CN109971669A - The detection method and application of bifidobacterium pseudocatenulatum and Residue of Antibiotics in Milk - Google Patents

The detection method and application of bifidobacterium pseudocatenulatum and Residue of Antibiotics in Milk Download PDF

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CN109971669A
CN109971669A CN201910104369.XA CN201910104369A CN109971669A CN 109971669 A CN109971669 A CN 109971669A CN 201910104369 A CN201910104369 A CN 201910104369A CN 109971669 A CN109971669 A CN 109971669A
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milk
standard
added
sample
antibiotic
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杜季梅
刘佳明
楼永良
叶盛
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Wenzhou Medical University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • 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
    • C12Q1/06—Quantitative determination
    • 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
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • 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 discloses a kind of bifidobacterium pseudocatenulatum DSQL-1, are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCCNo.12067 on 2 18th, 2016.And its application in screening Residues in Milk tetracycline, penicillin and erythromycin is disclosed, and disclose specific detection method.The screening methods of the method for the present invention are easy to operate, can be not required to special precision instrument, judgment method is easy, accurate, and sensitivity and accuracy are high, is suitble to Safety of Food Quality inspection, practical directly by observation indicator color discoloration interpretation result.

Description

The detection method and application of bifidobacterium pseudocatenulatum and Residue of Antibiotics in Milk
Technical field
The invention belongs to more particularly relate to a kind of bifidobacterium pseudocatenulatum in life science and field of food safety DSQL-1 and the method for quickly detecting Residues in Milk antibiotic using the bacterial strain.
Background technique
Residue of Antibiotics in Milk is a major issue of milk safety.Residue of Antibiotics in Milk not only can be to human body Various adverse effects are generated, such as cause human body to generate drug resistance, and intestinal bacilli illness, allergic reaction, teratogenesis, mutagenesis occur Etc. many reactions.In addition, may also suppress lactobacter growth, fermentation is caused to fail, brings economic loss to enterprise, and influence ox The outlet and trade of milk and Related product.
Antibiotic is widely used in treating and preventing cow disease, unavoidably causes Residue of Antibiotics in Milk.This Outside, application antibiotic treatment cow disease while obtaining effect, it is lack of standardization use antibiotic, such as increasing dosage, The extended treatment phase, or even to the behavior of milk cow in lactation period prolonged application antibiotic prophylaxis disease or even the illegal producer fresh Antibiotic anti-corrosion etc. is added in milk, so that Residue of Antibiotics in Milk is exceeded.
Antibiotics leftover detection is the important measures of milk foods security control and management.China's food hygienic standard at present Have specified in (GB/T 4789.27-2008) using the detection method of micro-biological process screening Residue of Antibiotics in Milk TTC method and bacillus stearothermophilus inhibit method.Wherein TTC method equipment is simple, expense is low, is suitble to screening and the base of large sample Laboratory operation and be widely used, which predominantly detects benzyl penicillin and streptomysin, the gentamicin and card of beta-lactam class That mycin.In addition, the testing principle and result of National Standard Method judge whether pressed down by residual antibiotic according to the growth of streptococcus thermophilus System and obtain, due to be added sample after 37 DEG C cultivate 2 hours, under this condition major part environment in bacterium (such as escherichia coli, The bacterium of the catalase-positives such as staphylococcus epidermis) fast-growth and the color of TTC can be caused to change, in operating process such as Appearance is polluted, and will lead to and false negative result occurs.
In addition, the national standard method that China uses at present does not include the detection to tetracycline, erythromycin, vancomycin etc..Four The speed of growth that ring element class antibiotic can prevent and treat infectious disease, improve feed efficiency, accelerate animal, in animal husbandry To extensive use.In the breeding process of milk cow, tetracycline is commonly used for feed addictive and prevention and treatment milk bovine mastitis, to lead Cause there may be tetracycline residue in milk.Erythromycin belongs to macrolide antibiotics, is also widely used for poultry, fowl, aquatic products and supports In growing, field of food safety mainly uses instrumental method to carry out the residual quantity of erythromycin in analyzing animal derived food.
The important method that detection is public health and food safety management is carried out to Residue of Antibiotics in Milk.In face of milk The substantial increase (Chinese milk crop in 2017 occupies third place in the world, 35.7billion kilograms) of yield, people's lives Horizontal raising and the demand to the increase of milk and Related product consumption figure and dairy management and milk related industry, A variety of remaining methods of common antibiotics in simple, quick, the effective detection milk of one kind are established to be of great significance.In milk most Main antibiotic risk is the once most common beta-lactam antibiotic mesh as caused by intramammary treatment and conventional therapy It is preceding to be still widely used, but with reasons such as the increases of antibody-resistant bacterium, " non-beta-lactam " drug is used in mastitis treatment Ratio it is higher and higher, it means that the risk of non-belt-lactam antibiotics residues is also with increasing, therefore, only detect β- The strategy of lactams is abnormally dangerous, such as common tetracycline medication will be unable to detect correct result.
