Chinese Journal of Animal Nutrition >

2026 , Vol. 38 >Issue 5: 3810 - 3822

DOI: https://doi.org/10.12418/CJAN2026.304

RESEARCH PAPER

Effects of Mixed Addition of Different Enzymes and Bacteria on in Vitro Rumen Fermentation Parameters and Microbial Composition of Sugarcane Top Silage in Buffalo

  • LIAO Pinfeng , 1 ,
  • ZENG Fanquan 1 ,
  • LI Mengwei 1 ,
  • LYU Tao 1 ,
  • BAI Haiyu 1 ,
  • WANG Keyao 1 ,
  • LAN Haijiao 2 ,
  • YANG Chengjian 1 ,
  • PENG Lijuan , 1, 3, *
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  • 1 Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology, Ministry of Agriculture and Rural Affairs (Guangxi), Guangxi Buffalo Research Institute, Nanning 530001, China
  • 2 Guangxi Nanning Kangtel Biotechnology Co., Ltd., Nanning 530001, China
  • 3 State Key Laboratory of Non-Food Biomass Energy Technology, Nanning 530001, China
*senior engineer, E-mail:

Received date: 2025-10-29

  Online published: 2026-05-14

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Abstract

The aim of this study was to investigate the effects of mixed addition of different enzymes and bacteria on in vitro rumen fermentation parameters and microbial composition sugarcane top silage in buffalo. The 5 different additives of Lactobacillus plantarum, Pediococcus pentosaceus, cellulase, xylanase and glucose oxidase were mixed into sugarcane top silage by a 5-factor and 3-level orthogonal design method, and six better sugarcane top silage (groups T1 to T6) were selected for the in vitro rumen fermentation experiment of buffalo. The results showed as follows: 1) the gas production of T3 group was significantly higher than that of other groups except group T4 (P<0.05), the hydrogen production of groups T3 and T4 was significantly higher than that of other groups (P<0.05), the acetic acid and total volatile fatty acids contents of groups T5 and T6 were significantly higher than those of other groups (P<0.05), and the ammonia nitrogen content of group T6 was significantly lower than that of groups T1, T2 and T5 (P<0.05). 2) The Firmicutes relative abundance of group T4 was significantly higher than that of other groups except group T5 (P<0.05), and the Bacteroidetes relative abundance of groups T1, T2 and T3 was significantly higher than that of other groups T4, T5 and T6 (P<0.05). The Succiniclasticum relative abundance of group T4 was significantly higher than that of other groups (P<0.05), the Rikenellaceae_RC9_gut_group relative abundance of group T2 was significantly higher than that of other groups (P<0.05), the Prevotella relative abundance of group T3 was significantly higher than that of other groups (P<0.05), and the Ruminobacter relative abundance of groups T4 and T6 was significantly higher than that of other groups (P<0.05). 3) The acetic acid content was extremely significantly positively correlated with the Ruminobacter relative abundance (P<0.01), and the ammonia nitrogen content was significantly positively correlated with Rikenellaceae_RC9_gut_group (P<0.05). In conclusion, the mixed addition of different enzymes and bacteria have a certain effect on in vitro rumen fermentation parameters and microbial composition of sugarcane top silage, the rumen fermentation quality and fermentation characteristics of sugarcane top silage of group T3 are better.

Key words： enzyme-bacteria mixture; sugarcane top silage; in vitro fermentation; microbial diversity

Cite this article

LIAO Pinfeng , ZENG Fanquan , LI Mengwei , LYU Tao , BAI Haiyu , WANG Keyao , LAN Haijiao , YANG Chengjian , PENG Lijuan . Effects of Mixed Addition of Different Enzymes and Bacteria on in Vitro Rumen Fermentation Parameters and Microbial Composition of Sugarcane Top Silage in Buffalo[J]. Chinese Journal of Animal Nutrition, 2026 , 38(5) : 3810 -3822 . DOI: 10.12418/CJAN2026.304

