筛选窖泥中尚未被纯培养的高丰度拟杆菌纲微生物,并在纯培养菌株层面和共培养层面探究其生理代谢特征及生态学功能。
采用传代培养提高窖泥拟杆菌纲微生物的相对丰度,在此基础上进行筛菌实验,并通过发酵实验解析主体拟杆菌的代谢特征及其与主体己酸菌的相互作用关系。
成功筛选到
探明了窖泥主体拟杆菌纲微生物
To isolate abundant uncultured
The relative abundance of
This study reveals the fundamental physiological and metabolic characteristics of
窖泥是用于浓香型、酱香型、凤香型和芝麻香型等8种香型白酒生产的重要厌氧菌种子库,是我国特有厌氧微生物资源宝库[
目前对于窖泥微生物的研究主要包括基于免培养方法的菌群结构分析和基于可培养方法的菌株筛选。菌群结构研究结果表明,窖泥中的微生物种类繁多,包括85个纲的225个属,其中的细菌主要包括梭菌纲(
获得良好的富集体系,寻找难培养微生物的生长因素,提高目标菌在菌群培养体系中的相对丰度,是实现难培养微生物纯培养化的重要前提[
本研究中所用的窖泥分别取自四川省和安徽省某浓香型白酒酒厂窖池。
乙酸、丙酸、丁酸、叔戊酸、戊酸、己酸、庚酸、乳酸(Sigma-Aldrich试剂公司);DNA提取试剂盒PowerSoil DNA(Qiagen公司)。其余常规化学试剂购自国药集团化学试剂有限公司。
NanoDrop 8000蛋白核酸测定分光光度计、厌氧培养箱(Thermo Fisher Scientific公司);气相色谱仪Agilent 7890B、高效液相色谱仪Agilent 1260 (Agilent公司);pH计(Mettler Toled公司);紫外分光光度计A380 (翱艺仪器(上海)有限公司);有机酸柱Aminex HPX-87H (Bio-Rad公司);气相色谱柱CP-WAX 57 CB (Agilent公司)。
本研究所用的拟杆菌
用于窖泥的传代培养及筛菌实验,培养基组成在Lu等[
参照Holdeman等[
酵母粉10 g,蛋白胨10 g,NH4Cl 0.5 g,MgCl2·6H2O 0.5 g,CaCl2 0.2 g,金属元素母液500 μL,磷酸盐母液5 mL,GYTS母液5 mL,微量元素母液350 μL,0.1%刃天青400 µL,7% NaHCO3溶液(过膜除菌) 25 mL,2.5%半胱氨酸溶液(过膜除菌) 5 mL,维生素溶液A (过膜除菌) 100 µL,维生素溶液B (过膜除菌) 100 µL。灭菌前将除NaHCO3溶液、半胱氨酸溶液、维生素溶液A以及维生素溶液B以外的培养基成分混合,调节pH至7.4±0.2,121 ℃灭菌20 min。灭菌后将剩余溶液按比例添加至培养基内。其中,金属元素母液成分为(1 L):ZnSO4·7H2O 0.1 g,MnCl2·4H2O 0.03 g,H3BO3 0.3 g,CoCl2·6H2O 0.2 g,CaCl2·2H2O 0.01 g,NiCl2·6H2O 0.02 g,Na2MoO4·2H2O 0.03 g,FeCl2·4H2O 1.5 g。磷酸盐母液成分为(1 L):K2HPO4 6 g,NaH2PO4·H2O 5 g。GYTS母液成分为(1 L):NaCl 5 g,MgCl2·6H2O 4 g,CaCl2 0.75 g,NH4Cl 2.5 g,KH2PO4 2 g,KCl 5 g。葡萄糖添加组实验中在上述MGF培养基中额外添加终浓度为20 g/L的葡萄糖。
