CN107446853B - Bacillus lysinate strain, enzyme preparation and application of enzyme preparation in degradation of pesticide residue - Google Patents
Bacillus lysinate strain, enzyme preparation and application of enzyme preparation in degradation of pesticide residue Download PDFInfo
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Abstract
The invention discloses a bacillus lysine-prolonging strain, an enzyme preparation and application thereof in degrading pesticide residues. The preservation number of the prolonged lysine bacillus (Lysinibacillus macrocrystals) FY-3 strain provided by the invention is CGMCC No. 13519. The invention also provides a method for screening, inducing and domesticating the strains and preparing the active enzyme for degrading pesticide residues, namely screening target bacteria by taking chlorpyrifos as a carbon source and inducing and domesticating the target bacteria in single pesticide and mixed pesticide. The strain is fermented, thallus is collected, cell breaking and centrifugation are carried out, the pesticide residue degrading enzyme preparation can be prepared from the supernatant, the pesticide residue in vegetables and fruits can be degraded, and the application potential in the aspect of degrading the pesticide residue of the vegetables and fruits is realized.
Description
Technical Field
The invention relates to the field of degrading pesticide residues by biotechnology, in particular to screening, induction and domestication of a bacillus Lysinibacillus macrocrystals strain and application of the bacillus Lysinibacillus macrocrystals strain in degrading pesticide residues on vegetables and fruits.
Background
With the rapid increase of varieties, yield and usage amount of pesticides in recent years, the phenomenon of mixed application of multiple pesticides occurs frequently and seriously in the agricultural production process, so that multiple pesticides appear in the same product. Eating vegetables and fruits with pesticide residues exceeding the standard can harm nerve centers and cause nerve conduction dysfunction; mutations can be induced, resulting in malformation and affecting the health of offspring; can induce liver enzyme change to make liver enlarged and necrotic; can invade kidney to cause pathological changes. The residual pesticide is accumulated in human body, and can cause some chronic diseases, such as muscle numbness, cough and the like, and even can induce vascular diseases, diabetes, cancer and the like after exceeding a certain limit.
The bioremediation technology is a biological engineering technology for eliminating and treating environmental pollution appeared and developed in the 80 s, and mainly utilizes the specific capability of organisms in decomposing toxic and harmful substances to remove pollutants in polluted environments such as soil so as to achieve the purpose of eliminating environmental pollution. The microorganism has strong adaptability and domestication capability, and the microorganism generates a corresponding enzyme system through a certain adaptation process or establishes a new enzyme system through gene mutation and the like to degrade new pesticide.
The improvement of food safety has great significance, but at present, reports of degrading mixed pesticide residues in vegetables and fruits by using bacterial strains or enzyme preparations prepared from the bacterial strains are not found.
Disclosure of Invention
The invention aims to provide a bacillus lysine prolonging strain obtained by screening, pesticide induction and domestication.
The bacillus protractus (lysine bacteria macroides) strain (marked as FY-3) has the preservation number of CGMCC No. 13519.
The method is characterized in that the bacillus lysine macroides strain is obtained by screening a target strain and inducing and domesticating the target strain by a single pesticide and a mixed pesticide, and comprises the following steps:
1) rejuvenating the screened target strain;
2) preparing culture media containing pesticides with different effective component concentrations by using chlorpyrifos pesticide, and carrying out streak inoculation and culture on the rejuvenated target strains in the step 1) and cultivating target strains capable of tolerating higher chlorpyrifos concentration through continuous induced cultivation (by continuously increasing the pesticide content);
3) the pesticide chlorpyrifos, carbendazim, imidacloprid, cyhalothrin and the like are mixed in concentration, diluted to a certain concentration and added into a culture medium to prepare the culture medium containing pesticides with different effective component concentrations. And then carrying out streak inoculation and culture on the target strain obtained in the step 2), and culturing through continuous induction culture (by continuously increasing the pesticide content) to obtain a strain with higher pesticide concentration tolerance, and finally separating to obtain a strain of Bacillus Lysinibacillus macrocrystals (marked as FY-3).
