微生物学报

原料Ingredients	含量Content (%)
¹⁾ The premix provided the following per kg of diets: VA 100 KIU, VD₃ 55 KIU, VE 400 IU, Mn 360 mg, Zn 580 mg, Fe 760 mg, Cu 200 mg, NaCl 10%−30%. ²⁾ Net energy of maintenance and weight gain were calculated value according to NRC (2016), while the others were measured values.
玉米Corn	32.77	38.47
豆粕Soybean meal	7.25	3.85
棉籽粕Cottonseed meal	5.26	5.58
菜籽粕Rapeseed meal	1.79	4.94
麦麸Wheat bran	2.85	1.51
预混料Premix¹⁾	2.97	2.80
碳酸氢钠NaHCO₃	1.19	1.12
食盐NaCl	0.59	0.56
全株青贮玉米Maize silage	37.84	34.36
小麦秸Wheat straw	7.50	6.81
合计Total	100.00	100.00
营养水平Nutrient levels²⁾
维持净能NEm (mcal/kg)	1.68	1.71
增重净能Neg (mcal/kg)	1.08	1.11
粗蛋白质CP	13.35	13.14
钙Ca	0.72	0.74
磷P	0.35	0.36

原料Ingredients	含量Content (%)
¹⁾ The premix provided the following per kg of diets: VA 100 KIU, VD₃ 55 KIU, VE 400 IU, Mn 360 mg, Zn 580 mg, Fe 760 mg, Cu 200 mg, NaCl 10%−30%. ²⁾ Net energy of maintenance and weight gain were calculated value according to NRC (2016), while the others were measured values.
玉米Corn	32.77	38.47
豆粕Soybean meal	7.25	3.85
棉籽粕Cottonseed meal	5.26	5.58
菜籽粕Rapeseed meal	1.79	4.94
麦麸Wheat bran	2.85	1.51
预混料Premix¹⁾	2.97	2.80
碳酸氢钠NaHCO₃	1.19	1.12
食盐NaCl	0.59	0.56
全株青贮玉米Maize silage	37.84	34.36
小麦秸Wheat straw	7.50	6.81
合计Total	100.00	100.00
营养水平Nutrient levels²⁾
维持净能NEm (mcal/kg)	1.68	1.71
增重净能Neg (mcal/kg)	1.08	1.11
粗蛋白质CP	13.35	13.14
钙Ca	0.72	0.74
磷P	0.35	0.36

项目 Items	对照组 Control	试验1组 Trial 1	试验2组 Trial 2	试验3组 Trial 3	P值 P-value
同行数据不同小写字母表示差异显著(P < 0.05)，相同或无字母表示差异不显著(P > 0.05). 下同 In same row, values with different small letters meant significant differences (P < 0.05), while with no letter or same letters meant no significant differences (P > 0.05). The same below.
Shannon指数Shannon index	6.50±0.13	5.79±0.49	5.91±0.23	6.50±0.23	0.232
Simpson指数Simpson index	0.97±0.00	0.93±0.02	0.95±0.01	0.95±0.01	0.370
Chao指数Chao index	775.00±10.86b	721.93±49.08b	669.90±24.72b	1 172.47±79.24a	< 0.001
ACE指数ACE index	788.50±13.14b	731.16±47.67b	681.81±24.31b	1 177.06±72.48a	< 0.001

项目 Items	对照组 Control	试验1组 Trial 1	试验2组 Trial 2	试验3组 Trial 3	P值 P-value
同行数据不同小写字母表示差异显著(P < 0.05)，相同或无字母表示差异不显著(P > 0.05). 下同 In same row, values with different small letters meant significant differences (P < 0.05), while with no letter or same letters meant no significant differences (P > 0.05). The same below.
Shannon指数Shannon index	6.50±0.13	5.79±0.49	5.91±0.23	6.50±0.23	0.232
Simpson指数Simpson index	0.97±0.00	0.93±0.02	0.95±0.01	0.95±0.01	0.370
Chao指数Chao index	775.00±10.86b	721.93±49.08b	669.90±24.72b	1 172.47±79.24a	< 0.001
ACE指数ACE index	788.50±13.14b	731.16±47.67b	681.81±24.31b	1 177.06±72.48a	< 0.001

项目 Items	对照组 Control	试验1组 Trial 1	试验2组 Trial 2	试验3组 Trial 3	P值 P-value
Shannon指数Shannon index	6.21±0.70	5.88±0.56	6.21±0.60	5.41±0.93	0.839
Simpson指数Simpson index	0.88±0.03	0.88±0.02	0.89±0.03	0.84±0.04	0.688
Chao指数Chao index	6 869.83±1 700.04	7 421.90±1 523.75	6 281.12±755.55	5 748.69±1 831.79	0.866
ACE指数ACE index	7 523.60±1 791.34	7 442.85±1 082.96	6 934.56±752.42	6 233.09±1 968.32	0.917

