The Potential Hazards Of Psychrotrophic Bacteria In Raw Milk

Milk is one of the ideal foods among consumers due to its high nutritional value,while it also serves as a good medium for the growth of a broad spectrum of microorganisms.In order to inhibit the growth of microorganisms,the cold chain system is widely used in the dairy industry.However,psychrotrophic bacteria can still grow at low temperatures and gradually become the dominant microflora in raw milk.In general,the secretion of heat-resistant spoilage enzymes and the formation of biofilms are two vital factors that restrain the development of dairy industry.Although psychrophilic bacteria can be killed by conventional heat treatment,the heat-resistant proteases and lipases produced by psychrophilic bacteria may still remain activity,which potentially affect the quality and shelf-life of dairy products.Biofilms formed by psychrophilic bacteria may lead to foodborne diseases,dairy spoilage,the loss of processing equipment and an increase in energy consumption.In this study,480 psychrophilic bacterial isolates from 16 raw milk samples were clustered and identified,and their potential risks based on the production of spoilage enzymes and biofilms formation were studied.Meanwhile,the regulation of AHLs on the growth,secretion of spoilage enzymes and biofilm formation of of psychrophilic bacteria was also explored.A number of 480 psychrophilic bacterial isolates in raw milk samples from 16 different regions were clustered into 85 different RAPD groups based on different RAPD patterns.The 16S rRNA results showed that Pseudomonas(58.8%),Acinetobacter(13.3%),Flavobacterium(6.0%),Sphingomonas(4.2%)and Serratia(3.1%)were the dominant genus.At the species level,Pseudomonas fluorescens(15.8%)proved to be the most dominant species,followed by Pseudomonas fragi(7.1%),Pseudomonas psychrophila(5.4%),Acinetobacter johnsonii(5%),Pseudomonas putida(4.6%)and Pseudomonas azotoformans(3.8%).Isolates belonging to the same species,such as P.fluorescens,P.fragi and P.psychrophila,have different RAPD profiles.In addition,some psychrotrophic bacteria have been identified as potential novel species in raw milk.Isolates of Pseudomonas,Acinetobacter,Flavobacterium,Chryseobacterium,Serratia,and Aeromonas proved to be the strong enzyme producers either at the room temperature or the low temperature.Proteases and lipases produced by psychrotrophic bacteria retained activity after heat treatment at 70,80,or 90?,and further thermodynamic parameters determination of proteases and lipase also confirmed that these enzymes have high thermal stabilities.The production of spoilage enzymes is a temperature-dependent process,as the incubation temperature siginificantly affected the enzymes production,while no significant effect on the heat resistance of enzymes was observed.Most psychrophilic bacterial isolates have the ability to form biofilms at 7?,with the OD595 values ranging from 0.087 to 1.324.Results showed that biofilm formation is a strain-dependent trait,and 25,10,19 RAPD groups were weak,moderate and high biofilm formers,respectively,based on results of the crystal violet staining.The biofilm formation ability of isolates belonging to Pseudomonas were significantly higher than that isolates of other genera.The composition of media is a key factor that affect the biofilm formation by bacteria.Psychrophilic bacteria were more likely to form biofilm on the surface on stainless steel in TSB(1.15-6.63 log CFU/cm2)than in skim milk(1.04-6.llog CFU/cm2).A moderate correlation(r=0.42)between the crystal violet assay of polystyrene and methods based on cell enumeration on coupons was observed,indicating the reliability of the two methods for evaluating the biofilm formation by bacteria.A marked variation in biofilm-forming capacity was observed within the strains belonging to the same species.Bacterial interactions significantly affected the spatial properties and the resistance to disinfectants of the formed biofilms.Pseudomonas libanensis enhanced the biofilm formation by Chryseobacterium oncorhynchi and Aeromonas hydrophila,and a synergistic effect was observed between P.libanensis and C.oncorhynchi.The resistance to three kinds of disinfectants(peroxyacetic acid,hydrogen peroxide and sodium hypochlorite)of the two dual-species biofilms(P.libanensis+A.hydrophila,P.libanensis+C.oncorhynchi)was significantly increased when compared to each single-species biofilm.Peracetic acid proved to be the most effective disinfectant to remove biofilms,followed by hydrogen peroxide and sodium hypochlorite.Both the plate counting method and the CLSM observation method effectively evaluated the disinfection treatment on the biofilms.The capacity of producing AHLs were screened by reporter plate method,and the positive detection rate of 28 isolates was 71.43%.LC-MS was further applied to identify the structure of AHLs produced by P.azotoformans(a)and Serrita liquefaciens,and it was found that P.azotoformans(a)produced C6-HSL,while S.liquefaciens produced C8-HSL.Exogenous addition of C6-HSL and C8-HSL significantly promoted the growth,spoilage enzymes production and biofilm formation of P.azotoformans(a)and S.liquefaciens.Meanwhile,exogenous addition of C6-HSL and C8-HSL also accelerated the spoilage of milk caused by P.azotoformans(a)and S.liquefaciens.The particle size of milk was significantly increased,and the acid flavor in milk is significantly increased.RNA sequencing results revealed that the addition of AHLs significantly regulated genes expressions of P.azotoformans(a)and S.liquefaciens,and up-regulated genes were more than down-regulated genes.AHLs play a regulatory role in the growth,production of spoilage enzymes and biofilm formation of psychrotrophic bacteria.Up-regulated genes(ribosomes,oxidative phosphorylation,glycolysis,TCA cycle,protein transport,amino acid metabolism,fatty acid metabolism,purine metabolism and resistant to stress)that closely relating to bacterial growth promotes bacterial growth and leads to higher concentrations of bacteria.Up-regulated genes relating to the biofilm formation(bacterial flagellar structure)and enzymes production also improved capacity to form biofilms and produce enzymes by psychrotrophic bacteria,respectively.In addition,the regulation of QS showed a dependence on types of AHLs and strains.