Impact of phenylalanine on Hanseniaspora vineae aroma metabolism during wine fermentation.

Hanseniaspora vineae exhibits extraordinary positive oenological characteristics contributing to the aroma and texture of wines, especially by its ability to produce great concentrations of benzenoid and phenylpropanoid compounds compared with conventional Saccharomyces yeasts. Consequently, in practice, sequential inoculation of H. vineae and Saccharomyces cerevisiae allows to improve the aromatic quality of wines. In this work, we evaluated the impact on wine aroma produced by increasing the concentration of phenylalanine, the main amino acid precursor of phenylpropanoids and benzenoids. Fermentations were carried out using a Chardonnay grape juice containing 150 mg N/L yeast assimilable nitrogen. Fermentations were performed adding 60 mg/L of phenylalanine without any supplementary addition to the juice. Musts were inoculated sequentially using three different H. vineae strains isolated from Uruguayan vineyards and, after 96 h, S. cerevisiae was inoculated to complete the process. At the end of the fermentation, wine aromas were analysed by both gas chromatography-mass spectrometry and sensory evaluation through a panel of experts. Aromas derived from aromatic amino acids were differentially produced depending on the treatments. Sensory analysis revealed more floral character and greater aromatic complexity when compared with control fermentations without phenylalanine added. Moreover, fermentations performed in synthetic must with pure H. vineae revealed that even tyrosine can be used in absence of phenylalanine, and phenylalanine is not used by this yeast for the synthesis of tyrosine derivatives.

The Role of Glucose-6-phosphate Dehydrogenase in the Wine Yeast Hanseniaspora uvarum.

Hanseniaspora uvarum is the predominant yeast species in the majority of wine fermentations, which has only recently become amenable to directed genetic manipulation. The genetics and metabolism of H. uvarum have been poorly studied as compared to other yeasts of biotechnological importance. This work describes the construction and characterization of homozygous deletion mutants in the HuZWF1 gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), which provides the entrance into the oxidative part of the pentose phosphate pathway (PPP) and serves as a major source of NADPH for anabolic reactions and oxidative stress response. Huzwf1 deletion mutants grow more slowly on glucose medium than wild-type and are hypersensitive both to hydrogen peroxide and potassium bisulfite, indicating that G6PDH activity is required to cope with these stresses. The mutant also requires methionine for growth. Enzyme activity can be restored by the expression of heterologous G6PDH genes from other yeasts and humans under the control of a strong endogenous promoter. These findings provide the basis for a better adaptation of H. uvarum to conditions used in wine fermentations, as well as its use for other biotechnological purposes and as an expression organism for studying G6PDH functions in patients with hemolytic anemia.

Gene loss and cis-regulatory novelty shaped core histone gene evolution in the apiculate yeast Hanseniaspora uvarum.

Core histone genes display a remarkable diversity of cis-regulatory mechanisms despite their protein sequence conservation. However, the dynamics and significance of this regulatory turnover are not well understood. Here, we describe the evolutionary history of core histone gene regulation across 400 million years in budding yeasts. We find that canonical mode of core histone regulation-mediated by the trans-regulator Spt10-is ancient, likely emerging between 320 and 380 million years ago and is fixed in the majority of extant species. Unexpectedly, we uncovered the emergence of a novel core histone regulatory mode in the Hanseniaspora genus, from its fast-evolving lineage, which coincided with the loss of 1 copy of its paralogous core histone genes. We show that the ancestral Spt10 histone regulatory mode was replaced, via cis-regulatory changes in the histone control regions, by a derived Mcm1 histone regulatory mode and that this rewiring event occurred with no changes to the trans-regulator, Mcm1, itself. Finally, we studied the growth dynamics of the cell cycle and histone synthesis in genetically modified Hanseniaspora uvarum. We find that H. uvarum divides rapidly, with most cells completing a cell cycle within 60 minutes. Interestingly, we observed that the regulatory coupling between histone and DNA synthesis was lost in H. uvarum. Our results demonstrate that core histone gene regulation was fixed anciently in budding yeasts, however it has greatly diverged in the Hanseniaspora fast-evolving lineage.

