CN116555078B - Bacillus subtilis for effectively degrading cypermethrin and application thereof - Google Patents
Bacillus subtilis for effectively degrading cypermethrin and application thereof Download PDFInfo
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- CN116555078B CN116555078B CN202310264488.8A CN202310264488A CN116555078B CN 116555078 B CN116555078 B CN 116555078B CN 202310264488 A CN202310264488 A CN 202310264488A CN 116555078 B CN116555078 B CN 116555078B
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- cypermethrin
- bacillus subtilis
- strain
- degradation
- pyrethroid
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Classifications
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- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
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- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A—HUMAN NECESSITIES
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- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/04—Pesticides, e.g. insecticides, herbicides, fungicides or nematocides
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
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Abstract
The invention relates to the field of reducing pollutants in the environment or food by utilizing microorganisms, in particular to bacillus subtilis which is separated from healthy broiler manure and has higher degradation capability on cypermethrin. The bacillus subtilis (Bacillus subtilis) strain number capable of effectively degrading cypermethrin is J6, and is registered and preserved in the China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms; the preservation number is CGMCC No. 26129, and the preservation date is 2022, 12 and 27. The strain can degrade cypermethrin and has good degradation effect on other pyrethroid pesticides and the main degradation product, namely 3-phenoxybenzoic acid. The bacillus subtilis J6 can develop corresponding biological agents for reducing pyrethroid in environment, feed and food; or the strain is developed into health-care food or medicine, so that the harm of pyrethroid pesticides to human body exposure is reduced.
Description
Technical Field
The invention relates to the field of reducing pollutants in environment or feed food by utilizing microorganisms, in particular to bacillus subtilis which is separated and screened from the cecum content of healthy broilers and has good degradation capability on cypermethrin.
Background
The pyrethroid pesticide has stable light and heat, wide pesticidal spectrum, high pesticidal activity, and high stomach toxicity and contact killing effect on farm and forest pests, such as lepidoptera, on cereal grain crops, melons, fruits and vegetables, and sanitary pests, such as mosquitoes, flies, cockroaches, and the like, so that the pyrethroid pesticide is widely applied to pesticide compounding and pesticide aerosol.
Pyrethroid pesticides have been considered as low-toxicity and safe-to-use pesticides in the past, but as the use amount of pyrethroids is increased, various problems such as environmental pollution, food safety, adverse effects on human health and the like caused by the pyrethroid are increasingly prominent, and the pyrethroid pesticides are attracting attention in various countries around the world. Because of the good stability and durability of pyrethroid pesticides in soil and water, background residues in the environment are continuously accumulated due to mass use, so that the quality of agricultural products and the food safety are directly affected, and the health of human beings is threatened.
Most of the pyrethroid degrading bacteria obtained at present are separated from the environment such as soil, sludge and the like polluted by pesticides, cannot be applied to removing pyrethroid pesticides in foods, and are difficult to be added into feeds and foods to relieve chronic damages of the pesticides to animals and human bodies. Pesticide residue is a long-standing and unavoidable potential safety hazard, but no effective removal method and effective removal way can be used for reducing pesticide residue in the food field, the livestock field and the human body, so that the development of probiotic strain resources with pesticide reducing capability is necessary.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide bacillus subtilis (Bacillus subtilis) J6 capable of effectively degrading cypermethrin and application thereof.
The strain provided by the invention is bacillus subtilis (Bacillus subtilis), the strain number J6 is preserved in China general microbiological culture Collection center, the preservation place is North Star Xiyu No.1 institute in the Korean region of Beijing city, the preservation number is CGMCC No. 26129, and the preservation date is 2022, 12 and 27.
The invention provides separation, screening and identification of animal-derived pesticide degrading bacterial strains.
Taking 0.1-0.5 g of the cecum content of a healthy animal, adding 2 mL g of an improved basic culture medium (the concentration of cypermethrin is 10-50 mg/L), fully swirling, transferring to a penicillin bottle, and placing at 37 ℃ and 160-rpm for shake culture for 2-5 d. After the cultivation is finished, adding 2 mL acetonitrile, ultrasonically extracting 30 min, uniformly mixing, transferring 1.5 mL to an EP tube, centrifuging 12000 rpm for 10 min, taking supernatant, passing through a 0.22 mu m organic filter membrane, collecting filtrate, measuring the concentration of cypermethrin by using High Performance Liquid Chromatography (HPLC), and calculating the degradation rate of the cecum content on the cypermethrin. The culture medium without cecum content was used as a control.
Weighing 0.1-1 g of the obtained cecum content sample with the cypermethrin degradation effect, adding 9-9.9 mL of physiological saline, carrying out gradient dilution after vortex mixing uniformly, and coating the diluted solution on a solid culture medium of an improved basic culture medium for 2-4 d by aerobic or anaerobic culture at 37 ℃. Selecting strains with different colony morphologies, carrying out repeated streak purification until the colony morphologies on the same plate are uniform, and carrying out inclined plane preservation. Inoculating the slant strain into 5mL of LB liquid medium in a loop, culturing for 2-4 d at 37 ℃, inoculating into 30 mL LB liquid medium according to 2% of inoculation amount, and culturing for 1-5 d at 37 ℃ and 160 rpm in a shaking way.
