| With the popular of natural products research, the studies of their functional ingredients, nutritional value and physiological activity have attracted a wide spread attention, the screening of low toxicity, no drug resistance of natural medicine as a new type of antibacterial agent become a research hotspot. It is reported that blueberry leaves polyphenols, chlorogenic acid,cinnamaldehyde and thymol have inhibitory effect on many kinds of bacteria.But the main compounds of blueberry leaves polyphenols which have antimicrobial activities and their antimicrobial mechanisms are still unclear. Therefore, this study selected antimicrobial active substances of these three kinds of plants , designed to track their antimicrobial active substances, and to explore its antibacterial mechanism and provide theoretical basis for the development of new type of antibacterial agent.The contents and results are as follows:1.Quantitative Analysis and Antimicrobial Activity of Polyphenols from Blueberry LeavesThe composition of polyphenols from blueberry leaves was quantitatively analyzed by HPLC-MS/MS and their antibacterial activities against foodborne pathogens were evaluated. The results showed that polyphenols from blueberry leaves were mainly composed of 5-O-caffeoylquinic acid, O-p-coumaroylquinic acid,3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, B-type proanthocyanidin dimer,Kampferol-3-O-hexiside, Quercetin-3-O-gluconide and Quercetin-3-O-pentoside. The minimum inhibition concentrations (MIC) against Salmonella and Bacillus subtilis were below 9.30 mg/mL, while others were all below 4.65 mg/mL. The antibacterial activity from strong to weak was: Staphylococcus aureus>Escherichia coli>Listeria monocytogene >Shigelta bogdii>Salmonella>Bacillus sublilis, which indicated the diverse bioactive polyphenols from blueberry leaves have many bioactive compounds,some of which have the broad-spectrum antibacterial activity and can be developed to a phytogenic bactericide.2.Study on antimicrobial activities of three plants extractsWith Zone of inhibition, antimicrobial diameter minimal inhibitory cincentration(MIC)and minimum bactericidal concentration(MBC)measured.The results showed that Plant bacteriostatic agent had a strong inhibitory effect against these 4 kinds of pathogenic bacteria,chlorogenic acid,cinnamaldehyde and thymol had inhibitory effect against Listeria monocytogene with Zone of inhibition(15.07±0.15)mm,(30.13±0.06)mn and (15.17±0.15)mm;with MIC 2500 ?g/mL,312.5 ?g/mL and 312.5 ?g/mL; with MBC 1250 ?g/mL,156.25 ?g/mL and 78.125 p.g/mL respectively. On the basis of plants extracts had showed strong antibacterial activity against Listeria monocytogene, it was used as the indicator bacteria for further study.3.The leakage of endocellular contents of Plant bacteriostatic agent on bacteria cellAfter Listeria monocytogenes treated by three plants extracts. The results showed that the OD260, electric conductivity,the glucose and alkaline phosphatase contents were markedly incresed,Besides, the composition of soluble membrane protein was markedly decresed after treated by plant extracts, which further confirmed that the three plants extracts could damage the structure of cell wall and membrane, which resulted in the increase of permeability of cell membrane and release of cell components, resulted in inhibiting the growth of cell ,and killing the cell.4. Toxicity of Plant bacteriostatic agent on cell membraneThe change of micromorphology, membrane damage and cFDA/PI double staining were analyzed by scanning electron microscope,fluorescence microscope and flow cytometry respectively.Besides, The membrane damage were explored by determining the change in MDA and SOD content. As the results showed,Listeria monocytogenes's membrane was damaged seriously;After treated by 1MBC chlorogenic acid,cinnamaldehyde and thymol, the apoptotic rate were21.9%, 95.6%and95.1%;The damage rate were 73.7%, 3.6% and 0.6% respectively.The antibacterial activity from strong to weak was:thymol > cinnamaldehyde >chlorogenic acid. |
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