CN106222235B - Method for rapidly screening and evaluating pesticide residues in tea - Google Patents
Method for rapidly screening and evaluating pesticide residues in tea Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The invention provides a method for rapidly screening and evaluating pesticide residues in tea. The microbiological method for screening carbamate pesticide residue in tea by using the butyric acid bacillus WZ001 as a working strain. And judging whether the residual quantity of the acephate in the food sample to be detected exceeds the standard or not according to the size of a bacteriostatic circle on the screening culture medium. The method for screening the carbamate pesticide residues in the tea has the characteristics of simplicity and convenience in operation, low cost, high sensitivity and suitability for sample screening, is superior to the existing national pesticide residue rapid screening standard, completely meets the requirement of the national food safety standard on the maximum pesticide residue limit in food, has good matrix interference resistance, and can effectively avoid the occurrence of false positive results.
Description
Technical Field
The invention relates to detection of pesticide residues in tea, in particular to biological safety assessment and application of carbamate pesticide residues in tea.
Background
Pesticide residue in tea is a food safety problem which is very concerned by the public, the existing pesticide residue detection in China generally adopts an instrument analysis method, and mainly adopts a large instrument for detection, namely gas chromatography, liquid chromatography and combination of gas quality and liquid quality, the instrument is expensive, an operator has to have considerable professional skills, the detection period is long, and the popularization of the instrument in the first-line supervision department is not facilitated. The instrumental analysis method can only detect single pesticides respectively, cannot comprehensively reflect the biological safety of foods, has long detection time, expensive equipment and complex operation, is not suitable for large-sample rapid screening and cannot evaluate the biological hazard of the pesticides.
The pesticide residue screening method is very effective in confirming whether the problem of pesticide residue exists in the sample; the application aims to ensure that the food to be detected does not contain pesticide residues exceeding the maximum residue limit, and the screening method reduces the detection range.
In order to solve the problems existing in pesticide residue detection, it becomes very important to establish a detection method which has good stability, strong anti-interference performance, simple and convenient operation, short detection time, easy learning and use and no need of professional technical knowledge. Many explorations have been made on pesticide residue screening using bioassay methods: in the later stage of the last 60 years, the Taiwan agricultural test adopts a bioassay method to carry out pesticide residue inspection, the principle is that highly sensitive houseflies are released into vegetable juice, and the death rate of the houseflies is qualified when the death rate is below 10% after 4-5 h. The microbiological method is a good screening method and has the following advantages: the microorganism has simple structure, high growth speed, sensitive reaction, low cost, relatively small occupied space and capability of making response quickly. Therefore, the microorganism detection technology has the characteristics of rapidness, simplicity, convenience and sensitivity, and can meet the requirement of screening large samples.
The carbamate pesticide has the structural characteristics that the molecule contains an N-methyl group, most of the carbamate pesticide is white crystals, is insoluble in water, is soluble in organic solvents such as acetone, dichloromethane, chloroform, acetonitrile and the like, is easily decomposed under alkaline and high-temperature conditions, mainly inhibits the activity of cholinesterase, and leads the hydroxyl group of serine at the center of the enzyme activity to be esterified by aminomethyl, thereby losing the hydrolyzing capability of the enzyme on acetylcholine. Are not degraded to the corresponding metabolites for a long time after administration. The metabolites thereof typically have the same or greater biological activity as the parent carbamate pesticide. With the improvement of living standard and health consciousness, people pay more and more attention to the problem of pesticide residue, and pay more and more attention to the measurement of the carbamate pesticide residue.
The spectrophotometry is used for measuring the pesticide residue, requires necessary sample enrichment to improve the sensitivity, is complicated to operate, is easy to be interfered by other substances, and is rarely used at present. High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) have been widely used for detecting the residual amount of carbamate pesticides. The method has the defects of complex instrument and equipment and complex sample pretreatment and measurement operation, is not suitable for screening a large number of samples, and has high cost, so that the popularization and the use of the method are limited. The microbiological method has the outstanding advantages of simplicity, convenience and economy, can be carried out under common experimental conditions without complex instruments and equipment, and can carry out quick primary screening on a large number of samples. However, the sensitivity and specificity of the working strain to the substance to be tested are the most critical problems affecting the accuracy and precision of the microbiological method. So far, no relevant report exists for screening out a butyric acid bacillus working strain with high sensitivity and high specificity of carbamate pesticide and the application of the butyric acid bacillus working strain in screening of carbamate pesticide residue in tea leaves.
