Chinese Journal of Animal Nutrition >

2024 , Vol. 36 >Issue 11: 7383 - 7393

DOI: https://doi.org/10.12418/CJAN2024.628

RESEARCH PAPER

Effects of Inoculation with Lactic Acid Bacteria on Aerobic Stability and Mycotoxin Contents in Whole-Plant Corn Silages

  • LIANG Yucheng , 1, 2 ,
  • WANG Wenbo 2 ,
  • YUAN Xianjun 2 ,
  • CAI Xinyu 2 ,
  • QI Bokang 2 ,
  • BAO Yuhong , 1, 3, *
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  • 1 Institute of Grass Science, Tibet Academy of Agriculture and Animal Husbandry Sciences, Lhasa 850000, China
  • 2 Institute of Ensiling and Processing of Grass, College of Prataculture Science, Nanjing Agricultural University, Nanjing 210095, China
  • 3 State Key Laboratory of Germplasm Resources and Genetic Improvement of Tibetan Barley and Yak, Lhasa 850000, China
* associate professor, E-mail:

Received date: 2024-05-15

  Online published: 2024-11-09

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Abstract

This experiment focused on the potential antibacterial and detoxifying activities of three strains of lactic acid bacteria as Lactobacillus paracasei LS2, Lactiplantibacillus plantarum S6-2 and Lactobacillus casei GD2-1, which were screened in the preliminary stage, and aimed to investigate their effects on the aerobic stability and mycotoxin contents in whole-plant corn silages. The experimental whole-plant corns from fields either infected (FI group) or not infected (NFI group) with fungi were ensiled with the following inoculant treatments: 1) no lactic acid bacteria inoculation control (CON treatment); 2) inoculation with Lactobacillus paracasei LS2 (LS2 treatment); 3) inoculation with Lactiplantibacillus plantarum S6-2 (S6-2 treatment); 4) inoculation with Lactobacillus casei GD2-1 (GD2-1 treatment), respectively. After 180 days of silage fermentation, the silage pits were opened, and the samples from each group were exposed to air for 4 days to analyze of fermentation parameters, chemical composition, microbial counts and mycotoxin contents in whole-plant corn silages. The results showed as follows: 1) regardless of fungal infection, LS2 treatment displayed the lowest pH in whole-plant corn silages after 4-day of aerobic exposure; and LS2 treatment in NFI group showed the highest lactic acid and acetic acid contents. 2) In NFI group, the numbers of yeasts and molds in S6-2 and GD2-1 treatments increased rapidly on the second day of aerobic exposure, after which GD2-1 treatment remained stable, while S6-2 treatment decreased slightly. In FI group, the numbers of yeasts and molds in each treatment showed an overall increasing trend during aerobic exposure. 3) Field fungal infection significantly increased the aflatoxin B1 (AFB1) content in whole-plant corn silages (P<0.05), all lactic acid bacteria inoculant treatments significantly decreased the AFB1 content (P<0.05), GD2-1 treatment significantly decreased the zearalenone content (P<0.05), and GD2-1 treatment significantly decreased the deoxynivalenol content (P<0.05). 4) Regardless of fungal infection, LS2 treatment maintained the longest aerobic stability time, and the aerobic stability was significantly higher than that in the other treatments (P<0.05). In conclusion, after aerobic exposure, the field fungal infection further degrades the quality of whole-plant corn silages and leads to premature aerobic spoilage. Regardless of fungal infection, the inoculation with Lactobacillus paracasei LS2 can maintain higher contents of lactic acid and acetic acid in whole-plant corn silages during aerobic exposure, and exhibit the highest aerobic stability.

Key words： whole-plant corn silages; lactic acid bacteria; aerobic stability; mycotoxins

Cite this article

LIANG Yucheng , WANG Wenbo , YUAN Xianjun , CAI Xinyu , QI Bokang , BAO Yuhong . Effects of Inoculation with Lactic Acid Bacteria on Aerobic Stability and Mycotoxin Contents in Whole-Plant Corn Silages[J]. Chinese Journal of Animal Nutrition, 2024 , 36(11) : 7383 -7393 . DOI: 10.12418/CJAN2024.628

