副干酪乳杆菌对青贮苜蓿有氧暴露品质和细菌多样性的影响

doi:10.13560/j.cnki.biotech.bull.1985.2021-0717

摘要/Abstract

摘要：

采用16S rRNA高通量测序技术,检测青贮苜蓿有氧暴露细菌群落动态变化,旨在为青贮苜蓿有氧暴露细菌多样性变化及其接种乳酸菌防止有氧变质提供依据。将未接种（CON）和接种副干酪乳杆菌（LCP）的苜蓿青贮发酵56 d,分析有氧暴露后0 d、7 d、14 d青贮发酵品质和细菌多样性的动态变化。结果表明,有氧暴露使未接种青贮苜蓿pH上升,乳酸含量下降。相比CON,接种副干酪乳杆菌在有氧暴露期间pH升高幅度更小,乳酸含量更高（P<0.05）。CON主要有乳杆菌属、肠杆菌属和肠球菌属为主,其中肠杆菌属和肠球菌属丰度逐渐下降;LCP主要以乳杆菌属为主,其丰度逐渐下降。此外,有氧暴露14 d时,LCP降低了醋杆菌属的丰度。关联性分析发现,乳杆菌属与pH呈负相关（P<0.05）,肠杆菌属、肠球菌属、魏氏菌属、Cedecea、Sporolactobacillus与pH呈正相关（P<0.05）,醋杆菌属Acetobacter与乳酸和乙酸负相关（P< 0.05）。综上,本研究发现醋杆菌属在长期有氧暴露的青贮苜蓿中大量存在,降低了乳酸和乙酸的含量。接种副干酪乳杆菌可提高有氧暴露青贮苜蓿乳杆菌属的丰度,降低长期（14 d）有氧暴露醋杆菌属的丰度。因此青贮苜蓿接种副干酪乳杆菌可改善短期有氧暴露青贮品质,提高其有氧稳定性,减缓有氧变质。

关键词: 青贮苜蓿, 有氧暴露, 副干酪乳杆菌, 细菌多样性

Abstract:

16S rRNA high-throughput sequencing technology was used to detect the dynamic variations of bacterial community in the silage alfalfa after aerobic exposure,aiming to provide a basis for the dynamic changes of bacterial diversity in the aerobic exposure of silage alfalfa and the inoculation of lactic acid bacteria to prevent aerobic deterioration. Alfalfa silage without inoculation（CON）and inoculated with Lactobacillus paracasei（LCP）were fermented for 56 d,and the dynamic changes of fermentation quality and bacterial diversity of the silage at 0,7,and 14 d after aerobic exposure. The results showed that aerobic exposure resulted in the increase of the pH of uninoculated silage alfalfa and the decrease of the lactic acid content. Compared with CON,inoculation of L. paracasei had a smaller increase in pH but higher lactic acid content during aerobic exposure（P<0.05）. CON was mainly composed of Lactobacillus,Enterobacter,and Enterococcus,among which the abundance of Enterobacter and Enterococcus gradually decreased;the LCP group was mainly dominated by Lactobacillus,and its abundance gradually decreased. In addition,abundance of Acetobacter appeared after 14 d of aerobic exposure. Correlation analysis revealed that Lactobacillus was negatively correlated with pH（P<0.05）. Enterobacter,Enterococcus,Wierella,Cedecea,Sporolactobacillus were positively correlated with pH. Acetobacter was negatively correlated with lactic acid and acetic acid（P<0.05）. In summary,this study found that Acetobacter was abundant in the silage alfalfa exposed to long-term aerobic exposure,reducing the content of lactic acid and acetic acid. Inoculation of L. paracasei may increase the abundance of Lactobacillus and reduce the abundance of Acetobacter in long-term（14 d）aerobic exposure. Therefore,inoculation of silage alfalfa with L. paracasei can improve the quality of short-term aerobic exposure silage,enhance its aerobic stability,and slow down aerobic deterioration.

