Yeast diversity of Ghanaian cocoa bean heap fermentations

The fermentation of the Theobroma cacao beans, involving yeasts, lactic acid bacteria, and acetic acid bacteria, has a major influence on the quality of the resulting cocoa. An assessment of the microbial community of cocoa bean heap fermentations in Ghana resulted in 91 yeast isolates. These were grouped by PCR-fingerprinting with the primer M13. Representative isolates were identified using the D1/D2 region of the large subunit rRNA gene, internal transcribed spacer sequences and partial actin gene sequences leading to the detection of 15 species. Properties of importance for cocoa bean fermentation, namely sucrose, glucose, and citrate assimilation capacity, pH-, ethanol-, and heat-tolerance, were examined for selected isolates. Pichia kudriavzevii ( Issatchenkia orientalis ), Saccharomyces cerevisiae , and Hanseniaspora opuntiae formed the major components of the yeast community. Hanseniaspora opuntiae was identified conclusively for the first time from cocoa fermentations. Among the less frequently encountered species, Candida carpophila, Candida orthopsilosis, Kodamaea ohmeri, Meyerozyma ( Pichia ) caribbica, Pichia manshurica, Saccharomycodes ludwigii , and Yamadazyma ( Pichia ) mexicana were not yet documented from this substrate. Hanseniaspora opuntiae was preferably growing during the earlier phase of fermentation, reflecting its tolerance to low pH and its citrate-negative phenotype, while no specific temporal distribution was recognized for P. kudriavzevii and S. cerevisiae .     

Yeast microbiota associated with spontaneous sourdough fermentations in the production of traditional wheat sourdough breads of the Abruzzo region (Italy)

The aims of this study were to describe the yeast community of 20 sourdoughs collected from central Italy and to characterize the sourdoughs based on chemical properties. A polyphasic approach consisting of traditional culture-based tests (spore-forming and physiological tests) and molecular techniques (PCR-RFLP, RAPD-PCR, PCR-DGGE) and chemical analysis (total acidity, acids, and sugar contents), was utilized to describe the yeast population and to investigate the chemical composition of the doughs. PCR-RFLP analysis identified 85% of the isolates as Saccharomyces cerevisiae, with the other dominant species being Candida milleri (11%), Candida krusei (2.5%), and Torulaspora delbrueckii (1%). RAPD-PCR analysis, performed with primers M13 and LA1, highlighted intraspecific polymorphism among the S. cerevisiae strains. The diversity of the sourdoughs from the Abruzzo region is reflected in the chemical composition, yeast species, and strain polymorphism. Our approach using a combination of phenotypic and genotypic methods identified the yeast species in the 20 sourdough samples and provided a complete overview of the yeast populations found in sourdoughs from the Abruzzo region.     

Phylogenetic analysis of a spontaneous cocoa bean fermentation metagenome reveals new insights into its bacterial and fungal community diversity

This is the first report on the phylogenetic analysis of the community diversity of a single spontaneous cocoa bean box fermentation sample through a metagenomic approach involving 454 pyrosequencing. Several sequence-based and composition-based taxonomic profiling tools were used and evaluated to avoid software-dependent results and their outcome was validated by comparison with previously obtained culture-dependent and culture-independent data. Overall, this approach revealed a wider bacterial (mainly γ-Proteobacteria) and fungal diversity than previously found. Further, the use of a combination of different classification methods, in a software-independent way, helped to understand the actual composition of the microbial ecosystem under study. In addition, bacteriophage-related sequences were found. The bacterial diversity depended partially on the methods used, as composition-based methods predicted a wider diversity than sequence-based methods, and as classification methods based solely on phylogenetic marker genes predicted a more restricted diversity compared with methods that took all reads into account. The metagenomic sequencing analysis identified Hanseniaspora uvarum, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus as the prevailing species. Also, the presence of occasional members of the cocoa bean fermentation process was revealed (such as Erwinia tasmaniensis, Lactobacillus brevis, Lactobacillus casei, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc mesenteroides, and Oenococcus oeni). Furthermore, the sequence reads associated with viral communities were of a restricted diversity, dominated by Myoviridae and Siphoviridae, and reflecting Lactobacillus as the dominant host. To conclude, an accurate overview of all members of a cocoa bean fermentation process sample was revealed, indicating the superiority of metagenomic sequencing over previously used techniques.     

