Effects of Extrusion Pretreatment on Fermentation Quality and Nutritional Value of Corn Stalk Microbial Ensilage by Bacteria-Enzyme Cooperative Fermentation

WANG Hongmei; MU Yuhui; SONG Yuejun; ZHANG Haiou; SI Bingwen

doi:10.12418/CJAN2025.229

2025 , Vol. 37 >Issue 4: 2739 - 2749

DOI: https://doi.org/10.12418/CJAN2025.229

RESEARCH PAPER

Effects of Extrusion Pretreatment on Fermentation Quality and Nutritional Value of Corn Stalk Microbial Ensilage by Bacteria-Enzyme Cooperative Fermentation

WANG Hongmei ^,¹ ,
MU Yuhui ² ,
SONG Yuejun ³ ,
ZHANG Haiou ¹ ,
SI Bingwen ^,¹^,^*

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¹ Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
² China Certification & Inspection Group Industrial Co., Ltd., Beijing 100028, China
³ Liaoyuan City Muxing Machinery Co., Ltd., Liaoyuan 136299, China

^* associate professor, E-mail: sibingwen@caas.cn

Received date: 2024-09-04

Online published: 2025-04-15

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Abstract

The purpose of this experiment was to study the effects of extrusion pretreatment on the degradation of lignocellulosic structure of corn stalk and the effects of bacteria-enzyme cooperative fermentation on sensory evaluation, fermentation quality and nutritional value of expanded corn stalk microbial ensilage. Four groups were set up according to the difference of outlet clearance of extruder with 5 replicates in each group, in which the control group was unexpanded corn stalk, and the outlet clearance of the three extrusion groups was 1.5 (E1.5 group), 2.5 (E2.5 group) and 4.0 mm (E4.0 group), respectively. According to the results of the extrusion pretreatment test, the optimized parameters of stalk extrusion pretreatment were determined, and the microbial ensilage test was further carried out. The microbial ensilage test was set up in 4 groups, which were control group (extruded corn stalk without any additives, CK group), extruded corn stalk+4 mg/kg Lactobacillus plantarum group (LP group), extruded corn stalk+1.0 g/kg cellulase group (EC group) and extruded corn stalk+4 mg/kg Lactobacillus plantarum+1.0 g/kg cellulase group (LP+EC group), with 5 replicates per group and 1 bag per replicate, respectively, and sealed for fermentation at 30 ℃ for 45 days. The results showed as follows: 1) compared with the control group, the contents of crude protein (CP), ether extract (EE) and water soluble carbohydrate (WSC) of corn stalk in extrusion groups were significantly increased (P<0.05), while the contents of neutral detergent fiber (NDF), acid detergent fiber (ADF), hemicellulose and cellulose were significantly decreased (P<0.05). Among them, E4.0 group had the best extrusion pretreatment effect and was used for subsequent microbial ensilage test. 2) Scanning electron microscopy showed that the cell wall structure of corn stalk was destroyed and degraded after expansion pretreatment, and the total bacterial count on stalk surface was significantly decreased (P<0.05). 3) Compared with CK group, the pH in extrusion pretreatment corn stalk microbial ensilage in experimental groups was reduced with sour flavor and better sensory quality, the comprehensive evaluation was excellent, and the total sensory evaluation scores were in the order of LP+EC group (87 points)>LP group (86 points)>EC group (82 points) from high to low. 4) Compared with CK group, the pH in microbial ensilage in experimental groups was significantly decreased (P<0.05); the acetic acid content and ammonia nitrogen (NH₃-N) to total nitrogen (TN) ratio in LP and LP+EC groups were significantly decreased (P<0.05), and the lactic acid content and lactic acid to acetic acid ratio were significantly increased (P<0.05). 5) Compared with CK group, the contents of NDF and ADF in microbial ensilage in experimental groups were significantly decreased (P<0.05), and the ferulic acid content was significantly increased (P<0.05); the contents of CP and EE in EC group and LP+EC group were significantly increased (P<0.05). Meanwhile, the contents of CP, EE and ferulic acid in LP+EC group were significantly higher than those in LP group (P<0.05), while the contents of NDF and ADF were significantly lower than those in LP group (P<0.05). In summary, when the outlet clearance of extruder is 4.0 mm, the extrusion pretreatment can degrade the lignocellulosic structure of corn stalk, release more WSC, and thus improve its nutritional value. When the expansion pretreatment is combined with microbial ensilage, the bacteria-enzyme cooperative fermentation has the best fermentation effect, which can effectively reduce the fiber content of corn stalk and release more active substance as ferulic acid, so as to improve the fermentation quality of corn stalk microbial ensilage.