A biostimulant yeast, Hanseniaspora opuntiae, modifies Arabidopsis thaliana root architecture and improves the plant defense response against Botrytis cinerea.

MAIN CONCLUSION: The biostimulant Hanseniaspora opuntiae regulates Arabidopsis thaliana root development and resistance to Botrytis cinerea. Beneficial microbes can increase plant nutrient accessibility and uptake, promote abiotic stress tolerance, and enhance disease resistance, while pathogenic microorganisms cause plant disease, affecting cellular homeostasis and leading to cell death in the most critical cases. Commonly, plants use specialized pattern recognition receptors to perceive beneficial or pathogen microorganisms. Although bacteria have been the most studied plant-associated beneficial microbes, the analysis of yeasts is receiving less attention. This study assessed the role of Hanseniaspora opuntiae, a fermentative yeast isolated from cacao musts, during Arabidopsis thaliana growth, development, and defense response to fungal pathogens. We evaluated the A. thaliana-H. opuntiae interaction using direct and indirect in vitro systems. Arabidopsis growth was significantly increased seven days post-inoculation with H. opuntiae during indirect interaction. Moreover, we observed that H. opuntiae cells had a strong auxin-like effect in A. thaliana root development during in vitro interaction. We show that 3-methyl-1-butanol and ethanol are the main volatile compounds produced by H. opuntiae. Subsequently, it was determined that A. thaliana plants inoculated with H. opuntiae have a long-lasting and systemic effect against Botrytis cinerea infection, but independently of auxin, ethylene, salicylic acid, or jasmonic acid pathways. Our results demonstrate that H. opuntiae is an important biostimulant that acts by regulating plant development and pathogen resistance through different hormone-related responses.

Exploring future applications of the apiculate yeast Hanseniaspora.

As a metaphor, lemons get a bad rap; however the proverb 'if life gives you lemons, make lemonade' is often used in a motivational context. The same could be said of Hanseniaspora in winemaking. Despite its predominance in vineyards and grape must, this lemon-shaped yeast is underappreciated in terms of its contribution to the overall sensory profile of fine wine. Species belonging to this apiculate yeast are known for being common isolates not just on grape berries, but on many other fruits. They play a critical role in the early stages of a fermentation and can influence the quality of the final product. Their deliberate addition within mixed-culture fermentations shows promise in adding to the complexity of a wine and thus provide sensorial benefits. Hanseniaspora species are also key participants in the fermentations of a variety of other foodstuffs ranging from chocolate to apple cider. Outside of their role in fermentation, Hanseniaspora species have attractive biotechnological possibilities as revealed through studies on biocontrol potential, use as a whole-cell biocatalyst and important interactions with Drosophila flies. The growing amount of 'omics data on Hanseniaspora is revealing interesting features of the genus that sets it apart from the other Ascomycetes. This review collates the fields of research conducted on this apiculate yeast genus.

High nitrogen concentration causes G2/M arrest in Hanseniaspora vineae.

Yeasts have been widely used as a model to better understand cell cycle mechanisms and how nutritional and genetic factors can impact cell cycle progression. While nitrogen scarcity is well known to modulate cell cycle progression, the relevance of nitrogen excess for microorganisms has been overlooked. In our previous work, we observed an absence of proper entry into the quiescent state in Hanseniaspora vineae and identified a potential link between this behavior and nitrogen availability. Furthermore, the Hanseniaspora genus has gained attention due to a significant loss of genes associated with DNA repair and cell cycle. Thus, the aim of our study was to investigate the effects of varying nitrogen concentrations on H. vineae's cell cycle progression. Our findings demonstrated that nitrogen excess, regardless of the source, disrupts cell cycle progression and induces G2/M arrest in H. vineae after reaching the stationary phase. Additionally, we observed a viability decline in H. vineae cells in an ammonium-dependent manner, accompanied by increased production of reactive oxygen species, mitochondrial hyperpolarization, intracellular acidification, and DNA fragmentation. Overall, our study highlights the events of the cell cycle arrest in H. vineae induced by nitrogen excess and attempts to elucidate the possible mechanism triggering this absence of proper entry into the quiescent state.

