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The genus Kloeckeraspora Niehaus (1932) was not accepted for a long time. This paper has tried to reinstate the genus. Two phylogenetic trees were constructed for three Kloeckeraspora and four representative Hanseniaspora species. In addition, the pair-wise sequence similarities were calculated. In the phylogeneetic tree based on the 26S rRNA gene D1/D2 domain sequences derived from the neighbour-joining method, the three species of the genus Kloeckeraspora represented an extremely long branch. The calculated pair-wise sequence similarities were abnormally low (86.2 - 88.5%) between the two genera. Incidentally, the sequence similarity between Vanderwatozyma polyspora and Saccharomyces cerevisiae used as references was 94.0%. The genus Kloeckeraspora should be unequivocally accepted and retained. However, the two genera were not yet perfectly taxonomic homogeneous-natured. Keywords: Kloeckeraspora osmophila, Kloeckerasora vineae, Kloeckeraspora occidentalis, Hanseniaspora valvyensis, Hanseniaspora uvarum, Hanseniaspora guilliermondii. Title: The Reinstatement of the Genus Kloeckeraspora Niehaus (1932) (Apiculate Yeast) Author: Taweesak Malimas, Huong Thi Lan Vu, Pattaraporn Yukphan, Somboon Tanasupawat, Yuzo Yamada International Journal of Novel Research in Life Sciences ISSN 2394-966X Vol. 10, Issue 2, March 2023 - April 2023 Page No: 32-37 Novelty Journals Website: www.noveltyjournals.com Published Date: 03-April-2023 DOI: https://doi.org/10.5281/zenodo.7794482 Paper Download Link (Source) https://www.noveltyjournals.com/upload/paper/The%20Reinstatement%20of%20the%20Genus-03042023-1.pdf, International Journal of Novel Research in Life Sciences, ISSN 2394-966X, Novelty Journals, Website: www.noveltyjournals.com

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.

Postharvest loss is a major constraint in the production of grapes, both in terms of quality and quantity. In this study, 33 yeasts were isolated from the fructosphere of grapes and tested for their antifungal activity against Aspergillus spp., which infects grapes during the postharvest stage. The results revealed that the yeast isolate YBB3 exhibited maximum inhibitory effect against Aspergillus spp. in dual plate assay (57% over control). Molecular characterization of YBB3 showed 99.31% identity with Hanseniaspora guilliermondii. Wound site colonization of H. guilliermondii in grape cv. Thompson Seedless showed 80% inhibition of Aspergillus spp. over the control in vitro. YBB3 produced volatile, non-volatile and thermostable compounds, which inhibited the mycelial growth of Aspergillus spp. In addition, YBB3 produced killer toxins, which were identified by the presence of blue colour-stained cells. Moreover, glycerol-based liquid bioformulations of H. guilliermondii (YBB3) showed maximum inhibitory effect against Aspergillus spp. (96%) over the control on inoculated wounded grapes berries. Thus, the yeast isolate H. guilliermondii identified and characterized in this study is worthy of further studies for the sustainable management of Aspergillus rot in grapes.

This study aimed to explore communities and the ethanol-fermenting ability of yeasts in fresh coconut, palmyra, and nipa palm saps. From the 90 samples of coconut, palmyra, and nipa palm saps, 204 yeast isolates were isolated and identified as 15 species in the phylum Ascomycota and a species (one strain) in Basidiomycota. Saccharomyces cerevisiae, Hanseniaspora guilliermondii, and Lachancea thermotolerans were found in the saps of all three palm species. Candida tropicalis and Pichia kudriavzevii were obtained from the coconut and palmyra palm saps, Hanseniaspora vineae, Lachancea fermentati, and Pichia manshurica were present in the coconut and nipa palm saps, whereas Torulaspora delbrueckii was found in the palmyra and nipa palm saps. The species with the highest occurrence in the saps of coconut, palmyra, and nipa palms was S. cerevisiae with 76.67%, 86.70%, and 100% frequency of occurrence, respectively. Using principal coordinates analysis for ordination, no marked difference was observed in the yeast communities from the saps of the three palm species. A total of 199 isolates were found to possess ethanol-fermentation ability when cultivated using shake flask in 160 g/L of glucose medium at 28°C for 48 h. Lachancea fermentati YSP-383, isolated from nipa palm sap, produced the highest amount of ethanol (76.74 g/L). Twenty-six isolates of Candida sanyaensis (1), C. tropicalis (1), H. guilliermondii (7), L. fermentati (8), L. thermotolerans (1), Pichia kudriavzevii (2), and S. cerevisiae (6) produced high amounts of ethanol ranging from 69.57 to 76.74 g/L. The result demonstrated that yeasts in the palm saps could play roles in the natural fermentation of palm saps.

