中国水稻秸秆年产量约1.9亿吨，但由于其粗纤维含量高，营养价值低和适口性差等缺点，导致秸秆饲料化比例不足秸秆综合利用率的20%。大量研究表明，秸秆经过微贮后营养价值和适口性得到改善。但水稻秸秆受可溶性碳水化合物含量低和硅元素含量高等因素限制，目前实际应用于水稻秸秆微贮的菌种资源有限。本试验采用单因素试验设计，利用三种不同异型发酵乳酸菌单独及组合对水稻秸秆进行微贮发酵研究，三株异型发酵乳酸菌筛选自青贮燕麦和氨化秸秆，分别为布氏乳杆菌R17（Lactobacillus buchneri）、短乳杆菌R33（Lactobacillus brevis）和肠膜明串珠菌HN13（Leuconostoc mesenteroides），研究单菌处理、双菌处理和三菌处理对微贮水稻秸秆的营养成分、发酵品质和体外降解率的影响，通过高通量测序技术比较不同处理间微生物群落的变化，为异型发酵乳酸菌微贮水稻秸秆提供一定的数据支撑。试验结果表明：

不同处理对微贮水稻秸秆营养成分的影响

R33组、R33+HN13组、R17+HN13组、R33+R17组和R33+R17+HN13组极显著提高了微贮水稻秸秆的干物质（DM）含量（P＜0.001），分别提高了4.80%、4.06%、5.16%、5.23%和5.34%，R17组和HN13组DM含量与对照组相比差异不显著（P＞0.05）。对照组中性洗涤纤维（NDF）和酸性洗涤纤维（ADF）含量分别为72.47%和47.01%，R33组、R33+HN13组、R33+R17组和R33+R17+HN13组四组NDF和ADF含量与对照组相比极显著降低（P＜0.001），R17组、HN13组和R17+HN13组三组NDF和ADF含量与对照组差异不显著（P＞0.05）。各处理对微贮水稻秸秆的粗蛋白质（CP）含量、碳（C）含量、可溶性碳水化合物（WSC）含量和酸性洗涤木质素（ADL）含量没有显著影响（P＞0.05）。

不同处理对微贮水稻秸秆发酵品质的影响

R33组、R33+HN13组、R33+R17组和R33+R17+HN13组pH值和氨态氮（NH₃-N）含量与对照组相比极显著降低（P＜0.001），R17组pH值和氨态氮（NH₃-N）含量与对照组相比显著降低（P＜0.05），HN13组和R17+HN13组pH值和氨态氮（NH₃-N）含量与对照组差异不显著（P＞0.05）。

R33组、R33+HN13组、R33+R17组和R33+R17+HN13组乳酸含量达到19.64g/kg与对照组相比差异极显著（P＜0.001），R17组、HN13组和R17+HN13组与对照组相比乳酸含量差异不显著（P＞0.05）。各处理组乙酸和丙酸含量与对照组相比差异不显著（P＞0.05）。R33组与R17组、HN13组和R17+HN13组三组相比丙酸含量显著提高（P＜0.05）。对照组丁酸含量为19.83 g/kg，R33组、R33+HN13组、R33+R17组和R33+R17+HN13组均未检测出丁酸，与对照组相比差异极显著（P＜0.001），HN13组显著降低了丁酸含量（P＜0.05），R17组和R17+HN13组丁酸含量与对照组相比差异不显著（P＞0.05）。

不同处理对水稻秸秆体外降解率的影响

12h时，R33组、R17组、HN13组、R33+HN13组和R33+R17+HN13组与对照组相比微贮水稻秸秆的体外降解率显著提高（P＜0.05），R17+HN13组和R33+R17组与对照组差异不显著（P＞0.05）。各处理组在24h后各时间点微贮水稻秸秆的体外降解率极显著提高（P＜0.001）。其中R33组提高幅度最大，在12h、24h、48h和72h体外降解率分别较对照组提高了10.68%、11.44%、9.27%和9.12%。

