The Inhibition Activity And Synergisticmechanism Of ε-Polylysine、Nisin And Natamycin

With the development of social economy, both green foods and health foods are becoming increasingly popular.It is evidenced that the chemical antimicrobial agents that are commonly used for the food preservationare potentially harmful to human body.Therefore, there is a huge demand for the safety and low-toxicity natural antimicrobial agents in recent years. Natural antimicrobial agents exhibit the better preservative quality, lower cost and broader antibacterial spectrum when used in the combination than used alone. Therefore, it is of practical significance to study the combined-utilization of the naturalantimicrobial agents.In this paper, Nisin, ε-PL and natmycin are examined for their antibacterial potency when used alone as well as when used in the combination. The results showed that combined-treatment of s-PL and nisin exhibited the strongest antibacterial effect. Furthermore, their synergistic antibacterial effects and involved mechanisms were investigated. The results are summarized as follows:1. Determination of antibacterial circle diameter and minimumantimicrobial concentration.ε-polylysine, nisin and natamycin showed inhibition ability against E. coli. S. aureus, B. subtilis, M. luteits, H. anomala, P.expansum and A.niger. FICI value showed that all drugs exhibited synergism in inhibiting the growth of the tested bacteria when used in the two-drug combination. The combination of ε-PL and nisin has significantly synergistic effectsagainst the four bacteria.2. Thebacillus subtiliswas used to evaluate the relationship between the antibacterial activity and time ofε-PL and nisin when used in the combination and when used alone. Results showed that antibacterial activity of ε-polylysine was rising gradually, and achieved active peak at 20h whilethe antibacterial activity of nisin reached the active peak at 3h and then declined after 6h.The combination of s-PL and nisin peaked at 3h, then decreased, and elevated after 9h, which showed a similar trend asε-PL.These findings suggestedthat the first activity peak might be closely associated with nisin while the nextis mainly attributed toε-PL.3. After incubation with different antibacterial agents, the bacteriostatic solution wasspectroscopicallydetermined at 630 nm, and observed under the scanning electron microscope and transmission electron microscope. Results showed that cell wall was damaged weakly by ε-PL but seriously destroyed by nisin. After treated together by ε-PL and nisin, bacteria cells lollapsed and the contour wass fuzzy, and the damage of cell wall was stronger than that when treated alone.4. The relative electric conductivity, protein leakage, ultraviolet absorption material leakage and ATPase activity were measured after the bacteria were treated individually or in combination by ε-PL and nisin. Results showed that the combination treatment group had the greater increase of all above indices compared to other groups, suggesting that the combined-treatment of ε-PL and nisin greatly enhanced the permeability of cell membrane. Furthermore, the fluorescence intensity in nisin and combined-treatment groups significantly higher than the other groups, indicating that both groupswere able to damage the cell membrane and increase the permeability of cell membrane; Both Na+K+-ATPase and Ca++ Mg++-ATPase activities in antibacterial agents treated groups were significantly decreased compared that in the control group.5. DNA agarose gel electrophoresis experimentsshowed that Bacteria DNA were ruptured and the content was decreasedboth in nisin and nisin and ε-PL treated groups.Ultraviolet spectrometry analysis illustrated that the absorption peak in ε-PLtreated group generated a redshift, and the peak value decreased significantly, suggesting that ε-polylysine might be embedded in the DNA.No significant difference was found between nisin treated group and control group.Fluorescence spectrum showed that there was an interaction between ε-PL and DNA whilenot between nisin and DNA. These findings indicated thatafter treated by nisin, DNA cleavageoccurred outside the cellwhen bacteria were dead. Moreover,therewas astrong interaction between ε-PL and DNA.6. The quenching effect of ε-PL to EB-DNA was weakened after the addition of phosphate, explaining there was aninteraction between ε-PL and phosphate groups. When ε-PL was added into the EB-DNAsystem, fluorescence intensity was decreased,suggesting that ε-PL and EB competed to combine with DNA. With the increasing of EB concentration in ε-PL-DNA complex system, the fluorescence intensity was elevated, which might be attributed to compete DNA combination betweene-PL and EB.In conclusion, the synergistic antibacterial mechanism of ε-PL and nisin was proposed as follows:nisin first destroyed cell membranes of the bacteria,thenε-PLentered the cell and combined with DNA, whichthereby hindered the DNA replication. Moreover.the combination of e-PL and nisin were able to inhibit ATPase activity.