甘蔗梢是甘蔗收成后产生的农副产品,含有丰富的碳水化合物和蛋白质,可用于饲喂反刍动物,是奶水牛粗饲料的重要来源。甘蔗梢在冬季大量产生,适口性差,容易霉变和变质,面临资源浪费。青贮可以降低甘蔗梢中的木质纤维素含量,更好地储存和运输,提高适口性,同时减少资源浪费。甘蔗梢青贮技术比较成熟,但存在厌氧菌发酵、酵母生长繁殖问题,导致青贮品质不稳定,因此会通过添加额外的添加剂如微生物制剂、酶制剂等来提高青贮质量。近年来,酶菌联合处理被广泛应用于改善粗饲料的青贮品质和瘤胃降解率。青贮发酵初期接种乳酸菌(lactic acid bacteria,LAB),可使LAB成为青贮中优势菌,LAB快速繁殖且产生大量乳酸,从而建立酸性厌氧环境,抑制梭菌、霉菌等有害微生物繁殖,使得青贮饲料品质有效改善。植物乳杆菌和戊糖片球菌均属于同型发酵LAB,其主要代谢产物是乳酸[1],相比异型发酵LAB,同型发酵LAB产酸速率更高、干物质(dry matter,DM)含量损失更少[2-3]。戊糖片球菌是青贮过程中的固有菌种,能显著提高青贮发酵速率,改善青贮饲料的品质[3-4]。甘蔗梢青贮发酵初期,由于LAB发酵的底物不充足,单独添加LAB制剂效果不够好,因此LAB制剂和酶制剂组合使用,可以促进LAB生长繁殖。纤维素酶不仅能够将纤维素分解为水溶性碳水化合物,为LAB发酵补充底物,还能够降解细胞壁成分,使饲料中的营养成分更易被动物消化吸收[5-6]。木聚糖酶将木聚糖水解为木糖,提高微生物发酵底物浓度,促进微生物发酵以及甘蔗梢青贮发酵,同时降低半纤维素含量,提高青贮饲料纤维利用率[7]。葡萄糖氧化酶能利用氧气将β-D-葡萄糖氧化,生成葡萄酸和过氧化氢,起到酸化、氧化的作用,能促进LAB繁殖,抑制有害菌生长,以保证青贮品质[8-9]。
研究表明,纤维素酶和LAB协同处理能够改善苎麻和象草混合青贮的青贮特性[10]。LAB和纤维素酶可以发挥细菌-酶协同作用,形成以LAB为主的共生微生物网络系统[11]。研究表明,酶菌协同处理相比于只添加枯草芽孢杆菌和乳酸杆菌,可以提高水稻秸秆的营养价值,显著增加瘤胃发酵的产气量,显著提高各挥发性脂肪酸(volatile fatty acids,VFA)和总挥发性脂肪酸(total volatile fatty acids,TVFA)产量[12]。研究表明,添加干酪乳杆菌TH14、纤维素酶和糖蜜提高了甘蔗梢青贮的饲料品质,促进了瘤胃气体的产生、体外消化率和瘤胃发酵[13-14]。以上研究表明,酶菌协同在一定程度上可提高青贮品质以及体外发酵品质。然而,关于酶菌混合添加处理的甘蔗梢青贮对水牛瘤胃微生物和发酵特性影响的研究仍较少。因此,本研究通过正交试验筛选出6组较优的甘蔗梢青贮,采用体外瘤胃发酵模型评估其对水牛体外瘤胃发酵参数及微生物组成的影响,以期为甘蔗梢的高效利用和水牛营养调控提供理论依据。

1 材料与方法

1.1 试验设计

试验所用甘蔗梢青贮由广西壮族自治区水牛研究所采用不同酶菌混合处理后所得,采用5因素3水平正交设计(表1),根据正交设计筛选出的6个较优的组合用于甘蔗梢青贮体外瘤胃发酵试验(表2)。瘤胃液采自广西壮族自治区水牛研究所种畜场3只健康状况良好、装有永久瘤胃瘘管的成年母水牛。
表1 甘蔗梢青贮酶菌混合正交设计

Table 1 Orthogonal design of bacteria and enzymes mixture of sugarcane top silage

项目
Items		 添加水平 Supplemental level
1 2 3
植物乳杆菌 Lactobacillus plantarum/(CFU/g) 1×105 1×106 1×107
戊糖片球菌 Pediococcus pentosaceus/(CFU/g) 1×105 1×106 1×107
纤维素酶 Cellulase/(U/g) 1×104 2×104 3×104
木聚糖酶 Xylanase/(U/g) 1×104 2×104 3×104
葡萄糖氧化酶 Glucose oxidase/% 0.1 0.2 0.3
表2 甘蔗梢青贮体外瘤胃发酵试验设计

