摘要
目的
为探索冻融条件对燕麦青贮在有氧暴露阶段的影响。
方法
将燕麦在冻融条件(20 ℃和-5 ℃每12 h交替1次,S组)下青贮60 d后进行有氧暴露监测,并以恒温20 ℃ (20组)作为对照。分别在青贮60 d和有氧暴露1、3、5 d取样进行发酵品质和营养品质的测定,同时进行16S rRNA基因和ITS测序。
结果
随着有氧暴露时间的延长pH值升高。与原冻融温度下的有氧暴露相比,室温下的有氧暴露导致pH值和氨态氮含量上升更快,乳酸和乙酸含量下降也更快。S组,特别是室温下的有氧暴露条件下,丙酸和丁酸的生成速度更快。随着有氧暴露的推进,肠杆菌和酵母菌数量不断增加,而乳酸菌数量不断减少(P<0.05)。细菌的Shannon指数和Simpson指数随着有氧暴露时间的延长而增加,乳酸菌的丰度不断降低。有氧暴露第3天时,细菌群落结构出现明显差异。与原温度下的有氧暴露相比,室温下的有氧暴露加速了微生物的更替。S组中指征腐败的酵母菌和霉菌的种类及数量更多。
结论
冻融条件加速了燕麦青贮在有氧暴露阶段的变败过程,尤其是在室温下进行有氧暴露时。本研究为高寒区燕麦青贮的高品质调制及存放提供理论指导。
发酵后的出料标志着青贮过程的结束。除了良好的发酵品质,有氧稳定性也是确保饲喂效果的关键因素。通过研究冻融对燕麦青贮发酵过程的影响,发现冻融不仅作用于发酵阶段的青贮品质,而且在开窖后对青贮的有氧稳定性也起着至关重要的作
冻融研究是发酵过程研究的重要组成部分。冻融引发的应激是一个复杂的过程,可能包括脱水作用、物理应激、低温应激和氧化应激
为全面了解冻融对燕麦青贮有氧稳定性的影响,本研究在燕麦青贮60 d后进行有氧暴露试验,旨在探究其在有氧暴露阶段的品质变化及微生物演替情况。
1 材料与方法
1.1 青贮调制
试验材料选用乳熟期的燕麦,留茬高度为10 cm。刈割后的燕麦使用揉丝机切割并揉丝成1-2 cm长度,经短暂晾晒后充分混匀。在所有材料充分混合后,随机取约700 g的材料装入无菌抽真空袋(直径×高度:20 cm×30 cm),并进行抽真空处理。试验设计采用双因素完全随机方式,温度分别是恒温20 ℃ (20组)和冻融条件(S组,在20 ℃和-5 ℃每12 h交替1次,即20 ℃/-5 ℃,温度切换时以每分钟0.3 ℃的速率变化直到稳定),取样时间点设定为青贮后的60 d,以及有氧暴露后的1、3、5 d,每个处理设置3个重复。
1.2 有氧稳定性监测
在青贮60 d后,从每个处理中均匀取出1 kg的样品放入2 L的灭菌烧杯中压实,并用两层灭菌棉纱布覆盖。将样品分别在其原始处理温度和室温(25±2) ℃条件下放置5 d。使用实时温度记录仪(神华科技股份有限公司)每5 min测量1次青贮核心区域(深度为10 cm)的温度,持续检测。分别在有氧暴露后的1、3、5 d分别取样,进行微生物计数和发酵品质分析。有氧稳定性的评估基于暴露于空气中的青贮饲料的温度超过基准环境温度2 °C所持续的时
1.3 青贮发酵品质和营养成分分析
样品于65 ℃烘箱中烘干至恒重,以测定其干物质含量(dry matter, DM)。粉碎后过0.6 mm筛用于测定其他营养成分。粗蛋白含量(crude protein, CP)采用杜马斯燃烧定氮法测
1.4 微生物计数
青贮微生物计数采用平板计数
1.5 微生物测序
将20 g的青贮样品中加入180 mL灭菌蒸馏水,在4 ℃振荡30 min,通过两层灭菌纱布过滤后4 ℃、10 000×g离心10 min,倒掉上清液,加入3 mL灭菌蒸馏水重制成混合菌液。采用CTAB方法对样品的基因组DNA进行提取,之后使用琼脂糖凝胶电泳检测提取出的DNA纯度和浓度。随后取适量DNA于离心管中,并用无菌水稀释至1 ng/μL。以稀释后的基因组DNA为模板,根据扩增区域选择使用带barcode的特异性引物、New England Biolabs公司的Phusio
