溶菌酶对瘤胃体外发酵、甲烷生成及微生物菌群结构的影响

Effects of lysozyme on in vitro fermentation, methanogenesis and microbial community structure of the rumen

摘要:

[目的] 通过体外静态模拟瘤胃发酵法研究溶菌酶对瘤胃发酵、甲烷生成及微生物菌群结构的影响。[方法] 采用单因素多水平试验设计，溶菌酶添加水平分别为0 (L-0，对照组)、0.1 mg/100 mL (L-0.1)、1 mg/100 mL (L-1)、10 mg/100 mL (L-10)和100 mg/100 mL (L-100)，定时测定产气量和甲烷产量，培养24 h后，发酵液用于发酵参数和微生物菌群数量的qPCR测定，其中L-0、L-1和L-100三个组发酵液同时进行16S rRNA基因Illumina高通量测序。[结果] 与对照组相比，低剂量溶菌酶添加(L-0.1组)不影响甲烷产量、氨氮浓度、干物质消失率、有机物消失率和总挥发性脂肪酸等瘤胃发酵参数(P>0.05)；随着剂量提高，L-1处理组甲烷产量、氨氮浓度显著降低(P<0.05)，丙酸浓度显著增加(P<0.05)，并且干物质消失率、有机物消失率和总挥发性脂肪酸不受影响(P>0.05)；而较高剂量组(L-10和L-100组)虽然甲烷产量显著降低，丙酸浓度显著增加(P<0.05)，但干物质消失率和有机物消失率也显著降低(P<0.05)。qPCR结果显示高剂量组(L-100组)总菌、原虫、甲烷菌数量与对照组相比显著降低(P<0.05)，而L-0.1、L-1和L-10组总菌、真菌和原虫数量与对照组相比均无显著变化(P>0.05)。高通量测序主成分分析(PCA)显示对照组与溶菌酶添加组间瘤胃细菌组成的明显区分，说明添加溶菌酶显著改变了瘤胃细菌菌群结构。溶菌酶通过增加月形单胞菌和琥珀酸弧菌等丙酸生成菌的相对丰度，使更多的氢被用于生成丙酸，导致甲烷产量降低；溶菌酶可抑制普雷沃氏菌和拟杆菌属等蛋白降解菌的生长，进而减少蛋白质过度降解，降低氨氮浓度。[结论] 添加适宜浓度(1 mg/100 mL)的溶菌酶可通过调控瘤胃微生态改变瘤胃发酵模式，降低瘤胃甲烷和氨的生成，短期内并不影响饲料消化。

Abstract:

[Objective] We studied the effects of lysozyme on in vitro rumen fermentation, methanogenesis and microbial community structure. [Methods] Lysozyme was added to in vitro ruminal cultures at 5 doses: with 0 (L-0, Control), 0.1 mg/100 mL (L-0.1), 1 mg/100 mL (L-1), 10 mg/100 mL (L-10), and 100 mg/100 mL (L-100). Total gas and methane production were measured at different time of incubation. Culture samples were collected at 24 h for analysis of fermentation parameters and functional microbial populations. In addition, samples of L-0, L-1, and L-100 collected at 24 h were also used subjected to metagenomics analysis of bacterial community using Illumina sequencing of 16S rRNA gene amplicons. [Results] Compared with control, methane production, ammonia concentration, dry matter digestibility, organic matter digestibility and total volatile fatty acid concentration were not influenced by L-0.1 (P>0.05). Methane production and NH₃-N concentration were reduced, and propionate concentration was increased by L-1 (P<0.05), whereas dry matter digestibility, organic matter digestibility or total volatile fatty acid were not affected (P>0.05). Methane production was reduced, and propionate concentration was increased by L-10 and L-100 (P<0.05), but dry matter digestibility and organic matter digestibility were also decreased significantly (P<0.05). The qPCR results showed that total bacteria, fungi and methanogens were significantly reduced by L-100 (P<0.05), but were not influenced (P>0.05) by L-0.1, L-1, or L-10. Principal Component Analysis of the sequencing data showed clear differences in the composition of the ruminal bacterial community between the control and the lysozyme treatments, demonstrating evident impact of the lysozyme addition. The abundance of propionate-producing bacteria (e.g., Selenomonas and Succinivibrio) was increased by lysozyme, resulting in more hydrogen being directed to production of propionate instead of methane. Moreover, the reduced ammonia concentration in L-1 was probably due to the lower abundance of proteolytic bacteria (e.g., Prevotella and Bacteroides) inhibited by lysozyme. [Conclusion] Appropriate lysozyme addition (1 mg/100 mL) can be used to modulate ruminal microbial ecology and reduce methanogenesis and ammoniagenesis by rumen microbiome without adversely affecting feed digestion or fermentation in short-term.s