Adding 4mL of acetonitrile into 1mL of culture medium, ultrasonically extracting for 30 min, uniformly mixing the extracting solution, centrifuging 1.5 mL liquid (12000 rpm,10 min), taking supernatant, passing through a 0.22 mu m organic filter membrane, collecting filtrate, measuring the concentration of cypermethrin by using High Performance Liquid Chromatography (HPLC), and calculating the degradation rate of the strain on the cypermethrin.
The HPLC detection method comprises the following steps: gemini 100A C18 chromatographic column (4.6 mm ×150 mm,5 μm), acetonitrile: ultrapure water=83:17 as mobile phase, flow rate of 1.0. 1.0 mL/min, column temperature of 25deg.C, sample injection amount of 10 μl, detector of ultraviolet detector, detection wavelength of 210 nm.
The formula of the degradation rate of cypermethrin is as follows:
(C0-C)/C0*100%
Wherein: c 0 is the concentration of cypermethrin (mg/L) in the control; c is the concentration of cypermethrin residue (mg/L) after addition of cecal content/strain.
Identification of strain morphology: the plate colony is round or elliptic, and has a slight grey-white yellow color, a central bulge, surface wrinkles and moist interior. Gram staining was positive and rod-like.
And (3) strain physiological and biochemical identification: the strain can ferment D-glucose, D-fructose, D-mannose, D-cellobiose, sucrose, trehalose, xylose and gentiobiose, hydrolyze starch, assimilate D-mannitol and N-acetylglucosamine, grow 7% NaCl, grow pH 5, and have positive V-P test, positive catalase, citrate and hydrolyzed gelatin, and not propionate.
And (3) identifying the strain J6 by molecular genetics.
Strain J6 was sequenced by 16S rDNA to obtain the following sequence:
CCCTTGGCCGTATTCTAATAATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGAACCGGG.
The sequencing result of the 16S rDNA gene of the strain is compared with the sequence in a GenBank database in Blast homology, the sequence of the strain is highly homologous with Bacillus subtilis gene sequence, the accession number is OQ195114, morphological and physiological biochemical indexes are combined, and finally the strain J6 is identified as bacillus subtilis.
The strain capable of efficiently adsorbing the cypermethrin is obtained by separating and screening the content of the cecum of healthy broilers, has the same degradation capability on the main degradation products of the cypermethrin, the deltamethrin, the fenvalerate and the pyrethroid pesticides, namely the 3-phenoxybenzoic acid, and can be applied to the reduction of residual cypermethrin in the environment and foods.
The culture medium used in the invention is as follows: improving a basal medium: inulin 4g, glucose 4g, beef extract 2g, peptone 2g, yeast powder 2g,NaCl22 g,K2HPO40.04 g,KH2PO40.04 g,MgSO4·4H2O 0.01 g,CaCl20.01 g,NaHCO32 g,L- cysteine 0.5g, tween-80 g, bile salt 0.5g, distilled water 1000 ml, and sterilization at 121 ℃ for 15min. LB medium: tryptone 10g, yeast extract 5g, naCl 10g, tween-80 2g, distilled water to 1000 mL, and sterilizing at 121 ℃ 20 min. Adding 2% agar to obtain corresponding solid culture medium.
Compared with the prior art, the invention has the following positive effects: the bacillus subtilis with the capability of degrading pyrethroid pesticides such as cypermethrin and intermediate products 3-phenoxybenzoic acid is obtained from the cecum content of healthy animals for the first time, and the bacillus subtilis is safe in source and can provide a good bacterial source for reducing and removing residual cypermethrin in the environment and food and in animals and human bodies.
Drawings
FIG. 1 shows the effect of culture temperature on the degradation of cypermethrin by the strain.
FIG. 2 shows the effect of substrate concentration on the degradation of cypermethrin by the strain.
FIG. 3 shows the effect of metal ions on the degradation of cypermethrin by the strain.
Description of the embodiments
Example 1: degradation characteristics of B.subtilis J6 on cypermethrin.
1) Effect of the culture temperature on degradation of cypermethrin by J6 Strain J6 seed solution was inoculated in an inoculum size of 2% (v/v) in LB medium with cypermethrin concentration of 50 mg/L, and shake-cultured at 20, 25, 30, 37 and 45℃at 160 rpm, respectively. The degradation rate of J6 to cypermethrin was determined by the methods described in [0010] and [0011] using cypermethrin working solutions containing no thalli at different temperatures as positive controls.
2) Effect of substrate concentration on degradation of cypermethrin by J6 strain J6 seed solution was inoculated in 2% (v/v) inoculum size to LB medium containing cypermethrin at concentration of 20, 50, 80, 100,150 mg/L, respectively, and shake-cultured at 37 ℃ and 160 rpm for 3d. The degradation rate of J6 for cypermethrin was determined as described in [0010] and [0011 ].
