Development of Genetic Modification Tools for Hanseniasporauvarum.

Apiculate yeasts belonging to the genus Hanseniaspora are commonly isolated from viticultural settings and often dominate the initial stages of grape must fermentations. Although considered spoilage yeasts, they are now increasingly becoming the focus of research, with several whole-genome sequencing studies published in recent years. However, tools for their molecular genetic manipulation are still lacking. Here, we report the development of a tool for the genetic modification of Hanseniaspora uvarum. This was employed for the disruption of the HuATF1 gene, which encodes a putative alcohol acetyltransferase involved in acetate ester formation. We generated a synthetic marker gene consisting of the HuTEF1 promoter controlling a hygromycin resistance open reading frame (ORF). This new marker gene was used in disruption cassettes containing long-flanking (1000 bp) homology regions to the target locus. By increasing the antibiotic concentration, transformants were obtained in which both alleles of the putative HuATF1 gene were deleted in a diploid H. uvarum strain. Phenotypic characterisation including fermentation in Müller-Thurgau must showed that the null mutant produced significantly less acetate ester, particularly ethyl acetate. This study marks the first steps in the development of gene modification tools and paves the road for functional gene analyses of this yeast.

Saccharomyces cerevisiae and Hanseniaspora uvarum mixed starter cultures: Influence of microbial/physical interactions on wine characteristics.

The growing trend in the wine industry is the revaluation of the role of non-Saccharomyces yeasts, promoting the use of these yeasts in association with Saccharomyces cerevisiae. Non-Saccharomyces yeasts contribute to improve wine complexity and organoleptic composition. However, the use of mixed starters needs to better understand the effect of the interaction between these species during alcoholic fermentation. The aim of this study is to evaluate the influence of mixed starter cultures, composed by combination of different S. cerevisiae and Hanseniaspora uvarum strains, on wine characteristics and to investigate the role of cell-to-cell contact on the metabolites produced during alcoholic fermentation. In the first step, three H. uvarum and two S. cerevisiae strains, previously selected, were tested during mixed fermentations in natural red grape must in order to evaluate yeast population dynamics during inoculated fermentation and influence of mixed starter cultures on wine quality. One selected mixed starter was tested in a double-compartment fermentor in order to compare mixed inoculations of S. cerevisiae/H. uvarum with and without physical separation. Our results revealed that physical contact between S. cerevisiae and H. uvarum affected the viability of H. uvarum strain, influencing also the metabolic behaviour of the strains. Although different researches are available on the role of cell-to-cell contact-mediated interactions on cell viability of the strains included in the mixed starter, to our knowledge, very few studies have evaluated the influence of cell-to-cell contact on the chemical characteristics of wine.

Proteomics profile of Hanseniaspora uvarum enhanced with trehalose involved in the biocontrol efficacy of grape berry.

This present study tested the extent to which 2% w/v trehalose enhanced the proteins expression profile of Hanseniaspora uvarum Y3. Furthermore, it explored the relative gene expression of stilbene synthase (StSy), one of the vital defense-related genes found in the skin of grapes. The proteomics profile revealed that 29 proteins were differentially expressed out of which 26 were significantly up-regulated and 3 were download-regulated. The pathogenesis related (PR) and other protein spots were visible at 97.4 kDa and 14.4 kDa. Peroxiredoxin TSA1 and superoxide dismutase were the main proteins involved in defense response and both proteins were significantly up-regulated. The carbohydrate and energy metabolism proteins were also significantly up-regulated. The results revealed that the treatments were associated with substantial increase in peroxidase activity compared to the control. StSy relative gene expression level was observed to increase by 2.5-fold in grapes treated with the pre-enhanced H. uvarum compared to the control.

Aroma modulation of Cabernet Gernischt dry red wine by optimal enzyme treatment strategy in winemaking.

