CN102517230B - Bifidobacterium breve and detection method of methamidopho pesticide residue in foodstuff - Google Patents

Bifidobacterium breve and detection method of methamidopho pesticide residue in foodstuff Download PDF

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CN102517230B
CN102517230B CN2011104114934A CN201110411493A CN102517230B CN 102517230 B CN102517230 B CN 102517230B CN 2011104114934 A CN2011104114934 A CN 2011104114934A CN 201110411493 A CN201110411493 A CN 201110411493A CN 102517230 B CN102517230 B CN 102517230B
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detection
acephatemet
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bifidobacterium breve
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CN102517230A (en
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刘佳明
杜季梅
孙晶
曹建明
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Shanghai Institute of Wenzhou Medical University
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Wenzhou Medical College
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/10—Enterobacteria
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Abstract

The invention relates to bifidobacterium breve and a method for detecting methamidopho pesticide residue in foodstuff by using bifidobacterium breve. According to the invention, a selected strain is an LJM-006 strain with a collection number of CGMCC No. 5418. The LJM-006 strain is hypersensitive to methamidopho. The methamidopho residue detection method established employing the strain as a working strain comprises the steps of: (1) strain activation, (2) strain suspension preparation, (3) detection tube preparation, (4) standard curve drawing and regression equation establishing, (5) sample pretreatment, (6) sample detection, and the like. The detection method provided by the invention is advantaged in simple operation, low cost, and high sensitivity. The result of detection is easy to determine. Compared to a high-efficiency liquid chromatography method, a result coincidence rate is high. Therefore, the method is suitable for sample detection. The method satisfies the requirement by the national pesticide residue detection department for large-batch sample screening.

Description

The detection method of methamidophos pesticide residue in bifidobacterium breve and food
Technical field
The present invention relates to the microorganism detection field of methamidophos pesticide, relate to a kind of bifidobacterium breve strain and carry out the method that in food, methamidophos pesticide residue detects take this bacterial strain as the work bacterial strain.
Background technology
Food is the basic substance that the mankind depend on for existence and development, and pesticide residue exceed standard becomes one of outstanding problem of China's present food security.Only since 2010, the food safety affair that a series of pesticide residue such as " Hainan poison cowpea ", " Qingdao detects 1930 kilograms of leek agricultural chemicals and exceeds standard " cause occurs in China; China every year occurs repeatedly because of the poisoning that causes that exceeds standard of Pesticide Residues amount, therefore, to Pesticide Residues in time, analyzing and testing is essential exactly, set up quick, easy, safe, the Detection Technologies of Pesticide Residues is the inevitable demand of food safety development accurately.
Acephatemet is a kind of organic phosphorous insecticide of efficient, the lasting period long, interior absorption is strong broad spectrum, is applicable to the crops such as vegetables, tea tree, paddy rice, wheat, can effectively prevent and treat various pests.But the excessive use of acephatemet makes its high residue in vegetable and fruit, brings huge threat for people's health, so China's uses of having limited the quantity of, and in detection food, the methamidophos residue amount is classified residual monitoring emphasis as.
At present, generally there are vapor-phase chromatography (GC), high performance liquid chromatography (HPLC), spectrophotometer, chemoluminescence method, enzyme to suppress method, capillary electrophoresis technique (CE) etc. for the food methamidophos pesticide residue.Detect with aforesaid method and have plant and instrument complexity, Sample pretreatment and the loaded down with trivial details defective of measurement operation, equipment cost is large, and testing cost is high, and is also high to testing staff's requirement, is not suitable for the great amount of samples screening, is unfavorable for applying in food inspection department of basic unit.Develop a kind of easy to operate, expense is low, the detection method of acephatemet is imperative in reliable, available a large amount of test sample.And that its primary ring is screening is a kind of to the general sensitivity of acephatemet and susceptibility are stable, detectability is low, promptly indicator grows.Up to now, there is no filter out acephatemet susceptibility is high, specificity is high bifidus bacillus work bacterial strain and in food methamidophos residue detect the relevant report of using.
