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 .     

Isolation of Yeasts from Guajillo Pepper (Capsicum annuum L.) Fermentation and Study of Some Probiotic Characteristics

Probiotics and Antimicrobial Proteins - Three yeast strains were isolated from the spontaneous fermentation of guajillo pepper: Hanseniaspora opuntiae, Pichia kudriavzevii, and Wickerhamomyces...     

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.     

Changes of diversity and population of yeasts during the fermentations by pure and mixed inoculation of Saccharomyces cerevisiae strains

Mixed inoculation of Saccharomyces cerevisiae strains is used in winemaking for achieving high sensory quality of the wine. However, information on the diversity and population of yeasts during inoculated fermentation is very limited. In this study, we evaluated the effect of mixed inocula with different inoculation timing on the yeast community during fermentations of Cabernet Sauvignon. Grape must was inoculated with pure cultures of S. cerevisiae RC212 or S. cerevisiae R312, and simultaneous and sequential inoculation of both strains. Wallersterin Laboratory Nutrient (WLN) medium and sequence of the 26S rDNA D1/D2 domain were used to compare the diversity of yeast species. Five species, including Candida diversa, Hanseniaspora opuntiae, H. uvarum, Issatchenkia orientalis and I. terricola, were identified in the grape must, with Issatchenkia sp. being predominant (67.5 %). Three to four species were involved in each fermentation treatment. The fermentations by mixed inocula presented more yeast species than by pure inocula. Interdelta sequence typing was used to identify S. cerevisiae strains. Ten genotypes were identified among 322 isolated S. cerevisiae strains. Their distribution varied among different stages of fermentations and different inoculation treatments. The inoculated strains were not predominant, while indigenous genotypes I, III, and V showed strong competitiveness during fermentation. In general, this study provided information on the change of population structure and genetic diversity of yeasts in fermentations inoculated with pure and mixed S. cerevisiae strains.     

葡萄酒有孢汉逊酵母属分子指纹图谱分析

为分析中国本土野生有孢汉逊酵母属Hanseniaspora酵母菌的种间差异和种内差异,构建分子指纹图谱,为优良有孢汉逊酵母资源的遗传多样性分析及开发利用提供科学依据。采用依赖于培养的经典方法包含WL营养培养基、26S rRNA基因D1/D2区域序列分析和5.8S-ITS-RFLP分析进行118株贵州紫云县刺葡萄自然发酵过程中分离的有孢汉逊酵母属酵母菌种水平的鉴定。采用Tandem repeat-tRNA（TRtRNA）指纹图谱法研究有孢汉逊酵母属酵母在两对微卫星引物扩增下的分子指纹图谱,并利用DPS软件分析不同分子指纹图谱类别之间的遗传发育关系。结果表明,118株贵州紫云县刺葡萄自然发酵过程中分离的野生有孢汉逊酵母属酵母菌,经依赖于培养的经典方法鉴定为3个种,包括Hanseniaspora opuntiae、H. uvarum和H. occidentalis。TRtRNA指纹图谱法的引物对一TtRNASC和5CAG扩增出5类图谱,而引物对二TtRNASC和ISSR-MB扩增出10类图谱,可以表现出所测118株有孢汉逊酵母属的种间差异和种内差异。其中种内差异主要表现在H. uvarum中。总体而言,贵州紫云县刺葡萄自然发酵过程中分离的有孢汉逊酵母属主要为H. opuntiae、H. uvarum和H. occidentalis,其中H. opuntiae和H. uvarum内部表现了不同的TRtRNA分子指纹图谱,表征了种内遗传多样性。     

Yeast Populations Residing on Healthy or Botrytis-Infected Grapes from a Vineyard in Attica, Greece

The yeast flora associated with healthy and Botrytis-infected grapes was assessed. Molecular identification methods assigned isolates to six genera and nine species. For the first time Hanseniaspora opuntiae was encountered as an inhabitant of the grape ecosystem. By using DraI, an informative restriction fragment length polymorphism pattern was generated to distinguish H. opuntiae from the closely related organism Hanseniaspora guilliermondii. Botrytis infection resulted in a larger population and greater diversity of yeasts enriched with fermentative or spoilage species.     