BACKGROUND The biotechnological potential of yeasts from nuts such as pistachio, not only for health applications but also for industry use, has been scarcely studied. Interest in the probiotic capability of yeasts has increased in the past years as well as their utilization as food or feed preservatives. Their capabilities as biocontrol against problematic (spoilage or toxigenic) microorganisms or as antioxidants have been revalued. As a result, both abilities would be desirable to develop a new potential probiotic microorganism which could be added to food or feed to improve their properties. RESULTS Molecular techniques allowed the identification of a total of seven different species and 15 strains. A screening of the probiotic potential of these strains was carried out. It was found that 65% of the strains resisted the gastrointestinal conditions as well as presented a generation time of

We explored how gut-associated yeasts influence olfactory behaviour and resource use in two pest species of Carpophilus beetle that co-exist in Australian stone fruits. Molecular analysis of yeasts isolated from the gut of C. davidsoni (prefers ripe fruits) and C. hemipterus (prefers overripe and rotting fruits) revealed that the predominant species were Pichia kluyveri and Hanseniaspora guilliermondii. In olfactory attraction and oviposition trials, adult beetles preferred H. guilliermondii over P. kluyveri, and follow up GC-MS analysis revealed unambiguous differences between the odour profiles of these yeasts. In contrast to behavioural trials, larval feeding assays showed that fruit substrates inoculated with P. kluyveri yielded significantly faster development times, higher pupal mass, and a greater number of adult beetles, compared to H. guilliermondii - in other words, the lesser preferred yeast (by foraging adults) was more suitable for larval survival. Moreover, whilst larvae of both species survived to adulthood when fed solely on P. kluyveri (i.e. without a fruit substrate), only larvae of C. davidsoni could develop on H. guilliermondii; and only C. davidsoni reached adulthood feeding on a yeast-free fruit substrate. We discuss how these findings may relate to adaptations towards early colonising of fruits by C. davidsoni, enabling differences in resource use and potentially resource partitioning in the two beetles. More broadly, consideration of microbial interactions might help develop host selection theory. Our results could pave the way to more powerful attractants to mass-trap and monitor Carpophilus pests in fruit orchards.

Wine production in Cyprus has strong cultural ties with the island’s tradition, influencing local and foreign consumers’ preferences and contributing significantly to Cyprus’ economy. A key contributor to wine quality and sensorial characteristics development is the microbiota that colonizes grapes and performs alcoholic fermentation. Still, the microbial patterns of wines produced in different geographic regions (terroir) in Cyprus remain unknown. The present study investigated the microbial diversity of five terroirs in Cyprus, two from the PGI Lemesos region [Kyperounta (PDO Pitsilia) and Koilani (PDO Krasochoria)], and three from the PGI Pafos region [Kathikas (PDO Laona Akamas), Panayia, and Statos (PDO Panayia)], of two grape varieties, Xynisteri and Maratheftiko, using high-throughput amplicon sequencing. Through a longitudinal analysis, we examined the evolution of the bacterial and fungal diversity during spontaneous alcoholic fermentation. Both varieties were characterized by a progressive reduction in their fungal alpha diversity (Shannon index) throughout the process of fermentation. Additionally, the study revealed a distinct separation among different terroirs in total fungal community composition (beta-diversity) for the variety Xynisteri. Also, Kyperounta terroir had a distinct total fungal beta-diversity from the other terroirs for Maratheftiko. Similarly, a significant distinction was demonstrated in total bacterial diversity between the PGI Lemesos region and the PGI Pafos terroirs for grape juice of the variety Xynisteri. Pre-fermentation, the fungal diversity for Xynisteri and Maratheftiko was dominated by the genera Hanseniaspora, Aureobasidium, Erysiphe, Aspergillus, Stemphylium, Penicillium, Alternaria, Cladosporium, and Mycosphaerella. During and post-fermentation, the species Hanseniaspora nectarophila, Saccharomyces cerevisiae, Hanseniaspora guilliermondii, and Aureobasidium pullulans, became the predominant in most must samples. Regarding the bacterial diversity, Lactobacillus and Streptococcus were the predominant genera for both grape varieties in all stages of fermentation. During fermentation, an increase was observed in the relative abundance of some bacteria, such as Acetobacter, Gluconobacter, and Oenococcus oeni. Finally, the study revealed microbial biomarkers with statistically significant higher relative representation, associated with each geographic region and each grape variety, during the different stages of fermentation. The present study’s findings provide an additional linkage between the grape microbial community and the wine terroir.