Summary of the invention
In view of this, the objective of the invention is to filter out and be difficult for being bacterial contamination impact and to the bifidobacterium strains of methamidophos pesticide sensitivity, and set up accordingly a kind of simple, quick, highly sensitive, can generally apply carry out the method that methamidophos pesticide residue in food detects take this bacterial strain as the work bacterial strain, the method adopts acephatemet the restraining effect of fermentation to be confirmed the methamidophos residue that whether contains in food samples, can effectively detect methamidophos pesticide residual in food.
For solving the problems of the technologies described above, the invention provides a strain high and be applied to bifidobacterium breve (Bifidobacterium breve) the LJM-006 CGMCC No.5418 that acephatemet detects to acephatemet susceptibility.
Bifidobacterium breve of the present invention (Bifidobacterium breve) LJM-006, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 28th, 2011, it is referred to as CGMCC (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is bifidobacterium breve (Bifidobacterium breve), and deposit number is CGMCC No.5418.
Bifidobacterium breve LJM-006 of the present invention is located away from Zhejiang Province's Healthy Youth human faecal mass.
Bifidobacterium breve LJM-006 bacterial strain of the present invention has following microbial characteristic:
(1) colonial morphology: LJM-006 bacterial strain bacterium colony on flat board is canescence or oyster white, and opaque, glossy, smooth surface, projection, quality are soft, neat in edge, diameter 1-1.5mm;
(2) individual morphology: be irregular G+ sporeless bacterium, straight-bar, knee, match head, dumbbell shape and wild goose shape are arranged, wherein take the wild goose shape as main;
(3) physiological and biochemical property: D-ribose (+); L-arabinose (-); Lactose (+); Cellobiose (-); Melizitose (+); Raffinose (+); Sorbyl alcohol (-); Starch (-); Sunmorl N 60S (-); Wood sugar (-); Seminose (+); Fructose (+); Semi-lactosi (+); Sucrose (+); Maltose (+); Trehalose (-); Melibiose (+); N.F,USP MANNITOL (+); Synanthrin (-); Salicin (-); F6PPK enzyme (+); Reaction (not growing on aerobic solid medium) to oxygen; Nitrate reduction (-); Catalase (-); Indole reaction (-);
(4) well-grown under anaerobism, do not grow in aerobic environment.Optimum growth temperature 37-41 ℃; Minimum growth temperature 25-28 ℃; The highest 43-45 ℃; Growth optimal pH 6.5-7.0; Do not grow at pH4.5-5.0 or 8.0-8.5.
The LJM-006 bacterial strain with belong to reference culture of the same race together and compare acephatemet susceptibility and improved more than 100 times.
The present invention also provides the detection method of methamidophos residue in a kind of food, said method comprising the steps of:
(1) bacterial strain activation: above-mentioned bifidobacterium breve LJM-006 inoculation on nutrient agar slant medium, is regulated the pH value to 6.8 of this substratum ~ 7.2, be optimized cultivation under 35 ~ 37 ℃, and carry out the transferred species succeeding transfer culture;
(2) collecting cells: get step (1) gained bifidobacterium breve LJM-006 and be inoculated in nutrient agar slant medium, 35 ± 2 ℃ of anaerobism were cultivated 24 hours, with sterile saline, lawn were washed, and made suspension, adjusted its OD 600Value to 0.25 ~ 0.35 is put in 2-8 ℃ of refrigerator and is preserved;
(3) detector tube preparation: by weight, get peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, sodium acetate 5g, K 2HPO 42g, MgSO 47 H 2O 0.5g, MnSO 44H 2O 0.2g, oligofructose 3g, dibasic ammonium citrate 2g, tween-80 1mL, distilled water 1L, heating for dissolving is proofreaied and correct pH value to 6.5,115 ℃ autoclaving 15-20 minute, cooling temperature is 50 ~ 65 ℃, and get rapidly 10 mL aforesaid liquids, inject 10 mL in vitro, as detector tube, uprightly standing under 0 ~ 4 ℃, preservation, standby under 0 ~ 4 ℃ after sealing;
