CN109971670B - Enterococcus faecalis, culture medium thereof, and method for detecting antibiotic residues in milk and application thereof - Google Patents
Enterococcus faecalis, culture medium thereof, and method for detecting antibiotic residues in milk and application thereof Download PDFInfo
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Abstract
The invention discloses enterococcus faecalis which is preserved in the China general microbiological culture Collection center (CGMCC) at 2016, 1 and 21, and the preservation number is CGMCC No.12088. And discloses a culture medium for screening residual tetracycline, erythromycin, penicillin, gentamicin, vancomycin and chloramphenicol in milk, and a specific detection method. The screening method of the method is simple to operate, can directly read the result by observing the solidification of the culture medium or the color change of the indicator, does not need special precise instruments, can simultaneously detect various antibiotic residues, is suitable for screening the antibiotic residues in food, and has strong practicability.
Description
Technical Field
The invention belongs to the fields of life science and food safety, and particularly relates to enterococcus faecalis and a method for rapidly detecting residual antibiotics in milk by using the same.
Background
Antibiotic residues in milk are an important issue for milk safety. With the rapid development of the livestock breeding industry, the milk yield is greatly improved. In order to prevent and treat diseases such as mastitis and the like, antibiotic veterinary drugs are increasingly widely applied in animal husbandry, and antibiotics are used in an irregular way while the effect of treating dairy cow diseases by applying the antibiotics is achieved, such as increasing the use dose, prolonging the treatment period, and even applying the antibiotics to dairy cows in the lactation period for a long time to prevent diseases, so that the antibiotics with higher content are always remained in milk.
Many dairy products in China do not meet the requirements of countries and regions needing import, and the overproof antibiotic residues are important limiting factors. Antibiotic residues in milk not only bring loss to milk production and related industries, but also bring a lot of potential hazards to the physical health of consumers. With the improvement of the living standard of people and the attention on biological safety and food safety, the antibiotic residue has become the key point of milk food safety concern. In order to meet increasingly strict food quality requirements at home and abroad and meet the requirements of markets and rapid customs clearance of import and export, the method for simply, rapidly and effectively detecting the residues of various antibiotics in milk has important significance.
The detection method of the residues of multiple antibiotic drugs in the relevant literature or methods is basically to detect each drug separately, and the analysis methods include chromatography, color/mass spectrometry, thin layer chromatography, immunoassay, microorganism inhibition method and the like. The general use of 33 veterinary drugs of 5 categories including sulfonamides, quinolones, beta-lactams, tetracyclines and chloramphenicols in China has large differences in chemical molecular structure and chemical properties, and is not easy to detect simultaneously. Meanwhile, the detection of various antibiotic residues in the milk is difficult to detect, and the detection methods for screening the antibiotic residues in the milk by using a microbiological method specified in the food sanitation standard (GB/T4789.27-2008) in China at present comprise a TTC method and a Bacillus stearothermophilus spore inhibition method. Among them, the TTC method is simple in equipment, low in cost, and suitable for screening of large samples and operation in basic laboratories and is widely used, and mainly detects penicillin G of β lactam, as well as streptomycin, gentamicin, and kanamycin. In addition, the detection principle and the result of the national standard method are obtained according to whether the growth of the streptococcus thermophilus is inhibited by residual antibiotics or not, because the streptococcus thermophilus is cultured for 2 hours at 37 ℃ after the sample is added, most of bacteria (such as escherichia coli, staphylococcus epidermidis and other catalase positive bacteria) in the environment can quickly grow under the condition and cause the color of TTC to change, and if pollution occurs in the operation process, a false negative result can be caused. The sensitivity on penicillin and chloramphenicol detection is about the same as that of a bacillus subtilis paper method, but the penicillin and chloramphenicol detection is less sensitive to streptomycin and not sensitive to neomycin, and the disinfectant can interfere with the test.
