动物营养学报 >

2025 , Vol. 37 >Issue 9: 6339 - 6347

DOI: https://doi.org/10.12418/CJAN2025.514

研究论文

不同青贮添加剂对桑枝叶青贮发酵品质、营养成分和微生物菌落数量的影响

  • 王红梅 , 1 ,
  • 母宇辉 2 ,
  • 何大彦 3 ,
  • 章海欧 1 ,
  • 司丙文 , 1, *
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  • 1 中国农业科学院饲料研究所,北京 100081
  • 2 中检集团实业有限公司,北京 100028
  • 3 湖北曼博瑞生物科技有限公司,荆州 558103
* 司丙文,副研究员,硕士生导师,E-mail:

王红梅(1983—),女,内蒙古通辽人,高级畜牧师,博士,从事动物营养与饲料研究。E-mail:

Office editor: 陈鑫

收稿日期: 2025-01-22

  网络出版日期: 2025-09-12

基金资助

国家重点研发计划(2021YFD1300300)

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Effects of Different Silage Additives on Fermentation Quality, Nutrient Composition and Number of Microbial Colonies of Mulberry Branches and Leaves Silage

  • WANG Hongmei , 1 ,
  • MU Yuhui 2 ,
  • HE Dayan 3 ,
  • ZHANG Haiou 1 ,
  • SI Bingwen , 1, *
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  • 1 Feed Research Institute of Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • 2 China Certification & Inspection Group Industrial Co., Ltd., Beijing 100028, China
  • 3 Hubei Manborui Biotechnology Co., Ltd., Jingzhou 558103, China
* associate professor, E-mail:

Received date: 2025-01-22

  Online published: 2025-09-12

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摘要

本试验旨在探讨不同青贮添加剂[植物乳杆菌(LP)、布氏乳杆菌(LB)、枯草芽孢杆菌(BS)、糖蜜(M)]对桑枝叶青贮的发酵品质、营养成分以及微生物菌落数量的影响。试验设对照(CON)组(无任何添加剂)、LP组(添加2.0 g/t LP)、LB组(添加2.0 g/t LB)、LP+BS组(添加1.0 g/t LP和1.0 g/t BS)和LP+LB+BS+M组(添加0.5 g/t LP、0.5 g/t LB、1.0 g/t BS和0.3% M),每组5个重复。在室温条件下,发酵60 d后开袋取样进行指标分析。结果表明:与CON组相比,添加剂组桑枝叶青贮乳酸菌(LAB)数量和乳酸(LA)含量显著提高(P<0.05),pH、氨态氮(NH3-N)和乙醇含量以及酵母菌数量显著降低(P<0.05),其中LP+LB+BS+M组LAB数量、LA和乙酸(AA)含量以及LA/AA最高,pH、丙酸(PA)、乙醇、NH3-N含量和酵母菌数量最低,且其大肠杆菌和霉菌数量均低于检测限[<2.00 lg(CFU/g)]。各组样品中均未检测到丁酸。各组干物质(DM)和粗脂肪(EE)含量均没有显著变化(P>0.05)。与CON组相比,LP+LB+BS+M组粗蛋白质(CP)含量显著提高(P<0.05);LB组、LP+BS组和LP+LB+BS+M组中性洗涤纤维(NDF)含量显著降低(P<0.05);LP组、LP+BS组和LP+LB+BS+M组酸性洗涤纤维(ADF)含量显著降低(P<0.05);LP+LB+BS+M组水溶性碳水化合物(WSC)含量显著提高(P<0.05),LP+BS组WSC含量显著低于CON组(P<0.05);LP组、LP+BS组和LP+LB+BS+M组总黄酮(TF)含量显著提高(P<0.05)。综上所述,添加LP+LB+BS+M可改善桑枝叶青贮饲料品质,释放更多的类黄酮活性物质。

关键词： 桑枝叶青贮; 青贮添加剂; 发酵品质; 营养成分; 菌落数量

本文引用格式

王红梅 , 母宇辉 , 何大彦 , 章海欧 , 司丙文 . 不同青贮添加剂对桑枝叶青贮发酵品质、营养成分和微生物菌落数量的影响[J]. 动物营养学报, 2025 , 37(9) : 6339 -6347 . DOI: 10.12418/CJAN2025.514