(4) drawing standard curve and set up regression equation: selection concentration is the acephatemet standard solution of 0mg/L, 0.25mg/L, 0.5 mg/L, 1.0 mg/L, 1.5 mg/L, 2.0 mg/L, 4.0 mg/L, the wherein negative contrast of 0mg/L.Get above standard solution 20 ~ 100 μ L and add in described detection tubule, 37 ℃ of anaerobism are cultivated 8 ~ 12h, the OD of working sample reaction tubes and negative control reaction tubes 500Value; The OD of working sample reaction tubes and negative control reaction tubes 500The concrete steps of value are: 1. collect thalline: get step (2) gained bacteria suspension 30 mL in each centrifuge tube, 4 ℃, centrifugal 10 min of 8,000 r/min abandon supernatant liquor, collect thalline, use the PBS damping fluid of pH 6. 5 to wash thalline 2 times; 2. bacterial cell disruption: add in centrifuge tube the PBS of 0.7 mL, pH 6.5 and 0.3 mL, 0.45% (mass volume ratio, CTAB W/V), mixing, effect 10 min place standby on ice after finishing; 3. get 2. liquid after bacterial cell disruption of above-mentioned steps, namely crude enzyme liquid 0.25 mL adds NaF (3 g/L), sodium iodoacetate (5 g/L) and the fructose-6-phosphate disodium (80 g/L) of each 0.25 mL, and mixing is placed in and hatches 30 min under 37 ℃; 4. after finishing, add hydrochloric acid-azanol (130 g/L) of 1. 5 mL, mixing, standing 10 min of room temperature; 5. TCA (15%, mass volume ratio), the HCl (4 mol/L) and the FeCl that then add each 1 mL 3(5%, mass volume ratio is with 0. 1 mol/L HCl preparations), mixing, after 4 ℃, centrifugal 15 min of 10,000 r/min, with 722 spectrophotometric determination OD values, detecting light wavelength is 500 nm, and the negative control reaction tubes replaces with distilled water, and all the other operations are the same.B/B 0(the B value is the OD of example reaction pipe 500Value, B 0The OD that is worth negative control reaction pipe 500Value) be ordinate zou Y, the logarithmic value of acephatemet concentration is that X-coordinate X(unit is mg/L), the drawing standard curve is set up methylamine phosphate content and B/ B 0Equation of linear regression: Y=-24.68x+66.72, the linearity range of acephatemet are 0.01 ~ 100mg/L, detect lower limit and reach 0.1mg/L, R 2Be 0.9902;
(5) sample pre-treatments: get food samples homogenate to be checked and organize 5g, with aseptic 1% phosphate buffered saline buffer (acidity is pH6.0 ~ 7.2) 20 mL dilutions, whirlpool mixing, in 80 ℃ of heating in water bath 5 min, in centrifugal 15 min of 5,000 r/min, get supernatant liquor as test sample liquid after cooling;
(6) sample detection: get step (5) gained test sample liquid 20 ~ 100 μ L and be added in described detection tubule, cultivated 8 ~ 10 hours in 35 ± 2 ℃ of anaerobism after airtight sealing, and the OD of working sample reaction tubes and negative control reaction tubes 500Value is according to the B/B of each sample of gained 0Value is obtained its corresponding acephatemet mass concentration on typical curve, or the logarithmic value X of substitution step (4) gained regression equation calculation sample quality concentration, asks its antilogarithm, is the mass concentration of contained acephatemet in sample.
Fructose-6-phosphate ketone solution enzyme (F6PPK) is the characteristic enzyme in the bifid pathways metabolism of genus bifidobacterium, glucose is fermented by special " fructose-6-phosphate branch road " (being called again the bifid branch road), this utilizes zymohexase and glucose-6-phosphate dehydrogenase (G6PD) obviously different from other bacterium.This enzyme catalysis fructose 6-phosphate decomposition is acetyl phosphate and erythrose-4-phosphate.Glucose carries out the bifid pathways metabolism without glycolytic pathway through the F6PPK enzyme and finally generates lactic acid and acetic acid, i.e. bifid branch road, fructose-6-phosphate branch road, and this enzyme has been widely used in evaluation and the live bacterial count of bifidus bacillus.
Work bacterial strain of the present invention is bifidobacterium breve LJM-006, on October 28th, 2011 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCC No.5418.
Food samples of the present invention comprises: the vegetable and fruits such as Chinese cabbage, eggplant, cucumber, pumpkin, wax gourd, apple, pear, peach.
