草业学报 ›› 2023, Vol. 32 ›› Issue (9): 222-230.DOI: 10.11686/cyxb2022395
• 研究论文 • 上一篇
田静1,2(), 曹彩霞3, 黄莉莹1, 吴娟燕1, 张建国1()
收稿日期:
2022-10-06
修回日期:
2022-12-15
出版日期:
2023-09-20
发布日期:
2023-07-12
通讯作者:
张建国
作者简介:
E-mail: zhangjg@scau.edu.cn基金资助:
Jing TIAN1,2(), Cai-xia CAO3, Li-ying HUANG1, Juan-yan WU1, Jian-guo ZHANG1()
Received:
2022-10-06
Revised:
2022-12-15
Online:
2023-09-20
Published:
2023-07-12
Contact:
Jian-guo ZHANG
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。
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.
菌株 Strain | 葡萄糖浓度Glucose concentration (g·L-1) | ||||
---|---|---|---|---|---|
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 |
表1 乳酸菌在不同葡萄糖含量培养基中培养48 h的OD600值
Table 1 OD600 values of lactic acid bacteria cultured in medium with different glucose concentration for 48 h
菌株 Strain | 葡萄糖浓度Glucose concentration (g·L-1) | ||||
---|---|---|---|---|---|
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 |
图1 乳酸菌在不同pH培养基中培养48 h的OD600不同小写字母表示不同菌种在同一pH条件下差异显著(P<0.05)。Different lowercase letters indicate significant differences among different strains at the same pH (P<0.05).
Fig.1 OD600 of lactic acid bacteria cultured in different pH medium for 48 h
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 | ± |
表2 菌株SCLN1的生理生化特性
Table 2 Physiological and biochemical properties of strain SCLN1
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 | + |
表3 菌株SCLN1可利用的糖
Table 3 Available sugars of strain SCLN1
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 | + |
项目Items | 柱花草Stylo | 苏丹草Sudan grass | 显著性Significance |
---|---|---|---|
干物质Dry matter (DM, % FM) | 34.17±0.33 | 34.33±0.26 | NS |
粗蛋白Crude protein (CP, % DM) | 15.25±0.32 | 8.16±0.34 | ** |
中性洗涤纤维Neutral detergent fiber (NDF, % DM) | 68.15±2.33 | 61.42±0.92 | ** |
酸性洗涤纤维Acid detergent fiber (ADF, % DM) | 39.04±1.43 | 31.78±1.03 | ** |
水溶性碳水化合物Water-soluble carbohydrate (WSC, % DM) | 4.74±0.12 | 9.26±0.34 | ** |
pH | 5.57±0.03 | 5.51±0.02 | NS |
缓冲能Buffering capacity (mEq·kg-1 DM) | 569.97±40.12 | 441.98±34.32 | NS |
乳酸菌Lactic acid bacteria (log10 cfu·g-1 FM) | 2.32±0.13 | 2.75±0.46 | NS |
好氧细菌Aerobic bacteria (log10 cfu·g-1 FM) | 4.96±0.25 | 7.16±0.09 | ** |
酵母Yeasts (log10 cfu·g-1 FM) | 4.26±0.00 | 5.66±0.16 | ** |
霉菌Molds (log10 cfu·g-1 FM) | 1.75±0.10 | 3.75±0.10 | ** |
表4 柱花草和苏丹草青贮前的化学特性和微生物数量
Table 4 The chemical characteristic and microbial population of stylo and sudan grass prior to ensiling
项目Items | 柱花草Stylo | 苏丹草Sudan grass | 显著性Significance |
---|---|---|---|
干物质Dry matter (DM, % FM) | 34.17±0.33 | 34.33±0.26 | NS |
粗蛋白Crude protein (CP, % DM) | 15.25±0.32 | 8.16±0.34 | ** |