Key words： corn stalk; extrusion; microbial ensilage; bacteria-enzyme cooperative; ferulic acid

Cite this article

WANG Hongmei , MU Yuhui , SONG Yuejun , ZHANG Haiou , SI Bingwen . Effects of Extrusion Pretreatment on Fermentation Quality and Nutritional Value of Corn Stalk Microbial Ensilage by Bacteria-Enzyme Cooperative Fermentation[J]. Chinese Journal of Animal Nutrition, 2025 , 37(4) : 2739 -2749 . DOI: 10.12418/CJAN2025.229

随着现代畜牧业的快速发展,饲料资源的开发与利用已成为制约行业可持续发展的关键因素。农作物秸秆作为一种非常规饲料资源,因其来源广泛、价格低廉而备受关注。我国农作物秸秆资源丰富,其中玉米秸秆占30%最为丰富,年产量为3.4亿t^[1]。然而,玉米秸秆因木质纤维素含量高、木质化程度高,导致适口性差且利用效率普遍较低,从而在畜牧业中的广泛应用受到限制^[2]。因此,玉米秸秆使用的第一步是破坏其顽固性木质纤维结构,然后找出一种经济的储存方法来支持稳定的全年供应。挤压膨化技术是一种高效分解木质纤维结构和提高生物质利用率的方法,是生物燃料和制浆生产中最常用的生物质加工方法之一^[3]。随着对挤压膨化机理的深入了解,膨化物料的后续储存越来越受到人们的关注,其中青贮被认为是保存新鲜饲料最为经济的方法之一。通过挤压膨化技术,秸秆的细胞结构得到有效破坏,木质纤维素的形态结构和分子间作用力被大幅削弱^[4],导致木质素、纤维素等成分得以降解,转化为易于消化吸收的糖类^[5];这一过程还显著增加其表面积,为微生物和纤维素酶提供更多的作用位点^[6],从而使得后续的发酵过程变得更加高效。

近年来,菌酶协同发酵技术以其独特的优势在饲料加工领域得到了广泛应用,该技术通过结合微生物发酵和酶解处理,不仅能够提高发酵效率,促进大分子物质的降解,还能够有效降解原料中的抗营养因子,改善饲料的适口性和营养价值,从而提高饲料的消化率和利用率。除了应用于蛋白质饲料^[7]和能量饲料^[8]外,菌酶协同发酵技术还应用于粗饲料基质,包括全株玉米青贮^[9]、玉米皮^[10]、玉米芯^[11]、玉米秸秆^[12]、花棒^[13]、棉花秸^[14]和苜蓿^[15]等。菌酶协同发酵所用的菌种以枯草芽孢杆菌、地衣芽孢杆菌、植物乳杆菌、戊糖片球菌和酵母菌为主,用以破坏木质纤维结构并释放胞内营养物质;所用的酶以纤维素酶、半纤维素酶和果胶酶等非淀粉多糖(non-starch polysaccharide,NSP)酶为主,用以将大分子碳水化合物分解为单糖或低聚糖,为动物和协同的微生物提供能源。不过,前人的研究很少关注菌酶作用下底物所产生的活性物质,特别是阿魏酸等功能物质。阿魏酸是一种天然活性物质,属于多酚类酚酸,常见于阿魏、当归及川芎等中药材中,也广泛分布于多种植物的细胞壁^[16]。阿魏酸具备多种生物学功能,如抗氧化、抗炎和抑菌,并能调节糖和脂代谢以及肠道菌群^[17⇓-19]。因此,本研究旨在通过膨化预处理,结合产阿魏酸酯酶植物乳杆菌与纤维素复合酶的协同发酵技术,探究其对玉米秸秆微贮饲料发酵品质、营养成分、抗营养因子降解以及活性物质释放等方面的影响,以期为玉米秸秆饲料的高效利用提供理论依据和技术支持。