项目 Items	对照组 Control	试验1组 Trial 1	试验2组 Trial 2	试验3组 Trial 3	P值 P-value
Shannon指数Shannon index	6.21±0.70	5.88±0.56	6.21±0.60	5.41±0.93	0.839
Simpson指数Simpson index	0.88±0.03	0.88±0.02	0.89±0.03	0.84±0.04	0.688
Chao指数Chao index	6 869.83±1 700.04	7 421.90±1 523.75	6 281.12±755.55	5 748.69±1 831.79	0.866
ACE指数ACE index	7 523.60±1 791.34	7 442.85±1 082.96	6 934.56±752.42	6 233.09±1 968.32	0.917

基于16S rDNA和ITS测序技术研究酿酒酵母细胞壁对育肥牛肠道微生物的影响

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王燕 ¹^,² , 陈志龙 ³ , 施安 ² , 李丹 ¹ , 李博 ¹ , 侯鹏霞 ¹^,²^,^* , 张恩平 ¹^,^*

微生物学报 | 研究报告 2024,64(8): 2844-2860

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微生物学报 | 研究报告 2024, 64(8): 2844-2860

基于16S rDNA和ITS测序技术研究酿酒酵母细胞壁对育肥牛肠道微生物的影响

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王燕¹^,², 陈志龙³, 施安², 李丹¹, 李博¹, 侯鹏霞¹^,²^,^*, 张恩平¹^,^*

作者信息

1 西北农林科技大学动物科技学院, 陕西杨凌 712100

2 宁夏农林科学院动物科学研究所, 宁夏银川 750000

3 宁夏农林科学院固原分院, 宁夏固原 756000

16S rDNA and ITS sequencing reveals the effects of cell walls of Saccharomyces cerevisiae on intestinal microbiota in finishing bulls

Yan WANG¹^,², Zhilong CHEN³, An SHI², Dan LI¹, Bo LI¹, Pengxia HOU¹^,²^,^*, Enping ZHANG¹^,^*

Affiliations

1 College of Animal Science and Technology, Northwest A & F University, Yangling 712100, Shaanxi, China

2 Institute of Animal Science, NingXia Academy of Agriculture and Forestry Sciences, Yinchuan 750000, Ningxia, China

3 Guyuan Branch of NingXia Academy of Agriculture and Forestry Sciences, Guyuan 756000, Ningxia, China

出版时间: 2024-04-12 doi: 10.13343/j.cnki.wsxb.20240052

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摘要

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【目的】本研究旨在通过16S rDNA和ITS测序技术探究酿酒酵母细胞壁对育肥牛肠道微生物的影响。【方法】选择体重550 kg左右西门塔尔杂交育肥牛40头，随机分为4组，每组10头牛，对照组饲喂基础饲粮，试验1、2、3组每日每头分别在基础饲粮中添加酿酒酵母细胞壁5、10、15 g。试验预试期10 d，正试期94 d，试验结束前7天于晨饲前采集肠道粪便。【结果】16S rDNA分析结果显示：(1) 试验3组Chao指数和ACE指数显著高于其他组(P < 0.05)。(2) 门水平上，厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidota)是主要优势菌门；属水平上，普雷沃氏菌属_9 (Prevotella_9)、栖粪杆菌属(Faecalibacterium)、琥珀酸弧菌属(Succinivibrio)、拟杆菌属(Bacteroides)和双歧杆菌属(Bifidobacterium)是主要优势菌属。(3) 经过线性判别分析效应大小(linear discriminant analysis effect size, LEfSe)，共检测到1个差异物种(LDA≥4.0, P < 0.05)在试验2组发挥重要作用。ITS分析结果显示：(1) 各组间α、β多样性无显著差异(P > 0.05)。(2) 门水平上，子囊菌门(Ascomycota)占比50.00%以上，是主要优势菌门；青霉属(Penicillium)、unidentified_Ascomycota_sp.、曲霉属(Aspergillus)、Orpinomyces和散囊菌属(Eurotium)为主要优势菌属。(3) 经过LEfSe分析，共检测到8个差异物种(LDA≥3.0, P < 0.05)，分别有3、3、2个差异物种在对照组、试验2组和试验3组发挥重要作用。【结论】本研究条件下，饲粮添加10−15 g/d酿酒酵母细胞壁提高了育肥牛肠道细菌群落的丰富度，显著增加了有益菌属Provetella_9、弯颈霉属(Tolypocladium)和Torulaspora的相对丰度，有利于优化肠道微生态环境。