中性洗涤纤维Neutral detergent fiber (NDF, % DM) | 68.15±2.33 | 61.42±0.92 | ** |
酸性洗涤纤维Acid detergent fiber (ADF, % DM) | 39.04±1.43 | 31.78±1.03 | ** |
水溶性碳水化合物Water-soluble carbohydrate (WSC, % DM) | 4.74±0.12 | 9.26±0.34 | ** |
pH | 5.57±0.03 | 5.51±0.02 | NS |
缓冲能Buffering capacity (mEq·kg-1 DM) | 569.97±40.12 | 441.98±34.32 | NS |
乳酸菌Lactic acid bacteria (log10 cfu·g-1 FM) | 2.32±0.13 | 2.75±0.46 | NS |
好氧细菌Aerobic bacteria (log10 cfu·g-1 FM) | 4.96±0.25 | 7.16±0.09 | ** |
酵母Yeasts (log10 cfu·g-1 FM) | 4.26±0.00 | 5.66±0.16 | ** |
霉菌Molds (log10 cfu·g-1 FM) | 1.75±0.10 | 3.75±0.10 | ** |
牧草 Grass | 处理 Treatments | 干物质 DM (% FM) | pH | 有机酸Organic acid (% DM) | 氨态氮 NH3-N (% TN) | 微生物Microorganism (log10 cfu·g-1 FM) | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
乳酸 Lactic acid | 乙酸 Acetic acid | 丙酸 Propionic acid | 丁酸 Butyric acid | 乳酸菌 LAB | 好氧细菌Aerobic bacteria | 酵母Yeasts | 霉菌Molds | |||||
柱花草 Stylo | CK | 26.70a | 5.36a | 2.00bc | 4.82ab | 3.99a | 3.64b | 30.50a | 4.75 | 4.78 | 2.73c | <2 |
SCLN1 | 24.97b | 4.67c | 4.16a | 2.98b | 2.11b | 1.76b | 11.24d | 5.61 | 5.74 | 5.47a | <2 | |
LM1 | 24.49b | 4.93b | 2.35abc | 3.26ab | 3.34ab | 3.35b | 13.17cd | 5.39 | 4.86 | 4.49ab | <2 | |
HT1 | 24.18b | 5.00b | 1.27bc | 3.47ab | 1.53b | 4.58ab | 19.23bc | 5.18 | 5.29 | 4.03b | <2 | |
SCLN1+LM1 | 24.52b | 4.72c | 2.93ab | 3.49ab | 1.75b | 4.29ab | 10.48d | 5.81 | 5.69 | 5.10ab | <2 | |
LM1+HT1 | 24.52b | 5.23a | 0.82c | 5.31a | 1.59b | 6.83a | 24.77ab | 5.37 | 5.05 | 4.46ab | <2 | |
标准差 Standard deviation | 0.196 | 0.056 | 0.319 | 0.294 | 0.285 | 0.451 | 1.867 | 0.139 | 0.163 | 0.232 | - | |
显著性Significance | ** | ** | * | NS | * | * | ** | NS | NS | ** | - | |
苏丹草 Sudan grass | CK | 27.65b | 5.19a | 1.82b | 2.09ab | 1.76b | 3.38ab | 29.01a | 5.38 | 5.66 | 4.28 | <2 |
SCLN1 | 28.70a | 4.10c | 8.29a | 1.77ab | 2.02ab | 0.00c | 12.69c | 5.68 | 4.99 | 4.13 | <2 | |
LM1 | 27.97ab | 4.72b | 2.40b | 2.21ab | 1.32b | 2.12b | 21.42b | 5.31 | 5.22 | 4.35 | <2 | |
HT1 | 27.77ab | 4.85b | 1.89b | 1.46b | 1.18b | 3.58ab | 15.92bc | 5.10 | 5.09 | 3.53 | <2 | |
SCLN1+LM1 | 26.98b | 4.79b | 2.46b | 2.75a | 2.99a | 3.63ab | 16.52bc | 5.45 | 5.42 | 4.08 | <2 | |
LM1+HT1 | 27.17ab | 5.07a | 1.24b | 2.47ab | 1.96ab | 3.94a | 20.61b | 5.15 | 5.06 | 3.60 | <2 | |
标准差 Standard deviation | 0.206 | 0.076 | 0.557 | 0.148 | 0.178 | 0.351 | 1.358 | 0.173 | 0.143 | 0.180 | - | |
显著性Significance | NS | ** | ** | NS | * | ** | ** | NS | NS | NS | - |
表5 添加耐低营养乳酸菌对柱花草和苏丹草青贮发酵品质的比较
Table 5 Comparison of the fermentation quality of stylo and sudan grass silage inoculated low-nutrient-tolerant lactic acid bacteria
