of strain MR17 reaches a stable period, strain MR17 grows slowly and the degradation curve also tends to be flat. After 60 hours of culture, strain MR17 enters the decline period, and there is no obvious change in the degradation curve and the strain growth curve; after 120 hours of culture, the degradation rate of β-cypermethrin by strain MR17 reaches 45.00%.
[0064] Example 3: Optimization of the degradation conditions of β-cypermethrin by strain MR17
[0065] (1) Effect of inoculum size on the degradation of β-cypermethrin by strain MR17
[0066] The seed solution prepared in step (1) of Example 2 was inoculated into 25 mL of LB liquid culture medium (pH 7.0) with a β-cypermethrin concentration of 50 mg / L at an inoculum size of 2% (v / v), 4% (v / v), 6% (v / v), 8% (v / v), and 10% (v / v) as an experimental group, and the LB liquid culture medium without the inoculated strain was used as a blank control. Each group of experiments was repeated three times, and the cultures were cultured at 30° C. and 180 rpm for 72 h. Samples were taken, and the concentration of β-cypermethrin was determined according to the method in step (3) of Example 2. Then, the degradation rate of β-cypermethrin was calculated according to formula (I):
[0067] Degradation rate (%) = [(C0-C) / C0] × 100% (I)
[0068] In formula (I), C is the concentration (mg / L) of β-cypermethrin remaining in the culture medium of the experimental group, and C0 is the concentration (mg / L) of β-cypermethrin remaining in the culture medium of the control group.
[0069] Effect of inoculum size on the degradation of β-cypermethrin by strain MR17 Figure 5 As shown by Figure 5 It can be seen that when the inoculation amount of strain MR17 is 2% (v / v), 4% (v / v), 6% (v / v), 8% (v / v), and 10% (v / v), the degradation rate of strain MR17 for 50 mg / L β-cypermethrin is 29.21%, 31.19%, 45.01%, 39.84%, and 34.83%, respectively. When the inoculation amount of strain MR17 is 6% (v / v) to 10% (v / v), the degradation rate of 50 mg / L β-cypermethrin can exceed 30%, especially when the inoculation amount of strain MR17 is 6% (v / v) to 8% (v / v), the degradation effect of β-cypermethrin is significantly better. When the inoculation amount of strain MR17 is 6% (v / v), the degradation effect of β-cypermethrin is best.
[0070] (2) Effect of temperature on the degradation of β-cypermethrin by strain MR17
[0071] The seed solution prepared in step (1) of Example 2 was inoculated in 25 mL of LB liquid culture medium (pH 7.0) with a β-cypermethrin concentration of 50 mg / L at an inoculum size of 6% (v / v) as an experimental group, and the LB liquid culture medium without the inoculated strain was used as a blank control. Each group of experiments was repeated three times, and the cultures were cultured at 25° C., 30° C., 37° C., 40° C. and 180 rpm shaking conditions for 72 h, and samples were taken. The concentration of β-cypermethrin was determined according to the method in step (3) of Example 2, and then the degradation rate of β-cypermethrin was calculated according to formula (I).
[0072] Effect of temperature on the degradation of β-cypermethrin by strain MR17 Figure 6 As shown by Figure 6 It can be seen that under the conditions of 25℃, 30℃, 37℃ and 40℃, the degradation rates of β-cypermethrin at a concentration of 50 mg / L by strain MR17 were 39.72%, 59.59%, 44.63% and 33.89% respectively. Different culture temperatures have a great influence on the degradation ability of strain MR17. When the temperature is 30-37℃, the degradation effect of β-cypermethrin by strain MR17 is relatively better, especially when the temperature is 30℃, the degradation effect of β-cypermethrin by strain MR17 is the best.
[0073] (3) Effect of culture medium pH on the degradation of β-cypermethrin by strain MR17
[0074] LB liquid culture medium with a β-cypermethrin concentration of 50 mg / L was prepared, and the pH values of each culture medium were adjusted to 5.0, 6.0, 7.0, 8.0, and 9.0, respectively. The seed solution prepared in step (1) of Example 2 was inoculated in 25 mL of the above LB liquid culture medium with different pH values at an inoculum amount of 6% (v / v) as an experimental group, and the LB liquid culture medium without the strain inoculation was used as a blank control. Each group of experiments was repeated three times, and the culture was cultured at 30° C. and 180 rpm for 72 h. Samples were taken, and the concentration of β-cypermethrin was determined according to the method in step (3) of Example 2, and then the degradation rate of β-cypermethrin was calculated according to formula (I).