In addition, the national standard method adopted in China does not comprise the detection of tetracycline, erythromycin, vancomycin and the like. The tetracycline antibiotics can prevent and treat infectious diseases, improve feed efficiency and accelerate the growth of animals, and are widely applied to animal husbandry. In the process of raising dairy cows, tetracycline is often used as a feed additive and to prevent mastitis in dairy cows, resulting in the possibility of tetracycline residues in the milk. Erythromycin belongs to macrolide antibiotics, is also widely used in livestock, poultry and aquaculture, and the food safety field mainly adopts an instrument method to analyze the residual quantity of the erythromycin in animal-derived food. Vancomycin belongs to glycopeptide antibiotics, contains sugar and a peptide chain structure in molecules, can effectively treat infection caused by methicillin-resistant staphylococcus aureus (MRSA) and methicillin-resistant staphylococcus epidermidis (MRSE), and particularly has good curative effect on bovine mastitis and other diseases. At present, the limit of vancomycin in milk is 10ug/kg in Japan, glycopeptide antibiotics are prohibited from being used in imported animal-derived foods in the United states, and the No. 560 bulletin of the Ministry of agriculture in China stipulates that vancomycin is a banned veterinary drug. With the increasing common infection of MRSA and MRSE and the minimal curative effect of other drugs, the illegal use of glycopeptide drugs such as vancomycin in the treatment of cow diseases is inevitable.
The microbial inhibition method has the characteristics of simple and convenient operation, low requirements on instruments, equipment and personnel, simple pretreatment of samples, short detection time and the like, and is always used for sieving a large quantity of samples. The invention mainly applies probiotics sensitive to beta-lactams, macrolides, tetracyclines and aminoglycosides, and establishes a microorganism inhibition method for rapidly screening and quantitatively detecting tetracycline, penicillin, gentamicin, erythromycin, vancomycin and chloramphenicol.
Disclosure of Invention
1. The invention aims to provide a novel method.
The invention screens out a enterococcus faecalis strain sensitive to antibiotics, and establishes a simple, rapid and sensitive screening method for detecting antibiotic residues in milk by taking the strain as a working strain.
2. The technical scheme adopted by the invention is as follows.
The Enterococcus faecalis (Enterococcus faecalis) is preserved in the China general microbiological culture Collection center (CGMCC) in 2016, 1 and 21 days, is preserved for short as CGMCC (China academy of sciences and microbiology, the address: no.1 Xilu No. 3 of Beijing Korean district, taiyang district, the code of the institute of microbiology, china academy of sciences, the postal code 100101), is classified and named as Enterococcus faecalis (Enterococcus faecalis), and the preservation number is 12088.
The enterococcus faecalis strains of the present invention have the following microbiological characteristics:
(1) Colony morphology: the colony diameter of enterococcus faecalis CGMCC No.12088 on TPY solid plate is 1.2-1.5mm, and the colony is round, convex, and has regular edge, milky white, opaque, glossy, soft and fine texture; the colony on the LB solid culture medium has the diameter of 5.0-12mm, is round, convex, neat in edge, milky white or grey white, opaque, glossy, soft and fine in texture and easy to emulsify;
(2) The shape of the thallus: gram-positive cocci, which are circular or elliptical in shape, mostly in double or scattered arrangement, without capsules and spores.
(3) Physiological and biochemical characteristics: the catalase is negative, galactose, sucrose and L-arabinose are fermented, fructose, maltose and xylose are not fermented, urea is not reduced, and the nitrate reduction experiment and the indigo substrate experiment are negative.
(4) The culture characteristics are as follows: good aerobic and anaerobic growth. The optimal growth temperature is 37-42 ℃, the lowest growth temperature is 15 ℃, and the highest growth temperature is 45 ℃; optimum pH7.0-7.5, pH below 6.0 or above 9.0, and poor growth.
And discloses the application of the enterococcus faecalis in screening residual tetracycline, erythromycin, penicillin, gentamicin, vancomycin and chloramphenicol in milk.