It is a second object of the present invention to provide a method for producing a biodegradable enzyme.
The method for producing the biodegradable enzyme provided by the invention comprises the following steps: fermenting the strain FY-3 of the Bacillus stearothermophilus, collecting the strain, breaking the cells, centrifuging, and extracting supernatant to obtain a biodegradable enzyme solution.
In the above-mentioned method for producing a biodegradable enzyme, the fermentation is conducted under conditions of 34-39 ℃ and 180-200rpm for 16-24 hours.
In the above method for producing a biodegradable enzyme, the fermentation medium used for the fermentation comprises the following components: each liter of the fermentation medium comprises 17-19g of tryptone, 1-4g of plant peptone, 2-5g of sodium chloride, 2-5g of dipotassium hydrogen phosphate and 2-5g of glucose, and the volume is complemented by water; the pH of the fermentation medium is 6.8 to 7.6, preferably 7.0 to 7.4.
In the above method for producing a biodegradable enzyme, the fermentation specifically comprises the steps of: the strain FY-3 of the bacillus protracted lysine (lysine bacteria macroides) is subjected to amplification culture to obtain seeds, and the seeds are inoculated into the fermentation medium to be cultured under the condition of fermentation.
The condition of the amplification culture is that the culture is carried out at 34-39 ℃ and 180-200rpm for 16-24 h.
The strain is a solution obtained by suspending the thallus of the bacillus protractus (lysine bacteria macroides) strain FY-3 in a phosphate buffer solution.
In the method for producing the biodegradable enzyme, after the fermentation, the fermentation product is centrifuged for 15-20min at the temperature of 4-6 ℃ at 12000-14000rpm, the thalli are collected, the thalli are subjected to high-pressure cell breaking after being resuspended, then the thalli are centrifuged for 50-65min at the temperature of 4-6 ℃ at 8000-10000rpm, the supernatant is collected, and the supernatant is filtered to obtain the biodegradable enzyme solution.
The third purpose of the invention is to provide a biodegradable enzyme preparation.
The biodegradable enzyme preparation of the present invention comprises the above-mentioned Bacillus lysinate strain FY-3 or the above-mentioned biodegradable enzyme as an active ingredient.
The fourth purpose of the invention is to provide the application of the bacillus protractus (lysine bacillus macrocoides) strain FY-3, the biodegradation enzyme and the biodegradation enzyme preparation in the degradation of pesticide residues, especially the degradation of pesticide residues on vegetables and fruits.
The using concentration of the biological degrading enzyme or the biological degrading enzyme preparation in the degradation of pesticide residues on vegetables and fruits is 2.5-5ppm, and the optimal concentration is 3.5-4.5ppm, which is different according to the specific vegetables and fruits.
The invention has the beneficial effects that:
experiments prove that the prolonged lysine bacillus macroides strain FY-3 obtained by induction and domestication can be used as a biodegradable enzyme after fermentation culture and intracellular enzyme extraction, and the biodegradable enzyme can act on pesticide residues in vegetables and fruits to degrade various pesticide residues, so that the effect of removing the pesticide residues is achieved, and the application potential is realized in the field of pesticide residue degradation.
Drawings
FIG. 1 shows the results of treating the pesticide residues of cherry tomatoes with a liquid biodegradable enzyme preparation based on Bacillus stearothermophilus strain FY-3.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The examples serve to better understand the invention, but do not limit it. The test materials used in the examples were all conventional biochemical reagents unless otherwise specified. In the examples,% is by mass unless otherwise specified. In the quantitative tests in the examples, three repeated experiments are set, and the results are averaged. The rotational speeds in the examples were rotational speeds at a centrifugal radius of 5.5 cm.
In the examples, the number of bacteria is determined by colorimetry, i.e. turbidity counting; absorbance OD of culture600Indicates the amount of bacteria grown.