关键词

酿酒酵母细胞壁 / 育肥牛 / 菌群结构 / 16S rDNA / ITS

Abstract

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[Objective] To explore the effects of cell walls of Saccharomyces cerevisiae on the intestinal microbiota in finishing bulls by 16S rDNA and ITS sequencing. [Methods] A total of 40 simmental crossbred finishing bulls weighing about 550 kg were randomized into 4 groups, with 10 bulls in each group. The control group was fed with a basic diet, and 5, 10, and 15 g cell walls of S. cerevisiae were added to the diet of each bull per day in trial 1, 2, and 3 groups, respectively. The preliminary trial and trial lasted for 10 days and 94 days, respectively. Intestinal feces were collected 7 days before the end of the trial. [Results] 16S rDNA: (1) The Chao and ACE indices in the trial 3 group were higher than those in other groups (P < 0.05); (2) Firmicutes and Bacteroidota were the dominant phyla, and Prevotella_9, Faecalibacterium, Succinivibrio, Bacteroides, and Bifidobacterium were the dominant genera; (3) The linear discriminant analysis effect size (LEfSe) revealed one differential species (LDA≥4.0, P < 0.05) playing an important role in the trial 2 group. ITS: (1) There was no significant difference in the alpha or beta diversity among groups (P > 0.05); (2) Ascomycota with the relative abundance above 50.00% was the dominant phylum. Penicillium, unidentified_Ascomycota_sp., Aspergillus, Orpinomyces, and Eurotium were the dominant genera; (3) LEfSe revealed 8 differential species (LDA≥3.0, P < 0.05), which included 3, 3, and 2 differential species playing an important role in the control, trial 2, and trial 3 groups, respectively. [Conclusion] Under conditions of this study, adding 10–15 g/d cell walls of S. cerevisiae in the basic diet increased the richness of intestinal microbiota and the relative abundance of beneficial bacteria Provetella_9, Tolypocladium, and Torulaspora, which were conducive to improve intestinal microecological environment of finishing bulls.

Key words

cell walls of Saccharomyces cerevisiae / finishing bulls / microbiota structure / 16S rDNA / ITS

引用本文

王燕, 陈志龙, 施安, 李丹, 李博, 侯鹏霞, 张恩平. 基于16S rDNA和ITS测序技术研究酿酒酵母细胞壁对育肥牛肠道微生物的影响. 微生物学报, 2024 , 64 (8) : 2844 -2860 . DOI: 10.13343/j.cnki.wsxb.20240052

Yan WANG, Zhilong CHEN, An SHI, Dan LI, Bo LI, Pengxia HOU, Enping ZHANG. 16S rDNA and ITS sequencing reveals the effects of cell walls of Saccharomyces cerevisiae on intestinal microbiota in finishing bulls[J]. Acta Microbiologica Sinica, 2024 , 64 (8) : 2844 -2860 . DOI: 10.13343/j.cnki.wsxb.20240052

正文

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肠道是重要的微生物消化场所^[1]，是反刍动物体内仅次于瘤胃的发酵器官^[2]。肠道菌群又称宿主的“外在器官”^[3]，在宿主营养代谢、能量平衡、免疫调节、疾病预防、肠道发育等方面均发挥重要作用^[4-11]，其能分泌乙酸、丙酸、丁酸等短链脂肪酸(short chain fatty acids, SCFAs)给肠道细胞提供营养^[12]；保持和促进肠道上皮屏障的结构完整性，限制致病菌的生长和定殖^[13]。粪便菌群与肠道菌群组成、相对丰度和功能相似，可一定程度反映肠道菌群变化^[14]。王丽华^[15]通过直肠取粪法采集粪便测定添加25-羟基维生素D₃对犊牛肠道微生物的影响；韩笑瑛^[16]采集直肠粪便测定日粮精料水平对奶山羊粪便菌群结构的影响；金宇航^[17]采集直肠内容物测定不同锌源对犊牛直肠微生物群落结构和多样性的影响。因此，可通过粪便研究反刍动物肠道菌群。

酿酒酵母细胞壁是从酿酒酵母中提取的功能性复合多糖(主要有效成分：甘露聚糖和β-葡聚糖^[18])，通过影响微生物在肠道的黏附、定殖^[19]及其代谢产物^[20]来调节菌群结构。酿酒酵母细胞壁主要成分之一的甘露聚糖可通过竞争性吸附作用，阻碍肠道内有害菌(大肠杆菌、沙门氏菌)繁殖，促进肠道内有益菌(乳酸杆菌、双歧杆菌)生长和定殖^[21]。因此，酿酒酵母细胞壁对肠道菌群的影响备受关注。饲粮添加啤酒酵母壁多糖^[22]、β-1, 3-葡聚糖^[23]、酵母多糖^[24]等多糖可显著降低鸡、断奶仔猪等单胃动物肠道致病菌(沙门氏菌、大肠杆菌、梭状芽孢杆菌)相对丰度，显著提高有益菌(双歧杆菌、乳酸杆菌、瘤胃杆菌属、毛螺菌)数量。Heinrichs等^[25]研究表明，甘露寡糖可优化牛胃肠道微生态环境，促进双歧杆菌等有益菌增殖，抑制大肠杆菌等致病菌；张洪伟等^[26]研究表明，饲粮添加不同剂量酵母培养物(0.1%、0.2%、0.3%)均可显著提高肉牛粪便中乳酸菌和双歧杆菌比例，显著降低大肠杆菌比例；周怿^[27]研究表明，饲粮添加75 mg/kg酵母β-葡聚糖显著抑制犊牛直肠内容物中大肠杆菌生长，显著促进乳酸杆菌生长。此外，本团队前期研究发现酿酒酵母细胞壁可显著提高育肥牛平均日增重，降低料重比，提高经济效益^[28]，提高肌内脂肪含量^[29]。基于前期研究和酿酒酵母细胞壁在经过瘤胃后仍具有生物活性^[30]的特点。本研究利用16S rDNA和内转录间隔区(internal transcribed spacer, ITS)测序技术分析酿酒酵母细胞壁对育肥牛肠道微生物的影响，探究酿酒酵母细胞壁调控育肥牛肠道菌群促进肉牛生长的机制，为酿酒酵母细胞壁的高效利用及育肥牛的营养调控提供理论基础。