The culture medium formula of the enterococcus faecalis for screening and detecting the residual tetracycline, erythromycin, penicillin, gentamicin, vancomycin and chloramphenicol in the milk comprises the following formula and preparation steps:
preparing a formula 1: 30g of peptone, 30g of yeast extract, 10g of beef extract, 1.0g of soluble starch, 40g of glucose, 10g of sodium chloride and K 2 HPO 4 6g，KH 2 PO 4 6g, dissolving with 1000mL of distilled water, adjusting the pH to 7.0-7.5, packaging, autoclaving at 121 ℃ for 20-30 minutes, cooling, and storing at 4 ℃ for later use;
preparing a formula 2: weighing 200g of high-quality soybean meal, dissolving the high-quality soybean meal in 1000mL of distilled water, heating and boiling for 30 minutes, and filtering by using multilayer gauze and qualitative filter paper;
preparing a formula 3: taking 150mL of filtrate prepared according to the formula 2, adding 50mL of liquid prepared according to the formula 1, adjusting the pH value to 7.0-7.2, carrying out autoclaving at 121 ℃ for 20-30 minutes, cooling, and storing at 4 ℃ for later use.
The invention also discloses a method for detecting residual tetracycline, erythromycin, penicillin, gentamicin, vancomycin and chloramphenicol in milk, which comprises the following steps:
(1) Strain activation: inoculating the enterococcus faecalis strain to an LB solid culture medium, and culturing at 40-42 ℃ for 18-24h;
(2) Preparing a bacterial suspension: taking a single colony of the enterococcus faecalis obtained in the step (1), and adjusting OD (origin-to-diameter) by using sterile normal saline 600nm 0.10-0.12 bacterial suspension; 0.1ml of the bacterial suspension was aspirated into 9.9ml of the medium prepared according to formulation 3, and cultured overnight at 42 ℃. The overnight culture can be stored at 4 ℃.
(3) Preparing an overnight culture into a working bacterial suspension of 1;
(4) Preparing non-antibiotic milk: weighing 10g-12g of non-antibiotic milk powder, and preparing 10% -12% solution with sterile distilled water; or dissolving in distilled water, sterilizing at 121 deg.C under high pressure for 15-30 min, and storing at 4 deg.C.
(5) Preparation of a standard tube: taking tetracycline, erythromycin, penicillin, gentamicin, vancomycin and chloramphenicol standard stock solution, and diluting with antibiotic-free milk to prepare antibiotic standard working solutions with different concentrations; 300 mu g/L, 200 mu g/L, 100 mu g/L, 50 mu g/L and 20 mu g/L of tetracycline standard working solution; erythromycin 160. Mu.g/L, 80. Mu.g/L, 40. Mu.g/L, 20. Mu.g/L, 10. Mu.g/L; ampicillin 20. Mu.g/L, 10. Mu.g/L, 4. Mu.g/L, 2. Mu.g/L, 1. Mu.g/L of standard working solution; standard working solution of gentamicin of 800. Mu.g/L, 400. Mu.g/L, 200. Mu.g/L, 100. Mu.g/L and 50. Mu.g/L; 400 ug/L, 200 ug/L, 150 ug/L, 100 ug/L, 80 ug/L, 60 ug/L, 40 ug/L, and 20 ug/L of vancomycin; standard working solutions of chloramphenicol 400. Mu.g/L, 300. Mu.g/L, 200. Mu.g/L, 100. Mu.g/L, 80. Mu.g/L, 60. Mu.g/L, 40. Mu.g/L, 20. Mu.g/L, 10. Mu.g/L; refrigerating at 4 deg.C for use;
(5) Sample preparation: taking 10mL of sample, putting the sample in a sterile test tube, carrying out water bath at 80 ℃ for 5-10 minutes, centrifuging the sample at 5000rpm for 5 minutes, and removing the precipitate;
