| 品种 Variety | 取样点 Sampling site | 收获时期 Harvest stage | 样品 Sample | 青贮时间 Silage duration/ month | 菌株总数 Total number | 序列比对结果 Sequence alignment result (> 99%) | 菌株数目 Total number |
| 雅玉8号(全株) Yayu No.8 (whole plant) | 四川宣汉 Xuanhan, Sichuan | 乳熟期 Milk stage | 原料 Raw material | 2 | 33 | Lactobacillus plantarum | 22 |
| Weissella cibaria | 1 | ||||||
| Pediococcus pentosaceus | 7 | ||||||
| Pediococcus acidilactici | 2 | ||||||
| Lactobacillus rhamnosus | 1 | ||||||
| 青贮Silage | 2 | 37 | Lactobacillus rhamnosus | 26 | |||
| Lactobacillus plantarum | 4 | ||||||
| Lactobacillus paracasei | 5 | ||||||
| Lactobacillus farraginis | 2 | ||||||
| 桂牧一号杂交象草 Hybrid elephant grass 'Guimu No.1' | 四川崇州 Chongzhou, Sichuan | 高于1.5米 Higher than 1.5 m | 原料 Raw material | 3 | 27 | Pediococcus pentosaceus | 2 |
| Lactobacillus plantarum | 22 | ||||||
| Lactobacillus salivarius | 2 | ||||||
| Pediococcus acidilactici | 1 | ||||||
| 青贮 Silage | 3 | 28 | Lactobacillus plantarum | 28 | |||
| 中单808号 (全株) Zhongdan No. 808 (whole plant) | 重庆云阳 Yunyang, Chongqing | 乳熟期 Milk stage | 原料 Raw material | 2 | 27 | Lactobacillus plantarum | 23 |
| Weissella cibaria | 2 | ||||||
| Weissella paramesenteroides | 2 | ||||||
| 青贮 Silage | 2 | 24 | Lactobacillus paracasei | 14 | |||
| Lactobacillus farraginis | 7 | ||||||
| Lactobacillus rhamnosus | 3 | ||||||
| 多玉3号 (全株) Duoyu No.3 (whole plant) | 贵州独山 Dushan, Guizhou | 乳熟期 Milk stage | 原料 Raw material | 2 | 30 | Lactobacillus plantarum | 23 |
| Pediococcus acidilactici | 5 | ||||||
| Pediococcus pentosaceus | 1 | ||||||
| Lactobacillus paracasei | 1 | ||||||
| 青贮 Silage | 2 | 21 | Lactobacillus plantarum | 20 | |||
| Lactobacillus paracasei | 1 |
引用本文:
关皓，曾泰儒，帅杨，闫艳红，张新全. 西南高温高湿地区青贮中天然乳酸菌群落结构特征及优质乳酸菌的筛选. 草业科学, 2019, 36(12): 3203-3213.
doi:
Citation: GUAN H, ZENG T R, SHUAI Y, YAN Y H, ZHANG X Q. Natural lactic acid bacterial community and screening of high-quality lactic acid bacteria in silage in southwest China. Pratacultural Science, 2019, 36(12): 3203-3213. doi:
Citation: GUAN H, ZENG T R, SHUAI Y, YAN Y H, ZHANG X Q. Natural lactic acid bacterial community and screening of high-quality lactic acid bacteria in silage in southwest China. Pratacultural Science, 2019, 36(12): 3203-3213. doi:
西南高温高湿地区青贮中天然乳酸菌群落结构特征及优质乳酸菌的筛选
摘要:
为提高湿热地区青贮饲料的品质，本研究首先分析了从西南高温高湿地区青贮饲料中分离到的227株天然乳酸菌种群结构，再根据乳酸菌在高温条件下的生长活性和产酸能力筛选到4株优质乳酸菌(LP149、LS358、LR753和LPA761)，并对菌株生理生化特性和生长、产酸曲线进行了测定。结果表明，天然植物乳杆菌(Lactobacillus plantarum)是高温高湿地区青贮料中最常见的乳酸菌菌株(占62.55%)，其次是鼠李糖乳杆菌(L. rhamnosus)和副干酪乳杆菌(L. paracasei)，分别占总乳酸菌的13.21%和9.25%。所筛选的4株菌株为革兰氏阳性和过氧化氢酶阴性的杆状菌，能够在pH 3.5和45 ℃的条件下生长。根据生理、生化特征和16S rRNA测序分析，LP149、LS358、LR753和LPA761分别被鉴定为植物乳杆菌、唾液乳杆菌(L. salivarius)、鼠李糖乳杆菌和副干酪乳杆菌。在37和45 ℃条件下的菌株生长和产酸曲线结果表明，筛选出的4株乳酸菌有良好的高温适应性(45 ℃)，不仅生长速度快，产酸能力强，耐盐性较好，对糖源利用范围广，而且对酸性环境有较好的适应性，可以作为后续进行高温青贮回填试验的候选菌株。
English
Natural lactic acid bacterial community and screening of high-quality lactic acid bacteria in silage in southwest China
Abstract:
