CN102590196A - Test method for rapidly screening antibacterial residue in animal food samples - Google Patents
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Abstract
The invention discloses a test method for rapidly screening antibacterial residue in animal food samples, which comprises the steps of: strain activation, bacterial suspension preparation, test tube preparation, sample preprocessing and sample testing. The test method utilizes the principle that antibacterial can inhibit the growth or reproduction of bacteria sensitive to the antibacterial, a sample to be tested is sucked and added into a test tube, and is cultured for a certain period of time, and according to the change of the color of the culture medium in the test tube, whether the sample contains antibacterial residue or not is judged. Compared with the prior art, the test method keeps the reliability of the TTC (triphenyltetrazolium chloride) test method, and solves the problem that in the TTC test method, the interference of autochthonous microorganisms or miscellaneous bacteria in the food sample leads to false positive and false negative results; and the test method has the characteristics of simplicity, little interference, high sensitivity, high accuracy and the like, and is applicable to the high-throughput rapid screening of antibacterial residue in the animal food samples.
Description
Technical field
The present invention relates to antibacterial medicine residue check and analysis technical field, belong to specifically that a kind of growth that utilizes bacterium receives that antibacterials in the test sample suppress and the detection method of penicillin medicament residue in the screening food samples fast.
Background technology
Antibacterial medicine residue is the serious problems of puzzlement food security always.Penicillin medicine is one type of important beta-lactam antibiotic; Because of it is efficient, low toxicity is that present veterinary science is used one of most popular medicine being widely used owing to such medicine clinically; Make its residual phenomena on animal derived food more and more serious, though penicillin medicine itself causes drug anaphylaxis easily to the very not strong toxicity of body; Be all kinds of allergic drugs the most simultaneously; If long-term edible such microbiotic of low concentration can make bacterium produce drug resistance, flora imbalance; Cause body immunity to reduce in addition; Return the animal derived food production and processing and bring imponderable loss therefore, foundation is directed against the residual fast detection method of penicillin medicine, and the moderate kit product of exploitation has important social and economic significance to control animal derived food hygienic quality, guarantee consumer health, assurance commercial production etc.
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. to food antibiotic is residual.Have instrument and equipment complicacy, sample pre-treatment and the loaded down with trivial details defective of measurement operation with the said method detection, equipment cost is big, 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 penicillin medicament residue is imperative in reliable, available a large amount of test sample.
The microorganism detection method is to come qualitative to the inhibiting effect of the physiological function of microorganism and metabolism or confirm that quantitatively the antibacterial material in the sample is residual according to antibacterial material, has fast, advantages such as sensitivity, cheapness.Usually in seized test sample, cultivate microorganism,, become muddy, opaque or owing to production by biological acid causes the indicator change color because microbial growth can be observed nutrient culture media if there is not microbiotic to exist in the sample to antibiotic sensitive; If had microbiotic or other antibacterial substances to exist could observe have inhibition zone to form on the nutrient culture media or nutrient culture media still transparent, or do not have change color.At present, the antibacterial medicine residue microorganism detection method of liquid-like such as milk mainly contains chlorinated triphenyl base four nitrogen methods, and has listed national standard in.But in the specific operation process of this method, disturbing factor is many; Food samples to be checked often has autochthonous microorganism or external microorganism, can not guarantee sample germ-free condition to be checked, and the existence of these microorganisms can have a strong impact on the microorganism detection method.
Raw milk such as just gathering contains bacterium, and under the suitable condition of temperature, bacterial reproduction is very fast, and the alternately phenomenon of differentiation appears in bacterium mutually.The pH of the raw milk of general firm collection is neutral, contains abundant lactose, helps in the raw milk lactic acid bacteria of existence naturally, and for example streptococcus lactis (Streptococcus lactis) breeds soon.Their lactose fermenterses produce lactic acid, the pH of cow's milk is reduced, and cause that proteins coagulation becomes the cheese shape.Low pH has finally also suppressed the breeding of streptococcus lactis, by can be tolerated more hang down pH bacterial classification lactobacillus (Lactobacillus) replace, they accomplish lactose fermentation, and pH is reduced more.Saccharomycete utilizes lactic acid and grows under this low pH condition.Along with the minimizing of lactose and lactic acid, pH rises once again, most of pseudomonas (Pseudomonas) and bacillus (Bacillus) growth and breeding.