Three new species of bipolar budding yeasts of the genus Hanseniaspora and its anamorph Kloeckera isolated in Thailand

In the course of a survey of yeast biodiversity in the natural substrates in Thailand, eight strains were found to represent three hitherto undescribed species of Hanseniaspora/Kloeckera . They were isolated from insect frass, flower, lichen, rotted fruit and rotted wood. Based on the morphological and physiological characteristics, and sequences of D1/D2 domain, six strains represent a single species of the genus Hanseniaspora , described as Hanseniaspora thailandica sp. nov. (type BCC 14938^T=NBRC 104216^T=CBS 10841^T), and another strain as Hanseniaspora singularis sp. nov. (type BCC 15001^T=NBRC 104214^T=CBS 10840^T). A further strain, which belongs to Kloeckera and does not produce ascospores, is described as Kloeckera hatyaiensis sp. nov. (type BCC 14939^T=NBRC 104215^T=CBS 10842^T). Strains belonging to H. thailandica sp. nov. differed by 17–19 nucleotide substitutions from Hanseniaspora meyeri , the closest species. DNA reassociation between the two taxa showed 30–48% relatedness. Kloeckera hatyaiensis sp. nov. and H. singularis sp. nov. differed by eight and 16 nucleotide substitutions with one gap from the nearest species, Hanseniaspora clermontiae and Hanseniaspora valbyensis , respectively.     

The phylogeny of the cactophilic yeasts based on the 18S ribosomal RNA gene sequences: the proposals of Phaffomyces antillensis and Starmera caribaea, new combinations.

The complete sequences of the 18S rRNA gene fragments of the type strains of the cactophilic yeast species, Pichia antillensis, Pichia caribaea, Phaffomyces opuntiae, Phaffomyces thermotolerans, Starmera amethionina var. amethionina, and Starmera amethionina var. pachycereana were determined and compared. The type strain of Phaffomyces opuntiae had two kinds of the 18S rRNA gene sequences of which base differences were counted to be 15 and of which the percent similarity was calculated to be 99.1. The type strains of P. antillensis, P. caribaea, and Starmera amethionina var. pachycereana had the Q-7 system. The phylogenetic analyses showed that the genera Phaffomyces and Starmera were monophyletic and distant from each other and from the other species examined of the ascogenous teleomorphic genera, and that P. antillensis and P. caribaea were included within the clusters of the genera Phaffomyces and Starmera, respectively. The two Pichia species were transferred to the genera Phaffomyces and Starmera as the new combinations, Phaffomyces antillensis and Starmera caribaea. The new family Phaffomycetaceae was proposed as the type genus Phaffomyces.     

Host-plant affinities of two biotypes of Dactylopius opuntiae (Homoptera: Dactylopiidae): enhanced prospects for biological control of Opuntia stricta (Cactaceae) in South Africa

1. Until recently, neither the phycitid moth Cactoblastis cactorum nor the cochineal insect Dactylopius opuntiae have been satisfactory biological control agents of Opuntia stricta in South Africa.
2. In marked contrast, both of these agents have kept O. stricta under biological control for many decades in Australia.
3. In an attempt to improve the situation in South Africa, a stock of D. opuntiae was obtained from O. stricta in Australia during 1996.
4. Host-specificity tests confirmed that the newly imported D. opuntiae from Australia is a different biotype to the one already established in South Africa.
5. The Australian ('stricta') biotype thrives on O. stricta but is unable to develop satisfactorily on O. ficus-indica , while the converse is true for the South African ('ficus') biotype, which thrives on O. ficus-indica but fares poorly on O. stricta .
6. The integrity of the host-plant specificity of the two biotypes of D. opuntiae has important implications for biological control of Cactaceae in South Africa, and has greatly enhanced prospects that O. stricta can be brought under biological control successfully.     