To improve the quality of silage in hot and humid areas, the community of 227 natural lactic acid bacteria isolated from silage in a hot and humid areas of Southwest China was analyzed. Four high-quality lactic acid bacteria (LAB) strains (LP149, LS358, LR753, and LPA761) were screened based on their growth activity and acid-producing ability under high temperature, and their physiological and biochemical characteristics, growth, and acid-production efficiency were determined. Lactobacillus plantarum was the most common LAB strain in the silage (62.55%), followed by L. rhamnosus and L. paracasei, accounting for 13.21% and 9.25% of the total strains, respectively. The four selected strains were gram-positive, catalase-negative, and rod shaped, and could grow at pH 3.5 and 45℃. According to the physiological and biochemical characteristics and 16S rRNA sequencing analysis, the four strains (LP149, LS358, LR753, and LPA761) were identified as L. plantarum, L. salivarius, L. rhamnosus, and L. paracasei, respectively. The results of growth and acid-production efficiency of the strains at 37 and 45 ℃ demonstrated that the four screened LAB strains exhibited good adaptability at high temperatures (45 ℃). They not only grow fast, with strong acid-producing ability and salt-tolerance, but also exhibited good adaptability to an acidic environment. This indicated they could be candidate strains for subsequent high temperature silage tests.
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Key words:
- silage /
- high temperature and humidity /
- lactic acid bacteria /
- community
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重庆欢乐生肖
图 1 重庆欢乐生肖 西南高温高湿地区玉米和狼尾草青贮前后乳酸菌种群结构
Figure 1. Community of lactic acid bacteria in corn and Pennisetum before and after ensiling in hot and humid areas of Southwest China
表 1 青贮前后样品信息及227株乳酸菌鉴定结果、分布详情
Table 1. 重庆欢乐生肖 Sample information before and after ensiling and identification and distribution of 227 strains of lactic acid bacteria
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表 2 乳酸菌的生理生化特性
Table 2. Physiological and biochemical characteristics of lactic acid bacteria
特性
Characteristic乳酸菌 Lacticacid bacteria LP149 LS358 LR753 LPA761 LP LS LR LPA LPC 来源
Source玉米
Maize杂交象草
Hybrid elephant grass玉米
Maize玉米
MaizeATCC
14917ATCC
11741ATCC
7469ATCC
334商业
Commercial采集地点
Collection site崇州，四川
Chongzhou, Sichuan宣汉，四川
Xuanhan, Sichuan宣汉，四川
Xuanhan, Sichuan宣汉，四川
Xuanhan, Sichuan未知
Unknown未知
Unknown未知
Unknown未知
Unknown未知
Unknown形状
Shape杆状
Rod杆状
Rod杆状
Rod杆状
Rod杆状
Rod杆状
Rod杆状
Rod杆状
Rod杆状
Rod革兰氏染色
Gram's stain+ + + + + + + + + 过氧化氢酶
Catalase test– – – – – – – – – 葡萄糖产气
Gas production– + + + – + + + – 乳酸的光学形式
Optical formDL L L L DL L L L DL 发酵类型
Fermentation type同型发酵
Homofermentation异型发酵
Heterofermentaion异型发酵
Heterofermetation异型发酵
Heteroermentation同型发酵
Homofermentation异型发酵
Heterofermentation异型发酵
Heterofermentation异型发酵
Heterofermentation同型发酵
Homofermentation+，90%及以上菌株有活性或显阳性；–，90%及以上菌株无活性或显阴性；W，有微弱活性；LP，ATCC 14917；LS，ATCC 11741；LR，ATCC 7469；LPA，ATCC 334；L: 左构型；DL: 左构型和右构型混合物；下同。
+, more than 90% of the strains were active or positive; –, more than 90% of the strains were inactive or negative; W, weakly active; LP, ATCC 14917; LS, ATCC 11741; LR, ATCC 7469; LPA, ATCC 334; L: Levo; DL: Dextro and levo; similarly for the following tables and figures.
