白酒大曲真菌群落结构多样性研究技术与进展

随着现代分子生物学技术在微生物群落结构多样性研究中的广泛应用,打破了传统微生物培养方法的局限性,提高了对于大曲中真菌群落结构多样性的认识。该文主要综述了研究大曲中真菌群落结构多样性的方法,传统可培养技术以及现代分子生物学技术的各种方法,主要包括磷脂脂肪酸技术（PLFA）、聚合酶链式反应（PCR）-变性梯度凝胶电泳（DGGE）技术、PCR-单链构象多态性（SSCP）技术、基因克隆文库技术、高通量测序（HTS）技术以及组学技术,以及近年来传统可培养技术和现代分子生物学技术手段在大曲中真菌群落结构多样性的应用研究进展,对深入研究大曲发酵过程中真菌多样性构成及变化规律奠定理论基础。     

PCR-DGGE技术在肉制品中微生物分析中的应用

<正>PCR-DGGE(变性梯度凝胶电泳)是一种分析微生物区系的分子指纹技术。本文概述了PCR-DGGE的基本原理、发展和在食品微生物分析中应用现状,着重阐述该技术在评价肉制品中的细菌群落组成和动态变化、肉品微生物鉴定等研究领域中的应用以及该技术存在的一些缺点,并进一步展望DGGE技术在今后肉制品研究领域的应用前景。变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE)     

液熏罗非鱼片加工过程中微生物群落的PCR-DGGE 分析

为弄清液熏罗非鱼片加工过程中微生物污染的源头,以进一步防控产品的微生物污染提供依据。应用基于细菌16S rDNA 的PCR-DGGE(PCR-denaturing gradient gel electrophoresis,PCR- 变性梯度凝胶电泳)技术分析液熏罗非鱼片主要加工关键环节的微生物群落结构,提取样品中的细菌总DNA,对细菌的16S rDNA 的V6～V8 区段进行PCR 扩增后,进行变性梯度凝胶电泳(DGGE),对DGGE 图谱进行微生物多样性分析,对主要条带进行序列分析并构建系统发生树。结果表明：10 个条带所代表的优势种很可能来源于以下几个属：巨型球菌属(Macrococus)、微球菌属(Micrococus)、肠道细菌属(Enterobacter)、假单胞菌属(Pseudomonas)、弧菌属(Vibrio)、突柄杆菌属(Prosthecobacter)、布特菌属(Buttiauxella),其中肠道细菌属、微球菌属、假单胞菌属和弧菌属细菌都具有使产品腐败的潜能。本研究表明：液熏罗非鱼片中呈现微生物多样性,PCR-DGGE 技术可用于研究液熏罗非鱼片加工过程中的微生物群落结构及变化,且具有一定的优越性。     

PCR-DGGE技术在肉品微生物生态学中的应用

人们一直通过传统的纯化、培养方法对肉和肉制品中的微生物进行研究,其操作过程复杂且无法准确描述完整的菌群结构。聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术因其具有快速、精确的特点在微生物生态学中得到了广泛应用。本文主要介绍PCR-DGGE技术的原理,从非发酵肉制品腐败和发酵肉制品成熟两个角度综述其在肉品微生物生态学研究中的应用,并对该技术今后在肉品微生物研究中的应用进行展望。     

PCR-DGGE技术概述及其在白酒领域的应用

PCR-DGGE(polymerase chain reaction-denaturing gradient gel electrophoresis)技术即为聚合酶链式反应-变性梯度凝胶电泳技术.它具有快速、灵敏度高、重复性好等特点,越来越多地被应用于微生物群落结构分析与鉴定,其在白酒酿造过程中微生物的鉴定领域中崭露头角.文中主要就其在白酒中的应用进行介绍.     

