Preparation And Antibacterial Effects Of Clostridium Perfringens Phage Endolysin

Clostridium perfringens（Cp）,is a spore producing pathogen that is an important zoonotic pathogen.It usually exists in soil,sewage,and the intestinal tract of humans and animals,which can cause food poisoning,necrotizing enteritis,gas gangrene and other serious infectious diseases.The irrational use of antibiotics and chemical disinfectants in the process of animal food production and slaughter leads to the frequent occurrence of drug-resistant bacteria,which not only threatens human health but also causes huge economic losses to animal food.With the gradual implementation of antibiotic reduction action,the infection rate of Cp in the process of animal food production showed an obvious increasing trend,which resulted in a significant increase in the detection rate of Cp in the slaughtering process.In order to effectively prevent Cp from infecting the body and reduce its contamination risk in animal food,a biological prevention and control method without side effects and residues is urgently needed.While improving the quality and safety of animal food,it also enhances the brand effect of biological source to meet people’s demand for green food.LysCP28,which can inhibit Cp,was identified from previously isolated Cp phage.The anti-bacterial effect of LysCP28 was identified,and prepared LysCP28compound preparation,to clarify the potential application as an antibacterial resource.By analyzing the genome sequence of Cp phage v B＿Cpe S＿BG3P of Siphoviridae,orf28 was predicted to have a hydrolase domain,which was expressed and purified by Escherichia coli BL21（DE3）to obtain soluble protein LysCP28.The anti-bacterial spectrum,thermal stability and optimum p H of LysCP28 were analyzed,and the anti-bacterial characteristics of LysCP28 were determined by scanning electron microscopy.The results showed that the LysCP28 lytic rate could reach 80.21%（77/96）,and the growth of Cp could be effectively inhibited within 2 h.The optimal reaction temperature was37℃ and the optimal reaction p H was about 7.LysCP28 was identified as a new Cp phage endolysin by sequence alignment and genetic evolution tree.At the same time,the inhibitory efficiency of LysCP28 on biofilm was analyzed.The minimum inhibitory concentration of LysCP28 was determined to be 18.75μg/m L.The anti-bacterial application of LysCP28 in fresh duck meat showed that LysCP28 could effectively inhibit Cp contamination in fresh duck meat,and the anti-bacterial effect was positive correlation with concentration of endolysin.500μg/m L LysCP28 reduced the Cp concentration by 3.37 log CFU/g within 24 h.The results of biofilm inhibition showed that LysCP28 could inhibit the growth of Cp biofilm,and A₅₇₀ decreased from 0.79 to 0.15（81.01%）.At the same time,endolysin LysCP28 also has the ability to inhibit the formation of biofilm after different time of Cp growth（1-3 d）.In conclusion,LysCP28 can not only effectively inhibit the formation of Cp biofilm,but also eliminate the already formed biofilm,which can effectively control the contaminated Cp in raw duck meat,and provide an important biological control method for the processing and preservation of poultry meat.Using sodium alginate,chitosan and diatomite to coat endolysin,an effective compound preparation of endolysin was prepared,and its antibacterial effect was determined and preliminary application was carried out.The results showed that the granulated product（1μg/m L）could be formed by sodium alginate coating.In vitro lytic activity showed that the endolysin complex had good anti-bacterial effect.In the simulation of artificial gastroenteric fluid complex sustained release aging up to 6 h and can withstand the degradation of gastric acid and bile.The endolysin complex was fed to broiler chicks continuously for 14 days.The results showed that the complex could effectively inhibit the growth of Cp in the intestinal tract of broilers.The Cp load in the intestinal tract was reduced by 1.83 log CFU/g,and the ratio of feed to gain was decreased by 17.91%.In conclusion,the endolysin complex can withstand acid and has a slow-release effect,effectively inhibit intestinal Cp load and improve the growth performance of broilers,thus providing a new antibacterial source for the antibacterial enhancement of poultry farming.