Chinese Journal of Animal Nutrition >

2025 , Vol. 37 >Issue 12: 8681 - 8696

DOI: https://doi.org/10.12418/CJAN2025.707

RESEARCH PAPER

Study on in Vitro Degradability, Cellulase Activities and Rumen Microbial Attachment of Corn Stover Co-Pretreated with Urea and Corn Steep Liquor

  • SHA Zhihang , 1 ,
  • HONG Haiyang 1 ,
  • JIANG Mingming 2 ,
  • REN Wenyi 1 ,
  • SU Xueying 1 ,
  • YE Xinyuan 1 ,
  • SU Xiaolong 1 ,
  • ZHANG Lili 1 ,
  • XU Xiaofeng 1 ,
  • MA Yulin , 1, *
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  • 1 College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China
  • 2 College of Animal Science and Technology, Heilongjiang Agricultural Economy Vocational College, Mudanjiang 157041, China
* associate professor, E-mail:

Received date: 2025-03-26

  Online published: 2025-12-13

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Abstract

This experiment aimed to investigate the effects of co-pretreatment with urea and corn steep liquor on in vitro degradability, cellulase activities and rumen microbial attachment of corn stover. A total of 20 kg corn stover was cut into 1 to 2 cm segments, and then randomly divided into 4 groups: control group (CON group: no additives), 3% urea group (UC group: supplemented with urea at 3% of corn stover dry matter), 5% corn steep liquor group (CCL group: supplemented with corn steep liquor at 5% of corn stover dry matter), and 3% urea+5% corn steep liquor group (UCC group: supplemented with both urea at 3% and corn steep liquor at 5% of corn stover dry matter). Each group had 5 replicates, with 1 kg of corn stover per replicate. Each replicate of corn stover was vacuum-sealed in a respiration-fermentation bag and fermented at room temperature. After 56 days, the samples were collected as fermentation substrates for a 72 h in vitro fermentation experiment to determine the in vitro degradability, analyze the cellulase activities in the fermentation broth, and assess the microbial diversity attached to the residue. The results showed as follows: 1) at both 24 and 72 h of in vitro fermentation, the UCC group showed significantly higher in vitro dry matter degradability (IVDMD), in vitro neutral detergent fiber degradability (IVNDFD), and in vitro acid detergent fiber degradability (IVADFD) compared with the CON group (P<0.05). 2) Compared with the CON group, the filter paper enzyme (FPA) activity in the 72 h fermentation broth of the UCC group was significantly increased(P<0.05). 3) At the family level, the dominant bacterial families in all groups were Prevotellaceae and Lachnospiraceae. Compared with the CON group, the UCC group had an increased relative abundance of Lachnospiraceae, but decreased relative abundances of Prevotellaceae and Ruminococcaceae. At the genus level, the dominant bacterial genera in all groups were Xylanibacter and Succiniclasticum. Compared with the CON group, the UCC group showed increased relative abundances of Succiniclasticum and [Ruminococcus]_gauvreauii_group, while the relative abundances of Xylanibacter and Ruminococcus were decreased. 4) LEfSe analysis showed that Saccharofermentans was significantly enriched in the CON group(P<0.05), Treponema was significantly enriched in the UC group (P=0.016), Ruminococcus was significantly enriched in the CCL group (P<0.05), and Syntrophococcus, Shuttleworthia, Eubacterium_ruminantium_group, Olsenella, Oribacterium and Eubacterium_cellulosolvens_group were significantly enriched in UCC group (P<0.05). 5) Correlation analysis showed that IVDMD72 h, IVNDFD72 h and IVADFD72 h were significantly or extremely significantly positively correlated with the relative abundances of Eubacterium_ruminantium_group, Shuttleworthia, Eubacterium_cellulosolvens_group, Olsenella, and Syntrophococcus (P<0.05 or P<0.01), and extremely significantly negatively correlated with the relative abundance of Saccharofermentans (P<0.001). In conclusion, these results indicate that 3% urea combined with 5% corn steep liquor pretreatment can effectively enhance the in vitro degradability of corn stover, stimulate the FAP activity, and promote the attachment of fiber-degrading bacteria.

Key words： corn stover; urea; corn steep liquor; co-pretreatment; in vitro degradability; cellulase; microbial diversity

Cite this article

SHA Zhihang , HONG Haiyang , JIANG Mingming , REN Wenyi , SU Xueying , YE Xinyuan , SU Xiaolong , ZHANG Lili , XU Xiaofeng , MA Yulin . Study on in Vitro Degradability, Cellulase Activities and Rumen Microbial Attachment of Corn Stover Co-Pretreated with Urea and Corn Steep Liquor[J]. Chinese Journal of Animal Nutrition, 2025 , 37(12) : 8681 -8696 . DOI: 10.12418/CJAN2025.707