Disclosure of Invention
The invention aims to establish a microbial method for screening carbamate pesticide residues in tea by using butyric acid bacillus WZ001 as a working strain.
The butyric acid bacillus WZ001 of the invention is separated from feces of healthy people in Zhejiang province, straight or bent bacillus, 0.6-1.2 multiplied by 3.0-7.0 mu m, terminal round, single, paired, short-chain, occasionally filamentous thallus, and is in the motion of periphytic flagellum, spore oval, eccentric to secondary terminal, and has no spore outer wall and accessory silk. Gram positive, becoming negative in old culture; the bacteria are often positive for starch grains. The cell wall contains DH-diaminopimelic acid; glucose is the only cell wall sugar. The form of the bacterium butyricum is consistent with that of the bacterium butyricum seen by a light mirror. According to Bergey's Manual of identification of bacteria, which is a typical characteristic of Clostridium, it can be judged that the bacterium is in the genus Clostridium. Certain sugars may be fermented to produce hydrogen and butyric acid. The visible bacterial colony in the solid culture medium is milk white, slightly convex in round shape, irregular in edge, 1-3 mm in diameter and slightly glossy in surface, bubbles are generated in the liquid culture medium, a bacterial strain sensitive to carbamate pesticides is separated through multiple times of separation and screening, and the bacillus butyricum (Clostridium butyricum) WZ001 is deposited in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms in 2014.01-22.23, which is abbreviated as CGMCC (address: the microbial research institute of China academy of sciences, No. 3, N.Y. 1 of North West Chen of the Yangxi of Beijing city, zip code 100880101) and is classified and named as the bacillus butyricum (Clostridium butyricum) with the preservation number of CGMCC No. 8.
The principle of screening and detecting the carbamate pesticide residue in the tea is that based on the obvious inhibition effect of the carbamate pesticide on the sensitive butyric acid bacillus WZ001, a tea sample is applied to a butyric acid bacillus layer screening culture medium, after anaerobic culture is carried out for 18-24 hours, a formed inhibition zone is determined, and whether the carbamate pesticide residue in a sample to be detected is qualified or not can be judged according to the size of the inhibition zone.
The invention relates to a microbiological method for screening carbamate pesticide residues in tea leaves by using butyric acid bacillus WZ001 as a working strain, which comprises the following steps:
(1) preparation of a screening culture medium:
10g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 5g of sodium acetate and K2HPO42g，MgSO4·7H2O0.5g，MnSO4·4H20.2g of O, 3g of fructo-oligosaccharide, 2g of diammonium citrate, 801mL of Tween-801, 15g of agar,dissolving the above components in 1L of distilled water under heating to adjust pH to 6.5, and autoclaving at 115 deg.C for 15-20 min;
(2) preparing a bacterial suspension:
inoculating the butyric acid bacillus WZ001 to a slant culture medium of nutrient agar, carrying out anaerobic culture at 35 +/-2 ℃ for 24 hours, washing down lawn by using sterilized normal saline to prepare suspension, controlling the light transmittance to be 0.7 at the wavelength of 580nm of a 721 spectrophotometer, and storing in a refrigerator at the temperature of 2-8 ℃;
(3) preparing a bacterium layer culture medium:
taking a flat plate with the diameter of about 90mm and the height of 16-17 mm, injecting 20mL of a heating and melting screening culture medium obtained in the step (1), uniformly spreading the flat plate in the bottom of the dish, and placing the flat plate on a horizontal table to solidify the flat plate to serve as a bottom layer; adding 0.5mL of the bacterial suspension obtained in the step (2) into the solidified bottom layer culture medium, and fully and uniformly mixing to uniformly spread the bacterial suspension on the bottom layer to be used as a bacterial layer;
(4) sample pretreatment:
taking 5g of homogenate tissue of a tea sample to be detected, diluting the homogenate tissue with 20mL of sterile 1% pH6.0 phosphate buffer solution, uniformly mixing by vortex, heating in a water bath at 80 ℃ for 5min, cooling, centrifuging at 5000r/min for 15min, and taking supernatant as a sample liquid to be detected;
(5) and (3) sample determination:
placing a circular filter paper sheet with the diameter of 13 +/-1 mm in a culture dish of the bacteria layer screening culture medium in the step (3), lightly pressing the circular filter paper sheet by using a pair of tweezers to enable the circular filter paper sheet to be in close contact with the culture medium, and dropwise adding 90 mu L of the sample liquid to be tested in the step (4) onto the circular filter paper sheet by using a micropipettor. Repeating at least 2 culture dishes for each sample, performing anaerobic culture at 35 +/-2 ℃ for 18-24 hours, and measuring the diameter of a bacteriostatic zone by a bacteriostatic zone computer measurement analyzer;
(6) and (4) judging a result:
and when the inhibition zone is more than or equal to 16mm, judging that the carbamate pesticide residue in the tea sample is positive (+).
