| Frozen drinks,especially ice cream and ice milk,are rich in dairy products,and are prone to microbial contamination.Foodborne diseases and outbreaks of ice cream contaminated by pathogenic bacteria have occurred in many foreign countries,indicating that there are some food safety risks of frozen drinks.However,microbial contamination in frozen drinks in our country has not been comprehensively reported and analyzed,and the contamination source is not yet clear.There is a lack of data on microbial control of frozen drinks.In order to obtain the current situation of microbial contamination in frozen drinks in our country,explore the sources of contamination and determine the key control points of production and processing,this study collected market-sold frozen drinks from 31 provinces to carry out microbial contamination study.The processing process studies were carried out in 12 production enterprises of different scales in 8 provinces.The main pathogenic bacteria isolated in the processing link were analyzed by whole genome sequencing(WGS).The main contents are:Part One Investigation of microbial contamination in frozen drinks in the marketObjective:This study investigated the microbial contamination of frozen drinks in the Chinese market.Methods:A total of 6818 frozen drinks samples were collected from different regions of China.Contamination by the aerobic plate count(APC),coliforms,Listeria monocytogenes(L.monocytogenes),Staphylococcus aureus,and Salmonella spp.,were detected in the samples.L.monocytogenes and Salmonella isolates were further analyzed for antibiotic susceptibility and multi-locus sequence typing(MLST).Results:The results showed that APC was more than 10~5 CFU/g in 3.52%(240/6818)and coliform was more than 100 CFU/g in 9.59%(654/6818)of all samples.The positive rates of S.aureus,L.monocytogenes,and Salmonella spp.were 0.56%(38/6818),0.62%(42/6818),and 0.10%(7/6818),respectively.Of these,S.aureus contamination was more than 100 CFU/g in six samples,and L.monocytogenes in the positive sample was in the range of 0.3 MPN/g～240MPN/g,with a median of 3.6 MPN/g.Ice cream had a significantly higher proportion of the APC and coliforms compared to ice milk and ice lolly(P<0.05).The hygienic status of the packaged samples was much better than that of the bulk samples(P<0.05).Catered samples were more frequently and heavily contaminated than samples from retail and wholesale outlets(P<0.05).No significant differences were observed in samples bought from urban and rural areas(P>0.05).For 42 L.monocytogenes strains isolated from frozen drinks,the resistance rate of nine antibiotics was 4.76%(2/42).By MLST,the L.monocytogenes strains were classified into 14 sequence types(STs),of which ST8 was most common(12 isolates).A total of 7 strains of Salmonella were isolated from frozen drinks,and Salmonella enteritidis was the dominant serotype(5 isolates),all of which were ST11.The antibiotic susceptibility test showed that Salmonella resistance was common and there were two multiple resistant strains.Conclusions:These above results indicate that a potential health risk to the public may be caused by frozen drinks,particularly in susceptible populations.Part Two The study of microbial contamination in frozen drinks productionObjective:To understand the microbial contamination in frozen drinks production and processing of,and to explore the high contamination points in the production process.Method:Twelve frozen drinks enterprises of different scales were selected in 8 provinces of China,and a total of 5183 samples were collected from different production areas(pre-treatment area,filling area and whole case packing area),including 1265 food samples(including raw materials,intermediate products and final products)and 3918 non-food samples(environment,personnel,instruments and equipment,etc.).The APC,Enterobacteriaceae,Listeria,L.monocytogenes and Salmonella were examined.Results:The hygienic condition of production water was poor,and 31.11%(28/90)of production water had APC more than 100 CFU/ml.The hygienic condition of other raw materials was good except production water.Individual intermediate products and final products had APC more than 10~5 CFU/g,and had Enterobacteriaceae more than 10~3 CFU/g.And 38.71%(36/93)of air samples in the filling area had airborne sedimentation bacteria more than 10CFU/dish,indicating that the production environment of enterprises was not clean enough and not effectively divided.For non-food samples,the packaging materials were in good sanitary condition,while