The phloem sap tapped from unopened inflorescence (spadix) of coconut palm using a novel collecting device, “coco-sap chiller,” has been branded Kalparasa® (henceforth as Kalparasa in the text) to distinguish its properties not found in sap harvested by traditional methods. To know its hitherto unidentified microbiome profile, we employed high-throughput sequencing to uncover the bacteriome and mycobiome in fresh and 12-h fermented samples. Fresh Kalparasa had a pH of 7.2, which dropped to 4.5 after 12 h, signifying fermentation of the sap. Diversity analysis indicated fresh Kalparasa having higher bacterial species than the fermented one. Contrary to this, fresh sap had lower fungal/yeast diversity than the fermented sample. Fresh Kalparasa had relatively higher abundance of probiotic-type Leuconostoc genus followed by equal proportions of Gluconobacter, Acetobacter, and Fructobacillus. The 12-h fermented Kalparasa showed a significant increase in Gluconobacter with a sharp decrease in Leuconostoc. Mycobiome data revealed fresh Kalparasa to be preponderant in Saccharomyces and Hanseniaspora genera of yeasts while the fermented sap had higher representation of Hanseniaspora and Cortinarius and lesser Saccharomyces. This suggested that the fermentation of Kalparasa was probably driven by symbiotic culture of bacteria and yeasts (SCOBY), particularly acetic acid bacteria and non-Saccharomyces yeasts. The bacteriome-function predictions highlighted the enrichment of glycerophospholipid, ABC transporters, purine, and pyrimidine metabolisms. Based on our findings, Kalparasa containing large population of Leuconostoc mesenteroides, Fructobacillus fructosus, Saccharomyces cerevisiae, and Hanseniaspora guilliermondii can be promoted as a healthy “unfermented” plant edible food containing live probiotic-type microbiome during its consumption.

In industrial winery, the use of mixed starter cultures composed of Saccharomyces and non-Saccharomyces yeasts is an approach of growing importance for winemakers to enhance the sensory and complexity of the wine without compromising the quality. In this work, the oenological properties and enzymatic activities of 196 non-Saccharomyces yeasts, belonging to Hanseniaspora guilliermondii and Hanseniaspora uvarum species, were investigated. This screening has allowed the selection of the best non-Saccharomyces yeast strain and the use of vinification for white grape of Campania Vitis vinifera cv Fiano, in an industrial scale. The experimental fermentations were performed in four different batches: batch A (H. guilliermondii BF1 and S. cerevisiae 404 in sequential inoculum); batch B only inoculum of S. cerevisiae 404; batch C without inoculation (spontaneous fermentation); batch D with the inoculum of H. guilliermondii BF1 strain. The results of chemical and sensorial analyses have showed that the best wine comes from batch A. In conclusion, the use of H. guilliermondii BF1 with good oenological properties and a strong β-glucosidase activity allowed to improve the sensorial complexity of the wine. Selected non-Saccharomyces yeast could be applied profitably to winemaking to enhance the quality of the wine using new fermentation technologies, and could be used in industrial winery.

Background Saccharomycodes ludwigii belongs to the poorly characterized Saccharomycodeacea family and is known by its ability to spoil wines, a trait mostly attributable to its high tolerance to sulfur dioxide (SO2). To improve knowledge about Saccharomycodeacea our group determined whole-genome sequences of Hanseniaspora guilliermondii (UTAD222) and S. ludwigii (UTAD17), two members of this family. While in the case of H. guilliermondii the genomic information elucidated crucial aspects concerning the physiology of this species in the context of wine fermentation, the draft sequence obtained for S. ludwigii was distributed by more than 1000 contigs complicating extraction of biologically relevant information. In this work we describe the results obtained upon resequencing of S. ludwigii UTAD17 genome using PacBio as well as the insights gathered from the exploration of the annotation performed over the assembled genome. Results Resequencing of S. ludwigii UTAD17 genome with PacBio resulted in 20 contigs totaling 13 Mb of assembled DNA and corresponding to 95% of the DNA harbored by this strain. Annotation of the assembled UTAD17 genome predicts 4644 protein-encoding genes. Comparative analysis of the predicted S. ludwigii ORFeome with those encoded by other Saccharomycodeacea led to the identification of 213 proteins only found in this species. Among these were six enzymes required for catabolism of N-acetylglucosamine, four cell wall β-mannosyltransferases, several flocculins and three acetoin reductases. Different from its sister Hanseniaspora species, neoglucogenesis, glyoxylate cycle and thiamine biosynthetic pathways are functional in S. ludwigii. Four efflux pumps similar to the Ssu1 sulfite exporter, as well as robust orthologues for 65% of the S. cerevisiae SO2-tolerance genes, were identified in S. ludwigii genome. Conclusions This work provides the first genome-wide picture of a S. ludwigii strain representing a step forward for a better understanding of the physiology and genetics of this species and of the Saccharomycodeacea family. The release of this genomic sequence and of the information extracted from it can contribute to guide the design of better wine preservation strategies to counteract spoilage prompted by S. ludwigii. It will also accelerate the exploration of this species as a cell factory, specially in production of fermented beverages where the use of Non-Saccharomyces species (including spoilage species) is booming.

