Hanseniaspora Opuntiae Research Articles

The environmental and intrinsic yeast diversity of Cuban cocoa bean heap fermentations

The environmental yeast diversity of spontaneous cocoa bean fermentations in east Cuba was investigated. Seven fermentations, 25 equipment- and handling-related samples, and 115 environmental samples, such as flowers, leaf and cocoa pod surfaces, as well as drosophilid insects, were analysed. The basic fermentation parameters temperature and pH were recorded during five fermentations for at least six days. A total of 435 yeast isolates were identified by a combination of PCR-fingerprinting of genomic DNA with the M13 primer and sequence analysis of DNA from representative isolates, using the internal transcribed spacer region, the D1/D2 region of the large subunit rRNA gene, and an actin gene-encoding fragment, as required. Among 65 yeast species detected, Pichia manshurica and Hanseniaspora opuntiae were the most frequently isolated species, obtained from five and four fermentations, followed in frequency by Pichia kudriavzevii from two fermentations. Saccharomyces cerevisiae was isolated only occasionally. Cocoa fermentation yeast species were also present on processing equipment. The repeated isolation of a preliminarily as Yamadazyma sp. classified species, a group of strains similar to Saccharomycopsis crataegensis from fermentations and equipment, and the isolation of fifteen other potentially novel yeast species in low numbers provides material for further studies. Environmental samples showed higher yeast diversity compared to the fermentations, included the most frequent fermentation species, whereas the most frequently isolated environmental species were Candida carpophila, Candida conglobata, and Candida quercitrusa. Potential selective advantages of the most frequently isolated species were only partly explained by the physiological traits tested. For instance, tolerance to higher ethanol concentrations was more frequent in strains of Pichia spp. and S. cerevisiae compared to Hanseniaspora spp.; the ability to also assimilate ethanol might have conferred a selective advantage to Pichia spp. In contrast, high glucose tolerance was common among strains of Hanseniaspora spp., Torulaspora delbrueckii, and Candida tropicalis, among which only Hanseniaspora spp. were frequently isolated.

Isolation, identification of yeast strains producing bioethanol and improvement of bioethanol production on cheese whey / Biyoetanol üreten maya türlerinin izolasyonu, tanılaması ve peynir altı suyunda biyoetanol üretiminin sağlıklaştırılması

Abstract Objective: In this study, bioethanol production on cheese whey (prehydrolysed enzymatically using β galactosidase by isolated yeast strains was investigated. Methods: The yeast strains were isolated from Algerian natural sources (soil and grape) and further were selected on the basis of high ethanol tolerance and high ethanol production on prehydrolysed cheese whey. The selected ones were identified by morphological, physiological and biochemical characteristics. Then, a molecular identification was carried out by amplification and sequencing the D1/D2 domain of 26S rDNA region. In addition, the operating parameters of fermentation such as temperature, pH and substrate concentration (mixture of glucose and galactose) were tested for efficient yeast strain. Results: Among the selected and identified yeast strains, three strain isolates were found to be able to produce bioethanol. These strains are Hanseniaspora opuntiae Z087A0VS, Candida tropicalis Z087B0VS and Candida tropicalis Z087D0VS with an identity of 99% and 100% respectively comparing with the stocked strains in data bank. Furthermore, Hanseniaspora opuntiae presents an ethanol tolerance ethanol up to 11% whereas the two other strains of Candida tolerate up to 12%. The fermentation parameters of most efficient strain were optimized, the temperature 30°C, pH 5 and sugar concentration (glucose and galactose) of 12.5% (w/v) are considered as optimum values for Candida tropicalis Z087B0VS. Conclusion: Candida tropicalis Z087B0VS can be considered as a good candidature for industrial bioethanol production.

From grape berries to wine: population dynamics of cultivable yeasts associated to "Nero di Troia" autochthonous grape cultivar.