[0075] Effect of pH on the degradation of β-cypermethrin by strain MR17 Figure 7 As shown by Figure 7 It can be seen that when the initial pH value of the culture medium is 5.0, 6.0, 7.0, 8.0 and 9.0, respectively, the degradation rate of β-cypermethrin at a concentration of 50 mg / L by strain MR17 is 30.69%, 41.87%, 45.02%, 27.35% and 24.14%, respectively; when the initial pH value of the culture medium is 6.0-7.0, the degradation effect of strain MR17 on β-cypermethrin is relatively better, especially when the initial pH value of the culture medium is 7.0, the degradation effect of strain MR17 on β-cypermethrin is the best.
[0076] It can be seen from this embodiment that strain MR17 has a good degradation effect on β-cypermethrin when the degradation temperature is 30-37°C, pH=6.0-7.0, and the inoculation amount is 6% (v / v)-8% (v / v). Among them, the best degradation conditions for β-cypermethrin are: degradation temperature 30°C, pH=7.0, and inoculation amount 6% (v / v).
[0077] Example 4: Degradation ability test of strain MR17 on different concentrations of β-cypermethrin and different pyrethroid pesticides
[0078] (1) Degradation ability of strain MR17 on different concentrations of β-cypermethrin
[0079] LB liquid culture medium (pH 7.0) with β-cypermethrin concentrations of 10, 25, 50, 75 and 100 mg / L was prepared, and the seed solution prepared in step (1) of Example 2 was inoculated in 25 mL of the above LB liquid culture medium with different β-cypermethrin concentrations at an inoculum size of 6% (v / v) as an experimental group, and the LB liquid culture medium without the inoculated strain was used as a blank control. Each group of experiments was repeated three times, and the cultures were cultured at 30° C. and 180 rpm for 72 h. Samples were taken, and the concentration of β-cypermethrin was determined according to the method in step (3) of Example 2, and then the degradation rate of β-cypermethrin was calculated according to formula (I).
[0080] The degradation ability of strain MR17 on different concentrations of β-cypermethrin is shown in Figure 8 As shown by Figure 8 It can be seen that after 72 hours of cultivation, the degradation rates of β-cypermethrin at concentrations of 10, 25, 50, 75, and 100 mg / L by strain MR17 were 39.91%, 34.57%, 66.93%, 53.84%, and 32.72%, respectively. When the initial concentration of β-cypermethrin was 50 mg / L, the degradation rate was the highest. As the concentration of β-cypermethrin increased, the degradation rate gradually decreased, but it was still able to effectively degrade β-cypermethrin at a concentration of 100 mg / L. In practical applications, the appropriate concentration of β-cypermethrin can be selected within 100 mg / mL based on factors such as degradation rate, cost, and degradation efficiency.
[0081] (2) Test on the degradation ability of strain MR17 on different pyrethroid pesticides
[0082] LB liquid culture medium (pH 7.0) with a concentration of 50 mg / L of β-cypermethrin, deltamethrin, bifenthrin, cypermethrin, cypermethrin, cypermethrin and flucythrinate was prepared respectively. The seed solution prepared in step (1) of Example 2 was inoculated in 25 mL of the above-mentioned LB liquid culture medium containing different pyrethroid pesticides at an inoculum amount of 6% (v / v) as an experimental group, and the LB liquid culture medium without inoculation of strains was used as a blank control. Each group of experiments was repeated three times, and the culture was cultured at 30° C. and 180 rpm for 72 hours. Samples were taken, and the concentration of β-cypermethrin was determined according to the method in step (3) of Example 2, and then the degradation rate of β-cypermethrin was calculated according to formula (I).
[0083] The degradation ability of strain MR17 on different pyrethroid pesticides is as follows Fig. 9 As shown by Fig. 9It can be seen that the degradation rates of strain MR17 for 50 mg / L cypermethrin, cypermethrin, bifenthrin, deltamethrin, cyfluthrin, and β-cypermethrin were 21.13%, 23.43%, 35.73%, 40.11%, 45.08%, and 50.81%, respectively. This shows that strain MR1 has a good degradation effect on different pyrethroid pesticides and can be used to degrade a variety of pyrethroid pesticides.
[0084] Example 5: Preparation of strain MR17 bacterial preparation and investigation of its performance
[0085] (1) Preparation of bacterial preparation protective materials
[0086] Prepare 10% suspension matrix: add 5.0 g skim milk powder into 50 mL deionized water, adjust pH to 7.0, pasteurize and sterilize with UV for 30 min, and set aside.
[0087] Preparation of 10% protective agent: corn starch, sucrose and glucose were used as protective agents respectively, 5.0 g of each protective agent was added into 50 mL of deionized water, and then pasteurized and sterilized by ultraviolet for 30 minutes for later use.