不同处理对水稻秸秆微生物群落的影响

各处理组显著改变了微贮水稻秸秆的微生物群落结构。在门水平，R33、R33+HN13、R33+R17和R33+R17+HN13的厚壁菌门（Firmicutes）相对丰度均达到80%以上，与对照组相比极显著提高（P＜0.001），未添加R33菌的处理组和对照组厚壁菌门的相对丰度差异不显著（P＞0.05），相对丰度百分比相差40%以上。R17、HN13、R17+HN13和对照组变形菌门(Proteobacteria)相对丰度显著高于其他处理。从属水平上看，R33、R33+HN13、R33+R17、R33+R17+HN13和R17五组乳酸杆菌属（Lactobacillus）相对丰度与对照组相比差异极显著（P＜0.001），前四组乳酸杆菌属（Lactobacillus）相对丰度在80%以上。其余菌属在R33、R33+HN13、R33+R17和R33+R17+HN13四组中相对丰度均在1%以下，与对照组差异显著（P＜0.05）。从微生物相对丰度与发酵指标相关性分析中可以得出，乳酸杆菌属与CP含量和乳酸含量呈正相关，与NDF含量、ADF含量、pH值、NH₃-N含量和丁酸含量呈负相关，考氏科萨克氏菌属、肠球菌属、明串珠菌属和产己酸菌属与DML呈正相关，与CP含量呈负相关。

综上所述，三种乳酸菌单独及组合处理均能不同程度改善水稻秸秆微贮发酵品质，其中R33菌处理对水稻秸秆微贮的效果优于其余各处理。R33能够大量产生乳酸显著降低pH值，抑制其他有害菌的生物活性，显著减少干物质的损失及NH₃-N和丁酸的产生，同时，显著降低NDF和ADF的含量，显著提高了微贮水稻秸秆的体外降解率。显著改变了微生物群落结构，提高乳酸杆菌属相对丰度，降低肠杆菌属和肠球菌属相对丰度。

关键词：乳酸菌微贮；水稻秸秆；发酵品质；微生物群落。

China has a huge production of rice straw, but due to its high fiber content, low nutritional level, and poor palatability, the utilization rate of rice straw by ruminants is low. Microbial fermentation can effectively improve the feeding value of rice straw and provide safe and inexpensive coarse feed for animal husbandry. This experiment used a single factor experimental design to use three different types of lactic acid bacteria, individually and in combination, for microbial fermentation fermentation of rice straw. Three strains of heterotypic fermentation lactic acid bacteria were selected from ensiled oats and ammoniated straw, namely R17 (Lactobacillus brucelli), R33 (Lactobacillus brevis), and HN13 (Streptococcus pneumoniae). An equal amount of distilled water was added as the control group, and single bacterial treatment groups were set up: R33, R17, and HN13; Dual bacterial treatment group: R33+HN13, R17+HN13, and R33+R17; Three bacterial treatment group: R33+R17+HN13. The main research findings are as follows:

The effect of different treatments on the nutritional composition of micro stored rice straw:

The R33 group, R33+HN13 group, R17+HN13 group, R33+R17 group, and R33+R17+HN13 group significantly increased the dry matter (DM) content of micro stored rice straw (P<0.001), increasing by 4.80%, 4.06%, 5.16%, 5.23%, and 5.34%, respectively. There was no significant difference in DM content between the R17 group and HN13 group and the control group (P>0.05). The content of neutral detergent fiber (NDF) and acidic detergent fiber (ADF) in the control group was 72.47% and 47.01%, respectively. The NDF and ADF content in the R33 group, R33+HN13 group, R33+R17 group, and R33+R17+HN13 group were significantly reduced compared to the control group (P<0.001), while the NDF and ADF content in the R17 group, HN13 group, and R17+HN13 group were not significantly different from the control group (P>0.05). Each treatment had no significant effect on the crude protein (CP) content, carbon (C) content, soluble carbohydrate (WSC) content, and acid washing lignin (ADL) content of micro stored rice straw (P>0.05).