The aim of this work was to study the biodiversity of yeasts isolated from the autochthonous grape variety called "Uva di Troia", monitoring the natural diversity from the grape berries to wine during a vintage. Grapes were collected in vineyards from two different geographical areas and spontaneous alcoholic fermentations (AFs) were performed. Different restriction profiles of ITS-5.8S rDNA region, corresponding to Saccharomyces cerevisiae, Issatchenkia orientalis, Metschnikowia pulcherrima, Hanseniaspora uvarum, Candida zemplinina, Issatchenkia terricola, Kluyveromyces thermotolerans, Torulaspora delbrueckii, Metschnikowia chrysoperlae, Pichia fermentans, Hanseniaspora opuntiae and Hanseniaspora guilliermondii, were observed. The yeast occurrences varied significantly from both grape berries and grape juices, depending on the sampling location. Furthermore, samples collected at the end of AF revealed the great predominance of Saccharomyces cerevisiae, with a high intraspecific biodiversity. This is the first report on the population dynamics of 'cultivable' microbiota diversity of "Uva di Troia" cultivar from the grape to the corresponding wine ("Nero di Troia"), and more general for Southern Italian oenological productions, allowing us to provide the basis for an improved management of wine yeasts (with both non-Saccharomyces and Saccharomyces) for the production of typical wines with desired unique traits. A certain geographical-dependent variability has been reported, suggesting the need of local based formulation for autochthonous starter cultures, especially in the proportion of the different species/strains in the design of mixed microbial preparations.

Yeasts isolated from figs (Ficus carica L.) as biocontrol agents of postharvest fruit diseases

Fresh fruit is highly perishable during postharvest life, mainly due to fungal growth. Thus, fungal control is an important goal for the fruit industry. In this work, a selection of antagonistic yeasts isolated from fig and breba crops were screened in vitro. The isolated yeasts were challenged with three moulds isolated from decayed figs and breba crops, identified as Penicillium expansum M639 and Cladosporium cladosporioides M310 and M624, and pathogenic moulds Botrytis cinerea CECT20518 and Monilia laxa CA1 from culture collections. Two yeast isolates, Hanseniaspora opuntiae L479 and Metschnikowia pulcherrima L672, were selected for their ability to inhibit the growth of aforementioned moulds. These yeasts reduced the radial growth of moulds on PDA by between 45.23% and 66.09%. Antagonistic activity was associated with the interaction of live yeast cells with moulds. M. pulcherrima L672 apparently parasitised C. cladosporioides isolates. In addition, challenges were assayed using wounded apples and nectarines, with significant reductions in percent infection and lesion size for all moulds tested. To our knowledge, this is the first report identifying H. opuntiae as an antagonist against different pathogenic moulds.

Autochthonous yeast populations from different brazilian geographic indications

Yeasts are versatile microorganisms which show heterogeneity in their abilities of aromatic molecules formation. The metabolic conversions may improve the production of a particular compound already formed by the microorganism or promote the production of a completely new biochemicals. These conversions depend on the environment. The microbiome of terroir is unique. If the term terroir is a set of physical properties of a vineyard that contribute to the specific characteristics of its wine, the microorganisms will undoubtedly form an integral part of this concept. There are yeasts, filamentous fungi and bacteria that can affect the quality of the wine. The aim of the present study was to identify the autochthonous yeast populations of grape berries collected from regions with Geographic Indications or under construction. The identification was carried out by an approach, combining Maldi-Tof-MS, PCR-RFLP of the internal transcribed spacer with 5.8S ribosomal DNA (rDNA) (ITS1-5.8S-ITS2) and sequences of the D1/D2 domain of the 26S rRNA gene. Some species are common to different GIs and in some of them other species are completely absent, besides some places are contiguous areas. In some areas, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Pichia myanmarensis and Hanseniaspora uvarum were the predominant species.

Non-Saccharomyces biodiversity in wine and the ‘microbial terroir’: a survey on Nero di Troia wine from the Apulian region, Italy

The aim of this work was to study the biodiversity of cultivable non-Saccharomyces yeasts isolated from the autochthonous wine variety ‘Uva di Troia’ from the North-Apulian region during vintages 2012 and 2013. Grapes were collected in vineyards from four different geographical areas on which four different wines with the status of geographical indication are produced. Different restriction profiles of ITS–5.8S rDNA regions, corresponding to those of Candida boidinii, Candida zemplinina, Hanseniaspora guilliermondii, Issatchenkia terricola, Zygosaccharomyces bailii, Hanseniaspora uvarum, Zygoascus hellenicus and Hanseniaspora opuntiae, were observed. The most abundant genera were represented by Hanseniaspora guilliermondii and Candida zemplinina. Significant differences among locations and vintages were observed. This first report on non-Saccharomyces diversity during the early steps of spontaneous alcoholic fermentation of Nero di Troia wines provides the basis for an improved management of non-Saccharomyces in typical Apulian wines, which will be important for the development of the local wine industry and to achieve an enhanced standard of safety in the final production.