Key words: silage alfalfa, aerobic exposure, Lactobacillus paracasei, bacterial diversity

王琦, 武之绚, 陈钟玲, 吴白乙拉, 胡宗福, 牛化欣. 副干酪乳杆菌对青贮苜蓿有氧暴露品质和细菌多样性的影响[J]. 生物技术通报, 2021, 37(9): 77-85.

WANG Qi, WU Zhi-xuan, CHEN Zhong-ling, WU Bai-yi-la, HU Zong-fu, NIU Hua-xin. Effects of Lactobacillus paracasei on the Quality and Bacterial Diversity of Silage Alfalfa After Aerobic Exposure[J]. Biotechnology Bulletin, 2021, 37(9): 77-85.

图/表 11

表1 有氧暴露苜蓿青贮品质

Table 1 Fermentation quality of alfalfa silage during aerobic exposure from 0 to 14 d

组别Group	鲜样Fresh sample	PO0		PO7		PO14		SEM	ANOVA
组别Group	鲜样Fresh sample	CON	LCP	CON	LCP	CON	LCP	SEM	I	S	I×S
DM /%	23.92	20.85	22.26	20.57	21.49	20.79	21.72	0.48	**	NS	NS
pH	6.27	5.46	4.55	5.44	4.74	6.43	4.83	0.01	**	**	**
LAB/（log₁₀ CFU/g FM）	5.53	7.22	7.68	6.56	7.34	5.38	6.28	0.06	**	**	**
Yeasts/（log₁₀CFU/g FM）	3.76	3.28	2.56	4.46	3.90	7.13	6.07	0.09	**	**	**
Molds/（log₁₀ CFU/g FM）	3.92	2.80	<2.00	3.08	3.51	6.24	4.68	0.09	—	—	—
LA/（g·kg^-1 DM）		31.67	48.61	11.56	26.85	4.73	15.49	1.04	**	**	**
AA/（g·kg^-1 DM）		18.32	15.88	11.55	12.52	7.43	10.41	0.63	**	**	**
PA/（g·kg^-1 DM）		3.75	2.64	1.56	1.24	0.82	0.68	0.28	*	**	NS
BA/（g·kg^-1 DM）		1.43	0.45	2.01	1.48	3.21	2.61	0.04	**	**	**

图1 青贮苜蓿菌群稀释曲线（A）和Shannon曲线（B） CONPO0、CONPO7和CONPO14分别表示对照组有氧暴露0 d、7 d和14 d;LCPPO0、LCPPO7和LCPPO14分别表示干酪乳杆菌接种处理组有氧暴露0 d、7 d和14 d Number of sampled reads

Fig.1 Rarefaction curves（A）and Shannon curves（B）of bacterial community in alfalfa silage CONPO0,CONPO7 and CONPO14 represent the control group aerobic exposure 0,7 and 14 d,respectively. LCPPO0,LCPPO7 and LCPPO14 represent the Lactobacillus casei inoculation treatment group aerobic exposure 0,7 and 14 d,respectively

表2 青贮苜蓿有氧暴露期间菌群多样性指数

Table 2 Alpha-diversity of bacterial community in the silage alfalfa during aerobic exposure

Sample\Estimators	Sobs	Shannon	Simpson	Ace	Chao
CONPO0_1	43.67	2.12	0.18	53.25	49.37
CONPO7_1	59.33	1.98	0.22	71.99	67.07
CONPO14_1	62.00	1.69	0.33	79.05	74.23
LCPPO0_1	43.00	1.21	0.41	79.80	55.789
LCPPO7_1	47.00	1.08	0.49	76.73	60.91
LCPPO14_1	44.00	1.33	0.38	92.71	67.73
SEM	2.66	0.10	0.03	5.65	3.22
P	0.092	0.001	0.004	0.552	0.263

图2 青贮苜蓿菌群Shannon指数和 Simpsom指数差异显著性分析

Fig. 2 Analysis on the difference between Shannon index and Simpson indexes of bacterial community in the silage alfalfa