Cabernet Gernischt (CG) is a famous Chinese wine grape cultivar, the red wine of which is known for its green trait, especially when produced from grapes cultivated in regions with monsoon climate. To modify CG wine aroma, three enzyme preparations (H. uvarum extracellular enzyme, AR2000, and pectinase) were introduced in different winemaking stages with Saccharomyces cerevisiae. Free and bound aroma compounds in young wines were detected using headspace solid-phase micro-extraction and gas chromatography-mass spectrometry, and aroma characteristics were quantified by trained panelists. Results showed that simultaneous inoculation of enzymes and yeasts improved wine aroma. Partial least-squares regression revealed that the green trait was due mainly to varietal compounds, especially C6 compounds, and could be partly weakened by fermentative compounds. Moreover, H. uvarum enzyme treatments enriched the acid fruit note of CG wine by enhancing the synergistic effect of varietal volatiles and certain fermentative compounds, such as esters and phenylethyls.

Effect of sequential fermentations and grape cultivars on volatile compounds and sensory profiles of Danish wines.

BACKGROUND: There has been an increasing interest in the use of selected non-Saccharomyces yeasts in co-culture with Saccharomyces cerevisiae. In this work, three non-Saccharomyces yeast strains (Metschnikowia viticola, Metschnikowia fructicola and Hanseniaspora uvarum) indigenously isolated in Denmark were used in sequential fermentations with S. cerevisiae on three cool-climate grape cultivars, Bolero, Rondo and Regent. During the fermentations, the yeast growth was determined as well as key oenological parameters, volatile compounds and sensory properties of finished rosé wines. RESULTS: The different non-Saccharomyces strains and cool-climate grape cultivars produced wines with a distinctive aromatic profile. A total of 67 volatile compounds were identified, including 43 esters, 14 alcohols, five acids, two ketones, a C13-norisoprenoid, a lactone and a sulfur compound. The use of M. viticola in sequential fermentation with S. cerevisiae resulted in richer berry and fruity flavours in wines. The sensory plot showed a more clear separation among wine samples by grape cultivars compared with yeast strains. CONCLUSION: Knowledge on the influence of indigenous non-Saccharomyces strains and grape cultivars on the flavour generation contributed to producing diverse wines in cool-climate wine regions. © 2017 Society of Chemical Industry.

De Novo Synthesis of Benzenoid Compounds by the Yeast Hanseniaspora vineae Increases the Flavor Diversity of Wines.

Benzyl alcohol and other benzenoid-derived metabolites of particular importance in plants confer floral and fruity flavors to wines. Among the volatile aroma components in Vitis vinifera grape varieties, benzyl alcohol is present in its free and glycosylated forms. These compounds are considered to originate from grapes only and not from fermentative processes. We have found increased levels of benzyl alcohol in red Tannat wine compared to that in grape juice, suggesting de novo formation of this metabolite during vinification. In this work, we show that benzyl alcohol, benzaldehyde, p-hydroxybenzaldehyde, and p-hydroxybenzyl alcohol are synthesized de novo in the absence of grape-derived precursors by Hanseniaspora vineae. Levels of benzyl alcohol produced by 11 different H. vineae strains were 20-200 times higher than those measured in fermentations with Saccharomyces cerevisiae strains. These results show that H. vineae contributes to flavor diversity by increasing grape variety aroma concentration in a chemically defined medium. Feeding experiments with phenylalanine, tryptophan, tyrosine, p-aminobenzoic acid, and ammonium in an artificial medium were tested to evaluate the effect of these compounds either as precursors or as potential pathway regulators for the formation of benzenoid-derived aromas. Genomic analysis shows that the phenylalanine ammonia-lyase (PAL) and tyrosine ammonia lyase (TAL) pathways, used by plants to generate benzyl alcohols from aromatic amino acids, are absent in the H. vineae genome. Consequently, alternative pathways derived from chorismate with mandelate as an intermediate are discussed.

Role of non-Saccharomyces yeasts in Korean wines produced from Campbell Early grapes: potential use of Hanseniaspora uvarum as a starter culture.