Analysis of culturable yeast diversity in spontaneously fermented orange wine, orange peel and orangery soil of a Ponkan plantation in China

A culturable diversity analysis of yeast species in spontaneous fermentation orange wine, orange peel and orangery soil of a Ponkan plantation in China was followed in this study. A total of 160 isolated yeast strains were identified at the species level through 5.8S-ITS-RFLP and sequence analysis of the 26S rDNA D1/D2 domain. The ten identified species were Barnettozyma californica, Candida humilis, Candida tropicalis, Clavispora lusitaniae, Hanseniaspora occidentalis, Hanseniaspora opuntiae, Hanseniaspora uvarum, Pichia kudriavzevii, Pichia terricola and Torulaspora delbrueckii. Barnettozyma californica and H. occidentalis were the most abundant species in orangery soil. Pichia terricola and H. opuntiae were the prevailing species in orange peel. Additionally, P. kudriavzevii, C. lusitaniae and T. delbrueckii were the dominant species during the beginning, middle and final stages of fermentation, respectively. As a first step in exploring untapped yeast resources of the region, it is important to investigate the yeast microbiota in spontaneous fermentations and screen indigenous yeast which will produce orange wine with regional characteristics.

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.

Isolation, selection and evaluation of yeasts for use in fermentation of coffee beans by the wet process

During wet processing of coffee, the ripe cherries are pulped, then fermented and dried. This study reports an experimental approach for target identification and selection of indigenous coffee yeasts and their potential use as starter cultures during the fermentation step of wet processing. A total of 144 yeast isolates originating from spontaneously fermenting coffee beans were identified by molecular approaches and screened for their capacity to grow under coffee-associated stress conditions. According to ITS-rRNA gene sequencing, Pichia fermentans and Pichia kluyveri were the most frequent isolates, followed by Candida Candida glabrata, quercitrusa, Saccharomyces sp., Pichia guilliermondii, Pichia caribbica and Hanseniaspora opuntiae. Nine stress-tolerant yeast strains were evaluated for their ability to produce aromatic compounds in a coffee pulp simulation medium and for their pectinolytic activity. P. fermentans YC5.2 produced the highest concentrations of flavor-active ester compounds (viz., ethyl acetate and isoamyl acetate), while Saccharomyces sp. YC9.15 was the best pectinase-producing strain. The potential impact of these selected yeast strains to promote flavor development in coffee beverages was investigated for inoculating coffee beans during wet fermentation trials at laboratory scale. Inoculation of a single culture of P. fermentans YC5.2 and co-culture of P. fermentans YC5.2 and Saccharomyces sp. YC9.15 enhanced significantly the formation of volatile aroma compounds during the fermentation process compared to un-inoculated control. The sensory analysis indicated that the flavor of coffee beverages was influenced by the starter cultures, being rated as having the higher sensory scores for fruity, buttery and fermented aroma. This demonstrates a complementary role of yeasts associated with coffee quality through the synthesis of yeast-specific volatile constituents. The yeast strains P. fermentans YC5.2 and Saccharomyces sp. YC9.15 have a great potential for use as starter cultures in wet processing of coffee and may possibly help to control and standardize the fermentation process and produce coffee beverages with novel and desirable flavor profiles.