Hanseniaspora menglaensis f.a., sp. nov., a novel apiculate yeast species isolated from rotting wood.

Two apiculate strains (NYNU 181072 and NYNU 181083) of a bipolar budding yeast species were isolated from rotting wood samples collected in Xishuangbanna Tropical Rainforest in Yunnan Province, southwest PR China. On the basis of phenotypic characteristics and the results of phylogenetic analysis of the D1/D2 domain of the large subunit (LSU) rRNA, internal transcribed spacer (ITS) region and the actin (ACT1) gene, the two strains were found to represent a single novel species of the genus Hanseniaspora, for which the name Hanseniaspora menglaensis f.a., sp. nov. (holotype CICC 33364T; MycoBank MB 847437) is proposed. In the phylogenetic tree, H. menglaensis sp. nov. showed a close relationship with Hanseniaspora lindneri, Hanseniaspora mollemarum, Hanseniaspora smithiae and Hanseniaspora valbyensis. H. menglaensis sp. nov. differed from H. lindneri, the most closely related known species, by 1.2 % substitutions in the D1/D2 domain, 2.5 % substitutions in the ITS region and 5.4 % substitutions in the ACT1 gene, respectively. Physiologically, H. menglaensis sp. nov. can also be distinguished from H. lindneri by its ability to assimilate d-gluconate.

PCR-based gene targeting in Hanseniaspora uvarum.

Lack of gene-function analyses tools limits studying the biology of Hanseniaspora uvarum, one of the most abundant yeasts on grapes and in must. We investigated a rapid PCR-based gene targeting approach for one-step gene replacement in this diploid yeast. To this end, we generated and validated two synthetic antibiotic resistance genes, pFA-hygXL and pFA-clnXL, providing resistance against hygromycin and nourseothricin, respectively, for use with H. uvarum. Addition of short flanking-homology regions of 56-80 bp to these selection markers via PCR was sufficient to promote gene targeting. We report here the deletion of the H. uvarum LEU2 and LYS2 genes with these marker genes via two rounds of consecutive transformations, each resulting in the generation of auxotrophic strains (leu2/leu2; lys2/lys2). The hereby constructed leucine auxotrophic leu2/leu2 strain was subsequently complemented in a targeted manner, thereby further validating this approach. PCR-based gene targeting in H. uvarum was less efficient than in Saccharomyces cerevisiae. However, this approach, combined with the availability of two marker genes, provides essential tools for directed gene manipulations in H. uvarum.

Insights into the transcriptional regulation of poorly characterized alcohol acetyltransferase-encoding genes (HgAATs) shed light into the production of acetate esters in the wine yeast Hanseniaspora guilliermondii.

Hanseniaspora guilliermondii is a well-recognized producer of acetate esters associated with fruity and floral aromas. The molecular mechanisms underneath this production or the environmental factors modulating it remain unknown. Herein, we found that, unlike Saccharomyces cerevisiae, H. guilliermondii over-produces acetate esters and higher alcohols at low carbon-to-assimilable nitrogen (C:N) ratios, with the highest titers being obtained in the amino acid-enriched medium YPD. The evidences gathered support a model in which the strict preference of H. guilliermondii for amino acids as nitrogen sources results in a channeling of keto-acids obtained after transamination to higher alcohols and acetate esters. This higher production was accompanied by higher expression of the four HgAATs, genes, recently proposed to encode alcohol acetyl transferases. In silico analyses of these HgAat's reveal that they harbor conserved AATs motifs, albeit radical substitutions were identified that might result in different kinetic properties. Close homologues of HgAat2, HgAat3, and HgAat4 were only found in members of Hanseniaspora genus and phylogenetic reconstruction shows that these constitute a distinct family of Aat's. These results advance the exploration of H. guilliermondii as a bio-flavoring agent providing important insights to guide future strategies for strain engineering and media manipulation that can enhance production of aromatic volatiles.

