| Antimicrobial peptides, a series of lipopeptide substances produced by therepresentatives of Bacillus, such as Bacillus subtilis, Bacillus amyloliquefaciens,Bacillus natto.(Usually concluding surfactin, iturin, fengycin subtilin and sublancinetc.). Its molecular usually exists in the form of structure of cyclic lipopeptide.Antimicrobial peptides owe the properties of antibacterial and biosurfactant. And it iswidely used in the fields of agriculture, medicine, cosmetics and food, oil exploration,environmental governance, etc. And it has attracted considerable attention.At present, the fermentation of antimicrobial lipopeptides is mainly based onliquid fermentation, but with high energy requirements, large cost, low output and thedifficulties of extraction and purification. Compared with the liquid fermentation, thesolid fermentation seems to perform an obvious advantage, for instance, byproduct orwaste of industrial and agricultural production are the main fermentation substratethat could reduce production costs, and moreover, not only the fermentationequipment is more simple, the concentration of unit matrix is higher, the yield ishigher, etc. And also avoid bubble problems which were difficult solved in liquidfermentation and greatly simplify extraction process, reduce costs, and so on.However, research about control conditions of solid-state fermentation ofantimicrobial lipopeptides rarely have been reported, and they greatly influence theproduction of antimicrobial lipopeptides, so it is essential to study the conditions ofsolid-state fermentation of antibacterial lipopeptides. However, the studies of solidfermentation of antimicrobial lipopeptides is still in an exploratory stage, for severalkey technologies and parameters of fermentation, extraction and purification areunclear, and further, lack of a systematic description of the various parameterschanged during the fermentation, thus affecting the overall fermentation efficiency and industrial fermentation control, and ultimately affect the industrial production ofantimicrobial lipopeptides. Consequently, the reseach was carried out to study thesolid-fermentation kinetics of antimicrobial lipopeptides, to systematically describethe changes of the values of various parameters during the whole fermentation process,thus providing the support for the efficient control of industrial production ofantimicrobial lipopeptides.In recent years, the breeding of Litopenaeus vannamei has been rapiddevelopment. With the rapid development of intensive farming, the deterioration ofwater environment lead to disease outbreaks. At present, One of the most importantmeasures for prevention and control of aquatic disease is adding large amounts ofantibiotics in feed.The abuse of antibiotics leads to its residues in shrimp body greatlyincreased, that not only reduce shrimp’s ability of resistant disease, form a viciousspiral, and finally threat to the safety of shrimp product, also will cause great harm tohuman health. Meanwhile, the sick rate of raw shrimp is higer, which threat tosubsequent processing safety of shrimp food. Therefore, the safety problems of sourcefood to be solved. Therefore, seeking a green, environmentally friendly and efficientadditive to replace antibiotics has become a hot in the area of improving the shrimp’sability of resist to disease and improving using security of shrimp product. In many ofthe additives, antimicrobial peptides as a new additive, with the advantages ofantibacterial, promoting growth, no residue, immune regulation, not easy to inducebacterial resistance and so on, have a certain application in aspects of poultry,livestock and aquaculture. However, due its the high cost, limited sources and lowactivity obtained by genetic engineering, so to find new substitutes is particularlyimportant.The research group has isolated a strain of high yield of antimicrobiallipopeptides from Bacillus NT-6, the yield of antimicrobial lipopeptides reached10g/(kg fermentation substrate) by solid-fermentation and it also has properties of highheat resistance, pH adapt to a wide range, widely antibacterial spectrum, goodsolubility and safe non-toxic. So the study of adding antibacterial lipopeptides intofeed as an alternative of antibiotics to improve the disease resistance and edible safetyof shrimp will have a broad application prospects.The research contents and resultsare as follows:1Optimization of parameters of solid-state fermentation by bags of antimicrobiallipopeptidesThe optimal fermentation conditions were determined by single factor test for bagasse content is15%, the ratio of material to water is5:5, temperature is30℃,fermentation time is48h and20mesh (10cm×40cm). On this basis, initiallydetermined the ratio of material to water, temperature, inoculation amount3factors asthe main influencing factors. Then the steepest ascent experiment, approximatemaximum response region, and with the help of the software Design Expert8, usingresponse surface method (Response surface Methodology, RSM) Box-Behnkendesign of experiments, further research and discussion on the best level factors to dothe main effects and interactions, and establishes the equation prediction model. Themodel equations of3-D and contour plots of three main factors, the value