在MGF基础上添加1.5 g/L CH3COONa·3H2O,碳源为终浓度20 g/L的葡萄糖或终浓度10 g/L的乳酸。
参照PowerSoil DNA提取试剂盒操作步骤对传代样本的总DNA进行提取。对原核生物16S rRNA基因的V4区选取引物515FmodF (5′-GTGYCAGCMGCCGCGGTAA-3′)和806RmodR (5′-GGACTACNVGGGTWTCTAAT-3′)进行扩增[
利用无菌生理盐水将上述获得的窖泥传代培养液进行梯度稀释(10–1–10–7),并取100 µL梯度稀释的菌液涂布于MCI固体培养基上,于37 ℃厌氧箱中培养5–10 d。待菌落长出后,挑取单菌落于MCI液体培养基中培养3–5 d。随后利用细菌16S rRNA基因的通用引物(27F:5′-AGAGTTTGATCCTGGCTCAG-3′,1492R:5′-GGTTACCTTGTTACGACTT-3′)对培养后的菌液进行PCR扩增。PCR扩增反应体系为:27F 0.5 µL,1492R 0.5 µL,PrimerStar Max Primer (TaKaRa) 12.5 µL,无菌水10.5 µL,菌液1 µL。PCR反应条件为:预变性98 ℃ 2 min;变性98 ℃ 10 s,退火55 ℃ 30 s,延伸72 ℃ 1 min,30个循环;72 ℃ 10 min。得到的PCR产物送至无锡天霖测序公司测序。将测序结果提交至GenBank进行在线BLAST比对分析,得到物种鉴定结果。对于目标菌株进行平板划线纯化,进一步获得纯培养菌株。
将窖泥样品以5% (
将获得的纯培养菌株
将
发酵结束后,利用紫外分光光度计测定发酵液在600 nm处的吸光值,以表征发酵体系的生长情况;利用pH计测定发酵上清液的pH值。
采用高效液相色谱仪(安捷伦1260 Infinity Ⅱ)对发酵体系的葡萄糖和乳酸进行分析。取300 μL发酵上清液,0.22 μm滤膜过膜后,在色谱柱为Aminex HPX-87H (Bio-Rad公司)、流动相为5 mmol/L稀硫酸溶液、流速为0.6 mL/min、柱温为60 ℃、检测器为示差检测器的条件下,检测葡萄糖和乳酸的含量。
向200 μL发酵上清液中加入50 μL内标(12.5 g/L叔戊酸,含5%体积比的浓盐酸,pH 2.5),混匀离心后取200 μL上清液利用气相色谱仪(Agilent 7890B)进行分析。色谱柱为CP-WAX 57 CB (Agilent公司),程序升温条件为:初始温度60 ℃,保持0.5 min,以20 ℃/min升到180 ℃,保持5.5 min。分流比为30:1,进样口温度为220 ℃,氢火焰离子检测器温度为220 ℃。
本实验室前期的研究结果表明,相较于营养丰富的培养基,寡营养条件能有效富集到窖泥中的主体拟杆菌纲微生物,且其相对丰度在富集前中期大于后期[
窖泥微生物传代过程中的菌群结构动态变化
The changes of microbial structures during the successive transfers of the enrichment cultures (populations with relative abundance < 3% were merged into others). A: class level; B: genus level. F1, F2, F3 and F4 indicated the successive culturing passages.