我国是一个农业大国,具有丰富的农作物秸秆资源。据统计,我国每年生产秸秆约10亿t,其中玉米秸秆居各类秸秆产量首位,年产量达3.1亿t[1]。玉米秸秆是反刍动物粗饲料的主要来源,提升其利用率对于增强反刍动物生产性能、降低饲料成本至关重要。然而,玉米秸秆中纤维素、半纤维素和木质素相互交织成复杂的聚合物,不利于瘤胃微生物的附着和酶降解作用的发挥,致使其在瘤胃中难以被降解[2-3]。因此,为了提升玉米秸秆的利用率,需采取高效的预处理手段,打破其纤维结构,进而促进瘤胃微生物的附着及酶降解作用的发挥。
玉米秸秆在瘤胃中被降解的首要条件是与微生物和酶进行接触,纤维结构、瘤胃微生物的数量以及纤维降解酶的活性等,都可能对玉米秸秆在瘤胃中的降解率产生影响[4-7]。尿素处理能够破坏秸秆的纤维结构,增加反刍动物瘤胃微生物与秸秆纤维的接触面积[8],有利于瘤胃微生物附着,从而提高秸秆的降解率[7]。玉米浆作为玉米淀粉生产的副产品,价格低廉且富含可溶性蛋白质、碳水化合物等营养物质,具有改善玉米秸秆营养品质,为瘤胃微生物提供更为有利的附着环境的作用[9]。
然而,目前仍缺乏尿素和玉米浆复合预处理玉米秸秆体外降解率、纤维素酶活性及瘤胃微生物附着研究的基础数据。因此,本试验旨在探究尿素和玉米浆复合预处理对玉米秸秆体外降解率、纤维素酶活性及瘤胃微生物附着的影响,以期为玉米秸秆高效、合理的饲料化利用提供一定的理论依据和参考。

1 材料与方法

1.1 试验材料

试验所用玉米秸秆取自黑龙江省牡丹江市西安区,玉米浆(干物质含量41.1%,粗蛋白质含量43.0%,粗灰分含量17.0%,亚硫酸盐含量0.28%)购自黑龙江骏华生物科技有限公司,尿素(总氮含量≥46.4%)购自黑龙江倍丰农资集团,呼吸发酵袋(35 cm×25 cm)购自浙江中诚塑业有限公司。

1.2 体外瘤胃发酵液制备

试验动物的处理和护理均按照宁夏大学科技伦理委员会的规定进行,并得到批准(NXU-2024-145)。试验所用的瘤胃液取自宁夏农垦贺兰山奶业奶牛场,选取3头健康、体重(679.00±15.32) kg、胎次[(3.20±0.25)胎]相近的中国荷斯坦奶牛为瘤胃液供体动物,在晨饲前从口腔采集瘤胃液。供体牛所喂基础饲粮组成及营养水平见表1。收集足够的新鲜瘤胃液,将其放入预先加热至39 ℃并充满二氧化碳(CO2)的保温瓶中,混合均匀后用4层纱布过滤。按照Menke等[10]的方法配制人工唾液,将配制好的人工唾液放置于39 ℃水浴锅中,并注入CO2,随后迅速按比例(人工唾液与瘤胃液配比为2∶1)加入制备好的瘤胃液,即可制得体外瘤胃发酵液。
表1 基础饲粮组成及营养水平(干物质基础)

Table 1 Composition and nutrient levels of the basal diet (DM basis) %

项目Items 含量Content
原料Ingredients
玉米青贮Corn silage 45.06
苜蓿青贮Alfalfa silage 4.12
啤酒糟Brewer’s grains 1.53
压片玉米Flaked maize 22.50
苜蓿Alfalfa 10.32
浓缩料Concentrated feed1) 14.30
甜菜颗粒Beet granules 2.00
小苏打NaHCO3 0.17
合计Total 100.00
营养水平Nutrient levels2)
净能NE/(MJ/kg) 5.94
粗蛋白质CP 11.69
中性洗涤纤维NDF 30.74
酸性洗涤纤维ADF 17.80
钙Ca 0.67
总磷TP 0.28

1)浓缩料营养成分含量为:干物质86%,粗蛋白质29.5%,粗纤维12%,粗灰分18%,钙1.3%,总磷0.6%,氯化钠1.3%,赖氨酸0.8%。每千克浓缩料含维生素A 12 000 IU,维生素D 3 300 IU,维生素E 87 IU,铜42 mg,锌181 mg,锰53 mg,铁56 mg,碘1.9 mg,钴0.41 mg。The nutrient contents of the concentrated feed: dry matter 86%, crude protein 29.5%, crude fiber 12%, crude ash 18%, calcium 1.3%, total phosphorus 0.6%, NaCl 1.3%, lysine 0.8%. One kg of concentrated feed contained VA 12 000 IU, VD 3 300 IU, VE 87 IU, Cu 42 mg, Zn 181 mg, Mn 53 mg, Fe 56 mg, I 1.9 mg, Co 0.41 mg.

2)净能为计算值[11],其他为测定值。NE was a calculated value[11], while the others were measured values.

1.3 玉米秸秆预处理

收集20 kg的玉米秸秆,将其切短至1~2 cm后,随机分为4组,即对照组(CON组:不添加任何物质)、3%尿素组(UC组:添加玉米秸秆干物质重量3%的尿素)、5%玉米浆组(CCL组:添加玉米秸秆干物质重量5%的玉米浆)、3%尿素+5%玉米浆组(UCC组:添加玉米秸秆干物质重量3%的尿素和5%的玉米浆),通过在玉米秸秆中加水的方式将各组玉米秸秆样品的水分含量均调整为45%,每组设立5个生物学重复。各组玉米秸秆样品经过充分混合后装入呼吸发酵袋,每袋装入1 kg,将呼吸发酵袋抽真空后,在室温[(25±5) ℃]条件下保存,在发酵56 d后开袋取样,分析样品的营养成分含量,详见表2。
表2 各组预处理玉米秸秆营养成分含量

Table 2 Nutritional component contents of pretreated corn straw in each group %

项目
Items		 组别Groups
CON UC CCL UCC
干物质DM 93.35 92.59 94.37 93.86
粗蛋白质CP 4.70 8.13 5.37 9.24
中性洗涤纤维NDF 74.64 74.71 74.99 70.21
酸性洗涤纤维ADF 54.21 51.37 55.46 45.99
粗灰分Ash 6.51 6.57 10.72 7.92

干物质以风干物质为基础测定,其余指标以干物质为基础测定。下表同。

DM was measured on the basis of air-dry matter, and other indicators were measured on the basis of dry matter. The same as below.

