Detection Of Antibiotics Residue In Milk By Lactic Acid Bacteria Inhibition Method

GB 19301-2010 National Food Safety Standard for Raw Milk:"the milk milked during the cow’s antibiotics using period and withdrawal period should not be used as raw milk", in other words, raw milk for dairy processing must not contain antibiotics. The methods used currently, enzyme linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), can not detect a variety of antibiotic residues simultaneously. GB/T 4789.27-2008 Microbiological Examination of Food Hygiene-examination of Residue of Antibiotics in Fresh Milk using Streptococcus thermophilus and Bacillus stearothermophilus as experimental strains, these strains’preservation, activation, culture requirement are very strict, thus they are not suitable for enterprises promotion. In practice we foud that:the milk containing antibiotic can not be fermented to yogurt. Thus, theoretically, the lactic acid bacteria inhibition method can be used to judge whether the raw milk containing antibiotics or not effectively. Direct vat set of lactic acid bacteria is highly commercialized, easy to be kept, activated and promoted. Thus, the lactic acid bacteria inhibition method to detect antibiotics residues in milk was established based on the principle that the milk can be fermented do not contain any antibiotics or its antibiotic content is below the detection limit. The lactic acid bacteria inhibition method was also used to determine the detection limits of 20 kinds of common antibiotics. The main contents and conclusions in three aspects were included as follows:Firstly, the lactic acid bacteria inhibition method was established. The optimal conditions of this method including the detected strain, the inoculation quantity, the fermentation temperature and the fermentation time were determined by single factor experiments, respectively. The optimal detected strain is Streptococcus thermophilus and Lactobacillus bulgaricus. The bacteria activated method was as follow:adding direct vat set lactic acid bacteria agents into the milk without antibiotics to ferment at 43℃ for 6 h and refrigerating for spare. The optimal operation steps and fermentation conditions were as follows: the milk was sterilized at 90℃ for 10 min and cooled to 43℃, after adding the bacteria 3～4 g/100g, the milk was continue to be ferment at 43 ℃. The method to decide the eligibility was as follows:after being fermented for 4h, milk without curd was the milk containing antibiotics, otherwise the milk excluding antibiotics.Secondly, the lactic acid bacteria inhibition method was used to determine the antibiotics detection limits of 20 kinds of common antibiotics. Determination of the antibiotics content performed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS.), the samples were prepared by adding antibiotics with different contents into milk. The detection limits were the antibiotics concentration of which the milk just cannot curd after fermented for 4h. Compared the detection limit of each antibiotic and the maximum residue limits (MRL) in milk, the results were as follows:the 20 kinds of antibiotics and their detection limits were ceftiofur 3 μg/kg, lincomycin 20 μg/kg, cephalexin 80 μg/kg, cefquinome 6 μg/kg, erythromycin 8 μg/kg, tylosin 45 μg/kg, tilmicosin 45μg/kg, oxytetracycline 60 μg/kg, tetracycline 50 μg/kg, chlortetracycline 50 μg/kg, amoxicillin 8 μg/kg, ampicillin and 8 μg/kg, benzathine penicillin 18 μg/kg, procaine penicillin 17 μg/kg, flumequine 900 μg/kg, streptomycin 700 μg/kg, sulfadimidine 500 μg/kg and gentamicin 600 μg/kg, enrofloxacin 700μg/kg and colistin 800μg/kg, respectively. The detection limits of the first 12 kinds of antibiotics were lower than their MRL in milk, which revealed that the lactic acid inhibition method could determine whether the 12 kinds of antibiotics residues in milk exceeded the regulatory limit. Additionally, the detection limits of the last 8 kinds of antibiotics were higher than their MRL in milk, indicating that 8 kinds of antibiotics residues may exceed the regulatory limits when the milk was considered as no antibiotics milk. The existence of this contradiction can not directly prove using lactic acid bacteria inhibition method to determine the existence of antibiotics may be a wrong judgement.Thirdly, the verification of lactic acid bacteria inhibition method and the comparison study of the GB/T 4789.27 were conducted.85 commercial milk samples and 26 commercial milk powder samples (mixed with distilled water in the ratio of 1:6 (w/w)) were examined by lactic acid bacteria inhibition method and these results were compared to the detection results of GB/T 4789.27-2008 Microbiological Examination of Food Hygiene-examination of Residue of Antibiotics in Fresh Milk. The results showed that 10 samples were positive and the disqualification rate was 9.0% detected by lactic acid bacteria inhibition method, while 13 samples were positive and the disqualification rate was 11.7% performed by GB/T 4789.27-2008, which indicated the results of these two methods has little difference. Therefore, Lactic acid bacteria inhibition method is novel, simple, fast, of low cost and can be easily promoted, it has great practical significance on monitoring antibiotic residues in milk, saving costs of detection, ensuring food safety of dairy products, protecting the health and safety of consumers.