Antimicrobial method have it is easy to operate, instrument and equipment and personnel requirement be not high, and sample pre-treatments are simple, as a result easily In observation and interpretation, the features such as detection time is short, it is used for the screening of batch samples always.The present invention mainly utilizes tetracycline The anaerobic type probiotics strain of the common antibiotics such as class, macrolides, beta-lactam sensitivity is established a kind of based on microorganism Inhibition method can in Rapid Screening and quantitative detection milk the antibiotic such as tetracycline, erythromycin and penicillin detection method.
Summary of the invention
1, goal of the invention.
The present invention filters out one plant of bifidobacterium pseudocatenulatum bacterium to antibiotic sensitives such as tetracycline, erythromycin, penicillin Strain, and thus establish one kind and simply, quickly, sensitively carry out Residue of Antibiotics in Milk detection by work bacterial strain of the bacterial strain Screening and quantitative detecting method.
2, the technical solution adopted in the present invention.
Bifidobacterium pseudocatenulatum DSQL-1 of the invention was deposited in Chinese microorganism strain preservation on 2 18th, 2016 Administration committee's common micro-organisms center is referred to as the (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, China CGMCC Institute of microbiology, the academy of sciences, postcode 100101) preservation, classification naming is bifidobacterium pseudocatenulatum (Bifidobacterium Pseudocatenulatum), deposit number is CGMCC No.12067.
Bifidobacterium pseudocatenulatum DSQL-1 bacterial strain of the invention has following microbial characteristic:
(1) colonial morphology: colony diameter 1.2- of the bifidobacterium pseudocatenulatum DSQL-1 bacterial strain on TPY solid plate It is 1.5mm, milky, circle, protrusion, neat in edge, opaque, there is gloss, it is soft, fine and smooth；
(2) thalli morphology: Grain stain positive sporeless bacterium, thallus is in the shape of a rod, has bending or bifurcation, it is seen that short Catenation has pleomorphism.
(3) physiological and biochemical property: catalase is negative, decomposition glucose, sucrose, maltose, fructose and galactolipin, regardless of fermentation L-arabinose, xylose, decomposing urea, nitrate reduction do not test negative, indole negative.
(4) cultural characteristic: anaerobic growth is good, aerobic not grow.35 DEG C -37 DEG C of optimum growth temperature, minimum growth temperature 20 DEG C, 45 DEG C of maximum growth temperature of degree；Optimal pH 6.5-7.0, pH are bad lower than 5.5 or higher than 9.0 growths.
Bifidobacterium pseudocatenulatum DSQL-1 of the invention can be applied to simple, quick, sensitive screening Residues in Milk four Ring element, erythromycin and penicillin.
Specific detection method are as follows:
(1) bacterial strain activates: by above-mentioned bifidobacterium pseudocatenulatum DSQL-1 strain inoculated in TPY solid medium, 37 DEG C Cultivate 24-48h；
(2) prepared by bacteria suspension: taking the resulting bifidobacterium pseudocatenulatum DSQL-1TPY bacterium colony of step (1), is matched with PBS adjustment Bacteria suspension processed and adjust to OD600 value be 0.10-0.12,4 DEG C preservation；
(3) detection is used preparation of culture medium: by weight, caseinhydrolysate 10g, pancreas soy peptone 5.0g, sodium chloride 5.0g, glucose 20.0g, yeast extract 2.0g, L-cysteine 0.5g, soluble starch 0.5g, oligofructose 2g, chlorination Magnesium 0.5g, calcium chloride 0.1g, zinc sulfate 0.2g, K2HPO4 2.0g, Tween-80 1.0ML, distilled water 1000mL, adjust pH to 6.5-6.8, heating are boiled 5 minutes, qualitative filter paper filtering, 115 DEG C high pressure sterilization 15-20 minutes after packaging, 4 DEG C of guarantors of cooling postposition It deposits, is spare；
(4) 0.2 ‰ methyl red solution preparations: 0.02g methyl red is weighed, is dissolved with 100mL dehydrated alcohol or 95% ethyl alcohol；
(5) prepared by nonreactive milk: weighing nonreactive milk powder, 10% solution is prepared with sterile distilled water；Or matched with distilled water After making 10% solution, after 121 DEG C of high pressure sterilizations, 4 DEG C are saved backup；
