Hanseniaspora Uvarum Research Articles

Isolation and Characterization of Cryotolerant Yeasts from Fiano di Avellino Grapes Fermented at Low Temperatures.

A fermentation of Fiano di Avellino grape must was carried out at 9°C with the aim of selecting cryotolerant yeast strains and testing their fermentative performances and volatile production following molecular characterization. A total of 20 yeast cultures were isolated at different fermentation stages. Based on molecular identification and characterization, Metschnikowia (M.) pulcherrima, Hanseniaspora (H.) uvarum, Staremerella (St.) bacillaris, Saccharomyces (S.) cerevisiae, S. kudriavzevii, and S. paradoxus were found to be the yeast species dominating the fermentation. S. paradoxus has been rarely isolated in vineyards and never in the cellar environment. Moreover, in this study, S. kudriavzevii is detected for the first time in vine-wine environments. Both S. kudriavzevii and S. paradoxus co-occurred with S. cerevisiae when grapes were micro-fermented at low temperatures. The growth kinetics of the three species were greatly affected by the fermentation temperature. As a consequence, Fiano wines obtained with S. kudriavzevii and S. paradoxus significantly differed from those made by S. cerevisiae in terms of chemical and volatile composition.

Sequential culture with aroma-producing yeast strains to improve the quality of Kyoho wine.

Despite many non-Saccharomyces yeasts being considered spoilage microorganisms, they can increase aroma and flavor diversity in alcoholic beverages. The purpose of this study was to investigate nontraditional inoculation strategies using aroma-producing yeast strains for Kyoho wine fermentation, followed by an instrumental analysis and sensory evaluation. The winemaking process was carried out using Saccharomyces cerevisiae Gr112, Hanseniaspora uvarum Pi235, and Pichia kluyveri Pe114. Multiple inoculation strategies were explored. In instrumental analysis results, mixed culture could promote the formation of esters (5.9-folds) and glycerol (1.3-folds) and reduce the content of ethanol (-0.5% [v/v]) in wine. The sensory analysis results suggested that the three yeast strains sequential inoculation treatment was associated with the aroma attributes "floral," "red fruity," and "tropical fruity." Co-cultivation contributed to an increase in complexity and aromatic intensity, with the three-strain inoculation treatment presenting a more distinctive appearance. PRACTICAL APPLICATION: The inoculation of S. cerevisiae improved the accumulation of volatile acids and esters by inhibiting the growth of non-Saccharomyces yeast strains. Inoculation of H. uvarum and P. kluyveri would effectively solve the defect of excessive content of higher alcohols in wines produced by S. cerevisiae. The suitable inoculation strategy between non-Saccharomyces yeasts could improve the overall quality of Kyoho wine whose starter might be widely used in fermentation industry.

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.

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.

Comparing the hierarchy of inter- and intra-species interactions with population dynamics of wine yeast cocultures.

In winemaking, the development of new fermentation strategies, such as the use of mixed starter cultures with Saccharomyces cerevisiae (Sc) yeast and non-Saccharomyces (NS) species, requires a better understanding of how yeasts interact, especially at the beginning of fermentation. Despite the growing knowledge on interactions between Sc and NS, few data are available on the interactions between different species of NS. It is furthermore still unclear whether interactions are primarily driven by generic differences between yeast species or whether individual strains are the evolutionarily relevant unit for biotic interactions. This study aimed at acquiring knowledge of the relevance of species and strain in the population dynamics of cocultures between five yeast species: Hanseniaspora uvarum, Lachancea thermotolerans, Starmerella bacillaris, Torulaspora delbrueckii and Sc. We performed cocultures between 15 strains in synthetic grape must and monitored growth in microplates. Both positive and negative interactions were identified. Based on an interaction index, our results showed that the population dynamics seemed mainly driven by the two species involved. Strain level was more relevant in modulating the strength of the interactions. This study provides fundamental insights into the microbial dynamics in early fermentation and contribute to the understanding of more complex consortia encompassing multiple yeasts trains.

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.