Example 1 isolation and identification of the Strain of Bacillus lysinibacillus protractus
First, obtaining of the Strain
1. Collection of soil samples
Collecting sludge from the discharge port of the pesticide factory.
2. Separation and screening of target strains
Weighing 10g of sludge in 100mL of inorganic salt liquid culture medium, wherein the inorganic salt liquid culture medium comprises the following components:
MgSO4·7H2O 0.2g；K2HPO40.1g；(NH4)2SO40.1g；CaSO40.04g；FeSO4·7H2o0.001g; 1L of deionized water; the pH was 7.0. Sterilizing at 121 deg.C for 30 min.
Adding chlorpyrifos with concentration of 100mg/L into inorganic salt liquid culture medium to obtain culture solution, shake culturing at 37 deg.C for 180r/min for one week, inoculating into fresh culture solution with 10% inoculum size, transferring once per week, and acclimating repeatedly. Finally, the culture medium is separated and purified by a plate dilution method, inoculated into LB culture medium containing 100ppm chlorpyrifos, and the best-growing inoculation inclined plane is selected, numbered and stored for domestication.
II, identification of the strains
The specific identification steps are as follows: extracting total DNA of the numbered preserved strains as a template, applying universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATG-3'), carrying out PCR amplification to obtain ITS, and obtaining a 129bp nucleotide sequence (shown in SEQ. ID. NO.1) by sequencing, wherein homology comparison analysis shows that: the number-preserved strain has the highest homology with the known species Bacillus stearothermophilus (Lysinibacillus macrocoides).
Example 2 Induction and acclimatization of extended lysine Bacillus Strain
Inducing and domesticating chlorpyrifos
1. The content interval of the effective components prepared from the chlorpyrifos is as follows: 300mg/L-800 mg/L. Tryptone agar containing varying concentrations of chlorpyrifos was prepared. Different amounts of chlorpyrifos were added to tryptone agar to make chlorpyrifos-containing medium. The method comprises the following specific steps: 1) chlorpyrifos is prepared into 4500mg/L pesticide standard solution. 13.3mL, 17.8mL, 22.2mL, 26.6mL, 31.1mL and 35.6mL of pesticide standard solution with the concentration of 4500mg/L are respectively added into 200mL of tryptone agar to prepare tryptone agar culture media containing 300mg/L, 400mg/L, 500mg/L, 600mg/L, 700mg/L and 800mg/L of chlorpyrifos. Plate manufacturing: the prepared tryptone agar containing 300mg/L-800mg/L chlorpyrifos is poured into a flat dish, 15 mL/dish, and the prepared tryptone agar is used after being cooled and solidified.
The tryptone agar comprises the following components: each L contained 17g of tryptone, 3g of phytone, 4.5g of sodium chloride and 15g of agar (pH 7.0. + -. 0.2).
2. All strains of interest were streaked separately onto plates at 300 mg/L.
3. The inoculated plate is placed in a constant temperature incubator at 37 ℃ and cultured for 24 h.
4. The cultured strain was then re-inoculated into 400mg/L plates. The inoculated plate was placed in a 37 ℃ incubator and incubated for 24 hours.
5. The culture is continued in plates of 500mg/L, 600mg/L, 700mg/L and 800mg/L step by step, and finally the target strain which can resist the organophosphorus pesticide (chlorpyrifos) of 800mg/L is obtained.
Second, mixed pesticide induction and domestication
1. Mixed pesticide diluting method
Chlorpyrifos, carbendazim, cyhalothrin and imidacloprid are respectively prepared into 6.2 percent solution by phosphate buffer solution (pH7.2).
2. And uniformly mixing 3.60mL of four pesticide solutions with the active ingredient content of 6.2% respectively to obtain the mixed pesticide.
3. 0.97mL, 1.30mL, 1.61mL, 1.94mL, 2.26mL and 2.58mL of the mixed pesticide are respectively added into 200mL of tryptone agar, and are uniformly mixed to prepare a mixed pesticide tryptone agar culture medium of 300mg/L, 400mg/L, 500mg/L, 600mg/L, 700mg/L and 800mg/L, and the mixed pesticide tryptone agar culture medium is prepared into a plate.