Table 2 Experimental design of in vitro rumen fermentation of sugarcane top silage

项目
Items		 组别 Groups
T1 T2 T3 T4 T5 T6




添加水平
Supplemental level		 植物乳杆菌 Lactobacillus plantarum 3 3 1 1 1 1
戊糖片球菌 Pediococcus pentosaceus 2 1 3 2 3 1
纤维素酶 Cellulase 1 3 2 2 3 3
木聚糖酶 Xylanase 3 2 2 2 3 3
葡萄糖氧化酶 Glucose oxidase 1 3 1 2 3 2

1.2 体外瘤胃发酵

体外培养前1天,向各发酵瓶中准确称取底物1 g(精料∶甘蔗梢青贮风干样=2∶8),每个样品设6个重复,每批次培养设3个空白(即只注入发酵培养液),接种缓冲液40 mL,通入二氧化碳(CO2)2~3 min后密封,放入39 ℃恒温箱保存,缓冲液配制参照甘洁[15]的方法。参照Marrez等[16]的方法,于晨饲前取瘘管牛瘤胃液4层100目尼龙纱布过滤,后按照与缓冲液1∶2的比例接种瘤胃液,全程通CO2保持厌氧环境,39 ℃恒温培养72 h。试验开始后分别记录和收集3、6、9、12、24、36、48、72 h的累积产气量。

1.3 样品采集

发酵结束后,发酵液用100目尼龙纱布过滤,测量滤液pH、VFA含量、氨态氮(NH3-N)含量和微生物多样性。将过滤后残渣与尼龙纱布一起65 ℃烘干,用于干物质消化率(dry matter digestibility,DMD)的测定。

1.4 指标检测

1.4.1 DMD

样品和发酵后残渣中DM含量参照GB/T 6435—2014的方法。DMD计算公式如下:

DMD(%)=[(样品中DM含量-降解后

残渣中DM含量)/样品中DM含量]×100。

1.4.2 发酵参数

pH采用pH计(PH8180-0-00,东莞万创电子制品有限公司)测定;参照Xie等[17]的方法测定,产气量采用注射器直接测量,氢气(H2)、甲烷(CH4)产量采用气相色谱(Agilent 8860,安捷伦科技有限公司)测定;有机酸含量参照Xie等[18]的方法,采用高效液相色谱仪(Agilent 1260,安捷伦科技有限公司)测定;NH3-N含量采用苯酚-次氯酸钠比色法[19]测定。

1.4.3 微生物多样性

发酵液总微生物DNA提取采用CTAB法[20]提取,利用16S V3~V4区的引物(338F:ACTCCTACGGGAGGCAGCAG;806R:GGACTACHVGGGTWTCTAAT)对DNA进行扩增。利用Illumina MiSeq PE300平台(上海美吉生物医药科技有限公司)对提纯、定量的DNA进行测序。使用FASTP软件(version 0.20.0)、FLASH软件(version 1.2.7)、QIIME 2软件(version 2024)对原始序列进行数据处理。使用美吉生物云平台(https://cloud.majorbio.com)对处理后的数据进行生物信息学分析。

1.5 数据统计

采用SPSS 26.0软件进行单因素方差分析,Duncan氏法进行多重比较,结果以“平均值±标准差”表示,P<0.05表示差异显著。

2 结果

2.1 不同酶菌混合添加对甘蔗梢青贮体外瘤胃发酵参数的影响

由表3可见,各组之间DMD、CH4产量及丙酸(propionic acid,PA)、丁酸(butyric acid,BA)含量均差异不显著(P>0.05)。T3组的产气量最高,显著高于除T4组外的其他各组(P<0.05)。T3和T4组的H2产量显著高于其他各组(P<0.05)。T1组的pH最高,T4组的pH最低,各组pH均处于正常水平。T5和T6组的TVFA、乙酸(acetic acid,AA)含量显著高于其他各组(P<0.05),且T4组的TVFA、AA含量显著高于T1、T2和T3组(P<0.05)。T6组的NH3-N含量最低,与T3和T4组差异不显著(P>0.05),显著低于T1、T2和T5组(P<0.05)。
表3 不同酶菌混合添加对甘蔗梢青贮体外瘤胃发酵参数的影响