Abstract

This study aimed to investigate the effects of different silage additives [Lactobacillus plantarum (LP), Lactobacillus brucei (LB), Bacillus subtilis (BS), molasses (M)] on fermentation quality, nutrient composition, and number of microbial colonies of mulberry branches and leaves silage. The experiment was divided into control (CON) group (without any additives), LP group (2.0 g/t LP), LB group (2.0 g/t LB), LP+BS group (1.0 g/t LP and 1.0 g/t BS) and LP+LB+BS+M group (0.5 g/t LP and 0.5 g/t BS) LB, 1.0 g/t BS and 0.3% M), five replicates per group. At room temperature, after 60 days of fermentation, samples were opened and taken for index analysis. The results indicated that compared with the CON group, the number of lactic acid bacteria (LAB) and the content of lactic acid (LA) in the additive groups increased significantly (P<0.05), while the pH, ammonia nitrogen (NH3-N) and ethanol contents and the number of yeast decreased significantly (P<0.05). Among them, LP+LB+BS+M group had the highest LAB quantity, LA, acetic acid (AA) content and LA/AA ratio, and the lowest pH, propionic acid (PA), ethanol, NH3-N content and yeast number, and the number of Escherichia coli and mold were below the detection limit [<2.00 lg(CFU/g)]. Butyric acid was not detected in any of the samples. The contents of dry matter (DM) and crude fat (EE) did not change significantly in all groups (P>0.05). Compared with CON group, the content of crude protein (CP) in LP+LB+BS+M group was significantly increased (P<0.05). The content of neutral detergent fiber (NDF) in LB, LP+BS and LP+LB+BS+M groups was significantly decreased (P<0.05). The content of acid detergent fiber (ADF) in LP, LP+BS and LP+LB+BS+M groups was significantly decreased (P<0.05). The content of water-soluble carbohydrate (WSC) in LP+LB+BS+M group was significantly increased (P<0.05), and the content of WSC in LP+BS group was significantly lower than that in CON group (P<0.05). The content of total flavonoids (TF) in LP, LP+BS and LP+LB+BS+M groups was significantly increased (P<0.05). In conclusion, the addition of LP+LB+BS+M can improve the quality of mulberry branches and leaves silage and release more flavonoid active substances.

Key words： mulberry branches and leaves silage; silage additives; fermentation quality; nutrient composition; number of microbial colonies

桑树(Morus alba L.)属于桑科,是多年生木本植物,主要生长于温带、热带和亚热带地区,其叶片是养蚕业的核心原料,占地上部生物量的64%以上。桑树不仅生长快速、适应力强,其叶和枝条含有丰富的营养成分,是理想的饲料资源[1]。在中国,桑树已有5 000多年的栽培历史。据统计,中国的桑树种植面积超过106 hm2,每年新鲜桑叶的生物量为25~30 t/hm2[2]。桑叶富含蛋白质(15%~35%)、维生素、矿物质(钙2.42%~4.71%、总蛋白质0.23%~0.97%)和代谢能(4.73~9.37 MJ/kg),且抗营养因子含量总体较低[3-4],但需关注特定品种及未加工桑叶中单宁等成分的潜在影响。此外,最新研究表明,桑叶还含有黄酮类化合物、生物碱、多糖和多酚等天然生物活性物质,具有降血糖、降脂、抗菌、抗氧化和抗炎等作用[5-6]。因此,桑叶被广泛用作动物生产中的优质蛋白质饲料,适用于猪、鸡、反刍动物和鱼等多种动物[7-10]。然而,桑叶的高水分和蛋白质含量使其不适合长期储存,尤其是在中国南方的雨季[11]。青贮桑叶具有贮藏期长、适口性好、营养价值高等优点,是一种有效的桑叶保鲜方法[12],可以克服牧草资源与动物生产之间的季节性不平衡。多项研究指出,足够数量的乳酸菌(LAB)、一定量的水溶性碳水化合物(WSC)和氮源是青贮成功的关键[13]。然而,附生LAB和氮源的不足会导致梭菌等有害细菌数量的增加[14]。近年来的研究强调,补充接种剂和足够的发酵底物可以提高青贮发酵质量[15]。大量研究已证实,LAB在提升桑叶青贮饲料的营养成分含量与保鲜效能方面具有显著潜力[11,16]。进一步的研究还揭示,芽孢杆菌凭借其卓越的性能,被归为第四代青贮接种剂,它能有效提升动物的生产性能[17],并显著改善青贮的发酵品质与有氧稳定性[18]。在青贮中添加枯草芽孢杆菌不仅能够迅速消耗氧气,有效抑制霉菌与腐败菌的繁殖,还确保LAB成为优势菌群,加快发酵进程,从而最大程度地保留饲料的营养价值[19]。此外,Sansinenea等[20]研究指出,枯草芽孢杆菌具备产生多样化抗菌化合物的能力,能有效抑制病原菌的生长,进一步增强青贮饲料的安全性。然而,关于在桑枝叶青贮中联合应用LAB、芽孢杆菌及糖蜜(molasses,M)的研究报道很少。因此,本研究探讨了单独或联合植物乳杆菌(Lactobacillus plantarum,LP)、布氏乳杆菌(Lactobacillus brucei,LB)、枯草芽孢杆菌(Bacillus subtilis,BS)及M添加对桑枝叶青贮发酵品质、营养成分和微生物菌落数量的影响,旨在为饲料桑的开发利用提供参考。

