Chinese Journal of Animal Nutrition >

2026 , Vol. 38 >Issue 8: 6317 - 6330

DOI: https://doi.org/10.12418/CJAN2026.504

RESEARCH PAPER

Effects of Compound Inoculant and Fermentation Time on Quality and in Vitro Rumen Fermentation Characteristics of Grape Pomace and Corn Straw Mixed Silage

  • TIAN Chen , 1 ,
  • LIU Jian 1, 2 ,
  • WANG Yunling 1, 2 ,
  • LI Hongya 3 ,
  • JI Shoukun 1, 2 ,
  • WANG Yaya 4 ,
  • LIU Zhongkuan 5 ,
  • ZHI Jianfei 5 ,
  • GAO Lijie , 1, 2, *
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  • 1 College of Animal Science and Technology, Hebei Agricultural University, Baoding 071000, China
  • 2 Hebei Technological Innovation Center for Forage Resources Development and Green Efficient Utilization, Baoding 071000, China
  • 3 College of Life Sciences, Hebei Agricultural University, Baoding 071000, China
  • 4 College of Mechanical and Electrical Engineering, Hebei Agricultural University, Baoding 071000, China
  • 5 Institute of Agricultural Resources and Environment, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang 050000, China
*professor, E-mail:

Received date: 2026-01-09

  Online published: 2026-08-13

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Abstract

This experiment was conducted to investigate the effects of compound inoculant and fermentation time on quality and in vitro rumen fermentation characteristics of grape pomace and corn straw mixed silage. Grape pomace and corn straw mixed at a mass ratio of 2∶1 were used as raw materials for ensiling. Two treatments were set up, including control group (CK group, no inoculant added) and compound inoculant group (NB group, inoculated with Bacillus subtilis YCNL1, Bacillus subtilis N2-10 and Limosilactobacillus reuteri L), with six replicates per treatment. The mixture was sealed and fermented for 45 days, and samples were collected on day 1, 3, 5, 7, 15, 25, 35 and 45 for analysis. The results showed as follows: 1) for the mixed silage system with fixed ratio of grape pomace to corn straw, the contents of dry matter (DM), crude protein (CP, except on day 3) and ether extract (EE) in NB group were higher than those in CK group at all fermentation time points, while the contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF, except on day 1) were lower in NB group. Meanwhile, the pH and ammonia nitrogen/total nitrogen (NH3-N/TN) of NB group were significantly lower (P<0.05), and the lactic acid content was significantly higher (P<0.05) compared with CK group. 2) Analysis of microbial community composition indicated that Proteobacteria was the dominant phylum in the early fermentation stage, while Firmicutes became predominant in the late fermentation stage at phylum level. At genus level, Acetobacter dominated in the early stage, and Lactobacillus was the dominant genus in the late stage of ensiling. 3) Results of in vitro rumen fermentation revealed that the in vitro dry matter degradability (IVDMD) and in vitro neutral detergent fiber degradability (IVNDFD) of mixed silage in NB group were significantly higher than those in CK group at all fermentation time points (P<0.05), and the in vitro acid detergent fiber degradability (IVADFD) of NB group was significantly increased on day 25 and 45 of fermentation (P<0.05). 4) The comprehensive evaluation based on membership function demonstrated that the NB group fermented for 45 days obtained the highest comprehensive membership function value of 0.61. In conclusion, for the mixed silage of grape pomace and corn straw at mass ratio of 2∶1, supplementation of compound inoculant could improve nutritional quality, fermentation quality and in vitro nutrient degradability of mixed silage; the mixed silage fermented for 45 days possessed the optimal comprehensive quality, which was recommended as the suitable fermentation time under this raw material ratio.

Key words： grape pomace; corn straw; compound inoculant; fermentation quality; in vitro rumen fermentation

Cite this article

TIAN Chen , LIU Jian , WANG Yunling , LI Hongya , JI Shoukun , WANG Yaya , LIU Zhongkuan , ZHI Jianfei , GAO Lijie . Effects of Compound Inoculant and Fermentation Time on Quality and in Vitro Rumen Fermentation Characteristics of Grape Pomace and Corn Straw Mixed Silage[J]. Chinese Journal of Animal Nutrition, 2026 , 38(8) : 6317 -6330 . DOI: 10.12418/CJAN2026.504