Proteolytic activity under white wine fermentation by Hanseniaspora vineae yeast strains

Apiculate yeasts from Hanseniaspora genus are predominant on the native flora of grapevines, while Hanseniaspora uvarum is well known for its abundant presence in grapes, it results generally, in detrimental quality effects on wine due to high production of acetic acid. By contrast, Hanseniaspora vineae is better adapted to fermentation, increasing flavor complexity in wines as it has been demonstrated in winemaking practices during the last decade. We obtained a collection of more than 22 different Hanseniaspora vineae strains from which we could detect, by a plating screening method, high and moderate protease activity. In this study, we tested these strains under real winemaking conditions on a Sauvignon blanc white wine must. Results obtained demonstrated that there is an interesting protease active diversity in all H. vineae strains when compared to conventional fermentations conducted by Saccharomyces cerevisiae. Four H. vineae strains showed a decrease in wine protein turbidity, up to three times lower than that observed in S. cerevisiae, which imply a significant decrease in the demand for bentonite before bottling. This attribute of some H. vineaestrains should be very attractive at the commercial level to reduce manipulations and flavor removal in the production of delicate white wines such as Sauvignon blanc.

Evolution of yeast biodiversity and volatile compounds during spontaneous fermentation of ‘Karasakiz’ grapes from different regions over two consecutive vintages

Spontaneous fermentation may produce complex and unique wines with geographical designations due to the region-specific yeast flora and variations in must characteristics. The current study focused on the yeast diversity and changes in volatile compounds during the spontaneous fermentation of ‘Karasakız’ grapes from three sub-regions of Çanakkale province, Turkey for two vintages (2019 and 2020). This is the first study on the diversity of autochthonous yeasts during wine fermentation of ‘Karasakız’ variety. In the present work, the strains belong to Hanseniaspora uvarum, Starmerella bacillaris, Pichia terricola, Metschnikowia pulcherrima and Zygoascus hellenicus by PCR-RFLP of the ITS region were identified . A total of 272 isolates were identified as S. cerevisiae. Yeast population and diversity changed related to the sub-region and the vintages. However, H. uvarum and S. bacillaris were common denominators of three regions evaluated. H. uvarum was dominant in the early stages of the fermentation, except for Bozcaada 2019 vintage. Bozcaada region also exhibited higher S. cerevisiae strain diversity compared to other regions. 1-Hexanol and 1-octanol gradually decreased during the fermentation of ‘Karasakız’ grapes, while the concentration of isoamyl alcohol, phenylethyl alcohol and ester compounds increased by the fermentation but with some fluctuation.

Synthesis of Aroma Compounds as a Function of Different Nitrogen Sources in Fermentations Using Non-Saccharomyces Wine Yeasts.

Non-Saccharomyces yeasts are prevalent at the onset of grape must fermentations and can have a significant influence on the final wine product. In contrast to Saccharomyces cerevisiae, the biosynthetic pathways leading to aroma compound formation in these non-conventional yeasts, in particular those that are derived from amino acid metabolism, remains largely unexplored. Within a synthetic must environment, we investigated the amino acid utilization of four species (Hanseniaspora uvarum, Hanseniaspora osmophila, Zygosaccharomyces rouxii, Starmerella bacillaris) and S. cerevisiae. We report on the differential uptake preferences for amino acids with H. uvarum displaying the most rapid uptake of most amino acids. To investigate the fate of amino acids and their direct contribution to aroma synthesis in H. uvarum, H. osmophila and Z. rouxii, musts were supplemented with single amino acids. Aroma profiling undertaken after three days showed the synthesis of specific aroma compounds by the respective yeast was dependent on the specific amino acid supplementation. H. osmophila showed similarities to S. cerevisiae in both amino acid uptake and the synthesis of aroma compounds depending on the nitrogen sources. This study shows how the uptake of specific amino acids contributes to the synthesis of aroma compounds in wine fermentations using different non-Saccharomyces yeasts.

Application of yeast in plant-derived aroma formation from cigar filler leaves.

