Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (9): 58-67.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0813
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CUI Xin-yu(), LI Rong-rong, CAI Rui, WANG Yan, ZHENG Meng-hu, XU Chun-cheng()
Received:
2021-06-27
Online:
2021-09-26
Published:
2021-10-25
Contact:
XU Chun-cheng
E-mail:cuixinyu1010@163.com;xucc@cau.edu.cn
CUI Xin-yu, LI Rong-rong, CAI Rui, WANG Yan, ZHENG Meng-hu, XU Chun-cheng. Isolation,Identification of Lactic Acid Degrading Bacteria in Alfalfa Silage and Their Degradation Characterization[J]. Biotechnology Bulletin, 2021, 37(9): 58-67.
指标 Index | 品种 Variety | 天数Days/d | SEM | P-value | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
3 | 7 | 14 | 21 | 28 | 56 | Variety(V) | Days(D) | V×D | |||||
乳酸Lactic acid /(g·kg-1 DM) | Khan | 6.76B | 9.82Ab | 11.66Aa | 10.41A | 10.37A | 9.14A | 0.333 | 0.036 | 0.000 | 0.067 | ||
Meizoo | 6.11BC | 13.89Aa | 10.11ABab | 10.49AB | 13.37A | 11.21AB | |||||||
Central | 6.50B | 10.55ABb | 8.91ABb | 9.20AB | 10.48AB | 11.50A | |||||||
乙酸Acetic acid /(g·kg-1 DM) | Khan | 15.68B | 20.71AB | 21.34A | 22.51A | 22.34A | 23.05A | 0.409 | 0.011 | 0.001 | 0.825 | ||
Meizoo | 16.35 | 17.73 | 18.71 | 19.10 | 20.22 | 22.26 | |||||||
Central | 16.48 | 17.70 | 18.85 | 19.08 | 19.21 | 19.01 | |||||||
丙酸Propanoic acid/(g·kg-1 DM) | Khan | 6.97a | 7.05 | 7.16 | 7.88 | 7.63 | 7.97 | 0.202 | 0.362 | <0.001 | 0.034 | ||
Meizoo | 4.63Bb | 8.21A | 8.24A | 8.41A | 8.59A | 8.77A | |||||||
Central | 4.25Bb | 7.33A | 8.44A | 8.49A | 8.48A | 8.95A | |||||||
丁酸Butyric acid/(g·kg-1 DM) | Khan | ND | ND | ND | ND | ND | 0.69 | 0.032 | <0.001 | <0.001 | <0.001 | ||
Meizoo | ND | ND | ND | ND | ND | 0.78 | |||||||
Central | ND | ND | ND | ND | ND | ND |
Table 1 Dynamic changes of organic acids content during alfalfa silage
指标 Index | 品种 Variety | 天数Days/d | SEM | P-value | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
3 | 7 | 14 | 21 | 28 | 56 | Variety(V) | Days(D) | V×D | |||||
乳酸Lactic acid /(g·kg-1 DM) | Khan | 6.76B | 9.82Ab | 11.66Aa | 10.41A | 10.37A | 9.14A | 0.333 | 0.036 | 0.000 | 0.067 | ||
Meizoo | 6.11BC | 13.89Aa | 10.11ABab | 10.49AB | 13.37A | 11.21AB | |||||||
Central | 6.50B | 10.55ABb | 8.91ABb | 9.20AB | 10.48AB | 11.50A | |||||||
乙酸Acetic acid /(g·kg-1 DM) | Khan | 15.68B | 20.71AB | 21.34A | 22.51A | 22.34A | 23.05A | 0.409 | 0.011 | 0.001 | 0.825 | ||
Meizoo | 16.35 | 17.73 | 18.71 | 19.10 | 20.22 | 22.26 | |||||||
Central | 16.48 | 17.70 | 18.85 | 19.08 | 19.21 | 19.01 | |||||||
丙酸Propanoic acid/(g·kg-1 DM) | Khan | 6.97a | 7.05 | 7.16 | 7.88 | 7.63 | 7.97 | 0.202 | 0.362 | <0.001 | 0.034 | ||
Meizoo | 4.63Bb | 8.21A | 8.24A | 8.41A | 8.59A | 8.77A | |||||||