采用SPSS (IBM SPSS Statistics 26)软件进行双因素方差分析,旨在探讨温度和青贮阶段及其交互作用对有氧暴露阶段所有指标的影响,并应用Tukey’s HSD检验方法确定差异显著性水平(P<0.05)。
2 结果与分析
2.1 发酵品质分析
青贮60 d及开袋后有氧暴露的发酵特性如
时间Time | 温度T/℃ | pH | 氨态氮 NH3-N (%) | 乳酸 Lactic acid (mg/g) | 乙酸 Acetic acid (mg/g) | 丙酸 Propionic acid (mg/g) | 丁酸 Butyric acid (mg/g) |
---|---|---|---|---|---|---|---|
Day 60 | 20 | 4.62k | 3.50f | 48.03a | 11.87c | ND | ND |
20/-5 | 4.92h | 4.24de | 28.06c | 12.74c | ND | ND | |
AE Day 1 | AE 20 | 4.67hi | 3.88ef | 41.36b | 18.26a | ND | ND |
AE 20/-5 | 5.24g | 4.19de | 19.95d | 15.62b | ND | ND | |
RT AE 20 | 4.73hi | 3.86ef | 18.44e | 18.64a | ND | ND | |
RT AE 20/-5 | 7.10e | 4.25de | 5.75h | 14.71b | 0.44d | 1.31d | |
AE Day 3 | AE 20 | 6.71f | 4.25de | 13.90f | 13.08c | ND | ND |
AE 20/-5 | 7.76d | 4.43de | 3.05jk | 9.84d | ND | ND | |
RT AE 20 | 8.03c | 4.61d | 3.48i | 12.19c | ND | ND | |
RT AE 20/-5 | 8.46b | 4.68d | 3.99i | 7.18e | 2.44b | 3.11b | |
AE Day 5 | AE 20 | 7.71d | 6.29c | 7.68g | 9.84d | ND | ND |
AE 20/-5 | 8.09c | 6.30c | 2.17kl | 6.44ef | ND | 1.15e | |
RT AE 20 | 8.45b | 7.61b | 1.89l | 9.22d | 1.35c | 2.13c | |
RT AE 20/-5 | 8.81a | 8.58a | 1.65l | 5.48f | 2.71a | 4.40a | |
SEM | 0.091 | 0.103 | 1.003 | 0.166 | 0.133 | 0.175 | |
Period | ** | * | ** | ** | * | ** | |
Temperature | ** | ** | ** | ** | NS | ** | |
Period×Temperature | NS | NS | ** | ** | NS | NS |
同列不同小写字母表示在0.05水平上差异显著(*:P<0.05;**:P<0.01);NS:不显著;SEM:标准误;DM:干物质;AE:有氧暴露;RT:室温;ND:未检测到。
The same column followed by different lowercase letters are significantly different at the 0.05 level (*: P<0.05; **: P<0.01); NS: Not significant; SEM: Standard error of mean; DM: Dry matter; AE: Aerobic exposure; RT: Room temperature; ND: Not detected.