近年来,随着畜牧业规模化、集约化程度不断提升,饲料资源供给压力日益加剧。常规饲料原料受耕地资源有限、生产成本攀升、进口依存度较高等因素制约,资源供给稳定性不足。因此,开发高效、低成本、可持续的非常规饲料资源,已成为现阶段畜牧业绿色发展的关键需求[1]。在国家大力推动节粮型养殖模式和资源循环利用的背景下,探索农业副产物的饲料化利用,不仅有助于缓解饲料资源紧缺、降低养殖成本,还可减少农业废弃物浪费,助力生态环境保护,具备良好的经济与生态价值。
葡萄渣是葡萄加工过程中产生的主要副产物,包括葡萄皮、籽、梗等成分,产量巨大且呈季节性集中[2]。研究表明,葡萄渣富含原花青素等多酚类物质,具有抗氧化、抗菌、改善脂代谢等多种生物活性[3];在反刍动物饲粮中适量添加葡萄渣,有助于改善瘤胃微生物区系、提升免疫力和提升生长性能[4-5]。Kafantaris等[6]研究发现,在希俄斯公羔羊饲粮中添加9%的葡萄渣能促进肠道益生菌(双歧杆菌)的生长,同时抑制大肠杆菌等有害菌的增殖。由此可见,葡萄渣是一类兼具营养价值与生物活性的优质非常规饲料,开发应用前景广阔。然而,葡萄渣含水量高、易腐败变质,自然发酵稳定性差,直接鲜喂或粗放利用会造成大量营养流失,严重制约了其在畜牧生产中的推广应用。玉米秸秆是我国来源稳定、产量巨大的粗饲料资源,但其粗纤维含量高、适口性差,单独饲喂时消化率低、营养配比不均衡[7]。若能将葡萄渣的营养优势与玉米秸秆的纤维资源优势相结合,通过适宜的发酵技术改善其物化特性和饲用价值,将为非常规饲料开发提供可行途径。
青贮发酵是缓解农业副产物季节性供给矛盾、提高原料营养稳定性和适口性的重要手段[8]。发酵时间与外源微生物菌剂添加是影响青贮发酵品质的重要因素。研究表明,枯草芽孢杆菌具备降解饲草粗纤维的能力,乳酸菌可快速降低青贮体系pH,单一菌种添加均可改善青贮发酵品质,且二者复配可发挥协同增效作用,同步提升青贮营养与发酵品质[9]。葡萄渣与玉米秸秆混贮体系具有高水分、高糖、纤维组分复杂等特点,发酵过程中微生物演替规律尚不清晰,不同发酵时间对其混贮品质的影响也缺乏系统研究。基于此,本研究在固定葡萄渣与玉米秸秆质量比2∶1的条件下,探究复合菌剂和发酵时间对葡萄渣与玉米秸秆混贮营养品质、发酵品质、微生物群落及体外瘤胃发酵特性的影响,旨在为葡萄加工副产物的高效利用及反刍动物饲料资源拓展提供数据支撑。

1 材料与方法

1.1 试验材料

本试验所用葡萄渣取自果汁加工厂榨汁副产物,含水量为75%,色泽为紫红色、无霉变,采集后立即真空包装运至实验室冷冻保存。玉米秸秆由干玉米秸秆经揉丝加工制得,长度为3~5 cm,色泽浅黄、无腐烂霉变,表面较粗糙,具备较好的结构性纤维。葡萄渣和玉米秸秆的营养成分及单宁含量见表1。复合菌剂包括枯草芽孢杆菌YCNL1(活菌数1×108 CFU/mL)、枯草芽孢杆菌N2-10(活菌数1×108 CFU/g)和罗伊氏黏液乳杆菌L(活菌数1×108 CFU/mL)。
表1 葡萄渣和玉米秸秆的营养成分及单宁含量(实测值)

Table 1 Nutrient and tannin contents of grape pomace and corn straw (measured values)

项目
Items		 葡萄渣
Grape pomace		 玉米秸秆
Corn straw
水分 Moisture/% FW 75.00 10.00
粗蛋白质 CP/% DM 13.90 2.00
中性洗涤纤维 NDF/% DM 45.70 65.00
酸性洗涤纤维 ADF/% DM 32.00 38.90
粗脂肪 EE/% DM 2.19 0.75
粗灰分 Ash/% DM 2.44 8.60
钙 Ca/% DM 0.58 0.08
磷 P/% DM 0.24 0.29
单宁 Tannin/% DM 1.20 ND

ND:未检出 not detected。

1.2 试验设计与混贮制作

以葡萄渣和玉米秸秆(质量比2∶1)为原料进行混贮,设置对照组(CK组,不添加菌剂)和复合菌剂组(NB组,添加复合菌剂)2个组,每组6个重复。混贮制备过程如下:将葡萄渣与玉米秸秆充分混匀,以混合原料干物质(DM)为基准添加2%硫酸铵作为辅助氮源,充分搅拌后完成原料预处理。准确称取300 g预处理后的新鲜混合原料,按照试验分组进行喷施处理:NB组均匀喷施复合菌剂(分别添加枯草芽孢杆菌YCNL1、N2-10及罗伊氏黏液乳杆菌L菌剂各1 mL,总喷施量为3 mL/份),CK组喷施等量无菌蒸馏水;处理完毕后将样品装入聚乙烯青贮袋,抽真空密封,置于室温避光条件下进行厌氧发酵贮存。分别于发酵1、3、5、7、15、25、35、45 d时进行取样,测定各组混合青贮饲料的营养品质、发酵品质、微生物组成及体外瘤胃发酵相关指标。

1.3 测定指标及方法

1.3.1 营养品质

葡萄渣、玉米秸秆及葡萄渣与玉米秸秆混贮中水分含量参照《饲料中水分的测定》(GB/T 6435—2014)测定,粗蛋白质(CP)含量参照《饲料中粗蛋白的测定 凯氏定氮法》(GB/T 6432—2018)测定,粗脂肪(EE)含量参照《饲料中粗脂肪的测定》(GB/T 6433—2025)测定,粗灰分(Ash)含量参照《饲料中粗灰分的测定》(GB/T 6438—2007)测定,中性洗涤纤维(NDF)参照《饲料中中性洗涤纤维(NDF)的测定》(GB/T 20806—2022)测定,酸性洗涤纤维(ADF)参照《饲料中酸性洗涤纤维的测定》(NY/T 1459—2022)测定。葡萄渣和玉米秸秆中钙含量参照《饲料中钙的测定》(GB/T 6436—2018)测定,磷含量参照《饲料中总磷的测定 分光光度法》(GB/T 6437—2018)测定,单宁含量参照《饲料中单宁的测定 分光光度法》(GB/T 27985—2011)测定。

1.3.2 发酵品质

开袋后称取20 g混贮样品,加入180 mL蒸馏水,4 ℃浸提48 h后,用4层纱布过滤分离残渣,滤液经离心后取上清液保存待测。使用PHS-3C型酸度计(上海仪电科学仪器股份有限公司)测定pH;采用苯酚-次氯酸钠比色法[10]测定氨态氮(NH3-N)含量,并计算NH3-N/总氮(TN);采用Scion456C气相色谱仪[天美仪拓实验室设备(上海)有限公司]测定乳酸、乙酸、丙酸和丁酸含量。