接种发酵和自然发酵中酿酒酵母菌株多样性比较

[目的]探究自然发酵和接种发酵两种发酵方式,对霞多丽葡萄发酵中酵母菌种多样性和酿酒酵母菌株遗传多样性的影响.[方法]以霞多丽葡萄为原料,分别进行自然发酵和接种不同酿酒酵母菌株(NXU 17-26、UCD522和UCD2610)的发酵,利用26S rDNA D1/D2区序列分析和Interdelta指纹图谱技术分别进行酵...     

Analysis of yeast diversity during spontaneous and induced alcoholic fermentations

The diversity of yeast species and strains was monitored by physiological tests and a simplified method of karyotyping of yeast chromosomes. During the first phase of investigated alcoholic fermentations, the yeast species Metschnikowia pulcherrima and Hanseniaspora uvarum were predominant, irrespective of the origin of the grape must. At the beginning of fermentation H. uvarum was even present in the case of induced fermentations with dried yeast. Middle and end phase of the alcoholic fermentation were clearly dominated by the yeast species Saccharomyces cerevisiae . In the case of spontaneous fermentations, several different strains of S. cerevisiae were present and competed with each other, whereas in induced fermentations only the inoculated strain of S. cerevisiae was observed. A competition of strains of S. cerevisiae also occurred during the fermentation with dried yeast product consisting of two different strains. An effect of H. uvarum on taste and flavour of wines can be postulated according to the frequency of its appearance during the first phase of fermentation. With the method of rapid karyotyping and supplementary physiological tests it was possible to make reliable assertions about the yeast diversity during alcoholic fermentation.     

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。     

Microbiota dynamics and diversity at different stages of industrial processing of cocoa beans into cocoa powder

We sampled a cocoa powder production line to investigate the impact of processing on the microbial community size and diversity at different stages. Classical microbiological methods were combined with 16S rRNA gene PCR-denaturing gradient gel electrophoresis, coupled with clone library construction, to analyze the samples. Aerobic thermoresistant spores (ThrS) (100°C; 10 min) were also isolated and characterized (identity, genetic diversity, and spore heat resistance), in view of their relevance to the quality of downstream heat-treated cocoa-flavored drinks. In the nibs (broken, shelled cocoa beans), average levels of total aerobic microorganisms (TAM) (4.4 to 5.6 log CFU/g) and aerobic total spores (TS) (80°C; 10 min; 4.3 to 5.5 log CFU/g) were significantly reduced (P < 0.05) as a result of alkalizing, while fungi (4.2 to 4.4 log CFU/g) and Enterobacteriaceae (1.7 to 2.8 log CFU/g) were inactivated to levels below the detection limit, remaining undetectable throughout processing. Roasting further decreased the levels of TAM and TS, but they increased slightly during subsequent processing. Molecular characterization of bacterial communities based on enriched cocoa samples revealed a predominance of members of the Bacillaceae, Pseudomonadaceae, and Enterococcaceae. Eleven species of ThrS were found, but Bacillus licheniformis and the Bacillus subtilis complex were prominent and revealed great genetic heterogeneity. We concluded that the microbiota of cocoa powder resulted from microorganisms that could have been initially present in the nibs, as well as microorganisms that originated during processing. B. subtilis complex members, particularly B. subtilis subsp. subtilis, formed the most heat-resistant spores. Their occurrence in cocoa powder needs to be considered to ensure the stability of derived products, such as ultrahigh-temperature-treated chocolate drinks.     