The working strain is the butyric acid bacillus WZ001 which is preserved in the common microorganism center of China general microbiological culture Collection center in 2014, 01, 22 and the preservation number is CGMCC No. 8808.
The invention has the advantages and effects that:
(1) the butyric acid bacillus WZ001 has high sensitivity and good specificity to carbamate pesticide, and can be used as a working strain for screening carbamate pesticide residues by a microbiological method;
(2) the detection method provided by the invention is simple and easy to implement, has the characteristics of high sensitivity, high accuracy and the like, the minimum detection limit in tea is 0.1mg/kg and is lower than the maximum residue limit of national standards, the recovery rates are all more than 70%, the variation coefficient is within 10%, the accuracy and precision requirements of screening of the tea carbamate pesticide residues are met, and the method is completely suitable for screening of the tea carbamate pesticide residues.
(3) The butyric acid bacillus WZ001 screened by the invention is sensitive to carbamate pesticides, not only can be used as a test bacterium of a microbiological method, but also can reflect the biological effect of pesticide residues, and has the advantages of sensitivity, rapidness, short culture time, reduction of interference from microbial pollution and the like.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to be limiting. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
EXAMPLE 1 screening of sensitive strains
The butyric acid bacillus WZ001 is separated and identified, straight or bent bacillus, 0.6-1.2 multiplied by 3.0-7.0 mu m, round end, single, paired, short-chain and occasionally filamentous thallus. With the circum-flagellum movement, the spore is oval, eccentric to the secondary, and has no spore outer wall and accessory silk. Gram positive, becoming negative in old culture; the bacteria are often positive for starch grains. The cell wall contains DH-diaminopimelic acid; glucose is the only cell wall sugar. The pattern of clostridium butyricum was observed with a light mirror. According to Bergey's Manual of bacteria identification, the genus is judged to be Clostridium butyricum. Certain sugars may be fermented to produce hydrogen and butyric acid. Visible bacterial colonies in the solid culture medium are milk white, the circular shape is slightly convex, the edge is irregular, the diameter is 1-3 mm, the surface is slightly glossy, and air bubbles are generated in the liquid culture medium. The screened butyric acid bacillus WZ001 has sensitivity to carbamate pesticides, and compared with the same genus standard strain, the sensitivity to carbamate pesticides is improved. The sensitivity of the strain to the carbamate pesticide is less than or equal to 0.1mg/L, the bacteriostatic diameter of the 0.1mg/L carbamate pesticide is more than 16mm, and the strain is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 8808.