图3 青贮苜蓿有氧暴露期间NMDS分析

Fig. 3 NMDS analysis of the silage alfalfa during aerobic exposure

图4 门水平上青贮苜蓿细菌菌群组成

Fig. 4 Composition of the silage alfalfa bacterial commu-nity at phylum level

图5 属水平上青贮苜蓿有氧暴露各阶段菌群演替

Fig. 5 Succession of bacterial community in the silage alfa-lfa at different stages of aerobic exposure at genus level

图6 种水平上青贮苜蓿有氧暴露各阶段菌群组成

Fig. 6 Composition of bacterial community in the silage alfalfa at different stages of aerobic exposure at species level

表3 青贮苜蓿有氧暴露各时段OTU组成

Table 3 OTU composition of the silage alfalfa in different exposure time and treatments（>1%）

Genus	OTU	CONPO0	CONPO7	CONPO14	LCPPO0	LCPPO7	LCPPO14
Acetobacter	OTU53	0	0.03	34.99	0	0.27	18.37
Lactobacillus	OTU123	0	0	0	4.08	0.35	0.46
Pediococcus	OTU58	0	0	0	0.52	1.2	0.13
Weissella	OTU26	4.17	3.71	1.58	0	0.01	0
Komagataeibacter	OTU125	0	0		0	0	5.58
Cedecea	OTU45	2.02	1.81	3.29	0	0	0
Lactobacillus	OTU127	0.65	0.48	0.19	0	0.01	2.29
Lactobacillus	OTU133	0.64	1.7	0.84	9.2	17.81	21.23
Lactobacillus	OTU41	2.63	34.63	28.38		0.18	3.97
Lysinibacillus	OTU57	0	0	3.81	0	0	0
ALCPaligenes	OTU35	0	0	0.36	0	0	0.03
Sporolactobacillus	OTU29	1.1	0.12	0.03	0	0	0
Lactobacillus	OTU27	8.52	10.71	8.47	0.06	0.05	0.13
Lactobacillus	OTU153	0.32		0.05	52.99	65.39	42.11
Garciella	OTU11	7.74	0	0	0	0	0
Lactobacillus	OTU54	4.3	3.17	2.54	0	0	0
Enterobacter	OTU38	32.58	19.35	6.59	0.94	1.19	0.43
Enterococcus	OTU21	13.12	6.84	3.19	0.04	0.03	0.03
Lactobacillus	OTU104	3.74	1.19	0.27	26.65	9.07	3.81
Lactobacillus	OTU76	1.23	8.69	1.47	0.07	0.05	0.06
Lactobacillus	OTU148	0	0	0	1.98	1.74	0.2
Lactobacillus	OTU115	0.01	0.01	0	2.54	1.69	0.85
Lactobacillus	OTU71	2.95	4.36	1.68	0	0	0
Enterococcus	OTU14	3.79	1.34	0.67	0	0	0

图7 LEfSe分析青贮苜蓿组间丰度差异显著的物种（LAD>4.0）不同颜色节点表示在对应组别中显著富集,且对组间差异存在显著影响的微生物类群;淡黄色节点表示在不同分组中均无显著差异,或对组间差异无显著影响的微生物类群

Fig. 7 LEfSe analysis of the bacteria taxon with significant difference between treatments（LAD>4.0） Nodes with different colors indicate microbial groups that are significantly enriched in the corresponding groups and have a significant impact on the differences between groups. Light yellow nodes indicate microbial groups that have no significant differences in different groups or have no significant impact on differences between groups

图8 青贮细菌菌群与青贮品质关联性热图分析 X轴和Y轴分别为环境因子和物种,通过计算获得相关性R值和P值。右侧图例是不同R值的颜色区间。*表示0.01< P≤0.05,**表示0.001<P ≤0.01,***表示P≤0.001

Fig. 8 Heatmap analysis of correlation between silage quality and bacterial community The X-axis and Y-axis are environmental factors and species,respectively,and the correlation R value and P value are obtained through calculation. The legend on the right is the color interval for different R values. * refers to 0.01<P≤0.05,** refers to 0.001<P≤0.01,*** refers to P≤0.001

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