1 材料与方法

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1.1 试验设计

选择体重550 kg左右西门塔尔杂交育肥牛40头，随机分为4组，每组10头牛。对照组饲喂基础饲粮，试验1、2、3组每日每头在基础饲粮中分别添加5、10和15 g酿酒酵母细胞壁(Phileo by Lesaffre公司，主要成分及含量：甘露聚糖≥20%，β-葡聚糖≥20%)。预试期10 d，正试期94 d。试验牛分圈饲养，按试验设计饲喂对应全混合日粮(total mixed ration, TMR)饲粮，每日2次(08:00和16:00)，自由饮水。试验饲粮根据国家农产品质量安全监督检验中心饲料标准(national research council, NRC) (2016)结合养殖场生产实际配制，基础饲粮组成及营养水平见表1。

1.2 粪样采集

饲养试验结束前7天，晨饲前，每组各随机挑选试验牛5头采集肠道粪便5 g左右(直肠采粪法)，分装入2 mL冻存管，立即投入液氮，带回实验室−80 ℃保存，用于肠道微生物16S rDNA和ITS扩增子测序。

1.3 高通量测序

利用Illumina NovaSeq测序平台对17个粪便样本(对照组、试验1组、试验3组各1个样品质检不合格，剔除)微生物区系DNA片段进行16S rDNA和ITS1扩增子测序。主要实验流程：(1) 基因组DNA提取与样品检测；(2) 目标片段PCR扩增，16S V3−V4引物对F (5′-CCTAYGGGRBGCASCAG-3′)和R (5′-GGACTACNNGGGTATCTAAT-3′)；ITS1-5F引物对F (5′-GGAAGTAAAAGTCGTAACAAGG-3′)和R (5′-GCTGCGTTCTTCATCGATGC-3′)。扩增条件：98 ℃ 1 min；98 ℃ 10 s，50 ℃ 30 s，72 ℃ 30 s，30次循环；72 ℃ 5 min；(3) PCR产物的混样和磁珠纯化回收；(4) 使用NEBNext^® Ultra^TM Ⅱ FS DNA PCR-free Library Prep Kit (New England Biolabs)进行文库构建；(5) 使用NovaSeq 6000进行PE250上机测序；(6) 生物信息学分析。以上检测由北京诺禾致源科技股份有限公司完成。

1.4 数据统计及分析

利用Uparse算法^[31]对样本进行effective tags聚类(97.00%相似性)，将序列聚类成操作分类单元(operational taxonomic unit, OTU)，选择出现频数最高序列作为OTUs代表性序列；对OTUs序列进行物种注释(16S rDNA：Silva 138.1数据库，Mothur算法；ITS：Unite数据库，blast算法)。使用QIIME软件(version 1.9.1)计算β多样性指数；R软件(version 2.15.3)绘制稀释曲线、等级聚类曲线和物种累积曲线；SPSS软件(version 26.0)分析α多样性指数。

2 结果与分析

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2.1 16S rDNA测序结果

2.1.1 测序深度分析

如图1A所示，随着测序深度增加，各组稀释曲线均趋于平坦，说明测序数据量渐进合理，已基本覆盖样品中细菌物种，满足后续分析要求。如图1B所示，等级聚类曲线显示试验3组物种丰富度和均匀度高于其他组。

2.1.2 OTUs数目分析

如图2A、2B所示，在97.00%相似度水平下，对照组，试验1、2、3组各有OTUs 1 130、1 098、1 085、1 985个；特有OTUs分别为62、101、46、890个。4组间共享OTUs 675条，试验组间共享OTUs 715个，说明添加酿酒酵母细胞壁育肥牛肠道细菌群落相似度较高。

2.1.3 α多样性分析

如表2所示，Shannon指数和Simpson指数各组间无显著差异(P > 0.05)；Chao指数和ACE指数试验3组显著高于其他组(P < 0.05)，说明添加15 g/d酿酒酵母细胞壁显著提高了育肥牛肠道细菌群落丰富度。

2.1.4 β多样性分析

如图3所示，基于Unweighted-UniFrac距离进行主坐标(principal co-ordinates analysis, PCoA)分析，PC1和PC2对组间差异贡献值分别为32.76%和12.69%。试验3组与其他组别明显区分无交叉，说明试验3组细菌群落结构与其他组别存在一定差异。