全株玉米青贮饲料因其营养价值高、保存效果好等特点在奶牛生产中大量使用,成为反刍动物饲粮的主要组成部分[1]。在全株玉米青贮过程中,乳酸快速大量生成,加之缓冲能力低导致pH快速下降,进而快速抑制不良微生物增殖[2]。但青贮饲料在开窖饲喂过程中,由于氧气的进入,厌氧环境被破坏,醋酸菌、酵母菌和霉菌等好氧微生物滋生,它们以青贮饲料中的乳酸和残留的水溶性碳水化合物(WSC)为底物快速增殖,代谢产生二氧化碳和热量,导致青贮饲料温度急速上升和养分大量损失[3]。全株玉米青贮饲料因其WSC和乳酸含量高,因此是一种发酵迅速但极易发生有氧腐败的青贮饲料。
近年来全球气候变暖引发的持续干旱、高温和洪涝等极端天气频发,我国大多地区青贮玉米在授粉和灌浆时正值高温和多雨季节,极易遭受霉菌侵染,导致霉菌毒素积累,影响全株玉米青贮饲料质量安全[4]。全株玉米在田间生长过程中虽然遭受了霉菌侵染,但青贮过程中产生的有机酸能抑制霉菌的生长或使它们处于休眠状态[5],而开窖后由于氧气的进入这些霉菌可能重新复活并快速增殖[6]。Wang等[7]研究表明,与自然生长组相比,田间霉菌侵染的全株玉米青贮饲料在有氧暴露期间,酵母菌和霉菌的数量更加快速的增殖,并始终保持更高水平。有氧暴露期间青贮饲料中产毒霉菌复苏,进一步导致毒素积累。Ferrero等[8]研究表明,黄曲霉菌(Aspergillus flavus)在有氧环境下快速增殖并加速了黄曲霉毒素B1(AFB1)的产生。Vandicke等[9]研究发现,在有氧条件下镰刀菌属(Fusarium)孢子可在玉米青贮饲料中萌发定殖,并代谢产生霉菌毒素。目前,为抑制有氧腐败,延长青贮饲料有氧稳定时间,前人进行了乳酸菌资源挖掘及其微生物制剂的大量研究工作。Kung等[10]研究表明,接种布氏乳杆菌(Lactobacillus buchneri)40788提高了玉米青贮饲料的乙酸含量,有效抑制了酵母菌等不良微生物活性,从而提高了玉米青贮饲料的有氧稳定性。Romero等[11]研究表明,布氏乳杆菌和戊糖片球菌(Pediococcus pentosaceus)复合接种剂可提高玉米青贮饲料中的乙酸含量,减少酵母菌和霉菌数量,从而延长有氧稳定性。然而,这些乳酸菌仅通过产生乙酸抑制好氧微生物,而对青贮饲料中已有毒素及有氧暴露过程中毒素的产生未见详细报道。本实验室前期筛选获得3株乳酸菌,其不仅具有抗真菌活性,而且兼具对主要毒素的脱毒潜力,但其对全株玉米青贮饲料有氧稳定性及其有氧暴露过程中毒素的影响尚不明确。
为此,本研究以实验室前期筛选的副干酪乳杆菌(Lactobacillus paracasei)LS2、植物乳杆菌(Lactiplantibacillus plantarum)S6-2和干酪乳杆菌(Lactobacillus casei)GD2-1这3株乳酸菌为研究对象,模拟青贮玉米田间遭受霉菌侵染,并设置自然生长的玉米为对照,分析3株乳酸菌对田间遭受霉菌侵染的全株玉米青贮饲料有氧稳定性和有氧暴露期间霉菌毒素含量的影响。

1 材料与方法

1.1 试验材料

青贮所用玉米种植于南京农业大学白马科学教研基地(北纬32°04',东经118°88'),包括8个试验小区。玉米处于灌浆期时,随机选取其中4个小区用于模拟人工霉菌侵染,用刀片划破玉米穗和茎,并喷施黄曲霉菌和禾谷镰刀菌(Fusarium graminearum)孢子悬液至破损处,隔1周后再进行一次人工侵染;另外4个小区玉米保持自然生长,各小区玉米于1/2乳线期收获制作青贮饲料。

1.2 试验设计

本试验采取完全随机试验设计,对田间人工遭受霉菌侵染(FI组)和霉菌未侵染(NFI组)全株玉米分别进行以下接种剂处理:1)未接种乳酸菌对照(CON处理);2)接种副干酪乳杆菌LS2(LS2处理);3)接种植物乳杆菌S6-2(S6-2处理);4)接种干酪乳杆菌GD2-1(GD2-1处理)。各菌株接种量均为1×106 CFU/g(鲜重基础)。FI组和NFI组的各小区每个处理制作3个青贮袋,每袋装500 g青贮饲料,4个小区为试验重复。