Effects of Hanseniaspora opuntiae C21 on the growth and digestive enzyme activity of juvenile sea cucumber Apostichopus japonicas

The effects of a diet containing Hanseniaspora opuntiae C21 on growth and digestive enzyme activity were estimated in juvenile Apostichopus japonicus. Groups of sea cucumbers were fed diets containing H. opuntiae C21 at 0 (control), 104, 105, and 106 CFU (colony-forming units)/g feed. Results showed that after 45 d the specific growth rate (SGR) of sea cucumbers fed a C21-supplemented diet at 10 4 CFU/g feed was significantly higher than that of the control (P < 0.05). Intestinal trypsin and lipase activities were significantly enhanced by C21 administration at 104 and 105 CFU/g feed compared with the control (P < 0.05). After feeding for 23–42 d, C21 was demonstrated by denaturing gradient gel electrophoresis to be present in the intestine of sea cucumbers. In addition, after feeding the C21-supplemented diets for 15 d, the sea cucumbers were switched to an unsupplemented diet and C21 was confirmed to be capable of colonizing the intestine for at least 31 d after cessation of feeding. In conclusion, C21 was shown to successfully colonize the intestine of juvenile A. japonicus via dietary supplementation, and improve growth and digestive enzyme activity.

Dynamics of microbial ecology during cocoa fermentation and drying: Towards the identification of molecular markers

This study aimed to evaluate molecular microbial markers associated with cocoa fermentation and drying steps. The dynamics of microbial populations were analyzed during postharvest processing (fermentation and sun drying) of cocoa from Ivory Coast. The variations in bacterial and yeast communities structure were monitored by a molecular technique (PCR-DGGE) using 16S rRNA and 26S rRNA profiles. Dominant microbial species were determined by sequencing. Our results showed that treatments of cocoa generated great variations in the microbial ecology in terms of number of species detected. The bacterial ecology quickly underwent dramatical changes during the fermentation step after two days. The dominance of Enterobacteria was first observed and then accompanied by Lactic acid bacteria and at the latest stages by acetic acid bacteria. Furthermore, Hanseniaspora opuntiae yeast species predominated during cocoa bean fermentation and drying processes. In addition, two bacterial species were only identified during the drying step of fermented cocoa beans and are potential candidates for biological markers of this process.

Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation

The potential impact of aromatic and pectinolytic yeasts on cocoa flavour was investigated using two defined mixed starter cultures encompassing strains of Pichia kluyveri and Kluyveromyces marxianus for inoculating cocoa beans in small scale tray fermentations. Samples for microbial and metabolite analysis were collected at 12–24hour intervals during 120h of fermentation. Yeast isolates were grouped by (GTG)5-based rep-PCR fingerprinting and identified by sequencing of the D1/D2 region of the 26S rRNA gene and the actin gene. Pulsed Field Gel Electrophoresis (PFGE) was conducted on isolates belonging to the species P. kluyveri and K. marxianus to verify strain level identity with the inoculated strains. Furthermore, Denaturing Gradient Gel Electrophoresis (DGGE) was performed to follow yeast and bacterial dynamics over time including the presence of the bacterial inoculum consisting of Lactobacillus fermentum and Acetobacter pasteurianus. Yeast cell counts peaked after 12h of fermentation with the predominant species being identified as Hanseniaspora opuntiae and Hanseniaspora thailandica. P. kluyveri and K. marxianus were found to compose 9.3% and 13.5% of the yeast population, respectively, after 12h of fermentation whilst PFGE showed that ~88% of all P. kluyveri isolates and 100% of all K. marxianus isolates were identical to the inoculated strains. Despite never being the dominant yeast species at any stage of fermentation, the un-conched chocolates produced from the two inoculated fermentations were judged by sensory analysis to differ in flavour profile compared to the spontaneously fermented control. This could indicate that yeasts have a greater impact on the sensory qualities of cocoa than previously assumed.

