Optimization And Application Of Fermented Soybean Meal By Bacillus

Since the rise and development of the high-density and intensive aquaculture, all kinds of problems also increase gradually. Disease caused by infection of viruses, such as white spot syndrome virus(WSSV), infection of bacteria, such as hepatopancreatic necrosis disease(AHPND), as well as diseases caused by the farming environment or poor nutrition, bring serious economic loss. Due to the development of drug resistance and environmental unfriendly by the traditional therapeutic method like antibiotics and disinfectants, the research of probiotic appeared. Because of the declining of capturing yield and the increasing price of fish meal, looking for effective vegetable protein to replace animal protein become an advanced research hotspot. This paper combined the two aspects innovatively, fermenting soybean meal use three strain of bacterium bacillus to replace some fish meal, expecting to obtain the fermented soybean meal which could include shrimp’s immunity boost and resist WSSV. The research mainly includes the following several aspects:1. Optimization of process conditions of fermented soybean by probioticIn order to obtain the optimal conditions of fermented soybean by probiotic, Bacillus firmus strain PC024, which was isolated from the farming environment of healthy shrimp Fenneropenaeus chinensis, was used for the fermentation of soybean meal. Univariate tests and the response surface methodology(RSM) were applied to optimize four fermentation factors, including temperature, time, water ratio, inoculation amount of bacteria. The optimum fermentation conditions were concluded as inoculation amount of bacteria 3.84×106 cfu/g, water ratio 1:0.96, fermentation time 100.3 h, fermentation temperature at 39.0 °C. Under the optimized condition, the bacteria concentration reached 1.23×1010 cfu/g after the fermentation which was 5.13% difference to the predicted value by the RSM model. This study provided a theoretical foundation for the following applications of fermented soybean in aquaculture production.2. Nutritional analysis of fermented soybean product by Bacillus firmus strain PC024To evaluate the effect of fermented soybean meal by Bacillus firmus(PC024), we researched the senses, fermentation yield, soluble protein content, crude protein, amino acids and so on of the fermented soybean product. Experimental results show: The odor and appearance of the soybean meal changed after fermentation, and the yield of the fermented soybean meal reached(93.89±0.01)%. The soluble protein content in the soybean meal increased from(39.16±0.01)% to(58.80±4.54)% after fermentation, and the concentrations of protein in the soybean meal increased from 50.71% to 55.03% after fermentation. The total content of 15 kinds of amino acids was increased to 132.3% of the original content, the largest increase in the proportion of five amino acids were arginine(168.6%), lysine(157.2%), serine(152.50%), threonine(139.04%) and glycine(138.4%). Analysis of the protein macromolecule by SDS-PAGE revealed that the natural protein macromolecule in soybean meal were effectively degraded and major bands at marker levels of approximately between 20 k Da and 30 k Da.The present work could provide technological supports for promising applications of probiotics in the feed nutrition and disease control for aquatic animals.3. Application of fermented soybean meal by Bacillus in feed and effect for disease resistance.In order to research a certain percentage replacement of fish meal by Bacillus fermented soybean meal and its impact on vannamei’s resistance to White Spot Syndrome Virus(WSSV), the study used three Bacillus(Bacillus firmus, Bacillus subtilis and Bacillus subtilis) which was isolated and saved by our laboratory. Replacing a proportion of fish meal by fermented soybean meal using Bacillus firmus, Bacillus licheniformis and Bacillus subtilis. Fermentation products replaced 20% and 100% fish meal respectively. With basal feed being the control group, every group was carried out in 5 duplications. The shrimp be fed with the probiotic fodder for 21 days were challenged with WSSV, using the method of reverse enema, and death status was recorded. The mortality rate showed that it were no significant difference with group basal fed(100.00%±0.00%) to be fed with group D20(83.33%±33.33%), group D100(85.00%±5.77%) and group K100(86.67%±6.67%). There were significant difference with group J20(63.33%±3.33%), group J100(78.33%±11.67%) and group K20(76.67%±3.33%). In conclusion, it could significantly reduce the mortality challenged by pathogeny that fed shrimp with probiotic fermented forage. Indeed, this probiotic fermented forage should be regard as the major object of study and be identified the optimal replace proportion to fish meal.4. The Bacillus firmus that has the highest resistance to WSSV was chosen to research the optimal proportion to replace fish meal.To further study the optimal proportion that Bacillus firmus PC024 replace fish meal, with the basis that it has resistance to WSSV, fermented soybean meal replaced 20%, 40%, 60%, 80% and 100% of fish meal to carry out breeding experiment. The growth rate and the cumulative mortality rate were analyzed to evaluate the growing performance and resistance to WSSV within 90 days post-administration. Results showed that growing performance of shrimps fed with fermented soybean meal that replaced 20%(FSBM20), 40%(FSBM40) and 60%(FSBM60) have no significance difference(P > 0.05) with group basal fed(FM). With the proportion of fermented soybean meal over 60% of fish meal, groups FSBM80 and FSBM100 slowed the growth in some degree and reduce the growth rate significantly(P < 0.05). Best ratio was 20%. The survival rate of groups that replaced 20%(20%±5%) fish meal and 40%(23%±4%) fish meal are significantly higher than control group(P > 0.05). There were no significante difference between group FSBM60, group FSBM80, group FSBM100 and FM. Best ratio was 40%. Therefore, fermented soybean meal using Bacillus firmus(PC024) could not only provide high quality protein source to ensure normal growth but also play a protective immune effects in some degree.