The Risk Assessment Of Benzylpenicillin G Residue In Heat-treated Animal Food Products

Benzylpenicillin G is a class of antibiotics,which works to destroy the cell wall of bacteria,especially to the gram-positive bacteria whilst only selective specificity to gram-negative cocci(meningococcus and gonorrhoeae etc.).Penicillin is widely used in medicine and animal husbandry due to its high anti-bacterial efficiency and relative low toxicity.However,the misuse and overuse of benzylpenicillin G in the livestock industry has resulted in the detection of its residue in animal derived products.Many detrimental side effects of ingestion of these residues exist,such as allergic reaction,antimicrobial resistance and effects on the diversity of GI microbiota,have been consistently reported.In clinical practice,other adverse effects of benzylpenicillin G are reported,such as neurotoxicity,abnormal liver function and ototoxicity.All these effects originated from benzypenicillin G residue in animal derived products would increase the potential risk to the public health.Benzylpenicillin G is unstable under numerous conditions as a result of its specific structure.Extreme acidic and alkaline conditions,metal ions,the presence of ?-lactamase all bring about a change to the structure and its function.One additional factor of benzylpenicillin G degradation in food is that of the change of temperature,which would generate kinds of products.Generally,different methods of cooking such as frying,stirring and stewing etc.utilize different temperatures,and is used for daily food preparation to promote tenderness,enhance taste and to destroy bacteria and other pathogens.Nonetheless,during the heating process,the benzylpenicillin G residue in animal derived products degrades to form different products,which has been reported by many scholars under numerous conditions,with different cooking methods and media.However,the toxicity of these thermo-derived products in food is rarely reported.In this study,we evaluated the toxicity of benzylpenicillin G residue in animal derived products after high temperature cooking.Based on the result of the acute toxicity study,benzylpenicillin G heated to cooking temperature(BPHCT)showed significant toxicity after 10 minutes of heating.Then we analyzed the degradation products of BPHCT using UPLC-MS/MS to accurately identify the degradation products.Later,we synthesized a major degradation product(benzylpenicilloic acid [BPNLA])and characterized it with UPLC,MS,1H-NMR and the starch-iodine reaction,which identified the target structure with a high purity?97%.Therefore,in this study,BPHCT and BPNLA were employed to investigate their toxicity in vivo and in vitro.In vivo: BPHCT and BPNLA were acutely toxic;the intraperitoneal values of LD50 were 933.04 mg/kg and 8.48 g/kg,respectively.The body weight of mice and the liver and kidney related serum biochemical parameters and organ ratios changed after long-term exposure to BPHCT.Necrosis of the hepatocyte,lymphocytic and histiocytic inflammatory infiltration and eosinophilic granular cytoplasm were observed in the liver parenchyma in the high dose group.In addition,multifocal lymphocytic,histiocytic and plasmacytic inflammatory infiltrations were observed in the parenchyma in the high dose group.Seminiferous tubules were loose with interstitial broadening.The decreased population of sustentacular cells,necrotic spermatids and spermatids remained within the lobules in the high dose group.Some mice in the high dose group showed neurologic symptoms.Here,BPHCT exhibits sperm toxicity and genotoxicity.In vitro: BPHCT and BPNLA were found to be cytotoxic,which could affect the viability of cells and inhibit proliferation in a dose and time dependent manner,and could induce cell apoptosis.The gene expressions of Caspase 3,8 and 9 were up-regulated and down regulated by the ratio of Bcl-2 to Bax after BPHCT or BPNLA treatment.The apoptotic cells were detected in the tissue of lungs,spleen and the GI of mice exposed to BPHCT.BPNLA could change cell cycle progression,making cell population accumulated in G1 phase and reduced in the S phase.In summary,we found that the toxicity of BPHCT is 3.75 times higher than its prototype in vivo and 2 to 55 times high than its prototype in vitro,showing the ability of inducing cell apoptosis and cell cycle progression.The toxic effect and mechanism between BPHCT and BPNLA are similar,which showed that BPNLA is one of the toxic components of BPHCT.Long-term exposure of BPHCT contaminated animal derived products results in clinical adverse effects,with toxicity to the liver and lung,sperm toxicity and genotoxicity as well.The antibiotics residues in animal derived products and real-situation cooking methods were taken into account to study the toxicity of antibiotics residues.It is believed that high temperature leads to degradation of heat unstable antibiotics,thus toxicity of the degradation products should be cautious.Through this experiment,we uncovered toxicological information on penicillin residue and communicated the evidence to consumers and relevant authorities of food security,to broadcast the potential toxic effects caused by penicillin residue in animal derived products.More attention should be given to the antibiotics residues after heat treatment and more investigation to discover its impact on other vital areas that pose a threat to public health(i.e.carcinogenic,teratogenic,mutagenic,infertility).Therefore,it is suggested that to combat the potential health hazard,the antibiotics used in livestock production should be under strict supervision by food standards agencies and governments should apply the strict withdrawal period and develop sensitive methods for antibiotics residues detection in order to ensure food safety.