The typical curve regression equation that the present invention uses is to obtain by following method: utilize the standard reserving solution configuration concentration of acephatemet to be respectively the standardized solution of 0.25mg/L, 0.5mg/L, 1.0mg/L, 1.5mg/L, 2.0mg/L, 4.0 mg/L, the wherein negative contrast of 0mg/L.Get above standard solution 20 μ L and add in the detection tubule, 37 ℃ of anaerobism are cultivated 8 ~ 12h, the OD of working sample reaction tubes and negative control reaction tubes 500Value (the F6PPK enzyme is lived) is with F6PPK enzyme inhibiting rate B/B alive 0(the B value is the OD of example reaction pipe 500Value, B 0The OD that is worth negative control reaction pipe 500Value) be ordinate zou Y, the logarithmic value of acephatemet concentration is X-coordinate X, and the drawing standard curve has obtained typical curve regression equation Y=-24.68x+66.72(Y:B/ B 0Ratio, B value are the OD of example reaction pipe 500Value, B 0The OD that is worth negative control reaction pipe 500Value, X: the logarithm concentration of contained acephatemet in food, its unit is mg/L), linearity range is 0.01 ~ 100mg/L, R 2Be 0.9902.
In the inventive method, repeat to measure for four times oneself vegetables or fruit sample of knowing of methylamine phosphate content, calculating its relative standard deviation is 7.80, shows that the detection tolerance range of the inventive method is higher.
Investigate the rate of recovery of the inventive method, add in the formaldehyde standardized solution vegetables that oneself knows in methylamine phosphate content or fruit sample of 40 μ L, 100mg/L, replicate analysis four times, the rate of recovery that calculates is 85. 94.
By measuring the volumetric soiutions of acephatemet minimum concentration, the lowest detection that obtains the inventive method is limited to 0.1mg/L.
The present invention has the following advantages and effect:
(1) bifidobacterium breve LJM-006 of the present invention has following characteristics: 1. high to acephatemet susceptibility, specificity good; 2. strain stability is good; 3. obligatory anaerobic bacteria, be difficult for being bacterial contamination impact in fermenting process;
(2) to be based on methamidophos pesticide inhibited to bifidus bacillus for the principle of the inventive method, and utilize characteristic, bifidus bacillus carries out fermentative processing to sample, follows the tracks of fermenting process, comes to have or not in judgement sample the residual of methamidophos pesticide according to its fermentation character; This detects determination methods economy, easy, quick, accurate, is fit to Safety of Food Quality and controls, and practicality is very strong;
(3) sample pre-treatments of the present invention is simple, a large amount of food samples of screening simultaneously;
(4) detection method of the present invention is simple and easy to do, have highly sensitive, the tolerance range high, the rate of recovery is all greater than 70%, the variation coefficient meets accuracy and the precision requirement of food methamidophos residue screening in 10%, be fit to the detection of methamidophos residue in food fully.
Description of drawings
Fig. 1 is the typical curve that acephatemet detects.
Embodiment
Be noted that following illustrating is all exemplary, be intended to the invention provides further invention.Except as otherwise noted, all Science and Technology terms used herein have the identical meanings of usually understanding with the technical field of the invention personnel.
The invention will be further described below in conjunction with specific embodiment, but illustrated embodiment is not as a limitation of the invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
Embodiment 1: the separation of bifidobacterium breve LJM-006 bacterial strain and evaluation
with the ight soil of Zhejiang Province's healthy young people as sample separation, on the MRS nutrient agar, under 37 ℃ of anaerobic conditions, 48h is coated with separation and Culture, obtain bifidobacterium breve LJM-006 of the present invention, be accredited as bifidobacterium breve, this bacterial strain on October 28th, 2011 in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms's common micro-organisms center, postcode 100101) preservation, Classification And Nomenclature is bifidobacterium breve (Bifidobacterium breve), preserving number is CGMCC No.5418.
The formula of MRS nutrient agar: peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, sodium acetate 5g, K 2HPO 42g, MgSO 47 H 2O 0.5g, MnSO 44H 2O 0.2g, oligofructose 3g, dibasic ammonium citrate 2g, tween-80 1mL, agar 15g, distilled water 1L transfers pH to 7.0, sterilizes 15 minutes for 115 ℃.
