Biodiversity, Spoilage Potential, And Survival Strategies Of Aerobic Spore-forming Bacteria In Powdered Milk

Aerobic spore forming bacteria are potential milk powder contaminants and are viewed as indicators of poor quality.Prior to this study,there was no knowledge on the nature,diversity,and prevalence of aerobic mesophilic and thermophilic spore forming bacteria in Chinese milk powders and their harmful impacts and survival strategies during milk powder manufacture.We aimed to explore the microbiota of Chinese milk powders and revealed the world's largest diversity of aerobic spore forming bacteria.In a study on twenty five milk powders,representative of whole China,based on the random amplified polymorphic DNA(RAPD)protocol,738 bacteria were isolated and it was found that Bacillus licheniformis is the most prevalent bacterium with the greatest diversity(?43%of the total isolates)followed by Geobacillus stearothermophilus and Anoxybacillus flavithermus.Actinomycetes appeared a new threat to the Chinese dairy industry due to the prevalence of newly reported species,Laceyella sacchari and Thermoactinomyces vulgaris.Out of the nineteen separate bacterial species(except five unidentified groups)recovered and identified from milk powders,twelve proved to belong to novel or previously unreported species in milk powders.In order to understand any harmful effects caused by this particular micro-flora on the quality and safety of milk powders,spoilage potential of these isolates was explored.Heat resistance of all spores produced by these bacteria at temperature ranging from 100 to 1250C was determined to evaluate the role of spore's heat resistance in bacterial survival strategies.We concluded that spores of two thermophiles(Geobacillus thermoleovorans group and G.stearothermophilus)and one mesophilic species(Breviacillusvis)have potential to survive up to 125? for 30 min.Spoilage potential in terms of bacterial ability to produce lipases,proteases,phospholipases and?-galactosidases was analysed for all strains,and all species with spoilage potential were highlighted.Paenibacillus macerans showed the highest proteolytic activity followed by members of the Bacillus cereus group,Br.brevis,Bacillus subtilis,G.thermoleovorans group and Virgibacillus proomii.The highest lipase producing strains belonged to B.licheniformis.Phospholipase activity was only shown by members of the B.cereus group and Brevibacillus parabrevis.Ten strains showed positive ?-galactosidase activity,while,4 strains showed positive haemolytic activity.B.licheniformis strains,despite belonging to one RAPD group or sub-group showed markedly different phenotypic characters which support the previous findings of heterogeneity in RAPD-based B.licheniformis groups.Evidence was provided that biofilm is an important survival strategy of the most prevalent bacteria in Chinese milk powders.Biofilm forming capacity of isolates from Chinese milk powders was determined on polystyrene and stainless steel(SS)surfaces.We concluded that only four species are of significance for biofilm development on the surface of stainless steel in the presence of skimmed milk,namely,B.licheniformis,G.stearothermophilus,G.thermoleovorans group and A.flavithermus.The maximum number of cells recovered from the biofilms developed on SS coupons in the presence of skimmed milk for these four species was as follows:4.8,5.2,4.5 and 5.3 log CFU/cm2,respectively.Number of cells recovered from bio films on 1 cm2 SS coupons increased in the presence of TSB for all mesophiles including B.licheniformis,while decreased for G.stearothermophilus,G.thermoleovorans group and A.flavithermus.The CV staining assay on polystyrene proved to be inadequate to predict cell counts on SS for the bacteria tested in our trial in the presence of either TSB or skimmed milk.Biofilm formation also proved to be a strain-dependent characteristic and interestingly significant variation in biofilm formation was observed within the same RAPD groups of B.licheniformis which supports the previously reported genetic and phenotypic heterogeneity within the same RAPD based groups.The results support the idea that biofilm formation is an important part of bacterial survival strategy as only the most prevalent isolates from milk powders formed good biofilms on SS in the presence of skimmed milk.The work on biofilms will broaden our knowledge on biofilm formation of a large number of dairy isolates and emphasise strain and substrate dependence.At the end,molecular regulation of bio film formation in the most prevalent bacterium in Chinese milk powders was determined to provide insights into biofilm formation.Transcriptional changes in the planktonic(logarithmic culture)and biofilm states(24 h old)were determined in a high and low biofilm forming B.licheniformis strains using RNA sequencing.Genes,representing 28.3%and 36.3%of the whole B.licheniformis gene content were upregulated in the biofilm state as compared to the planktonic state in high and low biofilm producers,respectively.The gene expression patterns of several groups of genes differed significantly(P<0.05)under the two growth conditions.The genes related to chemotaxis proteins(CheA,CheB,CheC,CheD,CheV,CheW,CheY,MotA,MotB)and flagellar assembley were exclusively upregulated in the planktonic phenotype as compared to biofilms in both strains.In terms of Kyoto Encyclopedia of Genes and Genomes(KEGG)based pathways,metabolic pathways were significantly(P<0.05;Q-values<0.05)downregulated in the planktonic state as compared to the biofilm state in both strains which shows that biofilm formation is an energetically expensive process.Lipid and sugar metabolism seemed to play an important role in the matrix production.Overall,several genes involved in adhesion,matrix production and matrix coating were either absent or less expressed in the biofilm state of low biofilm producer as compared to the high biofilm producer.Interestingly,the genes related to sporulation and extracellular polymeric substances were concomitantly expressed in the biofilm state of both strains which suggests that sporulation is coupled with the biofilm formation in this species similar to B.subtilis.Collectively,these results provide a comprehensive insight into biofilm formation in B.licheniformis that will be helpful for future research into mechanisms and targets.