Food samples to be checked can't adopt the method for sterilization, because can influence food quality like this; Asptic technique; Be microbiological basic demand, such reality is just just brought very big problem with microorganism detection method detection method: these disturb microbe-derived unclear, and are of a great variety; Have a strong impact on the growth (suppressing or promotion detection microbial growth) of bacterial detection; Perhaps disturb a large amount of indication results that have influence on acid base indicator that breed of microorganism, very big to the testing result influence, can produce false negative or false positive results.
Summary of the invention
The objective of the invention is to overcome the above-mentioned defective that prior art exists in promoting the use of process; A kind of specific large-sized analytic instrument equipment that do not need is provided; Has highly sensitive, with a high credibility, the simple screening method of method of application; Make it can effectively reduce in the animal foodstuff sample such as milk assorted bacterium such as autochthonous microorganism and disturb, reduce producing false negative or false positive results.
The object of the invention is realized through following technical scheme:
For solving the problems of the technologies described above, the present invention provides a strain high and be applied to bifidobacterium breve (Bifidobacterium breve) the LJM-006 CGMCC No.5418 of penicillin drug test to the penicillin drug susceptibility.
Bifidobacterium breve of the present invention (Bifidobacterium breve) LJM-006; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 28th, 2011; It abbreviates CGMCC (address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City as; Postcode 100101) preservation, classification called after bifidobacterium breve (Bifidobacterium breve), 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 milky, opaque, glossy, smooth surface, convexity, soft, the neat in edge of quality, diameter 1-1.5mm.
(2) individual morphology: being irregular G+ sporeless bacterium, straight-bar, knee, match head, dumbbell shape and wild goose shape are arranged, is main with the wild goose shape wherein.
(3) physiological and biochemical property: D-ribose (+); L-arabinose (-); Lactose (+); Cellobiose (-); Melezitose (+); Raffinose (+); Sorbierite (-); Starch (-); Gluconic acid sodium salt (-); Wood sugar (-); Mannose (+); Fructose (+); Galactose (+); Sucrose (+); Maltose (+); Trehalose (-); Close disaccharides (+); Sweet mellow wine (+); Synanthrin (-); Salicin (-); F6PPK enzyme (+); Reaction (on good gas-solid body nutrient culture media, not growing) to oxygen; Nitrate reduction (-); Catalase (-); Indole reaction (-).
Well-grown under the anaerobism is not grown in the 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 the penicillin drug susceptibility and improved more than 100 times.
The present invention also provides the detection method of antibacterial medicine residue in a kind of food, said method comprising the steps of:
(1) bacterial strain activation: bifidobacterium breve LJM-006 inoculation on nutrient agar slant medium, is regulated pH value to 6.8～7.2 of this nutrient culture media, under 35～37 ℃, be optimized cultivation, and change kind of subculture and cultivate;
(2) bacteria suspension preparation: 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 processed suspension, adjusted its OD
600Value to 0.25～0.35 is put in the 2-8 ℃ of refrigerator and is preserved;
(3) detector tube preparation: by weight, get peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium acetate 5g, K
2HPO
42g, MgSO
47H
2O 0.5g, MnSO
44H
2O 0.2g, FOS 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, chilling temperature is 50～65 ℃; And get the 10mL aforesaid liquid rapidly, and inject 10mL in vitro, for use as test bacterium liquid;
(4) sample pre-treatments: get animal foodstuff sample homogenization to be checked and organize 5g, with aseptic 1% phosphate buffer 20mL dilution, the whirlpool mixing, in 80 ℃ of water-bath heating 5min, the cooling back is in 5, and the centrifugal 15min of 000r/min gets supernatant as supplying test agent liquid;
(5) sample detection: with reference to the TTC method, residues of antibiotics amount in the check animal foodstuff.Getting step (4) gained supplies test agent liquid 20～100 μ L to be added in the said detector tube of step (3); Cultivated 8～10 hours in 35 ± 2 ℃ of anaerobism the airtight back of sealing; Add TTC 0.3mL; 30min are cultivated in 36 ± 1 ℃ of water-baths, judge test findings according to color status: positive when sample is the primary colors of breast, it is negative to take on a red color.
Work bacterial strain of the present invention is bifidobacterium breve LJM-006, on October 28th, 2011 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is CGMCCNo.5418.