3) Effect of metal ions on degradation of cypermethrin by J6 strain J6 seed solution was inoculated in an inoculum size of 2% (v/v) in LB medium containing 50 mg/L cypermethrin and a different metal salt (CuSO 4、MnSO4、ZnSO4、CdCl2、Pb (CH3COO)2) at a concentration of 0.01% (w/v), respectively, and cultured at 37 ℃ under 160 rpm shaking for 3d. The degradation rate of J6 for cypermethrin was determined as described in [0010] and [0011 ].
The degradation characteristics of strain J6 on cypermethrin are shown as follows: the degradation of the strain J6 to the cypermethrin is not greatly influenced by the temperature, and the degradation rate of the strain J6 is over 40 percent within the range of 20-45 ℃, and the degradation rate is at 37 ℃ at the highest; in the concentration range of 20-150 mg/L, the degradation rate is reduced along with the increase of the substrate concentration; cd 2+、Zn2+ and Cu 2+ have different degrees of inhibition effects on the degradation of cypermethrin by the strain J6, and Mn 2+ and Pb 2+ have no influence on the degradation effect. The results of this study provide a data reference for the degradation of residual cypermethrin in environmental and food applications of strain J6.
Example 2: the degradation effect of the bacillus subtilis J6 on other pyrethroid pesticides.
The seed solution of the strain J6 is inoculated into LB liquid culture medium containing 50 mg/L deltamethrin and fenvalerate according to the inoculum size of 2 percent, and is cultured for 3 days at 37 ℃ in a shaking way, and the degradation rate of deltamethrin and fenvalerate is measured by the method described in [0010] and [0011 ].
The strain J6 has better degradation capability on deltamethrin and fenvalerate, and the degradation rates are 65.48% and 14.68% respectively.
Example 3: the degradation effect of the bacillus subtilis J6 on the main intermediate product 3-phenoxybenzoic acid of the pyrethroid pesticide.
The strain is inoculated into LB liquid medium containing 50 mg/L3-phenoxybenzoic acid according to the inoculation amount of 2%, shaking culture is carried out at 37 ℃ for 24 h, 1mL of culture solution is respectively taken at 0 h and 24 h, 4mL of acetonitrile is added, ultrasonic extraction is carried out for 30 min, 1.5 mL is removed after uniform mixing, 10min is centrifuged at 12000 r/min, supernatant is taken, an organic filter membrane with the thickness of 0.22 μm is adopted, and the degradation rate of the filtrate to 3-phenoxybenzoic acid is collected and measured. Chromatographic conditions: gemini 100A C18 chromatographic column (4.6 mm ×150 mm,5 μm), mobile phase acetonitrile, pH 2.5 phosphoric acid water=55:45, flow rate 0.7: 0.7 mL/min, column temperature 25 ℃, sample injection amount 10 μl, detector ultraviolet detector, detection wavelength 210 nm.
The results showed that the degradation rate of strain J6 to 50 mg/L3-phenoxybenzoic acid was 76.12%.
Example 4: the bacillus subtilis J6 has the degradation effect on cypermethrin in cow milk.
Preparing cow milk: 20 g skimmed milk powder was dissolved in 1000: 1000 mL distilled water and sterilized 15: 15min at 115 ℃. A volume of cypermethrin is added to give a final cypermethrin concentration of 50 mg/L.
Seed solutions of the strain J6 were inoculated into cow milk having a cypermethrin concentration of 50 mg/L at a rate of 2% (v/v), respectively, and cultured under shaking at 37℃and 160 rpm. Samples were taken at 0h, 1 d, 2 d and 3d, respectively, and the cypermethrin degradation rates were determined as described in [0010] and [0011 ].
The degradation rates of the strain J6 on cypermethrin in 1d, 2d and 3d are 26.73%, 36.41% and 52.56% respectively, which shows that the strain J6 has good degradation effect on cypermethrin in cow milk.
The above examples are intended to illustrate embodiments of the invention and not to limit the scope of the invention, and modifications or variations may be made to the above description by those skilled in the art, all of which fall within the scope of the invention as defined in the appended claims. Aiming at the problems existing in the prior art, the invention aims to provide bacillus subtilis (Bacillus subtilis) J6 capable of effectively degrading cypermethrin and application thereof.
Claims (3)
1. A bacillus subtilis (Bacillus subtilis) J6 for effectively degrading cypermethrin is characterized by being preserved in China general microbiological culture Collection center, wherein the preservation place is North Star Xiyu No.1 institute of the Korean area of Beijing city, the preservation number is CGMCC No. 26129, and the preservation date is 2022, 12 and 27.
2. The use of bacillus subtilis (Bacillus subtilis) J6 according to claim 1, characterized in that: degradation of cypermethrin, fenvalerate, deltamethrin and 3-phenoxybenzoic acid.
3. The use of bacillus subtilis (Bacillus subtilis) J6 according to claim 1, characterized in that: is used for degrading residual cypermethrin in cow milk.
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