1.3.3 微生物群落

分别取发酵3、7、45 d的混贮样品(每组3个重复),装入5 mL无菌冻存管,于-80 ℃保存备测。采用DP302-02天根细菌基因组DNA提取试剂盒[天根生化科技(北京)有限公司]提取混贮样品中的细菌总DNA;以341F(5'-CCTACGGGNGGCWGCAG-3')和805R(5'-GACTACHVGGGTATCTAATCC-3')为特异性引物,对细菌16S rRNA基因V3~V4可变区进行PCR扩增[11]。扩增产物经纯化、文库构建与质量检测后,上机开展高通量测序;并基于测序数据完成物种注释及微生物群落组成分析。

1.3.4 体外瘤胃发酵特性

以安装永久性瘤胃瘘管的试验羊为瘤胃液供体,于晨饲前采集瘤胃液(相关程序已获得河北农业大学实验动物管理与伦理委员会批准,批准号为2026003),经4层纱布过滤后转移至塑料保温桶,持续通入二氧化碳(CO2)维持厌氧环境,置于39 ℃水浴锅中备用。参照Menke等[12]的方法制备人工瘤胃缓冲液。将瘤胃液与人工瘤胃缓冲液按体积比1∶2充分混匀配制发酵培养液,通入CO2至变色后,密封放入39 ℃水浴锅备用。开袋后取适量混贮样品,烘干至恒重,粉碎过20目标准筛,称取9 g过筛样品放入发酵袋中,加入300 mL发酵培养液并快速密封,置于39 ℃恒温摇床发酵培养48 h。
发酵结束后,取发酵液分装至10 mL离心管冷冻保存,用于测定体外瘤胃发酵参数(pH及乙酸、丙酸、丁酸含量,测定方法同1.3.2);收集发酵残渣,烘干后放入密封袋,用于测定DM、NDF、ADF含量(测定方法同1.3.1)。依据发酵前、后各样品DM、NDF、ADF含量测定结果,计算体外干物质降解率(IVDMD)、体外中性洗涤纤维降解率(IVNDFD)和体外酸性洗涤纤维降解率(IVADFD)。

1.3.5 混贮品质综合评价

采用隶属函数法对葡萄渣与玉米秸秆混贮品质及体外营养物质降解率进行综合评价。先计算各指标隶属函数值,将所有指标隶属函数值相加后取平均值作为综合隶属函数值,综合隶属函数值越高,混贮综合品质越优。隶属函数值计算公式如下:

UX(+)=(Xij-Ximin)/(Ximax-Ximin);

UX(-)=1-UX(+)。

式中:UX(+)为各正相关指标隶属函数值;UX(-)为负相关指标隶属函数值;Xij为某样品某指标平均测定值;Ximax为某样品某指标最大测定值;Ximin为某样品某指标最小测定值。

1.4 数据统计分析

试验数据经Excel 2021软件进行初步处理,使用SPSS 23.0软件进行两因素重复测量方差分析,并采用LSD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 结果与分析

2.1 复合菌剂和发酵时间对葡萄渣与玉米秸秆混贮营养品质的影响

由表2可知,复合菌剂对DM、CP、NDF和ADF含量有显著影响(P<0.05),发酵时间对DM、CP、EE、Ash、NDF和ADF含量有显著影响(P<0.05),二者的交互作用对CP、EE、NDF和ADF含量有显著影响(P<0.05)。在各发酵时间点,NB组DM、CP(除发酵3 d时)、EE含量均高于CK组,NDF和ADF(除发酵1 d时)含量均低于CK组;与CK组相比,NB组发酵1 d时NDF含量显著降低(P<0.05),发酵5 d时NDF和ADF含量显著降低(P<0.05),发酵7 d时EE含量显著升高(P<0.05),发酵15、25 d时CP含量显著升高(P<0.05),发酵35、45 d时DM含量显著升高(P<0.05)。上述结果提示,葡萄渣与玉米秸秆混贮中添加复合菌剂可有效降解纤维类物质,同时减少营养物质损耗,有利于提升混贮营养品质。
表2 葡萄渣与玉米秸秆混贮的营养品质

Table 2 Nutritional quality of grape pomace and corn straw mixed silage

项目
Items		 组别
Groups		 发酵时间 Fermentation time/d P值 P-value
1 3 5 7 15 25 35 45 T D T×D

干物质 DM/% FW		 CK 47.64±0.81ab 48.39±0.77a 47.54±0.62ab 47.85±0.43ab 47.15±0.30ab 46.65±0.92cd 44.10±0.79Be 46.22±0.50Bd
0.021
0.002
0.256
NB 48.46±2.52ab 49.19±3.26ab 49.12±1.72ab 48.21±0.55ab 47.24±0.47bc 46.83±0.88bc 46.66±0.60Ac 49.55±2.42Aa

粗蛋白质 CP/% DM		 CK 8.15±0.22b 7.46±0.58e 7.63±0.47de 8.09±0.39cd 8.13±0.44Bbc 8.37±0.52Bb 8.48±0.18b 9.60±0.11a
0.004
<0.001
0.012
NB 8.19±0.70d 7.44±0.63e 8.22±0.53cd 8.42±0.37c 9.68±0.24Aa 9.44±0.49Aab 9.29±0.72b 9.63±0.33a

粗脂肪 EE/% DM		 CK 2.27±0.16f 2.61±0.77de 2.76±1.05cd 2.77±0.48Bcd 2.95±1.11bc 3.20±0.42b 3.35±0.67ab 3.57±0.27a
0.083
<0.001
0.031
NB 2.40±0.92e 2.73±0.36d 3.36±0.85bc 3.29±0.64Ac 3.57±1.45ab 3.28±1.00bc 3.64±0.40a 3.38±1.38bc