(6) quantitative criterion control is standby: taking tetracycline, erythromycin and penicillin standard reserving solution, prepares antibiosis with nonreactive cream Plain standard working solution；500 μ g/L of tetracycline, 450 μ g/L, 400 μ g/L, 350 μ g/L, 300 μ g/L, 250 μ g/L, 200 μ g/L, The standard working solution of the standard working solution of 150 μ g/L, 100 μ g/L, 50 μ g/L, 0 μ g/L；100 μ g/L of erythromycin, 80 μ g/L, 60 μ The standard working solution of g/L, 40 μ g/L, 20 μ g/L, 10 μ g/L, 0 μ g/L；100 μ g/L of penicillin, 50 μ g/L, 40 μ g/L, 20 μ g/ L,10μg/L,5μg/L,4μg/L,2μg/L,1μg/L,0μg/L；；4 DEG C of refrigerations are spare；
(7) Specification Curve of Increasing: it is different dense to be separately added into step (5) by the detection culture medium 19.5mL for taking step (3) to prepare The 400 μ L of antibiotic standard working solution of degree, using 0 μ g/L as growth control；The 100 μ L of bacterium solution of step (2) preparation is added after mixing, 37 DEG C after mixing, Anaerobic culturel 6 hours；Only to add culture medium, the not pipe of added with antibiotic (0 μ g/L) standard pipe plus 100 μ LPBS For zeroing pipe, the absorbance value of each detection pipe under 600nm wavelength is measured, is vertical so that antibiotic concentration value to be added in standard series pipe Coordinate, absorbance value are abscissa, draw standard curve；
(8) preparation of samples: taking milk sample 10mL in sterile test tube, 80 DEG C water-bath -15 minutes 10 minutes, 5000rpm is centrifuged 5 minutes, abandons precipitating；Fresh cow milk after other are processed etc. can direct sample；
(9) control of screening positive control is standby: taking tetracycline, erythromycin and penicillin standard reserving solution, is prepared with nonreactive cream 1MRL antibiotic standard pipe: 100 μ g/L of tetracycline；40 μ g/L of erythromycin；4 μ g/L of penicillin；4 DEG C of refrigerations are spare；
(10) sample screening detects: taking the resulting 200 μ L of sample of step (8), is added to the resulting detection culture of step (3) 1.3mL in base, is added the bacterium solution 100 μ L of step (2) preparation, and 37 DEG C, Anaerobic culturel 6 hours；It takes out and step (4) preparation is added 0.02% methyl red indicator, 90 μ L observes result；With added with final concentration of 100 μ g/L tetracycline standard items milk sample, 40 The milk sample of μ g/L erythromycin standard items, 4 μ g/L penicillin standard items milk sample be Rapid Screening positive control instruction Pipe, using 0 μ g/L as growth control；
(11) sample amounts detect: taking the resulting 400 μ L of sample of step (8), be added to the resulting detection culture of step (4) Base 19.5mL, is added the bacterium solution 100 μ L of step (2) preparation, and 37 DEG C, Anaerobic culturel 8-10 hours；With only plus culture medium, be not added it is anti- The pipe of raw element (0 μ g/L) standard pipe plus 100 μ LPBS are zeroing pipe, measure 600nm wavelength absorbance value, read from standard curve Take residual antibiotic value.
Milk sample of the present invention includes: lactogenesis, liquid milk, reconstituted milk.
Ampicillin standard curve of the present invention is obtained by following methods: utilizing ampicillin stock solution The standard working solution that compound concentration is respectively, using 0 μ g/L as negative control.Take the ampicillin standard working solution of each concentration 400 μ L are added in the quantitative detection culture medium of 19.5mL and mix, and add detection bacterium solution 100 μ L, 37 DEG C of Anaerobic culturel 6-8h, The absorbance value for measuring each Concentration Testing pipe under 600nm wavelength, using antibiotic concentration value as ordinate, absorbance value is horizontal seat Mark draws standard curve.Obtaining standard curve regression equation is that (Y: ampicillin is dense in sample by Y=-0.006X+0.5713 Spend μ g/L, X: the OD600 value of reaction tube), the range of linearity is 1-80 μ g/L, R2It is 0.9943.
Tetracycline standard curve of the present invention is obtained by following methods: utilizing tetracycline stock solution compound concentration The tetracycline standard work of respectively 50 μ g/L, 100 μ g/L, 150 μ g/L, 200 μ g/L, 250 μ g/L, 300 μ g/L, 350 μ g/L Liquid, using 0 μ g/L as negative control.Method is the same, and obtaining standard curve regression equation is Y=-1.1574X+0.5362 (Y: sample Middle tetracycline concentration μ g/L, X: the OD600 value of reaction tube), the range of linearity is 50-350 μ g/L, R2It is 0.9947.