(6) Determination of detection limit: respectively taking 300 mu g/L, 200 mu g/L, 100 mu g/L, 50 mu g/L and 20 mu g/L of tetracycline standard milk samples, 160 mu g/L, 80 mu g/L, 40 mu g/L, 20 mu g/L and 10 mu g/L of erythromycin standard milk samples, 400 mu L of milk samples added with 20 mu g/L, 10 mu g/L, 4 mu g/L, 2 mu g/L and 1 mu g/L of penicillin standard milk samples, 800 mu g/L, 400 mu g/L, 200 mu g/L, 100 mu g/L and 50 mu g/L of daptomycin standard milk samples, 400 mu g/L, 200 mu g/L, 150 mu g/L, 100 mu g/L, 80 mu g/L, 60 mu g/L, 40 mu g/L and 20 mu g/L of vancomycin standard milk samples; 400 μ g/L, 300 μ g/L, 200 μ g/L, 100 μ g/L, 80 μ g/L, 60 μ g/L, 40 μ g/L, 20 μ g/L, 10 μ g/L of a chloramphenicol standard; and a milk sample with 0 mu g/L of antibiotic is used as a negative growth control, added into 600uL of the culture medium obtained by the formula 3, added with 30 mu L of the bacterial liquid prepared in the step 3, evenly mixed, put into 42 ℃ for culture for 3 hours, taken out for direct observation of the coagulation phenomenon or added with a 0.02% methyl red indicator for observation of the color change condition;
(7) Sample detection: respectively taking 400 mu L of the sample to be detected prepared in the step (5) and 400 mu L of nonreactive milk, and simultaneously taking 400 mu L of a milk sample containing 100 mu g/L tetracycline standard, 40 mu g/L erythromycin standard, 4 mu g/L penicillin standard, 200 mu g/L gentamicin standard, 100 mu g/L vancomycin standard and 80 mu g/L chloramphenicol standard; adding into the culture medium obtained by the formula 3 of 600 uL; adding 30 mu L of the bacterial liquid prepared in the step (3); mixing, culturing at 42 deg.C for 3 hr; taking out the sample, taking 0 μ g/L as negative (growth) control, and directly observing coagulation phenomenon or adding 0.02% methyl red indicator to observe color change;
the milk sample provided by the invention comprises: raw milk, liquid milk and reconstituted milk.
The method is a screening method and accords with the detection result of a national standard method.
3. The technical effect produced by the invention.
(1) The enterococcus faecalis strain of the present invention has the following characteristics: sensitive to various antibiotics such as tetracycline, erythromycin, penicillin, gentamicin, vancomycin, chloramphenicol, etc.; good growth after culture at 42 ℃, better acid production capacity and difficult contamination by mixed bacteria; the bacterial growth can enable the used self-prepared liquid culture medium to be solidified, and the result is easy to observe; the strain has good stability and is easy to store and operate; is probiotics, is friendly to human body and environment, and can not cause laboratory and environmental pollution.
(2) The principle of the method is that based on the inhibition effect of antibiotics on the used enterococcus faecalis, the enterococcus faecalis strain is used for fermenting the sample, the fermentation process is tracked, and whether the antibiotics residue exists in the sample is judged according to the fermentation characteristics.
(3) The screening method of the method is simple to operate, can directly judge and read the result by observing the solidification of the culture medium or the color change of the indicator, is simple, convenient and accurate in judgment method, is suitable for food quality safety inspection, and is high in practicability.
Drawings
FIG. 1 is a diagram showing the detection result of enterococcus faecalis on tetracycline labeling samples.
FIG. 2 is a diagram showing the result of the detection of an erythromycin-spiked sample by enterococcus faecalis.
FIG. 3 is a diagram showing the detection results of enterococcus faecalis on penicillin-labeled samples.
FIG. 4 is a graph showing the result of the detection of the sample for marking gentamicin by enterococcus faecalis.