Tandem repeat-tRNA (TRtRNA) PCR method for the molecular typing of non-<Emphasis Type="Italic">Saccharomyces</Emphasis> subspecies

There is a worldwide trend to understand the impact of non-Saccharomyces yeast species on the process of winemaking. Although the predominant species at the end of the fermentation is Saccharomyces cerevisiae, several non-Saccharomyces species present during the first days of the process can produce and/or release aromas that improve the bouquet and complexity of the final wine. Since no genomic sequences are available for the predominant non-Saccharomyces species selected from grapes or musts (Hanseniaspora uvarum, Hanseniaspora vineae, Hanseniaspora opuntiae, Metschnikowia pulcherrima, Candida zemplinina), a reproducible PCR method was devised to discriminate strains at the subspecies level. The method combines different oligonucleotides based on tandem repeats with a second oligonucleotide based on a conserved tRNA region, specific for ascomycetes. Tandem repeats are randomly dispersed in all eukaryotic genomes and tRNA genes are conserved and present in several copies in different chromosomes. As an example, the method was applied to discriminate native M. pulcherrima strains but it could be extended to differentiate strains from other non-Saccharomyces species. The biodiversity of species and strains found in the grape ecosystem is a potential source of new enzymes, fungicides and/or novel sustainable methods for biological control of phytopathogens.     

Molecular assessment of indigenous yeast population from traditional balsamic vinegar

AIMS: To identify and describe the indigenous yeast population involved in traditional balsamic vinegar (TBV) fermentation. METHODS AND RESULTS: Using the restriction analysis of the ribosomal region 5.8S (5.8S rRNA) and the internal transcribed spacers 1 and 2 (5.8S-ITS region) we were able to group 133 strains isolated from 17 cooked grape must samples into 10 different yeast species, included into 4 genera. Moreover, we sequenced the D1/D2 domains of the 26S rRNA and confirmed the reliability of each identification at species level. Most strains belonged to the genus Zygosaccharomyces. In particular, Zygosaccharomyces bailii was found in 41% of the samples, followed by Saccharomyces cerevisiae, Zygosaccharomyces pseudorouxii and Candida stellata. Strains belonging respectively to Zygosaccharomyces mellis, Zygosaccharomyces bisporus, Zygosaccharomyces rouxii, Hanseniaspora valbyensis, Hanseniaspora osmophila and Candida lactis-condensi species were also detected. Despite the great number of species recovered, the mtDNA restriction profiles showed low variability at strain level. Saccharomyces cerevisiae isolates with an higher degree of intraspecific variance were considered an exception. CONCLUSIONS: Many different indigenous yeast species were recovered and TBV yeasts population seems to be far more complex than what was reported in previous literature. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the indigenous yeast species of TBV cooked must.     

Mannose-containing Polysaccharides of the Apiculate Yeasts Nadsonia, Hanseniaspora, Kloeckera, and Saccharomycodes, and Their Use as an Aid in Classification

The mannose-containing polysaccharides formed by species of Nadsonia, Hanseniaspora, Kloeckera, and Saccharomycodes were extracted with hot aqueous alkali and purified by precipitation as their copper complexes. N. fulvescens and N. elongata formed galactomannans, while Hanseniaspora and Kloeckera species and S. ludwigii formed mannans. H. valbyensis, H. uvarum, and K. apiculata were a group which formed mannans which had identical H-1 regions in their proton magnetic resonance (PMR) spectra, and H. osmophila, K. africana, and K. magna mannas formed another group based on similar spectra. K. javanica formed a mannan with an H-1 spectral region which resembled that of the H. valbyensis group in some respects and that of the H. osmophila group in others. The H-1 portion of the PMR spectrum of S. lugwigii mannan was very complex and was unlike that of any other apiculate yeast studied.     

秦岭地区子囊菌酵母物种多样性研究

从陕西秦岭地区的果实和叶等不同基物上分离得到子囊菌酵母262株,利用26SrDNA的D1/D2区域序列分析并结合形态学特征对这些菌株进行了分类学研究,探讨了该地区子囊菌酵母的物种多样性及其分布。共鉴定出10属31种,其中优势属为假丝酵母属Candida,12种,(其中新种2个,另文发表)、酿酒酵母属Saccharomyces,5种、毕赤酵母属Pichia,5种和有孢汉逊酵母属Hanseniaspora,3种。对分离自陕西秦岭不同地区与海拔的同一个种的不同菌株进行了D1/D2序列分析比较,以探讨子囊菌酵母种内序列稳定性和变异幅度。     

