Chinese Journal of Animal Nutrition >

2024 , Vol. 36 >Issue 2: 1331 - 1339

DOI: https://doi.org/10.12418/CJAN2024.118

RESEARCH PAPER

Effects of Different Mixing Ratios of Broussonetia papyrifera L. and Moutai Distillers’ Grains on Quality and Microbial Composition of Their Silage

  • HE Xiangjiang , 1 ,
  • FAN Xueying 1 ,
  • CHEN Yulian , 1, * ,
  • CHENG Qiming 1 ,
  • LI Maoya 1 ,
  • LEI Yao 1 ,
  • WANG Jiachuhan 1 ,
  • ZHAO Yuanyuan 1 ,
  • CHEN Chao 1 ,
  • WANG Weidong 2 ,
  • WANG Zhijun , 3, *
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  • 1 College of Animal Science, Guizhou University, Guiyang 550025, China
  • 2 Agricultural and Animal Husbandry Technology Extension Center of Xing’an League, Ulanhot 137400, China
  • 3 Key Laboratory of Forage Cultivation, Processing and Highly Efficient Utilization, Ministry of Agriculture and Rural Affairs, Key Laboratory of Grassland Resources of the Ministry of Education, College of Grassland, Resources and Environment, Inner Mongolia Agricultural University, Hohhot 010021, China
* CHEN Yulian, experimentalist, E-mail: ;
WANG Zhijun, associate professor, E-mail:

Received date: 2023-09-14

  Online published: 2024-02-01

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Abstract

This experiment was conducted to investigate the effects of different mixing ratio of Broussonetia papyrifera L. and Moutai distillers’ grains on quality and microbial composition of their silage. A total of 6 different mixing ratios of Broussonetia papyrifera L. and Moutai distillers’ grains were set in the experiment as follows: 100% Broussonetia papyrifera L. (GCK group), 80% Broussonetia papyrifera L. and 20% Moutai distillers’ grains (group Ⅰ), 60% Broussonetia papyrifera L. and 40% Moutai distillers’ grains (group Ⅱ), 40% Broussonetia papyrifera L. and 60% Moutai distillers’ grains (group Ⅲ), 20% Broussonetia papyrifera L. and 80% Moutai distillers’ grains (group Ⅳ) and 100% Moutai distillers’ grains (JCK group), and there were 3 replicates in each treatment. After silage for 60 days, the nutrient contents, fermentation quality and microbial composition of the silage samples were determined. The results showed as follows: 1) the crude protein (CP) content of silage in group Ⅱ was significantly higher than that in groups GCK and Ⅰ (P<0.05), the neutral detergent fiber (NDF) content was significantly lower than that in GCK group (P<0.05), and the acid detergent fiber (ADF) content was significantly lower than that in groups GCK and Ⅰ (P<0.05). The contents of water soluble carbohydrate (WSC) and dry matter (DM) in mixed silage groups (groups Ⅰ to Ⅳ) were significantly lower than those in JCK group (P<0.05), and the contents of NDF and ADF were significantly lower than those in GCK group (P<0.05). 2) The pH of silage in group Ⅱ was significantly lower than that in groups GCK and Ⅰ (P<0.05), and the lactic acid (LA) content was significantly higher than that in groups Ⅲ and JCK (P<0.05). No butyric acid was detected in silage in all groups. 3) Proteobacteria was the dominant bacterium of silage in groups GCK, Ⅳ and JCK, while Firmicutes was the dominant bacterium in the other groups. The dominant bacterium of silage in group Ⅱ was Lactobacillus, with a relative abundance of 41.75%. The dominant fungus of silage in each group was Ascomycota; with the increase of mixing ratios of Moutai distillers’ grains, the relative abundance of Pichia of silage in group Ⅱ was lower than that in groups JCK and GCK, and which in group Ⅰ was higher than that in groups JCK and GCK. Considering the nutritional value, fermentation quality and microbial composition, the fermentation quality of silage mixed with 60% Broussonetia papyrifera L. and 40% Moutai distillers’ grains is better.

