生物技术通报 ›› 2021, Vol. 37 ›› Issue (9): 58-67.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0813
• 青贮微生物专题(专题主编:杨富裕 教授) • 上一篇 下一篇
崔欣雨(), 李荣荣, 蔡瑞, 王妍, 郑猛虎, 徐春城()
收稿日期:
2021-06-27
出版日期:
2021-09-26
发布日期:
2021-10-25
作者简介:
崔欣雨,女,硕士研究生,研究方向:饲草加工;E-mail: 基金资助:
CUI Xin-yu(), LI Rong-rong, CAI Rui, WANG Yan, ZHENG Meng-hu, XU Chun-cheng()
Received:
2021-06-27
Published:
2021-09-26
Online:
2021-10-25
摘要:
针对苜蓿青贮过程中乳酸降解问题选取3个品种的苜蓿进行青贮,以乳酸钠为唯一碳源分离乳酸降解菌。通过菌落形态观察、生理生化特征和16S rDNA序列分析对菌株进行鉴定,采用紫外分光光度法和气相色谱法测定菌株的乳酸降解率和降解性能。共获得75株乳酸降解菌,选取其中4株乳酸降解率高的RSM9、RSF15、RSF2和RSH16菌株,经鉴定分别为哈夫尼菌(Hafnia sp.)、变形菌(Proteus sp.)、肥杆菌(Obesumbacterium sp.)和柠檬酸杆菌(Citrobacter sp.),经30℃、pH值6.2、厌氧培养120 h后,乳酸降解率分别达到44.64%、33.86%、30.64%和33.35%。4株菌的酸类代谢产物主要是乙酸和丙酸,在pH<5.0的环境下能被有效抑制,除RSH16外均能分泌NAD-非依赖型乳酸脱氢酶和乳酸氧化酶。从苜蓿青贮过程中筛选得到4株乳酸降解菌,其代谢乳酸能使体系pH值升高,造成苜蓿青贮品质的下降。
崔欣雨, 李荣荣, 蔡瑞, 王妍, 郑猛虎, 徐春城. 苜蓿青贮中乳酸降解菌的分离、鉴定及降解性能研究[J]. 生物技术通报, 2021, 37(9): 58-67.
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 |
表1 苜蓿青贮过程中有机酸含量的动态变化
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 | + | + | + | + |
表2 乳酸降解菌的分组情况
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 | + | + | + | + |
图3 菌株菌落形态及革兰氏染色结果 A、a:RSM9的菌落形态及革兰氏染色结果;B、b:RSF15的菌落形态及革兰氏染色结果;C、c:RSF2的菌落形态及革兰氏染色结果;D、d:RSH16的菌落形态及革兰氏染色结果
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 |
表3 4株乳酸降解菌的代谢特征
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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