Antibacterial Mechanism Of Apis Laboriosa Honey And Its Application

Honey is a natural sweet substance made from the nectar,extra nectary secretions or honeydew collected by bees and mixed thoroughly with their own secretions,and is widely consumed because of its unique flavor and biological activities.Apis laboriosa honey（ALH）is a wild honey having antioxidant and antibacterial activities.The total phenols and total flavonoids of ALH samples were determined by hydroalcoholic extraction method.The DPPH free radical scavenging activity,ABTS free radical scavenging activity,FRAP total antioxidant capacity,superoxide anion free radical scavenging activity were determined using spectrophotometry.The inhibition ability of ALH against Salmonella typhimurium,Staphylococcus aureus,Escherichia coli and Listeria monocytogenes were evaluated by minimum inhibitory concentration（MIC）and bactericidal concentration（MBC）.Meanwhile,the pathways and mechanisms of ALH against Salmonella typhimurium were investigated based on Label-free proteomics.The preservation effect of ALH on orange juice was also determined.The main results were as follows:（1）Physicochemical properties,antioxidant and antibacterial activity of ALH:the analysis of physicochemical indicators of five ALH samples,The total phenolic content ranged from 49.18 to 23.16 mg GAE/100g and total flavonoid content ranged from 3.03 to 18.99 mg RE/100g.The analysis of antioxidant results showed that ALH had DPPH radical scavenging ability（from 16.69 to 64.37 mg TE/100g）,ABTS radical scavenging ability（from 32.82 to 105.03 mg TE/100g）,Fe²⁺complexing ability（from 38.54 to 105.03 mg TE/100g）,superoxide anion radical scavenging activity（from 76.10 to 91.26%）.A total of 73 phenolic compounds were identified by LC-MS/MS,including phenolic acids,flavonoids,and tannins.MIC and MBC of ALH against Salmonella typhimurium,Staphylococcus aureus,Escherichia coli and Listeria monocytogenes were 20～30%（w/v）,25～40%（w/v）,respectively.The antibacterial activity of ALH was likely equal to manuka Honey 5+.ALH1 was randomly selected for the study of inhibition mechanism,which IC₅₀was 2.97%（w/v）.（2）Proteomics study of S.typhimurium by ALH1 based on Label-free technique:There were totally 27265 peptides and 2733proteins were identified.There were 130（18.36%）up-regulated differentially expressed proteins and 578（81.64%）down-regulated differentially expressed proteins.According to the functional classification of GO,molecular functions mainly include oxidoreductase activity,transport activity,transmembrane transporter protein activity,matrix-specific transmembrane transporter protein activity;single organism process,redox process,single organism metabolic process in biological process;cell membrane in cellular component,overall component of membrane.By KEGG enrichment analysis,the differential protein was enriched in carbon metabolism,tricarboxylic acid cycle,microbial metabolism in different environment,bacterial chemotaxis pathways.According to the prediction of protein-protein interaction based on STRING DB,the differential proteins in Salmonella typhimurium cells had a very complex network interaction relationship after being treated with ALH1.The top 4 interactional proteins were aldehyde-alcohol,putative pyruvate-flavodoxin oxidoreductase,N-acetyl glucosamine specific PTS system components IIABC and malic enzyme.（3）RT-PCR analysis of the protein-regulated genes in Salmonella typhimurium affect by ALH:According to the results of proteomics,10genes were selected for RT-PCR analysis,which were icdA,gltA,fumC,nuoC,sucB,lpdA,motA,FliG,motB and pgi.Genes icdA,gltA,fumC,nuoC,sucB,lpdA,motB,pgi was down-regulated expression,and motA,FliG were up-regulated.（4）Application of Apis laboriosa honey in orange juice preservation:When ALH were added to unpasteurized freshly squeezed orange juice,the pH,titratable acid,and soluble solids content of the juice were preserved because of low pH,sugar content and organic acids of ALH.Additionally,its antibacterial properties prevent the growth of microorganisms and delays the spoilage of the juice.In conclusion,ALH has good antioxidant and antibacterial abilities.The antibacterial of ALH against S.typhimurium mainly through affecting the carbon metabolism,tricarboxylic acid cycle,microbial metabolism in diverse environments and bacterial chemotaxis pathways.Moreover,ALH has shown effectively inhibition effect on the growth of microorganisms in freshly squeezed orange juice.This finding provides valuable insights for the future development of ALH as a biological preservative,as well as other preservative materials.