Key words： Broussonetia papyrifera L.; Moutai distillers’ grains; nutrient contents; silage quality; microbial composition

Cite this article

HE Xiangjiang , FAN Xueying , CHEN Yulian , CHENG Qiming , LI Maoya , LEI Yao , WANG Jiachuhan , ZHAO Yuanyuan , CHEN Chao , WANG Weidong , WANG Zhijun . Effects of Different Mixing Ratios of Broussonetia papyrifera L. and Moutai Distillers’ Grains on Quality and Microbial Composition of Their Silage[J]. Chinese Journal of Animal Nutrition, 2024 , 36(2) : 1331 -1339 . DOI: 10.12418/CJAN2024.118

贵州省位于我国西南地区,拥有典型的喀斯特地貌,其主要特征是高海拔、多山及水土流失严重[1],不适合大量种植如苜蓿、玉米和黑麦草等传统牧草。而随着贵州畜牧业的快速发展,常规饲草饲料已经不能满足畜牧业的需求[2]。因此,开发利用非常规饲料资源以解决饲料短缺问题势在必行。
构树(Broussonetia papyrifera L.)是一种桑科、构树属的木本植物,适应性强,生长迅速,生产成本低且分布广泛[3]。构树的收获季节正值雨季,十分潮湿,难以制成干草长期保存,使用青贮调制的方式贮存构树已被证明是保存构树营养的最佳方式[4]。在青贮过程中,附生乳酸菌(lactic acid bacteria,LAB)将饲草中的可溶性碳水化合物(water soluble carbohydrate,WSC)发酵成有机酸[主要是乳酸(lactic acid,LA)],从而降低pH,抑制有害微生物[5]。已有研究表明,构树可以作为蛋白质饲料[6]。在牛和山羊的饲粮中添加适量的构树青贮饲料可以提高其生长性能、肉品质以及免疫和抗氧化功能[7-8]。由于构树富含蛋白质、维生素和氨基酸,我国已经种植了大约30万hm2的构树作为非常规动物饲料[9]。然而,新鲜构树由于缓冲能值高,且WSC含量和LAB数量较低(<105 CFU/g FM),导致构树单独青贮难以成功。
茅台酒糟是酿酒过程中由各种粮食谷物加工后所产生的固体废弃物[10],其中绝大部分的营养物质未被完全利用,如WSC、维生素和氨基酸等[11]。随着我国酿酒业的蓬勃发展,酒糟产量也逐渐增加。杨玉能等[12]研究表明,饲喂茅台酒糟生物料可以提高杂交牛的背部肌肉厚度,且通过改善肌内蛋白质和脂肪含量以及脂肪中各种脂质代谢分子表达量,使肉质得到改善。不过,酒糟由于含水量高,不易长期保存。张磊等[13]研究表明,将酒糟与其他优质原料采用适当比例进行混合青贮,可以提高酒糟及相应原料的营养物质利用率,这既能解决酒糟的保存问题,又可以提高青贮饲料营养价值,增强青贮饲料品质及适口性。因此,笔者猜想构树与茅台酒糟混合青贮可以制得优质青贮饲料。
目前,关于构树与茅台酒糟混合青贮的研究未见报道。本研究拟开展构树与茅台酒糟不同混合比例对其青贮品质的影响研究,通过综合分析其养分含量、发酵品质和微生物组成等指标,筛选出构树与茅台酒糟混合青贮的最适比例,为开发利用非常规饲料提供参考。

1 材料与方法

1.1 试验材料

本试验所用材料为取自贵州省安顺市长顺县的构树,以及取自贵州省仁怀市茅台镇茅台集团酒厂的茅台酒糟。2种原料通过自然风干晾晒6 h左右进行青贮。原料营养水平见表1。
表1 原料营养水平

Table 1 Nutrient levels of ingredients

项目
Items		 茅台酒糟
Moutai distillers’ grains		 构树
Broussonetia papyrifera L.
干物质 DM/(% FM) 68.21±0.33 54.95±0.31
粗蛋白质 CP/(% DM) 19.98±0.45 11.37±0.21
中性洗涤纤维 NDF/(% DM) 31.95±0.51 66.00±0.42
酸性洗涤纤维 ADF/(% DM) 20.41±0.61 42.00±0.19
可溶性碳水化合物 WSC/(% DM) 7.49±0.32 4.00±0.13