Introduction: There are various degrees of defects of cigar filler leaves after air drying. Methods: In order to improve the quality and plant-derived aroma content of cigar filler leaves, nine aroma-producing yeasts were applied in artificially solid-state fermentation of cigar filler leaves in this study. The differences with various yeasts application were compared by chemical composition and GC-MS analysis. Results and discussion: The results showed that 120 volatile components were identified and quantified in cigar filler leaves after fermentation, including aldehydes (25 types), alcohols (24 types), ketones (20 types), esters (11 types), hydrocarbons (12 types), acids (4 types) and other substances (23 types). Based on the analysis of odor activity value (OAV), the OVA of fruity and floral aroma components were higher. It was found that floral aroma are the representative aroma types of cigar filler leaves treated with Clavispora lusitaniae, Cyberlindera fabianii, Saccharomycosis fibuligera and Zygosaccharomyces bailii R6. After being inoculated with Hanseniaspora uvarum J1, Hanseniaspora uvarum J4 and Pichia pastoris P3, the OAV of fruity aroma in cigar filler leaves was the highest, followed by tobacco aroma and woody aroma. The correlation between volatile components of cigar filler leaves with different yeasts was revealed after PCA analysis. It was concluded that the quality of cigar filler leaves was improved, and cigar filler leaves fermented with different yeasts showed different flavor.

Ecological linkages between biotechnologically relevant autochthonous microorganisms and phenolic compounds in sugar apple fruit (Annona squamosa L.)

Our study investigated the potential of Annona squamosa (L.) fruit as a reservoir of yeasts and lactic acid bacteria having biotechnological implications, and phenolics capable of modifying the ecology of microbial consortia. Only a single species of lactic acid bacteria (Enterococcus faecalis) was identified, while Annona fruit seemed to be a preferred niche for yeasts (Saccharomyces cerevisiae, Hanseniaspora uvarum), which were differentially distributed in the fruit. In order to identify ecological implications for inherent phenolics, the antimicrobial potential of water- and methanol/water-soluble extracts from peel and pulp was studied. Pulp extracts did not show any antimicrobial activity against the microbial indicators, while some Gram-positive bacteria (Staphylococcus aureus, Staphylococcus saprophyticus, Listeria monocytogenes, Bacillus megaterium) were susceptible to peel extracts. Among lactic acid bacteria used as indicators, only Lactococcus lactis and Weissella cibaria were inhibited. The chemical profiling of methanol/water-soluble phenolics from Annona peel reported a full panel of 41 phenolics, mainly procyanidins and catechin derivatives. The antimicrobial activity was associated to specific compounds (procyanidin dimer type B [isomer 1], rutin [isomer 2], catechin diglucopyranoside), in addition to unidentified catechin derivatives. E. faecalis, which was detected in the epiphytic microbiota, was well adapted to the phenolics from the peel. Peel phenolics had a growth-promoting effect toward the autochthonous yeasts S. cerevisiae and H. uvarum.

Heterologous expression and functional characterization of Drosophila suzukii OR69a transcript variants unveiled response to kairomones and to a candidate pheromone

Drosophila suzukii is a main pest of berries on all continents. Population control relies on insecticides and a current research challenge is to develop alternative strategies. Methods based on behavior-modifying semiochemicals are widely used against other horticultural insects, and the functional characterization of chemoreceptors facilitates the identification of active compounds. Following heterologous expression of single olfactory receptors (ORs) in ab3A olfactory sensory neurons of D. melanogaster, we screened three transcript variants from the OR69a-locus (DsuzOR69aA, DsuzOR69aB, DsuzOR69aC), demonstrating binding to two possible kairomones (3-octanol and R-carvone) and to a possible fly-emitted volatile compound [(Z)-4-nonenal], although with different pharmacological qualities. By coupling Gas Chromatography to SSR (GC-SSR), these ligands enhanced ab3A-spiking at nanogram-aliquots in a complementary fashion among the different OR69a-variants, and we identified another possible kairomone, methyl salicylate, as the most active and specific ligand for the sole DsuzOR69aB. In testing headspaces collected from D. suzukii females and from the fly-associated yeast Hanseniaspora uvarum we did not observe activation from female headspaces but activation from yeast headspaces. In situ hybridization analysis on D. suzukii antennae suggests unique expression of OR69a-subunits in specific neurons, and points toward co-expression within the same neurons. The OR69a-subunits of D. suzukii constitute cation channels, which binding suggests kairomone specificity, even if effects coexist for a complementary binding of (Z)-4-nonenal. Methyl salicylate is the most active ligand and is specific to the sole DsuzOR69aB, inspiring future investigation to validate potentials of this compound for D. suzukii control strategies.

