Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 4: 2443 - 2454

DOI: https://doi.org/10.12418/CJAN2023.229

RESEARCH PAPER

Effects of Compound Bacteria Preparation on Fermentation Quality and Rumen Fermentation Characteristics of Phragmites australis Silage

  • HUANG Qi , 1, 2 ,
  • WANG Shuiping , 1, * ,
  • HUANG Qiaoshen 1 ,
  • LIU Yong 1 ,
  • YANG Xin 1 ,
  • ZHANG Yongkang 1 ,
  • LIU Yong 2 ,
  • LI Maochun , 3, *
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  • 1 Chongqing Key Laboratory of Herbivore Science, College of Animal Science and Technology, Southwest University, Chongqing 402460, China
  • 2 Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China
  • 3 Institute of Animal Husbandry and Veterinary Science, Yueyang Academy of Agricultural Sciences, Yueyang 414000, China
*WANG Shuiping, associate professor, E-mail: ;
LI Maochun, senior animal husbandry engineer, E-mail:

Received date: 2022-09-06

  Online published: 2023-04-12

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Abstract

The purpose of this study was to investigate the effects of compound bacteria preparation (including homogeneous Lactobacillus/heterogeneous Lactobacillus, Bacillus subtilis and Aspergillus niger as well as their compatibility) on nutrient composition, fermentation quality and rumen fermentation characteristics of Phragmites australis silage. Taking growing Phragmites australis from Dongting lake as silage material, five groups were set up, which were blank group (CK group), lactic acid bacteria group (LA group, supplemented with Lactobacillus plantarum, Pediococcus pentosaceus and Lactobacillus buchneri), lactic acid bacteria+Bacillus subtilis group (LA+BS group), lactic acid bacteria+Aspergillus niger group (LA+AN group) and lactic acid bacteria+Bacillus subtilis+Aspergillus niger group (LA+BS+AN group), and each group had 3 replicates. The supplemental level of lactic acid bacteria was 1.5×108 CFU/kg silage raw materials, and the supplemental levels of Aspergillus niger and Bacillus subtilis were 1.0×108 CFU/kg silage raw materials (all were based on fresh weight of silage raw materials). After 60 days of silage, the nutrient composition and fermentation quality of Phragmites australis silage were determined, and the rumen fermentation characteristics were evaluated by in vitro fermentation and in vivo degradation. The results showed as follows: 1) the contents of dry matter and ash in LA, LA+BS and LA+AN groups were significantly lower than those in CK group (P<0.05), and the crude protein content in LA+BS, LA+AN and LA+BS+AN groups was significantly higher than that in CK and LA groups (P<0.05). 2) The silage sensory evaluation scores of all groups were 60 to 70 points, and the ratings were good. The comprehensive scores of fermentation quality in LA+BS and LA+AN groups were higher, among which, the ammonia nitrogen to total nitrogen ratio, acetic acid content and acetic acid to total acid ratio in LA+AN group were significantly decreased compared with CK group (P<0.05), while the lactic acid content and lactic acid to total acid ratio were significantly increased compared with CK group (P<0.05). 3) The dry matter disappearance rate and total gas production of in vitro fermentation in LA+AN group were significantly higher than those in the other groups (P<0.05), and in vitro fermentation parameters such as volatile fatty acid contents in fermentation liquid were improved. 4) The rumen fermentation dry matter degradation rate in LA+AN and LA+BS+AN groups at 8, 24 and 48 h was significantly higher than that in the other three groups (P<0.05). In conclusion, the combined application of lactic acid bacteria and Aspergillus niger with supplemental levels in this experiment has the best effect on the quality of Phragmites australis silage and rumen fermentation characteristics.

Key words： Phragmites australis; compound bacteria preparation; silage; fermentation quality; rumen fermentation characteristics

Cite this article

HUANG Qi , WANG Shuiping , HUANG Qiaoshen , LIU Yong , YANG Xin , ZHANG Yongkang , LIU Yong , LI Maochun . Effects of Compound Bacteria Preparation on Fermentation Quality and Rumen Fermentation Characteristics of Phragmites australis Silage[J]. Chinese Journal of Animal Nutrition, 2023 , 35(4) : 2443 -2454 . DOI: 10.12418/CJAN2023.229

