CN107446856B - Providencia juveniles strain, enzyme preparation and application thereof in degrading pesticide residue - Google Patents
Providencia juveniles strain, enzyme preparation and application thereof in degrading pesticide residue Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- 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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- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
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Abstract
The invention discloses a providencia juveniles strain, an enzyme preparation and application thereof in degrading pesticide residues. The invention provides a Providencia juvenila (Providencia vermicola) FY-4 strain, and the preservation number of the strain is CGMCC No. 13520. The invention also provides a method for screening, inducing and domesticating the strains and preparing the pesticide residue degradation active enzyme, namely screening the 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, cells are broken, centrifugation is carried out, and the supernatant can be used for preparing the pesticide residue degrading enzyme preparation, can be used for degrading pesticide residues on vegetables and fruits, and has application potential in the aspect of degrading pesticide residues on vegetables and fruits.
Description
Technical Field
The invention relates to the field of degrading pesticide residues by biotechnology, in particular to screening and induced domestication of Providencia vermicola (Providencia vermicola) strains of larval larvae and application of the Providencia vermicola in removing pesticide residues in 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 providencia juveniles strain obtained by screening, pesticide induction and domestication.
The Providencia (Providencia vermicola) larval strain (marked as FY-4) provided by the invention has the preservation number of CGMCC No. 13520.
The Providencia (Providencia vermicola) larvas strain is obtained by screening a target strain, inducing and domesticating the target strain by using a single pesticide and a mixed pesticide, and comprises the following steps of:
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 pesticides carbendazim, carbofuran, 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 carrying out continuous induced cultivation (by continuously increasing the pesticide content), so as to obtain a strain with higher pesticide concentration tolerance, and finally separating to obtain a Providencia vermicola (Providencia vermicola) strain (marked as FY-4) as a larval.
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 Providencia (Providencia vermicola) strain FY-4, collecting the thallus, breaking the thallus, centrifuging, and extracting the supernatant to obtain the 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 15-19g of tryptone, 1-5g 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 6.5 to 7.5, preferably 6.8 to 7.2.
In the above method for producing a biodegradable enzyme, the fermentation specifically comprises the steps of: the Providencia juveniae (Providencia vermicola) strain FY-4 strain is subjected to amplification culture to obtain seeds, and the seeds are inoculated into the fermentation medium and cultured under the fermentation condition.
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 thallus of the Providencia (Providencia vermicola) strain FY-4 in a phosphate buffer solution.
In the method for producing the biodegradable enzyme, after the fermentation, the fermentation product is centrifuged for 15-25min at 12000-14000rpm and 4-6 ℃, 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 and 4-6 ℃, 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 active ingredient of the biodegradable enzyme preparation provided by the invention is the providencia juvenilis (providencia vermicola) strain FY-4 or the biodegradable enzyme.
The fourth purpose of the invention is to provide the application of the Providencia juveniae (Providencia vermicola) strain FY-4, the biodegradable enzyme and the biodegradable enzyme preparation in pesticide residue degradation, especially in 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 2.5-15ppm, and the optimal concentration is 2.5-7.5ppm, which is different according to the specific vegetables and fruits.
The invention has the beneficial effects that:
experiments prove that the providencia (providencia vermicola) strain FY-4 obtained by induction and domestication is fermented and cultured, and intracellular enzymes extracted from the strain FY-4 can be used as biodegradable enzymes which 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 providencia strain FY-4 has application potential in the field of pesticide residue degradation.
Drawings
FIG. 1 is a dendrogram of the development of Providencia juvenilis (Providencia vermicola) strain FY-4.
FIG. 2 is a chlorpyrifos tolerance curve for Providencia juveniles (Providencia vermicola) strain FY-4.
FIG. 3 is a mixed pesticide tolerance curve for Providencia juvenila (Providencia vermicola) strain FY-4.
FIG. 4 is a graph showing the optimum pH for a liquid biodegradable enzyme preparation based on Providencia (Providencia vermicola) strain FY-4.
FIG. 5 is a temperature profile of optimum action of a liquid biodegradable enzyme preparation based on Providencia (Providencia vermicola) strain FY-4.
FIG. 6 is a temperature stability curve of a liquid biodegradable enzyme preparation based on Providencia juveniae (Providencia vermicola) strain FY-4.
FIG. 7 shows the results of treating pakchoi with a liquid biodegradable enzyme preparation based on Providencia vera strain FY-4 for pesticide residues.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The examples are intended to facilitate a better understanding of the invention and are not intended to limit the invention. 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; using the absorbance OD of the culture600Indicates the amount of bacteria grown.
Example 1 isolation and identification of the Cure larva providencia Strain
First, obtaining of the Strain
1. Collection of soil samples
Collecting sludge from the discharge port of the pesticide factory.