Effect of Hanseniaspora uvarum-Saccharomyces cerevisiae Mixed Fermentation on Aroma Characteristics of Rosa roxburghii Tratt, Blueberry, and Plum Wines.

Hanseniaspora uvarum, a non-Saccharomyces cerevisiae species, has a crucial effect on the aroma characteristics of fruit wines, thus, attracting significant research interest in recent years. In this study, H. uvarum-Saccharomyces cerevisiae mixed fermentation was used to ferment Rosa roxburghii Tratt, blueberry fruit wine, and plum fruit wines using either a co-inoculated or a sequentially inoculated approach. The three fruit wines' volatile aroma characteristics were analyzed by headspace-solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The results showed that the mixed inoculation of H. uvarum and S. cerevisiae reduced the alcoholic content of Kongxinli fruit wine. Moreover, H. uvarum-S. cerevisiae fermented Rosa roxburghii Tratt, blueberry, and plum fruit wines and further enriched their flavor compounds. The overall flavor characteristics of sequentially inoculated fruit wines differed significantly from those fermented with S. cerevisiae alone, although several similarities were also observed. Sequential inoculation of H. uvarum and S. cerevisiae positively affected the mellowness of the wine and achieved a better harmony of the overall wine flavors. Therefore, H. uvarum-Saccharomyces cerevisiae mixed fermentation can improve the complexity of the wines' aromatic composition and empower them with a unique identity. In particular, H. uvarum-Saccharomyces cerevisiae blueberry wine produced by mixed fermentation had the widest variety and content of aroma compounds among the fermented wines. Therefore, H. uvarum-Saccharomyces cerevisiae mixed-fermentation inoculation in the three fermented fruit wines significantly increased the aroma compound variety and content, thus, enriching their aroma richness and complexity. This study is the first comparative evaluation of the aroma characteristics of different fruit wines fermented with a mixed inoculation of H. uvarum and S. cerevisiae and provides a preliminary guide for these fruit wines produced with non-Saccharomyces yeast.

Pulsed light reduces postharvest losses of Chinese bayberries by affecting fungal microbiota during cold storage

Non-thermal sterilization technology is considered a safe and effective approach in controlling postharvest losses of fruit. In this study, the effects of pulsed light (PL) on qualities of Chinese bayberries during storage at 10 °C, 95% RH for 12 d were investigated. At the same time, high-throughput sequencing was operated to identify fungal microbiota on Chinese bayberries. The results showed that PL treatment could inhibit the decay incidence and polyphenol oxidase (PPO) activity, maintain fruit firmness and enhance phenylalanine ammonia-lyase (PAL) activity, with no effect on titratable acid (TA), total soluble solids (TSS), malondialdehyde (MDA) content and superoxide dismutase (SOD) activity. Additionally, the PL treatment maintained alpha diversity and altered beta diversity. At the species level, the abundance of Monographella nivalis and Colletotrichum cordylinicota were reduced in PL samples, while Hanseniaspora uvarum and Diaporthe spp were enriched. M. nivalis and C. cordylinicota were positively correlated with weight loss and decay index according to canonical correspondence analysis (CCA). The data of microbial correlation network indicated that PL treatment reduced pathogenic fungal microbiotas and made fungal community more complex and steadier. This study demonstrated that PL affects fungal community by inhibiting plant pathogens, increasing the amounts of endophytes and consolidating microbial networks. It provides new insights into the dynamics of the microbiome of Chinese bayberries after PL treatment.

