【作者】 邓荟芬；

【导师】 揭雨成；

【作者基本信息】 湖南农业大学 ， 草学， 2021， 博士

【摘要】 本研究旨在通过添加酶制剂及微生物对苎麻进行生物降解,并通过代谢组学及微生物组学的研究,揭示苎麻青贮木质纤维素生物降解的规律,阐明降解机制,建立苎麻木质纤维素黑曲霉（Aspergillus Niger）降解的系统方法,进而研究苎麻黑曲霉生物降解青贮草粉对育肥猪生长和肉品质调控的影响。主要结果如下:1、饲用苎麻中添加酶制剂、微生物进行青贮,其中以每千克青贮料中添加100m L（即100m L/kg）浓度为5.73×10⁶cfu/m L黑曲霉菌液处理组处理效果较好。此处理组青贮时长60d可以显著降低苎麻青贮NDF（P<0.05）、ADF（P<0.05）以及ADL（P<0.05）的含量,即有效降解木质纤维素。苎麻经降解烘干粉碎后,过40目筛后絮状物含量约为0。2、添加黑曲霉菌液进行苎麻青贮的过程中,分别在青贮15 d、30 d和60 d开包取样测定青贮样品中的木质纤维素降解酶种类及酶活。漆酶和木聚糖酶活性在青贮第1天上升,然后持续下降;羧甲基纤维素酶（CMC）活性持续下降;锰过氧化物酶（Mn P）活性在15d前持续下降,15d后处于稳定状态。青贮60d进行检测,四种酶都仍然存在,CMC第60d比初始值下降了94.02%;锰过氧化物酶第60d比初始值下降了92.90%;漆酶活性第60d比初始值下降了52.27%;木聚糖酶第60d比初始值下降了88.11%。3、对对照组、青贮宝组、黑曲霉组苎麻青贮分别在青贮15 d,30 d和60 d取样,进行微生物组学分析,通过OTU数和稀释曲线等指标对比显示,黑曲霉组的物种多样性较丰富。且整个青贮过程中的优势菌门为厚壁菌门（Firmicutes）和变形菌门（Proteobacteria）,优势菌属为乳杆菌属（Lactobacillus）和片球菌属（Pediococcus）。对照组青贮过程中,肠杆菌是优势菌群之一,影响发酵品质,肠杆菌引起蛋白质降解产生氨态氮（NH₃-N）;在黑曲霉组青贮过程中,芽孢杆菌（Bacillales）和泛生菌（Pantoea）是其中的两个优势菌群,很多芽孢杆菌可以降解多种大分子化合物,如纤维素、淀粉、蛋白质等;泛生菌对纤维素和木质素有降解效果。两个优势菌群的存在,是黑曲霉组对木质纤维素降解效果较好的原因之一。4、对对照组、青贮宝组和黑曲霉组青贮15 d、30 d、60 d苎麻青贮样品进行代谢组学分析,筛选其差异代谢物,与对照组和青贮宝组相比,黑曲霉组中与木质纤维素降解相关的酚类、醇类、羧酸、糖类等物质显著增高（P<0.05）;黑曲霉组内青贮60d组中与木质纤维素降解相关的酚类、醇类、羧酸、糖类等物质比黑曲霉30d和黑曲霉组15d显著增高（P<0.05）。与木质纤维降解相关的主要代谢通路是色氨酸-酪氨酸-苯丙氨酸代谢通路、苯丙氨酸代谢通路、苯丙烷代谢通路、半乳糖代谢通路。不同处理中富集到不同代谢通路的差异代谢物水平都显示黑曲霉处理组的降解效果优于对照组,且黑曲霉组青贮60 d降解效果优于15 d和30 d。5、将苎麻青贮烘干粉碎过筛后,对基础日粮添加5%、10%、15%、20%和25%的苎麻青贮草粉进行育肥猪的饲喂,发现育肥猪日粮中添加一定量的苎麻青贮草粉可以提高猪肉的品质、营养价值和猪的免疫功能。在日粮中添加20%的苎麻青贮草粉不影响猪的生长性能,且肉质嫩瘦肉率高,具有较好的营养价值,能较好改善猪的血脂代谢,提高猪的免疫功能。结论:通过苎麻青贮试验筛选,发现苎麻青贮中100m L/kg的黑曲霉菌液可以达到充分降解木质纤维素的目的,消除烘干粉碎后的絮状物;青贮过程中黑曲霉产生的胞外酶逐渐减少,但始终有一定含量,对木质纤维素的降解发挥着作用。且该处理组青贮中微生物多样性丰富,优势菌属为乳杆菌属和片球菌属,其含有的芽孢杆菌、泛菌属对木质纤维素降解有促进作用。且此处理降解产物中出现大量与木质纤维素相关的酚类、醇类、羧酸及糖类物质,差异代谢物涉及到与木质纤维素降解的四条代谢通路,说明木质纤维素得到了降解。在日粮中添加不同比例的苎麻青贮草粉进行育肥猪饲喂,添加20%苎麻青贮草粉组不影响猪的生长性能,且肉质嫩、瘦肉率高,具有较好的营养价值,能较好改善猪的血脂代谢,提高猪的免疫功能。更多还原