粗灰分 Ash/% DM		 CK 2.27±0.16d 2.73±0.36c 3.36±0.85b 3.77±0.48a 3.76±0.40Aa 3.03±1.66bc 3.30±1.29b 3.36±0.88b
0.214
0.001
0.487
NB 2.40±0.92d 2.61±0.77cd 2.76±1.06cd 3.59±1.14b 3.54±0.86Bb 3.74±1.41ab 3.98±0.59a 3.09±1.01c

中性洗涤纤维 NDF/% DM		 CK 67.43±0.66Aab 65.38±1.17bc 67.55±1.42Aab 64.92±1.39c 67.93±1.76a 64.92±0.97c 63.80±1.10cd 60.29±2.91e
0.009
<0.001
0.018
NB 62.98±1.01Bbc 64.67±3.62ab 63.44±3.54Bb 64.41±2.59ab 64.95±3.05a 64.33±2.16ab 61.77±3.73c 58.04±4.40d

酸性洗涤纤维 ADF/% DM		 CK 41.31±3.56cd 47.93±2.70a 46.19±1.56Aab 39.33±3.38de 43.91±5.05bc 37.11±1.92e 39.61±4.12d 39.27±1.54de
0.006
<0.001
0.022
NB 47.96±6.52a 46.30±7.94a 41.11±3.16Bb 38.13±1.82b 40.04±2.86b 36.00±0.63e 37.88±0.98c 37.00±1.58cd

同行数据肩标不同小写字母表示同一组别不同发酵时间之间差异显著(P<0.05),同列数据肩标不同大写字母表示同一发酵时间不同组别之间差异显著(P<0.05)。T代表复合菌剂,D代表发酵时间,T×D代表复合菌剂与发酵时间的交互作用。下表(表6除外)同。

In the same row, values with different lowercase letter superscripts mean significant differences among different fermentation times within the same group (P<0.05); in the same column, values with different capital letter superscripts mean significant differences between different groups at the same fermentation time (P<0.05). T represents compound inoculant, D represents fermentation time, and T×D represents the interaction effect of compound inoculant and fermentation time. The same as below (except for Table 6).

2.2 复合菌剂和发酵时间对葡萄渣与玉米秸秆混贮发酵品质的影响

由表3可知,复合菌剂、发酵时间及二者的交互作用对pH、NH3-N/TN和乳酸含量均有显著影响(P<0.05)。在各发酵时间点,与CK组相比,NB组pH和NH3-N/TN均显著降低(P<0.05),乳酸含量均显著提高(P<0.05)。随着发酵时间的延长,NB组pH和NH3-N/TN在发酵45 d时降至最低,分别为3.85和1.56%;乙酸和丙酸含量随发酵时间的延长波动幅度较小;各组均未检出丁酸。
表3 葡萄渣与玉米秸秆混贮的发酵品质

Table 3 Fermentation quality of grape pomace and corn straw mixed silage

项目
Items		 组别
Groups		 发酵时间 Fermentation time/d P值 P-value
1 3 5 7 15 25 35 45 T D T×D

pH		 CK 4.28±0.15Aa 4.07±0.03Acd 4.37±0.01Aa 4.31±0.12Aab 4.19±0.01Abc 4.13±0.08Abc 4.03±0.03Ade 3.97±0.02Ae
0.003
<0.001
0.009
NB 4.20±0.05Ba 3.90±0.10Bde 4.03±0.04Bbc 4.13±0.04Bab 4.07±0.09Bbc 3.95±0.01Bcd 3.87±0.02Be 3.85±0.04Be

氨态氮/总氮 NH3-N/TN/%		 CK 3.04±0.12Aa 2.96±0.20Aab 2.44±0.27Ad 2.38±0.06Ade 1.88±0.08Af 2.75±0.15Ac 2.95±0.42Aab 2.10±0.45Aef
0.001
<0.001
0.014
NB 2.52±0.35Ba 2.14±0.42Bb 2.03±0.26Bbc 1.73±0.10Bcd 1.59±0.23Bd 1.64±0.36Bd 2.00±0.53Bbc 1.56±0.12Bd

乳酸 LA/%		 CK 1.30±0.05Be 1.83±0.11Bd 2.06±0.19Bbc 1.93±0.29Bbc 1.92±0.28Bcd 2.22±0.28Ba 2.14±0.10Bab 2.17±0.30Bab
<0.001
<0.001
0.006
NB 2.41±0.22Ad 2.50±0.28Acd 2.80±0.18Abc 2.89±0.29Aab 2.76±0.25Abc 2.77±0.30Abc 2.91±0.19Aa 2.88±0.14Aab

乙酸 AA/%		 CK 1.70±0.03 1.71±0.03 1.70±0.02 1.67±0.01 1.70±0.02 1.69±0.02 1.68±0.02 1.69±0.02
0.614
0.428
0.772
NB 1.70±0.02 1.69±0.02 1.70±0.01 1.70±0.02 1.70±0.01 1.70±0.03 1.68±0.02 1.71±0.03

丙酸 PA/%		 CK 0.19±0.03 0.22±0.01 0.20±0.03 0.18±0.01 0.21±0.02 0.19±0.02 0.20±0.03 0.19±0.03
0.297
0.183
0.465
NB 0.20±0.02 0.21±0.03 0.18±0.03 0.19±0.03 0.21±0.03 0.20±0.02 0.21±0.03 0.18±0.04