Erythromycin standard curve of the present invention is obtained by following methods: utilizing erythromycin stock solution compound concentration The standard working solution of respectively 100 μ g/L, 80 μ g/L, 60 μ g/L, 40 μ g/L, 20 μ g/L, 10 μ g/L are negative right with 0 μ g/L According to.Method is the same, obtain standard curve regression equation be Y=0.00604X+0.5545 (Y: erythromycin concentration μ g/L in sample, X: the OD600 value of reaction tube), the range of linearity is 10-100 μ g/L, R2It is 0.99977.
The Monitoring lower-cut that the present invention uses is obtained by following methods: compound concentration is 0 μ g/L, 0.25 μ g/L, 0.5 μ g/ L, the ampicillin standard solution of 1 μ g/L, 2 μ g/L, 4 μ g/L, 5 μ g/L, 10 μ g/L；Compound concentration be 5 μ g/L, 10 μ g/L, The tetracycline titer of 25 μ g/L, 50 μ g/L, 100 μ g/L, compound concentration are 5 μ g/L, 10 μ g/L, 20 μ g/L, 40 μ g/L, 80 μ The erythromycin titer of g/L, using 0 μ g/L as negative control.The 400 μ L of ampicillin standard working solution of each concentration is taken to be added It is mixed into the quantitative detection culture medium of 19.5mL, adds detection bacterium solution 100 μ L, 37 DEG C of Anaerobic culturel 6-8h, measure 600nm The absorbance value of each Concentration Testing pipe under wavelength, to generate the Cmin of bacteriostasis as minimum detection limit.Quantitative detection Lowest detection is limited to 4 μ g/L of ampicillin, 80 μ g/L of tetracycline, 40 μ g/L of erythromycin.
The rate of recovery of the method for the present invention is added various concentration antibiotic standard solution into known milk sample, repeats Analysis 3 times, obtained average recovery rate are ampicillin 98.2%, erythromycin 72.49%, tetracycline 106.7%.
The method of the present invention detects the accuracy rate of known mark-on tetracycline sample from 100%, mark-on ampicillin erythromycin Positive sample (be greater than or equal to MRL) and the accuracy rate of negative sample (lower than MRL) are 90% (36/40), false negative rate 0, False positive rate 20%.
3, technical effect caused by the present invention.
(1) bifidobacterium pseudocatenulatum DSQL-1 bacterial strain of the present invention has the following characteristics that erythromycin, Piperacillin, Fourth Ring Element, benzyl penicillin, to sensitivities such as ampicillins；Obligate anaerobic is not easy to be bacterial contamination in incubation；Strain stability is good, easily In preservation and operation；Compared with bifidobacterium breve (Bifidobacterium breve) DSQH-1 for being all probiotics, to Fourth Ring The detection accuracy rate of element is higher, more preferable to the sensitivity of erythromycin, and bacterial strain not will cause laboratory to human body and environmental-friendly And environmental pollution.
(2) principle of the method for the present invention is that have to inhibit to make to bifidobacterium pseudocatenulatum DSQL-1 bacterial strain based on antibiotic With using this characteristic, by bifidobacterium pseudocatenulatum DSQL-1 bacterial strain to sample progress fermentation process, tracking fermentation process and basis Fermenting characteristic comes in judgement sample with the presence or absence of antibiotic residue.
(3) screening methods of the method for the present invention are easy to operate, can directly pass through the solidification of observation culture medium or indicator discoloration Interpretation result, judgment method is easy, accurate, is suitble to Safety of Food Quality inspection, practical.
(4) quantitative detecting method of the method for the present invention is simple and easy to do, is not required to special precision instrument, sensitivity and accuracy Height is all larger than 95% to the rate of recovery of ampicillin and tetracycline, and the coefficient of variation is respectively less than 10%, meets antibiotic in milk Remain the accuracy and precision requirement of screening.
Detailed description of the invention
Fig. 1 is Bifidobacterium DSQL-1 to penicillin mark-on sample detection result figure.
Fig. 2 is Bifidobacterium DSQL-1 to erythromycin mark-on sample detection result figure.
Fig. 3 is Bifidobacterium DSQL-1 to tetracycline mark-on sample detection result figure.
Fig. 4 is ampicillin standard curve.
Fig. 5 is tetracycline standard curve.
Fig. 6 is erythromycin standard curve.