Table 3 Effects of mixed addition of different enzymes and bacteria on in vitro rumen fermentation parameters of sugarcane top silage

项目
Items		 组别 Groups P值
P-value
T1 T2 T3 T4 T5 T6
干物质消化率
DMD/%		 50.59
±1.68		 49.03
±1.28		 49.84
±1.03		 49.17
±1.53		 51.36
±1.98		 49.72
±1.01		 0.146
产气量
Gas/(mL/g)		 126.20
±2.95d		 127.60
±2.07d		 134.00
±2.45a		 133.6
±1.95ab		 130.80
±1.10bc		 128.80
±2.68cd		 <0.001
氢气
H2/(mL/g)		 3.96
±0.35c		 3.06
±0.38d		 8.26
±0.81a		 7.47
±0.86b		 4.50
±0.23c		 4.58
±0.26c		 <0.001
甲烷
CH4/(mL/g)		 26.66
±2.40		 27.99
±0.56		 28.09
±2.85		 28.91
±1.34		 26.97
±2.74		 26.52
±1.08		 0.384
pH 5.83
±0.04a		 5.82
±0.03ab		 5.75
±0.04abc		 5.60
±0.14c		 5.63
±0.18bc		 5.65
±0.20bc		 0.026
乙酸
AA /(mmol/L)		 18.31
±0.37c		 18.46
±0.43c		 19.02
±0.77c		 21.26
±1.57b		 26.62
±2.34a		 25.42
±0.95a		 <0.001
丙酸
PA/(mmol/L)		 20.34
±0.13		 20.17
±0.32		 19.83
±0.30		 20.15
±0.91		 19.98
±0.81		 19.97
±0.39		 0.742
丁酸
BA/(mmol/L)		 6.31
±0.15		 6.20
±0.15		 6.09
±0.28		 6.35
±0.83		 6.00
±0.26		 5.94
±0.13		 0.490
总挥发性脂肪酸
TVFA/(mmol/L)		 44.96
±0.61c		 44.82
±0.60c		 44.93
±0.76c		 47.76
±2.94b		 52.61
±2.84a		 51.34
±1.28a		 <0.001
氨态氮
NH3-N/(mg/dL)		 39.49
±2.30ab		 39.11
±4.93ab		 33.64
±3.80bc		 33.61
±4.51bc		 41.41
±1.32a		 28.28
±3.82c		 0.001

同行数据肩标无字母或相同字母表示差异不显著(P>0.05),不同小写字母表示差异显著(P<0.05)。下表同。

In the same row, values with no letter or the same letter superscripts mean no significant difference (P>0.05), while with different small letter superscripts mean significant difference (P<0.05). The same as below.

2.2 不同酶菌混合添加对甘蔗梢青贮体外瘤胃发酵微生物组成的影响

2.2.1 α多样性指数

由表4可知,各组之间Chao1指数、Ace指数、Shannon指数、Simpson指数和Coverage指数均差异不显著(P>0.05)。本次测序的Coverage指数均大于98%,说明测序结果能够反映样本微生物的真实情况。T1和T3组的Sobs指数显著高于T4组(P<0.05)。
表4 α多样性指数

Table 4 Alpha diversity indexes

项目
Items		 组别 Groups P值
P-value
T1 T2 T3 T4 T5 T6
Sobs指数
Sobs index		 2.21
±0.13a		 2.15
±0.13ab		 2.28
±0.06a		 2.00
±0.06b		 2.14
±0.14ab		 2.17
±0.17ab		 0.040
Chao1指数
Chao1 index (×103)		 2.64
±0.12		 2.57
±0.13		 2.67
±0.06		 2.41
±0.08		 2.52
±0.15		 2.59
±0.21		 0.064
Ace指数
Ace index (×103)		 2.71
±0.14		 2.63
±0.14		 2.76
±0.07		 2.47
±0.09		 2.61
±0.16		 2.67
±0.23		 0.071
Shannon指数
Shannon index		 5.36
±0.28		 5.31
±0.38		 5.55
±0.22		 5.00
±0.26		 5.13
±0.42		 5.29
±0.34		 0.174
Simpson指数
Simpson index		 0.04
±0.01		 0.04
±0.02		 0.03
±0.01		 0.06
±0.02		 0.05
±0.03		 0.04
±0.02		 0.426
Coverage指数
Coverage index		 98.68
±0.08		 98.74
±0.07		 98.69
±0.06		 98.78
±0.06		 98.75
±0.08		 98.69
±0.14		 0.407