1 材料与方法

1.1 试验材料

本试验所用干玉米秸秆及膨化机由吉林省辽源市牧兴机械有限公司提供。玉米秸秆产自吉林省长春市农安县,秸秆原料含水率为10.23%,粉碎长度为2~5 cm。产阿魏酸酯酶植物乳杆菌为中国农业科学院饲料研究所自主分离菌株,经发酵、冷冻干燥制得高活菌粉,活菌数为5.0×10⁹ CFU/g。纤维素复合酶(主要活性成分为纤维素酶和β-葡萄糖苷酶等)为市售产品,酶活性为5×10⁴ U/mL。

1.2 试验设计

1.2.1 膨化预处理试验

根据膨化机出料口间隙的不同,设置4个组,其中对照组为未膨化玉米秸秆,3个膨化组出料口间隙分别为1.5(E1.5组)、2.5(E2.5组)和4.0 mm(E4.0组),每组5个重复。根据膨化预处理试验结果,最终确定秸秆膨化预处理优化参数,并备用于微贮。

1.2.2 微贮试验

微贮试验采用完全随机设计,设置4个组,分别为对照组(膨化玉米秸秆无任何添加剂,CK组)、膨化玉米秸秆+植物乳杆菌组(LP组)、膨化玉米秸秆+纤维素酶组(EC组)以及膨化玉米秸秆+植物乳杆菌+纤维素酶组(LP+EC组),每组5重复,每个重复1袋,共20袋,30 ℃条件下密封发酵45 d。

1.3 试验方法

1.3.1 膨化预处理

将收获籽实后的玉米秸秆铡切、粉碎至2~5 cm,采用9P-150型多功能双螺杆膨化机按照试验设计3个加工参数(出料口间隙为1.5、2.5和4.0 mm)进行膨化预处理备用,在膨化过程中,适量添加水以防止秸秆焦糊。

1.3.2 微贮玉米秸秆制备

根据膨化最优参数预处理干秸秆20 kg,等分为4份,将菌剂(每千克干秸秆添加4.0 mg)和酶制剂(每千克干秸秆添加1.0 g)溶于蒸馏水,均匀喷洒在膨化玉米秸秆表面,边加水边搅拌,水分控制在60%左右。对照组添加等量蒸馏水,不添加任何添加剂,其余方法同上。将样品装入聚乙烯袋(30 cm×40 cm),每袋1.0 kg,用真空包装机(DZ-280/2SD)抽真空并封口,置于室温(25~37 ℃)条件下发酵45 d。原料样品贮存于-20 ℃备用。

1.3.3 指标测定

1.3.3.1 感官评定

玉米秸秆微贮结束后,按照青贮质量感官评分等级方法^[20],从pH、水分、气味、色泽和质地等方面对玉米秸秆微贮饲料进行感官评价。

1.3.3.2 发酵品质测定

称取膨化玉米秸秆微贮饲料样品20 g,加入180 mL蒸馏水,搅拌均匀,使用组织捣碎机(WARNING 8010S,美国)搅碎1 min,先后用4层纱布和定量滤纸过滤,滤得浸出液用pH计(梅特勒S210,瑞士)测定pH^[21]。采用高效液相色谱仪(Agilent 1200LC,美国)分析乳酸(lactic acid,LA)、乙酸(acetic acid,AA)、丙酸(propionic acid,PA)和丁酸(butyric acid,BA)含量。分析条件为:Agilent TC-C18色谱柱(250 mm×4.6 mm,5 μm),紫外检测器波长为210 nm,甲醇为流动相A,0.01 mol/L的磷酸二氢钾(KH₂PO₄)水溶液为流动相B,流速为0.7 mL/min,柱温为50 ℃。采用苯酚-次氯酸钠比色法^[22]测定氨态氮(ammonia nitrogen,NH₃-N)含量,并计算氨态氮/总氮(ammonia nitrogen/total nitrogen,NH₃-N/TN)值。

