Diversity of Mycobiota in Spanish Grape Berries and Selection of Hanseniaspora uvarum U1 to Prevent Mycotoxin Contamination.

The occurrence of mycotoxins on grapes poses a high risk for food safety; thus, it is necessary to implement effective prevention methods. In this work, a metagenomic approach revealed the presence of important mycotoxigenic fungi in grape berries, including Aspergillus flavus, Aspergillus niger aggregate species, or Aspergillus section Circumdati. However, A. carbonarius was not detected in any sample. One of the samples was not contaminated by any mycotoxigenic species, and, therefore, it was selected for the isolation of potential biocontrol agents. In this context, Hanseniaspora uvarum U1 was selected for biocontrol in vitro assays. The results showed that this yeast is able to reduce the growth rate of the main ochratoxigenic and aflatoxigenic Aspergillus spp. occurring on grapes. Moreover, H. uvarum U1 seems to be an effective detoxifying agent for aflatoxin B1 and ochratoxin A, probably mediated by the mechanisms of adsorption to the cell wall and other active mechanisms. Therefore, H. uvarum U1 should be considered in an integrated approach to preventing AFB1 and OTA in grapes due to its potential as a biocontrol and detoxifying agent.

The Impact of Hanseniaspora vineae Fermentation and Ageing on Lees on the Terpenic Aromatic Profile of White Wines of the Albillo Variety.

Hanseniaspora vineae is a non-Saccharomyces yeast that has a powerful impact on the sensory profile of wines. Its effect on the aromatic profile of non-aromatic grape varieties, such as Albillo Mayor (Vitis vinifera, L), during vinification is a useful biotechnology to improve sensory complexity. Fermentation in steel barrels using Hanseniaspora vineae and sequential inoculation with Saccharomyces cerevisiae have been used to study the formation of terpenes and cell lysis in the production of Albillo white wines. The GC-MS analysis profile shows a significant effect of H. vineae fermentation on the contents of terpenes (≈×3), mainly in linalool (>×3), ß-citronellol (>×4), geraniol (>×2) and α-terpineol (≈×2). The contents of several polyoxygenated terpenes and some volatile phenols with a spicy aroma were increased during fermentation. In summary, Hanseniaspora vineae releases a large number of cell wall polysaccharides during fermentation that affect wine palatability and structure. Hanseniaspora vineae is a powerful bio-tool to enhance the fruitiness, floral notes and freshness in non-aromatic white varieties.

Influence of Non-Saccharomyces on Wine Chemistry: A Focus on Aroma-Related Compounds.

Wine fermentation processes are driven by complex microbial systems, which comprise eukaryotic and prokaryotic microorganisms that participate in several biochemical interactions with the must and wine chemicals and modulate the organoleptic properties of wine. Among these, yeasts play a fundamental role, since they carry out the alcoholic fermentation (AF), converting sugars to ethanol and CO2 together with a wide range of volatile organic compounds. The contribution of Saccharomyces cerevisiae, the reference organism associated with AF, has been extensively studied. However, in the last decade, selected non-Saccharomyces strains received considerable commercial and oenological interest due to their specific pro-technological aptitudes and the positive influence on sensory quality. This review aims to highlight the inter-specific variability within the heterogeneous class of non-Saccharomyces in terms of synthesis and release of volatile organic compounds during controlled AF in wine. In particular, we reported findings on the presence of model non-Saccharomyces organisms, including Torulaspora delbrueckii, Hanseniaspora spp,Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia spp. and Candida zemplinina, in combination with S. cerevisiae. The evidence is discussed from both basic and applicative scientific perspective. In particular, the oenological significance in different kind of wines has been underlined.

Biodiversity of yeasts isolated during spontaneous fermentation of cool climate grape musts.

