Formation Mechanism And Control Strategies For Biofilm Of Dominant Thermophilic Bacteria During Milk Powder Processing

In milk powder processing,the occurrence of thermophilic bacteria is a worldwide issue to dairy industry.Thermophilic bacteria can proliferate rapidly at high temperatures,and the heat-resistant spoilage enzymes they produced may pose potential risks in quality and safety of milk powder products.A variety of thermophilic bacteria can form biofilms adhered on the inner wall of the pipelines,pasteurization equipment,heat exchanger,evaporators and so on,leading to microbial cross contamination.Especially in the wet-mix milk powder processing,temperatures used in the evaporation unit(50 ~oC～70 ~oC)are suitable for fast multiplication of thermophilic bacteria with the help of rich nutrients in milk,thus becoming a restrictive factor to continuous production.Milk powder processing samples were collected from different sites and time points,and were analyzed by high-throughput sequencing.Meanwhile,thermophilic bacteria were also isolated and identified.Approximately,10 CFU/m L thermophilic bacteria were detected before evaporating process,showing that pasteurization and UHT were not able to kill thermophiles and their spores completely.Evaporation process and the feed tank after it are the key points of prevention and control.Main species of thermophilic bacteria isolated in the manufacturing process were Bacillus licheniformis,Anoxybacillus flavithermus,Geobacillus stearothermophilus and Bacillus subtilis.The biofilm forming capacities and spoilage effects of the main thermophilic bacteria were determined in both single-species and mixed-species cultivation on the stainless steel surface on in skim milk at 55 ~oC.In single-species cultivation,the highest bacterial number recovered from surface biofilm,6.12 log CFU/cm~2,was detected for B.licheniformis,followed by G.stearothermophilus as 6.01 log CFU/cm~2.In four-species cultivation,the total cell numbers recovered from surface biofilm were 4.35 log CFU/cm~2,5.28 log CFU/cm~2,5.88 log CFU/cm~2and 6.15 log CFU/cm~2at 12 h,24 h,36 h and 48 h,respectively.B.licheniformis,accounting for 96.3%,was the most dominant species in the four-species biofilm after the incubation for 12 h.After 24 h,G.stearothermophilus occupied the highest proportion.Furthermore,the thermophilic bacteria had strong ability to decompose proteins and lactose in milk.Metabolomics analysis was performed to investigate dynamic changes of the metabolites and their roles during process of biofilm development of the dominant species B.licheniformis.Amino acid metabolism was quite active during early biofilm development from 8 to 12 h,among which,arginine biosynthesis pathway played an important role in biofilm construction.For the middle stage to mature stage during 16to 24 h,galactose metabolism and sphingolipid metabolism had crucial effects.It is a comprehensive metabolic network based on cooperation from in carbohydrate metabolism,lipid metabolism,amino acid metabolism and nucleotide metabolism during B.licheniformis biofilm development.Metabolites of a Lactobacillus plantarum strain were found to effectively inhibit biofilms formed by B.licheniformis.There was a 10-h delay on biofilm formation by the addition of L.plantarum cell free supernatant.Confocal laser scanning microscope images showed that L.plantarum metabolites decreased the number of B.licheniformis cells adhered to stainless steel and glass surfaces.3 k Da～10 k Da components from ultra-filtrates of L.plantarum cell free supernatant were proved to inhibit biofilm formation by B.licheniformis significantly,followed by components less than 3 k Da.Tandem mass tag-based quantitative proteomics analysis was employed to explore the biofilm formation mechanism of B.licheniformis and the inhibition mechanism from L.plantarum.It is indicated that bacterial chemotaxis,flagellar assembly and two-component system played important roles on biofilm formation by B.licheniformis.Spo0F,Kap B,Mot B,Fli G and Fli K are the key regulators for the biofilm prevention by L.plantarum,and intervention on two-component system is an important reason for L.plantarum to inhibit biofilm formation of B.licheniformis.Efficacies of B.licheniformis biofilm removal by ultrasound,enzymes and chemical disinfectants were evaluated,among which,benzalkonium chloride proved to be the most useful reagent.The minimum bactericidal concentration of benzalkonium chloride against B.licheniformis biofilm was 0.125%.A combined cleaning regime of0.0625%benzalonium chloride and CIP was designed.After different treatments,the living cells proportions of control group,0.0625%benzalonium chloride group,CIP group and combined group on stainless steel were 94.1%,59.2%,36.2%and 0.5%,respectively.Compared with the control group,the reductions of residual biofilm colonies were 1.61 log CFU/cm~2,3.03 log CFU/cm~2 and 3.89 log CFU/cm~2 in those three treatment groups respectively.All these results involved in community structure of thermophilic bacteria in milk powder processing,analysis of biofilm formation mechanism and inhibition mechanism of the major dominant bacteria,and exploration of efficient methods for biofilm prevention and control,contribute to improving quality of milk powder products and building a prevention and control system in microbial safety for dairy industry,which have important theoretical and practical significance.