1 材料与方法

1.1 试验材料

桑枝叶原料由湖北曼博瑞生物科技有限公司提供。本试验所用青贮菌剂分别为LP、LB和BS,有效活菌数≥1.0×1010 CFU/g,M有效成分含量≥98%。

1.2 青贮制备

试验选择株高约1.2 m的桑树,人工采集桑树的叶片和嫩枝,切碎至2~3 cm长度,将青贮添加剂溶液均匀地喷洒在桑枝叶原料表面,充分混匀后约取500 g样品装入聚乙烯厌氧袋(23 cm×30 cm)中,真空密封,置于室温避光发酵。发酵60 d后,开袋取样开展发酵品质、化学成分和微生物菌落总数测定分析。

1.3 试验设计

试验共设置5个组,分别为对照组(CON组):添加等量无菌水,不添加任何青贮添加剂;LP组:添加2.0 g/t LP;LB组:添加2.0 g/t LB;LP+BS组:添加1.0 g/t LP和1.0 g/t BS;LP+LB+BS+M组:添加0.5 g/t LP、0.5 g/t LB、1.0 g/t BS和0.3% M,每组设5个重复。

1.4 指标测定及方法

1.4.1 微生物菌落总数分析

准确称取10 g桑枝叶原料及其青贮的新鲜样品,用90 mL 0.9% NaCl无菌生理盐水匀浆,在37 ℃下,180 r/min摇匀1 h得到菌悬液。再用无菌生理盐水(0.9% NaCl)将菌悬液按10-1~10-7连续稀释。在MRS琼脂培养基上计数LAB,用厌氧罐厌氧袋37 ℃培养48 h;在孟加拉红琼脂培养基上28 ℃培养48 h后计数酵母(yeast)和霉菌(molds);在伊红-亚甲基蓝培养基上37 ℃培养48 h后计数大肠菌(CB)。所有微生物采用平板计数法,将数据转换为lg(CFU/g)用于微生物的定量分析。

1.4.2 发酵品质指标

称取桑枝叶青贮饲料样品20 g,加入180 mL蒸馏水,匀浆1 min,分别用4层纱布和定量滤纸过滤,采用精密pH计测定滤液pH。采用高效液相色谱法分析乳酸(LA)、乙酸(AA)、丙酸(PA)、丁酸(BA)的含量。分析条件:C18色谱柱(250 mm×4.6 mm×5 μm),紫外检测器波长为210 nm,流动相A和B分别为甲醇和0.01 mol/L的KH2PO4水溶液,流速0.7 mL/min,柱温50 ℃。采用苯酚-次氯酸钠比色法测定氨态氮(NH3-N)含量,并计算NH3-N/总氮(TN)。