1.3.3.3 营养成分含量测定

取适量玉米秸秆膨化前后和微贮前后的饲料样品,于65 ℃烘干48 h,室温回潮后测定初水分含量。烘干后的样品粉碎过40目筛,参照GB/T 6435—2014的方法,采用105 ℃恒重干燥法测定水分含量;参照GB/T 6432—2018的方法,使用全自动凯氏定氮仪(Foss 2300)测定粗蛋白质(CP)含量;参照GB/T 6433—2006的方法,采用索氏抽提法测定粗脂肪(EE)含量;参照GB/T 6438—2007的方法测定粗灰分(Ash)含量;采用Van Soest等^[23]的方法测定中性洗涤纤维(NDF)和酸性洗涤纤维(ADF)含量;采用蒽酮硫酸比色法^[24]测定可溶性碳水化合物(water soluble carbohydrate,WSC)含量。采用高效液相色谱法^[25]测定阿魏酸含量,分析条件:XBridge C18色谱柱(250 mm×4.6 mm,5 μm),检测波长为320 nm,流动相A为0.01%冰乙酸水溶液,流动相B为纯乙腈,流速为1.0 mL/min,柱温为30 ℃,按照标准曲线回归方程求得样品中阿魏酸含量。

1.3.3.4 细菌总数的测定

根据GB/T 13093—2023平板计数法测定饲料样品中的细菌总数,无菌称取试样10.0 g,放于含有90 mL无菌生理盐水的无菌均质杯子中,7 104×g均质5 min,制成1∶10的稀释液,并依次稀释得10^-3、10^-5、10^-7和10^-9的稀释液。每个稀释度吸取1 mL试样匀液移入无菌平皿内,及时将15 mL凉至45 ℃的培养基倾注平皿,小心转动平皿使试样与培养基充分混匀。每个稀释度做2个平皿,待琼脂凝固后,倒置平板于37 ℃恒温箱内培养72 h取出,并计数平板内菌落数目,菌落数乘以稀释倍数,即得每克试样所含细菌总数(CFU/g)。

1.3.3.5 秸秆微观结构电镜扫描分析

通过扫描电子显微镜评估膨化预处理前后的玉米秸秆结构,加速电压为3.0 kV,对所有样品表面进行喷金,以提高导电性,放大倍数为400倍和110倍。

1.4 数据统计分析

试验数据经过Excel 2019初步整理后,采用SAS 9.2软件对数据进行单因素方差分析(one-way ANOVA),并采用Duncan氏法进行组间多重比较,结果数据以“平均值±标准差(mean±SD)”形式表示,P<0.05为差异显著。

2 结果与分析

2.1 膨化预处理对玉米秸秆营养成分含量的影响

由表1可知,与对照组相比,膨化组玉米秸秆CP、EE和WSC含量显著提高(P<0.05),NDF、ADF、半纤维素和纤维素含量显著降低(P<0.05),干物质(DM)、Ash和酸性洗涤木质素(ADL)含量无显著差异(P>0.05)。在各膨化组间,E4.0组WSC含量显著高于其他2组(P<0.05),NDF和半纤维素含量显著低于其他2组(P<0.05),且ADF和纤维素含量显著低于E2.5组(P<0.05),该组膨化预处理效果最佳,因此选用出料口间隙为4.0 mm用于后续微贮试验。

表1 膨化预处理对玉米秸秆营养成分含量的影响(干物质基础)

Table 1 Effects of extrusion pretreatment on nutrient contents of corn stalk (DM basis) %

项目 Items	对照组 Control group	膨化组Extrusion groups			P值 P-value
项目 Items	对照组 Control group	E1.5	E2.5	E4.0	P值 P-value
干物质(风干基础) DM (air-dry basis)	70.15±0.24	70.94±1.45	69.90±0.62	70.08±0.50	0.055
粗蛋白质CP	4.43±0.08^c	5.55±0.06^a	5.43±0.05^b	5.40±0.04^b	<0.001
粗脂肪EE	0.47±0.01^b	0.52±0.04^a	0.52±0.02^a	0.56±0.01^a	0.007
粗灰分Ash	7.70±0.29	7.89±0.12	7.89±0.03	7.56±0.54	0.537
可溶性碳水化合物WSC	3.29±0.33^c	5.91±0.23^b	6.11±0.23^b	7.46±0.41^a	<0.001
中性洗涤纤维NDF	74.24±0.51^a	69.65±0.42^b	70.27±0.10^b	66.91±0.73^c	<0.001
酸性洗涤纤维ADF	40.88±0.59^a	37.97±0.64^c	39.30±0.44^b	36.56±0.82^c	<0.001
半纤维素HCL	34.03±1.10^a	31.44±1.03^b	30.10±0.41^b	29.05±0.68^c	0.003
纤维素CL	31.42±0.75^a	28.79±0.48^c	29.89±0.15^b	28.18±0.55^c	<0.001
酸性洗涤木质素ADL	7.53±0.30	6.74±0.23	6.98±0.45	6.27±0.39	0.097