Beer production is predominantly carried out by Saccharomyces species, such as S. cerevisiae and S. pastorianus. However, the introduction of non-Saccharomyces yeasts in the brewing process is now seen as a promising strategy to improve and differentiate the organoleptic profile of beer. In this study, 17 non-Saccharomyces strains of 12 distinct species were isolated and submitted to a preliminary sensory evaluation to determine their potential for beer bioflavouring. Hanseniaspora guilliermondii IST315 and H. opuntiae IST408 aroma profiles presented the highest acceptability and were described as having 'fruity' and 'toffee' notes, respectively. Their presence in mixed-culture fermentations with S. cerevisiae US-05 did not influence attenuation and ethanol concentration of beer but had a significant impact in its volatile composition. Notably, while both strains reduced the total amount of ethyl esters, H. guilliermondii IST315 greatly increased the concentration of acetate esters, especially when sequentially inoculated, leading to an 8.2-fold increase in phenylethyl acetate ('rose', 'honey' aroma) in the final beverage. These findings highlight the importance of non-Saccharomyces yeasts in shaping the aroma profile of beer and suggest a role for Hanseniaspora spp. in improving it.

Microorganisms produce hundreds of aroma-active compounds during fermentation process which affect the flavor of the final wine product. The current study explored the enological characteristics of non-Saccharomyces yeast strains and their influence on aroma compound complexity during fermentation process. Forty-two yeast strains were isolated from fruits by the DNA sequencing identification. Hanseniaspora uvarum Pi235, Pichia kluyveri Pe114, Saccharomyces cerevisiae Gr112 and Hanseniaspora guilliermondii Ki135 were selected for the following experiments due to the safety considerations and aroma diversity. Biochemical characteristics results showed that H. uvarum Pi235, P. kluyveri Pe114 and S. cerevisiae Gr112 exhibited different consumption preference in maltose, xylose and glycerol assimilation compared with their standard strains. Enological characteristics results showed that S. cerevisiae Gr112 exhibited best thermal tolerance, ethanol tolerance and β-glucosidase activity. Results of volatile aroma compounds analysis showed that non-Saccharomyces yeast strains were good at producing esters, such as ethyl acetate and 2-phenethyl acetate which were 11.8–25.4 and 4.1–15.2 folds higher than that from S. cerevisiae Gr112. Non-Saccharomyces yeast strains would provide more divers flavors in wines. The results will increase the applicability of non-Saccharomyces yeasts in the winemaking industry.

During spontaneous wine fermentations, most of the non-Saccharomyces yeasts present in grape musts show an early decline in their population. It was traditionally assumed that Saccharomyces cerevisiae (S.c.) prevalence was due to the higher resistance of this species to ethanol. However, wine fermentations performed with single cultures of non-Saccharomyces strains showed that those strains could withstand much higher ethanol levels [1]. It was then found that S.c. (strain CCMI 885) produced antimicrobial peptides (AMPs) that are responsible for the early death of the non Saccharomyces yeasts [2]. In previous work, we isolated, purified and sequenced those ntimicrobial peptides (AMPs) and found that they derive from the glyceraldehyde 3-phosphate dehydrogenase enzyme [3]. These GAPDH-derived AMPs compose the natural biocide secreted by S.c., which we named saccharomycin, and are effective against sensitive yeasts both in its natural/isolated and synthetic form. info:eu-repo/semantics/publishedVersion

In winemaking, non-Saccharomyces yeast species contribute important organoleptic complexity. Current interest focuses on abundant and dominant strains characteristically present in the early phase of spontaneous alcoholic fermentations. Non-Saccharomyces species are particularly relevant in Port wine production such that the fermentation is prematurely stopped, after the metabolism of only one half of the available sugar, through fortification with aguardente. This work aimed to isolate, identify and characterize non-Saccharomyces species present in spontaneously fermenting Port. To accomplish these goals, yeasts were isolated from a selection of frozen must samples (2012&ndash, 2016 harvests), using a pre-screening process choosing only the best candidates based on the organoleptic quality of the corresponding fortified wine. From five hundred non-Saccharomyces isolates, twelve species were identified. The three most abundant species, Hanseniaspora uvarum, Lachancea thermotolerans, and Metschnikowia pulcherrima, representing 89% of the isolates, exhibited particularly high diversity with high growth performance variability when exposed to typical stress conditions associated with common enological parameters. Less abundant species included Issatchenkia orientalis, Torulaspora delbrueckii, Hanseniaspora vineae, Hanseniaspora osmophila, Candida zemplinina, Rhodotorula mucilaginosa, Hanseniaspora guilliermondii, Issatchenkia occidentalis, and Zygosaccharomyces bisporus. This is the first study providing insights into the identification and characterization of non-Saccharomyces species responsible for spontaneous Port wine production.