LJM-006 bacterial strain of the present invention has following microbial characteristic:
(1) colonial morphology: LJM-006 bacterial strain bacterium colony on flat board is canescence or oyster white, and opaque, glossy, smooth surface, projection, quality are soft, neat in edge, diameter 1-1.5mm;
(2) individual morphology: be irregular G+ sporeless bacterium, straight-bar, knee, match head, dumbbell shape and wild goose shape are arranged, wherein take the wild goose shape as main;
(3) physiological and biochemical property: D-ribose (+); L-arabinose (+); Lactose (+); Cellobiose (-); Melizitose (+); Raffinose (+); Sorbyl alcohol (-); Starch (-); Sunmorl N 60S (-); Wood sugar (+); Seminose (-); Fructose (+); Semi-lactosi (+); Sucrose (+); Maltose (+); Trehalose (-); Melibiose (+); N.F,USP MANNITOL (+); Synanthrin (-); Salicin (-); F6PPK enzyme (+); Reaction (not growing on aerobic solid medium) to oxygen; Nitrate reduction (-); Catalase (-); Indole reaction (-);
(4) well-grown under anaerobism, do not grow in aerobic environment.Optimum growth temperature 37-41 ℃; Minimum growth temperature 25-28 ℃; The highest 43-45 ℃; Growth optimal pH 6.5-7.0; Do not grow at pH4.5-5.0 or 8.0-8.5.
Test 1: sensitivity test
Adopt scraps of paper agar diffusion (K-B) method, choose the acephatemet reference liquid of different concns, with the modified MRS nutrient agar, culture medium prescription is seen embodiment 1, take bifidobacterium breve LJM-006 of the present invention as the test bacterium, be placed in 37 ℃ of incubator anaerobism and cultivate the growing state of observing the LJM-006 bacterial strain after 24h, do the acephatemet sensitivity test with agar diffusion method of the paper, measure acephatemet to the bacteriostasis property of LJM-006 bacterial strain.
3 repetitions are done in above-mentioned test, to belong to bifidobacterium breve reference culture of the same race together as contrast.
Table 1: sensitivity test
Bacterial strain The susceptibility of acephatemet The bacteriostatic diameter of 1 mg/L acephatemet (mm)
LJM-006 bacterial strain of the present invention ≤0.1 mg/L 19
The bifidobacterium breve reference culture >10 mg/L 13
Test-results: bifidobacterium breve LJM-006 of the present invention has the susceptibility of height to acephatemet, its with belong to reference culture together and compare, susceptibility to acephatemet has improved more than 100 times, acephatemet susceptibility≤0.1mg/L of bifidobacterium breve LJM-006, lower than the residual quantity of national standard, can be used as the work bacterial strain of screening methamidophos residue fully.
Embodiment 2: the detection of green vegetables sample methamidophos residue
(1) bacterial strain activation: above-mentioned bifidobacterium breve LJM-006 inoculation on nutrient agar slant medium, is regulated the pH value 6.8 ~ 7.2 of substratum, be optimized cultivation under 35 ~ 37 ℃, and carry out the transferred species succeeding transfer culture;
(2) collecting cells: get step (1) gained bifidobacterium breve LJM-006 and be inoculated in nutrient agar slant medium, 35 ± 2 ℃ of anaerobism were cultivated 24 hours, with sterile saline, lawn were washed, and made suspension, adjusted its OD 600Value to 0.25 ~ 0.35 is put in 2-8 ℃ of refrigerator and is preserved;
(3) detector tube preparation: by weight, get peptone 10g, extractum carnis 10g, yeast extract paste 5g, glucose 20g, sodium acetate 5g, K 2HPO 42g, MgSO 47 H 2O 0.5g, MnSO 44H 2O 0.2g, oligofructose 3g, dibasic ammonium citrate 2g, tween-80 1mL, distilled water 1L, heating for dissolving is proofreaied and correct pH6.5,115 ℃ autoclaving 15-20 minute, cooling temperature is 50 ~ 65 ℃, get 10 mL aforesaid liquids, inject 10 mL in vitro, as detector tube, uprightly standing under 0 ~ 4 ℃, preservation, standby under 0 ~ 4 ℃ after sealing;