Biology and physiology of Hanseniaspora vineae: metabolic diversity and increase flavour complexity for food fermentation.

Apiculate yeasts belonging to the genus Hanseniaspora are predominant on grapes and other fruits. While some species, such as Hanseniaspora uvarum, are well known for their abundant presence in fruits, they are generally characterized by their detrimental effect on fermentation quality because the excessive production of acetic acid. However, the species Hanseniaspora vineae is adapted to fermentation and currently is considered as an enhancer of positive flavour and sensory complexity in foods. Since 2002, we have been isolating strains from this species and conducting winemaking processes with them. In parallel, we also characterized this species from genes to metabolites. In 2013, we sequenced the genomes of two H. vineae strains, being these the first apiculate yeast genomes determined. In the last 10 years, it has become possible to understand its biology, discovering very peculiar features compared to the conventional Saccharomyces yeasts, such as a natural and unique G2 cell cycle arrest or the elucidation of the mandelate pathway for benzenoids synthesis. All these characteristics contribute to phenotypes with proved interest from the biotechnological point of view for winemaking and the production of other foods.

A Versatile Toolset for Genetic Manipulation of the Wine Yeast Hanseniaspora uvarum.

Hanseniaspora uvarum is an ascomycetous yeast that frequently dominates the population in the first two days of wine fermentations. It contributes to the production of many beneficial as well as detrimental aroma compounds. While the genome sequence of the diploid type strain DSM 2768 has been largely elucidated, transformation by electroporation was only recently achieved. We here provide an elaborate toolset for the genetic manipulation of this yeast. A chromosomal replication origin was isolated and used for the construction of episomal, self-replicating cloning vectors. Moreover, homozygous auxotrophic deletion markers (Huura3, Huhis3, Huleu2, Huade2) have been obtained in the diploid genome as future recipients and a proof of principle for the application of PCR-based one-step gene deletion strategies. Besides a hygromycin resistance cassette, a kanamycin resistance gene was established as a dominant marker for selection on G418. Recyclable deletion cassettes flanked by loxP-sites and the corresponding Cre-recombinase expression vectors were tailored. Moreover, we report on a chemical transformation procedure with the use of freeze-competent cells. Together, these techniques and constructs pave the way for efficient and targeted manipulations of H. uvarum.

Co-inoculations of Lachancea thermotolerans with different Hanseniaspora spp.: Acidification, aroma, biocompatibility, and effects of nutrients in wine.

The use of non-Saccharomyces yeast in the winemaking industry and even more their co-inoculations to maximize their growth and to express phenotypic characteristic is gaining more and more relevance. This study aimed to shed light on the biocompatibilities between Lachancea thermotolerans and Hanseniaspora spp., using different types of nutrients and considering the effect on Yeast Assimilable Nitrogen (YAN), at low temperature (16 °C) and medium SO2 (50 mg/L), in white must. L. thermotolerans has been used for its positive effect on pH reduction and Hanseniaspora spp. for improving the sensory profile. The behaviour of these yeasts was evaluated in co-inoculation, always finishing the fermentation with the sequential inoculation of S. cerevisiae. Significant results were obtained on the population count (CFU/mL) in CHROMagar™, with higher populations of Hanseniaspora spp. with respect to L. thermotolerans. Fermentations with L. thermotolerans/H. vineae, showed inhibition of acidification, generating up to 0.41 g/L of lactic acid. On the contrary, a synergistic effect when L. thermotolerans/H. opuntiae was used, achieved 2.44 g/L of lactic acid and a pH reduction of up to 0.16 and always more significant with Nutrient Vit BlancTM. At the same time ethanol concentration decreased by 3.4 % and volatile acidity never exceeded 0.5 g/L. Aromatic composition was analysed and it was found that all fermentations retained more aromatic esters and that on day 7 the amount of 2-phenylethyl acetate was at least 3 times higher in all fermentations compared to the control (Sc + Nutrient Vit BlancTM) which had 5.96 mg/L. Less yellow intensity (-17.3 %) typical of oxidation were observed in all fermentations in which Nutrient Vit BlancTM had been used and in the sensory analysis the co-inoculations with H. vineae generated better scores.

