传统豆瓣酱微生物群落发酵演替规律及其功能分析

[目的]解析中国传统豆瓣酱发酵过程中的微生物群落演替规律和理化代谢物质变化,探讨不同发酵阶段影响豆瓣酱风味的核心功能微生物.[方法]采用高通量测序解析豆瓣酱发酵过程中的微生物群落结构和演替,并跟踪检测发酵过程中的理化代谢物质,然后分析微生物群落和理化代谢物质变化之间的相关性,最后在体外分离核心微生物并对其功能特性进行验...     

郫县豆瓣发酵过程的微生物多样性及溯源分析

[目的] 解析郫县豆瓣及其酿造半成品-蚕豆醅与辣椒醅微生物多样性和来源,探究郫县豆瓣酿造过程风味化合物特征。[方法] 采用高通量测序法测定蚕豆醅、辣椒醅与混合醅（蚕豆醅-辣椒醅混合物,发酵成熟形成郫县豆瓣）在酿造过程中的微生物群落结构;利用高效气相质谱与高效液相色谱高通量检测蚕豆醅及辣椒醅中基础理化指标及挥发性、非挥发性风味化合物浓度;利用多种生物信息学分析方法对混合醅酿造微生物及风味化合物进行溯源。[结果] 微生物方面：44%–59%的混合醅细菌来源于辣椒醅,5%–22%的混合醅细菌来源于蚕豆醅,其他混合醅细菌来源未知。同时,42%–77%的混合醅真菌来源于辣椒醅,2%–18%的混合醅真菌来源于蚕豆醅,其他混合醅真菌来源未知。另外,16个细菌属由辣椒醅特异性贡献;2个细菌属及2个真菌属由蚕豆醅特异性贡献。化合物方面：1-辛烯-3醇（1-octen-3-ol）、苯乙醛（phenylacetaldehyde）、异丁醛（isobutyraldehyde）、苹果酸（malic acid）与糠醛（furfural）仅由蚕豆醅贡献。辣椒素（capsaicin）、3-甲基-1-丁醇（3-methyl-1-butanol）、已醇（hexanol）与异丁醇（isobutanol）仅由辣椒醅贡献。[结论] 郫县豆瓣发酵中大部分微生物来源于辣椒醅,大部分发酵底物（氨基酸及葡萄糖）来源于蚕豆醅。两种发酵半成品均特异性贡献微生物及风味化合物,形成郫县豆瓣的独特风味密码。     

传统豆酱发酵过程中细菌多样性动态

细菌在豆酱发酵过程中起到非常重要的作用,并与豆酱的风味和质量密切相关,因此研究豆酱中细菌的多样性具有重要意义。以自然发酵的豆酱样品为研究对象,采用细菌16S rDNA的部分可变区的PCR-DGGE技术对自然发酵豆酱样品的细菌群落组成和优势菌群进行研究。结果表明,传统豆酱发酵过程细菌群体中既有原始种群的减少和增长,也有次级种群的增多和演变。在整个发酵过程中,初期和末期以不可培养细菌为主,初期细菌群体快速演替,细菌种群多样性指数在发酵42 d和56 d达到两次高峰。     

Biodiversity analysis of microbial community in the chem-bioflocculation treatment process

Total DNA was directly extracted from environmental samples and amplified with polymerase chain reaction (PCR) technique. The PCR products were fingerprinted via denaturing gradient gel electrophoresis (DGGE). Significant differences were observed in the microbial community structures between traditional treatment process and chem-bioflocculation process. The microbial community structure shift at different sampling locations in chem-bioflocculation process and on two typical operational conditions was studied. 16S rDNA V3 regions of some dominant species were sequenced and the species were identified. The microbial communities were stable in both the chem-bioflocculation process and the activated sludge process under various experimental conditions presented in this work. The attached growth treatment process was less stable when operational conditions changed.     

Determination of microbial diversity in meju,fermented cooked soya beans,using nested PCR‐denaturing gradient gel electrophoresis

Aims: To identify the microbiota in meju, fermented cooked soya beans, that may directly affect the microbial communities of Korean fermented soya bean foods. Methods and Results: Using conventional bacterial 16S rDNA, bacilli‐specific 16S rDNA or fungi 18S rDNA‐specific primers, PCR products were amplified through a series of PCRs using the DNA extracted from ten meju samples. The amplicons were analysed using denaturing gradient gel electrophoresis (DGGE), which showed that Enterococcus durans was commonly detected in nine of ten meju samples. Bacillus subtilis was shown to be the major strain of bacilli in the samples tested. Based on the DGGE analysis of fungi in meju, we determined that Absidia corymbifera, Aspergillus sp. and Candida rugosa were the main fungi in the tested samples. Conclusions: A variety of bacterial and fungal micro‐organisms were identified in meju samples, in addition to the micro‐organisms already known to be present. Significance and Impact of the Study: This is the first report showing the differences and similarities in the populations of micro‐organisms in meju samples using nested PCR‐DGGE, a culture‐independent method. The results may be applicable to the development of improved meju, in which the indigenous micro‐organisms required for fermentation can be standardized.     