Animal food sample of the present invention comprises: milk, pork, beef, chicken, egg etc.
Table 1 color status criterion
Determination methods: accurately cultivate the 30min observations,, continue to cultivate 30min again and do to observe for the second time as positive.Want during observation to avoid illumination to cross of a specified duration the interference rapidly.Have microbiotic to exist in the animal foodstuff sample, though then add bacterium liquid culture in the sample, because of the breeding of bacterium is suppressed, so indicator TTC do not reduce, and do not develop the color.In contrast, if there is not microbiotic to exist, then adds bacterium liquid and breed at once, TTC is reduced and shows red, and positive when that is to say colourless that sample is, it is negative to take on a red color.
The present invention to the residual sensitivity of penicillin in the animal foodstuff sample between 2-3ng/mL.
The present invention has the following advantages and effect:
1, the present invention has solved autochthonous microorganism in the food samples in this method or assorted bacterium disturbs the false positive that is caused, the problem of false negative result on the basis that has kept TTC detection method reliability; The used detection bacterium of the present invention is a strict anaerobes, the necessary anaerobic state of sweat, and the autochthonous microorganism of food samples or assorted bacterium are aerobic bacteria, can not under anaerobic state, grow effective like this interference of having avoided testing result;
2, high, highly sensitive to antibiotic drug test specificity, accuracy is good, detects the residual sensitivity of penicillin in the plain chocolate between 2-3ng/mL, is lower than national MRL standard, meets the residue detection requirement;
3, sample needs pre-treatment hardly, and tissues such as muscle, liver can be got animal tissue's liquid through modes such as squeezings and detect.
Embodiment
Be noted that following specifying all is exemplary, being intended to provides further invention to the present invention.Except as otherwise noted, all Science and Technology terms of using of this paper have with the present invention under the identical meanings of person skilled common sense.
Below in conjunction with specific embodiment the present invention is described further, but the embodiment that lifts not as to qualification of the present invention.If do not specialize the conventional means that used technological means is well known to those skilled in the art among the embodiment.
Embodiment 1: the separation and the evaluation of bifidobacterium breve LJM-006 bacterial strain
With the ight soil of Zhejiang Province's healthy young people as sample separation; On the MRS agar medium, 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 is on the October 28th, 2011 of (address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode 100101) preservation at China Committee for Culture Collection of Microorganisms common micro-organisms center; Classification called after bifidobacterium breve (Bifidobacterium breve), preserving number is CGMCC No.5418.
The prescription of MRS agar medium: peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium acetate 5g, K
2HPO
42g, MgSO
47H
2O 0.5g, MnSO
44H
2O 0.2g, FOS 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 milky, opaque, glossy, smooth surface, convexity, soft, the neat in edge of quality, diameter 1-1.5mm.
(2) individual morphology: being irregular G+ sporeless bacterium, straight-bar, knee, match head, dumbbell shape and wild goose shape are arranged, is main with the wild goose shape wherein.
(3) physiological and biochemical property: D-ribose (+); L-arabinose (+); Lactose (+); Cellobiose (-); Melezitose (+); Raffinose (+); Sorbierite (-); Starch (-); Gluconic acid sodium salt (-); Wood sugar (+); Mannose (-); Fructose (+); Galactose (+); Sucrose (+); Maltose (+); Trehalose (-); Close disaccharides (+); Sweet mellow wine (+); Synanthrin (-); Salicin (-); F6PPK enzyme (+); Reaction (on good gas-solid body nutrient culture media, not growing) to oxygen; Nitrate reduction (-); Catalase (-); Indole reaction (-).
Well-grown under the anaerobism is not grown in the 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 tests
Adopt scraps of paper agar diffusion (K-B) method; Choose the penicillin titer of variable concentrations, with the modified MRS agar medium, culture medium prescription is seen embodiment 1; With bifidobacterium breve LJM-006 of the present invention is the test bacterium; Place 37 ℃ of incubator anaerobism to cultivate the growing state of observing the LJM-006 bacterial strain behind the 24h, do the penicillin sensitivity test, measure the bacteriostasis property of penicillin the LJM-006 bacterial strain with agar diffusion method of the paper.
3 repetitions are done in above-mentioned test, are contrast to belong to bifidobacterium breve reference culture of the same race together.