Assessment of the yeast species composition of cocoa bean fermentations in different cocoa-producing regions using denaturing gradient gel electrophoresis

The yeast species composition of 12 cocoa bean fermentations carried out in Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-independently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments, obtained through polymerase chain reaction with universal eukaryotic primers, was carried out with two different commercial apparatus (the DCode and CBS systems). In general, this molecular method allowed a rapid monitoring of the yeast species prevailing during fermentation. Under similar and optimal denaturing gradient gel electrophoresis conditions, the CBS system allowed a better separated band pattern than the DCode system and an unambiguous detection of the prevailing species present in the fermentation samples. The most frequent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates a restricted yeast species composition of the cocoa bean fermentation process. Exceptionally, the Ivorian cocoa bean box fermentation samples showed a wider yeast species composition, with Hyphopichia burtonii and Meyerozyma caribbica among the main representatives. Yeasts were not detected in the samples when the temperature inside the fermenting cocoa pulp-bean mass reached values higher than 45 °C or under early acetic acid production conditions.     

Diversity of the total bacterial community associated with Ghanaian and Brazilian cocoa bean fermentation samples as revealed by a 16 S rRNA gene clone library

Cocoa bean fermentation is a spontaneous process involving a succession of microbial activities, starting with yeasts, followed by lactic acid bacteria and acetic acid bacteria. So far, all microbiological studies about cocoa bean fermentation were based on culture-dependent (isolation, cultivation, and identification), or, more recently, culture-independent (PCR-DGGE, or polymerase chain reaction denaturing gradient gel electrophoresis) methods. Using a metagenomic approach, total DNA was extracted from heap and box fermentations at different time points and from different locations (Ghana and Brazil, respectively) to generate a 16 S rDNA clone library that was sequenced. The sequencing data revealed a low bacterial diversity in the fermentation samples and were in accordance with the results obtained through culture-dependent and a second, culture-independent analysis (PCR-DGGE), suggesting that almost all bacteria involved in the fermentation process are cultivable. One exception was the identification by 16 S rDNA library sequencing of Gluconacetobacter species of acetic acid bacteria that were not detected by the two other approaches. The presence of Enterobacteriaceae related to Erwinia/Pantoea/Tatumella, as revealed by 16 S rDNA library sequencing, suggests an impact of these bacteria on fermentation.     

Effect of must characteristics on the diversity of Saccharomyces strains and their prevalence in spontaneous fermentations

Aims: The aim of this study was to investigate whether grapevine variety and must characteristics influence the diversity of Saccharomyces strains and their prevalence during spontaneous fermentations. Methods and results: Musts from different grapevine varieties, all of them autochthonous from Galicia, were used to perform spontaneous fermentations. Yeasts were isolated from the must and at the beginning, in the middle and at the end of fermentations. Those yeasts identified as Saccharomyces were characterized at the strain level by analysis of mtDNA‐RFLP. The results showed a low diversity of Saccharomyces strains, which was related to must sugar content and total acidity. Moreover, from a total of 44 different Saccharomyces strains, only eleven of them appeared at frequencies higher than 20% and were able to lead fermentations. A significant correlation between yeast strains and must acidity was observed, with the predominance of certain strains at high acidity values. Conclusions: Must characteristics, such as sugar content and acidity, influence the Saccharomyces strains diversity and the leader strains during fermentation. Significance and Impact of the Study: These results showed the adaptation of certain Saccharomyces strains to must with specific characteristics; this may be considered by winemakers for yeast inocula selection. Our findings have special relevance because this is the first study carried out in Galicia dealing with the influence of must properties on yeast strains that control fermentations.     