(4) drawing standard curve and set up regression equation: selection concentration is the acephatemet standard solution of 0mg/L, 0.25mg/L, 0.5 mg/L, 1.0 mg/L, 1.5 mg/L, 2.0 mg/L, 4.0 mg/L, the wherein negative contrast of 0mg/L.Get above standard solution 20 μ L and add in the detection tubule, 37 ℃ of anaerobism are cultivated 8-12h, the OD of working sample reaction tubes and negative control reaction tubes 500Value; The OD of working sample reaction tubes and negative control reaction tubes 500The concrete steps of value are: 1. collect thalline: get 30 mL in each centrifuge tube, 4 ℃, centrifugal 10 min of 8,000 r/min abandon supernatant liquor, collect thalline, use the PBS damping fluid of pH 6.5 to wash thalline 2 times; 2. bacterial cell disruption: add in centrifuge tube the PBS of 0.7 mL, pH 6.5 and 0.3 mL, 0.45% (mass volume ratio, CTAB W/V), mixing, effect 10 min place standby on ice after finishing; 3. get 2. liquid after bacterial cell disruption of above-mentioned steps, namely crude enzyme liquid 0.25 mL adds NaF (3 g/L), sodium iodoacetate (5 g/L) and the fructose-6-phosphate disodium (80 g/L) of each 0.25 mL, and mixing is placed in and hatches 30 min under 37 ℃; 4. after finishing, add hydrochloric acid-azanol (130 g/L) of 1. 5 mL, mixing, standing 10 min of room temperature; 5. TCA (15%, mass volume ratio), the HCl (4 mol/L) and the FeCl that then add each 1 mL 3(5%, mass volume ratio is with 0.1 mol/L HCl preparation), mixing, after 4 ℃, centrifugal 15 min of 10,000 r/min, with 722 spectrophotometric determination OD values, detecting light wavelength is 500 nm, and the negative control reaction tubes replaces with distilled water, and all the other operations are the same.B/B 0(the B value is the OD of example reaction pipe 500Value, B 0The OD that is worth negative control reaction pipe 500Value) be ordinate zou Y, the logarithmic value of acephatemet concentration is X-coordinate X, and the drawing standard curve is set up methylamine phosphate content and B/B 0Equation of linear regression: Y=-24.68x+66.72, the linearity range of acephatemet are 0.01 ~ 100 mg/L, detect lower limit and reach 0.1 mg/L, R 2Be 0.9902;
(5) sample pre-treatments: get green vegetables homogenate to be checked and organize 5g, cooling rear in centrifugal 15 min of 5,000 r/min in 80 ℃ of heating in water bath 5 min with aseptic 1% phosphate buffered saline buffer 20 mL dilutions, whirlpool mixing, get supernatant liquor as test sample liquid;
(6) sample detection: get step (5) gained test sample liquid 20 ~ 100 μ L and be added in described detection tubule, cultivated 8 ~ 10 hours in 35 ± 2 ℃ of anaerobism after airtight sealing, and the OD of working sample reaction tubes and negative control reaction tubes 500Value is according to the B/B of each sample 0Value is obtained its corresponding acephatemet mass concentration on typical curve, or the logarithmic value X of substitution regression equation calculation sample quality concentration, asks its antilogarithm, is the mass concentration of contained acephatemet in sample; Wherein, X is also the logarithmic value of contained acephatemet mass concentration in food, and its unit is mg/L, inhibiting rate when being 50% corresponding acephatemet mass concentration be 5.2mg/L.The linearity range of acephatemet is 0.01 ~ 100 mg/L, detects lower limit and reaches 0.1 mg/L, R 2Be 0.9902.
Test 1: false negative rate test
Take blank each 5g of food samples of agricultural chemicals of homogeneous, add the acephatemet reference liquid, make acephatemet concentration 1mg/L in food, measuring method is with embodiment 2, and replication 100 times is observed the probability that negative findings occurs, and calculates false negative rate.
Test-results: when in cucumber, methamidophos residue was 1mg/L, false negative rate was 0; When in wax gourd, methamidophos residue was 1mg/L, false negative rate was 2%.
Test 2: stability test
Prepare the detection tubule by the method in embodiment 2 and preserve successively 1 week, 2 weeks, 3 weeks, 4 weeks under 4 ℃, detect respectively and record its negative detection time, 10 hours, 10 hours, 10 hours, 10.5 hours, can change to some extent negative detection time along with the prolongation of time, change less negative detection time, and good stability is described.For the accuracy of warranty test, each test should be done a negative control, and it detects exactly the judgement time and is as the criterion with negative control.The inventive method detects that in food, the stability of methamidophos pesticide residue all is stabilized in 10 hours in front 3 weeks, to the 4th week be 10.5 hours, so the detection stability of this method is one month.