Table 2: sensitivity tests
| Bacterial strain | The susceptibility of penicillin | The bacteriostatic diameter of 2ng/mL penicillin (mm) |
| LJM-006 bacterial strain of the present invention | ≤1ng/mL | 19 |
| The bifidobacterium breve reference culture | ＞2ng/mL | 13 |
Test findings: bifidobacterium breve LJM-006 of the present invention has the susceptibility of height to penicillin; Its with belong to reference culture together and compare; Susceptibility to penicillin has improved more than 100 times; Penicillin susceptibility≤2-3ng/mL of bifidobacterium breve LJM-006 is lower than the residual quantity of national standard, can be used as the residual work bacterial strain of screening penicillin fully.
Embodiment 2: the detection of pork liver sample penicillin medicament residue
(1) bacterial strain activation: bifidobacterium breve LJM-006 inoculation on nutrient agar slant medium, is regulated pH value to 6.8～7.2 of this nutrient culture media, under 35～37 ℃, be optimized cultivation, and change kind of subculture and cultivate;
(2) bacteria suspension preparation: 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 processed suspension, adjusted its OD
600Value to 0.25～0.35 is put in the 2-8 ℃ of refrigerator and is preserved;
(3) detector tube preparation: by weight, get peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium acetate 5g, K
2HPO
42g, MgSO
47H
2O 0.5g, MnSO
44H
2O 0.2g, FOS 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, chilling temperature is 50～65 ℃; And get the 10mL aforesaid liquid rapidly, and inject 10mL in vitro, for use as test bacterium liquid;
(4) sample pre-treatments: get pork liver sample homogenization to be checked and organize 5g, with aseptic 1% phosphate buffer 20mL dilution, the whirlpool mixing, in 80 ℃ of water-bath heating 5min, the cooling back is in 5, and the centrifugal 15min of 000r/min gets supernatant as supplying test agent liquid;
(5) sample detection: with reference to the TTC method, residues of antibiotics amount in the check animal foodstuff.Getting step (4) gained supplies test agent liquid 20～100 μ L to be added in the said detector tube of step (3); Cultivated 8～10 hours in 35 ± 2 ℃ of anaerobism the airtight back of sealing; Add TTC 0.3mL, 30min is cultivated in 36 ± 1 ℃ of water-baths, observes as positive; In water-bath, cultivate 30min again and do to observe for the second time, every part of sample is done two parts.Remake negative and each portion of positive control in addition, the animal foodstuff sample added with antibiotic of the effective antibiotic-free of positive control and bacterium liquid and TTC.The effective antibiotic-free animal foodstuff of negative control sample adds bacterium liquid and TTC.
4% 2,3,5 monochlor(in)ate triphens, four nitrogen (TTC) WS: take by weighing 1g TTC, be melted in the 5mL sterile purified water, preserve in 7 ℃ of refrigerators in the dress brown bottle, face the time spent to be diluted to 5 times with sterile purified water.Become jade green or filbert like solution, then can not use again.
Test 1: false negative rate test
Take by weighing blank each 5g of animal foodstuff sample of homogeneous, add the penicillin titer, make penicillin concn 2-3ng/mL in the food, assay method is with embodiment 2, and replication 100 times is observed the probability that negative findings occurs, and calculates false negative rate.
Test findings: penicillin is residual in the milk when being 2-3ng/mL, and false negative rate is 0; Penicillin is residual in the pork when being 2-3ng/mL, and false negative rate is 2%.
Test 2: stability test
Prepare the detection tubule by the method among the embodiment 2 and under 4 ℃, preserve 1 week, 2 weeks, 3 weeks, 4 weeks successively; Detect and write down its negative detection time respectively; 10 hours, 10 hours, 10 hours, 10.5 hours; Can change to some extent negative detection time along with the prolongation of time, and 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 the judgement time exactly and is as the criterion with negative control.The inventive method detects that the stability of penicillin residues of pesticides all is stabilized in 10 hours in preceding 3 weeks in the food, to the 4th week be 10.5 hours, so the detection stability of the method is one month.
Test 3: the confirming of detectability
Use the PBS solution that contains variable concentrations (1ng/mL, 2ng/mL, 4ng/mL, 8ng/mL, 16ng/mL) penicillin standard items as analyte sample fluid respectively; Drip in testing tube; Carry out sample detection, analyzing and testing result confirms that the detection of detection method of the present invention is limited to 2-3ng/mL.