Use of sequentially inoculation of Saccharomyces cerevisiae and Hanseniaspora uvarum strains isolated from honey by-products to improve and stabilize the quality of mead produced in Sicily.

Mead is a beverage produced by alcoholic fermentation of honey-must. The starter yeasts that are commonly used for the alcoholic fermentation of honey-must are oenological Saccharomyces cerevisiae strains. The objective of the present work was, for the first time, to apply yeasts of honey by-products origin to evaluate the influences the taste-olfactory attributes of mead. For this purpose, three experimental productions were set up, which included: (i) single inoculation of S. cerevisiae; (ii) single inoculation of Hanseniaspora uvarum; (iii) sequential inoculation of H. uvarum/S. cerevisiae. Two control trials were performed, using a commercial strain of S. cerevisiae of oenological origin and a spontaneous fermentation. The results of the chemical parameters showed differences between the trials in terms of residual sugars, acetic acid, glycerol, ethanol and volatile organic compounds. Sensorial analysis also showed a high heterogeneity among trials. The attributes of sweetness, honey and floral were found in mead fermented with H. uvarum, whereas all meads obtained with S. cerevisiae were dry, balanced and without off-odors and off-flavours. The results obtained showed that the controlled application of conventional and non-conventional yeast strains isolated from honey by-products origin could be a promising approach to improve the quality of meads.

Influence of indigenous Hanseniaspora uvarum and Saccharomyces cerevisiae from sugar-rich substrates on the aromatic composition of loquat beer.

The demand for unique and exclusive food products and beverages is constantly on the increase. One of the products that mostly evolved to encounter market dynamics in the last decade is craft beer. For a long time, craft breweries have included fruit in beer production to enrich flavour and aroma profile of different beer styles. In this study, for the first time, the use of Saccharomyces and non-Saccharomyces yeast strains isolated from high-sugar matrices (manna and fermented honey by-products) were investigated to diversify fruit craft beer production, in order to improve the fermentation process and highlight the complexity of aroma profiles generated during alcoholic fermentation. Two yeast strains, Hanseniaspora uvarum YGA34 and Saccharomyces cerevisiae MN113, were tested as co-starters and starters for their beer production capacity. Commercial yeast strain US-05 was used as control. Loquat juice was added at the end of primary alcoholic fermentation in all trials. Interestingly, S. cerevisiae MN113 consumed sugars faster than control strain S. cerevisiae US-05, including maltose, even in the case of sequential inoculation. This strain showed an excellent ability to consume rapidly sugars present. All strains showed their concentrations ranged between 5 and 8 Log cycles during fermentation. The absence of off-odours and the improvement of aromatic perception were observed in experimental trials involving the use of S. cerevisiae MN113 as a monoculture and in sequential combination with H. uvarum YGA34. Esters and alcohols were the most abundant compounds emitted from the beers. The beers produced with sequential inoculation of H. uvarum YGA34 and S. cerevisiae MN113 or US-05 are characterised by a higher ester and lower alcohol concentration. These two unconventional yeast strains from high sugar matrices showed great technological properties, representing promising co-starters and starter during craft fruit beer production.

Hanseniaspora uvarum Attracts Drosophila suzukii (Diptera: Drosophilidae) With High Specificity.