芦苇是多年生禾本科植物,具有十分重要的生态和经济功能[1]。芦苇主要生长于湖泊或沼泽区域,分布区域辽阔,生物资源丰富,2017年,我国芦苇年产量高达300多万t,其中洞庭湖区芦苇产量约占1/3[2]。传统造纸行业以芦苇作为主要原材料,近年来,芦苇造纸产业由于其污染问题被全面关停,引发芦苇大面积弃收,造成资源严重浪费,带来严峻的生态问题,导致芦苇产业发展陷入困境[3]。随着我国草食畜牧业规模不断扩大,苜蓿、燕麦草等优质饲草资源供应日趋紧张,合理利用芦苇资源,将其作为一种非常规饲料资源进行饲料化高效利用不仅可实现资源循环利用,减少生态环境压力,还可扩大饲料资源库,缓解我国饲料资源紧缺的现状。
青绿芦苇是优良饲草,具有较高的营养价值及饲用价值,富含有机酸、多糖类和黄酮类化合物等活性物质,具有免疫调节、护肝、抑菌、抗衰老和抗氧化等多种生理活性,在提高生长性能和免疫性能方面发挥着重要作用[4⇓⇓-7]。青贮可以在保存芦苇高消化性营养物质的基础上,延长保存时间,提高消化率和适口性。研究发现,不同生长阶段的芦苇均可进行青贮,且最适时期为抽穗期[8]。已有研究表明,青贮添加剂的开发和利用在高品质青贮饲料生产和牧草产业发展中具有重要的作用,能显著提高饲料的性能和营养价值[9-10]。植物乳杆菌(Lactobacillus plantarum)、戊糖片球菌(Pediococcus pentosaceus)和布氏乳杆菌(Lactobacillus buchneri)是目前常用于提高青贮发酵品质的乳酸杆菌,可以增加青贮初期乳酸菌数量,促进乳酸发酵,以促进发酵体系前期快速产酸形成酸性环境,抑制腐败霉菌的生长和营养素消耗,对青贮发酵品质的保障十分重要[11⇓-13]。黑曲霉菌(Aspergillus niger)具有裂解大分子有机物和难溶无机物而便于吸收利用的效果[14];枯草芽孢杆菌(Bacillus subtilis)可有效分解粗纤维等抗营养因子,释放营养物质,促进可消化营养素的消化吸收[15]。不过,因单一菌制剂功能单一性和局限性,复合菌制剂功能开发成为当前青贮菌制剂领域研究的热点问题。
目前,芦苇青贮技术相对不成熟,相关研究较少,青贮后的品质和适口性较差,营养损失严重。且菌制剂调控芦苇青贮饲料的品质的研究尚不多见。因此,本研究选择复合菌制剂作为青贮添加剂,探究不同菌制剂搭配对青贮芦苇营养成分和发酵品质的影响,为改善芦苇青贮品质提供理论支撑,促进芦苇饲料化进程。

1 材料与方法

1.1 试验材料

本试验所用材料为湖南省岳阳市洞庭湖区的生长期全株芦苇,于2021年5月15日刈割后室温放置1.5 h,晾干表面水分后立即用粉碎机粉碎,粉碎长度为2~3 cm。取200 g芦苇原料在65 ℃烘箱中,烘干至恒重,粉碎后过40目筛,密封保存备用待测常规营养成分,其余立即制作青贮。芦苇营养成分见表1。
表1 芦苇营养成分(干物质基础)

Table 1 Nutrition composition of Phragmites australis (DM basis) g/kg

项目Items 含量Content
干物质DM 283.76
粗蛋白质CP 87.18
可溶性碳水化合物WSC 21.99
中性洗涤纤维NDF 786.05
酸性洗涤纤维ADF 461.87
粗脂肪EE 62.26
粗灰分Ash 106.64
总能GE/(MJ/kg) 16.41

1.2 试验设计

采用单因素试验设计,试验共设5个组,分别为空白组(CK组)、乳酸菌组(LA组,添加植物乳杆菌、戊糖片球菌和布氏乳杆菌)、乳酸菌+枯草芽孢杆菌组(LA+BS组)、乳酸菌+黑曲霉菌组(LA+AN组)、乳酸菌+枯草芽孢杆菌+黑曲霉菌组(LA+BS+AN组),每组3个重复。每个重复称取1 000 g左右样品,加入对应菌制剂[由威凯海思(山东)生物工程有限公司提供,活菌数均为1.0×1010 CFU/kg,植物乳杆菌、戊糖片球菌和布氏乳杆菌添加量均为1.5×108 CFU/kg青贮原料,枯草芽孢杆菌和黑曲霉菌添加量为1.0×108 CFU/kg青贮原料(均以青贮原料鲜重为基础)]。调制好后装入聚丙乙烯袋中(40 cm×60 cm),抽真空后密封,置于室温(25~30 ℃)避光保存,贮藏60 d后测定营养成分和发酵品质,并用体外发酵试验和体内降解试验评价青贮后5组青贮芦苇的瘤胃发酵特性。

