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Antifungal Mechanism of Antimicrobial Lipopeptide Produced by Bacillus Subtilis fmbJ Against Rhizopus Stolonifer

Author: TaoYang
Tutor: LuZhaoXin
School: Nanjing Agricultural College
Course: Of Food Science
Keywords: antimicrobial lipopeptides Rhizopus stolonifer surfactin fengycin antifungal mechanism
CLC: TS255.1
Type: Master's thesis
Year: 2010
Downloads: 20
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Abstract

Rhizopus stolonifer is the causal agent of soft rot in many fruit and vegetables. This fungus can cause a multitude of rotted fruits and vegetables in preservation, transportation and marketing which leads severe agricultural damage. For several years, synthetic fungicides have been used to control this microorganism. However, it has been shown that the compounds used in these fungicides can cause strain resistance. Moreover, since people are getting more and more concerned about pesticide residues, many pesticides are banned. Therefore, it is important to exploit safe, economical and effective methods to inhibit Rhizopus stolonifer. A number of antimicrobial substances can be produced by Bacillus subtilis during its growth and metabolism. Because the antimicrobial substances from Bacillus subtilis show a broad spectrum of inhibitory activity on pathogens, they have a potentienal application in agriculture, pharmacognosy and light industry.In the last few years, a food Bacillus subtilis fmbJ strain has been screened out in our lab, which could produce antibacterial active substances. These substances are stable to heat and some enzymes, and have very high antimicrobial activity and broad antibacterial spectrum. Previous studies have demonstrated that the substances mainly included surfactin and fengycin, both of cyclolipopeptide. The antifungal mechanism of these lipopeptides against Rhizopus stolonifer has not been elucidated and the interaction between surfactin and fengycin during antimicrobial process has also not been reported. Therefore, in the paper, the antifungal mechanism of the lipopeptides produced by Bacillus subtilis fmbJ against Rhizopus stolonifer was mainly studies. The interaction between surfactin and fengycin during the process of inhibiting Rhizopus stolonifer was also studied in different ways. Results were as followed: 1. ESI-MS/CID analysis showed surfactin appeared from 17.5min to 24.5min in the given HPLC condition, and fengycin appeared from 24.5min to 32.2min. The optimal time when lipopeptide productivity was highest was obtained by fermentation for different hours. The result showed that fermentation for 50 hours was the optimal time. And the concentration ratio of fengycin and surfactin in the lipopeptides produced after fermenting for 50 hours was 5:1.2. Both the two concentration ratios of lipopeptides could inhibit the hypha growth, spore germination and spore productivity of Rhizopus stolonifer. The minimal inhibitory concentration (MIC) of the 5:1 lipopeptide combination against Rhizopus stolonifer spores was fengycin 0.4mg/mL; the minimal fatal concentration (MFC) against Rhizopus stolonifer spores was fengycin 1.4mg/mL; the 50% effective concentration (EC50) against Rhizopus stolonifer hyphae was fengycin 27.68μg/mL. The MIC of the 1:1 lipopeptide combination against Rhizopus stolonifer spores was fengycin 2.0 mg/mL; the MFC against Rhizopus stolonifer spores was fengycin 6.5mg/mL; the EC50 against Rhizopus stolonifer hyphae was fengycin 98.12μg/mL. Scanning electron microscopy and Transmission electron microscopy were used to study changes in the morphology and inner structure of Rhizopus stolonifer after lipopeptide treatment. The results indicated the lipopeptides not only acted on the cell membrane of Rhizopus stolonifer, but also had an effect on the cellular organs. After comparing the antifungal activity of the two lipopeptide combination, it could conclude that the antifungal activity of fengycin decreased when surfactin was added.3. To understand the antifungal mechanism of these lipopeptides, the leakage of protein, nucleic acid and K+ of hyphae was monitored to determine the permeability of the cell membrane and propidium iodide (PI) staining was used to examine the integrity of the plasma membrane. The interaction of the lipopeptides in a model membrane was studied using 31P NMR. All these results indicated that the lipopeptides changed the permeability, damaged the integrity of cell membrane and discupted the structure of phosphoryl group. Moreover, surfactin could ease the disruption of phosphoryl group by fengycin.4. The effect of lipopeptide on the structure of Rhizopus stolonifer DNA in vitro was studied by UV scanning and gel retarding assay. The result indicated that the lipopeptide could not interact with Rhizopus stolonifer DNA in vitro. Flow cytometry and isotope labeling were used to study the effect of lipopeptide on replication of Rhizopus stolonifer DNA. It showed that the lipopeptide could change the cell cycle and inhibit the replication of Rhizopus stolonifer DNA. After comparing the utilization of 3H-TdR, it could conclude that surfactin could ease the inhibitory effect by fengycin on Rhizopus stolonifer DNA.

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