牧草 Grass | 处理 Treatments | 干物质 DM (% FM) | pH | 有机酸Organic acid (% DM) | 氨态氮 NH3-N (% TN) | 微生物Microorganism (log10 cfu·g-1 FM) | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
乳酸 Lactic acid | 乙酸 Acetic acid | 丙酸 Propionic acid | 丁酸 Butyric acid | 乳酸菌 LAB | 好氧细菌Aerobic bacteria | 酵母Yeasts | 霉菌Molds | |||||
柱花草 Stylo | CK | 26.70a | 5.36a | 2.00bc | 4.82ab | 3.99a | 3.64b | 30.50a | 4.75 | 4.78 | 2.73c | <2 |
SCLN1 | 24.97b | 4.67c | 4.16a | 2.98b | 2.11b | 1.76b | 11.24d | 5.61 | 5.74 | 5.47a | <2 | |
LM1 | 24.49b | 4.93b | 2.35abc | 3.26ab | 3.34ab | 3.35b | 13.17cd | 5.39 | 4.86 | 4.49ab | <2 | |
HT1 | 24.18b | 5.00b | 1.27bc | 3.47ab | 1.53b | 4.58ab | 19.23bc | 5.18 | 5.29 | 4.03b | <2 | |
SCLN1+LM1 | 24.52b | 4.72c | 2.93ab | 3.49ab | 1.75b | 4.29ab | 10.48d | 5.81 | 5.69 | 5.10ab | <2 | |
LM1+HT1 | 24.52b | 5.23a | 0.82c | 5.31a | 1.59b | 6.83a | 24.77ab | 5.37 | 5.05 | 4.46ab | <2 | |
标准差 Standard deviation | 0.196 | 0.056 | 0.319 | 0.294 | 0.285 | 0.451 | 1.867 | 0.139 | 0.163 | 0.232 | - | |
显著性Significance | ** | ** | * | NS | * | * | ** | NS | NS | ** | - | |
苏丹草 Sudan grass | CK | 27.65b | 5.19a | 1.82b | 2.09ab | 1.76b | 3.38ab | 29.01a | 5.38 | 5.66 | 4.28 | <2 |
SCLN1 | 28.70a | 4.10c | 8.29a | 1.77ab | 2.02ab | 0.00c | 12.69c | 5.68 | 4.99 | 4.13 | <2 | |
LM1 | 27.97ab | 4.72b | 2.40b | 2.21ab | 1.32b | 2.12b | 21.42b | 5.31 | 5.22 | 4.35 | <2 | |
HT1 | 27.77ab | 4.85b | 1.89b | 1.46b | 1.18b | 3.58ab | 15.92bc | 5.10 | 5.09 | 3.53 | <2 | |
SCLN1+LM1 | 26.98b | 4.79b | 2.46b | 2.75a | 2.99a | 3.63ab | 16.52bc | 5.45 | 5.42 | 4.08 | <2 | |
LM1+HT1 | 27.17ab | 5.07a | 1.24b | 2.47ab | 1.96ab | 3.94a | 20.61b | 5.15 | 5.06 | 3.60 | <2 | |
标准差 Standard deviation | 0.206 | 0.076 | 0.557 | 0.148 | 0.178 | 0.351 | 1.358 | 0.173 | 0.143 | 0.180 | - | |
显著性Significance | NS | ** | ** | NS | * | ** | ** | NS | NS | NS | - |
图3 添加耐低营养乳酸菌青贮柱花草和苏丹草的发酵品质评分CK:对照青贮饲料;SCLN1:添加植物乳杆菌;LM1:添加肠膜明串珠菌;HT1:添加鼠李糖乳杆菌;SCLN1+LM1:植物乳杆菌和肠膜明串珠菌混合添加;LM1+HT1:肠膜明串珠菌和鼠李糖乳杆菌混合添加。不同小写字母表示同种牧草不同处理间差异显著(P<0.05)。CK: Control silage; SCLN1: Silage inoculatedwith L. plantarum; LM1: Silage inoculatedwith L. mesenteroides; HT1: Silage inoculatedwith L. rhamnosus; SCLN1+LM1: Silage inoculatedwith L. plantarum and L. mesenteroides; LM1+HT1: Silage inoculatedwith L. mesenteroides and L. rhamnosus.Different lowercase letters differ significantly among treatments of the same grass at P<0.05.
Fig.3 Fermentation quality scores of stylo and sudan grass silage inoculated low-nutrient-tolerant lactic acid bacteria
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