1.4.3 营养成分指标

取适量桑枝叶青贮前后的样品,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的方法测定水分含量;采用凯氏定氮法测定粗蛋白质(CP)和TN含量;参照GB/T 6433—2025的索氏抽提法测定粗脂肪(EE)的含量;粗灰分(Ash)的含量参照GB/T 6438—2007的方法进行测定;采用蒽酮硫酸比色法测定可溶性碳水化合物(WSC)含量;采用Van Soest等[21]方法测定中性洗涤纤维(NDF)、酸性洗涤纤维(ADF)含量;缓冲能(BC)参照Herrmann等[22]的LA滴定法测定。
精确称取1.0 g新鲜样品浸泡于100 mL蒸馏水中,振荡30 min,用0.1 mol/L LA溶液滴定至pH 4.0,记录所需LA体积,并以100 mL水的滴定值进行空白校正,BC单位以g LA/kg DM表示;总黄酮(TF)含量根据Govardhan Singh等[23]的氯化铝比色法测定。
将约0.2 g桑枝叶青贮前后的冻干样品称入15 mL管中,然后用10 mL甲醇提取。在多功能酶标仪(Varioskan LUX,赛默飞)上测定反应溶液在510 nm处的吸光度。TF含量以芦丁当量(RE) mg RE/g DM表示,所有样品均设3个重复。

1.5 数据统计与分析

试验数据先用Excel 2019初步整理,再用SAS 9.2软件对数据进行单因素方差分析(one-way ANOVA),并用Duncan氏法对各测定数据进行多重比较。试验数据以“平均值±标准差”表示,P<0.05代表差异显著。

2 结果与分析

2.1 桑枝叶的营养成分

鲜桑枝叶化学成分结果见表1。新鲜桑枝叶的pH为6.27,干物质(DM)含量为28.65%,CP含量为13.30%,WSC含量为7.10%,NDF和ADF含量分别为51.94%和32.13%。BC值为27.67 g LA/kg DM,TF含量为27.38 mg RE/g DM。
表1 鲜桑枝叶化学成分

Table 1 Chemical composition of fresh mulberry branches and leaves

项目Items 含量Contents
干物质Dry matter/% FW 28.65±0.61
粗蛋白质Crude protein/% DM 13.30±0.72
粗脂肪Ether extract/% DM 3.59±0.24
可溶性碳水化合物
Water soluble carbohydrates/% DM		 7.10±0.56
中性洗涤纤维
Neutral detergent fiber/% DM		 51.94±2.51
酸性洗涤纤维
Acid detergent fiber/% DM		 32.13±2.73
缓冲能
Buffering capacity/(g LA/kg DM)		 27.67±1.58
总黄酮
Total flavonoid/(mg RE/g DM)		 27.38±1.21
pH 6.27±0.26

2.2 青贮前后桑枝叶表面微生物菌落数量变化

由表2可知,新鲜桑表面附着微生物种类及数量分别为LAB 4.45 lg(CFU/g),大肠杆菌6.58 lg(CFU/g),酵母3.85 lg(CFU/g)和霉菌4.13 lg(CFU/g)。在桑枝叶青贮中,与CON组相比,添加剂组LAB数量显著提高(P<0.05),酵母菌数量显著降低(P<0.05);添加剂组大肠杆菌数量明显降低,其中LP组和LP+LB+BS+M组大肠杆菌数量低于检测限[<2.00 lg(CFU/g)]。各组霉菌数量均低于检测限[<2.00 lg(CFU/g)]。
表2 青贮前后桑枝叶表面附生微生物菌落数量变化(鲜物质基础)

Table 2 Changes in the number of microbial colonies on mulberry branches and leaves before and after silage (FM basis) lg(CFU/g)

项目
Items		 青贮前
Pre-silage		 组别Groups (青贮后Post-silage) P值
P-value
CON LP LB LP+BS LP+LB+
BS+M
乳酸菌LAB 4.45±0.57 5.65±0.30c 7.30±0.01ab 7.13±0.08ab 6.78±0.30b 7.62±0.24a 0.002
酵母菌Yeast 3.85±0.36 7.79±0.12a 6.57±0.11b 6.52±0.12b 6.53±0.10b 5.47±0.22c <0.001
大肠杆菌CB 6.58±0.73 3.91±0.17 <2.00 2.71±0.08 2.43±0.12 <2.00 -
霉菌Molds 4.13±0.24 <2.00 <2.00 <2.00 <2.00 <2.00 -

同行数据肩标不同小写字母表示差异显著(P<0.05),相同小写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 lowercase letter superscripts mean significant difference (P<0.05), while with the same lowercase letter superscripts or no letter superscripts mean no significant difference (P>0.05). The same as below.