不同质量窖泥细菌群落的研究

采用多聚酶链式反应结合变性梯度凝胶电泳指纹分析技术(PCR-DGGE),对不同质量窖泥中细菌群落结构进行分析,从微生物角度说明了不同质量窖泥细菌种群的复杂性,同时,鉴定出了常见的芽孢杆菌、醋酸杆菌等微生物,以及发现可能的新菌种和尚未在窖泥研究中报道的瘤胃球菌。以质量最好的窖泥为参照,其他窖泥的质量与其跟参照窖泥的细菌群落间相似性指数总体呈正相关。这一规律为今后研究窖泥培养、养护和应用提供了理论依据。     

PCR-DGGE技术及其在食品菌相分析中的应用

传统培养、鉴定的方法研究微生物菌相变化的缺点突出,不易对微生物的变化情况进行实时的分析.近年来,使用聚合酶链式反应-变性梯度凝胶电泳技术(PCR-DGGE)分析食品中菌群变化情况的优点逐渐突显.本文介绍了PCR-DGGE技术的原理、优点和缺点,以及在食品菌相研究中的应用,并对该技术在食品微生物研究中的前景进行了展望.     

PCR-DGGE技术在微生物物种多样性研究中的局限性及其解决措施

变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)技术目前已经成为研究微生物物种多样性和动态变化的分子检测工具之一.该技术不依赖于微生物的分离培养,便能快速、准确地获取复杂样品中微生物的菌群组成及其遗传信息,因此被广泛应用于微生物生态学、食品微生物学等领域.但DGGE技术存在一定的局限性,有时会造成分析结果的偏差.本文简单概述了DGGE技术的基本原理及其在微生物群落分析中的应用,重点对造成DGGE结果偏差的因素,如DNA提取、PCR扩增、DGGE条件以及DGGE图谱分析等进行讨论,并提出相应的解决措施,旨在对DGGE检测结果的分析提供参考.     

变性梯度凝胶电泳(DGGE)技术在食品微生物研究中的应用

变性梯度凝胶电泳(DGGE)是一种分析微生物区系的分子指纹技术.概述DGGE的基本原理、发展和应用现状,着重阐述该技术在评价发酵食品的细菌群落组成和动态变化、区分发酵食品、控制食品质量、检测食源性致病微生物等食品微生物研究领域中的应用,进一步展望了DGGE技术在食品研究领域的应用前景.     

PCR-DGGE技术在废水生物处理中的应用

变性梯度凝胶电泳(PCR-DGGE)具有可靠性强、重复性好、方便快捷等优点,目前被广泛地应用于废水生物处理环境微生物群落多样性和动态分析。本文对PCR-DGGE技术的基本原理和流程,图谱的优化过程和图谱分析方法,及其在环境微生物生态学中的应用进行了综述,并对该技术自身存在的局限性和应用前景进行了评价。     

Omics in traditional vegetable fermented foods and beverages

Abstract

For a long time, food microbiota has been studied using traditional microbiological techniques. With the arrival of molecular or culture-independent techniques, a strong understanding of microbiota dynamics has been achieved. However, analyzing the functional role of microbial communities is not an easy task. The application of omics sciences to the study of fermented foods would provide the metabolic and functional understanding of the microbial communities and their impact on the fermented product, including the molecules that define its aroma and flavor, as well as its nutritional properties. Until now, most omics studies have focused on commercial fermented products, such as cheese, wine, bread and beer, but traditional fermented foods have been neglected. Therefore, the information that allows to relate the present microbiota in the food and its properties remains limited. In this review, reports on the applications of omics in the study of traditional fermented foods and beverages are reviewed to propose new ways to analyze the fermentation phenomena.     

Recent advances in molecular techniques to study microbial communities in food-associated matrices and processes

In the last two decades major changes have occurred in how microbial ecologists study microbial communities. Limitations associated with traditional culture-based methods have pushed for the development of culture-independent techniques, which are primarily based on the analysis of nucleic acids. These methods are now increasingly applied in food microbiology as well. This review presents an overview of current community profiling techniques with their (potential) applications in food and food-related ecosystems. We critically assessed both the power and limitations of these techniques and present recent advances in the field of food microbiology attained by their application. It is unlikely that a single approach will be universally applicable for analyzing microbial communities in unknown matrices. However, when screening samples for well-defined species or functions, techniques such as DNA arrays and real-time PCR have the potential to overtake current culture-based methods. Most importantly, molecular methods will allow us to surpass our current culturing limitations, thus revealing the extent and importance of the ‘non-culturable’ microbial flora that occurs in food matrices and production.     