1.4 体外发酵试验

体外发酵试验参照Liu等[12]方法进行,以上述各组预处理玉米秸秆作为体外发酵底物,进行体外瘤胃发酵。准确称量9 g玉米秸秆样品,装入发酵袋(1 000 mL,杭州安思科技有限公司生产)中,使用注射器抽取300 mL体外瘤胃发酵液注入发酵袋,密封,排气后于39 ℃恒温水浴摇床(频率为85 r/min)中分别发酵0.5、24、72 h后,过滤并收集全部残渣及过滤后的发酵液。

1.5 指标测定方法

1.5.1 营养成分含量

干物质(DM)、粗蛋白质(CP)、粗灰分(Ash)、钙(Ca)、总磷(TP)含量分别参照GB/T 6435—2014、GB/T 6432—2018、GB/T 6438—2022、GB/T 6436—2018、GB/T 6437—2018进行测定,中性洗涤纤维(NDF)、酸性洗涤纤维(ADF)含量参照Van Soest等[13]的方法测定。

1.5.2 体外降解率

各组预处理玉米秸秆体外发酵0.5、24或72 h后,将发酵袋置于冰水中终止发酵,发酵底物经过滤后用蒸馏水多次清洗残渣直至清洗液干净为止,置于65 ℃烘箱中烘干,测定DM、NDF、ADF含量,计算体外干物质降解率(IVDMD)、体外中性洗涤纤维降解率(IVNDFD)、体外酸性洗涤降解率(IVNDFD),计算公式如下:
IVDMD(%)=[(发酵底物DM含量-残渣DM含量)/发酵底物DM含量]×100;
IVNDFD(%)=[(发酵底物NDF含量-残渣NDF含量)/发酵底物NDF含量]×100;
IVADFD(%)=[(发酵底物ADF含量-残渣ADF含量)/发酵底物ADF含量]×100;

1.5.3 纤维素酶活性

使用北京索莱宝科技有限公司生产的试剂盒测定72 h发酵液中外切β-1,4-葡聚糖酶(CBH)活性;使用苏州科铭生物技术有限公司生产的试剂盒测定72 h发酵液中滤纸酶(FPA)、β-葡萄糖苷酶(β-GC)活性。

1.5.4 微生物多样性

将发酵72 h的残渣送至上海美吉生物医药科技有限公司进行16S rRNA基因扩增子测序。使用引物338F(5'-ACTCCTACGGGAGGCAGCAG-3')和806R(5'-GGACTACHVGGGTWTCTAAT-3')扩增16S rRNA基因的V3~V4区。在Illumina MiSeq PE300测序平台上测序,在美吉云平台上对微生物多样性进行可视化分析。

1.6 统计分析

所有试验数据均通过Excel 2019软件进行记录整理,以SAS 8.1统计软件进行方差分析,并采用Duncan氏法进行组间的多重比较,结果以平均值和均值标准误(SEM)表示。主坐标分析(PCoA)与非度量多维尺度(NMDS)分析基于Bray-Curtis距离算法构建。基于LEfSe分析获得的标志菌群与体外降解率进行皮尔森相关性分析。以P<0.05表示差异显著。

2 结果与分析

2.1 尿素和玉米浆复合预处理对玉米秸秆体外降解率的影响

由表3可知,UCC和CCL组IVDMD24 h显著高于CON组(P<0.05),UCC组IVDMD72 h显著高于CON组(P<0.05);UCC和CCL组IVNDFD24 h显著高于CON组(P<0.05),UCC组IVNDFD72 h显著高于其余各组(P<0.05);UC组IVADFD0.5 h显著高于CCL组(P<0.05),UCC组IVADFD24 h显著高于其余各组(P<0.05),且UCC组IVADFD72 h显著高于CON和CCL组(P<0.05)。
表3 尿素和玉米浆复合预处理对玉米秸秆体外降解率的影响

Table 3 Effects of co-pretreatment with urea and corn steep liquor on in vitro digestibility of corn stover %

项目
Items		 组别Groups SEM P值
P-value
CON UC CCL UCC
0.5 h体外干物质降解率IVDMD0.5 h 7.31 13.53 10.30 12.93 0.012 0.338
24 h体外干物质降解率IVDMD24 h 26.21c 31.08bc 40.94ab 42.47a 0.022 0.003
72 h体外干物质降解率IVDMD72 h 42.82b 47.20ab 42.97b 50.57a 0.011 0.009
0.5 h体外中性洗涤纤维降解率IVNDFD0.5 h 7.91 6.70 5.46 10.05 0.011 0.172
24 h体外中性洗涤纤维降解率IVNDFD24 h 24.44c 28.94bc 38.77ab 43.15a 0.071 0.002
72 h体外中性洗涤纤维降解率IVNDFD72 h 39.28b 45.28b 42.09b 54.60a 0.018 0.001
0.5 h体外酸性洗涤纤维降解率IVADFD0.5 h 10.90ab 14.35a 4.23b 8.66ab 0.010 0.039
24 h体外酸性洗涤纤维降解率IVADFD24 h 22.89c 32.20bc 32.76b 44.11a 0.010 0.001
72 h体外酸性洗涤纤维降解率IVADFD72 h 37.55b 47.42ab 39.23b 53.98a 0.012 0.006