Central | 4.25Bb | 7.33A | 8.44A | 8.49A | 8.48A | 8.95A | |||||||
丁酸Butyric acid/(g·kg-1 DM) | Khan | ND | ND | ND | ND | ND | 0.69 | 0.032 | <0.001 | <0.001 | <0.001 | ||
Meizoo | ND | ND | ND | ND | ND | 0.78 | |||||||
Central | ND | ND | ND | ND | ND | ND |
分组 Group | 菌株数(株) Amount(Strains) | 菌落形态Colo- nial morphology | 革兰氏染色 Gram stain | 葡萄糖发酵试验 Glucose fermentation test | 乳糖发酵试验Lactose fermentation test | 蔗糖发酵试验Sucrose fermentation test | 吲哚试验 Indole test | 甲基红试验 Methyl red test | VP试验Voges- Proskauer test | 柠檬酸盐试验 Citrate test |
---|---|---|---|---|---|---|---|---|---|---|
A | 7 | 白色圆形凸起 | - | 产酸产气 | + | 产酸 | - | + | - | + |
B | 10 | 白色圆形凸起 | - | 产酸产气 | + | 产酸 | + | + | + | - |
C | 6 | 白色圆形凸起 | - | 产酸产气 | + | 产酸 | - | + | + | + |
D | 3 | 白色圆形凸起 | - | 产酸 | - | 产酸 | + | - | - | - |
E | 3 | 黄色同心圆扁平 | - | 产酸 | - | 产酸 | + | + | - | - |
F | 3 | 白色不规则凹陷 | + | 产酸 | - | 产酸 | + | - | + | + |
G | 5 | 白色圆形凸起 | - | 产酸 | + | 产酸 | + | + | - | - |
H | 6 | 黄色圆形凸起 | + | 产酸产气 | - | 产酸产气 | + | + | + | + |
I | 1 | 白色圆形凸起 | - | 产酸产气 | - | 产酸 | + | + | + | + |
J | 1 | 黄色圆形凸起 | - | 产酸 | - | 产酸 | + | + | - | + |
K | 1 | 白色圆形凸起 | - | 产酸 | - | 产酸 | + | + | + | + |
L | 1 | 黄色不规则凸起 | + | 产酸 | - | 产酸 | + | + | - | - |
M | 2 | 白色圆形凸起 | - | 产酸产气 | - | 产酸 | + | + | + | - |
N | 1 | 白色圆形凸起 | - | 产酸产气 | - | 产酸产气 | + | - | + | + |
O | 2 | 白色圆形隆起 | + | 产酸 | + | 产酸 | + | + | + | - |
P | 7 | 白色圆形凸起 | + | 产酸 | + | 产酸 | + | + | - | - |
Q | 3 | 白色圆形凸起 | - | 产酸产气 | + | 产酸 | + | + | + | + |
R | 1 | 白色圆形隆起 | + | 产酸 | + | 产酸 | + | + | - | - |
S | 6 | 白色圆形隆起 | - | 产酸 | + | 产酸 | + | + | - | - |
T | 4 | 白色圆形凸起 | - | 产酸产气 | + | 产酸 | + | + | + | + |
U | 2 | 白色圆形凸起 | - | 产酸产气 | + | 产酸 | + | + | + | + |
Table 2 Grouping of lactic acid degrading bacteria
分组 Group | 菌株数(株) Amount(Strains) | 菌落形态Colo- nial morphology | 革兰氏染色 Gram stain | 葡萄糖发酵试验 Glucose fermentation test | 乳糖发酵试验Lactose fermentation test | 蔗糖发酵试验Sucrose fermentation test | 吲哚试验 Indole test | 甲基红试验 Methyl red test | VP试验Voges- Proskauer test | 柠檬酸盐试验 Citrate test |
---|---|---|---|---|---|---|---|---|---|---|
A | 7 | 白色圆形凸起 | - | 产酸产气 | + | 产酸 | - | + | - | + |
B | 10 | 白色圆形凸起 | - | 产酸产气 | + | 产酸 | + | + | + | - |
C | 6 | 白色圆形凸起 | - | 产酸产气 | + | 产酸 | - | + | + | + |
D | 3 | 白色圆形凸起 | - | 产酸 | - | 产酸 | + | - | - | - |
E | 3 | 黄色同心圆扁平 | - | 产酸 | - | 产酸 | + | + | - | - |
F | 3 | 白色不规则凹陷 | + | 产酸 | - | 产酸 | + | - | + | + |
G | 5 | 白色圆形凸起 | - | 产酸 | + | 产酸 | + | + | - | - |
H | 6 | 黄色圆形凸起 | + | 产酸产气 | - | 产酸产气 | + | + | + | + |
I | 1 | 白色圆形凸起 | - | 产酸产气 | - | 产酸 | + | + | + | + |
J | 1 | 黄色圆形凸起 | - | 产酸 | - | 产酸 | + | + | - | + |
K | 1 | 白色圆形凸起 | - | 产酸 | - | 产酸 | + | + | + | + |
L | 1 | 黄色不规则凸起 | + | 产酸 | - | 产酸 | + | + | - | - |