2.2 营养成分分析
如
时间Time | 温度T/℃ | 灰分Ash(%) | 中性洗涤纤维Neutral detergent fiber (%) | 酸性洗涤纤维Acid detergent fiber (%) | 粗蛋白 Crude protein (%) | 可溶性糖Water-soluble carbohydrates (%) |
---|---|---|---|---|---|---|
Day 60 | 20 | 10.14d | 56.98b | 35.97b | 8.67a | 4.96a |
20/-5 | 11.26c | 59.03b | 40.81ab | 8.93a | 4.84ab | |
AE Day 5 | AE 20 | 11.91b | 69.64a | 43.85a | 7.54b | 4.33ab |
AE 20/-5 | 11.45c | 67.83a | 41.95ab | 7.97b | 4.24ab | |
RT AE 20 | 12.79a | 70.25a | 41.01ab | 7.82b | 4.11b | |
RT AE 20/-5 | 12.47a | 67.22a | 37.26ab | 7.62b | 3.33c | |
SEM | 0.105 | 1.939 | 2.157 | 0.062 | 0.096 |
同列不同小写字母表示在0.05水平上差异显著;SEM:标准误;DM:干物质;AE:有氧暴露;RT:室温。
The same column followed by different lowercase letters are significantly different at the 0.05 level. SEM: Standard error of mean; DM: Dry matter; AE: Aerobic exposure; RT: Room temperature.
2.3 微生物计数
如
时间Time | 温度 T/℃ | 肠杆菌 Coliform bacteria | 乳酸菌 Lactic acid bacteria | 酵母菌 Yeasts | 霉菌 Molds |
---|---|---|---|---|---|
Day 60 | 20 | ND | 5.04de | ND | ND |
20/-5 | 4.42ef | 5.37c | ND | ND | |
AE Day 1 | AE 20 | ND | 5.41c | 3.82f | ND |
AE 20/-5 | 4.25f | 6.01a | 4.18ef | ND | |
RT AE 20 | ND | 4.32g | 3.00g | ND | |
RT AE 20/-5 | 4.3ef | 5.27cd | 4.42de | ND | |
AE Day 3 | AE 20 | ND | 5.39c | 5.31ab | ND |
AE 20/-5 | 6.21a | 5.77ab | 4.82c | 2.62d | |
RT AE 20 | 4.53d | 5.66b | 5.46ab | ND | |
RT AE 20/-5 | 5.12c | 5.76ab | 4.68cd | 3.93b | |
AE Day 5 | AE 20 | 2.15g | 4.96ef | 5.06bc | 2.54de |
AE 20/-5 | 6.24a | 4.79ef | 4.89c | 2.97c | |
RT AE 20 | 4.97c | 4.71f | 5.69a | 2.43e | |
RT AE 20/-5 | 5.61b | 4.35g | 5.45ab | 4.71a | |
SEM | 0.187 | 0.053 | 0.053 | 0.075 | |
Period | ** | ** | ** | ** | |
Temperature | ** | * | NS | ** | |
Period×Temperature | NS | ** | ** | ** |
同列不同小写字母表示在0.05水平上差异显著(*:P<0.05;**:P<0.01);NS:不显著;SEM:标准误;AE:有氧暴露;RT:室温;ND:未检测到。
The same column followed by different lowercase letters are significantly different (*: P<0.05; **: P<0.01). NS: Not significant; SEM: Standard error of mean; AE: Aerobic exposure; RT: Room temperature; ND: Not detected.
2.4 微生物演替分析
通过α多样性分析,发现细菌的ACE和Chao1这2个指数无显著差异,而Shannon指数和Simpson指数差异显著,20组和S组在室温下进行有氧暴露时,Shannon指数和Simpson指数不断增加,5 d后达到最高,且显著高于其他处理(P<0.05);而在原温度下进行有氧暴露时,Shannon指数和Simpson指数则先减小后增加。Shannon指数和Simpson指数与时间段和温度均呈极显著相关性(P<0.01),而时间段与温度的交互作用与Shannon指数无显著相关性,但与Simpson指数呈显著相关性(P<0.05)。真菌的α多样性指数在有氧暴露阶段均降低,且各处理间的差异显著(P<0.05)。ACE指数和Chao1指数与时间段、温度以及两者的交互作用均呈极显著相关性(P<0.01),Shannon指数和Simpson指数与时间段和温度极显著相关,但与两者的交互作用无显著相关性(
时间 Time | 温度 T/℃ | 细菌Bacteria | 真菌Fungi | ||||||
---|---|---|---|---|---|---|---|---|---|
Shannon | Simpson | Chao1 | ACE | Shannon | Simpson | Chao1 | ACE | ||
Day 60 | 20 | 3.37d | 0.81de | 283.48 | 281.74 | 4.26a | 0.87a | 549.32a | 562.44a |