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 尿素和玉米浆复合预处理对玉米秸秆体外发酵液中纤维素酶活性的影响

由表4可知,UCC组FPA活性显著高于其余各组(P<0.05),CCL组FPA活性显著高于CON组(P<0.05);与CON组相比,UC、CCL和UCC组β-GC、CBH活性均无显著变化(P>0.05)。
表4 尿素和玉米浆复合预处理对玉米秸秆体外发酵液中纤维素酶活性的影响

Table 4 Effects of co-pretreatment with urea and corn steep liquor on cellulase activities in in vitro fermentation broth of corn stover U/mg prot

项目
Items		 组别Groups SEM P值
P-value
CON UC CCL UCC
滤纸酶FPA 0.027c 0.034bc 0.042b 0.054a 0.081 0.003
β-葡萄糖苷酶β-GC 1.400 1.501 1.329 1.488 1.412 0.979
外切β-1,4-葡聚糖酶CBH 0.045 0.049 0.056 0.047 0.220 0.416

2.3 尿素和玉米浆复合预处理对玉米秸秆体外发酵瘤胃微生物附着的影响

2.3.1 瘤胃微生物序列数量

如图1所示,稀释曲线逐渐趋于平坦,说明本次样本测序数据量合理,包含绝大多数微生物信息。
图1 瘤胃微生物稀释曲线

Fig.1 Rumen microbiota dilution curve

2.3.2 瘤胃微生物Alpha多样性

由图2可知,各组之间Ace和Chao指数无显著差异(P>0.05);UCC组Shannon指数显著低于CON组(P<0.05),Simpson指数显著高于CON组(P<0.05)。上述结果表明,尿素和玉米浆复合预处理对残渣上附着的瘤胃微生物的丰富度无显著影响,但能够降低附着的瘤胃微生物的多样性。
图2 瘤胃微生物Alpha多样性分析

A:基于ASV水平的Ace指数;B:基于ASV水平的Chao指数;C:基于ASV水平的Shannon指数;D:基于ASV水平的Simpson指数。“*”表示P<0.05,“**”表示P<0.01,“***”表示P<0.001。

Fig.2 Alpha diversity analysis of rumen microbiota

A: Ace index based on ASV-level; B: Chao index based on ASV-level; C: Shannon index based on ASV-level; D: Simpson index based on ASV-level. “*” indicated P<0.05, “**” indicated P<0.01, and “***” indicated P<0.001.

2.3.3 瘤胃微生物Beta多样性

由PCoA(图3-A)可知,主坐标1和主坐标2的贡献率分别为20.48%和18.42%,UCC组与其余3组显著分离(P=0.001);由NMDS分析(图3-B)可知,UCC组与其余3组显著分离(P=0.001)。上述结果表明,UCC组与其余各组的物种多样性存在显著差异。
图3 瘤胃微生物Beta多样性分析

A:基于ASV水平的PCoA;B:基于ASV水平的NMDS分析。

Fig.3 Beta diversity analysis of rumen microbiota

A: PCoA based on ASV-level; B: NMDS analysis based on ASV-level.

2.3.4 瘤胃微生物组成

如图4-A所示,在科水平上,各组优势菌科均为普雷沃氏菌科(Prevotellaceae)和毛螺菌科(Lachnospiraceae)。与CON组相比,UC组普雷沃氏菌科相对丰度有所提高,但毛螺菌科和瘤胃球菌科(Ruminococcaceae)相对丰度有所降低;UCC组毛螺菌科相对丰度有所提高,普雷沃氏菌科和瘤胃球菌科相对丰度有所降低。如图4-B所示,在属水平上,各组优势菌属均为Xylanibacter和琥珀酸弧菌属(Succiniclasticum)。与CON组相比,UC组Xylanibacter相对丰度有所提高,但瘤胃球菌属(Ruminococcus)相对丰度有所降低;UCC组琥珀酸弧菌属、高氏瘤胃球菌群([Ruminococcus]_gauvreauii_group)、互营球菌属(Syntrophococcus)相对丰度有所提高,Xylanibacter和瘤胃球菌属相对丰度有所降低。
图4 瘤胃微生物组成

A:科水平;B:属水平。

Fig.4 Rumen microbial composition

A: family level; B: genus level.

2.3.5 差异物种分析

由表5可知,与CON组相比,UCC组厚壁菌门(Firmicutes)、放线菌门(Actinomycetota)、热脱硫杆菌门(Thermodesulfobacteriota)相对丰度显著升高(P<0.05),螺旋体门(Spirochaetota)相对丰度显著下降(P<0.05)。由表6可知,与CON组相比,UCC组毛螺菌科NK3A20群(Lachnospiraceae_NK3A20_group)、未分类的毛螺菌科(unclassified_f_Lachnospiraceae)、高氏瘤胃球菌群及互营球菌属相对丰度显著升高(P<0.05),而未分类的普雷沃氏菌科(unclassified_f_Prevotellaceae)、瘤胃球菌属相对丰度显著下降(P<0.05);UC组瘤胃球菌属相对丰度显著下降(P<0.05);CCL组NK4A214群(NK4A214_group)、瘤胃球菌属相对丰度显著升高(P<0.05)。
表5 优势菌门相对丰度(前10位)

