添加不同生物益生菌对木薯块根青贮品质和微生物菌群多样性的影响

潘佳慧; 喻珊; 蔡杰; 李开绵; 欧文军; 王志勇

doi:10.11829/j.issn.1001-0629.2021-0285

添加不同生物益生菌对木薯块根青贮品质和微生物菌群多样性的影响

1.
热带特色林木花卉遗传与种质创新教育部重点实验室 / 海南大学林学院热带作物学院，海南海口 570228
2.
中国热带农业科学院热带作物品种资源研究所，海南海口 571101

基金项目: 科技基础资源调查专项(2017FY100601)；现代农业产业技术体系建设专项资金资助(CARS-11-HNOWJ)

摘要: 以华南9号鲜木薯(Manihot esculenta)块根为材料，设4个处理组，试验组分别添加微生物发酵菌剂(C₁)、高效复合菌酶制剂(C₂)和生物饲料发酵剂(C₃)，对照组(CK)添加等体积蒸馏水，充分混合后密闭发酵30 d开封测定其营养成分、发酵品质及微生物群落结构，旨在探究添加不同生物益生菌对木薯块根的青贮品质和微生物多样性的影响。结果表明，添加生物益生菌后，各处理组的干物质和乳酸含量显著提升(P < 0.05)，可溶性糖、中性洗涤纤维含量和乳酸/乙酸无显著差异(P > 0.05)；C₂组粗蛋白含量显著低于CK组(P < 0.05)；C₁和C₃组的酸性洗涤纤维显著低于CK组(P < 0.05)，C₁组乙酸显著高于CK组(P < 0.05)；4个处理组的pH均在4.2以下，检测到少量丙酸，未检测到丁酸，且氨态氮/全氮均低于10%，属优良品质饲料。从微生物菌群结构看，菌群的丰度、多样性和分配的均匀程度均有所降低。在门水平上，添加生物益生菌后各处理组厚壁菌门(Firmicutes)丰度显著增加(P < 0.05)，蓝藻细菌门(Cyanophyta)、未分类细菌和其他细菌丰度均显著降低(P < 0.05)，C₃组变形菌门(Proteobacteria)显著降低(P < 0.05)；在属水平上，添加菌剂后乳杆菌属(Lactobacillus)和乳球菌属(Lactococcus)等有益菌属作为优势菌属丰度增高，未分类肠杆菌属和沙雷菌属(Serratia)等杂菌和有害菌属丰度则降低。综合分析可知，不同生物益生菌处理下的木薯块根青贮效果表现为C₃ > C₁ > C₂。

关键词:

English

Effect of different biological probiotics on the quality of cassava root silage and the impact on microbial flora diversity

1.
Key Laboratory of Genetics and Germplasm Innovation of Tropical Special Forest Trees and Ornamental Plants, Ministry of Education, College of Forestry & College of Tropical Crops, Hainan University, Haikou 570228, Hainan, China
2.
Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, 571101, Hainan, China

Received Date: 2021-05-09
Accepted Date: 2021-07-05
Available Online: 2021-08-30
Publish Date: 2021-11-14

Abstract: We performed an experiment using fresh cassava root (South China No. 9) tubers under four different treatments: microbial fermenting agent (C₁), high efficiency complex bacterial enzyme preparation (C₂), biological feed fermenting agent (C₃), and the control group (CK), with equal volumes of distilled water. The nutrient composition, fermentation quality, and microbial community structure were measured after 30 days of closed fermentation after the tuber material and bacterial agent were thoroughly mixed. The objective was to discover the effect of adding different biological probiotics on silage quality and microbial diversity of cassava tubers.The results showed that the dry matter and lactic acid content were significantly elevated in each treatment group after the addition of bio-probiotics (P < 0.05), and there were no significant differences in soluble sugars, neutral detergent fiber content, and lactic acid/acetic acid (P > 0.05). The crude protein content of the C₂ group was significantly lower than that of the CK group(P < 0.05). The acid detergent fiber content was significantly lower in the C₁ and C₃ groups than in the CK group (P < 0.05). The level of acetic acid in the C₁ group was significantly higher than that in the CK group (P < 0.05). The pH of all four treatment groups was below 4.2; a small amount of propionic acid was detected, but no butyric acid was detected, and ammoniacal nitrogen/whole nitrogen was below 10% in all the treatment groups. It shows that the four treatment groups are of excellent quality. The microflora structure was then analyzed and we found that the abundance, diversity, and uniformity of distribution of the flora were reduced. At the phylum level, the addition of bioprobiotics resulted in a significant increase in the abundance of the thick-walled phylum (P < 0.05), and a significant decrease in the abundance of the cyanobacterial phylum, unclassified bacteria, and other bacteria (P < 0.05). Among them, the phylum Proteobacteria was significantly reduced in group C₃ (P < 0.05). At the genus level, the addition of bio-probiotics increased the abundance of dominant genera, such as Lactobacillus and Lactococcus and decreased the abundance of miscellaneous bacteria, such as unclassified Enterobacter and Serratia. The combined analysis showed that the effect of adding different biological probiotics to cassava tubers was C₃ > C₁ > C₂.

