| Cold storage and transportation are important links to ensure the quality of raw cows’ milk.However,under low temperature conditions,psychrophilic bacteria can still grow and reproduce.And psychrophilic bacteria beco me the main microorganism along with the extended duration of refrigeration of raw cows’ milk.Some psychrotrophic bacteria have the ability to secrete heat-resistant lipase and protease whose activity cannot be completely destroyed by the normal heattreatment in dairy industry.Residual enzyme activity will decompose fat and protein in dairy products.The present work collected the raw cows’ milk samples from multiple farms in main milk production areas of North China and analyzed the diversity and dynamics of psychrotrophic bacteria in raw cows’ milk.The heat resistance of lipase and protease secreted by selected advantageous psychrophic bacteria was also studied,and a typical psychrotrophic bacterium was obtained.In addition,the loop-mediated isothermal amplification(LAMP)detection assay for detection of psychrotrophic bacteria was established and optimized.The aim of this study was to develop a more rapid approach for the detection of psychrotrophic bacteria and keeping the quality of raw cows’ milk.The raw cows’ milk samples were collected from three areas across the region: Beijing,Heihe and Harbin.The samples were divided into two parts which were stored at 0~5°C and 5~10°C,respectively.PCR-DGGE method was used to analyze the diversity of psychrotrophic bacteria in raw cows’ milk stored at two temperature conditions for 0,1 and 3 d,respectively.The bacterial structure and diversity of samples collected from different areas were significantly different.The similarity of different samples stored at the same temperature was lower than the same samples stored at the different temperatures.Nevertheless,bacteria affiliated to Pseudomonadales and Lactobacillales were found to be the major microflora prevailing in all three milk samples.The bacterial structure was also influenced by storage temperature.The increasement of psychrotrophic bacteria in samples stored at 5~10°C was higher than that stored at 0~5°C.As the storage time extended,the proportion of L.lactis decreased,while the proportion of Pseudomonas elevated.21 lipolytic isolates and 26 proteolytic isolates were obtained from the raw cows’ milk samples.The 16 S r DNA sequence analysis implicated that although the isolates were from samples of different areas,there was only one bacterial genus,Pseudomonas.Among these isolates,P.fragi was the most commonly found in the raw cows’ milk samples.All of the islated psychrotrophic bacteria were not heat-resistant,but most of the hydrolyzed enzyme secreted by them were heat-resistant.In these isolates,isolate 38 was the most potential lipase producer,and its lipase was heat-resistant;isolate 38 also have the highest proteolytic activity and its protease was the most heat-resistant.Isolate 38 was identified as Pseudomonas fluorescens by 16 S r DNA analysis.The LAMP systems were established according to the conserved sequence acquired from comparing the lipase gene and protease gene of isolate 38 with the P.fluorescens published in NCBI database,respectively.The systems were optimized by evaluating the effects of adjusting the following reaction variables: Mg2+ concentration,deoxynucleotide triphosphates concentration,Bst DNA polymerase concentration,inner/outer primer ratio,reaction time,and temperature.The LAMP and real-time LAMP systems of lipase gene and protease gene were specified for P.fluorescens.The other isolates cannot be detected by these two systems.Staphylococcus aureus,Salmonella typhimurium,Escherichia coli,Enterobacter sakazakii,Listeria monocytogenes and Shigella flexneri which are the common contamination in raw cows’ milk also had no signals.PCR detection was a control in study of the detection limit of LAMP and RT-LAMP.In detection of lipase gene from pure culture,the LAMP and RT-LAMP had the same detection limit which was 10 times lower than PCR,and the detection limit was 4×101 CFU/mL.In detection of protease gene from pure culture,the LAMP and RT-LAMP also had the same detection limit which was 10 times lower than PCR,and the detection limit was 3×101 CFU/mL.In detection of lipase gene from artificial contamination pasteurized milk,the LAMP and RT-LAMP also had the same detection limit which was 100 times lower than PCR,and the detection limit was 6.1×101 CFU/mL.In detection of protease gene from artificial contamination pasteurized milk,the LAMP and RT-LAMP had the same detection limit which was 100 times lower than PCR,and the detection limit was 8.9×101 CFU/mL.The time course of RT-LAMP was about 80 min,20 min shorter than LAMP.In actual production,the detection of lipase gene and protease gene in raw milk can be carried out simultaneously which can rapidly reflect the quantity of harmful bacteria in the milk and provide the guarantee for the quality of dairy products. |
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