20/-5 | 2.97e | 0.73g | 252.28 | 261.59 | 3.29b | 0.80a | 297.60bc | 195.57d | |
AE Day 1 | AE 20 | 2.96e | 0.79e | 227.27 | 233.76 | 1.28def | 0.41cd | 172.93def | 190.97d |
AE 20/-5 | 2.53f | 0.69h | 236.86 | 250.93 | 1.44cdef | 0.38cde | 206.11cde | 212.14d | |
RT AE 20 | 3.50cd | 0.84cd | 280.91 | 285.38 | 1.81cd | 0.48bc | 274.41bcd | 289.26c | |
RT AE 20/-5 | 3.00e | 0.74fg | 253.33 | 265.44 | 1.11ef | 0.27de | 175.26def | 195.83d | |
AE Day 3 | AE 20 | 3.02e | 0.79e | 226.15 | 241.89 | 1.72cde | 0.62b | 94.95efg | 137.5def |
AE 20/-5 | 2.98e | 0.78ef | 252.55 | 258.01 | 0.92f | 0.22e | 43.05g | 45.69g | |
RT AE 20 | 3.75bc | 0.87bc | 242.27 | 250.92 | 2.05c | 0.61b | 356.63b | 359.74b | |
RT AE 20/-5 | 3.95b | 0.88b | 248.86 | 266.45 | 1.15def | 0.32cde | 77.80fg | 85.70efg | |
AE Day 5 | AE 20 | 3.65cd | 0.87bc | 230.30 | 242.80 | 1.74cde | 0.63b | 55.79fg | 55.41g |
AE 20/-5 | 3.46d | 0.84cd | 243.80 | 259.22 | 1.16def | 0.28de | 69.30fg | 72.53fg | |
RT AE 20 | 4.44a | 0.91ab | 285.34 | 282.53 | 1.21def | 0.42cd | 84.38efg | 85.29efg | |
RT AE 20/-5 | 4.70a | 0.94a | 286.79 | 286.45 | 1.50cdef | 0.40cde | 148.38efg | 157.55de | |
SEM | 0.061 | 0.007 | 5.037 | 4.896 | 0.061 | 0.018 | 13.865 | 10.661 | |
Period | ** | ** | NS | NS | ** | ** | ** | ** | |
Temperature | ** | 0.003** | NS | NS | ** | ** | 0.001** | ** | |
Period×Temperature | 0.054 | 0.031* | NS | NS | NS | 0.05 | 0.005** | ** |
同列不同小写字母表示在0.05水平上差异显著(*:P<0.05;**:P<0.01);NS:不显著;SEM:标准误;AE:有氧暴露;RT:室温。
The same column followed by different lowercase letters are significantly different (*: P<0.05; **: P<0.01). NS: Not significant; SEM: Standard error of mean; AE: Aerobic exposure; RT: Room temperature.
S组中促生乳杆菌属(Levilactobacillus) 的相对丰度较高,而20组中广布乳杆菌属(Latilactobacillus)和芽孢杆菌属(Bacillus)的相对丰度则较为显著(

图1 有氧暴露过程中细菌和真菌群落分析。A:20组与S组在有氧暴露阶段的细菌相对丰度比较;B:20组与S组在有氧暴露各个阶段的细菌相对丰度;C:20组与S组在有氧暴露阶段的真菌相对丰度比较;D:20组与S组在有氧暴露各个阶段的真菌相对丰度;E:有氧暴露5 d后各个处理细菌韦恩图;F:有氧暴露5 d后各个处理真菌韦恩图。A表示原温度有氧暴露;RA表示室温下有氧暴露。
Figure 1 Bacterial and fungal community during aerobic exposure. A: Comparison of the relative abundance of bacteria during the aerobic exposure phase between group 20 and group S; B: The relative abundance of bacteria at various days of aerobic exposure between group 20 and group S; C: Comparison of relative abundance of fungi during the aerobic exposure phase between group 20 and group S; D: The relative abundance of fungi at various days of aerobic exposure between group 20 and group S; E: Venn plots of bacteria after 5 days aerobic exposure; F: Venn plots of fungi after 5 days aerobic exposure. A: Aerobic exposure under freeze-thaw; RA: Aerobic exposure at room temperature.