	同行数据肩标无字母或相同字母表示差异不显著(P>0.05),不同字母表示差异显著(P<0.05)。表3和表4同。
	In the same row, values with no letter or the same letter superscripts mean no significant difference (P>0.05), while with different letter superscripts mean significant difference (P<0.05). The same as Table 3 and Table 4.

2.2 膨化预处理对玉米秸秆细菌总数的影响

如图1所示,与对照组相比,膨化预处理后玉米秸秆细菌总数显著降低(P<0.05),3个膨化组细菌总数从膨化预处理前的1.69×10⁷ CFU/g分别降至为3.59×10⁵(E1.5组)、3.29×10⁵(E2.5组)和2.78×10⁵ CFU/g(E4.0膨化组)。同时,3个膨化组间玉米秸秆细菌总数无显著差异(P<0.05)

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图1 膨化预处理对玉米秸秆细菌总数的影响

数据柱标注不同小写字母表示差异显著(P<0.05)。

Fig.1 Effects of extrusion pretreatment on total bacterial count of corn stalk

Value columns with different small letters mean significant difference (P<0.05).

2.3 膨化前后玉米秸秆微观结构的变化

膨化预处理前后玉米秸秆扫描电镜观察微观结构如图2所示,未膨化的玉米秸秆外表面(图2-A)、内表面(图2-C)、叶片表面(图2-E)和玉米芯(图2-G)结构相对完整,表面比较光滑,轮廓清晰,平整;而膨化预处理后的玉米秸秆外表面(图2-B)、内表面(图2-D)、叶片表面(图2-F)和玉米芯(图2-H)均出现了大面积的破碎,并出现了规则或不规则的大小不等的孔状结构,结构有较大的破坏和降解情况。

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图2 膨化前后玉米秸秆微观结构的变化

A和B分别为膨化前后秸秆外表面结构(400×),C和D分别为膨化前后秸秆内表面结构(400×),E和F分别为膨化前后叶片表面结构(400×),G和H分别为膨化前后玉米芯结构(110×)。膨化机出料口间隙优化参数为4.0 mm。

Fig.2 Changes of microstructure of corn stalk before and after expansion

A and B were outer surface structure of stalk before and after expansion (400×), C and D were internal surface structure of stalk before and after expansion (400×), E and F were leaf blade surface structure before and after expansion (400×), and G and H were corn cob structure before and after expansion (110×), respectively. The optimized parameter of outlet clearance of extruder was 4.0 mm.

2.4 菌酶协同发酵对膨化预处理玉米秸秆微贮饲料感官评定的影响

通过上述对比试验,将膨化机出料口间隙参数优化至4.0 mm,并进行微贮试验。不同添加剂处理对玉米秸秆微贮饲料感官评定的影响结果见表2。与CK组相比,试验组膨化预处理玉米秸秆微贮饲料pH均降低,具有酸香味,感官品质较好。CK组微贮饲料感官评定总得分为74分,综合评价为良好;3个试验组感官评定总得分均超过80分,综合评价均为优等,且感官评定总得分从高到低依次为LP+EC组(87分)>LP组(86分)>EC组(82分)。

表2 菌酶协同发酵对膨化预处理玉米秸秆微贮饲料感官评定的影响

Table 2 Effects of bacteria-enzyme cooperative fermentation on sensory evaluation of extruded pre-treated corn stalk microbial ensilage