Example 2 screening of tea methomyl residue
(1) Preparation of a screening culture medium:
10g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 5g of sodium acetate and K2HPO42g，MgSO4·7H2O0.5g，MnSO4·4H20.2g of O, 3g of fructo-oligosaccharide, 2g of diammonium citrate, 801mL of Tween-801, 15g of agar and 1L of distilled water, heating, dissolving and correcting the pH value to 6.5, and sterilizing for 15-20 minutes at 115 ℃ under high pressure;
(2) preparing a bacterial suspension:
inoculating the butyric acid bacillus WZ001 to a slant culture medium of nutrient agar, carrying out anaerobic culture at 35 +/-2 ℃ for 24 hours, washing down lawn by using sterilized normal saline to prepare suspension, controlling the light transmittance to be 0.7 at the wavelength of 580nm of a 721 spectrophotometer, and storing in a refrigerator at the temperature of 2-8 ℃;
(3) preparing a bacterium layer culture medium:
taking a flat plate with the diameter of about 90mm and the height of 16-17 mm, injecting 20mL of a heating and melting screening culture medium obtained in the step (1), uniformly spreading the flat plate in the bottom of the dish, and placing the flat plate on a horizontal table to solidify the flat plate to serve as a bottom layer; adding 0.5mL of the bacterial suspension obtained in the step (2) into the solidified bottom layer culture medium, and fully and uniformly mixing to uniformly spread the bacterial suspension on the bottom layer to be used as a bacterial layer;
(4) sample pretreatment:
taking 5g of homogenate tissue of a tea sample to be detected, diluting the homogenate tissue with 20mL of sterile 1% pH6.0 phosphate buffer solution, uniformly mixing by vortex, heating in a water bath at 80 ℃ for 5min, cooling, centrifuging at 5000r/min for 15min, and taking supernatant as a sample liquid to be detected;
(5) and (3) sample determination:
placing a circular filter paper sheet with the diameter of 13 +/-1 rmm in a culture dish of the bacteria layer screening culture medium in the step (3), lightly pressing the circular filter paper sheet by using a pair of tweezers to enable the circular filter paper sheet to be in close contact with the culture medium, and dropwise adding 90 mu L of the sample liquid to be tested in the step (4) onto the circular filter paper sheet by using a micropipettor. Repeating at least 2 culture dishes for each sample, performing anaerobic culture at 35 +/-2 ℃ for 18-24 hours, and measuring the diameter of a bacteriostatic zone by a bacteriostatic zone computer measurement analyzer;
(6) and (4) judging a result:
and when the inhibition zone is more than or equal to 16mm, judging that the carbamate pesticide residue in the tea sample is positive (+).
Through detection, the linear range of the method for determining the methomyl pesticide residue in the tea is 0.05-2 mg/kg, the adding recovery rate of the method is 77-90%, and the precision of the method is 1-10%. The method is rapid and simple, the recovery rate and precision sensitivity indexes meet the requirement of pesticide residue analysis, and the method is suitable for monitoring the methomyl pesticide residue in the tea.
Example 2 screening of carbofuran residue in tea
(1) Preparation of a screening culture medium:
10g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 5g of sodium acetate, 42 g of K2HPO, MgSO 4.7H2O0.5g, MnSO 4.4H2O 0.2.2 g, 3g of fructo-oligosaccharide, 2g of diammonium citrate, 801ml of Tween, 15g of agar and 1L of distilled water, heating and dissolving the components to correct the pH value to be 6.5, and sterilizing under high pressure at 115 ℃ for 15-20 minutes;
(2) preparing a bacterial suspension:
the butyric acid bacillus WZ00l is inoculated in a slant culture medium of nutrient agar, anaerobic culture is carried out for 24 hours at the temperature of 35 +/-2 ℃, the lawn is washed down by sterilized normal saline to prepare suspension, the light transmittance is controlled to be 0.7 under the wavelength of 580nm of a 721 spectrophotometer, and the suspension is stored in a refrigerator at the temperature of 2-8 ℃;
(3) preparing a bacterium layer culture medium:
taking a flat plate with the diameter of about 90mm and the height of 16-17 mm, injecting 20mL of a heating and melting screening culture medium obtained in the step (1), uniformly spreading the flat plate in the bottom of the dish, and placing the flat plate on a horizontal table to solidify the flat plate to serve as a bottom layer; adding 0.5mL of the bacterial suspension obtained in the step (2) into the solidified bottom layer culture medium, and fully and uniformly mixing to uniformly spread the bacterial suspension on the bottom layer to be used as a bacterial layer;
(4) sample pretreatment:
taking 5g of homogenate tissue of a tea sample to be detected, diluting the homogenate tissue with 20mL of sterile 1% pH6.0 phosphate buffer solution, uniformly mixing by vortex, heating in a water bath at 80 ℃ for 5min, cooling, centrifuging at 5000r/min for 15min, and taking supernatant as a sample liquid to be detected;
(5) and (3) sample determination:
placing a circular filter paper sheet with the diameter of 13 +/-1 mm in a culture dish of the bacteria layer screening culture medium in the step (3), lightly pressing the circular filter paper sheet by using a pair of tweezers to enable the circular filter paper sheet to be in close contact with the culture medium, and dropwise adding 90 mu L of the sample liquid to be tested in the step (4) onto the circular filter paper sheet by using a micropipettor. Repeating at least 2 culture dishes for each sample, performing anaerobic culture at 35 +/-2 ℃ for 18-24 hours, and measuring the diameter of a bacteriostatic zone by a bacteriostatic zone computer measurement analyzer;
(6) and (4) judging a result:
and when the inhibition zone is more than or equal to 16mm, judging that the carbamate pesticide residue in the tea sample is positive (+).