DNA-based, culture-independent strategies for evaluating microbial communities in food-associated ecosystems

Culture-independent molecular techniques are now available to study microbial ecosystems. They are opening interesting perspectives to problems related to composition and population dynamics of microbial communities in various environmental niches (e.g., soil, water) and foods. In fermented food products, estimates of true microbial diversity is often difficult chiefly on account of the inability to cultivate most of the viable bacteria. The increasing knowledge of gene sequences and the concomitant development of new culture-independent molecular techniques are providing new and effective tools to compare the diversity of microbial communities and to monitor population dynamics in minimally disturbed samples. In this review, recent advances in these techniques are reported. Possible applications to food-associated microbial ecosystems are emphasised.     

Microbial diversity and succession during the manufacture and ripening of traditional, Spanish, blue-veined Cabrales cheese, as determined by PCR-DGGE

The diversity and dynamics of the dominant microbial communities arising during the manufacture and ripening of four batches of naturally fermented Cabrales cheese were investigated by the PCR-DGGE culture-independent technique. Total microbial DNA was extracted from cheese milk, curd and cheese samples and used as template material in PCR experiments to amplify the V3 region of the bacterial 16S rRNA gene, plus the D1 region of the eukaryotic 26S rRNA gene. These regions were then analysed using DGGE. Eukaryotic and bacterial bands were identified by isolation, reamplification and sequencing. The results were compared to those obtained in a previous microbial characterization of the same four batches using classical culturing methods. Great variability was recorded between batches by the PCR-DGGE technique. This was also shown by culturing, and underlines the uniqueness of artisanal products. Lactocococcus lactis subsp. lactis was dominant from the cheese milk stage until the end of ripening, whereas populations of certain Lactobacillus species appeared during ripening. Populations of species never isolated by culturing were found to be numerous by the PCR-DGGE method, in particular Lactococcus garvieae and Lactococcus raffinolactis. Other, completely unknown lactococci were also detected. The dominant eukaryotic populations from day 15 onwards were those of Penicillium roqueforti and Geotrichum candidum.     

Polyphasic study of microbial communities of two Spanish farmhouse goats' milk cheeses from Sierra de Aracena

The microbial communities present in 2 different types of farmhouse goats' milk cheese from the Aracena mountains (southwest Spain), Quesailla Arochena (hard cheese) and Torta Arochena (soft cheese), have been studied using both culture-dependent and culture-independent techniques. All bacterial isolates were clustered by using randomly amplified polymorphic DNA (RAPD) and identified by 16S rRNA gene sequencing, species-specific PCR and multiplex PCR. Thus a total of 26 different species were identified, the majority belonging to the lactic-acid bacteria (LAB), mainly represented by Lactococcus lactis and Lactobacillus species such as Lactobacillus plantarum and Lactobacillus paracasei, together with a significant proportion of enterococci. Amongst the non-lactic-acid bacteria (NLAB), which represented 37% of the isolates in Torta Arochena, enterobacteria were the most important, Hafnia alvei and Serratia liquefaciens being the predominant species in Quesailla Arochena and Torta Arochena respectively. Moreover, RAPD analysis of the isolates revealed that most of the genotypes were specific to one of the cheeses, although a few genotypes common to both cheeses were found.     

PCR–DGGE as a tool for characterizing dominant microbial populations in the Spanish blue-veined Cabrales cheese

The microbial populations of cheese milk and rennet extracts used in the production of traditional, Spanish, blue-veined Cabrales cheese were identified by PCR–DGGE analysis of the V3 region of the bacterial 16S rRNA gene and of the D1 region of the eukaryotic 26S rRNA genes. Ripe cheeses (60 days old) were examined in the same way. The results obtained by this culture-independent technique were compared to others previously obtained by conventional culturing methods. Rennet extracts were dominated by a number of Lactobacillus species, including Lb. plantarum, a non-starter lactic acid bacterium dominant during ripening. Lactococcus lactis was only found in one rennet extract. The cheese milk was clearly dominated by Lactococcus-like bacteria, with Lc. lactis in the greatest number. This bacterium was also dominant in the cheese samples (on both the surface and in the interior), in agreement with results obtained by culturing. The sequences of several bacterial DGGE bands from all samples showed less than 97% homology to known, cultured species. This indicates that unknown species are present in the Cabrales cheese environment and that culture-independent methods are needed to fully characterize this ecosystem.     

