| ?-Poly-L-lysine(s-PL)is a basic polyamide that consists of 25-30 residues of L-lysine with an ?-amino group-?-carboxyl group linkage.?-PL has been widely applied to food preservation because of its high thermostability,safety,andstrong antimicrobial activity.The antibacterial activity of ?-PL is closely related to its molecular weight and large molecular weight of ?-PL has a bitter taste,causing some limitations for applications in the food in the food industry.Although ?-PL has been recognized safety,the changes of microbial cells treated by different molecular weight of ?-PL are poorly understand.Moreover,it is now widely recognized that the antimicrobial mechanism of antimicrobial peptides involves cell membrane damage.However,the detailed antimicrobial mechanism of ?-PL with different molecular weight is unclear,and conclusive evidence for the proposed hypothese together with the molecular mechanism of action of ?-PL on the cytoplasmic membrane is still lacking.Furthermore,the study of action mode of ?-PL with different molecular weight is rarely reported.Therefore,in this study,a common yeast strain of Saccharomyces cerevisiae AS2399 was used as type strain,and the effect of ?-PL with different molecular weight on S.cerevisiae cells was investigated from multiple perspectives,such as cell morphology,cell membrane,cell wall and metabolome,in order to futher understanding of the antimicrobial mechanism of ?-PL with different molecular weight and provide more basic data for the applications of ?-PL form the perspective of food safety angle.?-PL product is separated into three components by tangential flow ultrafiltration system,average degree of polymerization are 8.02,15.21 and 21.83,respectively.And the number-average molecular weights are 1027.44,1967.77 and 2816.39,respectively,which provides a source of material for further research.By measuring the inhibitory activity of ?-PL of different molecular weight acting on S.cerevisiae,we found MICs are different claraly.When the concentration was 250?g/mL,?-PL with low molecular weight(<1KDa)had no inhibition to S.cerevisiae,?-PL with higher molecular weight(1-3KDa and>3KDa)and product significantly inhibited S.cerevisiae.Meanwhile ?-PL with higher molecular weight enhanced activity of cell membrane penetration,making the cell growth inhibited,thereby reducing the growth rate of cell density.?-PL with lower molecular weight(<1KDa)no similar effect.By the SEM we found cell morphology undergone various changes.Cells treated by ?-PL with higher molecular weight(1-3KDa and>3KDa)and product became rough,appeared depressed and micelles.The cell surface treated with ?-PL of low molecular weight(<1KDa)only had minor changes.The metabolomics approach was then utilized to compare the global metabolite profiles of S.cerevisiae in response to ?-PL of different molecular weight and alcohol at different levels.A total of 82 metabolites were analyzed by Principal component analysis(PCA),Hierarchical clustering analysis(HCA)and Partial least squares analysis(PLS).the results suggested that ?-PL treatment groups had a significantly different clustering compared with the control group,and cell exposure to ?-PL might have led to systematic changes in cell metabolism pathways.Futher analysis revealed that the ?-PL of different molecular weight stress led to different affections of primary metabolic pathways through the suppression of the tricarboxylic acid cycle and glycolysis.Meanwhile,some protective metabolites were accumulated differentle in the ?-PL treatment groups,.By measuring changes in the three main components of ?-1,3-glucans,mannose phosphate and chitin content and the distribution of chitin in the cell wall to study the effects of?-PL of different molecular weight to the cell wall.?-PL of different molecular weight had different effects on cell wall of S.cerevisiae,?-PL of high molecular weight(1-3KDa,>3KDa)and product made the changes of ?-1,3-glucan and mannose phosphate content more significantly than ?-PL of low molecular weight(<1KDa).While the effect of?-PL of different molecular weight let the content and distribution of cell wall chitin similar.We speculated that the impact of different molecular weight of s-PL aciting on S.cerevisiae cell wall may be the secondary damage the cell membrane produced.Finally,based on the above experimental results we obtained the possible inhibitory mechanism of s-PL with different molecular weight acting on S.cerevisiae.?-PL of low molecular weight(<1KDa)caused membrane phospholipid bilayer slight bending,and slight inhibition of the glycolytic pathway.Meanwhile the cell walls were weakly disturbed.?-PL of high molecular weight(1-3KDa,>3KDa)action on S.cerevisiae in the carpet model described.Cell membrane function was disturbed,?-PL of high molecular weight(1-3KDa,>3KDa)indirectly inhibited the intracellular central carbon metabolism,at the same time,damaged the cell wall structure and changed function.The growth and reproduction of S.cerevisiae were affected. |
PDF Full Text Request