| Most antimicrobial lipopeptides are the metabolites of Gram positive bacillus with its molecules composed of a hydrophilic peptide bond and lipophilic aliphatic hydrocarbon chain. Antimicrobial lipopeptides have many biological activities, such as the inhibition over many bacteria, tumors, mycoplasma and many other substances. Antimicrobial lipopeptides are safe, nontoxic and easily degradable which caused widespread concern in the pharmaceutical, food, biocontrol, oil exploration, environmental management,bio-pharmaceutical and other fields, showing a broad market development value.Antimicrobial lipopeptides are made up by a multi-component structure-similar categories lipopeptide compounds and each of them has various homologs, thus caused a complex product with difficult separation between the components and made it difficult to quantify this compound. At present, the antimicrobial lipopeptides can be quantified by weighing,measuring surface tension, analyzing its hemolytic activity and high performance liquid chromatography et al., but these methods can't meet the demand of actual production for their imprecise, and it's necessary to establish a new method for accurate quantification.Furthermore, the antimicrobial lipopeptide are usually produced by conventional fermentation methods with synthetic medium which can't afford practical applications for its low yield and high cost, and this limit the further applications, so we need to study a new method for fermentation to break the bottleneck. This research concerned the LC-MS quantification method of Surfactin and Iturin, discussed the impact of different fermentation conditions on antimicrobial lipopeptides, established a high-density Bacillus natto fermentation system with attapulgite and other large surface solid matrixes based on biofilm fermentation theory, analyzed the effects of several signaling molecules on fermentation system, aiming at improving the production of antimicrobial lipopeptide,establishing new fermentation system, providing a basis for the production and application of antimicrobial lipopetides. The results are as follows:1. A LC-MS quantification method of antimicrobial lipopeptide in fermentation broth of Bacillus was established by optimizing the conditions of MS, mobile phase,columns, selectivity of method: antimicrobial lipopeptide was eluted by Venusil XBP CN column with 7:3 5 mmol·L-1 ammonium acetate (0.1% formic acid) - acetonitrile as the mobile phase. With the optimized conditions, the linear relationship of antimicrobial lipopeptides was good with a level of 0.20?10.0?g·L-1 addition (R2>0.9995), spiked recoveries were 93.3% ?108.22%,the precision was 4.14% ?13.30%,and detection limit was 0.374 mg·L-1. The method was easy to operate with high degree of accuracy, overcome the shortcomings of traditional detection methods, provided practical basis for quantification of more components of antimicrobial lipopeptides and its actual producing and applications.2. This paper concerned the influence of several large surface solid matrixes on the antimicrobial lipopeptide production, aimed to explore the new method of antimicrobial lipopeptide yield increase. Attapulgite, activated carbon and gauze were added to the main medium of Bacillus natto with some designed amount. The inhibition rate to specific indicator were determined to evaluate the yield of antimicrobial lipopeptide and scanning electron microscopy was used to research the characteristics of bacteria adhensioned on different solid matrixes to analysis the mode of action of large surface matrix preliminarily.The optimal fermentation was found in the attapulgite experimental group.Measurement of inhibition rate of the best experimental group was 63.2%, 23.6%higher than control group, followed by activated carbon group with 11.9% higher than the control group and the gauze group with 9.6% higher than the control group; determination of biomass showed that cell concentration of control group was 44.7% lower than the best attapulgite experimental group, 38.5% lower than the activated carbon group and gauze group. Bacillus natto formed stable biofilm on attapulgite with a thick-dense tissue and attached with high density of bacteria, showed a special fold structure on actived carbon and attached with large number of biofilm-bacteria and got relatively thin but integrated biofilm on the surface of gauze fiber and also attached with lots of bacteria. Antimicrobial lipopeptide productivity by Bacillus natto was increased obviously by adding large surface solid matrixes into fermentation medium and attapulgite obtained the best productivity.The increased yield may due to the addition of the large surface solid matrixes which increased the biomass in the broth and the biofilm formed on the matrixes.3. The paper discussed the impacts of addition of some signaling molecules in Bacillus natto fermentation system on the production of antimicrobial lipopeptides.Quantify lipopeptides by LC-MS, detect the biomass in different fermentation systems bybacteria colony counting method, the results showed that: all of the six signaling molecules added to fermentation system can improve the production of lipopeptides with methanol extracts of solid-state fermentation having the most obvious promotion and standards of Surfactin and Iturin, methanol extracts of liquid-state fermentation having tiny impact; cells of Bacillus natto with addition of 15% attained optimal enhancement, and other five signal molecules got a optimum at 10% of added concentration; under the experimental conditions,the promotion of most signaling molecules showed a tendency of upgrade firstly and descending latter with 24h being the critical point as the cultivate time goes by. The biomass of fermentation broth has no directly relevant with the concentration of signaling molecules and the production of antimicrobial lipopeptides. Signaling molecules can promote the production of antimicrobial lipopeptides, the specific regulatory mechanisms and dose-effect relationship are worthy of further study.4. The research studied the effects of different fermentation conditions on antimicrobial lipopeptides to provide guidance for actual production and applications of antimicrobial lipopeptide. Analyzed the effect of different fermentation medium,temperatures, ventilations and other conditions on the yield and component of antimicrobial lipopeptides with LC-MS, the results showed that: under the experimental conditions, an optimal production was obtained at 30? with Landy medium; production of lipopeptide was improved with the increasing of incubator speed and attained maximum yield at 200r·min-1; the proportion of different components of lipopeptides changed in different fermentation methods: the propotinon of Iturin fermented with Landy reached 80.58%, sinnifibantly higher than other media; with fermentation temperature rises, Iturin component showing a trend of increase firstly and decrease secondly and reached a maximum value of 84.10% at 28 ?; the propotinon of Surfactin in solid state fermentation had a definite increase compared to liquid state fermentation with the component of 1008.20 and 1071.20 changed significantly. |
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