우리나라 해안 근접지역과 일부 내륙지역 야생화들로부터 분리한 국내 미기록 효모들의 특성

국내 해안 근접지역과 내륙 일부 지역에 서식하고 있는 야생화들로부터 분리, 동정한 효모들 중에서 국내 미기록 효모들을 선별하여 이들의 미생물학적 특성들을 조사하였다. 동해시의 야생화들에서 Candida silvae와 Cryptococcus terrestris, 안동시 부근의 야생화에서는 Auriculibuller fuscus의 국내에서 보고되지 않은 효모들을 분리하였다. 충남 내륙지역인 금산군에서는 Candida quercitrusa, Hanseniaspora opuntiae등의 미기록 효모들을 분리하였다. 또한 서해안 지역의 대천시에서는 Hannaella zeae, 태안시의 야생화에서는 Ca-ndida saopaulonensis, Sporobolomyces carnicolor, sporobolomyces phaffii등이 미기록 효모들로 분리, 선별하였고 예산군에서는 Candida oleophila와 Jaminaea angkoriensis 미기록 효모들을 분리하였다. 우리나라 서남부지역인 광주시 야생화에서는 Hanseniaspora vineae, 최남단인 완도군에서는 Pseudozyma hubeiensis, Pichia mexicana 등 모두 8곳에서 14종의 미기록 효모들을 분리하였다. 이들 미기록 효모들의 형태적, 배양학적 성질들이 조사한 결과 유포자 효모가 3종, 무포자 효모가 11종이었고 위균사 형성 효모가 7종이었으며 각기 다른 배양학적 특성을 보였다. Fourteen new records of yeasts were screened from one hundred thirty-three yeasts strains which were isolated from wild flowers in east, west and south coast near areas and inland areas, Korea. Candida silvae, Cryptococcus terrestris and Auriculibuller fuscus were newly isolated from wild flowers collected at Donghae and Andong, respectively. Candida quercitrusa, Hanseniaspora opuntiae, Sporobolomyces carnicolor, Candida saopaulonensis and Sporobolomyces phaffii were newly isolated from wild flowers in Geumsan-gun and Taean-gun in Chungcheongnam-do, respectively. Candida oleophila and Jaminaea angkoriensis were newly identified from flowers in Yesan-gun and Hannaella zeae was also newly identified from flowers of Daecheon. Hanseniaspora vineae and two other yeast strains including Pseudozyma hubeiensis were newly isolated and identified from flowers collected at Gwangju and Wando-gun, respectively. Morphological and physiological characteristics of their new recorded yeasts were investigated.

Identification and antifungal susceptibility of a large collection of yeast strains isolated in Tunisian hospitals

In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used as a rapid method to identify yeasts isolated from patients in Tunisian hospitals. When identification could not be exstablished with this procedure, sequencing of the internal transcribed spacer with 5.8S ribosomal DNA (rDNA) (ITS1-5.8S-ITS2) and D1/D2 domain of large-subunit (LSU rDNA) were employed as a molecular approach for species differentiation. Candida albicans was the dominant species (43.37% of all cases), followed by C. glabrata (16.55%), C. parapsilosis (13.23%), C. tropicalis (11.34%), C. dubliniensis (4.96%), and other species more rarely encountered in human diseases such as C. krusei, C. metapsilosis, C. lusitaniae, C. kefyr, C. palmioleophila, C. guilliermondii, C. intermedia, C. orthopsilosis, and C. utilis. In addition, other yeast species were obtained including Saccharomyces cerevisiae, Debaryomyces hansenii (anamorph known as C. famata), Hanseniaspora opuntiae, Kodamaea ohmeri, Pichia caribbica (anamorph known as C. fermentati), Trichosporon spp. and finally a novel yeast species, C. tunisiensis. The in vitro antifungal activities of fluconazole and voriconazole were determined by the agar disk diffusion test and Etest, while the susceptibility to additional antifungal agents was determined with the Sensititre YeastOne system. Our results showed low incidence of azole resistance in C. albicans (0.54%), C. tropicalis (2.08%) and C. glabrata (4.28%). In addition, caspofungin was active against most isolates of the collection with the exception of two K. ohmeri isolates. This is the first report to describe caspofungin resistant isolates of this yeast.

Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus predominate during well-performed Malaysian cocoa bean box fermentations, underlining the importance of these microbial species for a successful cocoa bean fermentation process