Three‐phase succession of autochthonous lactic acid bacteria to reach a stable ecosystem within 7 days of natural bamboo shoot fermentation as revealed by different molecular approaches

Microbial community structure and population dynamics during spontaneous bamboo shoot fermentation for production of ‘soidon’ (indigenous fermented food) in North‐east India were studied using cultivation‐dependent and cultivation‐independent molecular approaches. Cultivation‐dependent analyses (PCR‐amplified ribosomal DNA restriction analysis and rRNA gene sequencing) and cultivation‐independent analyses (PCR‐DGGE, qPCR and Illumina amplicon sequencing) were conducted on the time series samples collected from three independent indigenous soidon fermentation batches. The current findings revealed three‐phase succession of autochthonous lactic acid bacteria to attain a stable ecosystem within 7 days natural fermentation of bamboo shoots. Weissella spp. (Weissella cibaria, uncultured Weissella ghanensis) and Lactococcus lactis subsp. cremoris predominated the early phase (1–2 days) which was joined by Leuconostoc citreum during the mid‐phase (3 days), while Lactobacillus brevis and Lactobacillus plantarum emerged and became dominant in the late phase (5–7 days) with concurrent disappearance of W. cibaria and L. lactis subsp. cremoris. Lactococcus lactis subsp. lactis and uncultured Lactobacillus acetotolerans were predominantly present throughout the fermentation with no visible dynamics. The above identified dominant bacterial species along with their dynamics can be effectively utilized for designing a starter culture for industrialization of soidon production. Our results showed that a more realistic view on the microbial ecology of soidon fermentation could be obtained by cultivation‐dependent studies complemented with cultivation‐independent molecular approaches. Moreover, the critical issues to be considered for reducing methodological biases while studying the microbial ecology of traditional food fermentation were also highlighted with this soidon fermentation model.     

浓香型白酒窖池微生物群落结构特征

窖池是中国白酒,尤其是浓香型大曲酒生产颇具特色的固态生物反应器,窖龄与微生物群落结构关系密切且复杂,对产品质量影响非常显著.本研究以微生物细胞膜的特征组分磷酸脂肪酸(PLFA)为指标,研究了不同窖龄(5年、100年和300 年)窖池窖泥、糟醅和黄水的微生物群落结构特征.结果表明:窖泥中总PLFA含量最高,糟醅次之,黄水最低.PLFA的组成因窖龄而异,黄水中总PLFA含量随窖池窖龄的增长而减小.窖泥中直链饱和脂肪酸含量最高,为PLFA总量的50.7%～73.9%,其中300年窖池窖泥最高.窖泥中表征革兰氏阳性(G+)厌氧细菌的PLFA含量较高,而糟醅和黄水中均以表征革兰氏阴性(G-)厌氧菌的PLFA含量较高.100年窖泥中表征G+菌、G-菌和厌氧菌的PLFA含量高于其他窖龄相应样品.5年窖窖泥、糟醅和黄水中真菌PLFA含量均高于其他窖龄相应样品.经主成分分析,5年窖和100年窖中主要变异菌群是G+菌和真菌,300年窖中主要变异菌群是细菌.描述窖池微生物群落特征的多样性指数可以选用PLFA的频次、Simpson优势度指数和Shannon多样性指数.     

Effects of sampling location and time,and host animal on assessment of bacterial diversity and fermentation parameters in the bovine rumen

Aims: To investigate, using culture‐independent methods, whether the ruminal bacterial structure, population and fermentation parameters differed between sampling locations and time. Methods and Results: The detectable bacteria and fermentation parameters in the digesta from five locations in the rumen of three cows at three time points were analysed. The PCR‐denaturing gradient gel electrophoresis (PCR‐DGGE) profiles were similar among digesta samples from five locations (95·4%) and three time points (93·4%) within cows; however, a lower similarity was observed for samples collected from different host animals (85·5%). Rumen pH and concentration of volatile fatty acids (VFA) were affected by time points of sampling relative to feeding. Conclusions: The detectable bacterial structure in the rumen is highly conserved among different locations and over time, while the quantity of individual bacterial species may change diurnally in response to the feeding. Significance and impact of the study: This study supplies the fundamental understanding of the microbial ecology in the rumen, which is essential for manipulation of ruminal microflora and subsequent improvement in animal production.     