Test 3: the determining of detectability
Respectively with containing the PBS solution of different concns (0.1 mg/L, 0.25 mg/L, 0.5 mg/L, 1 mg/L, 1.5 mg/L, 2 mg/L, 4 mg/L) acephatemet standard substance as analyte sample fluid, drip in testing tube, carry out sample detection, the analyzing and testing result determines that the detection of detection method of the present invention is limited to 0.1mg/L.
Test 4: linear dependence Journal of Sex Research
Selecting concentration is the acephatemet standard solution of 0 mg/L, 0.25 mg/L, 0.5 mg/L, 1.0 mg/L, 1.5 mg/L, 2.0 mg/L, 4.0 mg/L, by above step, after processing again by step, the production standard curve, getting regression equation Y=-24.68x+66.72(Y is B/ B 0Ratio, B value are the OD of example reaction pipe 500Value, B 0The OD that is worth negative control reaction pipe 500Value, X is the logarithm concentration of contained acephatemet in food, its unit is mg/L), its linearly dependent coefficient r=0.9902 is satisfied the requirement to precision fully.The specific standards curve as shown in Figure 1.Through research, the acephatemet sample keeps higher dependent linearity in the concentration range of 0.01 ~ 100mg/L.
Test 5: determination of recovery rates
With the acephatemet standard substance with final concentration be respectively 0.5 mg/L, 1.5 mg/L, 2 mg/L add green vegetables sample and Plantula Brassicae chinensis sample to, by above its content of embodiment 2 step measurements, measurement result such as table 1, the rate of recovery of green vegetables sample is 78.5% ~ 91.2%, average 82.5%, the variation coefficient is 3.6% ~ 9.3%, and average 7.7%; The rate of recovery of Plantula Brassicae chinensis sample is 81.5% ~ 105.7%, and is average 89.5%, and the variation coefficient is 4.2% ~ 9.4%, and average 8.1%; Average coefficient of variation shows that all less than 15% detection method of the present invention has higher accuracy.Its average recovery rate is 99.11%, the standard deviation (RSD) of the corresponding rate of recovery〉be 0.23, it meets the precision requirement of quantitative analysis method fully.
Table 1 acephatemet adds recovery test
Figure 576300DEST_PATH_IMAGE001
Test 6: precision is measured
With the acephatemet standard substance with final concentration be respectively 0.5 mg/L, 1.5 mg/L, 2 mg/L add green vegetables sample and Plantula Brassicae chinensis sample to, by above its content of embodiment 2 step measurements, the results are shown in Table 2, the average variation within batch coefficient of green vegetables sample, Plantula Brassicae chinensis sample is respectively 9.08%, 9.03%, average interassay coefficient of variation is respectively 6.54%, 5.71%, average interassay coefficient of variation is all less than average variation within batch coefficient, and be no more than 15%, show that detection method of the present invention has higher precision.
The precision of table 2 acephatemet is measured
Figure 522959DEST_PATH_IMAGE002
Test 7: specific assay
When the inhibiting rate of acephatemet inhibition bifidus bacillus LJM-006 is 50%, corresponding concentration is 5.2mg/L(IC 50=5.2 mg/L), but acephatemet bifidus bacillus LJM-006 specificity is suppressed, all less than 0.5%, the results are shown in Table 3 with the right bifidus bacillus LJM-006 cross reacting rate % of other organophosphorus insecticides.
The cross reaction of table 3 acephatemet and other organophosphorus insecticides
Compound IC 50（mg/L） Cross reacting rate (%)
Acephatemet 1.5 100
Rogor 132 <0.5
Omethoate >260 <0.5
Acephate >260 <0.5
SD-1750 >260 <0.5
Thiophos >260 <0.5
Phorate >260 <0.5
Embodiment 3: the detection of cucumber sample methamidophos residue
The food samples that detects is cucumber, gets cucumber homogenate to be checked and organizes 5g, the acephatemet standard substance is added in 10 cucumber samples take final concentration as 100 mg/L, by above its content of embodiment 2 step measurements, according to the B/B of each sample 0Value is obtained its corresponding acephatemet mass concentration on typical curve, or the logarithmic value X of substitution regression equation calculation sample quality concentration, ask its antilogarithm, be the mass concentration of contained acephatemet in sample, the assay value that obtains acephatemet is 117.26 ± 0.79mg/L, and recovery of standard addition is 82.3% ~ 115.0%.
