利于高地芽孢杆菌YC-9生长和芽孢形成的碳源筛选

doi:10.13304/j.nykjdb.2021.0096

摘要/Abstract

摘要：

高地芽孢杆菌（Bacillus altitudinis）是烟叶发酵的优势菌之一。通过优化碳源提高高地芽孢杆菌菌株YC-9的生长量和芽孢产量：首先利用Biolog GenⅢ板测定菌株YC-9对71种碳源的代谢特征，然后采用全自动生长曲线仪分析不同碳源对芽孢生物量、生长周期及产孢率的影响，并通过摇瓶发酵确定最佳的产孢条件。结果表明，菌株YC-9可以较好地利用17种碳源，其中4种碳源可以同时促进其生长和产芽孢；在摇瓶水平上使菌株YC-9芽孢产量较多的碳源为L-苹果酸和α-D-葡萄糖，使其芽孢率最高的碳源为葡萄糖+肌苷，培养24 h芽孢总量为3.90×10⁷ cfu·mL^-1，芽孢率高达60.47%，比以葡萄糖为单一碳源时的芽孢率高约2倍。优化碳源可以显著提高高地芽孢杆菌菌株YC-9的生长、产芽孢率和产芽孢量。

关键词: 高地芽孢杆菌, Biolog, 生长曲线, 产芽孢率

Abstract:

Bacillus altitudinis is one of the most predominent bacteria during tobacco fermentation. This study aimed to impove the growth and spore-producing capability of Bacillus altitudinis YC-9 by optimizing the carbon sources. Firstly， the metabolic characteristics of strain YC-9 to 71 kinds of carbon sources were determined by Biolog Gen Ⅲ plate， then the effects of different carbon sources on the cell amount， growth cycle and spore yield were studied by using an automatic growth curves analyzer， and the optimal conditions for spore production were determined in shaking flasks. The results showed that strain YC-9 efficiently utilized 17 carbon sources， and 4 of them were favorable for both cell growth and spore production； L-malic acid and α-D-glucose were the better carbon sources for spore production in shake flasks， while glucose and inosine was the highest carbon source for the spore rate. The total amount of spores was 3.90 × 10⁷ cfu·mL^-1， and the spore rate was as 60.47%， which was about 2 times higher than that of glucose alone. The cell growth， spore yield， and spore-producing rate of Bacillus altitudinis YC-9 were significantly improved by using optimized carbon sources.

Key words: Bacillus altitudinis, Biolog, growth curve, spore-producing rate

中图分类号:

S182

马聪聪, 罗泽华, 蔡斌, 刘好宝, 王云山, 马锐, 顾金刚. 利于高地芽孢杆菌YC-9生长和芽孢形成的碳源筛选[J]. 中国农业科技导报, 2022, 24(7): 77-85.

Congcong MA, Zehua LUO, Bin CAI, Haobao LIU, Yunshan WANG, Rui MA, Jingang GU. Screening of Carbon Sources for Growth and Spore Formation of Bacillus altitudinis YC-9[J]. Journal of Agricultural Science and Technology, 2022, 24(7): 77-85.

图/表 8

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基质 Substrate	高度 Height	芽孢量 Spore yield	基质 Substrate	高度 Height	芽孢量 Spore yield	基质 Substrate	高度 Height	芽孢量 Spore yield
D-乳糖 D-lactose	170	+++++	N-乙酰-D-葡萄糖胺N-acetyl-D-glucosamine	205	++++	D-海藻糖 D-trehalose	192	+
L-岩藻糖 L-fucose	168	+++++	D-糖质酸 D-saccharic	155	+++	D-纤维二糖 D-cellobiose	196	+
L-鼠李糖 L-rhamnose	172	+++++	N-乙酰-β-D-甘露糖胺N-acetyl-β-D-annosamine	190	+++	β-甲基-D-葡萄糖苷β-methyl-D-glucoside	201	+
3-甲基-D-葡萄糖 3-methyl-D-glucose	189	+++++	N-乙酰-D-半乳糖胺N-acetyl-D-galactosamine	160	+++	D-葡萄糖-6-磷酸 D-glucose-6-phosphate	195	+
D-棉子糖 D-raffinose	190	+++++	D-乳酸甲酯 D-methyl lactate	155	+++	D-甘露醇 D-mannitol	211	+
水苏糖 Stachyose	152	+++++	α-酮戊二酸 α-ketoglutarate	182	+++	L-苹果酸 L-malic acid	222	+
D-松二糖 D-turanose	170	+++++	α-丁酮酸 α-ketobutyric acid	143	+++	L-精氨酸 L-arginine	196	+
蜜二糖 Melibiose	186	+++++	葡萄糖酸 Gluconic acid	182	+++	L-谷氨酸 L-glutamatic acid	208	+
D-山梨醇 D-sorbitol	181	+++++	吐温40 Tween 40	201	+++	L-天冬氨酸 L-aspartic acid	221	+
肌苷 Inosine	183	+++++	糊精 Dextrin	186	+++	D-天冬氨酸 D-aspartic acid	224	+
肌醇 Inositol	196	+++++	D-甘露糖 D-mannose	211	++	甘氨酸-L-脯氨酸 Glycine-L-proline	185	+
果胶 Pectin	215	+++++	D-苹果酸 D-malic acid	138	++	葡萄糖醛酸 Glucuronic acid	179	+
L-丙氨酸 L-alanine	186	+++++	柠檬酸 Citric acid	205	++	葡糖醛酰胺 Glucuronamide	132	+