1.3 指标测定

1.3.1 原料和青贮饲料常规营养成分含量

取200 g芦苇原料和青贮饲料,在65 ℃烘箱中烘干至恒重,粉碎后过40目筛,密封保存备用。依据国标[16]的方法测定营养成分含量,具体包括:样品在105 ℃烘箱中烘干,取出在干燥器中冷却30 min后测定干物质(dry matter,DM)含量;采用凯氏定氮法测定粗蛋白质(crude protein,CP)和总氮(total nitrogen,TN)含量;采用范式洗涤纤维法测定酸性洗涤纤维(acid detergent fiber,ADF)和中性洗涤纤维(neutral detergent fiber,NDF)含量;采用索氏提取法测定粗脂肪(ether extract,EE)含量;采用灼烧恒质量法测定粗灰分(Ash)含量;采用蒽酮-硫酸比色法测定可溶性碳水化合物(water-soluble carbohydrate,WSC)含量;采用精密全自动量热仪(5E-C5508,Kaiyuan Instrument,中国)测定总能(gross energy,GE)。

1.3.2 青贮发酵品质

青贮60 d后,开袋取出青贮饲料混合均匀后,准确称取50 g青贮饲料放入500 mL锥形瓶中,加450 mL无菌水后封口,振荡混匀后于4 ℃冰箱中浸提24 h,用涤纶布和定量滤纸过滤获得浸提液。使用pH计测定pH,使用高效液相色谱仪(Agilent-1290,美国)测定乳酸(lactic acid,LA)含量,使用高效气相色谱仪(Agilent-7890A,美国)测定乙酸(acetic acid,AA)、丙酸(propionic acid,PA)和丁酸(butyric acid,BA)含量,使用苯酚-次氯酸钠比色法测定氨态氮(NH3-N)含量。参照刘建新等[17]的评分标准进行感官评定和发酵品质评价,根据感官评定得分将青贮饲料划分为4个等级,分别为低劣(0~25分)、一般(26~50分)、良好(51~75分)和优质(76~100分);根据所测青贮饲料中的NH3-N、AA、PA和BA含量对发酵品质评价,将有机酸得分除以2,并加上NH3-N得分,即为综合得分,满分100分。将青贮饲料划分为5个等级,分别为极差(0~20分)、差(21~40分)、可(41~60分)、良(61~80分)和优(>80分)。

1.3.3 体外发酵试验

称取1.2 g底物于135 mL厌氧发酵瓶中。晨饲前采集瘤胃液,装入保温瓶带回实验室,6层纱布过滤后,量取600 mL滤液迅速加入提前准备好的2 400 mL人工瘤胃缓冲液中(39.5 ℃水浴,瘤胃液与人工瘤胃营养液体积比为1∶4),人工瘤胃缓冲液参照Menke等[18]的方法进行配制。将配好的缓冲液倒入其中,倒入发酵瓶中,每个发酵瓶60 mL,共计33个发酵瓶(每组6个重复,3个空白),期间不断通入二氧化碳(CO2),保证厌氧条件,然后用橡胶塞和铝制盖密封发酵瓶,置于39 ℃恒温气浴摇床中,转速保持为125 r/min,用采血针将气袋和发酵瓶连接好,开始发酵。分别于发酵过程中0、2、4、6、8、10、12、24、36、48和72 h测量发酵瓶中的产气值,并用空白值进行校对。72 h后终止发酵,采集固体和液体样品进行测定。
用pH计[Starter-300,奥豪斯仪器(上海)有限公司]测定每个发酵瓶中发酵液的pH。然后将发酵瓶充分摇匀,取2 mL发酵液,12 000 r/min、4 ℃下离心10 min,取1.35 mL上清液,加入0.15 mL 25%偏磷酸固定,静置15 min后,-20 ℃保存。样品在常温条件下解冻,然后12 000 r/min、4 ℃条件下离心10 min,取0.6 mL上清液装于测定瓶中,在气相色谱仪(Agilent-7890A,美国)测定挥发性脂肪酸(volatile fatty acids,VFA)组分[19]。同样取偏磷酸处理后的样品参照Weatherburn[20]的方法测定NH3-N含量。最后,剩余发酵液用0.441 mm的尼龙纱布过滤,置于105 ℃烘箱中烘干至恒重。根据底物重量和烘干之后的发酵底物重量,结合取样过程中发酵液体积的变化,参考Zhang等[21]描述的方法计算DM消失率。
应用非线性软件程序NLREG Version 5.4,按照Wang等[22]的模型对体外模拟瘤胃发酵产气曲线进行拟合,并计算起始底物降解速率(FRD0,/h)。模型及相关参数的计算公式如下:
V= V f [ 1 - e x p ( - k t ) ] 1 + e x p ( b - k t )
FDR0= k 1 + e x p ( b )。
式中:V为t时间点底物累积产气量(mL/g);FDR0为t=0时的底物降解速率;Vf为理论最大产气量(mL/g);k为产气速率(/h);b为曲线形状指标。
根据Wang等[22]描述的公式,计算VFA产氢量(net hydrogen produce per VFA,RNH2,‰),计算公式如下:
RNH2=[2(Cac+Cbu+Cibu)-(Cpr+Cva+Civa)]/CVFA。
式中:Cac、Cbu、Cibu、Cpr、Cva、Civa及CVFA分别为乙酸、丁酸、异丁酸、丙酸、戊酸、异戊酸及VFA含量(mmol/L)。