Hanseniaspora, a predominant yeast genus of grape musts, includes sister species recently reported as fast evolving. The aim of this study was to investigate the genetic relationships between the four most closely related species, at the population level. A multi-locus sequence typing strategy based on five markers was applied on 107 strains, confirming the clear delineation of species H. uvarum, H. opuntiae, H. guilliermondii, and H. pseudoguilliermondii. Huge variations were observed in the level of intraspecific nucleotide diversity, and differences in heterozygosity between species indicate different life styles. No clear population structure was detected based on geographical or substrate origins. Instead, H. guilliermondii strains clustered into two distinct groups, which may reflect a recent step toward speciation. Interspecific hybrids were detected between H. opuntiae and H. pseudoguilliermondii. Their characterization using flow cytometry, karyotypes and genome sequencing showed different genome structures in different ploidy contexts: allodiploids, allotriploids, and allotetraploids. Subculturing of an allotriploid strain revealed chromosome loss equivalent to one chromosome set, followed by an auto-diploidization event, whereas another auto-diploidized tetraploid showed a segmental duplication. Altogether, these results suggest that Hanseniaspora genomes are not only fast evolving but also highly dynamic.

Hanseniaspora, a predominant yeast genus of grape musts, includes sister species recently reported as fast evolving. The aim of this study was to investigate the genetic relationships between the four most closely related species, at the population level. A multi-locus sequence typing strategy based on five markers was applied on 107 strains, confirming the clear delineation of species H. uvarum, H. opuntiae, H. guilliermondii, and H. pseudoguilliermondii. Huge variations were observed in the level of intraspecific nucleotide diversity, and differences in heterozygosity between species indicate different life styles. No clear population structure was detected based on geographical or substrate origins. Instead, H. guilliermondii strains clustered into two distinct groups, which may reflect a recent step toward speciation. Interspecific hybrids were detected between H. opuntiae and H. pseudoguilliermondii. Their characterization using flow cytometry, karyotypes and genome sequencing showed different genome structures in different ploidy contexts: allodiploids, allotriploids, and allotetraploids. Subculturing of an allotriploid strain revealed chromosome loss equivalent to one chromosome set, followed by an auto-diploidization event, whereas another auto-diploidized tetraploid showed a segmental duplication. Altogether, these results suggest that Hanseniaspora genomes are not only fast evolving but also highly dynamic.

Saccharomyces yeast species are currently the most important yeasts involved in industrial-scale food fermentations. However, there are hundreds of other yeast species poorly studied that are highly promising for flavour development, some of which have also been identified in traditional food fermentations. This work explores natural yeast biodiversity in terms of aroma formation, with a particular focus on aromas relevant for industrial fermentations such as wine and beer. Several non-Saccharomyces species produce important aroma compounds such as fusel alcohols derived from the Ehrlich pathway, acetate esters and ethyl esters in significantly higher quantities than the well-known Saccharomyces species. These species are Starmera caribaea, Hanseniaspora guilliermondii, Galactomyces geotrichum, Saccharomycopsis vini and Ambrosiozyma monospora. Certain species revealed a strain-dependent flavour profile while other species were very homogenous in their flavour profiles. Finally, characterization of a selected number of yeast species using valine or leucine as sole nitrogen sources indicates that the mechanisms of regulation of the expression of the Ehrlich pathway exist amongst non-conventional yeast species.

The aim of this study was to evaluate the effect of indigenous yeasts on the aroma of fermented Chenin Blanc grape must by determining the volatile compounds, the odor activity value (OAV) and carrying out sensory analyses. The must, fermented by Hanseniaspora opuntiae/Saccharomyces cerevisiae combinations, presented higher concentrations of compounds such as ethyl acetate, ethyl hexanoate, 2-methyl-1-propanol and 2-phenylethanol, and a lower production of acids and acetaldehyde. This fermented must presented higher OAV values for compounds such as 2-methyl-1-propanol and ethyl acetate, 687.06 and 1264.16, respectively. 2-phenylethanol was produced by H. opuntiae and Hanseniaspora guilliermondii in combination with S. cerevisiae in amounts that resulted in OAV values of 5.63 and 4.62, respectively. In appropriate concentrations, these volatile compounds contribute positively to the aromatic quality of the fermented must. The highest mean acceptability and purchasing intention scores were obtained by the must fermented by H. opuntiae/S. cerevisiae. In the must fermented by H. guilliermondii/ S. cerevisiae, the absence of ethyl hexanoate and high concentrations of octanoic acid and acetaldehyde probably contributed to its low acceptability. Thus, it was suggested that the yeast genus Hanseniaspora in combination with S. cerevisiae, showed the potential to positively impact the wine aroma. Key words: Non-Saccharomyces yeasts, volatile compounds, odor activity value, acceptability test, Hanseniaspora, “Sao Francisco Valley region”.