2.3 复合菌剂和发酵时间对葡萄渣与玉米秸秆混贮微生物组成的影响

2.3.1 门水平微生物组成

由图1可知,葡萄渣与玉米秸秆混贮中微生物群落随发酵时间延长发生规律性演替:发酵前期(3、7 d)以变形菌门(Proteobacteria)为优势类群,发酵45 d时厚壁菌门(Firmicutes)成为绝对优势菌门,其余菌门如拟杆菌门(Bacteroidota)、放线菌门(Actinobacteriota)相对丰度整体偏低。
图1 门水平微生物群落组成

S3dCK1、S3dCK2、S3dCK3为发酵3 d时CK组的3个样品;S3dNB1、S3dNB2、S3dNB3为发酵3 d时NB组的3个样品;S7dCK1、S7dCK2、S7dCK3为发酵7 d时CK组的3个样品;S7dNB1、S7dNB2、S7dNB3为发酵7 d时NB组的3个样品;S45dCK1、S45dCK2、S45dCK3为发酵45 d时CK组的3个样品;S45dNB1、S45dNB2、S45dNB3为发酵45 d时NB组的3个样品。图2同。

Fig.1 Microbial community composition at phylum level

S3dCK1, S3dCK2 and S3dCK3 are three samples from the CK group at 3 days of fermentation; S3dNB1, S3dNB2 and S3dNB3 are three samples from the NB group at 3 days of fermentation; S7dCK1, S7dCK2 and S7dCK3 are three samples from the CK group at 7 days of fermentation; S7dNB1, S7dNB2 and S7dNB3 are three samples from the NB group at 7 days of fermentation; S45dCK1, S45dCK2 and S45dCK3 are three samples from the CK group at 45 days of fermentation; S45dNB1, S45dNB2 and S45dNB3 are three samples from the NB group at 45 days of fermentation. The same as Fig.2.

2.3.2 属水平微生物组成

由图2可知,发酵3 d时,CK组微生物群落结构复杂,多种菌属共存,且2组乳杆菌属(Lactobacillus)相对丰度均较低,尚未形成优势有益菌群;发酵7 d时,醋酸菌属(Acetobacter)相对丰度整体下降,乳杆菌属相对丰度升高,且NB组未分类肠杆菌目(unclassified_o_Enterobacterales)、泛菌属(Pantoea)等杂菌相对丰度较CK组降低,说明外源乳酸菌对发酵初期杂菌具有抑制作用;发酵45 d时,乳酸菌属相对丰度明显升高并成为绝对优势菌属,各类杂菌相对丰度大幅下降。
图2 属水平微生物群落组成

Fig.2 Microbial community composition at genus level

2.4 复合菌剂和发酵时间对葡萄渣与玉米秸秆混贮体外瘤胃发酵特性的影响

2.4.1 体外营养物质降解率

由表4可知,复合菌剂、发酵时间及二者的交互作用对IVDMD、IVNDFD和IVADFD均有显著影响(P<0.05)。NB组各发酵时间点混贮IVDMD和IVNDFD均显著高于CK组(P<0.05),发酵25、45 d时混贮IVADFD显著高于CK组(P<0.05)。随着发酵时间的延长,NB组和CK组均在发酵45 d时混贮IVDMD、IVNDFD和IVADFD达到最高。
表4 葡萄渣与玉米秸秆混贮的体外瘤胃发酵营养物质降解率

Table 4 In vitro rumen fermentation nutrient degradability of grape pomace and corn straw mixed silage%

项目
Items		 组别
Groups		 发酵时间 Fermentation time/d P值 P-value
1 3 5 7 15 25 35 45 T D T×D

体外干物质降解率 IVDMD		 CK 45.39±2.74Bde 45.72±2.74Bde 48.18±0.53Bab 43.51±1.91Be 47.13±2.80Bbc 46.98±3.90Bbc 46.42±2.84Bcd 50.01±1.52Ba
0.002
<0.001
0.011
NB 51.44±4.45Aab 51.44±3.84Aab 51.83±1.71Aab 47.64±2.60Ab 50.72±2.02Ab 51.74±0.92Aab 53.12±1.33Aa 55.16±2.24Aa

体外酸性洗涤
纤维降解率
IVADFD		 CK 41.81±1.38c 42.33±1.70bcd 42.07±1.15cd 42.31±1.15bcd 42.74±1.75bc 42.93±1.40Bb 42.33±1.34bcd 44.22±2.50Ba
0.004
<0.001
0.018
NB 42.88±1.16c 42.82±1.12c 42.39±0.87d 42.12±1.94d 42.06±1.92d 44.55±0.57Ab 42.84±1.15c 48.60±3.36Aa

体外中性洗涤
纤维降解率
IVNDFD		 CK 43.01±2.26Bd 43.60±0.95Bcd 47.09±1.95Babc 45.79±2.81Bbcd 45.86±2.23Bbcd 47.33±1.78Bab 45.87±0.99Bbcd 48.97±2.72Ba
0.001
<0.001
0.009
NB 48.26±3.22Ac 52.20±2.00Aabc 51.39±1.67Aabc 50.06±3.05Abc 51.83±0.87Aabc 52.50±0.64Aabc 53.23±1.68Aab 56.26±1.56Aa

2.4.2 体外瘤胃发酵参数

由表5可知,复合菌剂、发酵时间及二者的交互作用对发酵液pH、丙酸和丁酸含量及乙酸/丙酸均有显著影响(P<0.05)。各发酵时间点NB组发酵液pH及乙酸/丙酸与CK组均无显著差异(P>0.05);发酵45 d时NB组发酵液乙酸含量显著高于CK组(P<0.05);发酵5 d时NB组发酵液丙酸含量显著高于CK组(P<0.05);发酵1、15 d时NB组发酵液丁酸含量显著高于CK组(P<0.05)。
表5 葡萄渣与玉米秸秆混贮的体外瘤胃发酵参数