4. The target strain which can resist 800mg/L chlorpyrifos pesticide is transferred to a plate with 300mg/L mixed pesticide. The inoculated plate was placed in a 37 ℃ incubator and incubated for 24 hours.
5. Gradually increasing the concentration of the mixed pesticide in the plate, and continuing to induce and domesticate. Obtaining a mutant strain which can resist high-concentration mixed pesticide and is marked as FY-3.
The strain FY-3 has been preserved in China general microbiological culture Collection center (CGMCC for short, the address is No. 3 Xilu No.1 Beijing province of the rising area of Beijing) within 30 days at 12 months in 2016, the preservation number is CGMCC No.13519, and the strain is classified and named as Lysinibacillus macroides.
6. The domesticated FY-3 cells were washed from the petri dish with a sterile phosphate buffer, washed 3 times with repeated centrifugation and resuspension, weighed, and resuspended at a ratio of 0.1 g/500. mu.L in phosphate buffer (pH 7.2). Mixing sterilized 50% glycerol water solution and bacterial solution at a volume ratio of 1:1, subpackaging, and refrigerating at 4 deg.C in a refrigerator with 500 μ L tube.
EXAMPLE 3 preparation of biodegradable enzyme preparation Using Bacillus lentus Strain FY-3
First, fermentation broth obtaining
1. And (3) strain amplification culture: inoculating the refrigerated strain into 100mL of sterile TSB culture medium, and culturing at 37 ℃ and 180rpm for 24h to obtain first-grade seed; transferring the first-stage seeds to 1L of TSB culture medium according to the inoculation amount of 10%, and culturing at 37 ℃ and 180rpm for 24 hours to obtain second-stage seeds; transferring the second-level seeds to 10L of industrial culture medium according to the inoculation amount of 10%, and culturing at 37 ℃ and 180rpm for 24h to obtain third-level seeds.
2. The tertiary seeds were inoculated into a fermentor at 5% of the fermentation medium. Controlling the ventilation volume to be 2.5m3At 37 deg.C and pH7.2 + -0.4 at 180rpm, sampling at intervals, and determining OD600And pH, and adding proper amount of bacteria-free medium according to foam conditionAnd (4) defoaming agent. And (4) discharging the fermented product into a tank after 18 hours of fermentation.
3. Centrifuging the fermentation broth in the fermentation tank at high speed (14000rpm, 4 deg.C) for 15-20min, discarding liquid, and collecting thallus. The cells were resuspended at a ratio of 1:10(g/mL) in phosphate buffer to obtain a cell suspension.
4. And (3) crushing the heavy suspension of the thalli in the bacterial suspension by using a high-pressure cell crusher at the temperature of 4-6 ℃ under the pressure of 2.5-5.0MPa, and crushing twice to obtain a crushing solution.
5. The crushing liquid is centrifuged for 50min at 8000rpm and 4 ℃, and the supernatant is the crude enzyme liquid of the biodegradable enzyme.
6. And (3) filtering: filtering the crude enzyme solution with 6-8 layers of filter paper, and filtering with filter membrane with pore diameter of 0.45 μm to obtain clear biodegradable enzyme solution.
7. And (3) stabilizing treatment: diluting the biodegradable enzyme solution 100 times with phosphate buffer (pH7.2), adding 0.01-0.02% polyhexamethylene guanidine, mixing with sterilized glycerol, and storing at 4 deg.C.
The composition of the industrial medium (i.e., fermentation medium) was as follows: each liter of the fermentation medium consists of 17g of tryptone, 3g of plant peptone, 4.5g of sodium chloride, 3.5g of dipotassium hydrogen phosphate and 3.5g of glucose, and the volume of the fermentation medium is complemented by water.