[0088] (2) Preparation of strain MR17 bacterial preparation
[0089] The seed solution prepared in step (1) of Example 2 was inoculated in 25 mL of LB liquid medium at an inoculum volume of 6% (v / v), and cultured at 30°C and 180 rpm for 24 h. The obtained bacterial solution was centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was removed, the bacterial body was resuspended with sterile water and centrifuged, the supernatant was removed, the bacterial body was resuspended with sterile water and centrifuged again, the supernatant was removed, and the obtained bacterial body was mixed with 10% suspension matrix and 10% protective agent in a volume ratio of 1:1:1 to prepare a bacterial suspension for standby use. The obtained bacterial suspension was pre-frozen at -80°C for 12 h and then freeze-dried in a freeze dryer for 48 h to obtain orange-red crystals, which were the strain MR17 bacterial preparation, which were crushed to obtain the strain MR17 powder. The strain MR17 powder was placed in an aluminum film bag, and the aluminum film bag was vacuumed and stored at 0°C.
[0090] The total number of viable bacteria in the MR17 powder was determined and the survival rate of the strain was calculated according to the following operation.
[0091] Activation counting method: Take 0.1g of strain MR17 powder, add 1mL of sterile water and shake well to dissolve, complete rehydration, and dilute to 10 under sterile conditions. -6 , 10 -7 , 10 -8For each of the three gradients, 100 μL of the dilution was spread on LB solid medium, inverted and cultured at 30°C for 18 h, the colony growth was observed and the total number of viable bacteria was calculated. The survival rate of the strain was then calculated according to formula (II):
[0092] Survival rate (%) = [(N0-N) / N0] × 100% (II)
[0093] In formula (II), N is the total number of viable bacteria after freeze-drying, and N0 is the total number of viable bacteria before freeze-drying.
[0094] In this embodiment, the strain survival rate of the strain MR17 powder prepared using sucrose, glucose and corn starch as protective agents is shown in Table 1. As can be seen from Table 1, when sucrose is used as a protective agent, the survival rate of the prepared strain MR17 powder is the highest, reaching 39.14%.
[0095] Table 1 Effect of protective agent on the survival rate of strain MR17 powder
[0096]
[0097] (3) Degradation of β-cypermethrin using strain MR17 powder
[0098] Under sterile conditions, 0.1 g of each strain MR17 powder prepared in step (2) was weighed and dissolved in 100 mL of sterile water to obtain a reconstituted solution. Each reconstituted solution was inoculated into 25 mL of LB liquid culture medium with a β-cypermethrin concentration of 50 mg / L at an inoculum amount of 6% (v / v), and the LB liquid culture medium without the inoculated strain was used as a blank control. Each group of experiments was repeated 3 times, and the culture was shaken at 30° C. and 180 rpm for 72 h. Samples were taken, and the concentration of β-cypermethrin was determined according to the method in step (3) of Example 2, and then the degradation rate of β-cypermethrin was calculated according to formula (I). The results are shown in Table 2.
[0099] Table 2 Effect of protective agents on the degradation of β-cypermethrin by strain MR17 powder
[0100]
[0101] It can be seen from Table 2 that when sucrose, glucose and corn starch are used as protective agents, the prepared strain MR17 powder has a good degradation ability for β-cypermethrin, especially when sucrose is used as a protective material, the degradation rate of β-cypermethrin by the prepared strain MR17 powder is as high as 54.83%. When corn starch is used as a protective material, the degradation rate of β-cypermethrin by the prepared strain MR17 powder is also high, reaching 49.68%.
Claims
1. A strain of Bacillus amyloliquefaciens MR17 that efficiently degrades pyrethroid pesticides, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 8, 2024, with the deposit number CGMCC NO.31203.
2. A microbial preparation, characterized in that: The microbial preparation contains the Bacillus amyloliquefaciens MR17 according to claim 1.
3. Use of the Bacillus amyloliquefaciens MR17 according to claim 1 or the microbial preparation according to claim 2 in degrading pyrethroid pesticides.
4. The use according to claim 3, characterized in that: The Bacillus amyloliquefaciens MR17 described in claim 1 is inoculated into the sample to be degraded, or the microbial preparation described in claim 2 is added into the sample to be degraded, so as to degrade the pyrethroid pesticides in the sample to be degraded.
5. The use according to claim 3 or 4, characterized in that: The pyrethroid pesticide includes at least one of β-cypermethrin, deltamethrin, bifenthrin, cypermethrin, fenvalerate and cyfluthrin.
6. The use according to claim 3 or 4, characterized in that: When degrading pyrethroid pesticides, the degradation temperature is controlled at 30-37°C and the degradation pH is controlled at 6-7.
7. The use according to claim 3 or 4, characterized in that: When degrading pyrethroid pesticides, the degradation time is controlled to be 48 to 72 hours.
8. The use according to claim 3 or 4, characterized in that: When degrading pyrethroid pesticides, the concentration of pyrethroid pesticides in the sample to be degraded is controlled not to exceed 100 mg / L.