Specific embodiment
It is noted that detailed description below is all exemplary property, it is intended to provide further instruction to the present invention.Unless It is otherwise noted, all scientific and technical terms used herein have normally understood with the technical field of the invention personnel Identical meanings.
The present invention will be further described combined with specific embodiments below, but illustrated embodiment is not as to limit of the invention It is fixed.If specializing, the conventional means that technological means used in embodiment is well known to those skilled in the art.
The separation and identification of 1 bifidobacterium pseudocatenulatum bacterial strain of embodiment
Using the excrement of Zhejiang Province health young man as separation sample, 1g excrement is suspended with 10mL PBS and does 10 times of multiple proportions After dilution, it is inoculated in TPY solid medium, 37 DEG C, Anaerobic culturel 48 hours obtain Bifidobacterium DSQL- of the present invention 1, it is identified as bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum), on 2 18th, 2016 It is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: BeiChen West Road, Chaoyang District, BeiJing City 1 Institute 3, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCC No.12067.
TPY agar medium, TPY fluid nutrient medium are Qingdao GaoKeYuan Hai Bo Bioisystech Co., Ltd product.
Bifidobacterium pseudocatenulatum DSQL-1 bacterial strain of the invention has following microbial characteristic:
(1) colonial morphology: colony diameter 1.2- of the bifidobacterium pseudocatenulatum DSQL-1 bacterial strain on TPY solid plate It is 1.5mm, milky, circle, protrusion, neat in edge, opaque, there is gloss, it is soft, fine and smooth；
(2) thalli morphology: Grain stain positive sporeless bacterium, thallus are in rod shape, and one or both ends are expanded, and have bending Or bifurcation, form distinctive " Y ", " V " shape structure.
(3) physiological and biochemical property: catalase is negative, decomposition glucose, sucrose, maltose, fructose and galactolipin, regardless of fermentation L-arabinose, xylose, decomposing urea, nitrate reduction do not test negative, indole negative.
(4) cultural characteristic: anaerobic growth is good, aerobic not grow.35 DEG C -37 DEG C of optimum growth temperature, minimum growth temperature 25 DEG C, 45 DEG C of maximum growth temperature of degree；Optimal pH 6.5-7.0, pH are bad lower than 6.5 or higher than 8.5 growths.
(5)) sensitivity tests of bifidobacterium pseudocatenulatum DSQL-1 strains of the invention: scraps of paper agar is used (K-B) method of diffusion, chooses the different antibiotic scraps of paper, is test bacterium with bifidobacterium pseudocatenulatum DSQL-1 bacterial strain of the present invention, uses TPY Agar Plating is set 37 DEG C, Anaerobic culturel 24 hours and is observed, measures different antibiotic to the antibacterial of DSQL-1 bacterial strain Performance.3 repetitions are done in above-mentioned test, referring to CLSI standard determination.
Sensitivity tests result of the table 1DSQL-1 bacterial strain to different antibiotic
Experimental result: bifidobacterium pseudocatenulatum DSQL-1 bacterial strain of the present invention is to ampicillin, tetracycline, erythromycin, through the ages Mycin, chloramphenicol, gentamicin are sensitive, can be used as the work bacterial strain of multiple antibiotic residues in high-throughput screening milk.
Embodiment 2: the detection of antibiotic residue in milk sample
(1) bacterial strain activates: bifidobacterium pseudocatenulatum DSQL-1 strain inoculated described in claim 1 is trained in TPY solid Support base, 37 DEG C of culture 24-48h；
(2) prepared by bacteria suspension: taking the resulting bifidobacterium pseudocatenulatum DSQL-1TPY bacterium colony of step (1), is matched with PBS adjustment Bacteria suspension processed and adjust to OD600nm value be 0.10-0.12,4 DEG C preservation；
(3) detection is used preparation of culture medium: by weight, caseinhydrolysate 10g, pancreas soy peptone 5.0g, sodium chloride 5.0g, glucose 20.0g, yeast extract 2.0g, L-cysteine 0.5g, soluble starch 0.5g, oligofructose 2g, chlorination Magnesium 0.5g, calcium chloride 0.1g, zinc sulfate 0.2g, K2HPO42.0g, Tween-80 1.0ML, distilled water 1000mL, adjust pH to 6.5-6.8, heating are boiled 5 minutes, qualitative filter paper filtering, 115 DEG C high pressure sterilization 15-20 minutes after packaging, 4 DEG C of guarantors of cooling postposition It deposits, is spare；