Table 5 Relative abundances of dominant bacteria phyla (top 10) %

项目
Items		 组别Groups SEM P值
P-value
CON UC CCL UCC
厚壁菌门Firmicutes 51.37bc 48.82c 58.75ab 59.18a 2.847 0.008
拟杆菌门Bacteroidota 39.93ab 43.12a 32.63b 31.31b 2.412 0.005
放线菌门Actinomycetota 2.20b 1.87b 2.73ab 3.73a 0.470 0.023
热脱硫杆菌门Thermodesulfobacteriota 1.31b 1.47ab 1.68ab 1.72a 0.136 0.047
髌骨菌门Patescibacteria 1.51 1.28 1.69 1.70 0.183 0.084
螺旋体门Spirochaetota 1.59a 1.86a 0.97ab 0.52b 0.333 0.008
假单胞菌门Pseudomonadota 1.28 0.95 0.97 1.21 0.198 0.248
纤维杆菌门Fibrobacteres 0.57 0.47 0.38 0.34 0.080 0.069
疣微菌门Verrucomicrobia 0.14 0.09 0.08 0.16 0.051 0.486
未分类的细菌
Unclassified_k_norank_d_Bacteria		 0.06 0.05 0.07 0.06 0.024 0.651
表6 优势菌属相对丰度(前15位)

Table 6 Relative abundances of dominant bacteria genera (top 15) %

项目
Items		 组别Groups SEM P值
P-value
CON UC CCL UCC
Xylanibacter 15.76ab 17.69a 12.80ab 11.66b 1.588 0.027
未分类的穆里氏菌科Norank_f_Muribaculaceae 6.18 5.74 5.38 4.52 0.615 0.067
琥珀酸弧菌属Succiniclasticum 5.12 7.18 7.17 7.27 1.643 0.595
克里斯滕森菌科R-7群
Christensenellaceae_R-7_group		 4.26 4.13 4.76 3.78 0.368 0.165
毛螺菌科NK3A20群
Lachnospiraceae_NK3A20 _group		 4.18bc 4.18bc 4.89ab 5.37a 0.390 0.033
理研菌科RC9肠道群
Rikenellaceae_RC9_gut_group		 3.73 3.11 3.30 2.76 0.433 0.158
聚乙酸菌属Acetitomaculum 3.56 3.16 3.50 3.52 0.369 0.540
未分类的毛螺菌科Unclassified_f_Lachnospiraceae 3.15bc 2.89bc 3.63ab 4.07a 0.279 0.037
未分类的普雷沃氏菌科
Unclassified_f_Prevotellaceae		 2.98a 3.33a 2.42ab 2.19b 0.302 0.029
NK4A214群NK4A214_group 2.71b 2.33b 3.50a 2.49b 0.190 0.041
瘤胃球菌属Ruminococcus 2.13b 1.25d 2.60a 1.66c 0.127 0.005
高氏瘤胃球菌群
[Ruminococcus]_gauvreauii_group		 1.97b 2.06ab 2.32ab 2.95a 0.298 0.041
未命名的梭菌纲UCG-014
Norank_o_Clostridia_UCG-014		 1.84 1.56 1.79 1.78 0.090 0.050
普雷沃氏菌科YAB2003群
Prevotellacea_YAB2003_group		 1.78ab 2.40ab 1.05b 2.95a 0.539 0.038
互营球菌属Syntrophococcus 1.35b 1.39b 1.48b 3.40a 0.165 0.019

2.3.6 LEfSe分析

在属水平进行LEfSe分析,检验组间差异显著的物种,并用线性判别分析(LDA)得分来衡量差异物种影响效果的大小。差异菌属(LDA得分>3.5,P<0.05)的LDA得分图(图5)显示,产乙酸糖发酵菌属(Saccharofermentans)在CON组显著富集,密螺旋体属(Treponema)在UC组显著富集,瘤胃球菌属在CCL组显著富集,互营球菌属、萨特尔沃斯氏菌属(Shuttleworthia)、瘤胃艾氏真杆菌群(Eubacterium_ruminantium_group)、欧陆森氏菌属(Olsenella)、颤杆菌属(Oribacterium)、溶纤维真杆菌群(Eubacterium_cellulosolvens_group)在UCC组显著富集。
图5 差异菌属LDA得分图

Fig.5 LDA score diagram of differential bacteria genera

2.3.7 体外降解率与LEfSe分析获得的差异菌属相对丰度的相关性分析

由图6可知,IVDMD72 h、IVNDFD72 h、IVADFD72 h均与瘤胃艾氏真杆菌群、萨特尔沃斯氏菌属、溶纤维真杆菌群、欧陆森氏菌属、互营球菌属的相对丰度存在显著或极显著正相关关系(P<0.05或P<0.01),与产乙酸糖发酵菌属的相对丰度存在极显著负相关关系(P<0.001),IVDMD72 h和IVNDFD72 h与颤杆菌属的相对丰度存在显著正相关关系(P<0.05),IVDMD72 h和IVADFD72 h与瘤胃球菌属的相对丰度存在极显著负相关关系(P<0.01)。
图6 差异菌属相对丰度与体外降解率的相关性分析

IVDMD72 h:72 h体外干物质降解率;IVNDFD72 h:72 h体外中性洗涤纤维降解率;IVADFD72 h:72 h体外酸性洗涤纤维降解率。“*”表示P<0.05,“**”表示P<0.01,“***”表示P<0.001。

Fig.6 Correlation analysis between relative abundances of differential genera and in vitro digestibility

IVDMD72 h: 72 h in vitro dry matter degradability; IVNDFD72 h: 72 h in vitro neutral detergent fiber degradability; IVADFD72 h: 72 h in vitro acid detergent fiber degradability。 “*” indicated P<0.05, “**” indicated P<0.01, and “***” indicated P<0.001.