Since the early phase of the intercontinental dispersal of Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), fermentation baits have been used for monitoring. Self-made lures and commercial products are often based on wine and vinegar. From an ecological perspective, the formulation of these baits is expected to target especially vinegar flies associated with overripe fruit, such as Drosophila melanogaster (Meigen) (Diptera: Drosophilidae). Hanseniaspora uvarum (Niehaus) (Ascomycota: Saccharomyceta) is a yeast closely associated with D. suzukii and fruit, and furthermore attractive to the flies. Based on this relation, H. uvarum might represent a suitable substrate for the development of lures that are more specific than vinegar and wine. In the field, we therefore, compared H. uvarum to a commercial bait that was based on vinegar and wine with respect to the number of trapped D. suzukii relative to other drosophilids and arthropods. Trap captures were higher with the commercial bait but specificity for D. suzukii was greater with H. uvarum. Moreover, H. uvarum headspace extracts, as well as a synthetic blend of H. uvarum volatiles, were assayed for attraction of D suzukii in a wind tunnel and in the field. Headspace extracts and the synthetic blend induced strong upwind flight in the wind tunnel and confirmed attraction to H. uvarum volatiles. Furthermore, baited with H. uvarum headspace extract and a drowning solution of aqueous acetic acid and ethanol, 74% of field captured arthropods were D. suzukii. Our findings suggest that synthetic yeast headspace formulations might advance the development of more selective monitoring traps with reduced by-catch.

Localization, purification, and characterization of a novel ß-glucosidase from Hanseniaspora uvarum Yun268.

ß-Glucosidase is a key enzyme that hydrolyzes nonvolatile glycosylated precursors of aroma compounds and enhances the organoleptic quality of wines. In this study, a novel ß-glucosidase from Hanseniaspora uvarum Yun268 was localized, purified, and characterized. Results indicated that ß-glucosidase activity was mainly distributed within the cells. After purification via ammonium sulfate precipitation combined with chromatography, ß-glucosidase specific activity increased 8.36 times, and the activity recovery was 56.90%. The enzyme had a molecular mass of 74.22 kDa. It has a Michaelis constant (Km ) of 0.65 mmol/L, and a maximum velocity (Vmax ) of 5.1 nmol/min under optimum conditions; and Km of 0.94 mmol/L, and Vmax of 2.8 nmol/min under typical winemaking conditions. It exhibited the highest activity at 50°C and pH 5.0 and was stable at a temperature range of 20-80°C and pH range of 3.0-8.0. The enzyme has good tolerance to Fe3+ , especially maintaining 93.68% of its activity with 10 mmol/L of Fe3+ . Ethanol (<20%) and glucose (<150 g/L) inhibited its activity only slightly. Therefore, ß-glucosidase from H. uvarum Yun268 has excellent biochemical properties and a good application potential in winemaking. PRACTICAL APPLICATION: Winemaking is a biotechnological process in which exogenous ß-glucosidase is used to overcome the deficiency of endogenous ß-glucosidase activity in grapes. By localizing, purifying, and characterizing of ß-glucosidase from Hanseniaspora uvarum Yun268, it is expected to reveal its physical and chemical characteristics to evaluate its oenological properties in winemaking. The results may provide the basis for promoting the release of varietal aroma and improving wine sensory quality in the wine industry.

Field and greenhouse application of an attract-and-kill formulation based on the yeast Hanseniaspora uvarum and the insecticide spinosad to control Drosophila suzukii in grapes.

BACKGROUND: The invasive insect Drosophila suzukii (Matsumura) is an important pest of several red grape varieties. The yeast Hanseniaspora uvarum (Niehaus), which is associated with D. suzukii, strongly attracts flies and stimulates them to feed on yeast-laden food. In the present study, a formulation based on H. uvarum culture with spinosad insecticide was applied to the foliage of vineyards and control of D. suzukii was compared to applying spinosad to the whole plant. After successful H. uvarum and insecticide application in the vineyard, we tested additional H. uvarum-based formulations with spinosad in a greenhouse to determine their capacity to control D. suzukii. RESULTS: Application of the H. uvarum-spinosad formulation at 36.4 g of spinosad per hectare reduced the D. suzukii field infestation at the same rate as applying 120 g of spinosad per hectare and prevented spinosad residues on grapes. Leaves treated with H. uvarum and spinosad in the field and transferred to a laboratory assay caused high mortality to flies and reduced the number of eggs laid on fruits. Formulations with spinosad applied in the greenhouse showed that both H. uvarum culture and the yeast cell-free supernatant of a centrifuged culture increased fly mortality and reduced the number of eggs laid compared to the unsprayed control. CONCLUSION: In comparison to typical spinosad spray applications, the use of H. uvarum in combination with spinosad as an attract-and-kill formulation against D. suzukii reduces pesticide residues on the fruits by targeting the treatment to the canopy and decreasing the amount of insecticide per hectare without compromising control efficacy.