environment,personnel,equipment and tools found Enterobacteriaceae,Listeria,L.monocytogenes and Salmonella.The detection rate of Enterobacteriaceae in non-food samples was30.60%(1125/3677),and the detection rate of Enterobacteriaceae was 26.16%(537/2053)in the filling area.The Enterobacteriaceae was generally detected in the ground and contact materials in the filling area.The detection rate of L.monocytogenes in food samples was 0.71%(9/1265),including raw materials,intermediate products and final products.The detection rate of L.monocytogenes in non-food samples was 7.07%(260/3677),and the detection rate was 7.01%(144/2053)in the filling area.L.monocytogenes positive samples were found in the environment,equipment and personnel of the filling area,which was a potential safety hazard.Salmonella was not detected in food samples,but was occasionally detected in non-food samples with a positive rate of 0.44%(16/3677).The sanitary condition of small and medium-scale enterprises was significantly worse than that of large enterprises.The detection rate of Enterobacteriaceae in non-food samples from filling area of small and medium-sacle enterprises(39.26%and 34.72%)was much higher than that of large enterprises(9.51%).The ground,the steps of ladder,the soles of personnel,drains and movable equipment touching the ground parts are concentrated high-risk points in the frozen drinks production,which need to be the focus of daily monitoring.Conclusion:The contamination of Enterobacteriaceae and L.monocytogenes was widely distributed in the frozen drinks production,and the risk of cross contamination is high.Enterprises should strengthen the hygiene management and disinfection measures in production to ensure the edible safety of the final product.Part Three The analysis of gene characteristics of main pathogenic bacteria in frozen drinks productionObjective:To analyze the genetic characteristics of Listeria monocytogenes and Salmonella from frozen drinks production.Method:The269 strains of Listeria monocytogenes and 16 strains of Salmonella isolated from production were sequenced by whole genome sequencing technology,and the sequencing results were assembled and annotated by relevant software.STs,drug resistance genes and virulence genes were obtained from databases.And cg MLST was used for molecular typing and tracing analysis.Results:The 269strains of Listeria monocytogenes were divided into two lineages,and 95.17%(256 strains)were lineage II.There was 11 CC type,CC8 was the main type,accounting for 75.46%(203 strains);There were 12 STs,and ST8 was the dominant type,accounting for 75.46%(203 strains).The positive rate of drug resistance gene of L.monocytogenes was low,3.34%(9 isolates)carried drug resistance genes,mainly tetracycline resistant gene tet(8 isolates,2.97%).All strains carried LIPI-1,while none carried LIPI-4.Seven isolates(2.60%)carried LIPI-3,posing a potential risk of disease.By cg MLST analysis,L.monocytogenes strains were divided into 12 subgroups and 19 homologous or suspected homologous strains were identified.Compared with MLST,cg MLST cluster and trace the source more accurately.The sources of L.monocytogenes in frozen drinks production were complex,and many homologous strains were found in the same enterprise.There was serious cross-contamination in some enterprises,and homologous strains were isolated from food and various non-food samples in different production areas.Homologous or suspected homologous strains were also found in different enterprises.The 16 strains of Salmonella were divided into 3 STs,with ST197 as the main type(8 isolates),and the corresponding serotype was Salmonella Virchow.And 16 drug-resistant genes were identified.The positive rate of aminoglycoside resistance gene aac(6’)-Iaa was 100.00%,and the other 15 drug-resistant genes were only detected in 2 isolates,with the positive rate of 12.50%.A total of 249 virulence genes were identified.All 16 strains of Salmonella harbored avr A,inv B,sic A,sic P,sip A,sip B,sip C,spt P in SPI-1 and ssa B,ssa E,ssc A,ssc B in SPI-2,mgt CB,mis L in SPI-3 and sop B in SPI-5.All 16 strains of Salmonella didn’t harbor see L,see FG,ttr in SPI-2 and sii E in SPI-4.Conclusions:The results revealed the distribution characteristics of STs,drug resistance genes and virulence genes of Listeria monocytogenes and Salmonella in frozen drinks production,and provided a basis for the control of microbial contamination and the prevention and treatment of foodborne diseases in the production of frozen drinks. |
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