Study On The Antibacterial Mechanism And The Application Of Protocatechualdehyde Against Micrococcus Luteus And Listeria Monocytogenes

Protocatechualdehyde(PCA)is a natural antimicrobial agent mostly found in Salvia miltiorrhiza root.PCA has been found to have broad-spectrum antimicrobial effects as well as antioxidant,anti-inflammatory,and anti-cancer effects.Micrococcus luteus and Listeria monocytogenes are typical foodborne pathogenic bacteria.M.luteus can contaminate meat,aquatic products,soybean products,and other food products.L.monocytogenes can contaminate meat,dairy products,fruits,and vegetables,which can be harmful to human health.PCA has been widely used in food preservation because of its abundant resources,green health,safety,and non-toxic characteristics.This thesis aims to investigate the mechanism of membrane damage of PCA on M.luteus and L.monocytogenes and to evaluate its preservative effect in raw pork and cooked chicken breast,which provides theoretical support for the development of green,safe,and healthy natural food antimicrobial agents.The main findings of this thesis are as follows:(1)The antibacterial mechanism of PCA against M.luteus and L.monocytogenes was investigated by a series of in vitro antibacterial experiments.The results showed that the minimum inhibitory concentration(MIC)of PCA on M.luteus and L.monocytogenes were 1.25 mg/mL and 0.625 mg/mL,respectively.The abnormal changes in membrane potential,the decrease of intracellular ATP concentration,and intracellular pH concentration indicated that PCA affected the membrane integrity of M.luteus and L.monocytogenes.Confocal laser scanning microscope(CLSM)observation showed that PCA increased the membrane permeability of M.luteus and L.monocytogenes and induced the exudation of their contents,leading to cell death.Field emission gun scanning electron microscope(FEG-SEM)observation showed that PCA ruptured the cell membrane of M.luteus and L.monocytogenes,causing the contents to flow out,and giving the cells a concave morphology.(2)Since PCA has good antimicrobial activity against M.luteus and L.monocytogenes,PCA was applied as a natural antimicrobial agent in raw pork and cooked chicken breast,and physiological and biochemical parameters were examined to analyze its preservation effect.The results showed that PCA was found to inhibit the increase of M.luteus colony count in raw pork and L.monocytogenes colony count in cooked chicken breast,the growth rate of thiobarbituric acid reactive substances(TBARS)and moisture mobility.In addition,PCA improved the color and texture of raw pork and cooked chicken breasts,effectively extending their shelf life.(3)To further investigate the repressive ability of PCA,the molecular mechanism of PCA against M.luteus was investigated based on the transcriptome level.The results showed that PCA induced differential expression of 346 genes in M.luteus,of which 152 differentially expressed genes(DEGs)were up-regulated and 194 DEGs were down-regulated.The GO functional enrichment analysis of DEGs was mainly distributed in metabolic processes,cellular processes,localization,cellular anatomical entities,catalytic activity,and binding,mostly exhibiting gene down-regulation properties.The KEGG enrichment analysis showed that the important enrichment pathways of DEGs include phenylalanine metabolism,glyoxylate and dicarboxylic acid metabolism,ribosomes,ABC transporter,and other metabolic pathways.PCA inhibits the growth of M.luteus by destroying the integrity and structure of the cell wall and cell membrane of M.luteus,resulting in intracellular amino acid metabolism disorders,impaired energy metabolism pathways,and acidification of the intracellular environment.(4)The molecular mechanism of PCA against L.monocytogenes was investigated by transcriptome,and the results showed that PCA induced differential expression of 562 genes in L.monocytogenes,of which 249 DEGs were up-regulated and 313 DEGs were down-regulated.The GO functional enrichment analysis of DEGs was mainly distributed in metabolic processes,cellular processes,cellular anatomical entities,catalytic activity,and binding,mostly exhibiting gene up-regulation properties.The KEGG enrichment analysis showed that DEGs was significantly involved in metabolic pathways such as ABC transporter,phosphotransferase system(PTS),and purine metabolism.PCA interferes with various amino acid synthesis and metabolic pathways in L.monocytogenes cells,impairs DNA replication and repair systems,inhibits carbon catabolism,causes damage to cell membranes,and cannot maintain energy and nutrients needed for normal cell survival,thereby inhibiting the growth of L.monocytogenes.