(4) 0.2 ‰ methyl red solution preparations: 0.02g methyl red is weighed, is dissolved with 100mL dehydrated alcohol or 95% ethyl alcohol；
(5) prepared by nonreactive milk: weighing nonreactive milk powder, 10% solution is prepared with sterile distilled water；Or matched with distilled water After making 10% solution, after 121 DEG C of high pressure sterilizations, 4 DEG C are saved backup；
(6) standard regulation is standby: taking tetracycline, penicillin and erythromycin standard reserving solution, is released with nonreactive cream and be configured to difference Concentration antibiotic standard working solution (350 μ g/L of tetracycline, 300 μ g/L, 250 μ g/L, 200 μ g/L, 150 μ g/L, 100 μ g/L, The standard working solution of 50 μ g/L, 0 μ g/L；100 μ g/L of erythromycin, 80 μ g/L, 60 μ g/L, 40 μ g/L, 20 μ g/L, 10 μ g/L, 0 μ The standard working solution of g/L；80 μ g/L of ampicillin, 50 μ g/L, 40 μ g/L, 20 μ g/L, 10 μ g/L, 5 μ g/L, 4 μ g/L, 2 μ g/ L, the standard working solution of 1 μ g/L, 0 μ g/L)；
(7) Specification Curve of Increasing: it is different dense to be separately added into step (5) by the detection culture medium 1.3mL for taking step (3) to prepare The 200 μ L of antibiotic standard working solution of degree, using 0 μ g/L as growth control；The 30 μ L of bacterium solution of step (2) preparation is added after mixing, 37 DEG C after mixing, Anaerobic culturel 6 hours；Using not added with antibiotic, the standard pipe that bacterium solution is not added as blank control pipe, 600nm is measured The absorbance value of each detection pipe under wavelength, antibiotic concentration value to be added in standard series pipe as ordinate, absorbance value is cross Coordinate draws standard curve；
(8) preparation of samples: taking sample 10mL in sterile test tube, 85 DEG C water-bath 10-15 minutes；5000rpm is centrifuged 5 points Clock；
(9) sample screening detects: taking the resulting 200 μ L of sample of step (8), is added to the resulting 1.3ml detection of step (3) In culture medium, the 30 μ L of bacterium solution that step (2) are prepared is added, 37 DEG C, Anaerobic culturel 6 hours take out and step (4) preparation are added 0.02% methyl red indicator, 90 μ L observes result；With the penicillin standard of 100 μ g/L tetracyclines, 40 μ g/L erythromycin, 4 μ g/L Product solution standard solution is Rapid Screening positive control pipe instruction pipe, using 0 μ g/L as growth control；
(10) sample amounts detect: taking the resulting 400 μ L of sample of step (8), be added to the resulting detection culture of step (4) Base 19.5mL, is added the bacterium solution 100 μ L of step (2) preparation, and 37 DEG C, Anaerobic culturel 8-10 hours；The blank tube of bacterium solution is not added Zeroing measures 600nm wavelength absorbance value, and residual antibiotic value is read from standard curve.
Test 1: the determination of limit is detected
Respectively with having added various concentration antibiotic standard items (0 μ g/L of tetracycline, 10 μ g/L, 20 μ g/L, 50 μ g/L, 60 μ g/ L, 70 μ g/L, 80 μ g/L, 90 μ g/L, 100 μ g/L, 200 μ g/L, 400 μ g/L, erythromycin 0,5 μ g/L, 10 μ g/L, 20 μ g/L, 30 μg/L,40μg/L,60μg/L,80μg/L,160μg/L,320μg/L；0 μ g/L of ampicillin, 0.5 μ g/L, 1 μ g/L, 2 μ g/ L,3μg/L,4μg/L,5μg/L,10μg/L,20μg/L,50μg/L,100μg/L；) milk as sample to be tested, carry out sample Detection.As shown in Figure 1-3, analysis detection is as a result, determine that the lowest detection of the quantitative detection of detection method is limited to ammonia benzyl 4 μ g/L of penicillin, 80 μ g/L of tetracycline, 40 μ g/L of erythromycin.
Test 2:
False negative rate test
Take standard nonreactive milk sample 10mL, antibiotic titer be added, make antibiotic concentration 1.5MRL in milk, 1MRL, 0.5MRL and 0MRL, 10 samples of each concentration, for measuring method with embodiment 2, replication 3 times, be the positive with 1MRL Control, 0MRL are negative control, to observe the probability of negative findings appearance, calculate false negative rate.
Ampicillin false negative rate is 0 in test result milk；Tetracycline false negative rate is 0；Erythromycin false negative rate It is 0.
Test 3: false positive rate test
Take standard nonreactive milk sample 10mL, antibiotic titer be added, make antibiotic concentration 1MRL in milk, 0.75MRL, 0.5MRL, 0.25MRL and 0MRL, 10 samples of each concentration, measuring method is with embodiment 2, and replication 3 times, It is negative control by positive control, 0MRL of 1MRL, the probability that observation positive findings occur calculates false negative rate.