2.1.5 门和属水平微生物组成分析

四组共检出31个菌门，如图4A所示，优势菌门Firmicutes和Bacteroidota相对丰度之和占70.00%以上；四组共检出399个菌属，如图4B所示，优势菌属主要是Prevotella_9、Faecalibacterium、Succinivibrio、Bacteroides和Bifidobacterium等。

2.1.6 差异物种分析与标志物种

采用LEfSe^[32]分析探索组间差异微生物，当LDA值≥4.0时，认为该物种是生物标记物。如图5A、5B所示，属水平上，试验2组Prevotella_9显著丰富(LDA≥4.0, P < 0.05)，可作为潜在生物标记物。

2.2 ITS测序结果

2.2.1 测序深度分析

如图6A所示，各组箱形图位置呈现逐渐上升的趋势，随后逐渐趋于平稳，说明样本中物种不再随样本量增加而显著增多，抽样充分，可进行后续数据分析。如图6B所示，各组等级聚类曲线宽度和平滑度均较高，说明各组样本物种丰富度和均匀度均较高且相似。

2.2.2 OTUs数目分析

如图7A、7B所示，在97.00%相似度水平下得到每个样品的OTUs数目。对照组、试验1、2、3组共获得29 705个OTUS；各组特有OTUs 4 400、4 077、4 974、4 145个；共享OTUs 2 056个；试验组共获得25 305个OTUs，共享OTUs 2 456个。试验组间共享OTUs比例(9.71%)高于4组间共享OTUs比例(6.92%)。

2.2.3 α多样性分析

如表3所示，各组多样性指数Shannon、Simpson和丰富度指数Chao、ACE均无显著差异(P > 0.05)。

2.2.4 β多样性分析

如图8所示，基于Weighted-UniFrac距离进行主坐标(PCoA)分析，PC1和PC2对样本间变异贡献度分别为38.75%和18.23%，能够充分解释样本间变异，但对照组和试验组内样本没有聚集分布，说明组间肠道真菌β多样性无显著差异。

2.2.5 门和属水平微生物组成分析

四组共检出16个菌门，如图9A所示，Ascomycota、新毛孢菌门(Neocallimastigomycota)、担子菌门(Basidiomycota)和毛霉菌门(Mucoromycota)相对丰度大于1.00%，其中Ascomycota占比50.00%以上；四组共检出426个菌属，如图9B所示，优势菌属主要有Penicillium、unidentified_Ascomycota_sp.、Aspergillus、Orpinomyces和Eurotium等。

2.2.6 物种差异分析与标志物种

利用LEfSe进行多级差异物种判别分析，分析LDA≥3.0、P < 0.05具有显著作用的菌属。如图10A、10B所示，对照组Eurotium显著丰富；试验2组Tolypocladium显著丰富；试验3组Torulaspora显著丰富。

3 讨论与结论

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3.1 酿酒酵母细胞壁对育肥牛肠道细菌群落结构的影响

本研究利用16S rDNA测序技术对育肥牛肠道细菌群落结构、组成和丰富度进行了全面分析。17个样品的稀释曲线都趋于平缓，说明所采集粪便样本能覆盖绝大多数微生物群落种类。肠道菌群多样性在机体营养、免疫调控、微生态平衡中发挥重要作用^[33]。刘韶娜等研究报道高丰富度的微生物群落能使宿主更好地发挥肠道功能，更高效地消化降解摄入的饲料^[34]。本研究中，添加15 g/d酿酒酵母细胞壁育肥牛肠道细菌群落丰富度显著提高。周怿^[27]研究表明，早期断奶犊牛日粮中添加75 mg/kg酵母β-葡聚糖显著增加直肠微生物区系的总体多样性，与本研究结果相似，这可能是因为酵母β-葡聚糖不仅可以抑菌杀菌，还能促进肠道中有益微生物生长，使得肠道微生物菌群达到一个新的平衡。贺琴等^[35]研究表明，饲粮添加酵母壁多糖对断奶仔猪肠道菌群α多样性无显著影响。这与本研究的结果不一致，可能与试验动物、添加量及饲喂方式等不同有关。此外，通过PCoA分析发现，添加15 g/d酿酒酵母细胞壁育肥牛样品与其他组别完全区分，说明添加15 g/d酿酒酵母细胞壁育肥牛肠道细菌菌群结构与其他组别差异显著。

本研究侧重分析细菌门和属水平结构组成。Firmicutes许多成员是有益菌，例如乳酸杆菌、粪杆菌等，这些菌种中含有大量纤维分解菌属^[36]，降解纤维类物质产生SCFAs，参与胃肠道内营养物质发酵和能量物质代谢，提高饲料利用率^[37]；Bacteroidota细菌基因组中富含大量碳水化合物活性酶基因^[38]，降解饲料中非纤维类碳水化合物，例如分解利用多种膳食可溶性多糖，代谢产物以乙酸盐和丙酸盐为主^[39]。本研究结果显示，门水平上，Firmicutes和Bacteroidota是主要优势菌群(两者之和占比70.00%以上)，与前人研究结果一致^[40]。二者均参与宿主碳水化合物代谢、氨基酸代谢和SCFAs生成等生理过程，在能量生成和肠道健康维持中发挥重要作用^[41]。