表中数值<2.00表示该微生物菌落数量低于检测阈值[2.00 lg(CFU/g)]。Values in the table below 2.00 indicate that the microbial colony count is lower than the detection threshold [2.00 lg(CFU/g)].

2.3 桑枝叶青贮发酵品质

由表3可知,在室温下,桑枝叶青贮后发酵品质有所提高。发酵60 d后,所有添加剂组pH显著降低(pH<5.0,P<0.05),其中LP+LB+BS+M组的pH最低。与CON组相比,4个添加剂组LA含量均显著提高(P<0.05),NH3-N和乙醇含量显著降低(P<0.05),其中LP+LB+BS+M组的LA含量最高,NH3-N和乙醇含量最低;各组间乙酸(AA)含量没有显著差异(P>0.05)。LP组和LP+LB+BS+M组LA/AA比值显著提高(P<0.05),而其他2组与CON组之间没有显著差异(P>0.05)。LP组、LP+BS组和LP+LB+BS+M组PA含量显著低于CON组(P<0.05),而LB组的PA含量与CON组没有显著差异(P>0.05)。各组桑枝叶青贮样品中均未检测到丁酸。
表3 不同青贮添加剂对桑枝叶青贮60 d发酵品质的影响

Table 3 Effects of different silage additives on fermentation quality of mulberry branches and leaves silage after 60 days

项目
Items		 组别Groups P值
P-value
CON LP LB LP+BS LP+LB+BS+M
pH 5.13±0.05a 4.62±0.06c 4.88±0.02b 4.75±0.02bc 4.35±0.13d <0.001
乳酸LA/% DM 1.50±0.15d 3.40±0.08b 2.89±0.06bc 2.32±0.09c 4.94±0.66a <0.001
乙酸AA/% DM 0.85±0.17 1.00±0.23 1.47±0.31 1.18±0.16 1.49±0.23 0.126
丙酸PA/% DM 0.80±0.04a 0.46±0.15bc 0.67±0.10ab 0.52±0.03bc 0.39±0.02c 0.021
丁酸BA/% DM ND ND ND ND ND -
乳酸/乙酸LA/AA 1.82±0.54b 3.50±0.74a 2.02±0.46b 1.99±0.36b 3.33±0.08a 0.043
乙醇Ethanol/% DM 1.32±0.13a 0.59±0.01c 0.95±0.04b 1.08±0.02b 0.31±0.04d <0.001
氨态氮NH3-N/%TN 2.23±0.17a 1.56±0.08b 1.52±0.15bc 1.34±0.17bc 1.17±0.12c 0.005

ND表示未检出。

ND means not detected.

2.4 桑枝叶青贮营养成分

由表4可知,CON组与添加组间DM和EE含量没有显著差异(P>0.05)。与CON组相比,LP+LB+BS+M组CP含量显著提高(P<0.05),而其他组CP含量没有显著差异(P>0.05);LB组、LP+BS组和LP+LB+BS+M组NDF和ADF含量显著降低(P<0.05),而LP组的NDF和ADF含量与CON组没有显著差异(P>0.05)。LP+LB+BS+M组WSC含量显著提高(P<0.05),LP+BS组WSC含量显著低于CON组(P<0.05);LP组、LP+BS组和LP+LB+BS+M组TF含量显著提高(P<0.05),其中LP+LB+BS+M组TF含量最高,相较CON组提高1.3倍。
表4 不同青贮添加剂对桑枝叶青贮60 d营养成分的影响

Table 4 Effects of different silage additives on chemical composition of mulberry branches and leaves silage after 60 days

项目
Items		 组别Groups P值
P-value
CON LP LB LP+BS LP+LB+BS+M
干物质DM/% FM 25.89±0.69 26.69±0.04 26.73±0.95 25.72±1.83 27.24±0.08 0.550
粗蛋白质CP/% DM 12.80±0.45b 13.98±0.53ab 13.14±0.22b 13.37±0.57b 14.87±0.64a 0.048
粗脂肪EE/% DM 3.53±0.46 3.43±0.22 3.38±0.02 3.66±0.22 3.75±0.26 0.654
中性洗涤纤维NDF/% DM 48.41±1.08a 45.86±0.22ab 44.03±1.71bc 44.10±0.52bc 42.59±1.15c 0.018
酸性洗涤纤维ADF/% DM 29.81±1.46a 26.22±0.67b 27.62±0.95ab 25.38±0.91b 25.82±0.42b 0.027
水溶性碳水化合物
WSC/% DM		 0.84±0.09b 0.68±0.07bc 0.71±0.03b 0.53±0.02c 1.08±0.09a 0.003
总黄酮TF/(mg RE/g DM) 26.13±0.53c 32.13±0.78ab 28.87±0.73bc 31.94±2.21ab 33.82±0.99a 0.013

