耐低营养乳酸菌筛选及对难青贮牧草发酵品质的影响

doi:10.11686/cyxb2022395

摘要/Abstract

摘要：

水溶性碳水化合物（WSC）含量低和乳酸菌附着数量少的牧草难以成功青贮，通常添加糖类物质和乳酸菌改善其青贮发酵品质，但成本较高。本研究通过配制不同糖含量（0.002、0.02、0.2、2和20 g·L^-1）和pH（4.0和4.5）的培养基，筛选耐低营养且耐酸的乳酸菌，添加到不易青贮的柱花草和苏丹草中，研究其对改善发酵品质的效果。结果表明：初筛得到10株较耐低营养的乳酸菌，其中菌株SCLN₁和HT₁耐低营养和耐酸性均优、菌株LM₁为较耐低营养的耐酸菌株。SCLN₁经16S rDNA和生理生化特性鉴定后为植物乳杆菌，LM₁和HT₁为实验室保存菌株，分别为肠膜明串珠菌和鼠李糖乳杆菌。添加到柱花草和苏丹草青贮60 d后，未加菌的发酵品质均较差，pH高于5.0，乳酸含量低于2% DM，氨态氮（NH₃-N）含量分别高达30.50% TN和29.01% TN；添加乳酸菌显著改善了2种牧草的青贮发酵品质，尤其是单独添加菌株SCLN₁，显著降低了柱花草和苏丹草的pH（从5.36和5.19分别降至4.67和4.10）和NH₃-N含量（从30.50% TN和29.01% TN分别降至11.24% TN和12.69% TN），增加了乳酸含量（从2.00% DM和1.82% DM分别增加至4.16% DM和8.29% DM）；单独添加SCLN₁的效果显著优于其他菌株单独添加或与SCLN₁混合添加。

关键词: 耐低营养乳酸菌, 柱花草, 苏丹草, 青贮

Abstract:

Herbage with low water-soluble carbohydrate （WSC） content or low counts of lactic acid bacteria （LAB） is difficult to ensile. Sugars or LAB are usually added to improve the fermentation quality of silage， but the cost is relatively high. In this study， low-nutrient and acid-tolerant LAB were screened by adjusting the glucose content （0.002， 0.02， 0.2， 2 and 20 g·L^-1） and pH （4.0 and 4.5） of the medium， and inoculating them to stylo （Stylosanthes guianensis） and sudan grass （Sorghum sudanense） with poor ensiling conditions to investigate the silage fermentation quality. Ten low-nutrient-tolerant LAB strains were obtained. Low-nutrient and acid-tolerant strains SCLN₁ and HT₁， and an acid-tolerant strain LM₁ with relatively weak tolerance to low nutrient conditions were also obtained. SCLN₁ was identified Lactobacillus plantarum by 16S rDNA and physiological and biochemical characteristics， and LM₁ and HT₁ stored in the laboratory were identified as Leuconostoc mesenteroides and Lactobacillus rhamnosus， respectively. After ensiling for 60 days， uninoculated stylo and sudan grass silages had poor fermentation quality， with pH higher than 5.0， lactic acid less than 2% DM， and ammonia nitrogen （NH₃-N） reached up to 30.50% TN and 29.01% TN， respectively. Inoculation with LAB significantly improved the fermentation quality of silages of the two tested forage species， especially inoculation with SCLN₁. For stylo and sudan grass silages， inoculation with SCLN₁ alone significantly decreased pH values （from 5.36 and 5.19 to 4.67 and 4.10， respectively） and NH₃-N content （from 30.50% TN and 29.01% TN to 11.24% TN and 12.69% TN， respectively） and increased lactic acid content （from 2.00% DM and 1.82% DM to 4.16% DM and 8.29% DM， respectively）. The effect of inoculation with SCLN₁ alone was significantly better than that of inoculation with LM₁ and HT₁ alone or mixed with SCLN₁.

Key words: low-nutrient-tolerant lactic acid bacteria, stylo, sudan grass, silage

田静, 曹彩霞, 黄莉莹, 吴娟燕, 张建国. 耐低营养乳酸菌筛选及对难青贮牧草发酵品质的影响[J]. 草业学报, 2023, 32(9): 222-230.

Jing TIAN, Cai-xia CAO, Li-ying HUANG, Juan-yan WU, Jian-guo ZHANG. Screening low-nutrient-tolerant lactic acid bacteria and their effects on the fermentation quality of silages from poor materials[J]. Acta Prataculturae Sinica, 2023, 32(9): 222-230.