1.3 取样与指标测定

为模拟青贮饲料有氧暴露,所有青贮袋于青贮180 d后开袋,将各袋青贮饲料分别全部倒出取样,并将剩余青贮饲料装入新的5 L敞口的聚乙烯瓶中,使之保持蓬松,在有氧暴露第2和4天取样,分析全株玉米青贮饲料的发酵参数、化学组分和微生物数量,并测定有氧暴露最后1天(第4天)各组样品的霉菌毒素含量。有氧暴露期间将纽扣温度记录仪(DS9490R,上海沃第森电子科技有限公司)置于青贮饲料中心位置,每30 min记录1次温度以及环境温度。
每次取样时将青贮饲料全部倒出并充分混合均匀,取20 g样品放入100 mL锥形瓶中,并加入60 mL蒸馏水使之完全浸没,用保鲜膜封口,置于4 ℃冰箱中浸提24 h,将滤液经4层纱布和定性滤纸过滤获得浸提液,用于分析pH以及有机酸和氨态氮含量。另取10 g样品置于250 mL锥形瓶中,加入0.9%无菌生理盐水90 mL充分混合,于37 ℃恒温摇床上以120 r/min的速度振荡2 h。取1 mL菌体洗脱液进行10倍梯度稀释,将稀释液涂于培养基板进行微生物计数。取100 g青贮样品于65 ℃烘箱中烘干至恒重,测定干物质含量;并用高速粉碎机(FW110,天津市泰斯特仪器有限公司)粉碎,过1 mm筛,用于测定样品的粗蛋白质、淀粉、水溶性碳水化合物、中性洗涤纤维、酸性洗涤纤维和霉菌毒素含量。
采用高精度pH计(HI2222,Hanna Instruments,意大利)测定青贮饲料浸提液pH。采用高效液相色谱(Agilent 1260,Agilent,美国)测定有机酸和乙醇含量[12],测试条件:色谱柱为Carbomix® H-NP5(8%交联度,5 μm,7.8 mm×300 mm),流动相为2.5 mmol/L硫酸(H2SO4),柱温为55 ℃,流速为0.5 mL/min,进样量为10 μL,检测器为示差检测器)。采用苯酚-次氯酸盐比色法[13]测定氨态氮含量。采用凯氏定氮仪(Kjeltec 8400,FOSS,丹麦)测定样品中的粗蛋白质含量[14],粗蛋白质由总氮乘以6.25得出。采用硫酸-蒽酮比色法[15]测定水溶性碳水化合物含量。采用纤维分析仪(Ankom 220,Ankom Technology,美国)测定中性洗涤纤维和酸性洗涤纤维含量[16]。采用霉菌毒素酶联免疫检测试剂盒(AgraQuant®,Romer Labs,美国)测定AFB1、呕吐毒素(DON)和玉米赤霉烯酮(ZEN)含量。采用平板计数法对微生物进行计数,其中乳酸菌通过MRS琼脂培养基(上海盛思生化科技有限公司)在37 ℃厌氧条件下培养48 h后进行计数,酵母菌和霉菌通过马铃薯葡萄糖琼脂培养基(上海盛思生化科技有限公司)在30 ℃有氧条件下培养48 h后进行计数,好氧性细菌通过营养琼脂培养基(上海盛思生化科技有限公司)在37 ℃有氧条件下培养48 h后进行计数。

1.4 数据统计分析

采用SPSS 27.0软件对试验数据进行处理,采用单因素方差分析(one-way ANOVA)模型对全株玉米青贮饲料的有氧稳定时间进行分析;采用一般线性模型(GLM)对有氧暴露期间全株玉米青贮饲料发酵产物、微生物数量和霉菌毒素含量进行双因素方差分析,主效应包括霉菌侵染、接种剂及其交互效应;采用一般线性模型对有氧暴露期间全株玉米青贮饲料化学组分和发酵参数进行双因素方差分析,主效应包括接种剂、有氧暴露天数及其交互效应。采用Tukey’s法对各个处理间数据平均值进行多重比较,P<0.05表示差异显著。