Table 5 In vitro rumen fermentation parameters of grape pomace and corn straw mixed silage

项目
Items		 组别
Groups		 发酵时间 Fermentation time/d P值 P-value
1 3 5 7 15 25 35 45 T D T×D
pH CK 6.06±0.01e 6.15±0.04cd 6.04±0.04e 6.45±0.09a 6.50±0.12a 6.23±0.15bc 6.27±0.07b 6.25±0.06bc
0.012
<0.001
0.021
NB 6.14±0.02c 6.12±0.05c 6.14±0.02c 6.44±0.14a 6.51±0.06a 6.50±0.13a 6.20±0.09bc 6.27±0.05b
乙酸 AA/% CK 37.59±2.10d 37.66±1.31d 38.24±0.73cd 38.28±0.70cd 38.50±0.79bcd 38.57±1.16bc 39.46±1.36b 40.46±3.22Ba
0.087
<0.001
0.018
NB 38.07±3.89cd 36.91±0.38d 38.96±0.88cd 38.31±1.71cd 40.19±1.16bc 40.80±1.62bc 42.13±1.55ab 45.99±2.38Aa
丙酸 PA/% CK 8.69±0.43d 8.81±0.29cd 9.08±0.27Bcd 9.38±0.46cd 10.03±0.74bc 11.06±0.99b 10.78±1.83b 12.34±1.69a
0.006
<0.001
0.014
NB 8.93±0.49c 9.03±0.25c 10.02±0.95Abc 10.02±0.79bc 10.78±1.23b 12.18±0.79a 12.18±1.44a 12.26±1.86a
丁酸 BA/% CK 3.16±0.15Bc 4.16±0.44b 4.07±0.20b 3.94±0.42b 3.96±0.19Bb 4.65±0.35a 4.97±0.48a 4.78±0.41a
0.004
<0.001
0.011
NB 3.87±0.41Ab 4.18±0.30b 4.19±0.45b 4.06±0.17b 4.37±0.60Ab 5.19±0.33a 5.32±0.61a 5.31±0.47a
乙酸/丙酸
AA/PA		 CK 4.33±0.36a 4.27±0.19a 4.21±0.13a 4.09±0.25ab 3.88±0.29ab 3.59±0.32b 3.85±0.70ab 3.79±0.62ab
0.009
<0.001
0.019
NB 4.27±0.46a 4.08±0.07ab 3.91±0.39abc 3.83±0.33abcd 3.76±0.44abcd 3.35±0.18d 3.49±0.38cd 3.70±0.62bcd

2.5 葡萄渣与玉米秸秆混贮效果的综合评价

如表6所示,选取葡萄渣与玉米秸秆混贮品质及体外瘤胃发酵营养物质降解率相关指标开展隶属函数分析。其中,正向指标包括DM、CP、EE、乳酸含量及IVDMD、IVNDFD、IVADFD;负向指标包括pH、NH3-N/TN及NDF、ADF、Ash、乙酸、丁酸含量。对14项指标的隶属函数值平均值进行排序,结果显示发酵45 d时NB组综合隶属函数值最高,为0.61,表明添加复合菌剂发酵45 d的葡萄渣与玉米秸秆混贮综合饲用品质最优。
表6 葡萄渣与玉米秸秆混贮效果的隶属函数综合评价

Table 6 Membership-function-based comprehensive evaluation of grape-pomace-corn-straw mixed silage

发酵时间
Fermentation
time/d		 组别
Groups		 pH EE Ash NDF ADF CP NH3-N/TN DM LA AA PA IVDMD IVNDFD IVADFD 平均值
Mean		 排名
Rank
1 CK 0.39 0.39 0.40 0.45 0.53 0.28 0.49 0.54 0.41 0.46 0.50 0.10 0.53 0.30 0.39 14
NB 0.47 0.22 0.55 0.21 0.46 0.42 0.46 0.41 0.58 0.43 0.52 0.14 0.51 0.25 0.40 12
3 CK 0.55 0.28 0.33 0.34 0.46 0.23 0.29 0.58 0.29 0.36 0.69 0.10 0.47 0.39 0.37 16
NB 0.76 0.26 0.64 0.38 0.52 0.46 0.61 0.48 0.63 0.50 0.63 0.18 0.29 0.33 0.46 10
5 CK 0.29 0.20 0.42 0.48 0.71 0.43 0.25 0.48 0.03 0.46 0.47 0.66 0.59 0.33 0.40 13
NB 0.17 0.29 0.26 0.21 0.72 0.44 0.57 0.40 0.78 0.47 0.31 0.13 0.40 0.31 0.38 15
7 CK 0.35 0.29 0.29 0.39 0.79 0.41 0.52 0.46 0.35 0.76 0.34 0.25 0.29 0.29 0.41 11
NB 0.24 0.45 0.49 0.36 0.84 0.50 0.77 0.43 0.83 0.41 0.44 0.43 0.79 0.41 0.51 8
15 CK 0.55 0.49 0.31 0.18 0.61 0.42 0.71 0.36 0.34 0.67 0.60 0.53 0.84 0.36 0.49 9
NB 0.40 0.66 0.55 0.36 0.76 0.85 0.75 0.37 0.76 0.41 0.67 0.45 0.43 0.20 0.54 4
25 CK 0.70 0.59 0.66 0.36 0.92 0.48 0.77 0.33 0.49 0.53 0.42 0.54 0.41 0.26 0.52 7
NB 0.67 0.54 0.79 0.78 0.88 0.74 0.68 0.28 0.82 0.38 0.39 0.34 0.32 0.13 0.54 6
35 CK 0.84 0.60 0.48 0.43 0.85 0.83 0.29 0.08 0.45 0.64 0.54 0.45 0.68 0.26 0.54 5
NB 0.48 0.89 0.89 0.40 0.88 0.78 0.81 0.32 0.77 0.46 0.56 0.43 0.58 0.12 0.59 2
45 CK 0.81 0.63 0.63 0.64 0.79 0.51 0.63 0.55 0.47 0.58 0.41 0.46 0.64 0.25 0.56 3
NB 0.61 0.56 0.71 0.55 0.78 0.83 0.67 0.32 0.84 0.61 0.63 0.45 0.82 0.27 0.61 1