FIG. 5 is a diagram showing the result of detecting vancomycin with a labeled sample from enterococcus faecalis.
FIG. 6 is a diagram showing the result of detecting a chloramphenicol-labeled sample by enterococcus faecalis.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The technical measures used in the examples are, if specified otherwise, conventional measures known to the person skilled in the art.
Example 1 isolation and identification of enterococcus faecalis strains
Excrement of healthy men and young men in Zhejiang province is taken as a separation sample, 1g of excrement is suspended and diluted by 10mL of PBS and inoculated in TPY solid culture medium, anaerobic culture is carried out at 37 ℃ for 48 hours, and Enterococcus faecalis disclosed by the invention is obtained and identified as Enterococcus faecalis (Enterococcus faecalis), which has been preserved in the China general microbiological culture Collection management Committee general microbiological center (address: beijing city No.1 Xilu No. 3 of the rising district of Chaozhong, china academy of sciences, postal code 100101) in 2016 year 1 and 21 days, and the preservation number is CGMCC No.12088.
TPY agar culture medium and TPY liquid culture medium are products of Qingdao GaoKeyuan Haibo biotechnology limited.
The enterococcus faecalis strains according to the invention have the following microbiological characteristics:
(1) Colony morphology: the colony diameter of enterococcus faecalis on TPY solid plate is 1.2-1.5mm, and the colony is round, convex, regular in edge, milky white, opaque, glossy, soft and fine;
(2) The cell morphology is as follows: gram-positive cocci with circular or elliptical thalli, mostly in double or short-chain arrangement, without capsules or spores; the colony is white and protuberant, and the edge is smooth and tidy.
(3) Physiological and biochemical characteristics: galactose, sucrose and L-arabinose are fermented, fructose, maltose and xylose are not fermented, urea is not reduced, and nitrate reduction experiments and indigo substrate experiments are negative.
(4) The culture characteristics are as follows: good aerobic and anaerobic growth. The optimal growth temperature is 37-42 ℃, the lowest growth temperature is 20 ℃, and the highest growth temperature is 45 ℃; optimum pH7.0-7.5, poor growth at pH below 5.5 or above 8.5.
Susceptibility test of enterococcus faecalis according to the present invention to antibiotics
Paper agar diffusion (K-B) method is adopted to select different antibiotic paper sheets, the enterococcus faecalis is used as test bacteria, MH agar plate culture medium is used, anaerobic culture is carried out at 37 ℃ for 24 hours for observation, and the bacteriostasis performance of different antibiotics to the enterococcus faecalis strains is measured.
The above test was repeated 3 times and judged with reference to the CLSI standard.
TABLE 1 results of susceptibility tests of strains to different antibiotics
The invention shows that the enterococcus faecalis is sensitive to tetracycline and erythromycin, beta lactams (ampicillin, penicillin G, piperacillin and cefepime), vancomycin and chloramphenicol, and can be used as a working strain for screening antibiotic residues in milk.
Example 2: detection of antibiotic residues in milk samples
(1) Strain activation: inoculating the enterococcus faecalis to an LB solid culture medium, and culturing at 40-42 ℃ for 18-24h;
(2) Preparing a bacterial suspension: taking a single colony of the enterococcus faecalis obtained in the step (1), and adjusting OD600 to be bacterial suspension of 0.10-0.12 by using sterile normal saline; 0.1ml of the bacterial suspension was aspirated and added to 9.9ml of the medium prepared according to formulation 3, and incubated overnight at 42 ℃. The overnight culture can be stored at 4 ℃.