ofmaximum yield prediction value of antimicrobial peptides of8.74g/kg were: the ratioof material to water1.18, temperature29.78℃, inoculation amount5.17%. In orderto test the accuracy of the model, do the experiment under the optimal conditions, theyield of the antimicrobial lipopeptide was8.77g/kg and was very close to thepredicted values.2Optimization of parameters of solid-state fermentation by thin layer of antimicrobiallipopeptidesThe relative humidity, ventilation and thickness3factors were as the maininfluence factors, and with the help of the software Design Expert8, using responsesurface method (Response surface Methodology, RSM) Box-Behnken experimentaldesign, discussed the optimal level of main influence factor and researched its furtherinteraction, and established the prediction equation model. The model equations of3-D and contour plots of discovery, predictive value of antimicrobial lipopeptidemaximum yield was10.7552g/kg, and values of three main factors: relativehumidity87.68%, ventilation12.48m3/min, thickness3.11cm, respectively.3Study on kinetics of solid-state fermentation of antibacterial lipopeptidesDuring the process of solid state fermentation by thin layer of the antimicrobiallipopeptides, indexes of biomass, production of antimicrobial lipopeptides and contentof reducing sugar were determined timing, then fermentation parameters and thecorresponding model equations were obtained by Origin software for data processing.And the optimal fermentation time was48h, the minimum biomass Cx.0=9.56×106cfu/g, the maximum biomassCx.max=3.20×108cfu/g, the maximum specific growthrate of μm=0.16593h-1, the smallest antimicrobial lipopeptide productionCp.0=0.61275g/kg, maximum antimicrobial lipopeptide production Cp.max=10.42448g/kg, the maximum specific growth rate of πm=0.14697h-1, reducing sugar, the initial Cs.0=10.41958, b=0.46058. Three kinetic equations were all have a higher fittingdegree, explained that the parameters were reliable by and meanwhile providestheoretical support for the large-scale production of industrialization.4Effects of antibacterial lipopeptides on growth performance and parameters of non-specific immunity of litopenaeus vannameiThe antimicrobial lipopeptides were added into feed with0mg/(kg·feed)(group A0),50mg/(kg·feed)(group A1),100mg/(kg·feed)(group A2),150mg/(kg·feed)(group A3)and200mg/(kg·feed)(group A4), respectively to feed shrimp. The experimentalresults showed that the antimicrobial lipopeptide could significantly promote thegrowth and improve POD, AKP, LZM, T-SOD and T-AOC activity in serum andhepatopancreas of shrimp, thus improve the non-specific immunity of shrimp, thenultimately improve ability of the resist disease of shrimp.Finally, determine theoptimal concentration of antimicrobial lipopeptides is100mg/(kg·feed).Development and application of antimicrobial lipopeptides as feed additive inaquaculture will contribute to the healthy development of shrimp aquaculture forbreaking bottleneck problem of the disease prevention and control of shrimp farmingindustry, which have constrained industrial sustainable development, promoting thereplacement of resistant additives in aquatic feed to achieve shrimp green farming andto guarantee the safety of shrimp products at the source.5Control of NT-6antimicrobial lipopeptide on Vibrio in source of breeding ofLitopenaeus vannameiThe antimicrobial lipopeptides were added to feed with0mg/(kg·feed)(A0group),50mg/(kg·feed)(group A1),100mg/(kg·feed)(group A2) and200mg/(kg·feed)(group A3), respectively to feed shrimp. The number of Vibrio in water and shrimpwas texted before and after the exposure of shrimp, and texted the survival rate ofshrimp after exposure. Test results show that, the number of Vibrio alginolyticus and V.Parahaemolyticus in water and shrimp of experimental groups were lower than thecontrol group and lower than the threshold of pathogenic of Vibrio in water andshrimp body before exposure; and the number of Vibrio alginolyticus in water andshrimp of experimental groups were lower than the control group, but were higherthan the threshold of pathogenic of Vibrio in water and shrimp body after exposure.Indicating that antimicrobial lipopeptides can inhibit Vibrio in water and shrimp.Greatly reduce the number of various pathogenic bacteria in water, resulting inprevention and control of disease, improve the survival rate and the effect of improving disease resistance, while producing the carrier rate of shrimp andimproving the food safety of shrimp product.Bacillus antimicrobial lipopeptides, with the characteristics of efficient and broad-spectrum antibacterial activity, non-toxic side effects, no residue, no cause bacterialresistance and so on. And with the efficiencies of growth promoting, prevention andtreatment of diseases on shrimp, so its potential of application is huge.Accelerate thedevelopment and application of antimicrobial lipopeptides feed additive inaquaculture, promote the transition and upgrading of traditional culture model to themodern mode, not only can ensure food safety of shrimp in the source, but also canpromote aquatic feed additive update, achieve antimicrobial feed additives greensource, providing guarantee for food safety. |
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