2 结果与分析

2.1 3株乳酸菌对全株玉米青贮饲料有氧暴露期间发酵参数和化学组分的影响

由表1可知,乳酸菌接种剂与霉菌侵染对有氧暴露4 d后全株玉米青贮饲料pH、乳酸含量和乙酸含量存在显著交互效应(P<0.05)。与CON处理相比,无论是否遭受霉菌侵染,LS2处理均显著降低了pH(P<0.05),而GD2-1处理并未显著影响pH(P>0.05);LS2处理显著提高了NFI组中乳酸含量(P<0.05),各接种剂处理对FI组中乳酸含量均无显著影响(P>0.05);LS2和S6-2处理显著提高了NFI组中乙酸含量(P<0.05),而各接种剂处理对FI组中乙酸含量均无显著影响(P>0.05)。乳酸菌接种剂和霉菌侵染对全株玉米青贮饲料氨态氮含量均有显著影响(P<0.05)。与CON处理相比,无论是否遭受霉菌侵染,LS2处理显著降低了氨态氮含量(P<0.05),S6-2处理显著提高了氨态氮含量(P<0.05)。乳酸菌接种剂与霉菌侵染对全株玉米青贮饲料WSC和乙醇含量存在显著交互效应(P<0.05)。NFI组中S6-2和GD2-1处理WSC含量显著高于FI组中对应处理(P<0.05)。与CON处理相比,无论是否遭受霉菌侵染,LS2处理显著提高了乙醇含量(P<0.05);S6-2和GD2-1处理显著降低了FI组中乙醇含量(P<0.05)。乳酸菌接种剂与霉菌侵染对全株玉米青贮饲料乳酸菌以及酵母菌和霉菌数量存在显著交互效应(P<0.05)。与CON处理相比,S6-2和GD2-1处理显著提高了NFI组中乳酸菌数量(P<0.05),GD2-1处理显著提高了FI组中乳酸菌数量(P<0.05);各接种剂处理均显著提高了NFI组中酵母菌和霉菌数量(P<0.05),S6-2和GD2-1处理显著提高了FI组中酵母菌和霉菌数量(P<0.05)。
表1 接种乳酸菌和霉菌侵染对有氧暴露4 d后全株玉米青贮饲料发酵产物和微生物数量的影响

Table 1 Effects of inoculation with lactic acid bacteria and fungal infection on fermentation products and microbial counts in whole-plant corn silages after 4 days of aerobic exposure

项目
Items		 组别
Groups		 接种剂处理Inoculant treatment 平均值
Mean		 均值
标准误
SEM		 P值P-value
CON LS2 S6-2 GD2-1 F I F×I
pH NFI 6.33Ab 5.52Ac 6.61a 6.37b 6.21 0.082 <0.001 <0.001 <0.001
FI 6.09Bb 4.91Bc 6.45ab 6.28b 5.93
平均值Mean 6.21b 5.21c 6.53a 6.33b
乳酸
Lactic acid/
(g/kg DM)		 NFI 1.71Bb 5.38Aa 2.55Bb 1.99Bb 2.91 0.375 <0.001 0.043 <0.001
FI 3.25Aab 2.45Bb 4.88Aab 6.13Aa 4.18
平均值Mean 2.48 3.92 3.72 4.06
乙酸
Acetic acid/
(g/kg DM)		 NFI 0.37Bc 2.33Aa 0.91b 0.35Bc 0.99 0.124 <0.001 <0.001 <0.001
FI 0.68Aab 0.72Bab 0.80a 0.64Ab 0.71
平均值Mean 0.53b 1.53a 0.85ab 0.50b
氨态氮
Ammonia nitrogen/
(g/kg TN)		 NFI 51.3Ab 32.1Ac 66.6Aa 59.9ab 52.5 2.85 <0.001 <0.001 0.149
FI 40.8Bb 26.3Bc 56.8Ba 57.2a 45.3
平均值Mean 46.0b 29.2c 61.7a 58.6a
水溶性碳水
化合物
WSC/(g/kg DM)		 NFI 50.4Ab 55.5b 80.9Aa 72.2Aab 63.2A 3.36 <0.001 0.540 <0.001
FI 39.4B 49.4 36.3B 43.5B 43.7B
平均值Mean 44.9 52.4 58.6 57.9
乙醇
Ethanol/
(g/kg DM)		 NFI 0.77b 2.17Aa 0.99b 0.73Bb 1.17 0.103 0.852 <0.001 <0.001
FI 1.26b 1.67Ba 0.85c 0.91Ac 1.17
平均值Mean 1.02b 1.92a 0.92b 0.82b
乳酸菌
Lactic acid bacteria/
[log10(CFU/g FW)]		 NFI 2.00Bb 2.00Bb 4.39a 4.39Ba 3.19B 0.235 <0.001 <0.001 <0.001
FI 4.45Ab 4.66Aab 4.64ab 4.84Aa 4.65A
平均值Mean 3.23 3.33 4.51 4.61
酵母菌和霉菌
Yeasts and molds/
[log10(CFU/g FW)]		 NFI 5.37Bb 7.12Ba 7.61a 7.77a 6.97 0.167 <0.001 <0.001 <0.001
FI 7.14Ab 7.39Ab 7.98a 7.89a 7.60
平均值Mean 6.26b 7.25a 7.79a 7.83a