2.3.8 PICRUSt2功能预测

PICRUSt2功能预测可用于预测样本中微生物群落的功能信息。将16S扩增子测序结果与KEGG数据库进行比对,对尿素和玉米浆复合预处理玉米秸秆附着瘤胃微生物的代谢功能进行预测,排在前10的三级代谢通路见图7。各组(CON、UC、CCL、UCC组)一级代谢通路包括新陈代谢(metabolism)、遗传信息处理(genetic information processing)、环境信息处理(environmental information processing)、细胞过程(cellular processes)、人类疾病(human diseases)及有机系统(organismal systems)6类生物代谢功能,二级代谢通路包括全局概览通路(global and overview maps)、碳水化合物代谢(carbohydrate metabolism)、氨基酸代谢(amino acid metabolism)、辅助因子和维生素的代谢(metabolism of cofactors and vitamins)、能量代谢(energy metabolism)等46个子功能组。进一步对KEGG三级代谢通路进行分析,发现UCC组中代谢通路(metabolic pathways)、次生代谢物生物合成(biosynthesis of secondary metabolites)、氨基酸生物合成(biosynthesis of amino acids)、微生物多样环境代谢(microbial metabolism in diverse environments)、碳代谢(carbon metabolism)、核糖体(ribosome)、ABC转运蛋白(ABC transporters)、嘌呤代谢(purine metabolism)、双组分系统(two-component system)、丙酮酸代谢(pyrimidine metabolism)相对丰度均高于CON组。
图7 PICRUSt2功能预测三级代谢通路热图(前10)

Fig.7 PICRUSt2 function prediction metabolic pathway level 3 heat map (top 10)

3 讨论

3.1 尿素和玉米浆复合预处理对玉米秸秆体外降解率的影响

体外降解率可反映养分被消化利用的难易程度与微生物对饲料的降解效果,体外降解率越高,饲料的营养价值通常越高[14]。不同饲料的体外降解率会随着发酵时间的延长呈现不同程度的增加[15]。本试验中,各组IVDMD、IVNDFD、IVADFD均随发酵时间的延长而增加,其中,发酵24和72 h时,UCC组IVDMD、IVNDFD、IVADFD均显著高于CON组,发酵72 h时,UC和CCL组IVDMD、IVNDFD、IVADFD虽高于CON组但差异不显著,这表明尿素和玉米浆复合预处理玉米秸秆的效果优于尿素单独预处理。其原因可能是尿素预处理玉米秸秆,破坏了玉米秸秆的纤维结构,增加了瘤胃微生物与秸秆纤维的接触面积[8],同时,玉米浆的添加为瘤胃微生物提供了更多的营养物质[9],促进了瘤胃微生物的附着。Shaigani等[16]研究发现,玉米浆的添加可以提高小麦秸秆表面微生物的附着数量。乔金莲[17]研究发现,2.5%尿素+9%玉米浆+6%氢氧化钙复合处理水稻秸秆,可以显著提高水稻秸秆的DM、NDF和ADF降解率,并且效果优于单一处理,这进一步表明复合处理的效果优于单一处理。
增强牧草的IVNDFD可以提高泌乳奶牛的干物质采食量(DMI)[18]。Oba等[19]研究发现,饲喂褐色中脉玉米青贮饲料(IVNDFD高于对照的常规玉米青贮饲料)增加了泌乳奶牛的DMI。Demelash[20]通过体内和体外试验发现,在含有不同水平半纤维素的饲粮中添加酵母培养物和草酸可以显著提高IVDMD和IVNDFD,同时,饲喂这些饲粮的绵羊平均日增重和胴体重也显著增加。本试验中,发酵24和72 h时,UCC组IVDMD、IVNDFD、IVADFD均较CON组显著升高,表明尿素和玉米浆复合预处理的玉米秸秆具有提高反刍动物采食量及日增重的潜力,后续可通过奶牛体内饲喂试验进一步验证尿素和玉米浆复合预处理的效果。

3.2 尿素和玉米浆复合预处理对玉米秸秆体外发酵液中纤维素酶活性的影响

纤维素降解是一系列多酶协同作用的复杂过程,依赖于内切葡聚糖酶(EG)、CBH、和β-GC的共同作用[21]。EG为CBH提供更多游离末端以持续水解生成纤维二糖,而β-GC进一步将纤维二糖分解为葡萄糖,防止终产物积累对酶活性产生抑制[21]。FPA活性反映外切酶(CBH)与内切酶(EG)的协同作用强度,而CBH活性主要代表CBH的独立作用[21-22]。本试验中,UCC组FPA活性显著升高,表明尿素和玉米浆复合预处理可能优化了CBH和EG的协同作用。尿素预处理破坏了玉米秸秆的木质素-半纤维素复合结构,增加了纤维素的暴露面积,可能优先促进EG对无定形区的随机切割,从而为CBH提供更多末端并提升FPA活性。Chavan等[23]研究发现,在青霉菌NCIM 1228培养中,添加2.5%的玉米浆使FPA活性增加了3.82倍,表明玉米浆在一定浓度下能提升FPA活性。然而,在本试验中,UCC组CBH活性未显著升高,β-GC活性也无显著变化。CBH活性未显著升高的原因可能是结晶纤维素的顽固结构仍是EG活性的主要限制因素,导致CBH的底物供应不足[4,8];β-GC活性未显著变化可能与纤维二糖积累量不足有关,玉米浆提供的可溶性糖类可能被瘤胃微生物优先利用[24],导致纤维二糖生成量有限,未引发β-GC反馈抑制或表达诱导。此外,部分纤维降解菌可能通过胞内β-GC直接代谢纤维二糖,减少对胞外酶的需求[25-26]。LEfSe分析表明,UCC组富集的瘤胃真杆菌群和溶纤维真杆菌群均为高效纤维降解菌,其分泌的酶系可能以EG和CBH为主,而β-GC主要由普雷沃氏菌科等糖类发酵菌提供[27-28]。UCC组普雷沃氏菌科相对丰度降低,可能导致产生β-GC的菌源减少,抵消了纤维降解菌对β-GC活性的促进作用,最终表现为β-GC活性无显著变化。
尿素和玉米浆复合预处理通过提高底物可及性促进了EG与CBH的协同作用,坚韧性和菌群酶分泌特性的差异可能导致CBH和β-GC活性未同步增加。未来需结合宏基因组学解析关键菌群的纤维素酶基因表达谱,并测定纤维二糖动态积累量,以明确限速步骤的具体调控机制。