PCR-DGGE analysis of bacterial community dynamics in kava beverages during refrigeration

Aims: Kava beverages are highly perishable even under refrigerated conditions. This study aimed to investigate the bacterial community dynamics in kava beverages during refrigeration. Methods and Results: Four freshly made kava beverages were obtained from kava bars and stored at 4°C. On days 0, 3 and 6, the aerobic plate count (APC), lactic acid bacteria (LAB) count and yeast and mould count (YMC) of the samples were determined. Meanwhile, bacterial DNA was extracted from each sample and subjected to the polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE). Moreover, species‐specific PCR assays were employed to identify predominant Pseudomonas spp. involved in kava spoilage. Over the storage period, the APC, LAB count and YMC of the four kava beverages all increased, whereas their pH values decreased. The DGGE profile revealed diverse bacterial populations in the samples. LAB, such as Weissella soli, Lactobacillus spp. and Lactococcus lactis, were found in the kava beverages. Species‐specific PCR assays detected Pseudomonas putida and Pseudomonas fluorescens in the samples; Ps. fluorescens became dominant during refrigeration. Conclusions: LAB and Pseudomonas may play a significant role in the spoilage of kava beverages. Significance and Impact of the Study: This study provides important information that may be used to extend the shelf life of kava beverages.     

Effects of high‐ and low‐fiber diets on fecal fermentation and fecal microbial populations of captive chimpanzees

We examined fiber fermentation capacity of captive chimpanzee fecal microflora from animals (n=2) eating low‐fiber diets (LFDs; 14% neutral detergent fiber (NDF) and 5% of cellulose) and high‐fiber diets (HFDs; 26% NDF and 15% of cellulose), using barley grain, meadow hay, wheat straw, and amorphous cellulose as substrates for in vitro gas production of feces. We also examined the effects of LFD or HFD on populations of eubacteria and archaea in chimpanzee feces. Fecal inoculum fermentation from the LFD animals resulted in a higher in vitro dry matter digestibility (IVDMD) and gas production than from the HFD animals. However, there was an interaction between different inocula and substrates on IVDMD, gas and methane production, and hydrogen recovery (P<0.001). On the other hand, HFD inoculum increased the production of total short‐chain fatty acids (SCFAs), acetate, and propionate with all tested substrates. The effect of the interaction between the inoculum and substrate on total SCFAs was not observed. Changes in fermentation activities were associated with changes in bacterial populations. DGGE of bacterial DNA revealed shift in population of both archaeal and eubacterial communities. However, a much more complex eubacterial population structure represented by many bands was observed compared with the less variable archaeal population in both diets. Some archaeal bands were related to the uncultured archaea from gastrointestinal tracts of homeothermic animals. Genomic DNA in the dominant eubacterial band in the HFD inoculum was confirmed to be closely related to DNA from Eubacterium biforme. Interestingly, the predominant band in the LFD inoculum represented DNA of probably new or yet‐to‐be‐sequenced species belonging to mycoplasms. Collectively, our results indicated that fecal microbial populations of the captive chimpanzees are not capable of extensive fiber fermentation; however, there was a positive effect of fiber content on SCFA production. Am. J. Primatol. 71:548–557, 2009. © 2009 Wiley‐Liss, Inc.     

Nisin production of Lactococcus lactis N8 with hemin‐stimulated cell respiration in fed‐batch fermentation system

In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed‐batch fermentation in which respiration of cells was stimulated with hemin. Response surface model was able to explain the changes of the nisin production of L. lactis N8 in fed‐batch fermentation system with high fidelity (R² 98%) and insignificant lack of fit. Accordingly, the equation developed indicated the optimum parameters for glucose, hemin, and dissolved oxygen were 8 g L^?1 h^?1, 3 μg mL^?1 and 40%, respectively. While 1711 IU mL^?1 nisin was produced by L. lactis N8 in control fed‐batch fermentation, 5410 IU mL^?1 nisin production was achieved within the relevant optimum parameters where the respiration of cell was stimulated with hemin. Accordingly, nisin production was enhanced 3.1 fold in fed‐batch fermentation using hemin. In conclusion the nisin production of L. lactis N8 was enhanced extensively as a result of increasing the biomass by stimulating the cell respiration with adding the hemin in the fed‐batch fermentation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:678–685, 2015     

营养相互作用对传统发酵食品微生物群落构建的推动作用研究进展

传统发酵食品是由自然接种的多微生物组成的混菌体系,了解微生物群落自发式构建的机制是认识发酵机理和调控发酵的关键。尽管大量的测序数据已经对传统发酵食品中微生物群落的结构和功能有了较为清晰的认识,但是仍然不清楚微生物群落自发式构建的机制。本文提出微生物群落是分布式的代谢系统,微生物之间的营养相互作用推动了传统发酵食品微生物群落的自发式构建。本文主要阐述了营养相互作用的概念、发生的机理以及研究方法体系,整理了传统发酵食品中微生物之间营养相互作用的研究进展,并提出了未来的研究方向。通过营养相互作用推动的传统发酵食品微生物群落的自发式构建有助于定向控制发酵过程中的微生物种类、提高生产效率和改善发酵质量。