Optimization And Application Of Fermented Soybean Meal By Bacillus Licheniformis

The frequent occurrence of viral diseases in Litopenaeus vannamei severely affected the development of shrimp aquaculture around the world.The aquaculture industry is now suffering from the huge losses caused by aquatic animal diseases.Probiotics have been widely used in a series of methods for controlling aquatic animal diseases.Based on previous strain screening work in this laboratory,a strain of Bacillus licheniformis isolated from the digestive tract of Litopenaeus vannamei was obtained,codenamed LV005.Adding this strain to the feed of shrimps can effectively improve the ability of shrimps to resist WSSV infection.The nutritional value of soybean meal after it has been fermented by microorganisms can be improved,and it can be used as a substitute for fish meal in feed.The probiotic fermented soybean meal can be used as an economical,practical and efficient new probiotic feed additive technology.In this paper,the process of fermentation of soybean meal by Bacillus licheniformis was optimized,and the application prospect of fermented soybean meal in shrimps culture was examined.The following research contents were mainly included:1 Optimization of laboratorial scale fermentation of soybean mealBacillus licheniformis LV005 was used for solid fermentation of soybean meal and the effect of particle size and contained windrow on the laboratorial scale solid fermentation was optimized in this study.Comparing the effect of different blending methods on the counting of bacteria in fermented soybean meal.Comparing the effect of different fermentation vessels,soybean meal sizes and windrow thickness on the bacterial concentration of fermented soybean meal.In order to count the bacteria after fermentation,a 1 min vortex oscillation of the fermented soybean meal in PBS reached the best releasing of the bacteria in the soybean meal.Testing results indicated that the optimized conditions included the solid fermentation in a 4-layer newspaper-closed 50ml centrifuge tube with a windrow of 1.5 cm thickness of soybean meal particles through an 80mesh sieve.The bacteria in fermented soybean meal reached to 1.65×10¹⁰—1.76×10¹00 cfu/g.This study provides technical support for the laboratorial scale fermentation of soybean meal.2 Optimization of process conditions of fermented soybean meal by Bacillus licheniformisThrough the single factor experiment and response surface methodology on the solid fermented soybean meal process optimization,get the best fermentation condition:rate of raw material to water 1:0.8,salinity of soaking water 0,fermentation temperature 28.33?,fermentation time 50.90h,inoculum 4.80×10⁶ cfu/g,addition of sucrose 2.08%.In this condition,fermentation was carried out,and the concentration of bacteria in soybean meal was as high as?1.75±0.10?×10¹⁰cfu/g.Under the optimal fermentation conditions,the concentration of fermented soybean meal could reach the order of 10¹⁰cfu/g,indicating that Bacillus licheniformis LV005 can use nutrients from soybean meal to ferment.The use of Bacillus licheniformis LV005fermented soybean meal has a short cycle and high efficiency,laying the foundation for its application in production.3 Nutritional analysis of fermented soybean meal by Bacillus licheniformisEvaluation of the nutritional value of fermented soybean meal,soybean meal fermentation yield is high,up to 95.24%.The crude protein of fermented soybean meal increased by 14.57%,crude fat increased by 166.67%,crude fiber increased by 19.02%,acid soluble protein increased by 445.83%,total amino acid increased by 3.64%,essential amino acids of shrimps increased by2.67%,and taste amino acid increased by 3.72%.With an increase of 3.36%,SDS-PAGE electrophoresis demonstrated that the macromolecule protein in soybean meal was effectively degraded.This study preliminarily proved that the use of probiotics to ferment soybean meal can improve the nutritional value of soybean meal and improve the utilization rate.The probiotic fermented soybean meal can be used as an economical,practical and efficient new probiotic feed additive technology in production practice.4 Effect of different ratios of Bacillus licheniformis fermented soybean meal replaced fish meal on cultured shrimpsFermented soybean meal used 20%,60%,and 100%nitrogen instead of fishmeal in feed,and compared the effect of spraying Bacillus licheniformis feed and sterilized fermented soybean meal.The results showed that the substitution ratio of Bacillus licheniformis feed and fermented soybean meal was replaced.At no more than 60%,the feed had no significant effect on the growth performance of Litopenaeus vannamei?P>0.05?,and the replacement of fish meal by the full proportion of fermented soybean meal significantly reduced the growth performance of Litopenaeus vannamei?P<0.05?.The WSSV challenge experiment was conducted on each day group of Litopenaeus vannamei.The results showed that the replacement of fish meal with 20%and 60%of fermented soybean meal by Bacillus licheniformis LV005 can significantly improve the ability of Litopenaeus vannamei to resist WSSV infection.The results showed that replacing the fishmeal in the feed with the appropriate proportion of the resistant microorganism Bacillus licheniformis LV005 fermented soybean meal can not only save the cost of feed,but also improve the disease-resistant ability of the cultured shrimps,which has huge production and application value.The composition and structure of intestinal microflora in shrimps were compared between different feeding time and different culture time.The main bacterial gate levels of intestinal microflora in each group were found to be:Tenericutes,Proteobacteria,Firmicutes and Bacteroidetes.The average relative abundance of the intestinal microbiota of Trichomonas and Proteobacteria after the challenge with WSSV was significantly higher than that before the challenge,and the relative abundance of Salmonella and Bacteroides was significantly reduced.At each time point before the challenge,the microbial communities in the gut micro-organisms of shrimps in each group were significantly different and their community composition was more complex.The levels of major bacterial communities in the intestinal microflora of shrimps from each group were similar at various time points after challenge,mainly including Candidatus Bacilloplasma and Vibrio.Feeding different feeds had a greater impact on the composition and structure of the intestinal microflora of shrimps.The composition and structure of the intestinal microflora varied greatly during the different growth stages of shrimps fed the same diet.Feeding Bacillus licheniformis LV005 fermented soybean meal full replacement of fishmeal feed is beneficial to maintaining the stability of shrimps gut microflora.