Test 4: specific assay
The inhibiting rate that penicillin suppresses Bifidobacterium LJM-006 is that 50% o'clock corresponding concentration is 1.5ng/L (IC
50=1.5mg/L), but penicillin Bifidobacterium LJM-006 specificity is suppressed, with the right Bifidobacterium LJM-006 cross reacting rate % of other antibiotic all less than 0.5%, the result sees table 3.
The cross reaction of table 3 penicillin and other organophosphorus insecticides
| Compound | IC 50(ng/mL) | Cross reacting rate (%) |
| Penicillin | 1.5 | 100 |
| Tetracycline | 132 | ＜0.5 |
| Terramycin | ＞260 | ＜0.5 |
| Ofloxacin | ＞260 | ＜0.5 |
| Chloromycetin | ＞260 | ＜0.5 |
| Streptomysin | ＞260 | ＜0.5 |
The detection of embodiment 3 other samples
Tissues such as meat, liver, kidney are pressed extracting juice and are added an amount of phosphate buffer accent pH to 7.0 after can passing through weighing; To the different animal sample, add different damping fluids, with control pH.Other operation steps is with embodiment 2.
Claims (4)
1. the detection method of antibacterial medicine residue in the quick screening animal foodstuff appearance is characterized in that, said method comprising the steps of:
(1) bacterial strain activation: Bifidobacterium is inoculated on the nutrient agar slant medium, regulates pH value to 6.8～7.2 of this nutrient culture media, under 35～37 ℃, be optimized cultivation, and change kind of subculture and cultivate;
(2) bacteria suspension preparation: get step (1) gained Bifidobacterium and be inoculated in nutrient agar slant medium, 35 ± 2 ℃ of anaerobism were cultivated 24 hours, with sterile saline lawn were washed, and processed suspension, adjusted its OD
600Value to 0.25～0.35 is put in the 2-8 ℃ of refrigerator and is preserved;
(3) detector tube preparation: by weight, get peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium acetate 5g, K
2HPO
42g, MgSO
47H
2O 0.5g, MnSO
44H
2O 0.2g, FOS 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, chilling temperature is 50～65 ℃; And get the 10mL aforesaid liquid rapidly, and inject 10mL in vitro, for use as test;
(4) sample pre-treatments: get animal foodstuff sample homogenization to be checked and organize 5g, with aseptic 1% phosphate buffer 20mL dilution, the whirlpool mixing, in 80 ℃ of water-bath heating 5min, the cooling back is in 5, and the centrifugal 15min of 000r/min gets supernatant as supplying test agent liquid;
(5) sample detection: with reference to the TTC method, residues of antibiotics amount in the check animal foodstuff.Getting step (4) gained supplies test agent liquid 20～100 μ L to be added in the said detector tube of step (3); Cultivated 8～10 hours in 35 ± 2 ℃ of anaerobism the airtight back of sealing; Add TTC0.3mL; 30min are cultivated in 36 ± 1 ℃ of water-baths, judge test findings according to color status: positive when sample is the primary colors of breast, it is negative to take on a red color.
2. method according to claim 1 is characterized in that said Bifidobacterium is a bifidobacterium breve LJM-006 bacterial strain.
3. method according to claim 1 is characterized in that said antibacterials are penicillin.
4. method according to claim 1 is characterized in that said animal foodstuff is milk, pork, beef, chicken, egg etc.
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| CN105675604A (en) * | 2016-03-31 | 2016-06-15 | 山东五洲检测有限公司 | Method for detecting antibiotic residues in milk |
| CN105713859A (en) * | 2016-03-02 | 2016-06-29 | 温州医科大学 | Bifidobacterium breve and method for detecting various antibiotic residues in milk and application |
| CN106319026A (en) * | 2016-08-19 | 2017-01-11 | 界首市科创食品检测中心 | Method for detecting antibiotic in milk |
| CN109971669A (en) * | 2019-02-01 | 2019-07-05 | 温州医科大学 | The detection method and application of bifidobacterium pseudocatenulatum and Residue of Antibiotics in Milk |
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| CN109971669A (en) * | 2019-02-01 | 2019-07-05 | 温州医科大学 | The detection method and application of bifidobacterium pseudocatenulatum and Residue of Antibiotics in Milk |
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