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表 3 重庆欢乐生肖 乳酸菌糖发酵特性
Table 3. Characteristics of sugar fermentation of lactic acid bacteria
底物 Substrate LP149 LS358 LR753 LPA761 LP LS LR LPA LPC 半乳糖 Galactose + + + + + + + + + 菊粉 Inulin – + W – + W + + + 棉子糖 Raffinose + + + W + + + + + D-海藻糖 D-Trehalose + + + + + + + + + D-葡萄糖 D-Glucose + + + + + + + + + 麦芽糖 Maltose + + + + + + + + + 山梨醇 Sorbitol – – + + + + + + + D-甘露醇 D-Mannitol – – + + + + + + + 蜜二糖 Melibiose + + + + + + – + + 鼠李糖 Rhamnose + – W + – + – – – 乳糖 Lactose + + + W + + + + + 水杨苷 Salicin – W + + + + + + + 阿拉伯糖 Arabinose + + + + + – + + + 蔗糖 Saccharose + + + + + + + + + D-甘露糖 D-Mannose + + + + + + + + + 七叶苷 Esculin + – + + + + + + + D-果糖 D-Fructose + + + + + + + + + 纤维二糖 Cellobiose + – + + + – + + + 半固体琼脂 Semi-solid agar – – + + + + + + + 木糖 Xylose + + + – – + – – – 马尿酸 Hippuric acid + + + – – – + – – 苦杏仁苷 Amygdalin W W W – + W + + W
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表 4 重庆欢乐生肖 乳酸菌耐温性特征
Table 4. Temperature tolerance of lactic acid bacteria
温度处理
Temperature treatmentCZ149 XH358 XH753 XH761 LP LS LR LPA LPC 4 ℃ W W – – W W – W W 10 ℃ W W W – W W W W W 20 ℃ + + + + + + + + + 30 ℃ + + + + + + + + + 40 ℃ W + + + + + + + + 45 ℃ W + + + – + W – W 50 ℃ W W W W – – W – W
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表 5 37 ℃重庆欢乐生肖下乳酸菌耐盐性和耐酸性特征
Table 5. Salt and acid tolerance of lactic acid bacteria at 37 ℃
项目
Item处理
Treatment乳酸菌 Lactic acid bacteria LP149 LS358 LR753 LPA761 LP LS LR LPA LPC 耐盐处理
Salt tolerance treatment3% NaCl + + + + + + + + + 6.5% NaCl – – + – W – + + + 耐酸处理
Acid tolerance
treatmentpH 3 – – – – – – – – – pH 3.5 + + W + W – + W W pH 4 + + + + W W + W + pH 4.5 + + + + + + + + + pH 5 + + + + + + + + + pH 5.5 + + + + + + + + + pH 6 + + + + + + + + + pH 7 + + + + + + + + + pH 7.5 + + + + + + + + +
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重庆欢乐生肖 Yabo下载_Yabo官网下载 加拿大pc_加拿大pc官网 重庆欢乐生肖 重庆欢乐生肖 重庆欢乐生肖表 6 在45 ℃下乳酸菌耐盐性和耐酸性特征
Table 6. Salt and acid tolerance of lactic acid bacteria at 45 ℃
项目
Item处理
Treatment乳酸菌 Lactic acid bacteria LP149 LS358 LR753 LPA761 LP LS LR LPA LPC 耐盐处理
Salt tolerance treatment3% NaCl + + + W – + – – W 6.5% NaCl – – – – – – – – – 耐酸处理
Acid tolerance treatmentpH 3 – – – – – – – – – pH 3.5 – – – – – – – – – pH 4 – – – – – – – – – pH 4.5 + + + + – + – – pH 5 + + + + – + – – W pH 5.5 + + + + W + W W W pH 6 + + + + W W W W W pH 7 + + + + W + W W W pH 7.5 W + W + – + – – W
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