Several yeasts were isolated from Campbell Early grapes (Vitis labrusca cultivar Campbell Early), the major grape cultivar in Korea, grown in two different regions. PCR-RFLP analysis of the ITS I-5.8S-ITS II region showed that 34 isolates out of a total of 40 were in the same group. Phylogenetic analysis revealed that the major strain belonged to one species, Hanseniaspora uvarum, although they displayed some nucleotide mismatches between them. During spontaneous alcohol fermentation at 20 °C, the two grape musts containing 24 °Brix sugar exhibited similar fermentation patterns with differences in final alcohol production and yeast viable counts. PCR analysis of the yeasts randomly isolated during the fermentation using an intron splice site primer showed changes in yeast flora between 8 and 10 days of fermentation. We found that the dominant yeasts displaying various PCR patterns using the primer remained the same throughout the early stages of fermentation, as determined by molecular typing of their ITS regions using PCR-RFLP, and these yeasts were identical to those isolated from grape berries. Among the isolates, the strain designated SS6 was selected based on its potassium metabisulfite resistance, alcohol production (distillation method), and flavor (by sniffing test) of grape juice. When Campbell Early grape must was inoculated with H. uvarum SS6 cells, no differences in fermentation pattern were observed compared with that inoculated with cells of Saccharomyces cerevisiae W-3, an industrial wine yeast strain. However, SS6 wine showed higher levels of organic acid (especially lactic acid), aldehydes, and minor alcohols (except n-propyl alcohol), as well as a higher score in sensory evaluation, compared to those of W-3 wine.

Study of the volatile compounds produced by Debaryomyces hansenii NRRL Y-7426 during the fermentation of detoxified concentrated distilled grape marc hemicellulosic hydrolysates.

The volatile compounds produced by Debaryomyces hansenii NRRL Y-7426 during the fermentation of detoxified concentrated distilled grape marc hemicellulosic hydrolysates was analysed by GC-MS. Thirty-five compounds corresponding to ten groups of volatile compounds: terpenes, higher alcohols, C6 alcohols, aldehydes, volatile acids, acetates, ethyl esters, volatile phenols, sulphur compounds and hydrocarbons were identified. The supplementation with commercial nutrients increased the concentration of 2-phenylethanol, a commercial flavour and fragrance compound, with a rose-like odour, employed in cosmetics and food industries; and other positive compounds to the aroma such as terpenes and ethyl esters. Non-supplemented media produced the highest content in higher alcohols, volatile acids, sulphur compounds and in the majority of hydrocarbons detected, meanwhile supplementation with vinasses hardly produced volatile compounds. Only four volatile compounds contributed directly to the aroma according to the OAVs values higher than 1. Finally, a PCA analysis allowed accounting for 100 % of the variance.

Analysis and direct quantification of Saccharomyces cerevisiae and Hanseniaspora guilliermondii populations during alcoholic fermentation by fluorescence in situ hybridization, flow cytometry and quantitative PCR.

Traditionally, it was assumed that non-Saccharomyces (NS) yeasts could only survive in the early stages of alcoholic fermentations. However, recent studies applying culture-independent methods have shown that NS populations persist throughout the fermentation process. The aim of the present work was to analyze and quantify Saccharomyces cerevisiae (Sc) and Hanseniaspora guilliermondii (Hg) populations during alcoholic fermentations by plating and culture-independent methods, such as fluorescence in situ hybridization (FISH) and quantitative PCR (QPCR). Species-specific FISH probes labeled with fluorescein (FITC) were used to directly hybridize Sc and Hg cells from single and mixed cultures that were enumerated by epifluorescence microscopy and flow cytometry. Static and agitated fermentations were performed in synthetic grape juice and cell density as well as sugar consumption and ethanol production were determined throughout fermentations. Cell density values obtained by FISH and QPCR revealed the presence of high populations (107-108 cells/ml) of Sc and Hg throughout fermentations. Plate counts of both species did not show significant differences with culture-independent results in pure cultures. However, during mixed fermentations Hg lost its culturability after 4-6 days, while Sc remained culturable (about 108 cells/ml) throughout the entire fermentation (up to 10 days). The rRNA content of cells during mixed fermentations was also analyzed by flow cytometry in combination with FISH probes. The fluorescence intensity conferred by the species-specific FISH probes was considerably lower for Hg than for Sc. Moreover, the rRNA content of Hg cells, conversely to Sc cells, remained almost unchanged after boiling, which showed that rRNA stability is species-dependent.