Two spontaneous Malaysian cocoa bean box fermentations (one farm, two plantation plots) were investigated. Physical parameters, microbial community dynamics, yeast and bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the respective fermented dry cocoa beans. Similar microbial growth and metabolite profiles were obtained for the two fermentations. Low concentrations of citric acid were found in the fresh pulp, revealing low acidity of the raw material. The main end-products of the catabolism of the pulp substrates glucose, fructose, and citric acid by yeasts, LAB, and AAB were ethanol, lactic acid, acetic acid, and/or mannitol. Hanseniaspora opuntiae, Lactobacillus fermentum, and Acetobacter pasteurianus were the prevalent species of the two fermentations. Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus pentosus, and Acetobacter ghanensis were also found during the mid-phase of the fermentation processes. Leuconostoc pseudomesenteroides and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Tatumella saanichensis and Enterobacter sp. were present in the beginning of the fermentations and they could be responsible for the degradation of citric acid and/or the production of gluconic acid and lactic acid, respectively. The presence of facultative heterofermentative LAB during the fermentations caused a high production of lactic acid. Finally, as these fermentations were carried out with high-quality raw material and were characterised by a restricted microbial species diversity, resulting in successfully fermented dry cocoa beans and good chocolates produced thereof, it is likely that the prevailing species H. opuntiae, S. cerevisiae, Lb. fermentum, and A. pasteurianus were responsible for it.

Effects of dietary live yeast Hanseniaspora opuntiae C21 on the immune and disease resistance against Vibrio splendidus infection in juvenile sea cucumber Apostichopus japonicus

A feeding experiment was conducted to determine effects of Hanseniaspora opuntiae C21 on immune response and disease resistance against Vibrio splendidus infection in juvenile sea cucumbers Apostichopus japonicus. Sea cucumbers were fed with either diets containing C21 at 104, 105 and 106 CFU g−1 feed or a control diet for 30–50 days, respectively. After feeding for 30 days and 45 days, five sea cucumbers from each tank were sampled for immunological analyses. Results indicated that C21 significantly improved the phagocytic activity in coelomocytes of sea cucumbers (P < 0.05). Moreover, C21 administration significantly enhanced lysozyme (LSZ), phenoloxidase activity (PO), total nitric oxide synthase (T-NOS), superoxide dismutase (SOD), alkaline phosphatase (AKP) and acid phosphatase (ACP) activities in coelomic fluid, and LSZ, T-NOS, AKP and ACP activities in coelomocytes lysate supernatant (CLS) of sea cucumbers (P < 0.05). After feeding for 45 days, 10 sea cucumbers from each dose group were challenged with V. splendidus NB13. Cumulative incidence and mortality of sea cucumbers fed with C21 were found to be lower than those of control group. After feeding for 50 days, sea cucumbers in 104 CFU g−1 C21 treatment and control tanks were subjected to acute salinity changes (from 30 to 20) for 24 h in the laboratory, and the immunological parameters were measured to evaluate the immune capacities of the A. japonicus. Phagocytic, LAZ and T-NOS activities of C21-treated group were higher than those of control group, indicating that salinity stress tolerance of sea cucumber was enhanced by C21. The present results showed that a diet supplemented with C21 could stimulate the immune system of juvenile A. japonicus thus enhancing their resistance against V. splendidus.

Dynamic study of yeast species and Saccharomyces cerevisiae strains during the spontaneous fermentations of Muscat blanc in Jingyang, China

The evolution of yeast species and Saccharomyces cerevisiae genotypes during spontaneous fermentations of Muscat blanc planted in 1957 in Jingyang region of China was followed in this study. Using a combination of colony morphology on Wallerstein Nutrient (WLN) medium, sequence analysis of the 26S rDNA D1/D2 domain and 5.8S-ITS-RFLP analysis, a total of 686 isolates were identified at the species level. The six species identified were S. cerevisiae, Hanseniaspora uvarum, Hanseniaspora opuntiae, Issatchenkia terricola, Pichia kudriavzevii (Issatchenkia orientalis) and Trichosporon coremiiforme. This is the first report of T. coremiiforme as an inhabitant of grape must. Three new colony morphologies on WLN medium and one new 5.8S-ITS-RFLP profile are described. Species of non-Saccharomyces, predominantly H. opuntiae, were found in early stages of fermentation. Subsequently, S. cerevisiae prevailed followed by large numbers of P. kudriavzevii that dominated at the end of fermentations. Six native genotypes of S. cerevisiae were determined by interdelta sequence analysis. Genotypes III and IV were predominant. As a first step in exploring untapped yeast resources of the region, this study is important for monitoring the yeast ecology in native fermentations and screening indigenous yeasts that will produce wines with regional characteristics.

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.

Tandem repeat-tRNA (TRtRNA) PCR method for the molecular typing of non-Saccharomyces 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.

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.