2. Isolation and screening of 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 strain with the number preserved 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 718bp nucleotide sequence (shown in SEQ. ID. NO.1) through sequencing, wherein homology comparison analysis shows that: the number-saved strain has the highest homology with Providencia (Providencia vermicola) larvae, and the phylogenetic position is shown in fig. 1.
Example 2 Induction and acclimatization of Cure larva providencia strains
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 16g of tryptone, 5g of phytone, 3.5g of sodium chloride and 15g of agar (pH 7.3. + -. 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. The pesticide chlorpyrifos tolerance is shown in figure 2.
Second, mixed pesticide induction and domestication
1. Mixed pesticide diluting method
Carbendazim, carbofuran and cyhalothrin are respectively prepared into 6.2 percent solution by phosphate buffer solution (0.03mol/L, pH7.2).
2. 3.60mL of three pesticide solutions with the active ingredient contents of 6.2 percent are uniformly mixed 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 organophosphorus pesticide (chlorpyrifos) 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 to obtain a mutant strain capable of resisting the high mixed pesticide, which is recorded as FY-4. The tolerance of the mixed pesticide is shown in fig. 3.
The strain FY-4 is preserved in the China general microbiological culture Collection center (CGMCC for short, the address is No. 3 Xilu No.1 Hospital of Chaozhou, Chaoyang, Beijing) within 30 days at 12 months in 2016, the preservation number is CGMCC No.13520, and the strain is classified and named as Providencia juveniae (Providencia vermicola).
6. The domesticated FY-4 cells were washed from the petri dish with sterile phosphate buffer, washed 3 times with repeated centrifugation and resuspension, weighed, and resuspended in 0.1g of phosphate buffer at pH7.2 at a rate of 500. mu.L. 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 a biodegradable enzyme preparation Using Cure larva providencia Strain FY-4
Firstly, obtaining liquid biological degradation enzyme preparation
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. Inoculating the third-level seeds into a fermentation tank, wherein the inoculation amount is 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 a proper amount of a sterile defoaming agent in the middle according to the foam condition. 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. Crushing the heavy suspension thallus in the bacterial suspension by a high-pressure cell crusher at the temperature of 4-6 ℃ and under the pressure of 2.5-5.0MPa, and crushing twice to obtain a crushing liquid.
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 L of the fermentation medium consists of tryptone 16g,
5g of plant peptone, 3.5g of sodium chloride, 4.5g of dipotassium hydrogen phosphate, 4.5g of glucose and water, and the volume is complemented by water.
Secondly, study of enzymatic characteristics of biodegradation enzymes
1. Optimum pH value
Detecting the difference of enzyme activity under different pH conditions: phosphate buffer solutions (pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0) with different pH values are respectively used for preparing 100ppm chlorpyrifos solution, and the enzyme activity of the biodegradation enzyme solution is measured at the temperature of 37 ℃ under each pH value. Enzyme activity is defined as: hydrolyzing chlorpyrifos at 37 deg.C, and the enzyme amount required for 1 μmol chlorpyrifos degradation in 1min is one enzyme activity unit (U).
The highest enzyme activity is 100%, the ratio of the enzyme activity at other pH values to the highest enzyme activity is the relative enzyme activity, the pH value is the abscissa, and the relative enzyme activity is the ordinate, and the diagram is shown (fig. 4). The result shows that the optimum action pH of the biodegradation enzyme solution obtained after fermentation of the providencia juvenilis strain FY-4 as a liquid biodegradation enzyme preparation to chlorpyrifos is 7.0.
2. Optimum temperature of action
Detecting the difference of enzyme activity under different temperature conditions: the enzyme activity was measured at different reaction temperatures (30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃) and pH 7.0, respectively.
The highest enzyme activity is taken as 100%, the ratio of the enzyme activity at other temperatures to the highest enzyme activity is taken as the relative enzyme activity, the temperature is taken as the abscissa, and the relative enzyme activity is taken as the ordinate to construct a graph (fig. 5). The result shows that the optimal action temperature of the biodegradation enzyme solution obtained after fermentation of providencia fruticosa FY-4 strain of the juveniles as a liquid biodegradation enzyme preparation on chlorpyrifos is 40 ℃.
3. Temperature stability
The biological degradation enzyme solution obtained after fermentation of the larva providencia strain FY-4 is respectively placed in a water bath kettle at 30 ℃, 40 ℃ and 50 ℃ for heat preservation for a certain time (1h, 2h, 4h and 6h), then the enzyme activity of the biological degradation enzyme solution after heat preservation is measured under the conditions of pH 7.0 and 40 ℃, the enzyme activity (the highest enzyme activity) of the biological degradation enzyme solution is preserved at 4 ℃ to be 100%, the ratio of the enzyme activity of the biological degradation enzyme solution after heat preservation to the highest enzyme activity is the relative enzyme activity, the heat preservation time is used as a horizontal coordinate, and the relative enzyme activity is used as a vertical coordinate to be plotted (figure 6). The result shows that the enzyme activity of the biodegradable enzyme solution obtained after fermentation of the providencia juveniae strain FY-4 is gradually reduced along with the increase of the heat preservation time.