属水平上，Prevotella_9、Faecalibacterium、Succinivibrio、Bacteroides和Bifidobacterium是主要的优势菌属。Mizrahi等研究报道普雷沃氏菌属是高效降解半纤维素细菌^[42]；Bacteroides在维持肠道菌群平衡方面发挥重要作用^[43]；Faecalibacterium属于Firmicutes，是肠道中丁酸盐的生产者，丁酸盐可增强肠道上皮屏障的完整性，降低动物机体炎症反应^[44]；Succinivibrio能发酵多种糖类产生乙酸、琥珀酸及少量甲酸和乳酸；Bifidobacterium具有调节和维护肠道微生态平衡的功能。刘孟健等^[45]研究表明，饲粮添加0.3%−0.5%布拉氏酵母壁多糖显著提高了早期断奶羔羊盲肠中双歧杆菌和乳酸杆菌等益生菌数量，显著降低了沙门氏菌和大肠杆菌数量，与本研究结果不一致，可能是因为幼龄羔羊菌群发育不完善，酵母多糖能更好地发挥调节作用，而育肥中后期的牛菌群结构已基本趋于稳定。

利用LEfSe分析组间特异性差异物种发现，添加10 g/d酿酒酵母细胞壁育肥牛肠道Prevotella_9显著丰富。普雷沃氏菌属肠型有利于纤维的消化降解，促进机体吸收营养物质^[46]，有益于肠道健康^[47]。普雷沃氏菌属是降解碳水化合物产生SCFAs^[48]和调节免疫关键菌群^[49]。其中Prevotella_9是产乙酸和乳酸的一种有益菌，在发酵过程中有可能分泌β-葡萄糖苷酶和β-甘露糖苷酶降解葡甘露聚糖，β-葡聚糖可增加Prevotella_9的相对丰度^[50]，与本研究结果一致；此外，艰难梭菌感染患者肠道中Prevotella_9丰度显著降低^[51]，在多发性硬化症患者肠道中发现乳酸和乙酸含量与Provetella_9丰度呈正相关^[52]。说明酿酒酵母细胞壁通过增加肠道有益菌属Provetella_9相对丰度提高育肥牛对碳水化合物的降解能力，促进SCFAs产生，抑制致病菌，改善肠道健康，进而有利于营养物质的吸收和利用，促进育肥牛增重高达2.02 kg/d^[28]。

真菌与细菌通过互利共生、协同、拮抗等相互作用，来支持和保护彼此的生长发育，维持肠道黏膜屏障功能^[53]，直接参与宿主的食物消化、营养吸收、能量代谢、免疫应答等生理过程^[54]。与细菌相比，肠道内真菌数量和种类较少，但在肠道微生态平衡的维持中发挥不可替代的作用。因此，本研究进一步利用ITS测序方法分析育肥牛肠道真菌群落结构组成，初步揭示酿酒酵母细胞壁对育肥牛肠道真菌群落结构的影响，为更深入了解酿酒酵母细胞壁在育肥牛生长和肠道中的作用奠定基础。

3.2 酿酒酵母细胞壁对育肥牛肠道真菌群落结构的影响

真菌是摄食植物性饲料动物肠道首要定殖微生物，能产生纤维素酶、木质素酶和半纤维素酶等多种降解植物细胞壁酶类，粗纤维降解能力强大，对提高草食动物粗饲料利用率具有重要意义^[55]。厌氧真菌仅占肠道微生物数量7%−9%，但其能从动物摄入的植物性饲料中释放出超过50%的可发酵糖类物质^[56]。本研究17个测序样本覆盖率均达到99%以上，说明测序结果代表了样本真菌群落的真实情况。添加10 g/d酿酒酵母细胞壁育肥牛肠道真菌总的、特有OTUs数目均最多，说明酿酒酵母细胞壁会影响育肥牛肠道真菌数目，并且在本研究条件下添加10 g/d相对其他添加水平最有益。α和β多样性分析发现，各组间真菌群落物种组成相似度较高，说明酿酒酵母细胞壁对育肥牛肠道真菌组成和多样性方面无不良影响。罗世乾^[57]研究表明，酿酒活性干酵母细胞壁上的甘露聚糖对致病菌具有吸附能力，可减少致病菌对肠上皮细胞的黏附。本研究中添加15 g/d酿酒酵母细胞壁育肥牛肠道真菌α多样性指数均低于其他组，可能是因为酿酒酵母细胞壁有效抑制了部分肠道致病菌(沙门氏菌、大肠杆菌等)。