Biodiversity of native yeasts, especially in winemaking, has hidden potential. In order to use the value of non-Saccharomyces strains in wine production and to minimise the possibility of its deterioration, it is necessary to thoroughly study the yeast cultures present on grape fruits and in grape must, as well as their metabolic properties. The aim of the study was to characterise the yeast microbiota found during spontaneous fermentation of grape musts obtained from grape varieties 'Rondo', 'Regent' and 'Johanniter'. Grapes from two vineyards (Srebrna Góra and Zadora) located in southern Poland were used for the research. Succession of subsequent groups of yeasts was observed during the process. Metschnikowia pulcherrima yeasts were identified both at the beginning and the end of the process. Hanseniaspora uvarum, Wickerhamomyces onychis and Torulaspora delbrueckii strains were also identified during the fermentation. Torulaspora delbrueckii and Wickerhamomyces onychis strains were identified only in grape musts obtained from grapes of the Zadora vineyard. These strains may be characteristic of this vineyard and shape the identity of wines formed in it. Our research has provided specific knowledge on the biodiversity of yeast cultures on grapes and during their spontaneous fermentation. The research results presented indicate the possibility of using native strains for fermentation of grape musts, allowing to obtain a product with favourable chemical composition and sensory profile.

Fruit host-dependent fungal communities in the microbiome of wild Queensland fruit fly larvae.

Bactrocera tryoni (Froggatt), the Queensland fruit fly (Qfly), is a highly polyphagous tephritid fly that is widespread in Eastern Australia. Qfly physiology is closely linked with its fungal associates, with particular relationship between Qfly nutrition and yeast or yeast-like fungi. Despite animal-associated fungi typically occurring in multi-species communities, Qfly studies have predominately involved the culture and characterisation of single fungal isolates. Further, only two studies have investigated the fungal communities associated with Qfly, and both have used culture-dependant techniques that overlook non-culturable fungi and hence under-represent, and provide a biased interpretation of, the overall fungal community. In order to explore a potentially hidden fungal diversity and complexity within the Qfly mycobiome, we used culture-independent, high-throughput Illumina sequencing techniques to comprehensively, and holistically characterized the fungal community of Qfly larvae and overcome the culture bias. We collected larvae from a range of fruit hosts along the east coast of Australia, and all had a mycobiome dominated by ascomycetes. The most abundant fungal taxa belonged to the genera Pichia (43%), Candida (20%), Hanseniaspora (10%), Zygosaccharomyces (11%) and Penicillium (7%). We also characterized the fungal communities of fruit hosts, and found a strong degree of overlap between larvae and fruit host communities, suggesting that these communities are intimately inter-connected. Our data suggests that larval fungal communities are acquired from surrounding fruit flesh. It is likely that the physiological benefits of Qfly exposure to fungal communities is primarily due to consumption of these fungi, not through syntrophy/symbiosis between fungi and insect 'host'.

Hanseniaspora uvarum prolongs shelf life of strawberry via volatile production.

Gray mold caused by Botrytis cinerea led to severe postharvest losses for strawberry industry. In recent years, some studies have shown that postharvest diseases of strawberry can be controlled by using bacterial, fungal and yeast strains. The yeast strain Hanseniaspora uvarum was shown as an effective antagonist against B. cinerea growth. Here, we further investigated the volatile organic compounds (VOCs) production of H. uvarum and how this could impact on postharvest gray mold control of strawberry. A total of 28 VOCs were detected by GC-MS in the headspace of H. uvarum and strawberry with/without B. cinerea (SI and RSI ≥800). Among these VOCs, 15 VOCs were detected in both conditions, 4 VOCs were H. uvarum and strawberry without B. cinerea and the other 9 VOCs were only detected when B. cinerea was inoculated. Two VOCs, ethyl acetate and 1,3,5,7-cyclooctatetraene, enhanced by inoculation of B. cinerea. In in vitro assay, H. uvarum significantly inhibited mycelial growth and spore germination of B. cinerea via VOCs production. Moreover, in vivo assay showed that H. uvarum reduced B. cinerea infection of strawberry and maintained fruit appearance, firmness and total soluble solids via VOCs production. Collectively, our results showed that H. uvarum VOCs significantly controlled postharvest gray mold of strawberry and prolonged the storage time and shelf life.