【Abstract】 In the current study,the enzymatic degradation of ramie lignocellulose by the microorganism,and the efficacy of lignocellulolytic biodegradation in ramie silage was evaluated.The degradation mechanism of lignocellulos in remie by A.Niger was systematically elucidated by the metabolomics approach and microbiology.Lastly,we investigated the efficacy of biodegradation by A.Niger on silage by testing its effects on the growth and meat quality of finishing pigs that were fed with the silage in its powdered form.The methodology and results of the study are as described below:1.Enzyme additives and microbial inoculants were added to the ramie forage for the preparation of silage.A volume of 100 m L of inoculum containing A.Niger solution at a concentration of 5.73×10~6cfu/m L per kg of silage was regarded as the treatment group for optimization.The treatment groups were processed with or without fermentation.The group that was subjected to fermentation for 60 days showed to reduce the neutral detergent fiber(NDF),acid detergent fiber(ADF),and acid detergent lignin(ADL)of ramie silage significantly(p<0.05).In the case of treatment with fermentation where the ramie leaves hay was dried,crushed,and passed through mesh sieves about 40 times,about 0%of the flocculent content was retained.2.The lignocellulose-degrading enzymatic activities of A.Niger in the solution were measured after 1 day of fermentation,on day 15,day 30,and day 60 of ramie silage fermentation.Accordingly,the activities of laccase and xylanase showed to decrease on the first day of fermentation continuously same as seen for carboxymethyl cellulase(CMC)activity and manganese peroxidase(Mn P)activity;all of which continued to decrease until15 days.The activity,however,stabilized after day 15.By 60 days after fermentation,the activity of all the four enzymes in the study was still persistent,although there was a decrease in laccase activity by 52.27%,xylanase by 88.11%,CMC by 94.02%,and manganese peroxidase by 92.90%from that of the initial value.3.Aliquot Samples were taken on days 15,30,and 60 of fermentation from the different groups,namely,the silage group,A.Niger group,and control group,and the microbiology was conducted.By comparing factors such as OTU number and dilution curve,it was clear that the diversity of A.Niger group was richer;dominant phylum during fermentation process was to be Firmicutes and Proteobacteria,and dominant genera to be Lactobacillus and Pediococcus.One of the dominant phylum in the control group during the fermentation process was found to be Enterobacter affecting the fermentation quality incidentally due to its protein degradation ability that produces ammoniacal nitrogen(NH3-N).Followed by the A.Niger species,Bacillales and Pantoea were the other two dominant groups.Earlier reports show that many Bacillales can degrade a variety of macromolecules such as cellulose,starch,and proteins;Pantoea is effective in degrading cellulose and lignin.Evidently,the presence of these two dominant groups is one of the prime reasons for the effective degradation of lignocellulose by the A.Niger group.4.Metabolomic analysis was performed to screen the differential metabolites in ramie silage samples that were subjected to fermentation,and data were obtained during days 15,30,and 60 from the silage,A.Niger,and control groups.The results of this study indicated that the phenols,alcohols,carboxylic acids,and sugars associated with lignocellulose degradation were significantly higher(p<0.05)in the A.Niger group compared to the control and silage groups.Within the A.Niger group,these compounds were significantly higher(p<0.05)in the day 60 group compared to that of day 30 and day 15 groups.The metabolic pathways that are mainly associated with lignocellulose degradation were identified to be tryptophan-tyrosine-phenylalanine-,phenylalanine-,phenyl propane-,and galactose-metabolic pathway.Further,levels of the differential metabolites expressed in these metabolic pathways in different treatment groups showed that the degradation effect due to A.Niger treatment was better than the control group,and the degradation effect of fermentation in the A.Niger group was found to be better on day 60 than day 15 and day 30.5.Lastly,the dried,crushed,and powdered ramie silage was sieved and given as feed for finishing pigs at 5%,10%,15%,20%,and 25%quantity and tested for efficacy on meat quality.It was found that by adding a certain amount of ramie silage meal to feed the finishing pigs,the meat quality and nutritional value could be improved,and additionally,the animals showed the better immune function than earlier.Although the addition of 20%ramie silage feed to the animal diet did not visibly change their growth characteristics,their meat was nevertheless tender and lean with a better nutritional value indicating that the addition had impacted by improving the pigs’lipid metabolism and enhanced immune function.CONCLUSION:By screening the ramie silage in different fermentation conditions,it was found that the addition of A.Niger inoculum in solution at 100m L/kg of the silage achieved sufficient lignocellulose degradation and also eliminated flocculation when subjected to drying and crushing.The levels of extracellular enzymes produced by A.Niger in the silage showed to decrease gradually.Nonetheless,they retained certain levels throughout,indicating their role in lignocellulose degradation.Microbial diversity studies showed A.Niger treatment was rich in silage comparatively.However,it was seen that the dominant genera lactobacillus,lactococcus,bacillus,and pediococcus promote lignocellulose degradation.Further,a large number of phenols,alcohols,carboxylic acids,and sugars in connection with lignocellulose appeared in the degradation products in this treatment group.These differential metabolites were also found to be involved in various metabolic pathways of lignocellulose degradation,indicating their role in the process.Lastly,when different proportions of ramie silage meal were added to the finishing pigs feed,it was observed that the addition of 20%ramie silage meal did not affect the growth characteristics of pigs;however,their meat was tender and lean with a better nutritional value indicating better-improved lipid metabolism which was also supported by the enhanced immune function in these pigs.更多还原