一些研究报道，Ascomycota、Neocallimastigomycota、Basidiomycota和Mucoromycota在牛^[58]、绵阳^[59]、山羊^[60]等草食动物肠道中为优势菌门，与本研究结果一致。Ascomycota是真菌中最大的一类菌群^[61]，以腐生菌为主，可降解纤维素和木质素^[62]。马秀花等^[63]研究表明，在荞麦秸秆饲粮中添加1%和3%甘露聚糖滩羊瘤胃Ascomycota相对丰度高于对照组。本研究中，添加15 g/d酿酒酵母细胞壁育肥牛肠道中Ascomycota相对丰度最高，间接说明酿酒酵母细胞壁有利于饲粮中纤维的降解，与上述结果类似。

进一步分析真菌属水平结构发现，各组主要优势菌属为Penicillium、unidentified_Ascomycota_sp.、Aspergillus、Orpinomyces和Eurotium等。其中Penicillium是有益菌：(1) 能产生抗菌、抗肿瘤和抗病毒功能的活性次生代谢产物；(2) 分泌多种高活性的多糖、蛋白质、脂肪和木质素等多聚物的水解酶类；(3) 降解多种单环和多环芳香类环境污染物^[64]。Aspergillus对致病菌有一定抑制作用^[65]。Orpinomyces可生物氢化植物饲料的主要脂肪酸亚麻酸^[66]。目前研究较多的Eurotium是在茯砖茶发酵过程中的优势真菌，其具有提高机体免疫力、调节肠道菌群等功能。衣喆等^[67]研究表明，Eurotium发酵液通过拮抗肠道病原微生物定殖及集群生长以调节小鼠肠道菌群。本研究中Aspergillus和Eurotium均为优势菌属，这可能与饲养环境、饲粮等因素有关。此外，发现的优势菌属unidentified_Ascomycota_sp.暂未分类，需进一步探究。

通过LEfSe分析组间特异性差异物种。本研究中，添加10 g/d和15 g/d酿酒酵母细胞壁育肥牛肠道中Tolypocladium和Torulaspora分别显著丰富，对照组Eurotium显著丰富。关于反刍动物源Eurotium相关研究较少，尚不清楚其具体作用。Tolypocladium能产生多种杀虫和抗菌的有效成分(如环孢菌素、大团囊素、线肽素、四聚胺酸素等)^[68]。本研究中，添加15 g/d酿酒酵母细胞壁育肥牛肠道中德尔布有孢圆酵母(Torulaspora delbrueckii)显著丰富。Torulaspora属对志贺氏菌、白色念珠球菌、大肠杆菌、沙门氏菌、枯草芽孢杆菌、金黄色葡萄球菌具有稳定、优异的抑菌效果，是一种广谱抑菌酵母菌^[69]。董亚文^[70]研究表明，静脉注射给药灭活Torulaspora delbrueckii可提高单核-巨噬细胞吞噬功能，提高正常小鼠非特异性免疫功能。综上，添加10 g/d和15 g/d酿酒酵母细胞壁可提高育肥牛肠道有益真菌的相对丰度。

综上所述，本研究条件下，添加10−15 g/d酿酒酵母细胞壁提高了育肥牛肠道细菌群落的丰富度，显著增加了有益菌属Provetella_9、Tolypocladium和Torulaspora的相对丰度，有利于优化肠道微生态环境。

基金

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宁夏回族自治区农业科技自主创新项目(NGSB-2021-12-02)
陕西省农业关键核心技术攻关项目(2023NYGG005)
宁夏回族自治区优秀人才支持计划

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2024年第64卷第8期

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doi: 10.13343/j.cnki.wsxb.20240052

接收时间：2024-01-18
首发时间：2026-03-19
出版时间：2024-04-12

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收稿日期：2024-01-18
录用日期：2024-04-08

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by the Agricultural Science and Technology Independent Innovation Project of Ningxia Hui Autonomous Region(NGSB-2021-12-02)

宁夏回族自治区农业科技自主创新项目(NGSB-2021-12-02)

Key Agricultural Core Technology Project of Shaanxi Province(2023NYGG005)

陕西省农业关键核心技术攻关项目(2023NYGG005)

Excellent Talent Support Program of Ningxia Hui Autonomous Region

宁夏回族自治区优秀人才支持计划

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1 西北农林科技大学动物科技学院, 陕西杨凌 712100

2 宁夏农林科学院动物科学研究所, 宁夏银川 750000

3 宁夏农林科学院固原分院, 宁夏固原 756000

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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原料Ingredients	含量Content (%)
¹⁾ The premix provided the following per kg of diets: VA 100 KIU, VD₃ 55 KIU, VE 400 IU, Mn 360 mg, Zn 580 mg, Fe 760 mg, Cu 200 mg, NaCl 10%−30%. ²⁾ Net energy of maintenance and weight gain were calculated value according to NRC (2016), while the others were measured values.
玉米Corn	32.77	38.47
豆粕Soybean meal	7.25	3.85
棉籽粕Cottonseed meal	5.26	5.58
菜籽粕Rapeseed meal	1.79	4.94
麦麸Wheat bran	2.85	1.51
预混料Premix¹⁾	2.97	2.80
碳酸氢钠NaHCO₃	1.19	1.12
食盐NaCl	0.59	0.56
全株青贮玉米Maize silage	37.84	34.36
小麦秸Wheat straw	7.50	6.81
合计Total	100.00	100.00
营养水平Nutrient levels²⁾
维持净能NEm (mcal/kg)	1.68	1.71
增重净能Neg (mcal/kg)	1.08	1.11
粗蛋白质CP	13.35	13.14
钙Ca	0.72	0.74
磷P	0.35	0.36

