基于宏基因组研究植物乳酸菌或博落回提取物对山羊回肠微生物合成维生素B12的影响

【目的】探索研究反刍动物胃肠道微生物合成维生素B₁₂的方法，并评估植物乳酸菌或博落回提取物对断奶山羊回肠食靡微生物合成维生素B₁₂的影响。【方法】选取体重相近年龄相仿的断奶黑山羊20只，随机分为对照组(CON, n=7)、乳酸菌组(LAC, n=7)和博落回组(MAC, n=6)。CON组饲喂普通的日粮，LAC组饲喂基础日粮+10 g/d的植物乳酸菌(Lactobacillus plantarum P-8 strains, 4.0×10⁹ CFU/g)，MAC组饲喂基础日粮+0.3 g/d的博落回提取物(Macleaya cordata 3.75%)。试验结束后，采集回肠中段食靡样品。利用宏基因组测序技术，比对最新功能基因数据库VB12Path和公共数据库KEGG，分析植物乳酸菌和博落回提取物对山羊回肠食靡微生物合成维生素B₁₂的影响。【结果】结果显示，比对VB12Path数据库共注释到55个与维生素B₁₂合成相关的基因。与CON组相比，LAC组和MAC组中合成维生素B₁₂基因的丰富度和均匀度降低(P<0.05）。3组间基因的β多样性也有显著的差异(P<0.05)；比对KEGG数据库共注释到49个与维生素B₁₂合成相关的基因，LAC组的多样性与CON组没有差异，但MAC组的α多样性显著降低(P<0.05)。值得注意的是，比对VB12Path数据库和KEGG数据库均发现LAC组和MAC组中参与前咕啉2合成途径、参与无氧合成途径、有氧合成途径、参与重排转换途径以及腺苷钴胺素合成途径的部分基因(gltX、cbiT、cobT和btuD等)的丰度均显著地高于CON组(P<0.05)。【结论】2个数据库比对后的相似结果表明博落回提取物在对断奶山羊回肠微生物合成维生素B₁₂相关代谢上与植物乳酸菌的作用相似，均可以通过改变其多样性和提高部分关键基因的丰度，从而影响微生物合成维生素B₁₂的潜能，为后期博落回提取物和植物乳酸菌在畜牧养殖中的运用提供一定的理论支撑。此外，2个数据库比对的差异提示未来研究胃肠道微生物维生素B₁₂相关代谢时，应用多个数据库比对，能更全面精确地进行评价，为后期分析过程奠定研究基础和提供新的思路。

Lactobacillus plantarum or Macleaya cordata affects the biosynthesis of vitamin B12 by ileal microbiota of weaned goats: a metagenomics study

Objective] To explore the vitamin B₁₂ synthesis in ruminant gastrointestine and evaluate the effect of Lactobacillus plantarum (LAC) or Macleaya cordata (MAC) on the synthesis of vitamin B₁₂ by the ileal microbiota of weaned goats. Methods] Twenty weaned black goats with similar body weights and ages were randomly assigned into a control group (n=7), a LAC group (n=7), and a MAC group (n=6). The control group was fed with a normal diet, and the LAC and MAC groups with normal diets supplemented with LAC P-8 at 4.0×10⁹ CFU/g and 0.3 g/d MAC (3.75%), respectively. The contents in the middle section of ileum were collected for metagenomic sequencing. VB12Path and KEGG were employed to analyze the sequencing results. Results] A total 55 and 49 key genes associated with vitamin B₁₂ synthesis were identified in VB12Path and KEGG, respectively. The alpha and beta diversity of genes involved in vitamin B₁₂ synthesis were changed by the supplementation of LAC or MAC (P<0.05). Compared with that in the control group, the diversity of the LAC group showed little difference and that of the MAC group decreased (P<0.05). The abundance of genes involved in precorrin-2 synthesis pathway, aerobic and anaerobic synthesis pathways, salvage pathway, and Post-AdoCbi-P pathway (e.g., gltX, cbiT, cobT, and btuD) in LAC and MAC groups was higher than that in the control group. Conclusion] The supplementation of LAC or MAC enhanced the synthesis of vitamin B₁₂ by ileal microorganisms. This study expands our understanding and analytical method of the microbial vitamin B₁₂ synthesis.