Artisanal and experimental Pecorino Siciliano cheese: microbial dynamics during manufacture assessed by culturing and PCR-DGGE analyses

Traditional artisanal Pecorino Siciliano (PS) cheeses, and two experimental PS cheeses were manufactured using either raw or pasteurised ewes' milk with the addition of starter cultures. The bacterial diversity and dynamics of the different cheese types were evaluated both by culturing and characterisation of isolates, and a culture-independent approach based on the 16S ribosomal RNA (rRNA) gene. Following cultivation, artisanal and experimental cheese types showed similar microbial counts, and isolates belonging to Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecalis and Leuconostoc mesenteroides were identified by phenotypic characterisation and comparison of the restriction fragment length polymorphism (RFLP) of the 16S rRNA gene to that of reference species. The culture-independent fingerprinting technique PCR and denaturing gradient gel electrophoresis (DGGE) of V6 to V8 regions of the 16S rRNA gene of samples taken during artisanal PS cheese manufacture, from raw milk to the ripened cheese, indicated relevant shifts in the microbial community structure. The dominance of Streptococcus bovis and Lactococcus lactis species in the traditional artisanal PS was revealed by 16S rRNA gene sequencing. Comparison of DGGE profiles of samples from milk to ripened cheese, derived from artisanal procedure and the two experimental PS cheeses during production showed similar trends with the presence of intense bands in common. Nevertheless, the profiles of several artisanal cheeses from different farms appeared more diverse, and these additional species are probably responsible for the generally superior flavour and aroma development of traditional PS cheese.     

The diversity and evolution of microbiota in traditional Turkish Divle Cave cheese during ripening

The microbial diversity of traditional Turkish Divle Cave cheese was evaluated in three independent batches. Using molecular techniques, twenty three bacterial species were identified in the interior and outer part of the cheese on days 60 and 120. Bacilli and Gammaproteobacteria classes were predominant during early stages of ripening and Actinobacteria at later stages. Nineteen species of filamentous fungi and five yeast species were identified and the most frequently isolated species were Penicillium polonicum, Penicillium biforme, Penicillium roqueforti, Penicillium chrysogenum and Debaryomyces hansenii. The microbiota displayed similar communities among batches, but high diversity in different parts of the cheese during ripening. Novel cheese starter cultures could be developed after the technological characterisation of the isolated strains.     

Microbial composition during Chinese soy sauce koji-making based on culture dependent and independent methods

Koji-making is a key process for production of high quality soy sauce. The microbial composition during koji-making was investigated by culture-dependent and culture-independent methods to determine predominant bacterial and fungal populations. The culture-dependent methods used were direct culture and colony morphology observation, and PCR amplification of 16S/26S rDNA fragments followed by sequencing analysis. The culture-independent method was based on the analysis of 16S/26S rDNA clone libraries. There were differences between the results obtained by different methods. However, sufficient overlap existed between the different methods to identify potentially significant microbial groups. 16 and 20 different bacterial species were identified using culture-dependent and culture-independent methods, respectively. 7 species could be identified by both methods. The most predominant bacterial genera were Weissella and Staphylococcus. Both 6 different fungal species were identified using culture-dependent and culture-independent methods, respectively. Only 3 species could be identified by both sets of methods. The most predominant fungi were Aspergillus and Candida species. This work illustrated the importance of a comprehensive polyphasic approach in the analysis of microbial composition during soy sauce koji-making, the knowledge of which will enable further optimization of microbial composition and quality control of koji to upgrade Chinese traditional soy sauce product.