1.3.4 体内降解试验

选择3头体重相近、健康状况良好及安装永久性瘤胃瘘管的呼伦贝尔羊,按照中国科学院亚热带农业生态研究所试验羊场日常饲养管理方式饲养。称取3 g(精确至0.000 1 g)样品放入已知袋重的尼龙袋(8 cm×10 cm,300目,48 μm孔径)中,尼龙袋用绳子绑紧并绑在扎带上固定。在晨饲前将尼龙袋投入羊瘤胃中,每头瘘管羊每个时间点设有2个平行、3个重复(3头羊总计每个样品每个时间点6个尼龙袋),共设5个时间点,分别在投入8、16、24、48和72 h时取出,立即用清水洗去尼龙袋上的残渣,然后清洗尼龙袋至水澄清为止。将尼龙袋放入65 ℃烘箱48 h至恒重,称取尼龙袋及残渣的重量,并将尼龙袋中瘤胃未降解的残渣收集于自封袋,取样品残渣测定DM含量,计算DM降解率(dry matter degradation rate,DMD)。
A=[(B-C/B)×100]。
式中:A为待测饲料瘤胃DMD(%);B为待测饲料DM质量(g);C为残留物中DM质量(g)。

1.4 数据处理与分析

采用Excel 2019对数据进行整理,采用SPSS 26.0软件进行单因素方差分析(one-way ANOVA)和Duncan氏法多重比较检验,试验结果以“平均值±标准误(mean±SE)”表示,P<0.05表示差异显著。

2 结果与分析

2.1 复合菌制剂对芦苇青贮饲料常规营养成分的影响

由表2可知,添加不同复合菌制剂对芦苇青贮饲料的WSC、NDF、ADF和EE含量无显著影响(P>0.05)。LA组、LA+BS组、LA+AN组和LA+BS+AN组DM含量显著低于CK组(P<0.05),而CP含量(LA组除外)含量显著高于CK组(P<0.05);LA组、LA+BS组和LA+AN组Ash含量显著低于CK组和LA+BS+AN组(P<0.05)。
表2 复合菌制剂对芦苇青贮饲料常规营养成分的影响(干物质基础)

Table 2 Effects of compound bacteria preparation on conventional nutrient composition of Phragmites australis silage (DM basis) g/kg

项目
Items		 组别Groups P值
P-value
CK LA LA+BS LA+AN LA+BS+AN
干物质DM 273.58±0.09a 258.16±0.44c 264.34±0.12b 259.42±0.59c 266.53±1.47b <0.001
粗蛋白质CP 54.15±4.17b 51.86±0.37b 67.57±1.18a 75.62±2.79a 68.02±3.84a 0.009
可溶性碳水化合物WSC 6.25±0.27 6.91±0.13 7.29±0.53 7.89±0.29 6.45±0.55 0.138
中性洗涤纤维NDF 737.03±6.77 724.21±26.38 678.04±11.93 657.08±15.79 712.00±14.26 0.076
酸性洗涤纤维ADF 259.96±7.18 240.08±13.98 225.06±12.20 245.34±13.32 263.99±11.69 0.277
粗脂肪EE 70.46±4.73 47.56±3.27 46.25±3.47 46.18±2.00 43.97±8.61 0.055
粗灰分Ash 109.74±0.16a 99.26±1.34b 89.64±0.63c 100.28±0.03b 107.68±1.72a <0.001

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

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

2.2 复合菌制剂对芦苇青贮饲料发酵品质的影响

由表3可知,LA+BS组pH及NH3-N、乙酸、丙酸和丁酸含量均为最低,显著低于其他各组(LA+AN组NH3-N含量除外)(P<0.05),乳酸/乙酸值显著高于其他各组(P<0.05);LA+AN组NH3-N含量显著低于CK组(P<0.05),乳酸含量显著高于除LA组外的其他各组(P<0.05),而乙酸含量显著低于CK组、LA组和LA+BS+AN组(P<0.05);LA+BS+AN组乳酸和乙酸含量显著低于CK组(P<0.05),pH、丙酸和丁酸含量显著高于CK组(P<0.05)。
表3 复合菌制剂对芦苇青贮饲料发酵品质的影响

Table 3 Effects of compound bacteria preparation on fermentation quality of Phragmites australis silage

