The Screening Of Natural Plant Source Food Preservatives And The Research Of The Antimicrobial Mechanism Of Cinnamaldehyde

In order to select effective and safe natural antibacterial ingredients, more than30kinds of plant extracts were selected for their suitability as antibacterial agents. Astandard broth microdilution method was used to evaluate their antimicrobial activity,a cytotoxicity test was used to detect their safety, and a synergy assay was used todetermine which combinations have a synergistic effect. Then time-kill curves wereused to further verify their bactericidal capacity. As a result of these tests,cinnamaldehyde and carvacrol were identified first (MIC0.31mg/mL), thensubsequently verified to be the most effective and safe natural active substances. Itwas found that all of the11tested bacteria strains (E. coli, S. aureus, Y. regensburgei,S. intermedius, K. kristinae, L. garvieae, S. sanguinis, E. cloacae, S. haemolyticus, A.hydrophila, S. enteritidis) were sensitive to cinnamaldehyde and carvacrol. Thecombination of cinnamaldehyde with carvacrol also showed good synergisticantibacterial effect against7of the11tested bacterial strains (E. coil, S. aureus, Y.regensburge, S. intermedius, K. kristinae, S. haemolyticus, S. enteritidis). Thetime-kill assay verified synergism for the cinnamaldehyde/carvacrol combinationtoward Escherichia coli and Staphylococcus aureus. These results indicated that thecombination of cinnamaldehyde and carvacrol may serve as a promising naturallysourced food preservative with excellent bactericidal activity against common foodspoilage microorganisms.Gram-negative Escherichia coli (ATCC8735) and Gram-positiveStaphylococcus aureus (ATCC3101) were selected as model bacteria to determine theantimicrobial mechanism of cinnamaldehyde. Several techniques were utilized toinvestigate the effects of cinnamaldehyde on food-borne bacterial membranes. Theultraviolet (UV) absorption and electrical conductivity of the culture supernatant were used to determine membrane integrity. β-Galactosidase activity was determined todetect inner membrane permeability. Scanning electron microscopy (SEM) andtransmission electron microscopy (TEM) were performed to observe bacterialmorphology. Samples from both strains exposed to cinnamaldehyde showed higherUV absorptions, conductivity values, and β-Galactosidase activities compared withthe control group and displayed a rapid rise trend. Thereafter, the values stabilized at arelatively steady state. SEM shows that treated E. coli and S. aureus cell samplesexhibited rough cell membranes with particulate matter, and some of the S. aureuscells split due to deep wrinkle formation and distortion, unlike the control group.TEM shows that the bacteria treated with cinnamaldehyde exhibited numerousabnormalities, including cytoplasmic membrane separation from the cell wall, cellwall and cell membrane lysis, cytoplasmic content leakage, cytoplasmic contentpolarization, cell distortion, and cytoplasmic content condensation. These resultsindicate that bacterial cell morphology, membrane integrity, and permeability aredamaged when the E. coli and S. aureus cells are exposed to the minimum inhibitoryconcentrations of cinnamaldehyde (0.31mg/mL). In addition, the higher thecinnamaldehyde concentration, the more serious the bacterial membrane damage is.The E.coli ATCC8735was used in the genechip experiment to study theantimicrobial action of cinnamaldehyde. Thereafter, The PCR and western-blotexperiments were then used to vetify the result of genechip experiment. The genechipshows that126genes expressed differentially (2-fold difference in expression as asignificant criterion), where the number of up-regulated genes is83(65.9%) and thenumber of down-regulated genes is43(34.1%). The most important genes whichwere affected by cinnamaldehyde were7flagellin related genes,3cell membranesynthesis-related gene,2cytoplasmic protein-related gene,3chaperone protein genes,2secreted protein genes and ATP metabolism-related genes. The results of PCR andwestern-blot experiments coincide with the genechip study, which is to say, the thegenechip study is effective. These results indicate that flagellin protein and cellmembrane may be the preliminary antibacterial mechanism of cinnamaldehyde. Then,cinnamaldehyde would affect the synthesis and aggregation of cytoplasmic protein, and interfere ATP metabolism. These effect of cinnamaldehyde would lead bacterialmetabolic disorder and autolysis.