1.2 试验设计

本试验共设置6个不同的构树与茅台酒糟混合比例,分别为:100%构树(GCK组)、80%构树和20%茅台酒糟(Ⅰ组)、60%构树和40%茅台酒糟(Ⅱ组)、40%构树和60%茅台酒糟(Ⅲ组)、20%构树和80%茅台酒糟(Ⅳ组)以及100%茅台酒糟(JCK组)。以鲜重为基础,青贮60 d后,测定青贮样品的养分含量、发酵品质及微生物组成,每个处理设置3个重复。

1.3 青贮制备

将构树顶端50 cm左右的枝叶,切成2~3 cm的小段,按照试验设计比例将构树与茅台酒糟充分混匀后,称取330 g,装入聚乙烯袋中,用真空机抽真空密封青贮。青贮袋封口后置于室温下避光保存60 d。

1.4 测定指标及方法

1.4.1 样品处理

将10 g青贮样品与90 mL蒸馏水混合均匀,在4 ℃下浸提24 h后,通过4层纱布和定性滤纸过滤得到样品浸提液,置于-20 ℃条件下保存,用于氨态氮(ammonia nitrogen,NH3-N)和有机酸含量的测定。其余样品置于烘箱,65 ℃烘干至恒重,用于测定干物质(dry matter,DM)、粗蛋白质(crude protein,CP)、中性洗涤纤维(neutral detergent fiber,NDF)、酸性洗涤纤维(acid detergent fiber,ADF)和WSC含量。

1.4.2 养分含量的测定

CP含量采用凯氏定氮法利用定氮仪(KN680,ALVA)[14]测定;WSC含量采用蒽酮-硫酸法[15]测定;NDF和ADF含量采用滤袋分析法[16]利用全自动纤维测定仪(ANKOM A-200I)测定;DM含量参考张丽英[17]的方法测定。

1.4.3 发酵品质的测定

pH采用精密pH计进行测定。NH3-N、LA、乙酸(acetic acid,AA)、丙酸(propionic acid,PA)和丁酸(butyric acid,BA)含量参照DB15/T 1458—2018[18]方法使用DIONEX-2500型离子色谱分析仪测定。采用ASⅡ离子色谱柱,流动相由EG50淋洗液在线发生器产生(1%氢氧化钾溶液),流速0.8 mL/min;采用AS50自动进样器,进样量10 μL,在室温下进行测试。进样前样品稀释10~25倍,45 μm滤膜过滤。

1.4.4 微生物组成分析

将每个青贮处理的3个重复样品均匀地混合在一起,使用QIAamp Fast DNA Stool Mini Kit试剂盒提取青贮饲料中的微生物总DNA,使用细菌16S rRNA基因V3~V4区引物(F:CCTAYGGGRBGCASCAG;R:GGACTACNNGGGTATCTAAT),对以上获得的DNA模板进行序列扩增。真菌ITS扩增子测序引物为F:CTTGGTCATTTAGAGGAAGTAA;R:GCTGCGTTCTTCATCGATGC。使用2%凝胶电泳检测PCR产物,根据NovaSeq 6000的PE250模式pooling上机测序。微生物数据使用北京诺禾致源科技股份有限公司提供的云平台进行分析[19]。

1.5 数据统计分析

结果数据采用SPSS 26.0软件进行方差分析,并利用GraphPad软件制作图表;结果以“平均值±标准误”表示,P<0.05表示差异显著。

2 结果与分析

2.1 构树与茅台酒糟不同混合比例对其青贮饲料养分含量的影响

构树与茅台酒糟不同混合比例对其青贮饲料养分含量的影响见表2。在混合青贮组(Ⅰ组~Ⅳ组)中,Ⅱ组青贮饲料CP含量显著高于GCK组和Ⅰ组(P<0.05),NDF含量显著低于GCK组(P<0.05),ADF含量显著低于GCK组和Ⅰ组(P<0.05)。所有混合青贮组青贮饲料WSC和DM含量均显著低于JCK组(P<0.05),NDF和ADF含量均显著低于GCK组(P<0.05)。
表2 构树与茅台酒糟不同混合比例对其青贮饲料养分含量的影响

Table 2 Effects of different mixing ratios of Broussonetia papyrifera L. and Moutai distillers’ grains on nutrient contents of their silage