Yeasts Associated with Various Amazonian Native Fruits

Yeasts, commonly present on the surface of fruits, are of industrial interest for the production of enzymes, flavorings, and bioactive compounds, and have many other scientific uses. The Amazonian rainforest may be a good source of new species or strains of yeasts, but their presence on Amazonian fruits is unknown. The aim of this study was to identify and characterize yeasts isolated from Amazonian native fruits using molecular and phenotypic methods. In total, 81 yeast isolates were obtained from 10 fruits species. Rep-PCR showed 29 strain profiles. Using a combination of restriction-fragment length polymorphism (RFLP) of the 5.8S-ITS region and D1/D2 sequencing of the 26S rRNA gene, 16 species were identified belonging to genera Candida, Debaryomyces, Hanseniaspora, Kodamaea, Martiniozyma, and Meyerozyma. The most dominant species were Candida tropicalis, Debaryomyces hansenii, Hanseniaspora opuntiae, and Hanseniaspora thailandica. H. opuntiae and H. thailandica showed the highest number of the strain profiles. Phenotypic profiles were variable between species, and even among strains. Screening for hydrolases showed lipolytic activity in only one isolate, while proteolytic, cellulolytic and amylolytic capabilities were not detected. Yeast presence among fruits varied, with cidra (Citrus medica) and ungurahui (Oenocarpus bataua) having the highest number of species associated. This investigation broadens the understanding and possible biotechnological uses of yeast strains obtained from Amazonian native fruits.Key words: yeast diversity, fruit, Amazonia, PCR-RFLP, 5.8S-ITS     

Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera

Three molecular methods, RAPD-PCR analysis, electrophoretic karyotyping and RFLP of the PCR-amplified ITS regions (ITS1, ITS2 and the intervening 5.8S rDNA), were studied for accurate identification of Hanseniaspora and Kloeckera species as well as for determining inter- and intraspecific relationships of 74 strains isolated from different sources and/or geographically distinct regions. Of these three methods, PCR-RFLP analysis of ITS regions with restriction enzymes DdeI and HinfI is proposed as a rapid identification method to discriminate unambiguously between all six Hanseniaspora species and the single non-ascospore-forming apiculate yeast species Kloeckera lindneri. Electrophoretic karyotyping produced chromosomal profiles by which the seven species could be divided into four groups sharing similar karyotypes. Although most of the 60 strains examined exhibited a common species-specific pattern, a different degree of chromosomal-length polymorphism and a variable number of chromosomal DNA fragments were observed within species. Cluster analysis of the combined RAPD-PCR fingerprints obtained with one 10-mer primer, two microsatellite primers and one minisatellite primer generated clusters which with a few exceptions are in agreement with the groups as earlier recognized in DNA-DNA homology studies.     

Diversity and evolution of non-Saccharomyces yeast populations during wine fermentation: effect of grape ripeness and cold maceration

We have evaluated the effect of grape maturity and cold maceration prior to fermentation on the yeast ecology during wine fermentation. Non-Saccharomyces strains were selectively isolated and identified using two rapid PCR techniques, namely enterobacterial repetitve intergenic consensus-PCR and PCR-intron splice sites, in various wine fermentation conditions. These identifications were further complemented and confirmed by restriction fragment length poymorphism and sequencing analysis of the 5.8S-ITS and D1/D2 ribosomal regions, respectively. Eleven species belonging to five genera were identified. Candida stellata, Hanseniaspora uvarum and Hanseniaspora osmophila were the dominant species, representing almost 90% of the isolates. Minor strains presented different species of the genera Candida, Issatchenkia, Zygoascus and Zygosaccharomyces. Selective isolation made it possible to isolate some species that were hardly related to the wine-making process, such as Issatchenkia hanoiensis, a new species that has only been described recently.     

