Optimization Of Key Technology Of Fermented Mulberry Leaves And Its Regulation Mechanism Of Antioxidant And Lipid Metabolism In Mutton Sheep

Mulberry leaves（MLs）are rich in nutrients and bioactive ingredients.They are not only the main source of silkworm feed in sericulture industry,but also a traditional Chinese medicine with the same origin as medicine and food and a non-traditional feed with high nutritional value and application prospect.Fresh mulberry leaves with high water content,are easy to deteriorate and difficult to preserve for a long time,so the cycle of feed utilization is short.Drying and fermentation are usually adopted to prolong its preservation and service cycle in the production.The palatability of mulberry leaves and feed intake of domestic animals can be improved by fermentation.Although there are many researches on mulberry leaf fermentation,the results are uneven.Therefore,it is difficult to guide the formulation of feed production technology in MLs fermentation.Moreover,with the improvement of people’s living standards,threre is increasing market demand for mutton currently.Hence,it is of great significance to study the growth and production performance of mutton sheep.In this study,in order to optimize the fermentation process of MLs and explore the value of FMLs in feed utilization,the fermentation process of MLs is further explored and optimized,and the functional effects of fermented MLs（FMLs）on growth performance,antioxidantion,immunity,lipid metabolism,bacterial community in rumen and faeces of mutton sheep are studied.The main results are as follows:1.Optimization of key technologies for fermentation of MLs.According to the principle of single factor variable,a single variable study was conducted on the proportion of bacteria（single bacteria or different multi-bacteria combination）,fermentation temperature,inoculated size,moisture content of MLs and duration of fermentation.Based on the nutritional value,active ingredient content and total antioxidant activity of FMLs,the most effective fermentation parameters of MLs were obtained as follows:S.Cerevisiae≤L.plantarum,fermentation temperature（27-30℃）,the optimal inoculation volume of 5%with bacterial solution concentration of 10⁸CFU/m L,moisture content of MLs（50-68%）,duration of fermentation（14-30 days）.2.Study on bacterial community in FMLs.In view of the close relationship between the activity of fermented products and bacteria,16s sequencing method was used to study the composition of microbial community in FMLs.The results showed that the dominant genus of FMLs inoculated by L.plantarum is 88.60%Lactobacillus.The dominant genera of FMLs inoculated by S.cerevisiae and L.plantarum with a ratio of 1:1 are 26.94%Lactobacillus and 67.16%Enterococcus.Enterococcus（17.30%）and Carnobaterium（74.28%）are dominant genera in un-inoculated FMLs.The increase in moisture content of MLs may increase the abundance of Enterobacter.Inoculated fermentation could reduce p H and the content in ammonia nitrogen（NH₃-N）,both of which are related to the quality of FMLs,improve antioxidant activities and alter the diversity of bacterial community.In addition,relative quantitative PCR of bacteria reveals that L.plantarum plays a pivotal role in the dominant MLs fermentation,because its abundance is increased along with the increase in inoculation amount（p<0.05）.However,the content of S.cerevisiae in FMLs has nothing to do with its inoculation amount.3.FMLs can promote the growth and improve antioxidantive and immune properties of mutton sheep.Dietary supplementation of FMLs could increase feed intake,daily gain and growth hormone content of mutton sheep（p<0.05）,and decrease feed to gain ratio（p>0.05）.Moreover,the contents of superoxide dismutase（SOD）,catalase（CAT）,glutathione peroxidase（GSH-Px）and total antioxidant capacity（TAOC）in serum,muscle and adipose tissue were increased with FMLs treatment,while the content of malondialdehyde（MDA）was decreased（p<0.05）.Metabolomics and transcriptome analysis showed that the improvement of antioxidant performance was related to the downregulation of the oxidation of unsaturated fatty acids（PUFAs）and the up-regulation of tryptophan metabolic pathway.Additionally,the contents of immunoglobulin G（Ig G）and total immunoglobulin（IG）as well as the expression of immune-related genes were increased with FMLs treatment（p<0.05）,and the enhancement of immunity was found to be related to the up-regulation of glycolysis and mannose-6-phosphate metabolic pathways.4.FMLs have the lipid-lowering effect.Dietary supplementation of FMLs could decrease the content of crude fat in muscle（p<0.05）,the contents of triglyceride（TG）and free fatty acid（FFA）in serum,muscle and fat（p>0.05）,and significantly affected the content of hormones related to fat metabolism and the expression of related genes（p<0.05）.Immunofluorescence analysis showed that FMLs could promote the expression of mitochondrial marker protein（VDAC1）（p<0.05）.Widely target lipids and GSEA analysis show that FMLs could regulate the metabolism of glycolipids,phospholipids,ether lipids and sphingolipids,promote the degradation andβoxidation of fatty acids.5.FMLs can promote rumen digestion and reduce fecal odor.FMLs can improve the abundance of rumen beneficial bacteria Prevotella,norank＿f＿Bacteroidales＿UCG-001,Fibrobacter and Ruminococcus,decrease the abundance of harmful bacteria Rikenellaceae＿RC9＿gut＿group（p<0.05）,improve the contents of volatile fatty acids（VFAs）,digestive enzymes,bacterial proteins,reduce the content of NH₃-N（p<0.05）thus reducing the loss of nitrogen,and also reduce the excretion of dry matter in feces to promote the digestion and absorption of nutrients;In addition,FMLs can increase the abundance of fecal Unclassified＿f＿Lachnospiraceae,significantly reduce the abundance of fecal Christensenellaceae＿R-7＿group and NK4A214＿group（p<0.05）and reduce emissions of fecal odor or harmful volatile gas including NH₃-N,H₂S,3-methyl indole,p-cresol thus to reduce fecal.In conclusion,the alteration of fermentation conditions can affect the quality and microbial community of FMLs.FMLs can improve the growth performance,antioxidantive and immune properties of animals,promote the nutrients digestion and absorption in rumen,reduce the odor of faeces,and promote quality as well as healthy breeding and environmental protection.In addition,FMLs also have the lipid-lowering effect.Therefore,FMLs are an unconventional feed material with good application value in animal husbandry.