项目
Items		 组别Groups P值
P-value
CK LA LA+BS LA+AN LA+BS+AN
pH 3.99±0.01d 4.02±0.01c 3.94±0.00e 4.12±0.00a 4.04±0.01b <0.001
氨态氮NH3-N/(g/kg) 0.89±0.02a 0.84±0.05ab 0.60±0.06c 0.70±0.06bc 0.80±0.02ab 0.037
乳酸LA/(g/kg) 17.58±0.25bc 18.57±0.29ab 16.81±0.19cd 18.75±0.48a 16.37±0.10d 0.008
乙酸AA/(g/kg) 33.48±0.07a 34.07±0.99a 20.12±0.06d 29.57±0.22c 31.36±0.19b <0.001
丙酸PA/(g/kg) 3.23±0.01c 4.29±0.17a 1.72±0.01d 3.12±0.04c 3.96±0.02b <0.001
丁酸BA/(g/kg) 10.93±0.05b 12.05±0.34a 6.54±0.02c 11.83±0.05a 12.05±0.06a <0.001
乳酸/乙酸LA/AA 5.25±0.06c 5.45±0.07c 8.35±0.07a 6.34±0.12b 5.22±0.06c <0.001
由表4和表5可知,各组青贮饲料在色泽、气味、结构和水分得分上均无显著差异(P>0.05),且均无霉变现象,分级均到良好级别。LA+BS组NH3-N/TN、乙酸/总酸和丁酸/总酸值均显著低于CK组和LA组(P<0.05),乳酸/总酸值显著高于其他各组(P<0.05);LA+AN组NH3-N/TN和乙酸/总酸值均显著低于CK组和LA组(P<0.05),而乳酸/总酸值显著高于CK组和LA组(P<0.05)。CK组、LA组和LA+BS+AN组青贮饲料达到可等级,其中CK组的综合得分最低,LA+BS组和LA+AN组评级上均达到良等级,其中综合得分最高为LA+BS组,达70.5分。
表4 复合菌制剂对芦苇青贮饲料感官评定得分的影响

Table 4 Effects of compound bacteria preparation on sensory evaluation score of Phragmites australis silage

项目
Items		 组别Groups P值
P-value
CK LA LA+BS LA+AN LA+BS+AN
色泽得分Color score 16.17±1.17 17.25±0.75 15.33±1.20 16.00±0.50 16.50±1.50 0.851
气味得分Odor score 11.67±0.73 9.25±1.75 10.33±0.73 12.17±1.20 9.83±1.83 0.515
结构得分Structure score 9.67±0.17 9.75±0.25 9.50±0.00 9.50±0.00 9.50±0.00 0.431
水分得分Moisture score 17.00±0.29 17.25±0.25 16.17±0.44 17.17±0.44 17.00±0.29 0.313
pH得分pH score 14.50±0.00b 13.50±0.00c 16.00±0.00a 9.00±0.00e 12.00±0.00d <0.001
总得分Total score 69.00±1.76 67.00±1.00 67.33±1.17 63.83±2.03 64.83±0.60 0.170
青贮饲料分级Silage grading 良好 良好 良好 良好 良好
表5 复合菌制剂对芦苇青贮饲料发酵品质综合得分的影响

Table 5 Effects of compound bacteria preparation on fermentation quality comprehensive score of Phragmites australis silage

项目
Items		 组别Groups P值
P-value
CK LA LA+BS LA+AN LA+BS+AN
氨态氮/总氮 含量Content/% 10.35±1.02a 10.10±0.68ab 5.52±0.69c 5.84±0.72c 7.42±0.63bc
0.016
NH3-N/TN 得分Score 37 40 48 48 44
乳酸/总酸 含量Content/% 28.35±0.23cd 28.71±0.26c 38.67±0.19a 31.17±0.41b 27.38±0.23d
<0.001
LA/TA 得分Score 5 5 10 6 4
乙酸/总酸 含量Content/% 54.01±0.21a 52.66±0.21b 46.30±0.14d 49.17±0.25c 52.46±0.19b
<0.001
AA/TA 得分Score 8 8 11 10 8
丁酸/总酸 含量Content/% 17.64±0.02d 18.63±0.05c 15.03±0.06e 19.67±0.16b 20.16±0.04a
<0.001
BA/TA 得分Score 22 20 24 20 18
综合得分Total score 54.5 56.5 70.5 66.0 59.0
等级Rank 可 可 良 良 可