大别山五针松群落物种多样性及区系分析

设置22个20m×20m的样地,对大别山五针松群落的物种多样性和区系成分进行了研究。结果显示,8800m²的样地内共有维管植物71科152属209种。其中,蕨类植物4科5属6种,裸子植物3科3属4种,被子植物64科144属199种。蔷薇科、樟科、菊科、杜鹃花科、豆科和禾本科等为优势科,所含物种较多。分析结果表明,大别山五针松群落种子植物区系起源古老,地理成分复杂,67个科中,世界分布、热带和温带分布的科分别有21、31和15个,显示出由热带向温带过渡的特性。147个属中,热带成分共43属,占总属数（不包括15个世界广布属）的32.58%;温带成分共有89属,占总属数的67.42%,并以北温带为主。群落垂直结构分为乔木层、灌木层和草本层,其中,Margalef指数、Simpson和Shannon-Wiener指数均以灌木层最大,其次为乔木层,草本层最低。灌木层与草本层的Pielou指数相近,且均大于乔木层。     

四川大巴山巴山水青冈群落的物种多样性特征

巴山水青冈(Fagus pashanica)是川、陕、鄂、渝四省市交界处的特有植物。巴山水青冈林集中分布在四川省东北部的大巴山区。目前关于巴山水青冈群落的资料极其匮乏。作者在大巴山区通过对7个样地39个样方调查数据的分析, 探讨了巴山水青冈群落的物种多样性特征。主要结果有: (1) 巴山水青冈群落物种组成丰富, 共记录到维管束植物77科142属217种。乔木层物种集中于壳斗科、杜鹃花科、蔷薇科、山矾科等科; 灌木层物种集中于蔷薇科、忍冬科、壳斗科、槭树科等科; 草本层植物个体数量以苔草属(Carex)植物和普通鹿蹄草(Pyrola decorata)占绝对优势。(2) 群落乔木层物种多度对数呈倒“J”型分布, 灌木层与草本层的物种多度近似对数正态分布。群落物种丰富度由大到小依次为灌木层>草本层>乔木层; Shannon指数与Simpson指数的顺序为灌木层>乔木层>草本层; 均匀度指数的顺序为乔木层>灌木层>草本层。(3) 东坡、南坡的植物物种多样性高于北坡。乔木层Shannon指数与土壤第二层厚度回归关系显著; 草本层Shannon指数与土壤第一层厚度的回归关系显著; 灌木层的物种数和Shannon指数与土壤第一层有机质回归关系显著。(4) 群落建群种和乔木层主要树种重要值与物种多样性主要表现为负相关关系, 但与灌木层的物种多样性关系不显著(P≥0.05)。结果表明, 灌木层物种多样性主要受到环境因素的影响, 而乔木层和草本层物种多样性受到环境因子和群落自身特征的双重影响。     

Species diversity, invasion, and alternative community states in sequentially assembled communities

The relationship between resident species diversity and invasion is generally negative in experimental studies but takes various forms in observational studies of natural communities. We hypothesized that stochastic species colonization, which applies to natural communities but not to experimental communities generally assembled through simultaneous species introduction, may lead to nonnegative diversity-invasion relationships via incurring priority effects. To test this hypothesis, we manipulated both resident species diversity and colonization history in sequentially assembled communities of bacterivorous protist species. We found that, despite a significant effect of assembly history on invader abundance, invader abundance decreased with diversity. This result was largely driven by positive selection effects associated with the dominant influence of an invasion-resistant species, which shared the most similar resource use pattern with the invader, and by the overall weak priority effects observed for the resident communities. Increasing species diversity, however, significantly strengthened priority effects, providing the first experimental support for the idea that larger species pools promote alternative community states. We suggest that elucidating mechanisms regulating the strength of priority effects may help in understanding variation in diversity-invasion relationships among natural communities.     