3.3 尿素和玉米浆复合预处理对玉米秸秆体外发酵瘤胃微生物附着的影响

瘤胃微生物群落的复杂性对反刍动物的营养物质降解和内环境稳态至关重要,其中细菌是降解营养物质的关键微生物,它们之间的平衡对于维持瘤胃内环境和机体代谢稳定至关重要[29]。Alpha多样性指数体现了瘤胃微生物的丰富度和多样性。Ace和Chao指数反映群落的丰富度,Ace和Chao指数越高,群落丰富度就越高。较高的物种丰富度有利于保持瘤胃内微生态稳定[30]。本试验中,各组Ace和Chao指数无显著差异,表明尿素和玉米浆复合预处理玉米秸秆不会对瘤胃微生物群落丰富度产生不利的影响,对维持瘤胃内环境稳态有积极影响。Simpson和Shannon指数反映群落的多样性,Simpson指数越低,Shannon指数越高,表示群落多样性越高,在本试验中,UCC和UC组的Simpson指数显著高于CON组,而Shannon指数则显著低于CON组,这表明尿素和复合预处理可能降低了瘤胃微生物的多样性。孙美杰等[31]研究发现,在湖羊饲粮中添加10 g/kg的尿素会显著降低瘤胃微生物的多样性。其原因可能是尿素可以促进纤维分解菌的生长,而这种选择性繁殖可能会导致微生物群落结构的变化,从而影响整体多样性[32-33]。
在反刍动物瘤胃微生物中,厚壁菌门、拟杆菌门和变形菌门的数量较多,它们三者共同占据了细菌总门的近90%[34]。厚壁菌门在纤维分解和能量代谢中起核心作用[35-36],放线菌门擅长降解木质素、几丁质等难分解的有机物[37],并产生抗生素等物质,抑制病原菌的生长,有利于维持瘤胃微生态平衡[37-38],并且部分放线菌门参与尿素分解,将非蛋白氮转化为微生物蛋白,进而提高氮利用效率[37,39-40]。在本试验中,UCC组厚壁菌门、放线菌门的相对丰度显著高于CON组,这再次表明尿素和玉米浆复合预处理有利于玉米秸秆中纤维素的降解,与UCC组IVNDFD72 h显著高于其余各组的结果相一致。这可能与尿素与玉米浆复合预处理改变了玉米秸秆的纤维结构以及瘤胃液的pH有关[8,32,41-43]。厚壁菌门和放线菌门对瘤胃液pH敏感,且其相对丰度随pH降低而增加[32,44],玉米浆呈酸性[43],并且本课题组前期研究发现尿素和玉米浆复合预处理玉米秸秆会导致瘤胃液pH降低[45],因此厚壁菌门和放线菌门的相对丰度升高可能与瘤胃液pH下降有关。螺旋体门在降解多糖方面发挥作用,能够有效降解纤维素、果胶和磷酸酯等[35,46]。本试验中,UCC组螺旋体门相对丰度降低,在一定程度上会影响瘤胃纤维素的利用,其原因可能是厚壁菌门和放线菌门相对丰度升高,与螺旋体门存在竞争关系,具有分解纤维功能的细菌之间会相互竞争,纤维素、半纤维素物质会不断减少,这将会导致螺旋体门相对丰度显著减少[47],并且放线菌门合成抑菌物质,可能会抑制螺旋体门的生长[38-39,48]。Lee等[49]研究发现,在饲喂了含高山竹提取物饲粮的长白猪肠道微生物中,厚壁菌门和放线菌门相对丰度显著增加,而螺旋体门相对丰度显著降低。
本研究结果显示,在属水平上,各组的优势菌属均为Xylanibacter和琥珀酸弧菌属。毛螺菌科属于厚壁菌门,可提高瘤胃的效率,促进营养物质消化吸收,加快动物生长发育[50]。Askar等[51]研究发现,补饲尿素能提高绵羊瘤胃原生动物、真菌种群以及毛螺菌科的相对丰度。高氏瘤胃球菌群与反刍动物平均日增重具有正相关关系[52],互营球菌属与非纤维碳水化合物利用有关,有助于改善胃肠道消化、发育和免疫功能[53]。与CON组相比,UCC组毛螺菌科NK3A20群、未分类的毛螺菌科、高氏瘤胃球菌群、互营球菌属相对丰度显著升高。这表明尿素和玉米浆复合预处理有利于玉米秸秆在瘤胃中的消化和吸收。在本试验中,CCL组瘤胃球菌属的相对丰度显著高于其余各组,UC组瘤胃球菌属的相对丰度显著低于其余各组,这表明玉米秸秆中尿素的添加会抑制瘤胃球菌属的生长,而玉米浆的添加则可能缓解这一负面影响。Li等[54]研究发现,绵羊饲粮中添加低剂量的尿素(10 g/kg DM)会降低瘤胃中瘤胃球菌属的相对丰度,其原因可能是复合预处理改变了玉米秸秆的纤维结构,使其更易被其他菌属降解,而瘤胃球菌属对新底物的适应性较差。