Through detection, the detection limit of the method can reach 0.1-1 mg/kg, which is superior to the existing national standard for rapid screening of pesticide residues, completely meets the requirement of the national standard for food safety on the maximum residue limit of pesticides in food, has good matrix interference resistance, and can effectively avoid the occurrence of false positive results. In addition, the recovery rate of the pesticide is 78.3 to 116.2 percent by taking the main carbamate pesticides such as aldicarb sulfoxide, aldicarb sulfone, methomyl, carbofuran and the like as objects. The method is simple, convenient, quick, sensitive and reliable, and is suitable for quick and high-throughput screening of the carbamate pesticide in the tea leaves.
Claims (2)
1. The application of the butyric acid bacillus (Clostridium butyricum) WZ001 in screening of carbamate pesticide residues in tea leaves is disclosed, wherein the preservation number of the butyric acid bacillus WZ001 is CGMCC No. 8808.
2. The use of butyric acid bacteria WZ001 according to claim 1, wherein the screening of carbamate pesticide residues in tea leaves comprises the following steps:
(1) preparation of a screening culture medium:
10g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 5g of sodium acetate and K2HPO42g，MgSO4·7H2O0.5g，MnSO4·4H20.2g of O, 3g of fructo-oligosaccharide, 2g of diammonium citrate, 801ml of Tween-agar, 15g of agar and 1L of distilled water, heating, dissolving, correcting the pH value to be 6.5, and sterilizing for 15-20 minutes at 115 ℃ under high pressure;
(2) preparing a bacterial suspension:
inoculating the butyric acid bacillus WZ001 to a slant culture medium of nutrient agar, carrying out anaerobic culture at 35 +/-2 ℃ for 24 hours, washing down lawn by using sterilized normal saline to prepare suspension, controlling the light transmittance to be 0.7 at the wavelength of 580nm of a 721 spectrophotometer, and storing in a refrigerator at the temperature of 2-8 ℃;
(3) preparing a bacterium layer culture medium:
taking a flat plate with the diameter of 90mm and the height of 16-17 mm, injecting 20mL of a heating and melting screening culture medium obtained in the step (1), uniformly spreading the flat plate in the bottom of the dish, and placing the flat plate on a horizontal platform to solidify the flat plate to serve as a bottom layer; adding 0.5mL of the bacterial suspension obtained in the step (2) into the solidified bottom layer culture medium, and fully and uniformly mixing to uniformly spread the bacterial suspension on the bottom layer to be used as a bacterial layer;
(4) sample pretreatment:
taking 5g of homogenate tissue of a tea sample to be detected, diluting the homogenate tissue with 20mL of sterile 1% pH6.0 phosphate buffer solution, uniformly mixing by vortex, heating in a water bath at 80 ℃ for 5min, cooling, centrifuging at 5000r/min for 15min, and taking supernatant as a sample liquid to be detected;
(5) and (3) sample determination:
placing circular filter paper sheets with the diameter of 13 +/-1 mm in the culture dish of the bacteria layer screening culture medium in the step (3), lightly pressing the paper sheets by using forceps to enable the paper sheets to be in close contact with the culture medium, dropwise adding 90 mu L of the sample liquid to be tested in the step (4) on the circular filter paper sheets by using a micropipettor, repeating at least 2 culture dishes for each sample to be tested, carrying out anaerobic culture at 35 +/-2 ℃ for 18-24 hours, and measuring the diameter of a bacteriostatic ring by using a bacteriostatic ring computer measuring analyzer;
(6) and (4) judging a result:
and when the inhibition zone is more than or equal to 16mm, judging that the carbamate pesticide residue in the tea sample is positive (+).
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