项目 Items	组别Groups
项目 Items	CK	LP	EC	LP+EC
pH	3.95	3.87	3.90	3.85
pH得分pH score	19	21	20	21
水分Moisture/%	63.79	65.12	62.87	64.70
水分得分Moisture score	20	20	20	20
气味Smell	酒酸味	酸香味	酸香味	酸香味
气味得分Smell score	13	20	19	21
色泽Color	亮黄色	亮黄色	亮黄色	亮黄色
色泽得分Color score	14	16	15	16
质地Texture	松散不黏手	松散不黏手	松散不黏手	松散不黏手
质地得分Texture score	8	9	8	9
总得分Total score	74	86	82	87
综合评价Comprehensive evaluation	良好	优等	优等	优等

2.5 菌酶协同发酵对膨化预处理玉米秸秆微贮饲料发酵品质的影响

由表3可知,各组膨化预处理玉米秸秆发酵后pH均在4.2以下,且各试验组pH均显著低于CK组(P<0.05)。与CK组相比,LP组和LP+EC组乙酸含量和NH₃-N/TN值均显著降低(P<0.05),乳酸含量和乳酸/乙酸值均显著提高(P<0.05)。EC组乳酸含量显著高于CK组(P<0.05),但显著低于LP组和LP+EC组(P<0.05),乙酸含量和NH₃-N/TN值与CK组相比无显著差异(P>0.05)。各组均未检测到丙酸和丁酸。相比之下,LP+EC组发酵效果最佳,表现为pH、乙酸含量和NH₃-N/TN值最低,同时乳酸含量和乳酸/乙酸值最高。

表3 菌酶协同发酵对膨化预处理玉米秸秆微贮饲料发酵品质的影响

Table 3 Effects of bacteria-enzyme cooperative fermentation on fermentation quality of extruded pre-treated corn stalk microbial ensilage

项目 Items	组别Groups				P值 P-value
项目 Items	CK	LP	EC	LP+EC	P值 P-value
pH	3.95±0.02^a	3.87±0.02^bc	3.90±0.01^b	3.85±0.03^c	<0.001
乳酸LA/(g/kg DM)	36.85±0.95^d	50.57±1.70^b	44.10±3.08^c	54.75±1.48^a	<0.001
乙酸AA/(g/kg DM)	8.48±0.21^a	6.85±0.10^b	8.05±0.42^a	6.71±0.53^b	<0.001
乳酸/乙酸LA/AA	4.34±0.01^d	7.39±0.27^b	5.50±0.58^c	8.21±0.84^a	<0.001
氨态氮/总氮NH₃-N/TN/(g/kg DM)	46.21±1.70^a	37.56±1.42^b	44.35±1.59^a	36.39±2.40^b	<0.001

2.6 菌酶协同发酵对膨化预处理玉米秸秆微贮饲料营养成分含量的影响

由表4可知,与CK组相比,膨化预处理玉米秸秆发酵45 d后,试验组微贮饲料NDF和ADF含量显著降低(P<0.05),阿魏酸含量显著提高(P<0.05)。EC组和LP+EC组CP和EE含量显著高于CK组(P<0.05),而LP组CP和EE与CK组相比无显著差异(P>0.05)。LP+EC组CP、EE和阿魏酸含量显著高于LP组(P<0.05),NDF和ADF含量显著低于LP组(P<0.05)。

表4 菌酶协同发酵对膨化预处理玉米秸秆微贮饲料营养成分含量的影响(干物质基础)

Table 4 Effects of bacteria-enzyme cooperative fermentation on nutrient contents of extruded pre-treated corn stalk microbial ensilage (DM basis) %

项目 Items	组别Groups				P值 P-value
项目 Items	CK	LP	EC	LP+EC	P值 P-value
干物质(鲜样基础) DM (fresh sample basis)	39.21±0.68^ab	39.88±0.21^a	38.61±0.53^b	39.30±0.43^ab	0.025
粗蛋白质CP	5.74±0.35^c	5.50±0.08^c	6.63±0.14^a	6.09±0.05^b	<0.001
粗脂肪EE	0.81±0.03^b	0.82±0.03^b	0.96±0.11^a	0.93±0.03^a	0.012
中性洗涤纤维NDF	72.56±0.57^a	70.59±1.31^b	64.19±0.47^c	63.71±0.44^c	<0.001
酸性洗涤纤维ADF	41.63±0.48^a	40.27±0.73^b	36.91±0.76^c	36.24±0.20^c	<0.001
阿魏酸Ferulic acid/(mg/kg)	33.00±2.16^d	104.00±4.55^b	60.50±4.65^c	131.50±11.12^a	<0.001