(3) Preparing an overnight culture into a working bacterial suspension of 1;
(4) Preparation of a standard tube: taking tetracycline, erythromycin, penicillin, gentamicin, vancomycin and chloramphenicol standard stock solution, and diluting with phosphate buffer solution with pH8.0 to prepare antibiotic standard working solution with different concentrations; 300 mu g/L, 200 mu g/L, 100 mu g/L, 50 mu g/L and 20 mu g/L of tetracycline standard working solution; 160 mug/L, 80 mug/L, 40 mug/L, 20 mug/L and 10 mug/L of erythromycin; penicillin standard working solution with 20 mug/L, 10 mug/L, 4 mug/L, 2 mug/L and 1 mug/L; standard working solution of gentamicin of 800. Mu.g/L, 400. Mu.g/L, 200. Mu.g/L, 100. Mu.g/L and 50. Mu.g/L; 400 ug/L, 200 ug/L, 150 ug/L, 100 ug/L, 80 ug/L, 60 ug/L, 40 ug/L, and 20 ug/L of vancomycin; 400. Mu.g/L, 300. Mu.g/L, 200. Mu.g/L, 100. Mu.g/L, 80. Mu.g/L, 60. Mu.g/L, 40. Mu.g/L, 20. Mu.g/L and 10. Mu.g/L of chloramphenicol; refrigerating at 4 deg.C for use;
(5) Sample preparation: taking 10mL of sample, putting the sample in a sterile test tube, and carrying out water bath at 65 ℃ for half an hour;
(6) Sample detection: selecting 300 mu g/L, 200 mu g/L, 100 mu g/L, 50 mu g/L and 20 mu g/L tetracycline standard solutions, 160 mu g/L, 80 mu g/L, 40 mu g/L, 20 mu g/L and 10 mu g/L erythromycin standard solutions, 20 mu g/L, 10 mu g/L, 4 mu g/L, 2 mu g/L and 1 mu g/L penicillin standard solutions, 800 mu g/L, 400 mu g/L, 200 mu g/L, 100 mu g/L and 50 mu g/L daptomycin standard solutions, 400 mu g/L, 200 mu g/L, 150 mu g/L, 100 mu g/L, 80 mu g/L, 60 mu g/L, 40 mu g/L and 20 mu g/L vancomycin standard working solutions; 400. Mu.g/L, 300. Mu.g/L, 200. Mu.g/L, 100. Mu.g/L, 80. Mu.g/L, 60. Mu.g/L, 40. Mu.g/L, 20. Mu.g/L, 10. Mu.g/L of chloramphenicol standard working solution; and (3) taking the sample obtained in the step (5) as a negative growth control indicator tube for quick screening of the positive control tube indicator tube, uniformly mixing the samples in the culture medium obtained according to the formula 3, adding the mixture into the culture medium at 42 ℃ for culturing for 3 hours, taking out the mixture for direct observation or adding 0.02% methyl red indicator, and observing the solidification result by controlling the positive indicator tube and the negative indicator tube.
Test 1: determination of detection limit
The test was performed with antibiotics of different concentrations (300. Mu.g/L, 200. Mu.g/L, 100. Mu.g/L, 50. Mu.g/L, 20. Mu.g/L tetracycline, 160. Mu.g/L, 80. Mu.g/L, 40. Mu.g/L, 20. Mu.g/L, 10. Mu.g/L erythromycin, 20. Mu.g/L, 10. Mu.g/L, 4. Mu.g/L, 2. Mu.g/L, 1. Mu.g/L penicillin, 800. Mu.g/L, 400. Mu.g/L, 200. Mu.g/L, 100. Mu.g/L gentamicin, 400. Mu.g/L, 200. Mu.g/L, 150. Mu.g/L, 100. Mu.g/L, 80. Mu.g/L, 60. Mu.g/L, 40. Mu.g/L, 20. Mu.g/L vancomycin, 400. Mu.g/L, 300. Mu.g/L, 200. Mu.g/L, 100. Mu.g/L, 80. Mu.g/L, 60. Mu.g/L, 40. Mu.g/L, 20. Mu.g/L vancomycin, 400. Mu.g/L, and a negative test sample (as well as a test sample, as a standard sample, as shown in the test solution and a test sample, and a test solution, as well as a negative test solution, as shown in the following figure. The detection result is analyzed, and the lowest detection limit of the quantitative detection of the detection method is determined to be 4 mu g/L of penicillin, 5 mu g/L of tetracycline, 90 mu g/L of erythromycin, 400 mu g/L of gentamicin, 100 mu g/L of vancomycin and 80 mu g/L of chloramphenicol.