项目
Items		 干物质
DM/(% FM)		 粗蛋白质
CP/(% DM)		 中性洗涤纤维
NDF/(% DM)		 酸性洗涤纤维
ADF/(% DM)		 可溶性碳水化合物
WSC/(% DM)
组别 Groups
GCK 54.95±0.31c 9.17±0.18e 62.24±0.98a 39.34±0.37a 3.06±0.29b
Ⅰ 57.13±2.49c 12.19±0.18d 41.60±0.83b 34.06±1.79b 3.27±0.43b
Ⅱ 58.70±1.98bc 14.43±0.08c 36.66±1.52b 27.48±1.21c 3.20±0.13b
Ⅲ 59.42±3.72bc 15.71±0.59b 40.70±4.43b 29.86±2.39bc 3.83±0.10b
Ⅳ 62.56±1.50b 19.31±0.12a 30.33±0.35c 22.75±1.40d 3.51±0.19b
JCK 68.21±0.33a 19.11±0.69a 30.16±0.48c 17.97±0.66e 6.50±0.31a
P值 P-value <0.001 <0.001 <0.001 <0.001 <0.001

同列数据肩标不同小写字母表示差异显著(P<0.05)。下表同。

Values in the same column with different lowercase letter superscripts indicated significant difference (P<0.05). The same as below.

2.2 构树与茅台酒糟不同混合比例对其青贮饲料发酵品质的影响

构树与茅台酒糟不同混合比例对其青贮饲料发酵品质的影响见表3。所有混合青贮组青贮饲料pH显著低于GCK组(P<0.05),PA含量显著高于GCK组(P<0.05)。在混合青贮组中,Ⅱ组青贮饲料pH最低,显著低于Ⅰ组(P<0.05);LA含量最高,显著高于Ⅲ组(P<0.05),同时也显著高于JCK组(P<0.05)。Ⅳ组青贮饲料AA含量最低,显著低于其他混合青贮组(P<0.05)。Ⅲ组青贮饲料PA含量最低,显著低于其他混合青贮组(P<0.05);其他混合青贮组之间PA含量无显著差异(P<0.05),但均显著高于JCK组和GCK组(P<0.05)。各组青贮饲料均未检测出BA存在。Ⅲ组青贮饲料NH3-N含量显著低于GCK组和Ⅰ组(P<0.05)。
表3 构树与茅台酒糟不同混合比例对其青贮饲料发酵品质的影响

Table 3 Effects of different mixing ratios of Broussonetia papyrifera L. and Moutai distillers’ grains on fermentation quality of their silage

项目
Items		 pH 乳酸
LA/(% DM)		 乙酸
AA/(% DM)		 丙酸
PA/(% DM)		 氨态氮
NH3-N/(g/kg)
组别 Groups
GCK 6.30±0.04a 5.11±0.09a 0.58±0.02bc 1.72±0.24c 27.28±4.15a
Ⅰ 5.13±0.16b 4.61±0.35ab 1.15±0.14a 3.62±0.48a 26.91±1.69a
Ⅱ 4.50±0.01c 4.82±0.42ab 0.92±0.22ab 3.51±0.17a 25.87±2.38ab
Ⅲ 4.52±0.01c 3.31±0.03c 1.10±0.07a 2.48±0.19b 19.06±0.41bc
Ⅳ 4.52±0.06c 4.32±0.08b 0.31±0.13cd 3.78±0.04a 20.00±1.94abc
JCK 3.63±0.05d 3.10±0.02c 0.01±0.00d 0.72±0.05d 16.30±1.77c
P值 P-value <0.001 <0.001 <0.001 <0.001 <0.001

2.3 构树与茅台酒糟不同混合比例对其青贮饲料微生物组成的影响

2.3.1 青贮饲料细菌在门水平上的组成

基于门水平的构树与茅台酒糟混合青贮饲料细菌组成如图1所示。GCK组、Ⅳ组和JCK组青贮饲料的优势菌门为变形菌门(Proteobacteria),其余组的优势菌门为厚壁菌门(Firmicutes)。除此之外,各组还含有少量的拟杆菌门(Bacteroidota)和放线菌门(Actinobacteria),JCK组存在部分疣微菌门(Verrucomicrobiota)。Ⅰ组、Ⅱ组和Ⅲ组青贮饲料的厚壁菌门相对丰度相较于Ⅳ组均有所提高,同时变形菌门的相对丰度均有所降低。
图1 青贮饲料细菌在门水平上的组成