Artisanal cachacas are traditionally produced from spontaneous fermentation from sugarcane must in Brazil. The microbiological traits of such processes are still poorly understood, and this work aimed to assess the microbial population dynamics during one fermentation—a study of case—for the production of cachaca, as well as to correlate the chemical, and sensory profiles of the distillate. One fermentation was carried out by gradually increasing sugarcane must concentration, and the microbial communities were assessed by plating on selective and differential media. Distillation was performed in a simple copper pot still, and the distillate fractions analyzed by gas chromatograph. Acetic and lactic bacteria took part during the whole process, and the yeasts Meyerozyma guilliermondii, Pichia fermentans and Hanseniaspora guilliermondii were detected throughout fermentation. Saccharomyces cerevisiae emerged along the process, directly competing with H. guilliermondii during tumultuous fermentation. The spirit showed low levels of acetaldehyde, ethyl acetate and acetic acid, even as no methanol and ethyl carbamate traces. The cachaca produced stood out by fruity aromas, in contrast to other similar samples from uncontrolled processes. Sugarcane fermentations are traditionally performed in batch system, implying in complex interactions of microorganisms. Microbiota diversity contributes to the organoleptic complexity, but makes a point on rigorous monitoring to ensure high quality of the product. These findings shed light on better understanding the role of microbial population dynamics in spontaneous fermentation for cachaca’s production, even as bringing new information about the microorganisms implicated and their impact on the chemical quality of the product.

Properly conducted cocoa fermentation is an important step for the production of high-quality chocolate. The aim of this work was to investigate the effect of four cocoa varieties (CCN51, PS1030, FA13, and CEPEC 2004) inoculated with Saccharomyces cerevisiae CA11 on microbial communities and the profile of volatile compounds and sensory characteristics of chocolate. The S. cerevisiae population increased significantly (p S. cerevisiae and Hanseniaspora guilliermondii , while Lactobacillus casei and Gluconobacter oxidans were the predominant bacteria in the four different fermentations analyzed. Sixty-one volatile compounds—including aldehydes (11), ketones (10), esters (14), acids (8), alcohols (8), pyrazines (5), furans (3), and others (caffeine and heptadecane)—were detected and quantified by GC–MS in the different chocolates. The sensory analysis showed that caramel was perceptible in the chocolate of PS1030, while CEPEC2004 was related to astringency, bitterness, and chocolate flavor attributes. The chocolates produced from FA13 and CCN51 were more similar in terms of sour and chocolate aroma. A “temporal dominance of sensation” (TDS) analysis showed that although the bitter attribute was dominant, the fruity, sweet, sour, astringent, and cocoa attributes were also perceptible, depending on the cocoa variety. These results suggest that the cocoa varieties had an influence on the chocolate's quality, which should be considered to obtain chocolate with different sensory characteristics or for better standardization of the process, even when using yeast as a starter culture.

In this study, the effect of sequential inoculation with non-Saccharomyces (Hanseniaspora guilliermondii) and Saccharomyces cerevisiae yeast on the distinctive characteristics of the Campanino white wine was investigated. For this purpose, three independent winemaking experiments were carried out on an industrial scale (batches A, B and C). In detail, the first one was carried out using the sequential inoculation technique while the other two, using a S. cerevisiae single-strain starter or no inoculation representing the control batches. Microbiological and chemical parameters and sensorial profiles of the wines were defined. Interestingly, the results showed that when sequential cultures (H. guilliermondii in a sequential mixture with S. cerevisiae) were used, a better wine aroma and quality was observed. More specifically, the wine obtained by sequential inoculation showed lower acetic acid values and enhanced volatile profiles than the wine from the control batches. Finally, sensorial analysis confirmed that the sequential cultures led to an improvement in wine flavour. Therefore, results suggest that the sequential inoculation using non-Saccharomyces and Saccharomyces yeast represents a biotechnological practice that can improve the quality features of traditional white wine. It has been shown for the first time that on an industrial scale H. guilliermondii could be used in sequential inoculum with S. cerevisiae in making white Campanino wine., Research in our labs is funded by Grants AGL2015-64522-C2-R (Spanish Ministry of Economy and Competitiveness), ALIBIRD-CM 2013 S2013/ABI-2728 (Comunidad de Madrid) and within the framework of the project by Molise Region (Rural Development Programme 2007–2013; Measure 1.2.4. Cooperation for development of new products, processes and technologies in the agriculture and food sector and in forestry).

Table grapes are of increasing interest for wine production in China. In this study, 480 yeast isolates were isolated from the tumultuous stage during the spontaneous fermentation of six table grape varieties, which were cultivated in an ecological environment that was not industry-influenced, in Central China. The 26S rDNA D1/D2 domain sequence analysis was more efficient for yeast species identification than the 5.8S-ITS region RFLP analysis in the present study. All the tested strains belonged to nine species from six genera: Hanseniaspora guilliermondii, H. opuntiae, H. uvarum, Pichia terricola, Kazachstania hellenica, K. zonata, P. occidentalis, Saccharomyces cerevisiae and Zygosaccharomyces bailii. The yeast species and populations differed notably among the grape varieties. S. cerevisiae was found in the samples of four grape varieties (Vitis amurensis, Iona, Moldova and V. davidii), but not detected in Cuihong and Alimandeng Rose. Interdelta sequence fingerprinting analysis was used to discriminate between 128 S. cerevisiae isolates. Eight S. cerevisiae genotypes (G1 to G8) were distinguished. Genotypes G1, G2, and G3 were the most dominant strains, accounting for 32.03%, 24.22% and 28.13% of the isolates respectively. This study shows the diversity of yeast species associated with spontaneous fermentations of different table grape varieties grown in an ecological environment without any wine industry effect or footprint.