Fermentation behaviour and metabolic interactions of multistarter wine yeast fermentations.

Multistarter fermentations of Hanseniaspora uvarum, Torulaspora delbrueckii and Kluyveromyces thermotolerans together with Saccharomyces cerevisiae were studied. In grape musts with a high sugar content, mixed trials showed a fermentation behaviour and analytical profiles of wines comparable to or better than those exhibited by a pure culture of S. cerevisiae. Sequential trials of T. delbrueckii and K. thermotolerans revealed a sluggish fermentation, while those of H. uvarum exhibited an unacceptable increase in ethyl acetate content (175 ml l(-1)). A low fermentation temperature (15 degrees C) of multistarter trials of H. uvarum resulted in a stuck fermentation that was not due to a deficiency of assimilable nitrogenous compounds since lower amounts of these compounds were used. Sequential fermentation carried out by H. uvarum at 15 degrees C confirmed the high production of ethyl acetate. The persistence and level of non-Saccharomyces yeasts during multistarter fermentations under stress conditions (high ethanol content and/or low temperature) can cause stuck fermentations.

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.

Study of beta-glucosidase production by wine-related yeasts during alcoholic fermentation. A new rapid fluorimetric method to determine enzymatic activity.

AIMS: The beta-glucosidase activity is involved in the hydrolysis of several important compounds for the development of varietal wine flavour. The aim of the present study was to investigate the production of beta-glucosidase in a number of wine-related yeast strains and to measure and identify this activity over the course of grape juice fermentation. METHODS AND RESULTS: beta-glucosidase activity was measured as the amount of 4-methylumbelliferone released from 4-methylumbelliferyl-beta-d-glucopyranoside substrate. Intact cells of some grape and wine-spoilage yeasts showed beta-glucosidase activity much higher than those observed in wine yeasts "sensu stricto". During fermentation, three Saccharomyces cerevisiae strains, one Hanseniaspora valbyensis strain and one Brettanomyces anomalus strain showed beta-glucosidase activity both intra- and extracellularly. CONCLUSIONS: In the studied strains, beta-glucosidase activity was at its maximum when the cells were in the active growth phase. However, a lowering of medium pH to values around 3 during fermentation led to total loss of activity. SIGNIFICANCE AND IMPACT OF THE STUDY: During the course of this study, a new, rapid and reproducible method to assay beta-glucosidase activity was developed. The fact that Saccharomyces and non-Saccharomyces yeast strains are able to express beta-glucosidase activity during the alcoholic fermentation sheds new light on the contribution of these yeasts in the aroma expression of wines.

Controlling grape must fermentation in early winemaking phases: the role of electrochemical treatment.

AIMS: To contribute to an understanding of the phenomena related to the effect of low electric current (LEC) in grape must fermentation during laboratory and pilot plant scale winemaking, with selected co-culture yeasts (Saccharomyces cerevisiae strain 404 and Hanseniaspora guilliermodii strain 465). METHODS AND RESULTS: LEC (10, 30, 50 and 100 mA) was applied to fresh grape must as an alternative method to the usual addition of SO2. Parameters such as polarity, treatment duration (24-96 h) and type of inoculum yeast were varied one at a time. LEC decreased the survival time and increased the death rate of H. guilliermondii strain 465 in co-cultures, whereas it did not affect the growth and survival of S. cerevisiae strain 40. A final comparison was made of the main physico-chemical parameters on wine obtained after the different tests. CONCLUSIONS: The results have demonstrated that the low voltage treatment using a pair of graphite electrodes had a positive effect on grape juice fermentation (yeast microflora) during the early stages of winemaking, even with the potential of being an alternative method to the usual addition of SO2. SIGNIFICANCE AND IMPACT OF THE STUDY: These results could be of significant importance in developing new winemaking technologies for an innovative yeast fermentation control process for 'biological wine'.