| Biofilms in nature are defined as a microbially derived sessile community that primarily consists of viable and nonviable microorganisms,which are irreversibly attached to a substratum,interface,or each other.Bacterial cells are embedded in their self-produced matrix called extracellular polymeric substances（EPS）.The formation of biofilms on food processing surfaces could lead to undesirable outcomes,such as enhancing bacterial resistance to antimicrobials,increasing production of secondary metabolites,and serving as potential contamination sources for food products.However,there is limited qualitative literature on the bacterial biofilm in the milk powder production system.Therefore,a systematic characterization of the distribution of food contact-attaching bacteria in a commercial milk powder processing factory was evaluated in this study.The main research objectives and results are summarized as follows:1.The microbial diversity of the microbiota in the milk powder processing systemResidual bacteria in the milk powder processing system after clean-in-place（CIP）processes were collected in this study.Bacteria were isolated from eleven different sampling sites and identified based on 16S r DNA sequence analysis.Forty-five strains were obtained and subsequently identified as Acinetobacter spp.（8）,Bacillus spp.（22）,Clostridium spp.（1）,Enterobacter spp.（2）,Microbacterium spp.（1）,Micrococcus spp.（2）,Moraxella spp.（2）,Pseudomonas spp.（2）,Serratia spp.（3）,and Staphylococcus spp.（2）.Bacterial contamination was present in all sampling sites and the distribution of these strains was uneven in the milk powder processing system.Of Gram-positive isolates,78%were Bacillus spp.,while 47%of Gram-negative isolates were Acinetobacter spp.At the same time,there was a difference in the distribution of the microbiota:47%of the isolated strains were isolated from milk tanks,while 18%were isolated from pasteurizer heat-exchangers.2.Biofilm-forming ability of the isolates recovered in the milk powder processing systemThe biofilm-forming abilities of the forty-five strains grown on AISI 304 stainless steel surfaces were evaluated by the crystal violet staining method and the CIP tolerance of these biofilms was investigated.All of the strains in this study showed biofilm-forming abilities when grew on stainless steel surfaces and presented moderate hot-alkali and hot-acid resistance,indicating that biofilm formation is an important part of bacterial survival strategy in the milk powder processing system.Moreover,according to the D-values,strains were found to be more resistant to hot-alkali than hot acid,suggesting that hot acid washing might be used to reduce colonization by hot-alkali resistant strains.Meanwhile,the effects of temperatures and growth media on the production of biofilms by these strains on polystyrene surfaces were assessed.The results showed that biofilm formation was a strain-dependent characteristic and influenced by several environmental factors such as temperature and nutrient availability,and there was no uniform tendency.3.Effects of carbon sources on the biofilm-forming capability and metabolic activity of B.licheniformis and B.cereusTo clarify the effects of carbon sources on the biofilm-forming capability of Bacillus licheniformis（Site 1 EF105377）and Bacillus cereus（Site 3 AJ577290）,which showed a strong ability to form biofilms on stainless steel surfaces,the production of biofilms formed by B.licheniformis and B.cereus under different nutrient conditions（glucose,maltose,lactose and skim milk）was evaluated by crystal violet staining method.The results indicated that B.licheniformis and B.cereus were able to form biofilm in all conditions(OD570 values>0.067).Furthermore,both glucose and maltose could improve biofilm formation and metabolic activities of B.licheniformis and B.cereus.Skim milk could increase the biofilm biomass of B.licheniformis and B.cereus and the metabolic activity of B.licheniformis,while lactose could only increase the metabolic activity of B.licheniformis.These results indicated that lactose is not the main component of skim milk that effected the biofilm-forming ability and metabolic activity of B.licheniformis and B.cereus.4.Rearrangement of carbon metabolism during biofilm development of B.licheniformis under different carbon sourcesTo clarify the metabolic control mechanism of carbon sources on the biofilm formation of B.licheniformis,the overall regulation of genes at the transcription level in biofilms formed by B.licheniformis under different carbon sources was investigated using the Illumina Hi Seq sequencing platform,and the most enriched pathways were further analyzed in each category.The differentially expressed genes mainly distributed in amino acid metabolism,pyruvate metabolism,myo-inositol metabolism,fatty acid metabolism,flagella assembly,and bacterial chemotaxis.The carbon flux toward the tricarboxylic acid pathway decreased through different metabolic pathways under different carbon sources.（1）Genes encoding acetoin synthesis by pyruvate via branched-chain amino acid metabolism pathway in the glucose group were up-regulated.（2）Genes related to the myo-inositol degradation and genes related to fatty acid degradation in the maltose group were down-regulated.（3）Genes related to fatty acid degradation in the lactose group were down-regulated.（4）For the skim milk group,genes related to the acyl-Co A derivative degradation were down-regulated.Thus,the carbon flux toward the tricarboxylic acid pathway decreased by a decreased concentration of the acetyl-Co A under different carbon sources.We inferred that carbon flux toward the extracellular polysaccharide synthesis pathway increased,contributing to an increase in the biofilm biomass of B.licheniformis.Moreover,B.licheniformis showed the highest utilization ratio of glucose,with up-regulated genes related to chemotaxis proteins and flagellar assembly.5.Rearrangement of carbon metabolism during biofilm development of B.cereus under different carbon sourcesTo clarify the metabolic control mechanism of carbon sources on the biofilm formation of B.cereus,the overall regulation of genes at the transcription level in biofilms formed by B.cereus under different carbon sources was investigated using the Illumina Hi Seq sequencing platform,and the most enriched pathways were further analyzed in each category.The differentially expressed genes mainly distributed in glycolysis and pentose phosphate pathway,tricarboxylic acid cycle,amino acid metabolism,and fatty acid metabolism.Results showed that genes related to the glycolytic pathway and/or pentose phosphate pathway were up-regulated in four kinds of carbon sources,while genes related to the tricarboxylic acid pathway were not up-regulated.Thus,we inferred that carbon flux toward the extracellular polysaccharide synthesis pathway increased,contributing to an increase in the biofilm biomass of B.cereus.（1）Genes related to acetoin synthesis by pyruvate via branched-chain amino acid metabolism pathway in the glucose group were up-regulated.（2）Genes related to valine,leucine,and isoleucine synthesis by pyruvate in the maltose group were up-regulated.（3）Genes related to acetoin synthesis by pyruvate via branched-chain amino acid metabolism pathway in the lactose group were up-regulated.Then,the carbon flux toward the tricarboxylic acid pathway decreased by a decreased concentration of the acetyl-Co A in the glucose,maltose,and lactose groups.（4）For the skim milk group,the concentration of pyruvate increased via the glycolysis pathway,while genes related to the tricarboxylic acid pathway were not up-regulated.These results revealed a theoretical foundation for further research on the mechanisms and targets of novel control measures and contributed to the microbiological safety in the milk powder processing system. |
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