3 讨论

3.1 新鲜桑枝叶化学成分与微生物菌落数量

原料附生 LAB 数量和 WSC含量是预测青贮饲料发酵是否充分和是否需要使用添加剂的重要因素。 实现优质青贮发酵的附生LAB最小数量为5 lg(CFU/g FM)[24],最低WSC含量为7.0% DM[25]。然而,还有其他重要因素,如DM含量、BC值和附生LAB组成也会影响最终的发酵质量[26]。理论上,桑枝叶WSC含量(7.10% DM)足以在青贮过程中启动LAB繁殖和随后的LA发酵。然而,在本研究中,新鲜桑枝叶中较低DM含量(28.65% vs. 30%~35%优质青贮理想DM含量[27])、较高的BC(27.67 g LA/kg DM)和霉菌[4.13 lg(CFU/g FM)]、大肠杆菌[6.58 lg(CFU/g FM)]和酵母[3.85 lg(CFU/g FM)]的分布可能限制了青贮过程中乳酸发酵的进程。低水平的LAB和WSC可能导致LA积累减少,pH升高,从而促进青贮前期有害微生物的生长。因此,快速提高LAB的优势度可能有利于生产优质桑树青贮饲料。在青贮饲料中添加糖或接种剂是实现优质青贮的必要条件。一般来说,大肠菌群是LAB的主要竞争对手,使青贮饲料中氨基酸含量降低,导致青贮饲料营养流失[28]。在本研究中,由于pH的降低,大肠菌群数量减少,其中LP+LB+BS+M组的LAB数量最高,酵母菌和大肠杆菌数量最低,说明这些微生态制剂与碳源(糖蜜)的协同作用,能够更为高效地促进LA的生成,从而迅速降低环境的pH,对大肠菌群和酵母的生长有抑制作用。

3.2 不同青贮添加剂对桑枝叶青贮发酵品质的影响

青贮饲料的pH对其质量至关重要,pH 4.2常被视作发酵良好的一个基准点,尤其对于高水分含量的发酵饲料,更低的pH可以保证充分的发酵和长期保存[27]。这种pH的降低主要是由于有机酸的累积所致。本试验中,添加剂组pH均降至5.0以下,其中LP+LB+BS+M组pH(4.35)相对较低。与此同时,与CON组相比,LP+LB+BS+M组的LA含量也表现出最高水平。LA(pKa 3.86)主要是由同型发酵LAB通过转化碳水化合物而产生,其酸性强度比其他主要有机酸(如AA pKa 4.75,PA pKa 4.87)高10~12倍,对青贮饲料中pH的快速降低起到了显著作用[13]。LP组和LP+LB+BS+M组的LA/AA大于3.0,达到了理想值,表明以同型LAB发酵为主导,同时保证适当的AA对真菌具有抑制作用,有助于青贮饲料的有氧稳定性[29]。所有青贮料中BA含量均低于检测限,说明梭状芽孢杆菌等不良微生物没有大量繁殖[30]。酵母和大肠杆菌可将葡萄糖转化为乙醇。与CON组相比,LP组和LP+LB+BS+M组青贮中乙醇含量较低,可能是由于pH的快速下降限制了产乙醇的微生物,这与Cao等[31]研究结果一致。NH3-N含量是衡量蛋白质分解程度的关键指标,在较低的pH环境下,蛋白酶的活性会受到抑制甚至失活[32]。以往研究表明,保存良好的青贮饲料NH3-N含量应低于TN的10%[33]。然而,本研究中,所有青贮饲料的NH3-N含量(1.17%~2.23%TN)均低于该推荐标准,且添加剂组NH3-N含量均显著低于CON组。这结果进一步证明了添加剂的重要作用,它们通过降低环境的pH,有效抑制了其他微生物(如梭状芽孢杆菌)的生长和蛋白水解活性[34]。这不仅有助于保持饲料的营养价值,还减少了因微生物活动过度而导致的饲料变质风险。