Tetracycline is that false positive rate is 0；Erythromycin 0.75MRL false positive rate is 20% (6/30)；Ampicillin 0.75MRL false positive rate is 26.7% (8/30), and 0.5MRL false positive rate is 6.67% (2/30.
Test 4:
Determination of recovery rates
Antibiotic standard items are added in milk sample with various concentration, it is measured by above 2 step of case study on implementation and contains Amount, every a sample is 3 parts parallel, measurement result such as table 2-4, and the average recovery rate of each antibiotic is tetracycline 106.7%, erythromycin 72.49%, ampicillin 98.2%.
The measurement of the 2 tetracycline standard items rate of recovery of table
The measurement of the 3 erythromycin standard items rate of recovery of table
The measurement of the 4 ampicillin standard items rate of recovery of table
Test 5: milk sample antibiotic residue Screen test
By antibiotic (25 μ g/L of tetracycline, 50 μ g/L, 100 μ g/L, 200 μ g/L, 10 μ g/ of erythromycin containing various concentration L, 25 μ g/L, 50 μ g/L, 100 μ g/L, 40 μ g/L of ampicillin, 20 μ g/L, 10 μ g/L, 5 μ g/L, 4 μ g/L, 2 μ g/L, 1 μ g/ L,0.5μg/L,0μg/L；) standard solution milk as sample to be tested (using 0 μ g/L as negative control), be added in equal volume In the resulting screening detection pipe of step (4), addition step (2) resulting bacterium solution, 37 DEG C, Anaerobic culturel 6-8 hours；Instruction is added Agent occurs red being that antibiotic residue is lower than national standard, is that antibiotic residue is exceeded in yellow.Screening method starts yellow occur Minimum concentration be 60 μ g/L of tetracycline, 20 μ g/L of erythromycin, 3 μ g/L of ampicillin, determine yellow concentration be tetracycline 80 μ g/L, 40 μ g/L of erythromycin, 4 μ g/L of ampicillin.Highest in Screen test result and the animal food of China's approval Residue limits (milk: 4 μ g/L of ampicillin, 40 μ g/L of erythromycin, 100 μ g/L of tetracycline) meet or quite, can meet big Measure the screening of sample.
Test 5: antibiotic residue quantitative detection in milk sample
To contain various concentration standard antibiotic (350 μ g/L of tetracycline, 300 μ g/L, 250 μ g/L, 200 μ g/L, 150 μ g/L, The standard working solution of 100 μ g/L, 50 μ g/L；100 μ g/L of erythromycin, 80 μ g/L, 60 μ g/L, 40 μ g/L, 20 μ g/L, 10 μ g/L Standard working solution；80 μ g/L of ampicillin, 50 μ g/L, 40 μ g/L, 20 μ g/L, 10 μ g/L, 5 μ g/L, 4 μ g/L, 2 μ g/L, 1 μ The standard working solution of g/L；) milk as sample to be tested (using 0 μ g/L as negative control), be added to step (4) resulting inspection In test tube, be added step (2) resulting bacterium solution, 40 DEG C Anaerobic culturel 12-16 hours；It is returned to zero with culture medium plus antibiotic-free milk, measurement The OD600nm of sample cell negative control pipe reads antibiotic concentration value (μ g/L) from standard curve.Measure result such as Fig. 4-6 And shown in table 5-7.
The measurement of 5 ampicillin standard items repeatability of table
The measurement of 6 tetracycline standard items repeatability of table
The measurement of 7 erythromycin standard items repeatability of table

Claims (4)

1. a kind of bifidobacterium pseudocatenulatum DSQL-1 was preserved in Chinese microorganism strain preservation management committee on 2 18th, 2016 Member's meeting common micro-organisms center, deposit number CGMCCNo.12067.
2. bifidobacterium pseudocatenulatum DSQL-1 described in claim 1 is in screening Residues in Milk tetracycline, penicillin and red mould Application in element.