图/表 8

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菌株 Strain	葡萄糖浓度Glucose concentration （g·L^-1）
菌株 Strain	0.002	0.02	0.2	2	20
SCLN₁	0.726±0.01a	0.728±0.01a	0.704±0.01b	0.902±0.01bc	2.157±0.01d
LA₁	0.709±0.01a	0.780±0.00a	0.765±0.01a	1.052±0.07ab	1.645±0.01f
LA₂	0.083±0.00f	0.087±0.00e	0.206±0.01g	0.599±0.02de	2.521±0.01a
LA₃	0.297±0.01c	0.433±0.05c	0.473±0.01c	0.473±0.20e	2.483±0.01a
LA₄	0.260±0.01d	0.285±0.01d	0.317±0.00e	0.821±0.02bcd	1.959±0.01e
LA₅	0.135±0.00e	0.167±0.02e	0.260±0.02f	0.721±0.15cde	2.117±0.00d
LA₆	0.095±0.01f	0.132±0.01e	0.373±0.01d	0.937±0.01bc	2.325±0.09bc
LM₁	0.154±0.00e	0.153±0.00e	0.203±0.00g	0.654±0.00de	2.119±0.00d
CCZZ₁	0.306±0.01c	0.459±0.09c	0.765±0.02a	0.912±0.01bc	2.236±0.00cd
HT₁	0.585±0.02b	0.590±0.01b	0.688±0.01b	1.231±0.03a	2.416±0.05ab

菌株 Strain	葡萄糖浓度Glucose concentration （g·L^-1）
菌株 Strain	0.002	0.02	0.2	2	20
SCLN₁	0.726±0.01a	0.728±0.01a	0.704±0.01b	0.902±0.01bc	2.157±0.01d
LA₁	0.709±0.01a	0.780±0.00a	0.765±0.01a	1.052±0.07ab	1.645±0.01f
LA₂	0.083±0.00f	0.087±0.00e	0.206±0.01g	0.599±0.02de	2.521±0.01a
LA₃	0.297±0.01c	0.433±0.05c	0.473±0.01c	0.473±0.20e	2.483±0.01a
LA₄	0.260±0.01d	0.285±0.01d	0.317±0.00e	0.821±0.02bcd	1.959±0.01e
LA₅	0.135±0.00e	0.167±0.02e	0.260±0.02f	0.721±0.15cde	2.117±0.00d
LA₆	0.095±0.01f	0.132±0.01e	0.373±0.01d	0.937±0.01bc	2.325±0.09bc
LM₁	0.154±0.00e	0.153±0.00e	0.203±0.00g	0.654±0.00de	2.119±0.00d
CCZZ₁	0.306±0.01c	0.459±0.09c	0.765±0.02a	0.912±0.01bc	2.236±0.00cd
HT₁	0.585±0.02b	0.590±0.01b	0.688±0.01b	1.231±0.03a	2.416±0.05ab

特性Characteristics	生长状况Growth situation	特性Characteristics	生长状况Growth situation
形状Shape	杆状Rod	NaCl生长Growth in NaCl
革兰氏染色Gram stain	+	3.0%	+
过氧化氢酶反应Catalase reaction	-	6.5%	±
发酵类型Type of fermentation	同型Homo	10.0%	-
生长温度Growth temperature		pH生长Growth at pH
10 ℃	±	3.5	±
15 ℃	+	4.0	++
45 ℃	+	4.5	++
50 ℃	±	7.5	+
		8.0	±

特性Characteristics	生长状况Growth situation	特性Characteristics	生长状况Growth situation
形状Shape	杆状Rod	NaCl生长Growth in NaCl
革兰氏染色Gram stain	+	3.0%	+
过氧化氢酶反应Catalase reaction	-	6.5%	±
发酵类型Type of fermentation	同型Homo	10.0%	-
生长温度Growth temperature		pH生长Growth at pH
10 ℃	±	3.5	±
15 ℃	+	4.0	++
45 ℃	+	4.5	++
50 ℃	±	7.5	+
		8.0	±

可利用的糖Available sugar	生长状况Growth situation	可利用的糖Available sugar	生长状况Growth situation
D/L-阿拉伯糖Arabinose	±	七叶灵Esculin	+
D-木糖Xylose	±	水杨苷Salicin	+
甲基-βD吡喃木糖苷Methyl-βD xylopyranoside	±	D-纤维二糖Cellobiose	+
D-葡萄糖Glucose	+	D-麦芽糖Maltose	+
D-果糖Fructose	+	D-蔗糖Sucrose	+
D-甘露糖Mannose	+	菊粉Inulin	±
L-鼠李糖Rhamnose	+	糖原Glycogen	±
肌醇Inositol	±	D-龙胆二糖Gentian disaccharide	+
甘露醇Mannitol	±	D-松二糖Turanose	±
N-乙酰葡萄糖苷Acetyl-glucosamine	+	D-塔格糖Tagatose	+
苦杏仁苷Amygdalin	+	葡萄糖酸盐Potassium gluconate	±
ARBULIN	+