基质 Substrate	高度 Height	芽孢量 Spore yield	基质 Substrate	高度 Height	芽孢量 Spore yield	基质 Substrate	高度 Height	芽孢量 Spore yield
D-乳糖 D-lactose	170	+++++	N-乙酰-D-葡萄糖胺N-acetyl-D-glucosamine	205	++++	D-海藻糖 D-trehalose	192	+
L-岩藻糖 L-fucose	168	+++++	D-糖质酸 D-saccharic	155	+++	D-纤维二糖 D-cellobiose	196	+
L-鼠李糖 L-rhamnose	172	+++++	N-乙酰-β-D-甘露糖胺N-acetyl-β-D-annosamine	190	+++	β-甲基-D-葡萄糖苷β-methyl-D-glucoside	201	+
3-甲基-D-葡萄糖 3-methyl-D-glucose	189	+++++	N-乙酰-D-半乳糖胺N-acetyl-D-galactosamine	160	+++	D-葡萄糖-6-磷酸 D-glucose-6-phosphate	195	+
D-棉子糖 D-raffinose	190	+++++	D-乳酸甲酯 D-methyl lactate	155	+++	D-甘露醇 D-mannitol	211	+
水苏糖 Stachyose	152	+++++	α-酮戊二酸 α-ketoglutarate	182	+++	L-苹果酸 L-malic acid	222	+
D-松二糖 D-turanose	170	+++++	α-丁酮酸 α-ketobutyric acid	143	+++	L-精氨酸 L-arginine	196	+
蜜二糖 Melibiose	186	+++++	葡萄糖酸 Gluconic acid	182	+++	L-谷氨酸 L-glutamatic acid	208	+
D-山梨醇 D-sorbitol	181	+++++	吐温40 Tween 40	201	+++	L-天冬氨酸 L-aspartic acid	221	+
肌苷 Inosine	183	+++++	糊精 Dextrin	186	+++	D-天冬氨酸 D-aspartic acid	224	+
肌醇 Inositol	196	+++++	D-甘露糖 D-mannose	211	++	甘氨酸-L-脯氨酸 Glycine-L-proline	185	+
果胶 Pectin	215	+++++	D-苹果酸 D-malic acid	138	++	葡萄糖醛酸 Glucuronic acid	179	+
L-丙氨酸 L-alanine	186	+++++	柠檬酸 Citric acid	205	++	葡糖醛酰胺 Glucuronamide	132	+

碳源 Carbon source	芽孢率Spore rate /%
碳源 Carbon source	24 h	36 h	48 h
α-D-葡萄糖 α-D-glucose	9 c	82 a	88 a
L-苹果酸 L-malic acid	5 c	93 a	93 a
D-果糖D-fructose	14 c	2 c	70 b
D-海藻糖D-trehalose	50 a	48 b	47 c
D-纤维二糖D-cellobiose	28 b	53 b	40 c
甘油 Glycerol	47 a	1 c	7 d
D-甘露醇D-mannitol	4 c	4 c	5 d
水杨苷Salicin	5 c	2 c	1 d

碳源 Carbon source	芽孢率Spore rate /%
碳源 Carbon source	24 h	36 h	48 h
α-D-葡萄糖 α-D-glucose	9 c	82 a	88 a
L-苹果酸 L-malic acid	5 c	93 a	93 a
D-果糖D-fructose	14 c	2 c	70 b
D-海藻糖D-trehalose	50 a	48 b	47 c
D-纤维二糖D-cellobiose	28 b	53 b	40 c
甘油 Glycerol	47 a	1 c	7 d
D-甘露醇D-mannitol	4 c	4 c	5 d
水杨苷Salicin	5 c	2 c	1 d

基质 Substrate	高度 Height	芽孢量 Spore yield	基质 Substrate	高度 Height	芽孢量 Spore yield	基质 Substrate	高度 Height	芽孢量 Spore yield
L-焦谷氨酸 L-pyroglutamic acid	175	+++++	甲酸 Formic acid	132	++	半乳糖二酸 Galactaric acid	160	+
L-丝氨酸 L-serine	150	+++++	乙酸 Acetic acid	216	++	半乳糖醛酸内酯 Galacturonic acid lactone	169	+
半乳糖醛酸 Galacturonic acid	194	+++++	D-丝氨酸 D-serine	150	++	丙酸 Propionic acid	162	+
α-羟基丁酸 α-hydroxybutyric acid	146	+++++	L-组氨酸 L-histone	165	++	乙酰乙酸 Acetoacetic acid	228	+
β-羟基-D，L-丁酸 β-hydroxy-D，L-butyric acid	162	+++++	D-阿拉伯醇 D-arabinol	185	++	丙酮酸甲酯 Methyl pyruvate	134	+
N-乙酰神经氨酸N-acetylneuraminic acid	165	+++++	明胶 Gelatin	191	++	水杨苷 Salicin	204	+
D-果糖-6-磷酸D-fructose-6-phosphate	195	+++++	γ-氨基丁酸 γ-aminobutyric acid	209	+	甘油 Glycerol	217	+
D-半乳糖 D-galactose	196	++++	D-蔗糖 D-sucrose	186	+	奎宁酸 Quinic acid	203	+
D-麦芽糖 D-maltose	161	++++	D-果糖 D-fructose	211	+	ρ-羟基-苯乙酸ρ-hydroxy-phenylacetic acid	151	+
龙胆二糖 Gentiobiose	188	++++	α-D-葡萄糖 α-D-glucose	223	+	溴代丁二酸 Bromosuccinic acid	170	/
L-乳酸 L-lactic acid	162	++++	D-岩藻糖 D-fucose	189	+