同列数据肩标不同大写字母表示差异显著(P<0.05),同行数据肩标不同小写字母表示差异显著(P<0.05)。NFI:霉菌未侵染;FI:霉菌侵染。CON:对照;LS2:副干酪乳杆菌LS2;S6-2:植物乳杆菌S6-2;GD2-1:干酪乳杆菌GD2-1。F:霉菌侵染效应;I:接种剂效应;F×I:霉菌侵染与接种剂的交互效应。图2同。

Values in the same column with different capital letters mean significant difference (P<0.05), and values in the same row with different small letters mean significant difference (P<0.05). NFI: non-fungal infection; FI: fungal infection. CON: control; LS2: Lactobacillus paracasei LS2; S6-2: Lactiplantibacillus plantarum S6-2; GD2-1: Lactobacillus casei GD2-1. F: effect of fungal infection; I: effect of inoculant; F×I: interaction between fungal infection and inoculant.

图1展示了全株玉米青贮饲料有氧暴露期间pH、乳酸和乙酸含量的动态变化,无论是否遭受霉菌侵染,乳酸菌接种剂与有氧暴露天数对pH、乳酸和乙酸含量均存在显著交互效应(P<0.05)。FI组和NFI组各处理pH在有氧暴露前2 d均未见明显上升,之后快速升高;在有氧暴露第4天时,2组CON、S6-2和GD2-1处理pH均迅速上升至6.0以上,且显著高于LS2处理(P<0.05)。NFI组中LS2处理乳酸含量在有氧暴露第2天时略有升高;而FI组LS2处理在有氧暴露第2天时明显降低,其他处理乳酸含量在有氧暴露前2 d保持稳定,之后均快速降低。NFI组各处理乙酸含量在有氧暴露前2 d均缓慢上升,随后快速下降;而FI组LS2处理乙酸含量在有氧暴露前2 d快速下降,之后缓慢下降,其他各处理乙酸含量均缓慢下降。
图1 接种乳酸菌和霉菌侵染对全株玉米青贮饲料有氧暴露过程中pH、乳酸和乙酸含量变化的影响

NFI:霉菌未侵染;FI:霉菌侵染;I:接种剂效应;D:有氧暴露天数效应;I×D:接种剂与有氧暴露天数的交互效应。CON:对照;LS2:副干酪乳杆菌LS2;S6-2:植物乳杆菌S6-2;GD2-1:干酪乳杆菌GD2-1。下图同。

Fig.1 Effects of inoculation with lactic acid bacteria and fungal infection on changes of pH and contents of lactic acid and acetic acid in whole-plant corn silages during aerobic exposure

NFI: non-fungal infection corn; FI: fungal infection; I: effect of inoculant; D: effect of aerobic exposure days; I×D: interaction between inoculant and aerobic exposure days. CON: control; LS2: Lactobacillus paracasei LS2; S6-2: Lactiplantibacillus plantarum S6-2; GD2-1: Lactobacillus casei GD2-1. The same as below.

