Optimization Of Cultivation Processes For Antimicrobial Lipopeptide Produced By Bacillus Natto In Solid-State Fermentation And Application In Fresh-Keeping Ofprawn

Antimicrobial lipopeptide are a series of lipopeptide substance produced byrepresented Bacillus strains of Bacillus subtilis，Bacillus amyloliquefaciens and Bacillusnatto, which including surfactin, iturin, fengycin, subtilin and so on. At present, theproduction of antimicrobial lipopeptide is mostly by submerged liquid fermentations, butwith high energy requirements, large cost and low output. However, solid-statefermentation has several advantages over submerged fermentation in that it requiresinexpensive equipment and uses cheap materials, such as agricultural byproducts. It yieldsa high quantity of biomass and is cost-effective. And avoids blistering run material.Therefore, SSF has found several potential applications in the production of antimicrobiallipopeptide.Shrimp belonging to the phylum Arthropoda, gill Amon, crustaceans, soft A subclass,Decapoda, swimming suborder, Penaeidae shrimp genus, have a lot of types. In whichLitopenaeus vannamei, popular name Penaeus vannamei, is one of the highest of theproduction of three shrimp in today’s world aquaculture. Fresh shrimp because of its highmoisture and protein content, easily are corrupted by bacterial invasion and contaminationin the process of fishing, transportation, processing and storage. In order to extend its shelflife, the traditional physical, chemical preservation method is most commonly used, butdue to its constraint in serviceable range, operative technique and security issues, so it isdifficult to widely popularized, but a kind of safe, non-toxic biological preservationtechnology is a hot topic of research in the food field. Antimicrobial lipopeptides due to itsspecial chemical composition and the amphiphilic molecular structure have a startlingrange of antimicrobial activities that can include action against most Gram-negative andGram-positive bacteria, fungi, enveloped viruses, and eukaryotic parasites. and a goodadaptability to the external environment. Through oral acute toxicity in mice and foundthat its LD50greater than5000mg/kg body weight, indicating that antimicrobial peptide itshave high food safety. Therefore, Antimicrobial lipopeptide as a biological preservativewill have a wide range of applications in shrimp preservation.The content of this research and findings are as follows: 1. Firstly, by making single factor test on the impact of the production of antimicrobiallipopeptide by solid state fermentation including medium composition andfermentation conditions, initially determine the related8factors including glucose,sodium glutamate, KCL, MgSO4, K2HPO4, inoculum size, fermentation temperatureand fermentation time, and then the methodology of Plackett-Burman design wasundertaken to screen the key factors rapidly. By analyzing the statistical regressionand the prediction profiler, the temperature and the concentrations of MgSO4andK2HPO4were found to be the most important factors for the production ofantimicrobial lipopeptide. On this basis, in order to approach the maximum responseregion, make steepest ascent experiment. At last, a three-level three-factorBox-Behnken design was applied to optimize the production of antimicrobiallipopeptide. The critical factors selected for the investigation were mentioned above.By analysis of the3-D plots and their corresponding plots, the optimum values of thetemperature and the concentrations of MgSO4and K2HPO4for obtaining the mostproduction of antimicrobial lipopeptide were30.33℃,0.97g/kg, and1.50g/kg,respectively. Compared with the liquid fermentation the optimized cultivationconditions allowed antimicrobial lipopeptide production to be increased3-4times.2. After the end of the solid-state fermentation, the crude extracts of the antimicrobiallipopeptide are extracted by acid precipitation, By separating and purifing of theMacroporous the resin X-5column, collect the elution peak. Then the TSQ QuantumAccess triple quadrupole mass spectrometer will be applied to detect concentratedelution peak with antibacterial activity, By analysis of the positive ion spectra,compared with other reports, initially identify the antimicrobial lipopeptide group arecomposed of surfactin and iturin A.3. The antibacterial activities of antimicrobial lipopeptide on spoilage organisms weredetermined by Oxford cup assay. Then the quality changes of Litopenaeus vannameiduring storage at (4±1)℃were investigated, including the pH, total volatile basicnitrogen (TVB-N), aerobic plate count (APC) and sensory assessment. The resultsshowed that antimicrobial lipopeptide can effectively inhibit8strains of spoilageorganisms isolated from Litopenaeus vannamei. During storage at (4±1)℃, With theextension of storage time, The gradually increasing value of pH, TVB-N and APC ofLitopenaeus vannamei was observed during the7days storage. However, incubated0.5mg/ml antimicrobial lipopeptide can effectively slow down the value increasing,which extends the shelf-life of Litopenaeus vannamei by2-3days.