 The effect of different treatments on the fermentation quality of micro stored rice straw

The pH value and ammonia nitrogen (NH₃-N) content of R33 group, R33+HN13 group, R33+R17 group, and R33+R17+HN13 group were significantly reduced compared to the control group (P<0.001), while the pH value and ammonia nitrogen (NH₃-N) content of R17 group were significantly reduced compared to the control group (P<0.05). The pH value and ammonia nitrogen (NH₃-N) content of HN13 group and R17+HN13 group were not significantly different from the control group (P>0.05).

The lactate content in the R33 group, R33+HN13 group, R33+R17 group, and R33+R17+HN13 group reached 19.64g/kg, which was significantly different from the control group (P<0.001). However, there was no significant difference in lactate content between the R17 group, HN13 group, and R17+HN13 group and the control group (P>0.05). The content of acetic acid and propionic acid in each treatment group showed no significant difference compared to the control group (P>0.05). Compared with the R17 group, HN13 group, and R17+HN13 group, the propionic acid content in R33 group was significantly increased (P<0.05). The content of butyric acid in the control group was 19.83 g/kg. No butyric acid was detected in the R33 group, R33+HN13 group, R33+R17 group, and R33+R17+HN13 group, which was significantly different from the control group (P<0.001). The HN13 group significantly reduced the content of butyric acid (P<0.05), while the R17 group and R17+HN13 group had no significant difference in butyric acid content compared to the control group (P>0.05).

The effect of different treatments on the in vitro digestibility of rice straw

 At 12 hours, the in vitro digestibility of rice straw in the R33 group, R17 group, HN13 group, R33+HN13 group, and R33+R17+HN13 group significantly increased compared to the control group (P<0.05), while the differences between the R17+HN13 group and R33+R17 group and the control group were not significant (P>0.05). After 24 hours, the in vitro digestibility of rice straw micro stored in each treatment group was significantly improved at each time point (P<0.001). The R33 group showed the greatest improvement, with in vitro digestibility increased by 10.68%, 11.44%, 9.27%, and 9.12% compared to the control group at 12h, 24h, 48h, and 72h, respectively.

The effect of different treatments on the microbial community of rice straw

 Each treatment group significantly altered the microbial community structure of micro stored rice straw. At the phylum level, the relative abundance of Firmicutes in R33, R33+HN13, R33+R17, and R33+R17+HN13 reached over 80%, which was significantly higher than the control group (P<0.001). The relative abundance difference of Firmicutes between the treatment group without R33 bacteria and the control group was not significant (P>0.05), with a relative abundance percentage difference of over 40%. The relative abundance of Proteobacteria in the R17, HN13, R17+HN13 and control groups was significantly higher than other treatments. At the subordinate level, the relative abundance of Lactobacillus in the R33, R33+HN13, R33+R17, R33+R17+HN13, and R17 groups was significantly different from the control group (P<0.001), with the relative abundance of Lactobacillus in the first four groups exceeding 80%. The relative abundance of other bacterial genera in the R33, R33+HN13, R33+R17, and R33+R17+HN13 groups was below 1%, with significant differences compared to the control group (P<0.05). From the correlation analysis between the relative abundance of microorganisms and fermentation indicators, it can be concluded that Lactobacillus is positively correlated with CP content and lactate content, and negatively correlated with NDF content, ADF content, pH value, NH3-N content, and butyric acid content. Saccharomyces, Enterococcus, Streptomyces, and Caproic acid producing bacteria are positively correlated with DML and negatively correlated with CP content.

In summary, the three types of lactic acid bacteria, both individually and in combination, can improve the fermentation quality of rice straw micro storage to varying degrees. Among them, the R33 bacterial treatment has a better effect on rice straw micro storage than the other treatments. R33 can significantly reduce pH by producing a large amount of lactic acid, inhibit the biological activity of other harmful bacteria, significantly reduce dry matter loss and the production of NH₃-N and butyric acid. At the same time, it significantly reduces the content of NDF and ADF, and significantly improves the in vitro digestion rate of micro stored rice straw. Significantly altered the microbial community structure, increased the relative abundance of lactobacilli, and decreased the relative abundance of Escherichia and Enterococcus genera.

Key words: Lactic acid bacteria microbial fermentation; rice straw; fermentation quality;Microbial community.

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