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图 1 木薯块根青贮物种分布韦恩图

Figure 1. Venn diagram of cassava root tuber silage species distribution

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图 2 基于门水平青贮品质指标与细菌菌群相关性分析热图

DM，干物质； WSC可溶性糖；CP，粗蛋白；NDF，中性洗涤纤维；ADF，酸性洗涤纤维；LA，乳酸；AA，乙酸；PA，丙酸；BA，丁酸；AN/TN，氨态氮/全氮；r介于−1和1之间，r < 0 时为负相关，r > 0 时为正相关。颜色深浅表示相关性系数大小；下同。

Figure 2. Heat map of correlation analysis between silage quality indicators and bacterial flora based on phylum level

DM, dry matter; WSC, water soluble carbohydrates; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; LA, lactic acid; AA, acetic acid; PA, propionic acid; BA, butyric acid; AN/TN, ammonia nitrogen/total nitrogen; r is between −1 and 1. When r < 0, there is a negative correlation, and when r > 0, there is a positive correlation. Color shades indicate the magnitude of the correlation coefficient; this is applicable for the following figure as well.

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图 3 基于属水平青贮品质指标与细菌菌群相关性分析热图

Figure 3. Heat map of correlation analysis between silage quality indicators and bacterial flora based on genus level

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表 1 添加不同生物益生菌对木薯块根青贮营养成分和发酵品质的影响

Table 1 Effect of adding different biological probiotics on the nutrient composition and fermentation quality of cassava root silage

项目 Item	指标 Parameter	处理 Treatment				显著性 Significance
项目 Item	指标 Parameter	CK	C₁	C₂	C₃	显著性 Significance
营养成分 Nutrients	干物质(DM) Dry matter/%	33.62 ± 0.28b	34.97 ± 0.20a	34.92 ± 0.18a	35.06 ± 0.20a	***
	可溶性糖(WSC) Water soluble carbohydrates/%	37.98 ± 1.24ab	32.62 ± 2.30b	40.14 ± 3.03a	41.48 ± 5.27a	NS
	粗蛋白(CP) Crude protein/%	2.41 ± 0.07a	2.31 ± 0.03ab	2.23 ± 0.04b	2.32 ± 0.09ab	*
	中性洗涤纤维(NDF) Neutral detergent fiber/%	11.95 ± 0.48	12.84 ± 2.61	13.76 ± 2.55	12.30 ± 1.33	NS
	酸性洗涤纤维(ADF) Acid detergent fiber/%	5.24 ± 0.33a	4.37 ± 0.36b	4.92 ± 0.22ab	4.48 ± 0.17b	*
发酵指标 Fermentat ion index	pH	3.66 ± 0.00b	3.66 ± 0.15b	3.68 ± 0.01a	3.63 ± 0.01c	***
	乳酸(LA) Lactic acid/%	5.69 ± 0.11b	7.02 ± 0.47a	6.53 ± 0.55a	6.40 ± 0.09a	*
	乙酸(AA) Acetic acid/%	0.58 ± 0.07b	0.73 ± 0.04a	0.67 ± 0.08ab	0.64 ± 0.01ab	NS
	丙酸(PA) Propionic acid/%	0.04 ± 0.02	0.04 ± 0.02	0.04 ± 0.02	0.04 ± 0.02	NS
	丁酸(BA) Butyric acid/%	ND	ND	ND	ND
	乳酸/乙酸(LA/AA) Lactic acid/acetic acid	9.89 ± 1.10	9.68 ± 0.23	9.76 ± 0.78	9.97 ± 0.08	NS
	氨态氮/全氮(AN/TN) Ammonia nitrogen/total nitrogen	5.85 ± 0.46b	5.94 ± 0.21b	6.69 ± 0.35a	6.06 ± 0.04b	*
ND，未检测到。同行不同小写字母表示差异显著(P < 0.05)；，P < 0.05；，P < 0.01；*，P < 0.001, NS，不显著；乳酸、乙酸、丙酸、丁酸以干物质基础计算；下表同。　ND, not detected. Different lowercase letters within the same row indicate significant differences at the 0.05 level; , P < 0.05；, P < 0.01；*, P < 0.001, NS, no significant effect; Lactic acid, acetic acid, propionic acid, and butyric acid were calculated on a dry matter basis; this is applicable for the following tables as well.