2.3 复合菌制剂对芦苇青贮饲料体外发酵特性的影响

由图1可知,在芦苇青贮的过程中添加复合菌制剂对青贮后底物体外发酵总产气量有明显影响,发酵0~6 h时,LA+BS组的总产气量高于其他各组,随后的8~72 h LA+AN组总产气量均高于其他各组。由表6可知,LA+AN组DM消失率、72 h总产气量和潜在最大产气量均显著高于其他4组(P<0.05);LA+BS组总产气速率显著低于CK组、LA+AN组和LA+BS+AN组(P<0.05),但FRD0显著高于CK组和LA+AN组(P<0.05)。由表7可知,LA+BS组和LA+AN组体外发酵pH显著高于其他3组(P<0.05),LA组pH显著低于其他4组(P<0.05);与CK组和LA组相比,LA+BS组和LA+AN组NH3-N和总挥发性脂肪酸含量、乙酸/丙酸值和RNH2显著降低(P<0.05);与CK组相比,其他各组丙酸含量显著提高(P<0.05),丁酸、异丁酸、戊酸和异戊酸含量显著降低(P<0.05),其中LA+AN组降低效果最显著,显著低于其他4组(P<0.05);各组间乙酸含量无显著差异(P>0.05)。
图1 复合菌制剂对芦苇青贮饲料体外发酵总气体生成曲线的影响

Fig.1 Effects of compound bacteria preparation on total gas production curve of Phragmites australis silage in in vitro fermentation

表6 复合菌制剂对芦苇青贮饲料体外发酵底物降解和产气参数的影响

Table 6 Effects of compound bacteria preparation on substrate degradation and gas production parameters of Phragmites australis silage in in vitro fermentation

项目
Items		 组别Groups P值
P-value
CK LA LA+BS LA+AN LA+BS+AN
DM消失率
DM disappearance rate/(g/kg)		 412.31±15.72b 405.81±1.58b 389.64±6.57b 453.89±5.11a 404.61±5.60b <0.001
72 h总产气量
Total gas production in
72 h/(mL/g)		 89.08±2.51b 92.50±2.97b 94.38±1.52b 115.21±2.43a 90.16±2.10b <0.001
潜在最大产气量
Potential maximum
gas production/(mL/g)		 91.24±3.07c 96.41±3.42bc 100.20±1.97b 117.36±2.79a 92.13±2.35bc <0.001
总产气速率
Total gas production rate/(/h)		 0.08±0.00a 0.07±0.00bc 0.06±0.00c 0.08±0.00ab 0.07±0.00ab 0.001
起始底物降解速率
FRD0/[mmol/(g·h)]		 0.01±0.00b 0.02±0.00ab 0.02±0.00a 0.02±0.00b 0.02±0.00ab 0.008
表7 复合菌制剂对芦苇青贮饲料体外发酵参数的影响

Table 7 Effects of compound bacteria preparation on in vitro fermentation parameters of Phragmites australis silage

项目
Items		 组别Groups P值
P-value
CK LA LA+BS LA+AN LA+BS+AN
pH 6.39±0.01b 6.30±0.01c 6.46±0.01a 6.45±0.01a 6.38±0.01b <0.001
氨态氮NH3-N/(mmol/L) 16.33±0.06a 16.48±0.20a 15.31±0.34bc 14.79±0.44c 15.77±0.03ab 0.002
总挥发性脂肪酸
TVFA/(mmol/L)		 70.26±0.51b 79.35±0.96a 60.01±1.50d 63.40±0.29c 68.78±0.88b <0.001
乙酸/丙酸Acetate/propionate 2.39±0.01a 2.35±0.00b 2.25±0.01d 2.23±0.01d 2.32±0.01c <0.001
VFA产氢量RNH2/‰ 1 133.16±2.09a 1 124.10±0.50ab 1 107.27±5.77c 1 098.60±1.69c 1 118.03±2.25b <0.001
挥发性脂肪酸VFA/‰
乙酸Acetate 615.74±0.87 618.48±0.26 615.43±2.27 615.58±0.22 618.22±1.01 0.297
丙酸Propionate 257.59±0.68e 262.57±0.29d 272.99±1.04b 276.70±0.69a 266.82±0.77c <0.001
丁酸Butyrate 9.99±0.08a 9.49±0.03b 8.12±0.21c 7.61±0.06d 9.24±0.10b <0.001
异丁酸Iso-butyrate 85.32±0.29a 80.33±0.34b 78.85±0.65c 76.20±0.36d 78.13±0.17c <0.001
戊酸Valerate 17.23±0.06a 15.86±0.03b 13.77±0.29d 12.75±0.13e 14.68±0.24c <0.001
异戊酸Iso-valerate 14.13±0.04a 13.61±0.15b 12.60±0.10c 10.82±0.06d 12.49±0.04c <0.001

2.4 复合菌制剂对芦苇青贮饲料体内降解特性的影响

由表8可知,LA+AN组和LA+BS+AN组8、24和48 h的DMD显著高于其他3组(P<0.05),且16 h的DMD显著高于LA+BS组(P<0.05);各组间72 h的DMD无显著差异(P>0.05)。
表8 复合菌制剂对芦苇青贮饲料DMD的影响