Fig.1 Composition of bacteria in silage at phylum level

2.3.2 青贮饲料细菌在属水平上的组成

基于属水平的构树与茅台酒糟混合青贮饲料细菌组成如图2所示。与混合青贮组相比,GCK组或JCK组青贮饲料的乳杆菌属(Lactobacillus)相对丰度较低,其他(others)和假单胞菌属(Pseudomonas)相对丰度较高,因此单独青贮不易成功。Ⅱ组青贮饲料的优势菌属为乳杆菌属,相对丰度为41.75%。随着茅台酒糟混合比例的提高,乳杆菌属的相对丰度呈现先升高后降低的趋势,当茅台酒糟混合比例达到40%时乳杆菌属相对丰度最高,说明Ⅱ组的青贮效果较好。
图2 青贮饲料细菌在属水平上的组成

Fig.2 Composition of bacteria in silage at genus level

2.3.3 青贮饲料真菌在门水平上的组成

基于门水平的构树与茅台酒糟混合青贮饲料真菌组成如图3所示。各组青贮饲料真菌的优势菌门为子囊菌门(Ascomycota),其中JCK组青贮饲料的子囊菌门相对丰度最高。随着茅台酒糟混合比例的提高,混合青贮组青贮饲料中子囊菌门相对丰度逐渐提高。
图3 青贮饲料真菌在门水平上的组成

Fig.3 Composition of fungi in silage at phylum level

2.3.4 青贮饲料真菌在属水平上的组成

基于属水平的构树与茅台酒糟混合青贮饲料真菌组成如图4所示。JCK组青贮饲料的哈萨克斯坦酵母属-假丝酵母属分支(Kazachstania-Candida_clade)相对丰度达20.00%,而Ⅱ组青贮饲料未检测到哈萨克斯坦酵母属-假丝酵母属分支。
图4 青贮饲料真菌在属水平上的组成

Fig.4 Composition of fungi in silage at genus level

随着茅台酒糟混合比例的提高,青贮饲料的毕赤酵母属(Pichia)相对丰度呈现先降低后升高的趋势,Ⅱ组青贮饲料的毕赤酵母属相对丰度低于JCK组和GCK组,Ⅰ组青贮饲料的毕赤酵母属相对丰度高于JCK组和GCK组。

3 讨论

3.1 构树与茅台酒糟不同混合比例对其青贮饲料养分含量的影响

研究发现,新鲜原料中足够的WSC含量(6%~8% DM)对于保证青贮发酵质量至关重要[20]。新鲜构树中NDF和ADF含量较高,WSC含量低于6%,难以很好的青贮;而酒糟中WSC含量较高(7.49% DM),故而本研究中添加茅台酒糟与构树混合青贮弥补了新鲜构树原料WSC含量的不足,以促进LAB发酵。
本研究中,所有混合青贮组青贮饲料的WSC含量均显著低于JCK组,这是因为新鲜酒糟本身WSC含量较高。随着茅台酒糟混合比例的提高,各混合青贮组青贮饲料CP含量呈上升趋势,这与陈冬梅等[21]的研究中,随着白酒糟混合比例的提高,青贮饲料CP含量呈上升趋势的研究结果一致。本研究中,所有混合青贮组青贮饲料NDF和ADF含量显著低于GCK组;任海伟等[22]研究报道,白酒糟与菊芋渣混合青贮,青贮饲料的NDF和ADF含量变化与本研究类似,这说明构树与茅台酒糟混合青贮有利于青贮原料中纤维的降解,从而提高青贮饲料的饲用价值[23]。