M | 2 | 白色圆形凸起 | - | 产酸产气 | - | 产酸 | + | + | + | - |
N | 1 | 白色圆形凸起 | - | 产酸产气 | - | 产酸产气 | + | - | + | + |
O | 2 | 白色圆形隆起 | + | 产酸 | + | 产酸 | + | + | + | - |
P | 7 | 白色圆形凸起 | + | 产酸 | + | 产酸 | + | + | - | - |
Q | 3 | 白色圆形凸起 | - | 产酸产气 | + | 产酸 | + | + | + | + |
R | 1 | 白色圆形隆起 | + | 产酸 | + | 产酸 | + | + | - | - |
S | 6 | 白色圆形隆起 | - | 产酸 | + | 产酸 | + | + | - | - |
T | 4 | 白色圆形凸起 | - | 产酸产气 | + | 产酸 | + | + | + | + |
U | 2 | 白色圆形凸起 | - | 产酸产气 | + | 产酸 | + | + | + | + |
Fig.3 Colony morphology and Gram staining of each strain A,a:Colony morphology and gram staining results of RSM9. B,b:Colony morphology and gram staining results of RSF15. C,c:Colony morphology and gram staining results of RSF2. D,d:Colony morphology and gram staining results of RSH16
指标 Index | RSM9 | RSF15 | RSF2 | RSH16 | SEM | P-value |
---|---|---|---|---|---|---|
pH值 | 6.76A | 6.52B | 6.47B | 6.56B | 0.287 | 0.001 |
OD600 | 0.989A | 0.268B | 0.200C | 0.220C | 0.099 | <0.001 |
iLDH酶活力Enzymatic activity of iLDH/(U·L-1) | 112.91B | 276.81A | 36.42B | 61.92B | 29.215 | <0.001 |
LOX酶活力Enzymatic activity of LOX/(U·L-1) | 61.92AB | 54.63B | 109.27A | ND | 10.532 | 0.039 |
乙酸Acetic acid/(mg·L-1) | 1482.01A | 541.04B | 469.76B | 546.53B | 127.295 | <0.001 |
丙酸Propionic acid/(mg·L-1) | 48.19AB | 40.06BC | 21.99C | 66.11A | 5.352 | 0.004 |
异丁酸Isobutyric acid/(mg·L-1) | 13.47 | ND | ND | ND | 1.947 | 0.004 |
丁酸Butyric acid/(mg·L-1) | 178.47 | ND | ND | ND | 23.382 | <0.001 |
异戊酸Isovaleric acid/(mg·L-1) | 19.67 | ND | ND | ND | 2.605 | <0.001 |
戊酸Valeric acid/(mg·L-1) | 65.24 | ND | ND | ND | 8.630 | <0.001 |
己酸Caproic acid/(mg·L-1) | 12.14B | 27.90AB | ND | 29.371A | 3.319 | 0.030 |
Table 3 Metabolic characteristics of 4 strains of lactic acid degrading bacteria
指标 Index | RSM9 | RSF15 | RSF2 | RSH16 | SEM | P-value |
---|---|---|---|---|---|---|
pH值 | 6.76A | 6.52B | 6.47B | 6.56B | 0.287 | 0.001 |
OD600 | 0.989A | 0.268B | 0.200C | 0.220C | 0.099 | <0.001 |
iLDH酶活力Enzymatic activity of iLDH/(U·L-1) | 112.91B | 276.81A | 36.42B | 61.92B | 29.215 | <0.001 |
LOX酶活力Enzymatic activity of LOX/(U·L-1) | 61.92AB | 54.63B | 109.27A | ND | 10.532 | 0.039 |
乙酸Acetic acid/(mg·L-1) | 1482.01A | 541.04B | 469.76B | 546.53B | 127.295 | <0.001 |
丙酸Propionic acid/(mg·L-1) | 48.19AB | 40.06BC | 21.99C | 66.11A | 5.352 | 0.004 |
异丁酸Isobutyric acid/(mg·L-1) | 13.47 | ND | ND | ND | 1.947 | 0.004 |
丁酸Butyric acid/(mg·L-1) | 178.47 | ND | ND | ND | 23.382 | <0.001 |
异戊酸Isovaleric acid/(mg·L-1) | 19.67 | ND | ND | ND | 2.605 | <0.001 |
戊酸Valeric acid/(mg·L-1) | 65.24 | ND | ND | ND | 8.630 | <0.001 |
己酸Caproic acid/(mg·L-1) | 12.14B | 27.90AB | ND | 29.371A | 3.319 | 0.030 |
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