Bacterial Diversity And Community Structure In The Chicken Follicles During Slaughter And The Mechanism Of Its Limitation In Decontamination Of Carcass Surface

Fresh chicken is susceptible to bacterial contamination,which can lead to spoilage and shortened shelf life.Some foodborne pathogens contamination will increase the food safety risk of chicken that may result in food recall and significant economic losses.Therefore,the development of appropriate control strategies of bacterial contamination and investigation of the potential contamination is of great significance to facilitate the poultry industry.Although the development of automation technolgy has largely improved the production efficiency of chicken slaughtering,the research on the microbial control technolgy is still seriously off track,which lead to the significant cross-contamination during the slaughtering.Current technolgy already cannot satisfy the rapidly developed automated chicken slaughtering process.Therefore,it is critical to clarify the mechanism of bacterial contamination during slaughtering and corresponding bacteria reduction technolgy.The complex microscopic structure of the chicken's skin surface is prone to contamination and bacterial growth and difficult to clean,making it difficult forthe slaughterline to control bacterial contamination on the carcass surface.The role and mechanism of chicken skin hairfollicles in the process of microbial contamination during slaughteris not clearyet,which hinderthe development of strategies to reduce the bacterial contamination on chicken skin.Therefore,the objective of this study is to clarify the impact of featherfollicles on the bacterial diversity and community of fresh chicken skin during slaughtering and the related mechanism.In this study,the morpholgical parameters(follicle diameter,depth,volume and area density)of chicken skin follicles in different parts were determined in orderto monitorthe basic morpholgy of chicken skin follicles.Moreover,the p H data of carcass and skin during slaughtering were collected,which can be utilized to evaluate the effects of postmortem rigormortis on featherfollicle shrinkage.HE staining(hematoxylin-eosin staining)and paraffin tissue sectioning were used to study the changes of the internal structure of chicken skin follicles during the critical process of slaughtering(bleeding,defeathering,evisceration and pe-chilling).In addition,microbial cultivation method was applied to validate the contamination routes and bioinformatic results of the bacteria(total numberof colonies,fecal coliform,Psychrophilic bacterium,Escherichia coli and staphylococcus aureus)on chicken carcass chicken and in waterduring the critical steps of slaughtering(pre-chilling,evisceration,defeathering,scalding,etc.).Experiments have also been carried OTU to investigate the impact of concentration,time,p H,and otherfactors of sodium hypochlorite peracetic acid on the bacteria residue of chicken skin.16S rRNA High throughput sequencing(HTS)and biopsy sampling method with a circumferential skin drill were utilized to conducte the in-situ sequencing to profile the change microbial community and diversity in chicken follicles and environment throughout the slaughtering.Fourcategories of samples were periodically collected at the critical operation points within the automatic slaughtering and systematically analyzed,which included: skin follicles,carcass appendants(feather,crop and bowel contents),watersamples(scalding waterand chilling waterat three chillertanks),and the surface fouling of 5 kinds of production facilities(de-feathering,evisceration,convey belts and the wall of the 3 chiller).Bioinformatic tools were applied to perform the OTU analysis.With the utilization of various analytical approaches including ?-diversity,?-diversity and environmental factorcorrelation analysis,etc,the bacterial community diversity and evolution,cross-contamination,sourcetracking and the effectiveness and of disinfectants have been investigated.This study found the chicken skin featherfollicles formed some internal closed cavity((?):200?m × D:1040?m)within the precooling process through skin tissue paraffin section and HE staining microscopy.During defeathering process,the follicles adsorbed waste waterand mixed debris from the carcass surface by vacuum.The surface skin of the shrinking follicle has hydrophobic fat components that can prevent the water-soluble disinfectant from entering the follicle,and the follicle is rich in organic compounds that can consume chemical disinfectants,so the featherfollicles had the structure and organic interferers that could provide disinfectant resistance foradsorbed bacteria.The cavity structure inside the follicle has the function of containing bacteria and resisting reducing bacteria foradsorbed bacteria,improving the permeability of decontamination technology can reduce the bacterial survival rate in chicken skin,and verify the influence