DM:干物质 dry matter;CP:粗蛋白质 crude protein;EE:粗脂肪 ether extract;Ash:粗灰分 crude ash;NDF:中性洗涤纤维 neutral detergent fiber;ADF:酸性洗涤纤维 acid detergent fiber;LA:乳酸 lactic acid;AA:乙酸 acetic acid;PA:丙酸 propionic acid;NH3-N/TN:氨态氮/总氮 ammonia nitrogen/total nitrogen;IVDMD:体外干物质降解率 in vitro dry matter degradability;IVNDFD:体外中性洗涤纤维降解率 in vitro neutral detergent fiber degradability;IVADFD:体外酸性洗涤纤维降解率 in vitro acid detergent fiber degradability。

3 讨论

3.1 复合菌剂和发酵时间对葡萄渣与玉米秸秆混贮营养品质的影响

枯草芽孢杆菌为需氧或兼性厌氧的革兰氏阳性菌,能产生纤维素酶、半纤维素酶、淀粉酶等多种降解酶,分解秸秆类饲料中的纤维素和半纤维素[13]。DM、CP、EE、NDF、ADF含量等是评价青贮饲料营养品质的重要指标[14]。本试验结果表明,NB组在发酵45 d时混贮DM含量最高,同时CP含量处于较高水平,与高克杰等[15]的研究结果相符。此外,随着发酵时间的延长,混贮CP含量呈上升趋势,这可能是由于发酵后期混贮体系pH下降,微生物代谢活性减弱,从而减少了蛋白质降解损耗[16];同时微生物可利用原料中非蛋白氮合成菌体蛋白,进而提升CP含量[17]。枯草芽孢杆菌可分泌纤维素酶、葡聚糖酶、蛋白酶、几丁质酶等多种水解酶,有效降解植物细胞壁组分[18-19]。
Larae等[20]研究表明,在玉米青贮中单独接种枯草芽孢杆菌,或将其与植物乳杆菌联合接种,均能降低青贮NDF和ADF含量;Ning等[21]研究表明,将枯草芽孢杆菌添加至蛋白桑青贮中,发酵14 d后青贮粗纤维含量显著低于对照组。本试验中,NB组在发酵3~45 d期间NDF和ADF含量均低于同期CK组,与上述研究结果相符,表明复合菌剂有效发挥了纤维降解作用。此外,乳酸菌作为常用青贮微生物添加剂,可同步改善青贮发酵品质、营养品质及瘤胃降解特性等[22]。通过添加乳酸菌,可使秸秆类高纤维原料的组织结构变得更为蓬松,易被动物消化[23]。

3.2 复合菌剂和发酵时间对葡萄渣与玉米秸秆混贮发酵品质的影响

pH检测操作简便、成本低廉,是直观反映青贮发酵品质的重要指标。优质青贮饲料pH通常维持在3.8~4.2,较低的pH可使青贮体系保持稳定,不易发生腐败变质[24]。本试验结果表明,2组混贮发酵过程中pH均存在显著变化,且均在发酵45 d时降至各组最低值,表明发酵至45 d时混贮体系酸化程度最高、发酵环境趋于稳定。各个发酵时间点NB组混贮的pH均显著低于同期CK组,原因在于复合菌剂中的乳酸菌可快速生成乳酸,促使混贮体系pH更快下降。NH3-N含量是评价青贮发酵品质的重要指标。一般来说,NH3-N含量越低,青贮发酵品质越好[25];NH3-N含量越高,意味着发酵过程中肠杆菌等有害细菌含量越高[26-27],不利于饲料品质提升。在本试验整个发酵过程(1~45 d)中,NB组的NH3-N/TN均显著低于CK组,说明复合菌剂的加入很好地抑制了蛋白质的分解,这可能是由于乳酸菌大量增殖竞争性抑制了其他有害菌的生长,使以蛋白质为底物的微生物代谢活动受到抑制,进而降低了NH3-N生成量。随着发酵时间的延长,NB组发酵45 d时NH3-N/TN达到最低,说明添加复合菌剂发酵45 d更利于混贮营养物质留存。
乳酸菌是促进青贮发酵进程的核心功能微生物。本试验所用复合菌剂中含有罗伊氏黏液乳杆菌,使得NB组发酵体系初始乳酸菌丰度更高,可更快地占据发酵主导地位,因此NB组混贮乳酸含量在整个发酵过程中整体高于CK组。乳酸是青贮有机酸中含量最高、酸性最强的组分[28],其含量越高,越利于青贮体系稳定与发酵品质提升。本试验中,NB组发酵5 d时乳酸含量便达到较高水平,后期无显著上升,表明发酵5 d时混贮体系内乳酸菌代谢趋于饱和;而CK组乳酸含量直至发酵25 d时才逐步稳定。由此可见,发酵初期外源添加乳酸菌能够加速发酵环境稳定,保障青贮品质。适宜含量的乙酸、丙酸可改善青贮品质,提升青贮有氧稳定性[29-30]。乙酸主要由异型发酵乳酸菌代谢产生,适量乙酸可抑制青贮霉变,但若积累过量则会产生刺激性异味,降低发酵品质。本试验中,CK组与NB组乙酸含量均处于适宜区间,且组内随发酵时间延长波动幅度较小,未出现乙酸大量累积现象。推测发酵初期青贮袋内残存微量氧气,好氧微生物短暂增殖代谢生成少量乙酸,待体系形成厌氧环境后,好氧菌代谢活动停止,乙酸不再持续产生。