3 讨论

3.1 膨化预处理对玉米秸秆营养成分含量和微观结构的影响

研究表明,秸秆膨化预处理后会变得柔软蓬松,膨化预处理能有效降解秸秆木质纤维结构,改善其适口性,提高动物消化吸收利用率^[26-27]。本研究结果显示,玉米秸秆原料含有较高的木质纤维素成分,其中NDF含量为74.24%,ADF含量为40.88%,半纤维素含量为34.03%,纤维素含量为31.42%,以及ADL含量为7.53%。经过膨化预处理后,玉米秸秆的木质纤维结构得到有效降解,其中E4.0组效果最佳。与未膨化玉米秸秆相比,E4.0组膨化玉米秸秆NDF和ADF含量分别显著降低了9.87%和10.57%,而WSC含量则显著提高了126.75%。这与Nie等^[28]的研究结果相似,该研究发现发酵前膨化预处理能够显著降低玉米秸秆NDF和ADF含量,显著提高WSC含量。本研究结果表明,与对照组相比,E4.0组玉米秸秆中大部分半纤维素(从34.03%降至29.05%)和纤维素(从31.42%降至28.18%)被降解为WSC(从3.29%提高至7.46%),这与研究报道的蒸汽爆破物料中半纤维素和纤维素含量大量降低^[5]相符。本研究发现,膨化预处理对玉米秸秆ADL的降解作用不显著,但其数值呈现出降低的趋势。据报道,ADL的熔点较低,约为140 ℃,当生物质加热时,它会变得柔软,有时会融化^[29]。膨化挤压过程时间较短,加之ADL结构坚固,使得在挤压过程中,螺杆的快速旋转伴随温度上升,更可能促使ADL软化并破坏其结构,而并非显著提高其脱除量^[30]。ADL与半纤维素以共价键形式结合,构成稳定的网络结构,将纤维素分子包埋在其中,形成一种天然屏障^[27]。膨化预处理后,玉米秸秆中WSC含量的提高可能归因于膨化过程对ADL封闭结构的破坏,使得部分半纤维素和纤维素在酸性和高温条件下水解为寡糖或单糖^[31]。在膨胀过程中氮是不可能改变的,因此CP的绝对含量也不可能改变,但由于高温、热能和水蒸气的作用,膨胀过程中会损失淀粉、糖和维生素等物质,导致氮元素比例提高^[30]。此外,本研究还发现,膨化过程中玉米秸秆的EE含量有所提高,这可能是由于脂肪细胞的破裂以及部分水解酶在膨化过程中失活,导致原本束缚在细胞内的脂肪释放出来,从而使得膨化后的秸秆中EE含量相对提高^[32]。从纤维微观结构来看,膨化预处理后,玉米秸秆的细胞壁结构受到一定程度的断裂破坏,形成了多孔结构,进而使纤维结构变得疏松且柔软。这种转变显著有利于后续阶段酶与微生物的作用过程,使得酶能更轻易地接触到内部的作用位点,同时微生物的接触面积也得到增加^[6]。

3.2 菌酶协同发酵对膨化预处理玉米秸秆微贮饲料感官评定的影响

pH和感官指标是评估玉米秸秆微贮饲料质量的关键参数。通过合理规范的微贮处理,秸秆发酵料pH在3.65~4.00,水分控制在60%左右,呈亮黄色,具有酸香味,质地蓬松不黏手,综合评价其品质可达到优等标准^[33]。本研究结果表明,添加菌剂和酶制剂微贮玉米秸秆,能够降低玉米秸秆微贮饲料pH并有效改善其感官指标,使得综合评价等级从良好提升至优等,这与前人研究结果^[34]一致。由此说明,纤维素酶能够破坏秸秆中的纤维素结构,从而释放更多的可溶性糖分,为乳酸菌提供更丰富的营养底物,促进其生长繁殖成为优势菌群,并抑制有害菌,同时产生挥发性脂肪酸,降低pH,并赋予发酵料酸甜芳香味道,改善其适口性,提高玉米秸秆饲料的利用价值。