如图2所示,有氧暴露期间乳酸菌接种剂与有氧暴露天数对全株玉米青贮饲料WSC、氨态氮和乙醇含量均存在显著交互效应(P<0.05)。除FI组LS2处理和NFI组S6-2处理WSC含量有波动以外,其他各处理WSC含量均随有氧暴露天数的延长而逐渐下降,其中各接种剂处理WSC含量始终高于CON。NFI组CON和LS2处理氨态氮含量在有氧暴露第2天时达到最大值,之后快速下降;S6-2处理氨态氮含量随着有氧暴露天数的延长先下降后略有升高;而GD2-1处理氨态氮含量始终缓慢上升直至有氧暴露第4天。FI组GD2-1处理氨态氮含量在有氧暴露第2天达到最大值,之后快速降低;而LS2处理氨态氮含量在有氧暴露第2天时快速下降至最低值,随后略有升高;S6-2处理氨态氮含量在有氧暴露期间始终呈现上升趋势;CON处理氨态氮含量在有氧暴露期间无明显变化。NFI组各处理乙醇含量随有氧暴露天数的延长均缓慢下降,其中NFI组LS2处理乙醇含量在有氧暴露期间始终高于其他各处理;FI组LS2处理乙醇含量在有氧露前2 d快速下降,而其他3个处理缓慢下降,之后各处理乙醇均快速消失。
图2 接种乳酸菌和霉菌侵染对全株玉米青贮饲料有氧暴露过程中WSC、氨态氮和乙醇含量变化的影响

Fig.2 Effects of inoculation with lactic acid bacteria and fungal infection on changes of contents of WSC, ammonia nitrogen and ethanol in whole-plant corn silages during aerobic exposure

如图3所示,全株玉米青贮饲料有氧暴露期间乳酸菌、酵母菌和霉菌数量的动态变化结果显示,无论是否遭受霉菌侵染,乳酸菌接种剂与有氧暴露天数对乳酸菌、酵母菌和霉菌数量均存在显著交互效应(P<0.05)。NFI组各处理乳酸菌数量在有氧暴露第2天时均呈上升趋势,随后S6-2和GD2-1处理乳酸菌数量保持稳定,而CON和LS2处理略有上升;FI组除LS2处理乳酸菌数量略有波动以外,其余各处理乳酸菌数量在有氧暴露期间均呈缓慢上升趋势。NFI组CON、S6-2和GD2-1处理酵母菌和霉菌数量在有氧暴露第2天时快速上升,之后GD2-1处理保持稳定,而CON和S6-2处理酵母菌和霉菌数量略有下降;FI组各处理酵母菌和霉菌数量在有氧暴露期间呈现波动状态,但整体呈现上升趋势。
图3 接种乳酸菌和霉菌侵染对全株玉米青贮饲料有氧暴露过程中乳酸菌、酵母菌和霉菌数量变化的影响

Fig.3 Effects of inoculation with lactic acid bacteria and fungal infection on changes of numbers of lactic acid bacteria, yeasts and molds in whole-plant corn silages during aerobic exposure

2.2 3株乳酸菌对全株玉米青贮饲料霉菌毒素含量的影响

由表2可知,乳酸菌接种剂和霉菌侵染对全株玉米青贮饲料AFB1含量有显著影响(P<0.05)。其中,FI组AFB1含量显著高于NFI组(P<0.05),各乳酸菌接种剂处理AFB1含量均显著低于CON处理(P<0.05)。乳酸菌接种剂对全株玉米青贮饲料ZEN含量有显著影响(P<0.05),其中GD2-1处理ZEN含量显著低于CON处理(P<0.05)。乳酸菌接种剂对全株玉米青贮饲料DON含量有显著影响(P<0.05),其中LS2和GD2-1处理DON含量显著低于CON处理(P<0.05)。
表2 接种乳酸菌和霉菌侵染对有氧暴露4 d后全株玉米青贮饲料霉菌毒素含量的影响

Table 2 Effects of inoculation with lactic acid bacteria and fungal infection on mycotoxin contents in whole-plant corn silages after 4 days of aerobic exposure μg/kg

项目
Items		 组别
Groups		 接种剂处理Inoculant treatment 平均值
Mean		 均值
标准误
SEM		 P值P-value
CON LS2 S6-2 GD2-1 F I F×I
黄曲霉毒素B1
AFB1		 NFI 3.93 3.80 3.31 2.94 3.50B 0.130 0.006 0.001 0.058
FI 4.88 3.48 4.08 3.61 4.01A
平均值Mean 4.41a 3.64b 3.70b 3.27b
玉米赤霉烯酮
ZEN		 NFI 199 171 188 152 178 8.12 0.345 0.033 0.301
FI 223 224 169 148 191
平均值Mean 211a 198ab 179ab 150b
呕吐毒素
DON		 NFI 0.254 0.197 0.250 0.199 0.225 0.010 0.713 0.007 0.838
FI 0.284 0.192 0.254 0.194 0.231
平均值Mean 0.269a 0.194b 0.252ab 0.197b

