苜蓿青贮中乳酸降解菌的分离、鉴定及降解性能研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0813

摘要/Abstract

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。

关键词: 苜蓿, 乳酸降解菌, 分离鉴定, 发酵品质, 降解性能

Abstract:

In order to solve the lactic acid degradation problem during alfalfa silage,three varieties of alfalfa were selected for silage,and sodium lactate was used as the sole carbon source to isolate microorganisms capable of degrading lactic acid. The morphological observation,physiological and biochemical characteristics and 16S rDNA sequence analysis were employed to identify the strains,and the UV spectrophotometry and gas chromatography were used to study degradation rate and characteristics of the strains. A total of 75 strains of lactic acid degrading bacteria were obtained,among which RSM9,RSF15,RSF2 and RSH16 with high degradation rate were selected. They were identified as Hafnia sp.,Proteus sp.,Obesumbacterium sp. and Citrobacter sp. The degradation rates of lactic acid reached 44.64%,33.86%,30.64% and 33.35% after 120 h of anaerobic culture at 30℃ and pH 6.2,respectively. The acid metabolites of the 4 strains were mainly acetic acid and propionic acid,and 4 strains were effectively inhibited at pH<5.0. NAD-independent lactate dehydrogenases and lactate oxidase were detected in all strains except for RSH16. Four strains of lactic acid degrading bacteria are screened from the process of alfalfa silage,as well as,their metabolism of lactic acid can increase the pH of the system and cause the decline of alfalfa silage quality.

Key words: alfalfa, lactic acid degrading bacteria, isolation and identification, fermentation quality, degradation characterization

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.

图/表 8

参考文献 34

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指标 Index	品种 Variety	天数Days/d						SEM	P-value
指标 Index	品种 Variety	3	7	14	21	28	56	SEM	Variety（V）	Days（D）	V×D
乳酸Lactic acid /（g·kg^-1 DM）	Khan	6.76^B	9.82^Ab	11.66^Aa	10.41^A	10.37^A	9.14^A	0.333	0.036	0.000	0.067
	Meizoo	6.11^BC	13.89^Aa	10.11^ABab	10.49^AB	13.37^A	11.21^AB
	Central	6.50^B	10.55^ABb	8.91^ABb	9.20^AB	10.48^AB	11.50^A
乙酸Acetic acid /（g·kg^-1 DM）	Khan	15.68^B	20.71^AB	21.34^A	22.51^A	22.34^A	23.05^A	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	6.97^a	7.05	7.16	7.88	7.63	7.97	0.202	0.362	<0.001	0.034
	Meizoo	4.63^Bb	8.21^A	8.24^A	8.41^A	8.59^A	8.77^A
	Central	4.25^Bb	7.33^A	8.44^A	8.49^A	8.48^A	8.95^A
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

指标 Index	品种 Variety	天数Days/d						SEM	P-value
指标 Index	品种 Variety	3	7	14	21	28	56	SEM	Variety（V）	Days（D）	V×D
乳酸Lactic acid /（g·kg^-1 DM）	Khan	6.76^B	9.82^Ab	11.66^Aa	10.41^A	10.37^A	9.14^A	0.333	0.036	0.000	0.067
	Meizoo	6.11^BC	13.89^Aa	10.11^ABab	10.49^AB	13.37^A	11.21^AB
	Central	6.50^B	10.55^ABb	8.91^ABb	9.20^AB	10.48^AB	11.50^A
乙酸Acetic acid /（g·kg^-1 DM）	Khan	15.68^B	20.71^AB	21.34^A	22.51^A	22.34^A	23.05^A	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	6.97^a	7.05	7.16	7.88	7.63	7.97	0.202	0.362	<0.001	0.034
	Meizoo	4.63^Bb	8.21^A	8.24^A	8.41^A	8.59^A	8.77^A
	Central	4.25^Bb	7.33^A	8.44^A	8.49^A	8.48^A	8.95^A
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	+	+	+	+

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	+	+	+	+

指标 Index	RSM9	RSF15	RSF2	RSH16	SEM	P-value
pH值	6.76^A	6.52^B	6.47^B	6.56^B	0.287	0.001
OD₆₀₀	0.989^A	0.268^B	0.200^C	0.220^C	0.099	<0.001
iLDH酶活力Enzymatic activity of iLDH/（U·L^-1）	112.91^B	276.81^A	36.42^B	61.92^B	29.215	<0.001
LOX酶活力Enzymatic activity of LOX/（U·L^-1）	61.92^AB	54.63^B	109.27^A	ND	10.532	0.039
乙酸Acetic acid/（mg·L^-1）	1482.01^A	541.04^B	469.76^B	546.53^B	127.295	<0.001
丙酸Propionic acid/（mg·L^-1）	48.19^AB	40.06^BC	21.99^C	66.11^A	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）	12.14^B	27.90^AB	ND	29.371^A	3.319	0.030