3.3 菌酶协同发酵对膨化预处理玉米秸秆微贮饲料发酵品质的影响

研究表明,优质的秸秆微贮富含乳酸,同时展现出较低的pH、丁酸及NH₃-N含量^[33]。而NH₃-N/TN值越高,往往意味着饲料中蛋白质和氨基酸的分解程度越高,表示饲料质量有所下降^[35]。本研究结果表明,膨化预处理玉米秸秆经菌酶协同微贮后,乳酸含量和乳酸/乙酸值提高,乙酸含量、pH和NH₃-N/TN值降低,且未检测出丙酸和丁酸。毛建红^[36]研究发现,添加复合酶和双菌微贮玉米秸秆后,也同样提高了乳酸含量,降低了pH和NH₃-N/TN值,但不同得是提高了乙酸含量和降低了乳酸/乙酸值。该研究中所用菌剂除了有同型发酵菌——植物乳杆菌外,还包含了异型发酵菌——布氏乳杆菌,所以发酵过程中促进了乙酸的形成;而本研究中只添加了植物乳杆菌与纤维素酶联用,说明通过添加酶制剂能够将秸秆中纤维结构性的碳水化合物降解为可溶性糖,以获得更多的发酵底物,促进乳酸菌发酵^[37],且同型发酵乳酸菌占据主导地位,会产生大量的乳酸,进而提高饲料发酵品质^[38]。

3.4 菌酶协同发酵对膨化预处理玉米秸秆微贮饲料营养成分含量的影响

已有研究表明,酶制剂与乳酸菌联用微贮秸秆能够改善其营养价值,尤其是降解木质纤维素含量,进而促进秸秆消化吸收利用^[34-35]。本研究结果表明,菌酶协同微贮能够提高膨化预处理玉米秸秆CP和EE含量,降低NDF和ADF含量,且效果优于单用菌剂或酶制剂。谭树义等^[39]研究表明,添加复合酶制剂和乳酸菌可显著提高玉米秸秆微贮饲料CP含量,而降低NDF和ADF含量,这与本研究结果一致。由此说明,在发酵过程中,纤维素酶与乳酸菌能产生协同作用,纤维素酶能将秸秆原料中的结构性碳水化合物有效分解为可溶性营养物质,为乳酸菌发酵提供碳源,而乳酸菌可将糖分转化为乳酸^[40],从而提高微贮玉米秸秆的营养价值。另外,本研究还发现,所用产阿魏酸酯酶植物乳酸杆菌在与纤维素酶协同作用下,能够显著提高微贮玉米秸秆活性物质——阿魏酸含量,且与单独添加菌株或纤维素酶相比,二者协同处理对玉米秸秆阿魏酸含量的提升幅度更大。Wang等^[41]筛选到一株能够分泌一种新型阿魏酸酯酶的嗜酸乳杆菌(Lactobacillus acidophilus),与木聚糖酶和α-L-阿拉伯糖苷酶协同作用能够促进木质纤维素的降解,进而提高阿魏酸的释放量。Li等^[42]在玉米秸秆发酵中验证了这一结论。这表明在饲料发酵中,乳酸菌所产的阿魏酸酯酶能与纤维素酶协同作用,同时降低植物细胞壁中木质纤维素空间位阻以及水解促进纤维多糖与阿魏酸连接的酯键,进而释放更多的游离阿魏酸单体或阿魏酸二聚体^[43],这对改善秸秆微贮饲料提质增效、降低生产成本具有重要意义。

4 结论

本研究结果表明,膨化机出料口间隙最适宜设定参数为4.0 mm,此时能有效降低玉米秸秆中的NDF、ADF、半纤维素和纤维素含量,并将其转化为易于消化吸收的WSC,从而提升其营养价值。膨化预处理再与微贮结合处理,菌酶协同发酵的效果最佳,能够显著提高阿魏酸和CP含量以及乳酸/乙酸值,同时显著降低NH₃-N/TN值以及NDF和ADF含量,有效改善玉米秸秆微贮饲料的发酵品质。

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