四川大巴山米心水青冈（Fagus engleriana）群落的物种多样性特征

四川省大巴山处于秦岭南部的北亚热带,水青冈属植物分布较集中.该处米心水青冈(Fagus engleriana)林分布面积较大,生长良好,保存相对完好,具有重要的地域代表性.分析了米心水青冈群落的物种组成、区系地理成分、物种多样性以及群落自身特征与物种多样性的灰色关联度,得到以下主要结果:(1)在4个样地28个10m×10m群落样方中,一共记录到维管束植物197种,隶属140属、74科. 群落乔木层物种主要由壳斗科、蔷薇科、杜鹃花科、桦木科、槭树科等科组成;灌木层物种主要由蔷薇科、忍冬科、杜鹃花科等科组成;草本层主要由莎草科、菊科、百合科等科植物组成.(2)地理区系成分中,温带成分分别占群落总属数、总种数的65.38%和64.32%,处于主导地位;热带和亚热带成分占总属数、总种数的23.85%、24.32%.(3)群落中物种数目-多度在不同的层次变化趋势不同.(4)群落的物种丰度以灌木层最高、乔木层最低.Shannon指数、Simpson指数、均匀度指数以草本层最高、灌木层最低.(5)乔、灌、草3层的物种多样性均受到乔、灌两层植物的多度与盖度的影响,说明群落自身的特征与群落物种多样性是紧密相关的.     

Diversity and dynamics of bacterial populations during spontaneous sorghum fermentations used to produce ting, a South African food

Ting is a spontaneously fermented sorghum food that is popular for its sour taste and unique flavour. Insight of the microbial diversity and population dynamics during sorghum fermentations is an essential component of the development of starter cultures for commercial production of ting. In this study, bacterial populations associated with spontaneous sorghum fermentations were examined using a culture-independent strategy based on denaturing gradient gel electrophoresis and sequence analysis of V3-16S rRNA gene amplicons, and a culture-dependent strategy using conventional isolation based on culturing followed by 16S rRNA and/or pheS gene sequence analysis. The entire fermentation process was monitored over a 54 h period and two phases were observed with respect to pH evolution and microbial succession. The first phase of the process (0-6 h) was characterized by relatively high pH conditions and the presence of Enterococcus mundtii, albeit that this species was only detected with the culture-dependent approach. The second phase of the fermentation process (12-54 h) was characterized by increased acidity and the predominance of a broader range of lactic acid bacteria, including Lactococcus lactis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, Weissella cibaria, Enterococcus faecalis, and a close relative of Lactobacillus curvatus, as well as some members of the Enterobacteriaceae family. The Lb. curvatus-like species was only detected with PCR-DGGE, while the majority of the other species was only detected using the culture-dependent approach. These findings highlighted the fact that a combination of both approaches was essential in revealing the microbial diversity and dynamics during spontaneous sorghum fermentations.     

胶州湾海域鱼类群落种类组成及多样性

为了解胶州湾海域鱼类群落结构特征,根据2016—2017年间对胶州湾海域进行的4个航次底拖网调查数据,采用相对重要性指数、生态多样性指数和典范对应分析(canonical correspondence analysis,CCA)、非线性多维标度排序(non-metric multidimensional scaling,NMDS)等方法分析了胶州湾海域鱼类群落的种类组成和多样性特征。结果表明:调查共采集到鱼类46种,隶属2纲10目30科41属,以硬骨鱼纲鱼类为主(45种,97.83%)。其中,鲈形目(Perciformes)最多(22种,47.83%),其次是鲉形目(Scorpaeniformes),占15.22%。种类数季节变化明显,以夏季最高,23种;秋季最低,16种。优势种组成以赤鼻棱鳀(Thryssa kammalensis)、褐菖鲉(Sebastiscus marmoratus)、褐牙鲆(Paralichthys olivaceus)、大泷六线鱼(Hexagrammos otakii)、许氏平鮋(Sebastes schlegeli)和矛尾鰕虎鱼(Chaeturichthys stigmatias)等鱼类为主。多样性分析显示,鱼类物种多样性存在明显的季节差异。多样性指数(H′)季节变化范围为1.668—2.453,以夏季最高,春季最低;均匀度指数(J′)季节变化范围为0.577—0.808,以秋季最高,春季最低;丰富度指数(D′)季节变化范围为2.431—3.123,以冬季最高,秋季最低。典范对应分析表明,水温、盐度、水深和pH是影响胶州湾海域鱼类群落物种组成的主要环境因子,且水温和pH是影响鱼类群落结构及多样性时空变化的主要因子。与历史调查资料相比,由于人类活动对胶州湾生态系统的干扰,鱼类群落结构发生了较大变化,优势种组成更替明显,多样性水平降低,鱼类群落结构趋向简单化。     