2.2.2 Venn图

由图1可知,6组(T1、T2、T3、T4、T5和T6组)的操作分类单元(OTU)数目分别为2 908、2 744、3 036、2 632、2 952和2 955个;6组共用2 177个OTU,占OTU总数的34.33%;6组独有的OTU数目分别为731、567、859、455、775和778个,分别占OTU总数的11.53%、8.94%、13.54%、7.17%、12.22%和12.27%。
图1 Venn图

Fig.1 Venn diagram

2.2.3 门水平上的微生物组成

由图2可知,在门水平上,主要优势菌群为以下5个菌门:厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidota)、疣微菌门(Verrucomicrobiota)、螺旋体门(Spirochaetota)、变形菌门(Proteobacteria)。由表5可知,T4组的厚壁菌门相对丰度与T5组差异不显著(P>0.05),显著高于T1、T2、T3和T6组(P<0.05)。T1、T2、T3组的拟杆菌门相对丰度显著高于T4、T5和T6组(P<0.05)。T5组的疣微菌门相对丰度与T4组差异不显著(P>0.05),显著高于T1、T2、T3和T6组(P<0.05)。T4、T6组的螺旋体门相对丰度显著高于T1、T2、T3和T5组(P<0.05)。T4组的普雷沃氏菌门相对丰度显著高于其他各组(P<0.05)。
图2 门水平上的微生物相对丰度分布图

Firmicutes:厚壁菌门;Bacteroidota:拟杆菌门;Verrucomicrobiota:疣微菌门;Proteobacteria:变形菌门;Spirochaetota:螺旋体门;Patescibacteria:髌骨菌门;Cyanobacteria:蓝细菌门;Others:其他。

Fig.2 Relative abundance distribution map of microbiota at phylum level

表5 不同酶菌混合添加对甘蔗梢青贮体外瘤胃发酵微生物相对丰度的影响

Table 5 Effects of mixed addition of different enzymes and bacteria on relative abundances of microbiota in in vitro rumen fermentation of sugarcane top silage%

项目
Items		 组别 Groups P值
P-value
T1 T2 T3 T4 T5 T6
门水平
Phylum level		 厚壁菌门
Firmicutes		 46.32
±4.62bc		 45.50
±2.52bc		 42.77
±2.88c		 50.91
±0.74a		 48.09
±1.92ab		 45.61
±2.90bc		 0.004
拟杆菌门
Bacteroidota		 39.36
±1.29a		 40.55
±2.46a		 41.39
±1.68a		 29.62
±1.88c		 34.28
±2.83b		 36.06
±2.60b		 <0.001
疣微菌门
Verrucomicrobiota		 5.88
±0.66cd		 5.50
±0.43d		 5.37
±1.19d		 7.42
±0.37ab		 7.78
±0.29a		 6.75
±0.92bc		 <0.001
螺旋菌门
Spirochaetota		 3.30
±0.27c		 3.60
±0.27c		 3.94
±0.96c		 6.31
±0.45a		 4.84
±0.57b		 5.95
±0.52a		 <0.001
普雷沃氏菌门
Proteobacteria		 1.28
±0.24b		 1.15
±0.10b		 1.18
±0.12b		 1.51
±0.16a		 1.23
±0.12b		 1.24
±0.12b		 0.015
属水平
Genus level		 琥珀酸菌属
Succiniclasticum		 22.87
±1.11c		 21.78
±0.91c		 19.24
±0.59d		 29.75
±1.38a		 27.56
±2.26b		 23.61
±1.77c		 <0.001
理研菌科RC9
肠道群
Rikenellaceae_
RC9_gut_group		 14.02
±0.48b		 16.08
±1.37a		 11.59
±0.73c		 9.82
±1.04d		 13.15
±1.47b		 11.34
±0.46c		 <0.001
未定级F082
Norank_f_F082		 13.53
±1.12ab		 13.60
±0.41a		 13.14
±0.52ab		 9.80
±1.11c		 12.03
±0.98b		 13.02
±1.70ab		 <0.001
普雷沃氏菌属
Prevotella		 7.56
±0.88b		 6.71
±0.53b		 18.14
±0.86a		 6.92
±0.42b		 5.32
±0.31c		 7.27
±0.87b		 <0.001
未定级WCHB1-41
Norank_f_norank_o_
WCHB1-41		 3.15
±0.08c		 3.42
±0.37c		 3.08
±0.38c		 4.94
±0.58b		 5.72
±0.57a		 4.53
±0.75b		 <0.001
反刍杆菌属
Ruminobacter		 2.60
±0.15d		 2.84
±0.31cd		 3.20
±0.48c		 5.38
±0.36a		 4.41
±0.47b		 5.44
±0.44a		 <0.001
克里斯滕森菌科
R-7群
Christensenellaceae_
R-7_group		 1.92
±0.08ab		 2.15
±0.30a		 1.96
±0.19a		 1.64
±0.25b		 1.98
±0.30a		 1.93
±0.11ab		 0.042
未定级vadinBE97
Norank_f_vadinBE97		 2.35
±0.29a		 1.45
±0.28c		 1.63
±0.21c		 1.96
±0.25b		 1.61
±0.23c		 1.65
±0.13bc		 <0.001
未定级UCG-011
Norank_f_UCG-011		 1.83
±0.19a		 1.42
±0.21b		 1.68
±0.18ab		 1.50
±0.19b		 1.55
±0.37ab		 1.84
±0.13a		 0.031
NK4A214群
NK4A214_group		 1.44
±0.21		 1.51
±0.38		 1.57
±0.56		 1.26
±0.10		 1.48
±0.11		 1.45
±0.09		 0.681