Monitoring of Saccharomyces cerevisiae, Hanseniaspora uvarum, and Starmerella bacillaris (synonym Candida zemplinina) populations during alcoholic fermentation by fluorescence in situ hybridization.

Various molecular approaches have been applied as culture-independent techniques to monitor wine fermentations over the last decade. Among them, those based on RNA detection have been widely used for yeast cell detection, assuming that RNA only exists in live cells. Fluorescence in situ hybridization (FISH) targeting intracellular rRNA is considered a promising technique for the investigation of wine ecology. For the present study, we applied the FISH technique in combination with epifluorescence microscopy and flow cytometry to directly quantify populations of Saccharomyces cerevisiae, Hanseniaspora uvarum, and Starmerella bacillaris during alcoholic fermentations. A new specific probe that hybridizes with eight species of Hanseniaspora genus and a second probe specific for Starm. bacillaris were designed, and the conditions for their application to pure cultures, mixed cultures, and wine samples were optimized. Single and mixed fermentations were performed with natural, concentrated must at two different temperatures, 15 °C and 25 °C. The population dynamics revealed that the Sacch. cerevisiae population increased to 10(7)-10(8)cells/ml during all fermentations, whereas H. uvarum and Starm. bacillaris tended to increase in single fermentations but remained at levels similar to their inoculations at 10(6)cells/ml in mixed fermentations. Temperature mainly affected the fermentation duration (slower at the lower temperature) but did not affect the population sizes of the different species. The use of these probes in natural wine fermentations has been validated.

Inhibitory activity of tea polyphenol and Hanseniaspora uvarum against Botrytis cinerea infections.

AIMS: To investigate the effect of tea polyphenol (TP) and Hanseniaspora uvarum alone or in combination against Botrytis cinerea in grapes and to evaluate the possible mechanisms involved. METHODS AND RESULTS: TP alone was effective in controlling grey mould in grape at all concentrations. TP at 0.5 and 1.0% in combination with H. uvarum (1 x 10(6) CFU ml(-1)) showed a lower infection rate of grey mould. TP at 0.01% or above significantly inhibited the spore germination of B. cinerea. TP at 0.1% showed inhibition ability on mycelium growth of B. cinerea. The addition of TP did not affect the growth of H. uvarum in vitro and significantly increased the population of H. uvarum in vivo. CONCLUSIONS: TP exhibited an inhibitory effect against B. cinerea and improved the biocontrol efficacy of H. uvarum. The inhibitory effects of spore germination and mycelial growth of B. cinerea and the increased populations of H. uvarum in vivo may be some of the important mechanisms of TP. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggested that TP alone or in combination with biocontrol agents has great potential in the commercial management of postharvest diseases of fruits.

The zymocidial activity of Tetrapisispora phaffii in the control of Hanseniaspora uvarum during the early stages of winemaking.

AIMS: The yeast strain Tetrapisispora phaffii DBVPG 6706 (formerly Kluyveromyces phaffii) secretes a killer toxin (Kpkt) that has antimicrobial activity against apiculate yeasts. The aim of this study was to evaluate the killer activity of Kpkt towards Hanseniaspora uvarum under winemaking conditions. METHODS AND RESULTS: The zymocidial activity of Kpkt on H. uvarum was assayed in microfermentation trials inoculated with free and immobilized T. phaffii cells. The microbial evolution and fermentation profiles of the wines were evaluated to determine the effects of Kpkt on apiculate yeasts, in comparison with SO(2). The results indicate that the fungicidal activity of Kpkt against H. uvarum is stable for at least 14 days in wine, and the zymocin can control the proliferation of apiculate yeasts. The analytical composition of wines with the inoculum of T. phaffii immobilized cells did not differ from the wines with SO(2). In contrast to wines without this control of apiculate yeasts, an increase in ethyl acetate was seen. CONCLUSIONS: Tetrapisispora phaffii is an excellent candidate for the biological control of undesired proliferation of apiculate yeasts during the first steps of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Tetrapisispora phaffii cells in an immobilized form can be used as a biocontrol agent to reduce the need for SO(2) addition.

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.