2.3 3株乳酸菌对全株玉米青贮饲料有氧稳定性的影响

如图4所示,NFI组和FI组LS2处理保持稳定时间(有氧稳定性)最长分别达到77.0和55.0 h,均显著高于其他处理(P<0.05)。NFI组S6-2和GD2-1处理有氧稳定性显著低于CON处理(P<0.05),而FI组S6-2和GD2-1处理有氧稳定性均显著高于CON处理(P<0.05)。
图4 接种剂对全株玉米青贮饲料有氧稳定性的影响

数据柱标记不同大写字母表示差异显著(P<0.05)。

Fig.4 Effects of inoculants on aerobic stability of whole-plant corn silages

Data columns marked with different capital letters indicated significant difference (P<0.05).

图5展示了全株玉米青贮饲料有氧暴露期间的温度变化曲线,FI组CON处理温度升高早于NFI组CON处理,NFI组S6-2和GD2-1处理均提前发生了有氧腐败。无论是否遭受霉菌侵染,LS2处理全株玉米青贮饲料都维持了最长的有氧稳定时间。在NFI组中,S6-2和GD2-1处理全株玉米青贮饲料均在48 h内开始发生有氧腐败,并显示出较大的温度变化;在FI组中,各乳酸菌接种剂处理全株玉米青贮饲料维持有氧稳定时间均长于CON处理。
图5 接种乳酸菌和霉菌侵染对全株玉米青贮饲料有氧暴露过程中温度变化的影响

AT:环境温度

Fig.5 Effects of inoculation with lactic acid bacteria and fungal infection on changes of temperatures in whole-plant corn silages during aerobic exposure