原料Ingredients

含量Content (%)

试验前期50 d Pre-test period 50 d

试验后期44 d Post-test period 44 d

¹⁾ The premix provided the following per kg of diets: VA 100 KIU, VD₃ 55 KIU, VE 400 IU, Mn 360 mg, Zn 580 mg, Fe 760 mg, Cu 200 mg, NaCl 10%−30%. ²⁾ Net energy of maintenance and weight gain were calculated value according to NRC (2016), while the others were measured values.

玉米Corn

32.77

38.47

豆粕Soybean meal

7.25

3.85

棉籽粕Cottonseed meal

5.26

5.58

菜籽粕Rapeseed meal

1.79

4.94

麦麸Wheat bran

2.85

1.51

预混料Premix¹⁾

2.97

2.80

碳酸氢钠NaHCO₃

1.19

1.12

食盐NaCl

0.59

0.56

全株青贮玉米Maize silage

37.84

34.36

小麦秸Wheat straw

7.50

6.81

合计Total

100.00

营养水平Nutrient levels²⁾

维持净能NEm (mcal/kg)

1.68

1.71

增重净能Neg (mcal/kg)

1.08

1.11

粗蛋白质CP

13.35

13.14

钙Ca

0.72

0.74

磷P

0.35

0.36

项目 Items	对照组 Control	试验1组 Trial 1	试验2组 Trial 2	试验3组 Trial 3	P值 P-value
同行数据不同小写字母表示差异显著(P < 0.05)，相同或无字母表示差异不显著(P > 0.05). 下同 In same row, values with different small letters meant significant differences (P < 0.05), while with no letter or same letters meant no significant differences (P > 0.05). The same below.
Shannon指数Shannon index	6.50±0.13	5.79±0.49	5.91±0.23	6.50±0.23	0.232
Simpson指数Simpson index	0.97±0.00	0.93±0.02	0.95±0.01	0.95±0.01	0.370
Chao指数Chao index	775.00±10.86b	721.93±49.08b	669.90±24.72b	1 172.47±79.24a	< 0.001
ACE指数ACE index	788.50±13.14b	731.16±47.67b	681.81±24.31b	1 177.06±72.48a	< 0.001

项目
Items

对照组
Control

试验1组
Trial 1

试验2组
Trial 2

试验3组
Trial 3

P值
P-value

同行数据不同小写字母表示差异显著(P < 0.05)，相同或无字母表示差异不显著(P > 0.05). 下同
In same row, values with different small letters meant significant differences (P < 0.05), while with no letter or same letters meant no significant differences (P > 0.05). The same below.

Shannon指数Shannon index

6.50±0.13

5.79±0.49

5.91±0.23

6.50±0.23

0.232

Simpson指数Simpson index

0.97±0.00

0.93±0.02

0.95±0.01

0.370

Chao指数Chao index

775.00±10.86b

721.93±49.08b

669.90±24.72b

1 172.47±79.24a

< 0.001

ACE指数ACE index

788.50±13.14b

731.16±47.67b

681.81±24.31b

1 177.06±72.48a

< 0.001

项目 Items	对照组 Control	试验1组 Trial 1	试验2组 Trial 2	试验3组 Trial 3	P值 P-value
Shannon指数Shannon index	6.21±0.70	5.88±0.56	6.21±0.60	5.41±0.93	0.839
Simpson指数Simpson index	0.88±0.03	0.88±0.02	0.89±0.03	0.84±0.04	0.688
Chao指数Chao index	6 869.83±1 700.04	7 421.90±1 523.75	6 281.12±755.55	5 748.69±1 831.79	0.866
ACE指数ACE index	7 523.60±1 791.34	7 442.85±1 082.96	6 934.56±752.42	6 233.09±1 968.32	0.917

项目
Items

对照组
Control

试验1组
Trial 1

试验2组
Trial 2

试验3组
Trial 3

P值
P-value

Shannon指数Shannon index

6.21±0.70

5.88±0.56

6.21±0.60

5.41±0.93

0.839

Simpson指数Simpson index

0.88±0.03

0.88±0.02

0.89±0.03

0.84±0.04

0.688

Chao指数Chao index

6 869.83±1 700.04

7 421.90±1 523.75

6 281.12±755.55

5 748.69±1 831.79

0.866

ACE指数ACE index

7 523.60±1 791.34

7 442.85±1 082.96

6 934.56±752.42

6 233.09±1 968.32

0.917