| Nowadays,the rapid development of aquaculture industry has increased the demand for fish meal in the feed industry,but the fish meal is in short supply at home and abroad.In order to ensure the production of farmed animals,the research on the development of new fish meal alternative protein source has become one of the important means to solve the high feed cost.In our country,soybean as a common planting crop,not only has good economic value,but also has rich nutritional value,therefore,using soybean to make feed not only saves cost but also ensures nutrition supply.At the same time,soybeans are rich in some components that interfere with the digestion,absorption and utilization of nutrients,and these components can even harm the body’s health.Solving these active components is the key to make full use of soybeans.The diversification of processing methods provides a way to solve this problem.Fermentation,as an economical and pollution-free processing method,has a significant digestion and deactivation effect on these active ingredients.In this paper,the optimization of soybean fermentation process and the effects of fish meal replacement on intestinal microorganisms,digestive enzymes and intestinal structure of Procambarus clarkii were studied through soybean solid stated fermentation,in order to provide scientific basis for the application of fish meal replacement by soybean in Procambarus Clarkii feed.Firstly,in order to determine the optimal process parameters of soybean liquid fermentation and evaluate the fermentation quality,Aspergillus oryzae,Saccharomyces cerevisiae,Bacillus subtilis and Lactobacillus plantarum were used as fermentation microorganisms,soybean and bran were used as fermentation substrate,and the optimal conditions of soybean liquid fermentation process were explored from four parameters:fermentation time,fermentation temperature,initial fermentation moisture and proportion of bran in fermentation substrate.1 kg fermentation matrix was evenly mixed with fermentation microbial liquid and water,then filled into the fermentation breathing bag and sealed,and placed in an incubator for constant temperature fermentation.Each experimental treatment level was repeated 4 times.The effect of liquid fermentation process parameters was evaluated using effective acidity,crude protein content,acid-soluble polypeptide content and urease activity as evaluation indexes.The results show that: The urease activity decreased gradually with the extension of fermentation time and there was no significant difference between the 6 and 7 day groups(P>0.05).The urease activity was the lowest at 30℃(P<0.05).The effective acidity and urease activity showed a downward trend with the increase of initial moisture.There was no significant difference in the contents of crude protein and acid-soluble polypeptide when 25%,30%,35% water was added(P>0.05).The effective acidity and urease activity reached the lowest point when the proportion of bran in fermentation substrate was increased to 20%(P<0.05).The results showed that the optimum time of soybean fermentation was 5 days,the optimum initial moisture content was 30%,the optimum temperature was 30℃,and the optimum proportion of matrix bran was 20%.The fermentation process increased the contents of protein,acid soluble polypeptide and other components,reduced the activities of urease and other antinutritional factors,and optimized the amino acid structure.Secondly,because the above indexes could not accurately describe the effect of fermentation time,the effect of fermentation time was explained from the aspect of fermentation microorganisms.In order to determine the distribution changes of microbial flora,the interaction between nutrient quality and microorganisms and the evolution behavior of microorganisms during liquid fermentation of soybean,this study used complex fermentation microorganisms to ferment soybean,and conducted high-throughput sequencing of microorganisms in fermented products at 0 d,3 d,4 d,5 d,6 d and 7 d,respectively.The results show that: The number of OTUs in samples of 4-day fermentation was the lowest,and Shannon and Chao1 indices both showed a trend of first rising and then declining.The abundance of Cyanobacteria and Proteobacteria in exogenous water decreased after 3 days of fermentation,but they were still dominant species.After 4 days of fermentation,the abundance of beneficial microorganisms such as Firmicutes increased,and functional prediction showed that pathways related to limiting amino acid and carbohydrate metabolism were activated(P<0.05).The results showed that fermentation increased the abundance of beneficial microorganisms such as Firmicutes in soybean,and Firmicutes played an important role in nutrient composition changes such as protein,acid-soluble polypeptide and ash,weakened the interspecific interaction,and activated the negative feedback loop of microbial community.Finally,juvenile Procambarus clarkii were fed with fermented soybean instead of fish meal to determine the effects of fermented soybean instead of fish meal on intestinal microorganisms,digestive enzymes and intestinal structure of Procambarus clarkii.In this study,juvenile Procambarus clarkii(group specifications written)were used as experimental animals,and fermented soybeans were designed to replace fish meal in 5 groups: 0%(group F0),25%(group F1),50%(group F2),75%(group F3)and 100%(group F4).A total of 300 healthy Procambarus clarkii with uniform size were randomly divided into 5 groups with 3replicates per group and cultured in a circulating tank(60×40×20 cm)for 16 days.The results showed that,compared with F0 group,the diversity of dominant species in the intestinal microorganisms of Procambarus clarkii increased,and Firmicutes were the main dominant species.There were no significant differences in digestive enzyme activities among all groups(P>0.05),and intestinal tissue sections showed that intestinal injury was aggravated with the increase of fermented feed content.The results showed that fermented soybean feed increased the abundance of Firmicutes and other probiotics,and activated related metabolic pathways in the digestive system to compensate for the decreased digestive function caused by intestinal injury.Conclusions: The best results were obtained by using compound probiotics in liquid fermentation of soybean at 30℃ supplemented with 30% initial water and 20% bran for 5 days.The protein content of fermented soybean was improved,and the abundance of Firmicutes and Bacteroidetes was increased after feeding Procambarus clarkii. |
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