Example 4 use of biodegradable enzyme preparation for degradation of pesticide residue in vegetables (fruits)
A representative vegetable pakchoi is purchased from a vegetable wholesale market (Shouguang) and tested, firstly, a biodegradable enzyme solution (enzyme preparation) obtained in example 3 is prepared into a test solution with the concentration of the enzyme preparation being 2.5-5ppm by pure water, the pakchoi is soaked in the test solution for 3-5min under a constant-temperature water bath at 37-43 ℃, and then the pakchoi is sent to a third party detection mechanism (Nonah's force commercial product detection (Qingdao) Co., Ltd.) for detection. The pesticide residue degradation types (carbendazim, carbofuran, 3-hydroxy carbofuran, cyhalothrin, difenoconazole, dimethomorph, imidacloprid and the like) and the effects of the pakchoi are shown in figure 7.
Sequence listing
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Claims (10)
1. Providencia juvenia (A) for larvaeProvidencia vermicola) A strain characterized by: the preservation number of the strain is CGMCC No. 13520.
2. A method for preparing a biodegradable enzyme, which is characterized by comprising the following steps: the method comprises the following steps: providencia juvenia (A) and providencia juvenia (B)Providencia vermicola) 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. 13520.
3. The method for producing a biodegradable enzyme according to claim 2, wherein: the culture medium adopted by the fermentation comprises 15-19g/L of tryptone, 1-5g/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 6.5-7.5.
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 15-25min 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. A biodegradable enzyme formulation characterized by: the active ingredient of the enzyme preparation is a biodegradable enzyme prepared by the method of claim 2.
8. Providencia larvae as claimed in claim 1, (b) a providencia larvae productProvidencia vermicola) The application of the strain in degrading pesticide residue is characterized in that: the pesticide is one or more selected from carbendazim, carbofuran, 3-hydroxy carbofuran, cyhalothrin, difenoconazole, dimethomorph and chlorpyrifos.
9. Use of a biodegradable enzyme preparation according to claim 7 for degrading pesticide residues, characterized in that: the pesticide is one or more selected from carbendazim, carbofuran, 3-hydroxy carbofuran, cyhalothrin, difenoconazole, dimethomorph and chlorpyrifos.
10. An agent for degrading a pesticide residue, characterized in that: the active ingredient of the agent is providencia juvenilis (A)Providencia vermicola) A strain having a preservation number of CGMCC No.13520 or a biodegradable enzyme prepared by the method of claim 2.
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| CN107446856B true CN107446856B (en) | 2020-08-11 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994488A (en) * | 2011-12-20 | 2013-03-27 | 辽宁中科生物工程有限公司 | Pesticide degradation enzyme preparation |
| CN104531582A (en) * | 2014-12-29 | 2015-04-22 | 北京市农林科学院 | Strain for degrading pendimethalin pesticide and fungicide and application of strain |
| CN104894000A (en) * | 2015-02-09 | 2015-09-09 | 暨南大学 | Application of Providencia sp. 2D in remediation of dibutyl phthalate contaminated soil |
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2017
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994488A (en) * | 2011-12-20 | 2013-03-27 | 辽宁中科生物工程有限公司 | Pesticide degradation enzyme preparation |
| CN104531582A (en) * | 2014-12-29 | 2015-04-22 | 北京市农林科学院 | Strain for degrading pendimethalin pesticide and fungicide and application of strain |
| CN104894000A (en) * | 2015-02-09 | 2015-09-09 | 暨南大学 | Application of Providencia sp. 2D in remediation of dibutyl phthalate contaminated soil |
Non-Patent Citations (3)
| Title |
|---|
| Isolation and characterization of a chlorpyrifos-degrading bacterium from agricultural soil and its growth response;M Surekha Rani et al;《African Journal of Microbiology Research》;20080229;26-31 * |
| Isolation of a selected microbial consortium from a pesticide-contaminated mix-load site soil capable of degrading the herbicides atrazine and alachlor;Anastasia EM Chirnside et al;《Soil Biology & Biochemistry》;20071231;第39卷(第12期);3056-3065 * |
| Isolation, identification and acclimatization of Atrazine-resistant soil bacteria;E EI-Bestawy et al;《Annals of Agricultural Science》;20130905;第58卷(第2期);119-130 * |
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