玉米秸秆的体外降解率是评估玉米秸秆营养价值的重要指标[11]。在本试验中,尽管UCC组中普雷沃氏菌科和瘤胃球菌属相对丰度降低,但通过LEfSe分析发现,瘤胃艾氏真杆菌群及溶纤维真杆菌群等纤维降解菌显著富集,并与IVDMD72 h、IVNDFD72 h、IVADFD72 h存在显著正相关关系。这一现象表明,尿素和玉米浆复合预处理可能通过功能冗余性维持纤维降解效率。溶纤维真杆菌群可分泌木聚糖酶,靶向降解半纤维素网络[55],而瘤胃真杆菌群能高效解构结晶纤维素并产生丁酸[56],二者的协同作用提高了纤维素与半纤维素的降解效率。基于PICRUSt2功能预测及KEGG代谢通路分析,UCC组中碳代谢、氨基酸生物合成、ABC 转运蛋白、核糖体、丙酮酸代谢等代谢通路相对丰度高于CON组。UCC组中碳代谢的增强表明糖酵解、三羧酸(TCA)循环等产能途径可能增强,为降解酶分泌与催化提供更多ATP;核糖体、氨基酸生物合成通路相对丰度增加,加速mRNA转录与翻译,可能促进纤维降解酶的大量合成,快速响应纤维底物刺激,增强降解纤维能力;ABC转运蛋白通路相对丰度增加,则加快了UCC组中瘤胃微生物与外界的物质交换,这有可能进一步加强其对降解纤维的能力;丙酮酸代谢通路的相对丰度升高,则证明溶纤维真杆菌群和瘤胃真杆菌群可能具有将葡萄糖通过丙酮酸节点转化为短链脂肪酸(SCFA)的能力。结合UCC组FPA活性显著升高,推测溶纤维真杆菌群和瘤胃艾氏真杆菌群可能具备“纤维素→葡萄糖→短链脂肪酸”的全链路代谢能力。这一代谢链路的完整性可能是UCC组体外降解率显著提升的关键机制,同时也解释了溶纤维真杆菌群和瘤胃真杆菌群作为“核心菌群”的生态位优势——通过底物降解与产物转化的耦合,避免中间代谢产物的积累抑制酶活性。此外,互营球菌属与非纤维碳水化合物的利用相关,可能通过改善微生物代谢分工优化瘤胃发酵环境[53],这一结果与UCC组IVNDFD72 h和IVADFD72 h显著升高的结果相对应。这表明,尿素和玉米浆复合预处理通过理化协同效应(尿素破坏纤维结构、玉米浆提供氮源)选择性富集了高效纤维降解菌群,从而提升了玉米秸秆的可降解性。但本试验未直接测定溶纤维真杆菌群和瘤胃艾氏真杆菌群的代谢产物或功能基因,对其功能的推断主要基于文献报道、纤维素酶活性及PICRUSt2功能预测的间接推断,未来需结合宏基因组或代谢组学进一步验证其功能。
通过LEfSe分析发现,产乙酸糖发酵菌属在CON组显著富集,且其相对丰度与IVDMD72 h、IVNDFD72 h、IVADFD72 h均呈极显著负相关。产乙酸糖发酵菌属是一种严格厌氧的乙酸生成菌,其代谢特征以糖类发酵为主,可能更依赖于可溶性碳水化合物而非结构性纤维底物[57],而瘤胃球菌属细菌通过分泌纤维素体(cellulosome)分解植物纤维,将纤维素转化为可吸收的糖类[21]。在本试验中,尿素和玉米浆复合预处理可能通过破坏木质素-半纤维素的交联结构(UCC组ADF含量低于CON组),增加纤维素的可及性,从而改变了瘤胃微生物的底物偏好性。这种结构变化可能更有利于纤维降解菌(如瘤胃艾氏真杆菌群、溶纤维真杆菌群)的附着与增殖,而产乙酸糖发酵菌属和瘤胃球菌属因底物竞争或生态位挤压导致相对丰度下降。郭孟娇等[58]研究发现,碱性过氧化氢处理水稻秸秆会导致瘤胃中产乙酸糖发酵菌属和瘤胃球菌属相对丰度下降。此外,玉米浆的添加可能通过提供氮源和可溶性糖类,间接抑制了依赖复杂底物的菌群生长。但本试验未直接测定产乙酸糖发酵菌属和瘤胃球菌属的代谢产物或功能基因,其与IVDMD72 h、IVNDFD72 h、IVADFD72 h的负相关性可能受多种因素(如种间竞争、代谢产物抑制等)影响,未来需结合宏基因组或代谢组学进一步验证其功能。

4 结论

在本试验条件下,3%尿素和5%玉米浆复合预处理能够提高玉米秸秆体外发酵72 h的IVDMD、IVNDFD、IVADFD,同时提高FPA活性以及毛螺菌科NK3A20群、高氏瘤胃球菌群及互营球菌属相对丰度。

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