2.2.4 属水平上的微生物组成

由图3可知,在属水平上,瘤胃微生物主要优势菌属有解琥珀酸菌属(Succiniclasticum)、理研菌科RC9肠道群(Rikenellaceae_RC9_gut_group)、未定级F082(norank_f_F082)、普雷沃氏菌属(Prevotella)、未定级WCHB1-41(norank_f_norank_o_WCHB1-41)、反刍杆菌属(Ruminobacter)等17个属。由表5可知,T4组的琥珀酸菌属相对丰度显著高于其他各组(P<0.05),T3组的琥珀酸菌属相对丰度显著低于其他各组(P<0.05)。T2组的理研菌科RC9肠道群相对丰度显著高于其他各组(P<0.05)。T4组的未定级F082相对丰度显著低于其他各组(P<0.05)。T3组的普雷沃氏菌属相对丰度显著高于其他各组(P<0.05),T5组的普雷沃氏菌属相对丰度显著低于其他各组(P<0.05)。T5组的未定级WCHB1-41相对丰度显著高于其他各组(P<0.05)。T4和T6组的反刍杆菌属相对丰度显著高于其他各组(P<0.05)。T4组的克里斯滕森菌科R-7群(Christensenellaceae_R-7_group)相对丰度显著低于T2、T3和T5组(P<0.05)。T1组的未定级vadinBE97(norank_f_vadinBE97)相对丰度显著高于其他各组(P<0.05)。T1和T6组的未定级UCG-011(norank_f_UCG-011)相对丰度显著高于T2和T4组(P<0.05)。
图3 属水平上的微生物相对丰度分布图

Succiniclasticum:琥珀酸菌属;Rikenellaceae_RC9_gut_group:理研菌科RC9肠道群;norank_f_F082:未定级F082;Prevotella:普雷沃氏菌属;norank_f_norank_o_WCHB1-41:未定级WCHB1-41;Ruminobacter:反刍杆菌属;Christensenellaceae_R-7_group:克里斯滕森菌科R-7群;norank_f_vadinBE97:未定级vadinBE97;norank_f_UCG-011:未定级UCG-011;NK4A214_group:NK4A214群;Saccharofermentans:糖发酵菌属;Sphaerochaeta:球孢菌属;Candidatus_Saccharimonas:暂定种糖单孢菌属;norank_f_norank_o_Clostridia_UCG-014:未定级梭菌目UCG-014;norank_f_norank_o_Gastranaerophilales:未定级嗜胃肠厌氧目;norank_f_Bacteroidales_UCG-001:未定级拟杆菌目UCG-001;Eubacterium_ruminantium_group:瘤胃真杆菌属反刍动物群;Others:其他。