EnglishThis study aimed to know the biodiversity of non-Saccharomyces yeasts in Brazilian apples must with potential to improve of the aromatic quality of ciders. The strains were isolated from thirty-five (35) Gala and Fuji apple musts from different locations from south region of Brazil. Forty-five (45) strains were isolate and identified by PCR analysis. Results indicated ten (10) species: Candida oleophila, Candida parapsilosis, Candida tropicalis, Clavispora lusitaniae, Hanseniaspora guilliermondii, Hanseniaspora uvarum, Lodderomyces elongisporus, Pichia anomala, Pichia fermentans and Rhodotorula mucilaginosa. The genus Rhodotorula sp., Lodderomyces sp. and Clavispora sp. constituted 71.2% of the strains identified. The following strains, C. oleophila, R. mucilaginosa, P. fermentans, H. uvarum and H. guilliermondii were selected in qualitative tests due the fruity aroma production by trained team in the aromatic assessment of cider. Key words: apiculate yeast; biodiversity; fruity aroma; PCR analysis portuguesEste estudo teve como objetivo conhecer a biodiversidade de leveduras nao-Saccharomyces em macas com potencial para a melhoria da qualidade aromatica da sidra brasileira. As cepas foram isoladas de trinta e cinco (35) mostos de maca Gala e Fuji de diferentes locais da regiao Sul do Brasil. Quarenta e cinco (45) cepas foram isoladas e identificadas por analise de PCR. Os resultados indicaram dez (10) especies: Candida oleophila, Candida parapsilosis, Candida tropicalis, Clavispora lusitaniae, Hanseniaspora guilliermondii, Hanseniaspora uvarum, Lodderomyces elongisporus, Pichia anomala, Pichia fermentans e Rhodotorula mucilaginosa. Tres desses generos (Rhodotorula sp., Lodderomyces sp. e Clavispora sp.) juntos constituiram 71,2% das cepas identificadas. Entre estas cepas, C. oleophila, R. mucilaginosa, P. fermentans, H. uvarum e H. guilliermondii foram selecionadas em teste qualitativo devido a producao de aroma frutado, indicando potencial para a producao de compostos aromaticos na sidra. Palavras-chave: levedura apiculada; biodiversidade; aroma frutado; analise por PCR

Saccharomyces cerevisiae produces antimicrobial peptides (AMPs) during alcoholic fermentation that are active against several wine-related yeasts (e.g. Hanseniaspora guilliermondii) and bacteria (e.g. Oenococcus oeni). In the present study, the physiological changes induced by those AMPs on sensitive H. guilliermondii cells were evaluated in terms of intracellular pH (pHi), membrane permeability and culturability. Membrane permeability was evaluated by staining cells with propidium iodide (PI), pHi was determined by a fluorescence ratio imaging microscopy (FRIM) technique and culturability by a classical plating method. Results showed that the average pHi of H. guilliermondii cells dropped from 6.5 (healthy cells) to 5.4 (damaged cells) after 20 min of exposure to inhibitory concentrations of AMPs, and after 24 h 77.0% of the cells completely lost their pH gradient (∆pH=pHi-pHext). After 24h of exposure to AMPs, PI-stained (dead) cells increased from 0% to 77.7% and the number of viable cells fell from 1×10(5) to 10 CFU/ml. This means that virtually all cells (99.99%) became unculturable but that a sub-population of 22.3% of the cells remained viable (as determined by PI staining). Besides, pHi results showed that after 24h, 23% of the AMP-treated cells were sub-lethally injured (with 0

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.

This study aimed to enhance the bio-ethanol production from date molasses as a cheap and renewable resource by local species of non-Saccharomyces yeasts by optimization the production’s conditions. Hanseniaspora guilliermondii KKUY-0036 and H. uvarum KKUY-0078 were used based on their ability to ferment the date molasses efficiently. They were identified by the sequencing of D1/D2 domain of the 26S rRNA gene. Their identity was confirmed by comparing the obtained sequence with similar sequences allocated in the GenBank. To enhance the ethanol productivity, temperature, pH, fermentation period, molasses concentration and addition of some elements were optimized. Results revealed that the two yeast species exhibited their maximum productivity of ethanol at 30°C when they were grown on 20-25% of the date molasses after 96-120 h of incubation. The highest ethanol concentration was achieved in weak acidic medium (pH 4-6). Addition of zinc, magnesium and manganese induced the production of ethanol by the two yeasts and the optimum concentrations were 0.6, 0.2-0.3 and 0.03 g LG1, respectively. The study introduces both H. guilliermondii KKUY-0036 and H. uvarum KKUY-0078 as new ethanol-producers that ferment date molasses efficiently and will greatly reduce the biofuel production cost.