Characterization, ecological distribution, and population dynamics of Saccharomyces sensu stricto killer yeasts in the spontaneous grape must fermentations of southwestern Spain

Killer yeasts secrete protein toxins that are lethal to sensitive strains of the same or related yeast species. Among the four types of Saccharomyces killer yeasts already described (K1, K2, K28, and Klus), we found K2 and Klus killer yeasts in spontaneous wine fermentations from southwestern Spain. Both phenotypes were encoded by medium-size double-stranded RNA (dsRNA) viruses, Saccharomyces cerevisiae virus (ScV)-M2 and ScV-Mlus, whose genome sizes ranged from 1.3 to 1.75 kb and from 2.1 to 2.3 kb, respectively. The K2 yeasts were found in all the wine-producing subareas for all the vintages analyzed, while the Klus yeasts were found in the warmer subareas and mostly in the warmer ripening/harvest seasons. The middle-size isotypes of the M2 dsRNA were the most frequent among K2 yeasts, probably because they encoded the most intense K2 killer phenotype. However, the smallest isotype of the Mlus dsRNA was the most frequent for Klus yeasts, although it encoded the least intense Klus killer phenotype. The killer yeasts were present in most (59.5%) spontaneous fermentations. Most were K2, with Klus being the minority. The proportion of killer yeasts increased during fermentation, while the proportion of sensitive yeasts decreased. The fermentation speed, malic acid, and wine organoleptic quality decreased in those fermentations where the killer yeasts replaced at least 15% of a dominant population of sensitive yeasts, while volatile acidity and lactic acid increased, and the amount of bacteria in the tumultuous and the end fermentation stages also increased in an unusual way.     

Population dynamics and metabolite target analysis of lactic acid bacteria during laboratory fermentations of wheat and spelt sourdoughs

Four laboratory sourdough fermentations, initiated with wheat or spelt flour and without the addition of a starter culture, were prepared over a period of 10 days with daily back-slopping. Samples taken at all refreshment steps were used for determination of the present microbiota. Furthermore, an extensive metabolite target analysis of more than 100 different compounds was performed through a combination of various chromatographic methods including liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. The establishment of a stable microbial ecosystem occurred through a three-phase evolution within a week, as revealed by both microbiological and metabolite analyses. Strains of Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus rossiae, Lactobacillus brevis, and Lactobacillus paraplantarum were dominating some of the sourdough ecosystems. Although the heterofermentative L. fermentum was dominating one of the wheat sourdoughs, all other sourdoughs were dominated by a combination of obligate and facultative heterofermentative taxa. Strains of homofermentative species were not retrieved in the stable sourdough ecosystems. Concentrations of sugar and amino acid metabolites hardly changed during the last days of fermentation. Besides lactic acid, ethanol, and mannitol, the production of succinic acid, erythritol, and various amino acid metabolites, such as phenyllactic acid, hydroxyphenyllactic acid, and indolelactic acid, was shown during fermentation. Physiologically, they contributed to the equilibration of the redox balance. The biphasic approach of the present study allowed us to map some of the interactions taking place during sourdough fermentation and helped us to understand the fine-tuned metabolism of lactic acid bacteria, which allows them to dominate a food ecosystem.     

Short Communication: Characterization and succession of yeast populations associated with spontaneous fermentations during the production of Brazilian sugar-cane aguardente

A succession of yeasts was observed during fermentation of aguardente with Saccharomyces cerevisiae being the predominant species. Candida sake, Kluyveromyces marxianus var. drosophilarum and apiculate yeasts were also frequent. Transient yeast species were found in variable numbers, probably due to the daily addition of sugar-cane juice. Killer yeasts were isolated and may have a role in the exclusion of some transient and contaminant species.     

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...