Embodiment 4: the detection of eggplant sample methamidophos residue
The food samples that detects is eggplant, gets eggplant homogenate to be checked and organizes 5g, the acephatemet standard substance is added in 10 eggplant samples take final concentration as 0.1 mg/L, by above its content of embodiment 2 step measurements, according to the B/B of each sample 0Value is obtained its corresponding acephatemet mass concentration on typical curve, or the logarithmic value X of substitution regression equation calculation sample quality concentration, ask its antilogarithm, be the mass concentration of contained acephatemet in sample, the assay value that obtains acephatemet is 0.11 ± 0.29mg/L, and recovery of standard addition is 89.3% ~ 105.7%.
Embodiment 5: the detection of apple sample methamidophos residue
The food samples that detects is apple, gets apple homogenate to be checked and organizes 5g, the acephatemet standard substance is added in 10 apple samples take final concentration as 50mg/L, by above its content of embodiment 2 step measurements, according to the B/B of each sample 0Value is obtained its corresponding acephatemet mass concentration on typical curve, or the logarithmic value X of substitution regression equation calculation sample quality concentration, ask its antilogarithm, be the mass concentration of contained acephatemet in sample, the assay value that obtains acephatemet is 52.11 ± 0.59mg/L, and recovery of standard addition is 83.3% ~ 108.3%.
Embodiment 6: the detection of pear sample methamidophos residue
The food samples that detects is pear, gets pear homogenate to be checked and organizes 5g, the acephatemet standard substance is added in 10 pear samples take final concentration as 10mg/L, by above its content of embodiment 2 step measurements, according to the B/B of each sample 0Value is obtained its corresponding acephatemet mass concentration on typical curve, or the logarithmic value X of substitution regression equation calculation sample quality concentration, ask its antilogarithm, be the mass concentration of contained acephatemet in sample, the assay value that obtains acephatemet is 11.16 ± 0.52mg/L, and recovery of standard addition is 89.3% ~ 104.2%.
Embodiment 7: the method significant difference is analyzed (adopting F value determining method)
Method by the embodiment of the present invention 2, to buying the acephatemet interpolation test of carrying out different concns in the green vegetables sample that does not contain acephatemet (confirming not contain acephatemet through high performance liquid chromatography) in market, take same batch of 4 parallel sample, measure by above step with the inventive method, adopt simultaneously vapor-phase chromatography that same batch of sample measured, comparing result also adopts the F method of inspection to carry out computational analysis to two kinds of method acquired results, to judge whether there is significant difference between two methods.Detected result sees Table 4.
Table 4 detection method of the present invention and high performance liquid chromatography detected result are relatively
Sample number Present method measurement result (%) Gas chromatography determination result (%)
1 98.8 99.2
2 98.3 98.6
3 99.1 98.7
4 98.5 99.1
On average 98.7 98.9
Standard deviation 0.35% 0.24%
The F value is calculated judgement (F calculations=S2 greatly/S2 is little):
F calculates=2.16, look into the F table and obtain F table=9.28 (degree of confidence 95%), as seen F calculation＜F table, show no difference of science of statistics between these two kinds of detection methods of vapor-phase chromatography and microbial method of the present invention, can replace mutually between two kinds of methods and can not produce significant difference.
Microbial method of the present invention can be measured at visible region, and present method accuracy and precision are all fine, meets the Detecting Pesticide requirement.Operating process of the present invention is simple and convenient, and detected result is accurate, good reproducibility, and economical and practical, method can directly be applied to methylamine phosphate content analysis and the quality control of food accurately and rapidly, so the present invention has actual popularization economic worth.
The above is only the preferred embodiments of the present invention, should be understood that; for the those of ordinary skill in present technique; under the prerequisite that does not break away from core technology feature of the present invention, can also make some improvements and modifications, these retouchings and improvement also should belong to scope of patent protection of the present invention.

Claims (1)

1. a strain bifidobacterium breve, it is characterized in that, described bacterial strain is bifidobacterium breve (Bifidobacterium breve) LJM-006, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 28th, 2011, and deposit number is CGMCC No.5418.
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