Identification And Proteolytic Activity Of Pseudomonas Spp. Isolated From Different Raw Milks At Storage Temperatures

Spoilage of milk associated with the contamination of dairy products by psychrotrophic microorganisms is a particular concern for the dairy industry,which causes significant economic losses for the food industry.Milk is usually stored at low temperatures for 2 to 5 days prior to heat treatment.During storage,the microbiota shifts towards psychrotrophic microorganisms,which leads to the poor quality of raw milk.The major of psychrotrophic microorganisms have been identified as members of Pseudomonas,making it one of the most important bacteria in dairy.Pseudomonas spp.can grow in different environment and frequently link to the spoilage of food,especially raw milk.The growth of the Pseudomonas is often related with the production of extracellular enzymes(e.g.proteases and lipases).Proteases secreted by Pseudomonas during cooled stored are heat-stable extracellular peptidases that can retain their activity after pasteurization or an ultrahigh temperature(UHT)treatment.These residual enzyme activities can cause coagulation and degradation of milk and dairy products over time.Therefore,Pseudomonas spp.in raw milk should be strictly controlled.Characterization of Pseudomonas spp.and associated proteolytic property in raw milk stored at low temperatures.The aim of this study was to identify the Pseudomonas spp.isolated from raw milk and investigate associated proteolytic property stored at low temperatures.Raw milk samples(n=87)were collected from 87 dairy farms in Shaanxi Province of China.Pseudomonas spp.were identified using Pseudomonas specific 16S,universal 16S rRNA and rpoB gene sequencing.The proteolytic properties of Pseudomonas spp.were performing using milk agar,quantitative trinitrobenzenesulfonic acid(TNBS)assay,and the identification of alkaline metallopeptidases gene(aprX).Total 143 isolates from all 87 samples were confirmed as Pseudomonas,which were identified as 14 Pseudomonas species.Of these,40(28.0%)isolates revealed proteolysis on milk agar at 2?,74(51.8%)at 4?,104(72.7%)at 7?,and 102(71.3%)at 10?.However,proteolytic activity produced by 45(31.5%)isolates were exceeded 2 ?mol of glycine equivalents per mL at 7?,followed by 43(30.1%)at 10?,18(12.6%)at 4?,and 7(4.9%)at 2?.The results demonstrated proteolytic activity of Pseudomonas spp.present in milk and indicated their spoilage potential at different temperatures.Identification and proteolytic activity quantification of Pseudomonas spp.isolated from different raw milks at storage temperatures.The objectives of this study were to identify Pseudomonas spp.isolated from goat,buffalo,camel,and yak milks of five provinces in China,and to measure proteolytic activity of Pseudomonas spp.under storage temperatures.Raw milk samples of goat(n=50),buffalo(n=25),camel(n=25),and yak(n=25)were collected from five provinces in China.Pseudomonas spp.were analyzed by Pseudomonas specific 16S,universal 16S rRNA,and rpoB gene sequence analyses.Proteolytic activity on milk agar,quantification via the trinitrobenzenesulfonic acid(TNBS)assay at 2?,4?,7?,10? and 25?,as well as alkaline peptidase gene(aprX)identification were performed to ascertain proteolytic activity of these isolates.Pseudomonas spp.were found in 46 samples out of total 125 samples.A total of 67 Pseudomonas spp.were identified.Of Pseudomonas isolates,we obtained extracellular peptidase activity from seven(10.4%)at 2?,17(25.4%)at 4?,24(35.8%)at 7?,39(58.2%)at 10?,and 41(61.2%)at 25?.The results revealed that a large diversity of Pseudomonas spp.were present in different non-bovine raw milks with the ability to produce peptidases at storage temperatures.However,proteolytic activity varied widely among the peptidase positive isolates.A majority isolates from yak milk had high preoteolytic activity.Therefore,to control Pseudomonas spp.growth and to prevent milk spoilage during the storage and transportation,adequate storage temperatures and time should be established for raw milks originated from different animal species.In addition to the total bacterial counts of raw milk,psychrotrophic microorganisms should also be tested.Low storage temperature and short periods before processing(within 48 h)could reduce the heat-stable peptidase production of Pseudomonas spp,but the milking hygiene should also be properly controlled.This is of great significance for reducing the economic loss of the dairy industry caused by Pseudomonas spp.