Test 2: antibiotic residue screening test for milk sample
Antibiotics with different concentrations (300. Mu.g/L, 200. Mu.g/L, 100. Mu.g/L, 50. Mu.g/L, 20. Mu.g/L tetracycline, 160. Mu.g/L, 80. Mu.g/L, 40. Mu.g/L, 20. Mu.g/L, 10. Mu.g/L erythromycin, 20. Mu.g/L, 10. Mu.g/L, 4. Mu.g/L, 2. Mu.g/L, 1. Mu.g/L penicillin, 800. Mu.g/L, 400. Mu.g/L, 200. Mu.g/L, 100. Mu.g/L, 50. Mu.g/L gentamicin, 400. Mu.g/L, 200. Mu.g/L, 150. Mu.g/L, 100. Mu.g/L, 80. Mu.g/L, 60. Mu.g/L, 40. Mu.g/L, 20. Mu.g/L vancomycin; 400 mu g/L, 300 mu g/L, 200 mu g/L, 100 mu g/L, 80 mu g/L, 60 mu g/L, 40 mu g/L, 20 mu g/L and 10 mu g/L chloramphenicol) as a sample to be tested (0 mu g/L is used as a negative control), adding the milk in the same volume into the screening detection tube obtained in the step (4), adding the bacterial liquid obtained in the step (2), and carrying out anaerobic culture at 40 ℃ for 6 hours; if the milk is coagulated or red, the antibiotic residue is lower than the national standard, and if the milk is not coagulated and is yellow, the antibiotic residue exceeds the standard. The lowest concentration at which coagulation and redness began to appear by the screening method was 5. Mu.g/L tetracycline, 90. Mu.g/L erythromycin, 4. Mu.g/L ampicillin, 400. Mu.g/L gentamicin, 100. Mu.g/L vancomycin, and 80. Mu.g/L chloramphenicol. The screening test result is in accordance with or equivalent to the highest residue limit (milk: ampicillin 4 mug/L, erythromycin 40 mug/L and tetracycline 100 mug/L) in the animal food approved by China, can meet the screening of a large number of samples, and can prompt the existence of gentamicin, vancomycin and chloramphenicol.
Claims (4)
1. Enterococcus faecalis (Enterococcus faecalis) is preserved in China general microbiological culture Collection center (CGMCC) in 2016 (2.18.4), with the preservation number of CGMCC No.12088.
2. The use of enterococcus faecalis according to claim 1 for screening milk for residual tetracycline, erythromycin, penicillin, gentamicin, vancomycin, and chloramphenicol.
3. The culture medium formula of enterococcus faecalis in the screened milk containing residual tetracycline, erythromycin, penicillin, gentamicin, vancomycin and chloramphenicol, which is prepared by the following steps:
preparing a formula 1: 30g of peptone, 30g of yeast extract, 10g of beef extract, 1.0g of soluble starch, 40g of glucose, 10g of sodium chloride and K 2 HPO 4 6g，KH 2 PO 4 6g, dissolving with 1000mL of distilled water, adjusting the pH to 7.0-7.5, packaging, autoclaving at 121 ℃ for 20-30 minutes, cooling, and storing at 4 ℃ for later use;
preparing a formula 2: weighing 200g of high-quality soybean meal, dissolving the high-quality soybean meal in 1000mL of distilled water, heating and boiling for 30 minutes, and filtering by using multiple layers of gauze and qualitative filter paper;
preparing a formula 3: taking 150mL of filtrate prepared according to the formula 2, adding 50mL of liquid prepared according to the formula 1, adjusting the pH value to 7.0-7.2, carrying out autoclaving at 121 ℃ for 20-30 minutes, cooling, and storing at 4 ℃ for later use.