Fig.3 Relative abundance distribution map of microbiota at genus level

2.3 体外瘤胃发酵参数与属水平微生物相对丰度的相关性分析

由图4可知,产气量与理研菌科RC9肠道群相对丰度呈极显著负相关(P<0.01)。H2产量与未定级梭菌属UCG-014和反刍杆菌属相对丰度呈显著正相关(P<0.05),与理研菌科RC9肠道群相对丰度呈显著负相关(P<0.05)。pH与未定级梭菌属UCG-014相对丰度呈显著负相关(P<0.05),与暂定种糖单孢菌属和反刍杆菌属相对丰度呈极显著负相关(P<0.01),与理研菌科RC9肠道群相对丰度呈显著正相关(P<0.05)。AA含量与反刍杆菌属相对丰度呈极显著正相关(P<0.01)。BA含量与糖发酵菌属、NK4A214群和理研菌科RC9肠道群相对丰度呈显著正相关(P<0.05)。TVFA含量与暂定种糖单孢菌属相对丰度呈显著正相关(P<0.05),与反刍杆菌属相对丰度呈极显著正相关(P<0.01)。NH3-N含量与暂定种糖单孢菌属、球孢菌属和普雷沃氏菌属相对丰度呈显著负相关(P<0.05),与理研菌科RC9肠道群相对丰度呈显著正相关(P<0.05)。
图4 体外瘤胃发酵参数与属水平微生物相对丰度的相关性热图

DMD:干物质消化率 dry matter digestibility;Gas:产气量 gas production;H2:氢气 hydrogen;CH4:甲烷 methane;AA:乙酸 acetic acid;PA:丙酸 propionic acid;BA:丁酸 butyric acid;TVFA:总挥发性脂肪酸 total volatile fatty acids;NH3-N:氨态氮 ammonia nitrogen;Lachnospiraceae_NK3A20_group:毛螺菌科NK3A20群;norank_f_norank_o_Clostridia_UCG-014:未定级梭菌目UCG-014;Candidatus_Saccharimonas:暂定种糖单孢菌属;Sphaerochaeta:球孢菌属;Saccharofermentans:糖发酵菌属;NK4A214_group:NK4A214群;norank_f_UCG-011:未定级UCG-011;norank_f_vadinBE97:未定级vadinBE97;Christensenellaceae_R-7_group:克里斯滕森菌科R-7群;Ruminobacter:反刍杆菌属;norank_f_norank_o_WCHB1-41:未定级WCHB1-41;Prevotella:普雷沃氏菌属;norank_f_F082:未定级F082;Rikenellaceae_RC9_gut_group:理研菌科RC9肠道群;Succiniclasticum:琥珀酸菌属;Others:其他。

*代表显著相关(P<0.05),**代表极显著相关(P<0.01)。* represented significant correlation (P<0.05), and ** represented extremely significant correlation (P<0.01).

Fig.4 Correlation heatmap between in vitro rumen fermentation parameters and relative abundances of microbiota at genus level

3 讨论

3.1 不同酶菌混合添加对甘蔗梢青贮体外瘤胃发酵参数的影响

DMD反映饲料在发酵体系中的降解程度,是评估反刍动物瘤胃利用粗饲料的重要指标[21]。本试验中,甘蔗梢青贮体外发酵的DMD在49.03%~51.36%,各组之间没有显著差异,表明不同酶菌混合添加对甘蔗梢青贮体外发酵的DMD无显著影响。产气量是评价瘤胃发酵体系中饲料利用程度的一项重要参数,可以间接反映出反刍动物对青贮饲料营养物质的消化和降解特性,也可以体现饲料营养价值的高低[22-24]。饲料的营养价值越高,发酵性越强,产气量就越高[25]。本试验中,T1组的产气量最低,可能是植物乳杆菌数量过高,抑制其他瘤胃微生物的活性,降低瘤胃发酵产气量;T3组的产气量最高。然而,有研究发现使用LAB对玉米秸秆青贮的产气量没有影响[26],这可能是本试验中不同酶菌组合的相互作用。纤维素酶通过释放细胞壁化合物来刺激瘤胃微生物的生长,增加纤维分解活性,促进细胞壁的分解,提高碳水化合物和蛋白质的降解速率,从而提高体外发酵产气量[27]。瘤胃内H2主要被甲烷菌用于CH4合成[28],当CH4生成受到抑制后,H2产量往往会显著升高[29],本试验中,T3组的H2产量显著高于其他各组,但各组之间CH4产量没有显著差异,在数值上,H2产量高的组别CH4产量也随之增加。瘤胃内pH正常范围在5.5~7.5[30],本试验中,各组的pH均处于正常的范围内,这是饲料能够在瘤胃中降解利用的前提[31],同时有助于瘤胃微生物的生长繁殖。
瘤胃液的VFA含量取决于发酵底物的来源、微生物种群和瘤胃环境,是评价瘤胃健康和发酵状态的重要指标[32]。本试验中,T5和T6组的AA和TVFA含量显著高于其他各组,而各组之间PA和BA含量无显著差异,这可能是不同组间参与PA、BA生成的底物或者微生物没有差异。NH3-N是饲料中非蛋白氮降解的主要中间产物,其含量越高,蛋白质的分解程度越大,发酵质量越差[33]。而且,NH3-N在动物体内的吸收和利用能力远低于真正的蛋白质,因此NH3-N含量越高,饲料的营养价值就越低[34]。T5组的NH3-N含量最高,其次是T1和T2组,T6组最低,表明T6组具有更强的瘤胃发酵特性。

