Table_1_Effects of intrinsic tannins on proteolysis dynamics, protease activity, and metabolome during sainfoin ensiling.XLSX

Condensed tannins (CT) from sainfoin have a high capacity to inhibit proteolysis. A previous study reported that CT from sainfoin can inhibit lactic acid bacteria activity and decrease ammonium-nitrogen (N) content during sainfoin ensiling; however, no study has focused on the metabolome of ensiled sainfoin. The objective of the present study was to investigate the effects of CT [following supplementation of deactivated CT with polyethylene glycol (PEG)] on protease activity, keystone bacteria, and metabolome during sainfoin ensiling. According to the results, PEG amendment increased non-protein N, amino acid, and soluble protein contents significantly (in the 49.08–59.41, 116.01–64.22, and 23.5–41.94% ranges, respectively, p < 0.05) during ensiling, whereas neutral detergent-insoluble protein and acid detergent-insoluble protein were decreased significantly (in the 55.98–64.71 and 36.58–57.55% ranges, respectively, p < 0.05). PEG supplementation increased aminopeptidase and acid protease activity after 3 days of ensiling (p < 0.05) and increased carboxypeptidase activity during the entire ensiling process (p < 0.05). The keystone bacteria changed following PEG addition (Stenotrophomonas, Pantoea, and Cellulosimicrobium in the control vs. Microbacterium, Enterococcus, and Brevundimonas in the PEG-treated group). In total, 510 metabolites were identified after 60 days of sainfoin ensiling, with 33 metabolites annotated in the Kyoto Encyclopedia of Genes and Genomes database. Among the metabolites, phospholipids were the most abundant (72.7% of 33 metabolites). In addition, 10 upregulated and 23 downregulated metabolites were identified in the PEG-treated group when compared with the control group, after 60 days of ensiling (p < 0.05). Pediococcus (correlated with 20 metabolites, R2 > 0.88, p < 0.05) and Lactobacillus (correlated with 16 metabolites, R2 > 0.88, p < 0.05) were the bacteria most correlated with metabolites. The results suggested antagonistic effects between Lactobacillus and Pediococcus during ensiling. The decreased proteolysis during sainfoin ensiling was mainly attributed to the inhibition of protease activity by CT, particularly carboxypeptidase activity. In addition, proteolysis decreased partly due to CT inhibiting Pediococcus activity during ensiling, with Pediococcus being significantly and positively correlated with dopamine after 60 days of ensiling (R2 = 0.8857, p < 0.05).

红豆草来源的缩合单宁（CT）具有较强的蛋白水解抑制能力。既往研究表明，红豆草缩合单宁可在红豆草青贮过程中抑制乳酸菌活性并降低铵态氮（N）含量，但目前尚无针对青贮红豆草代谢组的相关研究。本研究旨在探究经聚乙二醇（PEG）灭活的CT对红豆草青贮过程中蛋白酶活性、关键类群细菌及代谢组的影响。研究结果显示，青贮期间添加PEG可显著提升非蛋白氮、氨基酸及可溶性蛋白含量（分别为49.08%~59.41%、116.01%~64.22%及23.5%~41.94%，p < 0.05），而中性洗涤不溶蛋白与酸性洗涤不溶蛋白则显著降低（分别为55.98%~64.71%及36.58%~57.55%，p < 0.05）。青贮3天后，添加PEG可提升氨肽酶与酸性蛋白酶活性（p < 0.05），且整个青贮过程中羧肽酶活性均显著升高（p < 0.05）。添加PEG后，青贮体系的关键类群细菌发生改变：对照组关键类群为寡养单胞菌属、泛菌属与纤维微菌属，而PEG处理组则为微杆菌属、肠球菌属与短波单胞菌属。红豆草青贮60天后，共鉴定出510种代谢物，其中33种可注释至京都基因与基因组百科全书（KEGG）数据库。在上述注释代谢物中，磷脂占比最高，占33种代谢物的72.7%。与对照组相比，PEG处理组青贮60天后共鉴定出10种上调代谢物与23种下调代谢物（p < 0.05）。片球菌属（与20种代谢物呈显著相关，R²>0.88，p < 0.05）与乳杆菌属（与16种代谢物呈显著相关，R²>0.88，p < 0.05）是与代谢物相关性最强的细菌类群。研究结果表明，青贮过程中乳杆菌属与片球菌属之间存在拮抗作用。红豆草青贮过程中蛋白水解程度降低，主要归因于CT对蛋白酶活性的抑制，尤其是对羧肽酶活性的抑制。此外，CT通过抑制青贮过程中片球菌属的活性，也部分降低了蛋白水解程度；青贮60天后，片球菌属与多巴胺呈显著正相关（R²=0.8857，p < 0.05）。