细菌LEfSe分析(

图2 有氧暴露阶段细菌LEfSe分析。A:60 d开袋;B:有氧暴露1 d;C:有氧暴露3 d;D:有氧暴露5 d。
Figure 2 Bacterial variations in samples during aerobic exposure using LEfSe analysis. A: 60 days silage; B: Aerobic exposure for 1 day; C: Aerobic exposure for 3 days; D: Aerobic exposure for 5 days.
根据真菌LEfSe分析(

图3 有氧暴露阶段真菌LEfSe分析。A:60 d开袋;B:有氧暴露1 d;C:有氧暴露3 d;D:有氧暴露5 d。
Figure 3 Fungal variations in samples during aerobic exposure using LEfSe analysis. A: 60 days silage; B: Aerobic exposure for 1 day; C: Aerobic exposure for 3 days; D: Aerobic exposure for 5 days.
通过matestate分析(

图4 细菌(A)和真菌(B)群落的matestate分析
Figure 4 Matestate analysis of bacterial (A) and fungal (B) communities. *: P<0.05.
通过细菌与发酵指标的相关性分析(

图5 有氧暴露阶段20组(A)和S组(B)的细菌群落与发酵指标的相关性分析
Figure 5 The correlation analysis of silage quality and differential bacteria communities at 20 ℃ (A) and freeze-thaw condition (B) during aerobic exposure. *: P<0.05; **: P<0.01.
通过真菌与发酵指标的相关性分析(

图6 有氧暴露阶段20组(A)和S组(B)的真菌群落与发酵指标的相关性分析
Figure 6 The correlation analysis of silage quality and differential fungal communities at 20 ℃ (A) and freeze-thaw condition (B) during aerobic exposure. *: P<0.05, **: P<0.01.
3 讨论
3.1 有氧暴露后青贮发酵品质和营养成分的变化
青贮饲料的有氧稳定性是确保其营养价值不受损失、有效抑制霉菌孢子及霉菌毒素污染,进而为动物提供优质饲料的关键。青贮60 d后,S组的pH值显著高于20组,且2个处理下的pH值均高于优质青贮通常要求的pH 4.2,这可能受到贮存温度和原料附生乳酸菌数量的共同影响。研究表明低温(<20 ℃)会抑制青贮发酵,导致pH值偏高、pH下降速率减
3.2 有氧暴露后细菌多样性的变化及其影响
青贮60 d后,20组和S组中的乳酸菌相关OTUs占主导地位,但其丰度相对较低,并非绝对优势菌,这与前人的研究结果不一
3.3 有氧暴露后真菌多样性的变化及其影响
青贮开袋前后的真菌群落结构存在显著差异。与发酵指标呈正相关的Blumeria和Vishniacizyma等真菌迅速减少,毕赤酵母(Pichia)、Wickerhamomyces和Issatchenkia等成为优势菌种。在大多数青贮饲料有氧暴露后,酵母菌是导致其腐败的主要原因,其中具有消耗乳酸作用的酵母,如Issatchenkia、Saccharomyces、Candida和毕赤酵母(Pichia),通常是青贮饲料有氧腐败的主要引发
4 结论
有氧暴露后,酵母和霉菌迅速代谢乳酸,导致pH值不断升高,乳酸和乙酸含量降低。同时,细菌和真菌的种群结构也发生了变化,S组的群落更替较快,尤其是在室温下有氧暴露时,S组中的好氧腐败菌更多。这表明冻融加速了有氧变败过程,尤其是在室温下有氧暴露。
作者贡献声明
李海萍:研究构思和设计,论文撰写和修改;贾志锋:数据收集和处理;刘文辉:数据收集和处理;马祥:数据收集与处理;周青平:论文修改;关皓:研究构思和设计。
利益冲突
作者声明不存在任何可能会影响本文所报告工作的已知经济利益或个人关系。
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