3.2 不同酶菌混合添加对甘蔗梢青贮体外瘤胃发酵微生物组成的影响

反刍动物瘤胃液中优势菌门为厚壁菌门和拟杆菌门[35]。本研究中,体外发酵后优势菌门与其一致,证明酶菌混合添加后的甘蔗梢青贮不会破坏水牛瘤胃液中优势菌门。瘤胃内纤维物质的降解主要由厚壁菌门微生物参与,部分由拟杆菌门微生物降解[36],而碳水化合物的分解主要由拟杆菌门微生物进行[37]。本研究中,体外发酵后不同组间厚壁菌门和拟杆菌门相对丰度呈现差异,可能是由于甘蔗梢青贮过程中酶菌的添加量不同导致的。此外,普雷沃氏菌门也可以协同降解木质纤维[36]。研究发现,瘤胃拟杆菌门相对丰度与VFA含量呈正相关[38],与pH呈负相关[39]。在属水平上,本研究观察到琥珀酸菌属、理研菌科RC9肠道群、普雷沃氏菌属、反刍杆菌属、克里斯滕森菌科R-7群为优势菌属。据报道,瘤胃内普雷沃氏菌属通常将半纤维素、淀粉、蛋白质、肽和果胶发酵成琥珀酸盐、丙酸盐和乙酸盐[40-41]。反刍杆菌属在瘤胃液中与淀粉的降解相关[42],反刍杆菌属所属的琥珀酸弧菌科是琥珀酸盐的主要生产者[43]。琥珀酸菌属是核心瘤胃微生物群落中的一个组成部分,属于革兰氏阴性菌,具有以琥珀酸盐为底物发酵产生丙酸盐的能力[39,44-45]。在丙酸盐的形成过程中,琥珀酸途径和丙烯酸酯途径可以竞争性地抑制瘤胃中的CH4生成[46]。不同普雷沃菌属的功能差异很大,有些表型会产生大量CH4[47],而其他表型则不会大量产生CH4[48]。在本研究中,普雷沃氏菌属相对丰度与CH4产量没有显著的相关性,而表现出与NH3-N含量有显著的负相关性。理研菌科RC9肠道群属于理研菌科RC9家族,瘤胃中的纤维素水平与理研菌科RC9肠道群的活性有关[49],同时与VFA含量呈负相关[50],这与本试验研究结果一致。克里斯滕森菌科R-7群在碳水化合物和氨基酸降解为乙酸盐和氨方面起着重要作用,AA和BA是发酵终产物[51]。反刍杆菌属相对丰度与H2产量及AA、TVFA含量呈显著或极显著正相关,与pH呈极显著负相关,这与Wei等[52]研究结果一致。本试验中,T3组的普雷沃氏菌属相对丰度最高;T4、T6组的理研菌科RC9肠道群相对丰度较低,反刍杆菌属相对丰度较高,这表明从微生物变化及发酵产物综合分析,T3、T4和T6组的底物发酵品质较好。

4 结论

综上所述,甘蔗梢青贮中添加1×105 CFU/g植物乳杆菌、1×107 CFU/g戊糖片球菌、2×104 U/g纤维素酶、2×104 U/g木聚糖酶和0.1%葡萄糖氧化酶,具有较好的发酵品质和发酵特性。

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