Effects of three indigenous non-Saccharomyces yeasts and their pairwise combinations in co-fermentation with Saccharomyces cerevisiae on volatile compounds of Petit Manseng wines.

The combined use of selected Saccharomyces cerevisiae and non-Saccharomyces strains is becoming an effective way to achieve wine products with distinctive aromas. The purpose of this study was to further improve the wine aroma complexity through optimizing inoculation protocols of multi-starters. The three indigenous non-Saccharomyces strains (Torulaspora delbrueckii, Hanseniaspora vineae, and Lachancea thermotolerans) and their pairwise combinations (co-inoculation) were sequentially inoculated with S. cerevisiae in Petit Manseng grape must, respectively. Results evidenced a higher divergence in aroma compounds produced by two different non-Saccharomyces species compared to single species. Especially for the combination of T. delbrueckii and L. thermotolerans, the concentrations of most ethyl esters were further increased, contributing to a higher score of 'pineapple' note in agreement with sensory analysis. Our results highlighted that the inoculation of more than one non-Saccharomyces species is a potential strategy to improve the aroma diversity and quality of industrial wines.

Bioprospecting of indigenous yeasts involved in cocoa fermentation using sensory and chemical strategies for selecting a starter inoculum.

Cocoa fermentation is the key and most relevant process in the synthesis of aroma and flavor precursor molecules in dry beans or raw material for producing chocolate. Because this process occurs in an uncontrolled manner, the chemical and sensory quality of beans can vary and be negatively affected. One of the strategies for the standardization and improvement of the sensory quality of chocolate is the introduction of microbial starter cultures. Among these, yeasts involved in fermentation have been studied because of their pectinolytic and metabolic potential in the production of volatile compounds. This study was aimed at isolating and characterizing, both sensory and chemically, yeasts involved in cocoa fermentation that could be used as starter cultures from two agro-ecological regions for the cultivation of cocoa in Colombia. The microbiological analyses identified 22 species represented mostly by Saccharomyces cerevisiae, Wickerhamomyces anomalus and Pichia sp. The preliminary sensory analysis of eight of these species showed that Hanseniaspora thailandica and Pichia kluyveri presented sensory profiles characterized by high intensity levels of fruity notes, which could be ascribed to the production of ethyl acetate, isoamyl acetate, and 2-phenylethyl acetate.

Diversity of non-Saccharomyces yeasts of grape berry surfaces from representative Cabernet Sauvignon vineyards in Henan Province, China.

Non-Saccharomyces yeasts are important players during winemaking and may come from grapes grown in vineyards. To study the diversity of non-Saccharomyces yeasts on grape berry surfaces, 433 strains were isolated from different Cabernet Sauvignon vineyards grown in Henan Province. Our results demonstrated that these strains were classified into 16 morphotypes according to their growth morphology on Wallerstein Laboratory agar medium, and were identified as seven species from four genera-Hanseniaspora opuntiae, Hanseniaspora vineae, Hanseniaspora uvarum, Pichia occidentalis, Pichia kluyveri, Issatchenkia terricola and Saturnispora diversa-based on a series of molecular biological experiments. Hanseniaspora opuntiae was obtained from all sampling sites except Changyuan County, while Pichia kluyveri and Saturnispora diversa were only found in sites of Zhengzhou Grape Resource Garden and Minquan County, respectively. The site Minquan was home of the greatest species richness, while only one single species (Hanseniaspora opuntiae) was detected at NAPA winery from Zhengzhou or at Anyang County. Finally, this study suggested that the geographic distribution and diversity of non-Saccharomyces yeast populations on Cabernet Sauvignon grape berries were likely to be determined by a combination of grape varieties and environmental factors.