ambient temperature。

3 讨论

青贮饲料有氧腐败是指在青贮饲料开窖或取饲过程中,青贮饲料暴露于有氧环境中,好氧性微生物如酵母菌和霉菌等快速增殖,造成营养损失并发生霉变的现象[5]。青贮饲料有氧暴露过程中,活性被抑制的好氧性微生物能快速复苏,以WSC或乳酸为底物快速增殖,随着微生物代谢的加强pH快速升高并伴随着温度的上升[17]。因此,pH和温度变化通常被用作评估青贮饲料有氧腐败的标准之一,且通常认为青贮饲料温度高于环境温度2 ℃[18]或pH超过初始青贮饲料0.5时认为青贮饲料发生有氧腐败[19]。在本研究中,各处理全株玉米青贮饲料有氧暴露期间pH在有氧暴露第4天上升,乳酸和乙酸含量呈现下降趋势,其中LS2处理无论是否遭受侵染pH上升均缓于其他组,这可能与其较低的氨态氮含量有关。Kung等[20]研究发现,氢氧化铵因其碱性特征,能够延缓紫花苜蓿青贮过程中pH的下降;根据Wang等[7]的试验,接种布氏乳杆菌和植物乳杆菌的玉米青贮饲料有氧暴露期间乳酸和乙酸含量呈现下降趋势,这与本试验结果一致。
本试验中,无论是否遭受霉菌侵染,除NFI组LS2处理外,其他各处理乳酸含量均在有氧暴露前2 d保持稳定,之后呈现出下降趋势,这与本研究中pH快速上升趋势一致。本研究中,NFI组LS2处理乳酸含量在有氧暴露第2天时有短暂上升,之后快速下降,这可能是由于有氧暴露前期青贮饲料表面好氧性微生物快速增殖,消耗了氧气,为青贮饲料内部造成了微厌氧环境,为乳酸菌代谢提供了条件,导致乳酸含量短暂上升。Yuan等[21]在紫花苜蓿青贮饲料有氧暴露前3 d也观测到了乳酸含量的短暂上升,之后逐渐下降。
本研究中,FI组LS2处理酵母菌和霉菌数量较高,致使该处理乳酸含量在有氧暴露期间快速下降。青贮饲料中乳酸菌是一类优势菌群,它们在青贮过程中能够高效地产生乳酸,从而降低饲料的pH,形成酸性环境,抑制有害微生物的生长。然而,当青贮饲料受到霉菌侵染时,乳酸菌的生长受到芽孢杆菌属(Bacillus)和类芽孢杆菌属(Paenibacillus)等菌群的影响,导致青贮结束时乳酸和乙酸的含量较低,对不良微生物的抑制作用较弱[22]。本试验中,无论是否遭受霉菌侵染,各处理乙酸含量在整个有氧暴露期间均呈下降趋势。人工侵染的黄曲霉菌和禾谷镰刀菌通过分泌酸性代谢产物和酶类,干扰乳酸发酵过程,导致乳酸和乙酸含量的下降。青贮饲料中接种乳酸菌降低了发酵期间WSC的损耗,因此各接种剂处理WSC含量均高于CON处理。Ranjit等[23]研究发现,玉米青贮时组合接种植物乳杆菌和戊糖片球菌提高了发酵效率,保留了更多的WSC。
丝状真菌侵染饲料时,它们可能会形成一种特殊的生态位,为酵母菌提供庇护和生存空间。这种空间结构的形成为酵母菌在青贮环境中的存活提供了有利条件。因此,即使在有氧暴露的初期,酵母菌也能在丝状真菌的帮助下快速增殖,S6-2和GD2-1处理酵母菌和霉菌数量随有氧暴露天数的延长呈现先下降后上升的趋势,这可能是由于乳酸菌的发酵作用,产生了大量的乳酸和其他有机酸,抑制了不良微生物的活性,随着有氧暴露时间的延长,乳酸和乙酸等有机酸逐渐被挥发代谢,pH升高,使得酵母菌和霉菌又逐渐滋生[24]。Carvalho等[25]试验表明,乙酸等有机酸对酵母菌和霉菌具有抑制作用,另外乳酸菌能够通过引发酵母菌细胞膜的去极化作用,进而与真菌形成共聚集现象,这种相互作用会对酵母菌的生长产生阻碍作用。
本研究中,有氧暴露4 d后,FI组AFB1含量高于NFI组,田间侵染的黄曲霉菌和禾谷镰刀菌在有氧条件下可复苏并由于酸性环境的存在,导致霉菌毒素等次级代谢产物的产生,如黄曲霉菌在代谢糖和乳酸的过程中产生的次级代谢产物主要是黄曲霉毒素,而禾谷镰刀菌则产生多种次级代谢产物,包括镰刀菌素和玉米赤霉烯酮等[26]。各乳酸菌接种剂处理均显著降低了AFB1含量,这可能是由于乳酸菌通过产生乳酸等有机酸,使环境的pH降低,创造了不利于霉菌生长和毒素产生的酸性环境,从而降低了毒素的产生。GD2-1处理显著降低了ZEN含量,而LS2和GD2-1处理降低了DON含量,可能是归因于接种菌株的脱毒效应。Cheli等[27]报道表明,乳酸菌可能将DON转化为毒性更低的脱氧雪腐镰刀菌烯醇-3-葡萄糖苷,并将ZEN转化为α-玉米赤霉烯醇和β-玉米赤霉烯醇。
在有氧暴露期间,NFI组S6-2和GD2-1处理维持有氧稳定时间均有所提前,可能是因为NFI组S6-2和GD2-1处理有较高的乳酸和WSC含量,为有氧暴露期间酵母菌和霉菌发酵提供了底物,加速了有氧腐败。Filya[28]研究表明,在小麦、高粱和玉米青贮中单独接种同型发酵植物乳杆菌显著提高了乳酸含量,加速了有氧腐败。无论是否遭受霉菌侵染,LS2处理均维持了最长的有氧稳定时间,可能与其在青贮结束时较高的乙酸含量有关,乙酸可以有效抑制酵母菌和霉菌,进而提高有氧稳定性。Kung等[5]研究表明,接种布氏乳杆菌40788提高了苜蓿青贮56 d后pH和乙酸含量,并有效延长了苜蓿青贮饲料有氧稳定时间。

4 结论

① 青贮饲料有氧暴露后,有害微生物如酵母菌和霉菌快速增殖,导致青贮饲料品质下降并发生有氧腐败,而田间霉菌侵染使全株玉米青贮饲料品质进一步下降并导致有氧腐败提前。
② 接种副干酪乳杆菌LS2的全株玉米青贮饲料在有氧暴露期间始终保持着较高的乳酸和乙酸含量。
③ 接种各乳酸菌接种剂降低了全株玉米青贮饲料AFB1含量,接种干酪乳杆菌GD2-1降低了ZEN含量,接种副干酪乳杆菌LS2和干酪乳杆菌GD2-1降低了DON含量。
④ 无论是否遭受霉菌侵染,接种副干酪乳杆菌LS2的全株玉米青贮饲料均维持了最高的有氧稳定性。

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