Yeast diversity and persistence in botrytis-affected wine fermentations

Culture-dependent and -independent methods were used to examine the yeast diversity present in botrytis-affected ("botrytized") wine fermentations carried out at high ( approximately 30 degrees C) and ambient ( approximately 20 degrees C) temperatures. Fermentations at both temperatures possessed similar populations of Saccharomyces, Hanseniaspora, Pichia, Metschnikowia, Kluyveromyces, and Candida species. However, higher populations of non-Saccharomyces yeasts persisted in ambient-temperature fermentations, with Candida and, to a lesser extent, Kluyveromyces species remaining long after the fermentation was dominated by SACCHAROMYCES: In general, denaturing gradient gel electrophoresis profiles of yeast ribosomal DNA or rRNA amplified from the fermentation samples correlated well with the plating data. The direct molecular methods also revealed a Hanseniaspora osmophila population not identified in the plating analysis. rRNA analysis also indicated a large population (>10(6) cells per ml) of a nonculturable Candida strain in the high-temperature fermentation. Monoculture analysis of the Candida isolate indicated an extreme fructophilic phenotype and correlated with an increased glucose/fructose ratio in fermentations containing higher populations of CANDIDA: Analysis of wine fermentation microbial ecology by using both culture-dependent and -independent methods reveals the complexity of yeast interactions enriched during spontaneous fermentations.     

Killer toxin of Hanseniaspora uvarum

The yeast Hanseniaspora uvarum liberates a killer toxin lethal to sensitive strains of the species Saccharomyces cerevisiae. Secretion of this killer toxin was inhibited by tunicamycin, an inhibitor of N-glycosylation, although the mature killer protein did not show any detectable carbohydrate structures. Culture supernatants of the killer strain were concentrated by ultrafiltration and the extracellular killer toxin was precipitated with ethanol and purified by ion exchange chromatography. SDS-PAGE of the electrophoretically homogenous killer protein indicated an apparent molecular mass of 18,000.Additional investigations of the primary toxin binding sites within the cell wall of sensitive yeast strains showed that the killer toxin of Hanseniaspora uvarum is bound by -1, 6-d-glucans.     

田间施药对自然发酵葡萄酒酵母菌群落结构的影响

【背景】酵母菌是葡萄酒发酵过程中一类非常重要的微生物,其多样性及群体组成对葡萄酒的质量有重要贡献。影响葡萄酒中酵母菌组成的因素有很多,但目前尚未见葡萄园田管理对葡萄酒酵母菌群落结构影响方面的报道。【目的】探索田间施药对自然发酵葡萄酒酵母菌群落结构的影响。【方法】采用分离培养、常规分子生物学鉴定和Illumina MiSeq宏基因组测序结合的方法分析不同样品中的酵母菌群落结构情况。【结果】从不使用内吸收型化学农药的葡萄样品自然发酵液中分离鉴定出Pichia、Hanseniaspora、Schizosaccharomyces、Candida、Saccharomyces、Zygoascus、Issatchenkia等7个属8个种的酵母菌,宏基因组测序结果表明有Pichia(29.42%)、Saccharomyces(21.91%)、Issatchenkia(17.99%)、 Hanseniaspora(12.10%)、 Candida(7.47%)、 Zygosaccharomyces(5.32%)、Schizosaccharomyces (3.07%)、Aureobasidium (0.29%)等属的酵母菌参与发酵;使用常规化学农药的葡萄样品自然发酵液中分离鉴定出Pichia、Hanseniaspora、Schizosaccharomyces、Candida、Cryptococcus等5个属6个种的酵母菌,宏基因组测序结果表明有Pichia (41.66%)、Hanseniaspora (21.54%)、Candida(19.11%)、 Zygosaccharomyces(7.78%)、 Schizosaccharomyces(4.04%)、 Cryptococcus(3.21%)、Saccharomyces (1.12%)、Aureobasidium (0.49%)等属的酵母菌参与发酵。【结论】两样品中酵母菌比例有显著差异,表明在酿酒葡萄的园田管理中化学农药的使用对自然发酵葡萄酒的酵母菌群落结构有较大影响。     

基于28S rDNA序列构建侧耳属系统发育树

通过对侧耳属18个分类单元的28S rDNA序列进行分析,构建了侧耳属较为完整的系统发育树。分子系统学资料显示:Coremiopleurotus组和侧耳属内单、二系菌丝系统分别为多系起源的;Pleurotus组单系菌丝种类、被划分在Tuberregium组的具核侧耳和Lentodiellum组的P. levis能够分别与侧耳属内其他成员进行区分;红侧耳、P. calyptratus、P. opuntiae三者关系密切,而金顶侧耳应作为白黄侧耳的种下分类单元。