扩增子测序结果表明,不同来源窖泥在原始窖泥组成及传代过程中的微生物组成变化存在一定的趋异性,但是总体上纲水平的优势微生物基本相同,包括厚壁菌门(
通过富集培养共获得41个属,主要分布于梭菌纲(
以在任一窖泥中相对丰度大于1%的标准界定为优势微生物,进一步分析窖泥优势拟杆菌纲微生物在传代进程中的相对丰度变化,结果如
传代过程中优势拟杆菌纲微生物相对丰度的变化
The relative abundance of dominant
Family genus | Distillery 1/% | Distillery 2/% | |||||||||
Pit mud | F1 | F2 | F3 | F4 | Pit mud | F1 | F2 | F3 | F4 | ||
ND: not detected. | |||||||||||
17.09 | 39.24 | 52.39 | 43.74 | 38.94 | 24.72 | 20.63 | 37.59 | 32.66 | 34.27 | ||
16.15 | 35.68 | 43.57 | 17.62 | 16.64 | 17.31 | 14.05 | 35.07 | 21.43 | 15.45 | ||
10.37 | 22.33 | 23.99 | 11.29 | 9.61 | 6.60 | 4.04 | 12.03 | 8.53 | 7.02 | ||
3.49 | 6.84 | 9.90 | 3.56 | 2.18 | 10.32 | 9.32 | 20.01 | 10.99 | 8.30 | ||
2.22 | 4.39 | 3.85 | 0.66 | 1.03 | 0.01 | 0.01 | 0.01 | 0.00 | 0.00 | ||
0.17 | 1.84 | 7.37 | 25.77 | 22.13 | 5.32 | 0.22 | 0.05 | 11.09 | 18.79 | ||
0.13 | 1.72 | 5.20 | 10.86 | 6.57 | 5.22 | 0.21 | 0.04 | 11.0% | 18.78 | ||
ND | ND | ND | ND | ND | 1.10 | 6.34 | 2.48 | 0.13 | 0.03 | ||
ND | ND | ND | ND | ND | 1.10 | 6.34 | 2.48 | 0.13 | 0.03 | ||
0.02 | ND | 0.01 | 0.04 | 0.26 | 4.49 | 0.06 | ND | ND | ND | ||
0.02 | ND | 0.01 | 0.04 | 0.26 | 4.49 | 0.06 | ND | ND | ND | ||
0.02 | ND | 0.01 | 0.04 | 0.26 | 4.49 | 0.06 | ND | ND | ND |
综上,通过寡营养培养手段对窖泥进行短期传代培养,成功提高了体系中优势拟杆菌纲微生物的相对丰度,为纯培养菌株的筛选奠定了基础。
经上述传代培养后,窖泥富集体系内拟杆菌纲微生物的相对丰度显著提高。进一步通过稀释涂布方法,对所获得的窖泥传代体系进行筛菌。总计对挑取的89个单菌落进行寡营养液体培养及PCR扩增与测序鉴定,结果如
基于16S rRNA基因序列的菌株生物鉴定比对结果
BLAST results based on the 16S rRNA gene sequences of isolated strains
Closest type species | Phylum | Class | Occurrence number | Frequency of occurrence/% |
29 | 32.60 | |||
15 | 16.90 | |||
9 | 10.10 | |||
7 | 7.90 | |||
6 | 6.70 | |||
6 | 6.70 | |||
5 | 5.60 | |||
3 | 3.40 | |||
2 | 2.20 | |||
2 | 2.20 | |||
1 | 1.10 | |||
1 | 1.10 | |||
1 | 1.10 | |||
1 | 1.10 | |||
1 | 1.10 |
结果表明,所筛的菌株主要隶属于窖泥两大高丰度细菌类型——梭菌纲和拟杆菌纲,与扩增子测序结果相符合,表明传代富集结合寡营养筛菌是从窖泥中获得优势微生物的可行方法。进一步通过平板划线纯化,成功获得一株
Growth, substrate utilization and main metabolites of
结果表明,
上述
葡萄糖碳源条件下
The fermentation characteristics for the co-culture of
由
结合生长和产物的变化,在以葡萄糖为发酵底物的共培养体系内2株单菌表现为交替生长,即前期以
乳酸是窖泥体系中含量最高且长期存在的有机酸碳源[
乳酸碳源条件下
The fermentation characteristics for the co-culture of
如
上述葡萄糖条件和乳酸条件的共培养结果表明,无论是否存在底物竞争效应,
浓香型白酒酿造窖泥在长期发酵过程中富集出了大量的土壤稀有菌,是我国特有的传统发酵食品微生物资源宝库[
之前的研究表明,在不同窖泥中广泛分布着以
The model of metabolic substrate interaction in
本研究对拟杆菌纲微生物
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