Study On The Diversity And Main Hydrolase Properties Of Psychrotrophic Bacteria In Raw Milk

As the main raw material of dairy products,the quality of raw milk is a key factor affecting the whole dairy processing chain.Psychrotrophic bacteria can be the dominate organism in refrigerated raw milk.Heat-resistant proteases and lipases secreted by them may have potential adverse effects on the quality and shelf-life of dairy products.For instance,heat-resistant enzymes may cause severe quality issues such as the reduction of nutritional value,a bitter or rancid flavor and the gelation of UHT milk,shortening the shelf-life of dairy products.The divrsity of psychrotrophic bacterial microbiota in raw milk is contingent on several elements,such as seasonality,storage conditions,geographical areas,farm management practices and feeding patterns.Therefore,the research on diversity and main hydrolase properties of psychrotrophic bacteria is a major point.This study took the psychrotrophic bacteria of raw milk as the object.On the one hand,the traditional cultivation method was used to determine the concentration and taxonomic levels of psychrotrophic bacteria.SMRT sequencing technology was combined to further reveal the diversity and seasonal differences of psychrotrophic bacteria.On the other hand,the enzyme activity was determined by agar diffusion assays combined with azocasein and p-nitrophenol method.The heatresistance of enzymes was investigated after heat treatment by simulating pasteurization and ultrahigh temperature instantaneous sterilization temperature.The results were as follows:(1)The concentration of psychrotrophic bacteria(PBC)showed seasonal differences.The mean concentration of psychrotrophic bacteria in 20 raw milk samples was 3.73 log CFU/m L.PBC in summer was 3.92 log CFU/m L,while in winter was 3.54 log CFU/m L.In addition,PBC in summer was significantly higher than that in winter(p<0.05).(2)The diversity of psychrotrophic bacteria obtained by traditional cultivation method was high.A total of 600 strains were classified into 45 genera(120 species),2 potential novel genera and 3 unknown species.The genus with the highest isolation frequency was Pseudomonas(25.5%),and it was detected in 18 of 20 samples,indicating that Pseudomonas was distributed in raw milk commonly.The species with the highest isolation frequency was P.fluorescens(5.2%).Psychrotrophic bacteria strains in summer group represented 40 genera(83 species),while winter group included 27 genera(75 species),indicating more complex psychrotrophic bacterial communities in the summer group.Pseudomonas predominated among the psychrotrophic microbiota in two groups.L.raffinolacti showed the highest relative abundance in summer samples,while L.piscium showed the highest relative abundance in winter samples.(3)SMRT sequencing was combined to analyse the composition of psychrotrophic bacteria microbiota.The results showed that Proteobacteria was the dominant bacterium,and the relative abundance of Brevundimonas and B.vesicularis in raw milk was the highest.And the relative abundance of Firmicutes(28.6%)and Bacteroidetes(12.2%)in summer group was higher than that in winter group(Firmicutes,7.1%;Bacteroidetes,7.1%).At genus and species level,the dominate OTUs(Operational Taxonomic Unit)in summer group were: Enterococcus(15.6%) and E.faecalis(15.6%),while in winter group were: Brevundimonas(14.6%)and B.vesicularis(13.9%).(4)There were significant differences in community structures of psychrotrophic bacteria among groups.Abundance difference analysis indicated that 103 OTUs showed significant different among two groups,including 3 phyla,22 genera and 44 species.The results of alpha diversity analysis showed that the indices of Shannon and Simpson in summer group were higher than that in the winter group significantly(p<0.05),indicating the diversity of psychrotrophic bacteria in summer group was higher than that in winter group.In addition,beta diversity analysis showed that summer group was clearly separated from winter group,which indicated the significant divergences in the community structures of psychrotrophic bacteria among two seasons.(5)About 68.8% psychrotrophic bacteria had the ability to secrete proteases or lipases.The results of agar diffusion assays showed that 18 dominant genera were able to secrete protease or lipase except Enterococcus and Moraxella.Pseudomonas and Lactococcus had the strongest enzyme-secreting ability.(6)The main hydrolases secreted by dominant psychrotrophic bacteria had strong heat resistance.Enzyme activity tests showed that P.fluorescens secreted the highest protease activity(10.8 ??/(h·m L))and A.guillouiae secreted the highest lipase activity(7.3 ?mol/(min·m L)).The results of heat-resistance study showed that the hydrolases secreted by more than 70% of dominant psychrotrophic bacteria had heat resistance.The protease secreted by P.fluorescens and the lipase secreted by chryseobacterium carnipullorum showed the strongest heat resistance.