3. a kind of detection method of Residue of Antibiotics in Milk, which is characterized in that the described method comprises the following steps:
(1) bacterial strain activates: by bifidobacterium pseudocatenulatum DSQL-1 strain inoculated described in claim 1 in TPY solid medium, 37 DEG C of culture 24-48h；
(2) prepared by bacteria suspension: taking the resulting bifidobacterium pseudocatenulatum DSQL-1TPY bacterium colony of step (1), is adjusted with PBS and prepare bacterium Suspension and adjust to OD600 value be 0.10-0.12,4 DEG C preservation；
(3) detection is used preparation of culture medium: by weight, caseinhydrolysate 10g, pancreas soy peptone 5.0g, sodium chloride 5.0g, Glucose 20.0g, yeast extract 2.0g, L-cysteine 0.5g, soluble starch 0.5g, oligofructose 2g, magnesium chloride 0.5g, calcium chloride 0.1g, zinc sulfate 0.2g, K2HPO4 2.0g, Tween-80 1.0ML, distilled water 1000mL, adjust pH to 6.5-6.8, heating are boiled 5 minutes, qualitative filter paper filtering, 115 DEG C high pressure sterilization 15-20 minutes after packaging, 4 DEG C of guarantors of cooling postposition It deposits, is spare；
(4) 0.2 ‰ methyl red solution preparations: 0.02g methyl red is weighed, is dissolved with 100mL dehydrated alcohol or 95% ethyl alcohol；
(5) prepared by nonreactive milk: weighing nonreactive milk powder, 10% solution is prepared with sterile distilled water；Or it is prepared with distilled water After 10% solution, after 121 DEG C of high pressure sterilizations, 4 DEG C are saved backup；
(6) quantitative criterion control is standby: taking tetracycline, erythromycin and penicillin standard reserving solution, prepares antibiotic mark with nonreactive cream Quasi- working solution；500 μ g/L of tetracycline, 450 μ g/L, 400 μ g/L, 350 μ g/L, 300 μ g/L, 250 μ g/L, 200 μ g/L, 150 μ g/ L, the standard working solution of the standard working solution of 100 μ g/L, 50 μ g/L, 0 μ g/L；100 μ g/L of erythromycin, 80 μ g/L, 60 μ g/L, 40 The standard working solution of μ g/L, 20 μ g/L, 10 μ g/L, 0 μ g/L；100 μ g/L of penicillin, 50 μ g/L, 40 μ g/L, 20 μ g/L, 10 μ g/ L,5μg/L,4μg/L,2μg/L,1μg/L,0μg/L；；4 DEG C of refrigerations are spare；
(7) Specification Curve of Increasing: the detection culture medium 19.5mL for taking step (3) to prepare is separately added into step (5) various concentration 400 μ L of antibiotic standard working solution, using 0 μ g/L as growth control；The 100 μ L of bacterium solution of step (2) preparation is added after mixing, mixes 37 DEG C afterwards, Anaerobic culturel 6 hours；Only to add, culture medium, the pipe of added with antibiotic (0 μ g/L) standard pipe plus 100 μ LPBS are not to adjust Zero pipe, measures the absorbance value of each detection pipe under 600nm wavelength, is vertical sit so that antibiotic concentration value to be added in standard series pipe Mark, absorbance value is abscissa, draws standard curve；
(8) preparation of samples: taking milk sample 10mL in sterile test tube, 80 DEG C water-bath -15 minutes 10 minutes, 5000rpm Precipitating is abandoned in centrifugation 5 minutes；Fresh cow milk after other are processed etc. can direct sample；
(9) control of screening positive control is standby: taking tetracycline, erythromycin and penicillin standard reserving solution, prepares 1MRL with nonreactive cream Antibiotic standard pipe: 100 μ g/L of tetracycline；40 μ g/L of erythromycin；4 μ g/L of penicillin；4 DEG C of refrigerations are spare；
(10) sample screening detects: taking the resulting 200 μ L of sample of step (8), is added in the resulting detection culture medium of step (3) 1.3mL, is added the bacterium solution 100 μ L of step (2) preparation, and 37 DEG C, Anaerobic culturel 6 hours；It takes out and step (4) preparation is added 0.02% methyl red indicator, 90 μ L observes result；With added with final concentration of 100 μ g/L tetracycline standard items milk sample, 40 The milk sample of μ g/L erythromycin standard items, 4 μ g/L penicillin standard items milk sample be Rapid Screening positive control instruction Pipe, using 0 μ g/L as growth control；
(11) sample amounts detect: taking the resulting 400 μ L of sample of step (8), be added to the resulting detection culture medium of step (4) 19.5mL, is added the bacterium solution 100 μ L of step (2) preparation, and 37 DEG C, Anaerobic culturel 8-10 hours；Only to add culture medium, antibiosis be not added The pipe of 0 μ g/L standard pipe of element plus 100 μ LPBS are zeroing pipe, measure 600nm wavelength absorbance value, read from standard curve Residual antibiotic value.
4. the detection method of Residue of Antibiotics in Milk according to claim 3, it is characterised in that: the milk sample For lactogenesis, liquid milk or reconstituted milk.
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