Screening Of Mulberry Leaf Tannin-Degrading Bacteria,Fermentation Process And Application Of Fermented Mulberry Leaves In Carp Culture

Mulberry trees are widely planted and have a high yield in our country,and their main utilization is sericulture.Due to seasonal restrictions,a large number of mulberry leaves have not been used effectively.Therefore,mulberry leaves can be used for livestock and poultry breeding after proper treatment to alleviate the problem of feed shortage,reduce the feeding cost and promote the development of breeding industry.The presence of anti-nutritional factors such as tannins in mulberry leaves restricts the application of mulberry leaves in livestock and poultry feed to a certain extent.By using microbial fermentation,large molecules of protein are degraded into small peptides,soluble proteins and free amino acids,which are beneficial to the digestion and absorption of animals and at the same time can effectively reduce the content of anti-nutritional factors in mulberry leaves and improve the quality of mulberry leaves.In this experiment,two strains were selected from the strains isolated from pickles and preserved in the laboratory,which could effectively reduce the tannin content of mulberry leaves.The strains were identified by colony and cell morphology observation,physiological and biochemical tests and 16S r DNA sequence analysis.The fermentation conditions of mulberry leaves were optimized by single-factor test and response surface experiment,and the fermented mulberry leaves were tested for carp culture using cage.The main results were as follows:1.Two strains of Lactobacillus sp.and Saccharomyces sp.which named strain R₃ and strain J₁ were screened from sauerkraut and the strains preserved in the laboratory that could effectively degrade tannins.Strain R3 was identified as Lactobacillus paracasei and strain J₁as Saccharomyces cerevisiae through morphological observation,physiological and biochemical tests and molecular biological identification,the phylogenetic tree was constructed after blast sequence alignment.2.Effects of different strain ratios,additions of glucose,sodium acetate,（NH₄）₂SO₄,C₆H₁₄N₂O₇,KH₂PO₄,solid water ratio,fermentation time,inoculum amount and temperature on the degradation of mulberry leaf tannins by the compound fermentation of Lactobacillus paracasei R₃ and Saccharomyces cerevisiae J₁ were investigated by single-factor test method,and the culture conditions were optimized by response surface analysis.The results showed that the optimal fermentation conditions were:solid water ratio 0.9,inoculum level 9.8%,and fermentation time 44.6 h.Under these conditions,the tannin degradation rate reached 50.49%.The verification test showed that the response surface model could accurately predict the influence of various factors on tannin degradation rate.3.0%（W₁）,5%（W₂）,10%（W₃）,15%（W₄）and 20%（W₅）of fermented mulberry leaves（FML）were added to the crucian carp feed to prepare the experimental feed with equal nitrogen and energy.750 healthy and lively crucian carp were randomly divided into 5 groups with 5 replicates in each group and 30 fish in each replicate.According to the random block design,they were put into the cage hanging in the cement pool for 7 days,and were formally fed for 8 weeks.At the end of the experiment,the samples were taken and relevant indexes were determined.The results showed that the weight gain rate,specific growth rate and fatness increased by 45.31%,0.14%and 0.48%in the treatment of 5%FML and the feed coefficient,feeding rate,visceral body ratio,intestinal body ratio,liver body ratio and visceral fat ratio were decreased by 9.65%,0.11%,0.31%,0.14%,0.02%and 0.18%compared with the control group.It was indicated that the addition of fermented mulberry leaves in the feed could promote the growth performance,anti-oxidation ability of serum and the activity of intestinal digestive enzyme.There was no significant difference between the addition of 5%FML and the blank control group.With the increase of FML（over 10%）,the growth performance,serum antioxidant capacity and intestinal digestive enzyme activity decreased,and the homeostasis of intestinal flora was destroyed.