3.3 复合菌剂和发酵时间对葡萄渣与玉米秸秆混贮微生物组成的影响

固态发酵是复杂的系统,由多种微生物共同参与并影响着最终的发酵品质。通过高通量测序可以较为完整地展现不同发酵时间下发酵微生物群落结构,从而辅助判定核心的功能微生物种类[31]。本试验中,门水平微生物群落分析结果显示,发酵前期各组优势菌门为变形菌门,占据较高比例,其次为厚壁菌门。变形菌门菌群会与乳酸菌竞争营养底物,影响青贮发酵品质[32]。与CK组相比,NB组混贮变形菌门相对丰度更低、厚壁菌门相对丰度更高,说明添加枯草芽孢杆菌和乳酸菌可促进厚壁菌门增殖,这与王新宇等[33]的研究结论相符。发酵45 d时,优势菌门转变为厚壁菌门,该菌门中主要包括芽孢杆菌属(Bacillus)、乳杆菌属等功能菌群,可降解原料中的蛋白质、纤维素等物质;伴随厚壁菌门相对丰度上升,变形菌门相对丰度同步下降,表明添加复合菌剂并延长发酵时间可以减少有害微生物的数量[34]。属水平微生物群落分析结果显示,发酵3、7 d时CK组与NB组均以醋酸菌属为主,乳杆菌属占比偏低,同时存在多种杂菌,且CK组杂菌相对丰度更高;发酵45 d时,乳杆菌属相对丰度明显升高并成为绝对优势菌属,醋酸菌属相对丰度明显降低。这是由于葡萄渣原料含糖量较高,含有少量天然酵母,发酵初期袋内残留微量氧气,酵母有氧代谢生成乙醇,醋酸菌属可利用乙醇快速增殖并产生乙酸;同时残存氧气会抑制乳杆菌属生长,因此发酵前期菌群结构呈现醋酸菌属相对丰度高、乳杆菌属相对丰度低的特征。发酵45 d时,体系形成稳定厌氧环境,pH达到较低水平,乳杆菌属具备更强生存竞争优势,将大量糖转为乳酸,同时低pH与厌氧环境抑制醋酸菌属生长,最终发酵后期形成乳杆菌属占绝对优势、醋酸菌属生长受抑制的群落结构。研究报道,青贮发酵后期乳杆菌属通常占据优势地位[35],本试验结果与之相符。

3.4 复合菌剂和发酵时间对葡萄渣与玉米秸秆混贮体外瘤胃发酵特性的影响

瘤胃pH是反映瘤胃发酵内环境稳定性的重要指标之一,pH过高或过低都会影响瘤胃正常发酵。正常情况下,反刍动物瘤胃pH为6.0~7.0[36]。本试验体外瘤胃发酵结果显示,NB组发酵液pH整体高于CK组,该变化规律与Morm等[37]研究结果相符;但2组发酵液pH均处于正常生理区间内,说明经复合菌剂发酵的混贮饲料不会破坏瘤胃稳态。
IVDMD、IVNDFD、IVADFD是衡量反刍动物对饲料的消化程度及饲料营养价值的关键指标[38],其中IVDMD与反刍动物采食量呈正相关[39]。本试验中,NB组混贮的IVDMD、IVNDFD、IVADFD均高于同期CK组,表明复合菌剂处理可提升饲料瘤胃降解效率,有利于提高动物采食量;发酵45 d时的IVDMD、IVADFD最高,说明发酵45 d时复合菌剂的改良效果最优。
瘤胃微生物分解碳水化合物产生的挥发性脂肪酸,可为反刍动物提供70%以上的能量,同时参与乳脂合成、调控饲料转化效率[40]。乙酸是瘤胃中主要的挥发性脂肪酸,其含量越高,通常代表结构性碳水化合物在瘤胃中的降解程度越充分。本试验体外发酵结果显示,NB组发酵液乙酸含量整体高于CK组,且发酵45 d的混贮提升效果最为突出。推测复合菌剂所含乳酸菌于青贮阶段发生异型发酵,优化了混贮碳水化合物组成,更利于瘤胃微生物分解底物生成乙酸,该推论与董文卓等[41]的研究结果相符,即乳酸菌可通过糖类异型发酵改善饲料碳水化合物的瘤胃降解特性。瘤胃液乙酸/丙酸可表征瘤胃发酵类型,比值越低,机体能量利用效率越高[42]。本试验结果表明,NB组发酵液的乙酸/丙酸均低于CK组,但组间差异未达显著水平。分析原因,复合菌剂中的乳酸菌虽能通过异型发酵适度改良碳水化合物结构,但调控效果有限。随着混贮发酵时间增加,2组样品体外发酵液乙酸/丙酸呈降低趋势,提示延长混贮贮存时间可能有利于提升饲粮的能量利用效率,而复合菌剂的具体调控机制仍需进一步研究。

4 结论

在葡萄渣与玉米秸秆质量比2∶1的混贮体系中,添加复合菌剂可提高DM、CP、EE含量,降低NDF、ADF含量及pH和NH3-N/TN,促进乳酸积累,同时可改善混贮体外发酵营养物质降解率。隶属函数综合评价结果显示,添加复合菌剂发酵45 d的葡萄渣与玉米秸秆混贮综合隶属函数值最高,混贮效果最好。

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