3.3 不同青贮添加剂对桑枝叶青贮营养成分的影响

与CON组相比,LP+LB+SB+M组CP含量显著提高。这一提升可能归因于以下3个主要原因:首先,微生物会通过呼吸作用消耗桑枝叶中的部分有机物,导致产物总量减少,进而产生蛋白质“浓度效应”,使得CP含量相对提高[35];其次,青贮过程中CP含量的变化与pH密切相关,pH直接影响饲料中蛋白酶(羧肽酶和酸性蛋白酶)的活性,酸化降低青贮蛋白水解和脱羧,抑制蛋白水解酶活性,降低NH3-N含量,从而有助于保存更多的CP[36];最后,部分CP的增加还可能来源于接种菌体及其代谢产物中的氨[37]。研究发现,青贮原料中存在足够的WSC含量(6%~8% DM)对于保证青贮发酵质量至关重要[38]。在本研究中,新鲜桑枝叶WSC含量(7.10% DM)足以促进LAB发酵。青贮60 d后,LP+BS组青贮中WSC含量显著低于CON组,而LP+LB+SB+M组反之。WSC含量的减少,很可能是因为植物乳杆菌与枯草芽孢杆菌的结合使用,使LAB转化WSC为LA的能力增强,这与Guo等[39]研究结果一致。相反,WSC含量的增加则可能是由于额外添加的M所带来的,M作为碳源补充,直接提升了青贮料中的WSC含量。另外,发酵结束后,LP及其与BS结合处理的青贮中NDF和ADF含量低于未接种青贮,这可能一方面是由于青贮过程中更易消化的细胞壁组分的酸水解。阿拉伯糖是木质素与阿拉伯木聚糖交联的关键成分,对酸水解敏感,它的增溶可改变细胞壁的可降解性[40];另一方面可能是由于BS产生的纤维素酶的作用,它将纤维素等复合物降解为碳水化合物[41]。
很多研究表明,桑树之所以具有降血糖、降脂、抗菌、抗氧化和抗炎等多重功效,与其富含的多种生物活性成分密切相关,如酚类和生物碱,特别是类黄酮[42-43]。在本研究中,新鲜桑原料中的TF含量为27.38 mg RE/g DM,这结果低于He等[44]研究的桑枝叶中TF含量(66.20 mg RE/g DM),而高于Bao等[43]研究的黑桑果中TF含量(15.08 mg RE/g DM)。这种TF含量的差异可能是由于桑的品种、取样组织部位以及生长时间的不同所导致的。本研究中发现,含有LP的3个组桑枝叶青贮中TF含量均显著高于CON组,表明LP在发酵过程中表现出更高的类黄酮转化或释放效率,在桑枝叶发酵过程中也发现了类似的结果[44]。与自然发酵相比,添加LAB诱导了更广泛的酶促反应,如糖苷键断裂、糖转移、羟基取代、甲氧基取代和开环,发酵过程中产生的酶如糖苷酶、脱氢酶和甲基转移酶促进了类黄酮的转化[45],如Gao等[46]在研究LAB对苜蓿青贮黄酮生物转化的影响发现,LP能够完全降解水仙素,表现出显著改变类黄酮组成的独特能力,这可能是由菌株特异性酶-鼠李糖苷酶促成的,该酶可有效切割类黄酮中的糖苷键,突出了LAB的菌株特异性酶促能力。Mueller等[47]研究也进一步证实了鼠李糖苷酶对类黄酮水解的能力对菌株具有高度特异性。总体而言,发酵后桑枝叶TF含量的增加不是由于植物本身的生物合成,而主要是发酵过程中通过微生物和酶作用释放结合的黄酮的结果。未来应研究黄酮类化合物的结构-活性关系,从而更深入地了解发酵影响其生物活性的机制。

4 结论

综上所述,LP+LB+SB+M组桑枝叶青贮饲料品质显著提升,主要表现为LA含量增加与pH下降,协同抑制腐败菌增殖,使NH3-N含量降低;NDF和ADF含量下降能够有效促进纤维的降解消化;同时,TF含量提升表现出更高的抗氧化潜力。因此,LP、LB、BS和M的联合使用是提高桑枝叶青贮品质的一种高效且可行的途径。

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