mechanism of chicken skin follicle on bacterial contamination in chicken skin.According to the analysis of OTU clustering and annotation information,Proteobacteria(58.1%),Firmicutes(28.9%),Actinomycetes(7.3%)and Bacteroidetes(4.0%)are the dominant flora of chicken skin and process-related contacts(potential pollution vectors)in the slaughtering process of high-speed automated broilers.Scalding,defeathering,evisceration and pre-chilling all significantly increased the bacterial community species abundance of carcass chicken skin samples(SAB,SAD,SAE,SAC)and significantly increased with the development of slaughterprocess(P<0.05).The presence of microenvironment in the slaughtering line increases the risk of carcass contamination.This study forthe first time found that scalding wateris the source of contamination of the three types of bacillus producing bacteria in the skin of broilers,which may lead to the spoilage of heat-resistant bacillus products in meat products.The waterafterprechilling tank is the main source of Cryophilic bacillus and Pseudomonas which lead to the shortened shelf life of the carcass.It was found that the chlorine content and temperature of pre-chilling watersignificantly affected the community structure of prechilling water,and chlorine-tolerant bacteria appeared in the pre-chilling waterof commercial slaughterhouses.The results are as follows:(1)Chicken featherfollicles can form a closed cavity inside the skin tissues.reducing the bacteriostatic effect of disinfectants on the surface microorganisms of the carcass,and acting as a "storage pond" forcontaminating bacteria,increasing the difficulties in cross-contamination control and bacterial reduction during the slaughter.The maximum innerdiameterof chicken featherfollicles is 2.3×16.74 mm.The cavity inside the featherfollicles is rugged,and the featherfollicles can provide anti-sterilizing space to bacteria adsorbed inside the featherfollicles.With the increase of post-mortem time and the decrease of the temperature of the pre-chilling water,skin featherfollicles begin to shrink.Fat-rich featherfollicles reduce the contact between the disinfectant solution and the bacteria inside the featherfollicles,resulting in bacteria in the chicken skin remained relatively stable underthe condition of sterilization of various disinfectants,and the colony structure of chicken skin featherfollicles do not show significance before and afterthe pre-chilling process.Aiming at the morpholgical changes of the featherfollicles and the protective mechanism against bacteria,this article proposes an inference based on the morpholgical changes of the featherfollicles.That is,when the featherfollicles are not closed,spraying bacteriostat on the skin surface of the carcass,using the diastolic negative pressure to suck the bactericide into the inside of the featherfollicle,then massaging the carcass with the help of the mechanical force of plucking machine.This method can solve the technical bottleneck that the disinfectant cannot penetrate the skin featherfollicle afterthe featherfollicle is closed.In conventional production,we can promote the penetration of disinfectant into the featherfollicle by adjusting the p H of the disinfectant,increasing the concentration of the disinfectant,extending the contact time,and stirring.It also provides research directions and basic data forthe future application of physical Penetration technolgy(rradiation,ultrasonic steam,etc.).(2)The microenvironment of slaughterhouse process increases the risk of chicken carcass contaminationSodium hypochlorite in pre-chilling waterhas bactericidal effect on most bacteria.However,pre-chilled waterand plant production waterwith OTU disinfectant have become a source of contamination of spoilage bacteria such as Psychrotrophs and Pseudomonas.The high temperature environment of the scalding pool has potential to breed three types of heatresistant bacteria that can produce spores: Anoxybacillus,Ureibacillus,and Tepidimicrobium,which remain in the skin of the chicken to contaminate carcass afterplucking.Contamination caused by heat-resistant spores increases the risk of spoilage of cooked meat products made from chicken skin.(3)Bacterial reduction technolgy forpre-chilling and disinfection in broilerslaughterneeds new bacteriostatic factors and rotation mechanismAt present,pre-chilling waterand facility dirt in the production environment have breeding bacteria that are resistant to sodium hypochlorite,which may lead to more severe food spoilage and food safety issues.At present,the long-term use of sodium hypochlorite in the sterilization mode of the broilerslaughterindustry in China cause a serious risk of bacterial disinfectant resistance.Multiple simultaneous sterilization measures orregularrotation of the existing antibacterial technolgy can be introduced to reduce the numberof bacteria,control bacterial resistance and realize sustainable development of poultry industry.