Analysis Of Risk Key Points Of Microorganism In Raw Milk And Characteristics Of Pseudomonas

Milk is the perfect food for human beings as it is rich in a variety of nutrients and bioactive substances,which plays an important role in human nutrition and health.However,milk is susceptible to microorganism contamination during milking,transportation,storage and processing,which poses a serious threat to the quality and safety of milk and milk products.In this study,the 16 S rRNA gene sequencing and source tracker modeling were used to analyze the risk key points of microorganism contamination in raw milk,and the diversity of psychrotrophic bacteria in raw milk in different regions was also investigated via culture method and Pac Bio sequencing technology.The culture method and Pac Bio sequencing technology were used to analyze the diversity of psychrotrophic bacteria in the raw milk in different regions.The functional genes of Pseudomonas isolated from raw milk were verified,and the functional genes of Pseudomonas isolates were analyzed by whole-genome sequencing.The main results of current study as follows:1.Analysis of Risk Key Points of microbial in Raw MilkThe differences of the microbial community structure between raw milk and dairy farm environment,including cow trough water,teat dip cup,teat,teat liner,dairy hall air,cowshed air,feces,feed,and bedding,was analyzed using 16 S rRNA gene sequencing analysis.The relative abundances of Acinetobacter and Pseudomonas in the raw milk of Zhengzhou dairy farm were higher at the genus level,and the relative abundance of psychrotrophic bacteria was as high as 95.9%.The relative abundances of EscherichiaShigella and Serratia in raw milk of Qiqihar dairy farm were high at the genus level,and the relative abundance of psychrotrophic bacteria accounted for 35.1%.The 16 S rRNA gene sequencing and source tracker modeling were used to investigated the ten key risk points,such as cow trough water,teat dip cup,teat,teat liner,dairy hall air,cowshed air,feces,feed,and bedding,in Heilongjiang province and Henan province,China.Analysis showed that milk cups and medicated bath cups were the most important risk points for microbial contamination of raw milk samples in Zhengzhou dairy farm and Qiqihar dairy farm?2.Diversity of psychrotrophic bacteria in raw milk in different regions The community structure and diversity of psychrotrophic bacteria in raw milk in Heilongjiang,Inner Mongolia,Gansu,Henan,Anhui,Jiangsu,Chongqing and Hunan provinces of China were investigated via traditional culture method and Pac Bio singlemolecule real-time sequencing.The isolated psychrotrophic community structure was with high diversity,composing of 21 genera and 59 species.Pseudomonas accounted for 58.9% of the total genera,and Pseudomonas azotoformans accounted for 16.9 % at the species level.Combining the construction of a raw milk psychrophilic strain database by traditional culture and the Pac Bio single-molecule real-time sequencing method,it was found that regional differences were found significantly among the eight regions except samples from Heilongjiang and Inner Mongolia were similar.Additionally,there were significant differences between among Henan,Anhui and Jiangsu samples with different sampling dates.Therefore,different control strategies must be implemented according to regions and seasons.3.Characterization and proteolytic activity of psychotrophic Pseudomonas species isolated from raw milk samplesA total of 116 Pseudomonas strains isolated from raw milk in Heilongjiang,Inner Mongol,Gansu,Henan,Anhui,Jiangsu,Chongqing and Hunan province were identified,and apr X gene were identified in 60.3% Pseudomonas strains.Around 68.9%of Pseudomonas isolates showed proteolytic ability at 4 °C,while 81.9% and 85.3% had proteolytic ability at 10 °C and 25 °C,respectively.There were differences in the proteolytic activity among Pseudomonas strains from different regions.In addition,the proteolytic proteases produced by Pseudomonas strains were heat-resistant.4.Whole-genome comparisons of Pseudomonas sp.strains from raw milk Whole-genome sequencing technology was used to analyze the gene function,antibiotic resistance,and proteolytic activity characteristics of Pseudomonas in raw milk.Pseudomonas strain P1,P2,P3,P3,P4,P5,P6,and P7 were identified as Pseudomonas brenneri,Pseudomonas lurida,Pseudomonas fluorescens,Pseudomonas lurida,Pseudomonas fluorescens,Pseudomonas fluorescens and Pseudomonas fragi,respectively.All Pseudomonas contained protease genes and resistance genes.Pseudomonas with strong protease hydrolysis ability contains more carbohydrate transport and metabolism function genes,signal transduction mechanisms genes,secondary metabolite synthesis,transport and metabolism genes.Pseudomonas with higher multidrug resistance index also carried genes for polysaccharide lyase and carbohydrate esterase.This study can provide data support for the detection of protease genes and reduce the risk of transmission of antibiotic resistance genes,which provides a basis for improving the quality of milk and milk products.