Aims The aim of this study was to evaluate non-Saccharomyces yeasts isolated from spontaneous fermenting musts of Touriga Nacional (TN), one of the most important Portuguese red grape variety, to improve and diversify TN wines. Methods and results Two hundred and seventy nine isolates were assigned to 11 yeast species by conventional molecular and growth tests. Starmerella bacillaris was the most frequently detected yeast species, followed by Hanseniaspora guilliermondii and Hanseniaspora uvarum. Twenty-three isolates from 10 species were selected for oenological study, namely fermentation performance, physicochemical and quantitative descriptive sensory analysis of the wines produced. A significant species effect was observed for most of the variables evaluated, some species generating wines with quite interesting aromas. Conclusions Candida diversa and S. bacillaris isolates produced wines with higher overall quality, higher balance and more intense and diverse aroma. Furthermore, S. bacillaris isolates generated wines with enhanced TN typical aroma, such as bergamot, violet and rock-rose, and were thus regarded as the most promising for improving TN wines. Significance and impact of study This study revealed the diversity of wine aroma profiles generated by non-Saccharomyces yeast isolates. This knowledge is particularly important given the growing trend from industry to use non-Saccharomyces yeasts as a tool for improving and diversifying the sensory characteristics of wine.

The increase of infections due to non-Candida albicans species made it very necessary to conduct adequate characterization to be able to identify the species of Candida isolated from traditional fermented foods. In this study, based on their hue on Candida Chromogenic Agar medium, a total of 136 yeast strains were isolated from tchapalo and bangui. Molecular identification based on PCR-RFLP of internal transcribed spacers of rDNA (ITS) and sequencing of the ITS and the D1/D2 regions allowed us to assign these isolates to seven species: Candida tropicalis, Candida inconspicua, Candida rugosa, Saccharomyces cerevisiae, Kluyveromyces marxianus, Hanseniaspora guilliermondii, Trichosporon asahii. With the respect to each beverage, six species were found among with four species are regarded as opportunistic pathogens. From these, C. tropicalis, C. inconspicua and K. marxianus were the most commonly encountered. The enzyme activities of the potential pathogens assessed using API ZYM system showed that almost strains had esterase, esterase lipase, valine and cystine arylamidase, alpha chymotrypsin, alkaline phosphatase and naphthol phosphohydrolase activities. The activity of α-glucosidase was found only in C. tropicalis and C. inconspicua strains isolated from tchapalo while β-glucosidase activity was found in all strains from tchapalo and only in C. inconspicua isolated from bangui.

International audience; Although differences in yeast biodiversity have often been found between vineyards subjected to organic protection or conventional protection, little is known about the effect of copper and sulfur fungicides (the only fungicides allowed in organic farming) on yeast populations. The sensitivity to copper and sulfur of 158 yeast isolates of seven different species (Aureobasidium pullulans, Hanseniaspora guilliermondii, Hanseniaspora uvarum, Metschnikowia sp., Pichia membranifaciens, Saccharomyces cerevisiae and Starmerella bacillaris) was evaluated. The species Aureobasidium pullulans and Starmerella bacillaris appeared to be more resistant to copper than the other species tested. The species Aureobasidium pullulans, Hanseniaspora guilliermondii and Metschnikowia sp. had the highest sulfur resistance. Thus, only isolates of the species Aureobasidium pullulans exhibited high resistance to both antifungal agents. These results may explain the lower diversity of yeasts present on berries and the strong dominance of the species Aureobasidium pullulans for vineyards protected by copper or sulfur-based fungicides compared to other vineyards reported by several studies.

In this work, we disclose the genome sequence and a corresponding manually curated annotation of the non- Saccharomyces yeast Hanseniaspora guilliermondii UTAD222, a strain shown to have interesting oenological traits for the production of wines with improved aromatic properties.

Article History The aim of this study was to determine the intermediate chemical compounds during the fermentation of spoilage date juice (SDJ) by newly isolated yeast strain Hanseniaspora guilliermondii KKUY-0045. This yeast strain was identified based on sequence of the variable D1/D2 domain of the large subunit (26S) ribosomal DNA and comparing it with similar sequences in the Genbank database. Phylogenetic analysis using rDNA sequences indicated that strain KKUY-00045 shared one cluster with Hanseniaspora guilliermondii. In 7-L fermenetor, this strain produced 87.74 g/L of ethanol after 3 days of incubation. The chemical analysis of the cell free extract of the yeast strain grown on the SDJ by using GC-MS revealed the presence of some important chemical compound such as pregnane and nonacosane that could be used in the medical and pharmaceutical industries. The study recommends extensive studies to discover the chemicals associated with fermentation process and their industrial role.