Table 8 Effects of compound bacteria preparation on DMD of Phragmites australis silage g/kg

降解时间
Degradation time/h		 组别Groups P值
P-value
CK LA LA+BS LA+AN LA+BS+AN
8 354.36±5.71c 353.51±3.85c 319.29±22.12c 374.76±16.90b 384.51±16.49a <0.001
16 368.12±2.67ab 364.65±6.35ab 363.79±7.69b 424.57±7.69a 461.42±9.45a 0.044
24 371.28±7.83d 368.02±14.07d 409.27±15.70c 491.30±13.24b 542.09±3.58a <0.001
48 420.17±13.33b 412.67±12.32b 417.59±11.26b 528.67±15.04a 546.67±14.20a <0.001
72 451.99±19.6 453.05±32.68 455.5±25.65 547.12±21.08 532.14±27.57 0.060

3 讨论

3.1 复合菌制剂对芦苇青贮饲料营养成分的影响

本研究发现,添加乳酸菌能够显著降低芦苇青贮饲料中的DM含量,而与黑曲霉菌和枯草芽孢杆菌联用后能够提高其DM含量。CP含量也出现了同样的现象,与CK组相比,添加乳酸菌具有降低CP含量的趋势(无显著差异),添加枯草芽孢杆菌和黑曲霉菌后显著提高了CP含量。与本试验结果类似,闫琦等[23]研究发现,添加乳酸菌可以显著降低菊芋茎叶青贮饲料CP含量。任付平[24]研究发现,在青贮玉米青贮过程中利用黑曲霉、绿色木霉和枯草芽孢杆菌等处理后青贮饲料DM和CP含量都有所增加。这可能是由于植物本身的有害微生物以及各种酶对饲料大分子养分的降解,从而导致养分和蛋白质的损耗,而补充枯草芽孢杆菌和黑曲霉菌能帮助乳酸菌更快地营造酸性厌氧环境,抑制有害微生物的繁殖,并降低青贮发酵早期植物自身呼吸作用对CP的消耗,从而降低蛋白质等大分子的损耗[14-15],且较高的DM含量也可以抑制丁酸菌和大肠杆菌等有害菌的生长繁殖[25]。饲料纤维含量与反刍动物采食量和营养物质消化率呈负相关,NDF和ADF含量越低,饲料营养价值越高[26]。本试验也发现,添加黑曲霉菌有降低NDF含量的趋势,这与黑曲霉菌分泌的多种酶如纤维素降解酶存在密切相关,许多研究已经证明了黑曲霉菌在纤维降解方面的作用[27⇓-29]。而唐振华等[30]研究发现,无论是黑曲霉菌还是黑曲霉菌组合其他菌种在饲料青贮发酵中并没有显著降低NDF含量,这可能是添加量不足导致其产生的高活性纤维素酶、木聚糖酶等活性不足导致。