3.2 构树与茅台酒糟不同混合比例对其青贮饲料发酵品质的影响

本研究中,构树与茅台酒糟混合青贮处理对其青贮饲料pH以及LA、AA、PA和NH3-N含量有显著影响。其中,所有混合青贮组青贮饲料pH都显著低于GCK组,这与张远凌[24]的研究结果相似,表明混合青贮能够降低构树青贮pH,提高构树青贮成功率。除Ⅲ组外,所有混合青贮组青贮饲料LA含量均显著高于JCK组,其中Ⅱ组青贮饲料LA含量最高,且显著高于Ⅲ组,证明该组青贮发酵效果较好。尉小强等[25]研究表明,青贮中PA含量越高,有氧稳定性越好。本试验中,所有混合青贮组青贮饲料PA含量均较高,且显著高于GCK组和JCK组,这说明混合青贮组有氧稳定性可能更好。而PA含量较高的原因可能是混合调制的青贮饲料在60 d之前就已经发酵完全[26],酒糟比例越高可能发酵完全的越快,故而PA含量随酒糟比例的提高而提高。NH3-N含量可以反映青贮过程中蛋白质和氨基酸的降解程度[27]。本研究中,Ⅱ组青贮饲料NH3-N含量低于GCK组,说明Ⅱ组的蛋白质水解程度小于构树单独青贮。如果青贮饲料中检测到BA,则说明青贮饲料中有梭菌存在,这会加剧青贮中DM的损失,本研究中所有混合青贮组均未检测到BA,说明构树与茅台酒糟混合青贮有利于青贮饲料养分含量的保存[28]。从发酵指标综合来看,构树与茅台酒糟混合比例为6:4时的青贮饲料发酵品质较好。

3.3 构树与茅台酒糟不同混合比例对其青贮饲料微生物组成的影响

微生物作为青贮发酵过程中的关键因素,将直接影响青贮饲料发酵品质的好坏。本研究基于门水平的青贮饲料细菌组成中,混合青贮组(Ⅳ组除外)的优势菌门为厚壁菌门,而JCK组和GCK组的优势菌门为变形菌门,这与陶莲等[29]的研究结果类似。由此说明构树与茅台酒糟混合青贮后有益菌相对丰度提高,且抑制了有害菌的生长,从而降低了青贮饲料养分的消耗。从属水平上来看,单独青贮组的优势菌属为假单胞菌属和其他,而乳杆菌属和肠球菌属(Enterococcus)等有益菌相对丰度较低,这说明这2种原材料单独青贮时都不易成功[30]。构树与茅台酒糟混合青贮后优势菌属为乳杆菌属,且随着茅台酒糟混合比例的提高,乳杆菌属相对丰度呈现先提高后降低的变化趋势,当茅台酒糟混合比例达到40%时乳杆菌属相对丰度最高,这说明Ⅱ组青贮效果较好;而茅台酒糟混合比例过高时,乳杆菌属相对丰度出现下降,这说明茅台酒糟混合比例不宜过高。
基于门水平的青贮饲料真菌组成中,青贮饲料的优势菌门为子囊菌门,其中JCK组子囊菌门相对丰度最高。随着茅台酒糟混合比例的提高,混合青贮组青贮饲料的子囊菌门相对丰度逐渐提高。从属水平上来看,JCK组青贮饲料的哈萨克斯坦酵母属-假丝酵母属分支相对丰度达20.00%,这可能是因为茅台酒糟原料中酵母菌属相对丰度较高所致;而混合青贮组和GCK组未检测到哈萨克斯坦酵母属-假丝酵母属分支,这说明混合青贮有利于减少哈萨克斯坦酵母属-假丝酵母属分支的存在。随着茅台酒糟混合比例的提高,青贮饲料的毕赤酵母属相对丰度呈现先降低后升高的趋势,Ⅱ组青贮饲料的毕赤酵母属相对丰度低于JCK组和GCK组,这说明混合青贮可以减少毕赤酵母属的存在;而Ⅰ组青贮饲料的毕赤酵母属相对丰度高于单独青贮组,这说明构树混合比例不宜过高。
从微生物组成来看,当构树与茅台酒糟混合比例为6:4时,混合青贮饲料中乳杆菌属等有益菌相对丰度较高,假单胞菌属等有害菌相对丰度较低,哈萨克斯坦酵母属-假丝酵母属分支相对丰度较低,更适宜青贮。

4 结论

构树与茅台酒糟混合青贮不仅提高了构树的利用率,丰富了饲料来源,而且提升了青贮饲料的营养价值和发酵品质,同时提高了青贮饲料中乳杆菌属等有益菌的相对丰度,降低了假单胞菌属等有害菌的相对丰度。从营养价值、发酵品质和微生物组成等因素综合考虑,60%构树与40%茅台酒糟混合青贮发酵品质较好。

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