Example 4 use of biodegradable enzyme preparation for degradation of pesticide residue in vegetables (fruits)
A representative vegetable cherry tomato is purchased from a vegetable wholesale market (Shouguang) and tested, wherein a biodegradable enzyme solution (enzyme preparation) obtained in example 3 is added into pure water to obtain a test solution, the concentration of the enzyme preparation is 3.5-4.5ppm, the cherry tomato is soaked in the test solution for 3-5min in a constant-temperature water bath at 37-43 ℃, and the test solution is sent to a third-party detection mechanism (Nonah force commercial available detection (Qingdao) Co., Ltd.) for detection. The degradation effect of the pesticide residues such as chlorpyrifos, carbendazim, diethofencarb, imidacloprid, iprodione, procymidone, propamocarb, pyrimethanil and the like in cherry tomatoes is shown in figure 1.
Sequence listing
<110> Sai' an Roots Biotechnology Ltd
<120> lysine-extended bacillus strain, enzyme preparation and application thereof in degrading pesticide residue
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>129
<212>DNA
<213>Lysinibacillus macroides
<400>1
ctttgcactt cggcggctgg ctccaaaggt tacctcaccg acttcgggtg ttacaaactc 60
tcgtggtgtg atgggcggtg tgtacaaggc ccgggaacgt attcaccgcg gcctgttgat 120
cctcgattt 129
<210>2
<211>19
<212>DNA
<213> Artificial Synthesis ()
<400>2
tccgtaggtg aacctgcgg 19
<210>3
<211>19
<212>DNA
<213> Artificial Synthesis ()
<400>3
tcctccgctt attgatatg 19
Claims (10)
1. A prolonged lysine bacillus (B)Lysinibacillus macroides) A strain characterized by: the preservation number of the strain is CGMCC No. 13519.
2. A method for preparing a biodegradable enzyme, which is characterized by comprising the following steps: the method comprises the following steps: bacillus lentus (B.) (Lysinibacillus macroides) After the bacterial strain is fermented, thallus is collected, broken cells are centrifuged, and supernatant is extracted to obtain a biodegradable enzyme solution, wherein the preservation number of the bacterial strain is CGMCC No. 13519.
3. The method for producing a biodegradable enzyme according to claim 2, wherein: the culture medium adopted by the fermentation comprises 17-19g/L of tryptone, 1-4g/L of plant peptone, 2-5g/L of sodium chloride, 2-5g/L of dipotassium hydrogen phosphate and 2-5g/L of glucose, and the pH value of the culture medium is 6.8-7.6.
4. The method for producing a biodegradable enzyme according to claim 2, wherein: the fermentation conditions were 34-39 ℃ at 180-.
5. The method for producing a biodegradable enzyme according to claim 2, wherein: after the fermentation, the fermentation product is centrifuged for 15-25 min at 12000-14000rpm, the thalli are collected, the thalli are subjected to high-pressure cell breaking after being resuspended, then the thalli are centrifuged for 50-65min at 8000-10000rpm, the supernatant is collected, and the supernatant is filtered to obtain the biodegradable enzyme solution.
6. The method for producing a biodegradable enzyme according to claim 5, wherein: the specific step of filtering is that the supernatant is filtered by filter paper and a filter membrane of 0.45 mu m in sequence to obtain clear liquid, namely the biodegradable enzyme solution.
7. The Bacillus lentus of claim 1, (b) aLysinibacillus macroides) The application of the strain in-vitro degradation of pesticide residues.
8. The application of the biodegradable enzyme preparation in-vitro degradation of pesticide residues is characterized in that: the active ingredient of the enzyme preparation is the biodegradable enzyme prepared according to claim 2.
9. A biodegradable enzyme formulation characterized by: the active ingredient of the enzyme preparation is the biodegradable enzyme prepared according to claim 2.
10. An agent for degrading a pesticide residue, characterized in that: the active ingredient of the agent is Bacillus lysinate: (Lysinibacillus macroides) The strain or the biodegradable enzyme prepared by the method in claim 2 has a preservation number of CGMCC No. 13519.
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