4. A method for detecting residual tetracycline, erythromycin, penicillin, gentamicin, vancomycin and chloramphenicol in milk comprises the following steps:
(1) Strain activation: inoculating the enterococcus faecalis strain according to claim 1 into an LB solid medium, and culturing at 40-42 ℃ for 18-24h;
(2) Preparing a bacterial suspension: taking a single colony of the enterococcus faecalis obtained in the step (1), and adjusting the OD600nm of the single colony to be 0.10-0.12 by using sterile normal saline; sucking 0.1ml of bacterial suspension, adding the bacterial suspension into 9.9ml of culture medium prepared according to the formula 3, culturing overnight at 42 ℃, and storing the overnight culture at 4 ℃;
(3) Preparing an overnight culture into a working bacterial suspension with a ratio of 1;
(4) Preparation of a standard tube: taking tetracycline, erythromycin, penicillin, gentamicin, vancomycin and chloramphenicol standard stock solution, and diluting with phosphate buffer solution with pH8.0 to prepare antibiotic standard working solution with different concentrations; 300 mu g/L, 200 mu g/L, 100 mu g/L, 50 mu g/L and 20 mu g/L of tetracycline standard working solution; 160 mug/L, 80 mug/L, 40 mug/L, 20 mug/L and 10 mug/L of erythromycin standard working solution; ampicillin 20. Mu.g/L, 10. Mu.g/L, 4. Mu.g/L, 2. Mu.g/L, 1. Mu.g/L of standard working solution; standard working solution of gentamicin of 800. Mu.g/L, 400. Mu.g/L, 200. Mu.g/L, 100. Mu.g/L and 50. Mu.g/L; 400 ug/L, 200 ug/L, 150 ug/L, 100 ug/L, 80 ug/L, 60 ug/L, 40 ug/L, and 20 ug/L of vancomycin; standard working solutions of chloramphenicol 400. Mu.g/L, 300. Mu.g/L, 200. Mu.g/L, 100. Mu.g/L, 80. Mu.g/L, 60. Mu.g/L, 40. Mu.g/L, 20. Mu.g/L, 10. Mu.g/L; refrigerating at 4 deg.C for use;
(5) Sample preparation: taking 10mL of sample raw milk, putting the sample raw milk into a sterile test tube, carrying out water bath at 85 ℃ for 15-20 minutes, centrifuging at 5000rpm for 5 minutes, and discarding the supernatant;
(6) Sample detection: selecting 300 mu g/L, 200 mu g/L, 100 mu g/L, 50 mu g/L, 20 mu g/L tetracycline, 160 mu g/L, 80 mu g/L, 40 mu g/L, 20 mu g/L, 10 mu g/L erythromycin, 20 mu g/L, 10 mu g/L, 4 mu g/L, 2 mu g/L, 1 mu g/L penicillin standard solution, 800 mu g/L, 400 mu g/L, 200 mu g/L, 100 mu g/L, 50 mu g/L gentamycin standard, 400 mu g/L, 200 mu g/L, 150 mu g/L, 100 mu g/L, 80 mu g/L, 60 mu g/L, 40 mu g/L, 20 mu g/L vancomycin standard working solution; 400 mug/L, 300 mug/L, 200 mug/L, 100 mug/L, 80 mug/L, 60 mug/L, 40 mug/L, 20 mug/L and 10 mug/L of chloramphenicol standard working solution, the solution is an indication tube of a rapid screening positive control tube, 0 mug/L is a negative growth control indication tube, the sample obtained in the step (5) is taken out and put into a culture medium obtained according to the formula 3, after being mixed evenly, the mixture is put into 42 ℃ for culturing for 3 hours, and then is taken out for directly observing the coagulation phenomenon, or 0.02 percent of methyl red indicator is added for observing the color change.
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