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表 2 添加不同生物益生菌木薯块根青贮品质的隶属函数值

Table 2 Affiliation function values of cassava tuber silage quality under different bio-probiotic treatments

处理 Treatment	R₁	R₂	R₃	R₄	R₅	R₆	R₇	R₈	R₉	平均值 Mean	排序 Rank
CK	0.00	0.60	1.00	1.00	0.00	0.40	0.00	0.00	1.00	0.44	Ⅲ
C₁	0.94	0.00	0.44	0.51	1.00	0.40	1.00	1.00	0.98	0.70	Ⅱ
C₂	0.90	0.85	0.00	0.00	0.37	0.00	0.21	0.60	0.00	0.33	Ⅳ
C₃	1.00	1.00	0.50	0.81	0.87	1.00	0.53	0.40	0.96	0.79	Ⅰ
R₁ – R₉分别代表各处理组干物质、可溶性糖、粗蛋白、中性洗涤纤维、酸性洗涤纤维、pH、乳酸、乙酸、氨态氮/全氮的隶属函数值。　R₁ – R₉ represent the membership function values of dry matter, soluble sugar, crude protein, neutral detergent fiber, acid detergent fiber, pH, lactic acid, acetic acid, and ammonia nitrogen/total nitrogen in each treatment group, respectively.

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表 3 不同样本的Alpha多样性指数

Table 3 Alpha diversity analysis of different samples

处理 Treatment	样本序列数 Number of sample sequences	序列分类单元 OTUs	Shannon	Chao	Ace	Simpson	Shannoneven	覆盖度 Coverage
CK	39 794	141.33	1.84	174.94	171.57	0.32	0.37	0.999
C₁	44 614	136.67	1.72	155.95	158.85	0.35	0.35	0.999
C₂	42 869	141.00	1.76	163.47	166.31	0.33	0.36	0.999
C₃	44 824	135.67	1.68	160.13	158.06	0.36	0.34	0.999

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表 4 基于门水平的木薯块根青贮微生物群落结构比例

Table 4 Microbial community structure of cassava tuber silage based on phylum level

菌门 Phylum	处理 Treatment				显著性 Significance
菌门 Phylum	CK	C₁	C₂	C₃	显著性 Significance
厚壁菌门 Firmicutes	76.76 ± 0.98c	81.20 ± 0.68ab	79.59 ± 1.81b	82.30 ± 0.33a	***
变形菌门 Proteobacteria	19.12 ± 1.27a	16.91 ± 1.05ab	18.72 ± 1.83a	15.59 ± 0.30b	*
蓝藻细菌门 Cyanobacteria_Chloroplast	2.28 ± 0.74a	1.09 ± 0.31b	0.98 ± 0.21b	1.19 ± 0.06b	*
未分类细菌 Unclassified_Bacteria	1.50 ± 0.66a	0.54 ± 0.09b	0.45 ± 0.01b	0.66 ± 0.19b	*
其他 Others	0.34 ± 0.05a	0.26 ± 0.02b	0.25 ± 0.02b	0.26 ± 0.02b	*

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表 5 基于属水平的木薯块根青贮微生物群落结构比例

Table 5 Microbial community structure of cassava tuber silage based on genus level

菌属 Genus	处理 Treatment				显著性 Significance
菌属 Genus	CK	C₁	C₂	C₃	显著性 Significance
乳杆菌属 Lactobacillus	56.28 ± 2.07c	60.19 ± 0.56a	57.32 ± 1.40bc	59.33 ± 0.70ab	*
乳球菌属 Lactococcus	14.10 ± 0.71c	15.13 ± 0.44bc	16.30 ± 1.02ab	16.86 ± 0.56a	**
沙雷菌属 Serratia	6.93 ± 0.36a	5.45 ± 0.32bc	6.04 ± 0.47b	5.09 ± 0.07c	***
链球菌属 Streptophyta	2.28 ± 0.74a	1.09 ± 0.31b	0.98 ± 0.21b	1.19 ± 0.06b	*
明串珠菌属 Leuconostoc	1.79 ± 0.25a	1.25 ± 0.23b	1.11 ± 0.14b	1.27 ± 0.18b	*
魏斯氏菌属 Weissella	0.76 ± 0.09	0.97 ± 0.20	1.05 ± 0.20	1.09 ± 0.20	NS
肠球菌属 Enterococcus	1.08 ± 0.14a	0.87 ± 0.11b	0.92 ± 0.09ab	0.85 ± 0.01a	NS
不动杆菌属 Acinetobacter	0.84 ± 0.12	0.69 ± 0.04	1.19 ± 0.95	0.68 ± 0.04	NS
未分类属肠杆菌 Unclassified_Enterobacteriaceae	10.85 ± 0.76a	10.41 ± 0.75ab	11.06 ± 0.69a	9.42 ± 0.19b	NS
未分类属乳酸杆菌 Unclassified_Lactobacillales	1.04 ± 0.05	0.99 ± 0.04	1.02 ± 0.02	0.99 ± 0.06	NS
未分类的细菌 Unclassified_Bacteria	1.50 ± 0.66a	0.54 ± 0.09b	0.45 ± 0.01b	0.66 ± 0.19b	*
其他 Others	2.56 ± 0.17	2.41 ± 0.12	2.55 ± 0.13	2.57 ± 0.09	NS

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添加不同生物益生菌对木薯块根青贮品质和微生物菌群多样性的影响

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Effect of different biological probiotics on the quality of cassava root silage and the impact on microbial flora diversity

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