3.2 复合菌制剂对芦苇青贮饲料发酵品质的影响

感官评定能够在一定程度上反映发酵过程中的环境控制和青贮饲料的发酵品质,本试验在芦苇青贮过程中分别添加了乳酸菌(植物乳杆菌、戊糖片球菌和布氏乳杆菌)、枯草芽孢杆菌和黑曲霉菌之后,各组的感官评定得分均无显著差异。谢婷霞等[31]在皇竹草青贮过程中添加乳酸杆菌发现对其色泽和pH评分有明显改善作用。吴进东[32]也发现了类似结果,但其研究表示不同种类的添加剂对青贮饲料的感官评定指标影响并不大。本试验中,各组青贮饲料感官评定得分均在60~70分,评级均为良好,表示本试验发酵过程中无氧环境的控制等较好,且各菌制剂添加对青贮饲料的感官评定无有害影响。NH3-N含量、有机酸含量和pH是反映发酵品质的重要指标之一[33],其中NH3-N含量可以反映原料中CP的降解程度,NH3-N/TN值与青贮饲料的蛋白质降解程度呈正相关[34]。本试验中,LA+BS组和LA+AN组NH3-N含量显著低于CK组,且最低的为LA+BS组,说明枯草芽孢杆菌或黑曲霉菌单独与乳酸菌联用时能够更有效地保存芦苇中的蛋白质,且枯草芽孢杆菌在这方面具备更好的效果。枯草芽孢杆菌是一种兼性厌氧菌,已有研究表明,在青贮过程中添加枯草芽孢杆菌可以改善饲料的发酵品质,并提高其发酵后小分子蛋白质和游离氨基酸的含量等[24,35]。与本试验类似,多项研究结果表明,枯草芽孢杆菌[36]或者黑曲霉菌[37-38]的添加可以显著降低青贮的NH3-N/TN值。
pH是进行青贮饲料品质评价的简单有效的指标,较低的pH可以抑制大部分有害菌的繁殖,提高发酵品质,延长青贮饲料的保质期[39]。本研究中各组pH在3.8~4.2,青贮品质良好,且枯草芽孢杆菌的添加可以显著降低发酵过程中的pH。有机酸含量中,乳酸占比越高,丙酸和丁酸占比越低,青贮发酵品质越好[33]。乳酸的存在可以抑制饲料中好氧微生物的繁殖,防止饲料在发酵过程中腐败变质[24]。本研究中,LA组和LA+AN组乳酸含量相较于CK组有所提高,且LA+AN组乳酸含量较LA组高,而LA+BS组和LA+BS+AN组乳酸含量较LA组出现显著降低。与CK组和LA组相比,LA+BS组、LA+AN组和LA+BS+AN组乙酸含量显著降低。LA+BS组和LA+AN组乳酸/乙酸值显著高于CK组、LA组和LA+BS+AN组。这说明乳酸菌的添加可以提高饲料中乳酸含量,且与黑曲霉菌联用后可以促进这种提高效应,而与枯草芽孢杆菌的加入会破坏这种提高效应,同时枯草芽孢杆菌和黑曲霉菌与乳酸菌的联用会降低发酵过程中乙酸的产生。LA组、LA+AN组和LA+BS+AN组丁酸含量显著高于CK组和LA+BS组,LA+BS组丁酸含量显著低于其他各组,说明枯草芽孢杆菌和乳酸菌的联用能够抑制发酵过程中丁酸和丙酸的产生,而黑曲霉菌加入会抑制这种作用。
综上所述,枯草芽孢杆菌和黑曲霉菌单独与乳酸菌联用作为添加剂均能在一定程度上改善青贮饲料的发酵品质,且是通过不同的原理改善发酵品质,黑曲霉菌在降低发酵过程中的NH3-N含量、提高乳酸含量方面更具优势,而枯草芽孢杆菌在降低发酵过程中的pH及NH3-N、乙酸和丁酸含量方面更具优势。黑曲霉菌为好氧微生物,一般在发酵初期迅速繁殖并消耗大量氧气,加速无氧环境的形成,使发酵环境向乳酸菌等厌氧菌的适宜环境转变。研究表明,其在发酵过程中能产生纤维素酶、木聚糖酶、果胶酶和木质素降解酶等多种酶,因此能快速降解饲料中的纤维素,为乳酸菌繁殖提供所需营养的营养物质,从而提高乳酸的含量使pH降低[40]。枯草芽孢杆菌也是一种有益的微生物菌制剂,能够有效改善青贮发酵品质。与本研究相似,穆胜龙等[41]研究发现枯草芽孢杆菌和布氏乳杆菌联用可以显著降低甘蔗尾青贮饲料中的NH3-N含量,提高有氧稳定性,从而改善青贮品质。

3.3 复合菌制剂对芦苇青贮饲料瘤胃发酵特性的影响

在体外发酵试验中,黑曲霉菌与乳酸菌的组合能够显著提高DM消失率、72 h总产气量和潜在最大产气量,而其余几种组合并没有太好的提升效果,这说明黑曲霉菌与乳酸菌的组合能够提高瘤胃对营养物质的利用率。瘤胃pH能够影响其微生物菌群,从而影响瘤胃发酵效果,本试验中LA+AN组和LA+BS组出现发酵液pH升高的现象(但均在正常瘤胃液变化范围之内),可能与发酵过程中的VFA产生有关,其发酵液中具有更低的总挥发性脂肪酸含量和RNH2,导致发酵液pH偏高。作为碳水化合物在瘤胃中的发酵产物,瘤胃VFA是反刍动物重要的能量来源,本试验中,LA+AN组丙酸含量高于其他组,乙酸/丙酸值及丁酸、异丁酸、戊酸和异戊酸含量低于其他组。在尼龙袋试验中发现,LA+AN组和LA+BS+AN组8、24和48 h的DMD均有一定程度的提高,表明黑曲霉菌与乳酸菌的组合或3种菌的组合添加能够提高青贮芦苇在瘤胃中的利用率。本试验结果表明,黑曲霉素与乳酸菌联用能够在一定程度上提高营养物质在瘤胃中的消化利用率,改善瘤胃的发酵环境,从而提高发酵效果。

4 结论

乳酸菌与枯草芽孢杆菌或者与黑曲霉菌联用对芦苇青贮饲料的营养成分、青贮发酵品质、瘤胃体外发酵特性和体内消化均有一定的改善作用。综合考虑,在本试验添加量下,乳酸菌和黑曲霉菌联用对芦苇青贮品质、瘤胃体外发酵和体内消化的改善效果最好。

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