Exploring the Efficacy of Biocontrol Microbes against the Fungal Pathogen Botryosphaeria dothidea JNHT01 Isolated from Fresh Walnut Fruit.

Biological control by antagonistic microorganisms are an effective and environmentally friendly approach in postharvest disease management. In order to develop a biocontrol agent for fresh walnut fruit preservation, the potential biocontrol effects of Bacillus amyloliquefaciens RD.006 and Hanseniaspora uvarum FA.006 against the main fungal pathogen of walnuts were evaluated. Botryosphaeria species showed the highest detection, and the JNHT01 strain showed the strongest pathogenicity. Bot. dothidea JNHT01 caused gray mold and brown rot on fresh walnuts, and its incidence rate reached 100% after an 8 days incubation. The growth of this fungal strain can be promoted by lighting, with a maximum growth rate achieved at a pH of 7 and at 28 °C. B. amyloliquefaciens RD.006 and H. uvarum FA.006 supernatants at a concentration of 1-15% v/v showed antifungal activity. The mycelial growth inhibition rates of Bot. dothidea JNHT01 were 23.67-82.61% for B. amyloliquefaciens RD.006 and 1.45-21.74% for H. uvarum FA.006. During Bot. dothidea JNHT01 growth, the biomass, nucleic acid leakage, and malondialdehyde content gradually increased, while the DPPH scavenging capacity and SOD activity decreased. The B. amyloliquefaciens RD.006 and H. uvarum FA.006 strains showed antifungal activity by damaging fungal cell membranes and reducing fungal antioxidant activity. Moreover, the antifungal effect of B. amyloliquefaciens RD.006 was higher than that of H. uvarum FA.006. Hence, the RD.006 strain of B. amyloliquefaciens can be considered a potential biocontrol agent for the management of postharvest walnut diseases caused by Bot. dothidea.

Influence of vintage, geographical location and agricultural management on yeast populations in Galician grape musts (NW Spain)

The influence of vintage, geographical location and farming system on yeast populations was evaluated in grape musts from Galicia (NW Spain) in three consecutive years. Grape samples were taken in organic and conventional vineyards from four Denominations of Origin (DOs) during the 2013, 2014 and 2015 vintages. Cultivable yeast populations were characterised at the quantitative and qualitative level. The general results show the existence of significant differences for yeast counts in musts among vintages and DOs, but not between farming systems. Given the geographical location of the grapes, the influence of vintage was significant in all DOs; however, the farming system only had a significant impact on viable yeasts in Rías Baixas, although the number of yeasts tended to be higher in organic samples than in conventional ones in most cases. Species richness was location dependent, with Rías Baixas and Ribeira Sacra showing the highest values. In addition, the type of yeasts varied between Denominations of Origin. From a total of 39 different yeast species identified, Metschnikowia spp., Hanseniaspora uvarum and Aureobasidium spp. were the prevailing species. These major yeasts were found to be widely distributed. However, species such as Issatchenkia terricola, Starmerella bacillaris and different species of Candida, Pichia and Zygosaccharomyces genera were mainly found in Rías Baixas and Ribeira Sacra DOs. In contrast, Lachancea thermotolerans was isolated in Monterrei and Ribeiro DOs only. Accordingly, the ANOSIM and PERMANOVA analyses evidenced significant differences in species richness among different DOs and, to a lesser extent, among vintages, but not between farming systems.

The Efficacy of Yeast Phagostimulant Baits in Attract-and-Kill Strategies Varies between Summer- and Winter-Morphs of Drosophila suzukii.

Drosophila suzukii (Matsumura), is a globally invasive pest of soft and stone fruit. To survive winter in temperate zones it enters a reproductive diapause in a morphologically distinct phenotype. Phagostimulant baits can be combined with insecticides in attract-and-kill strategies for control. We investigated the effectiveness of single yeast species and combinations of co-fermented yeast phagostimulant baits when combined with insecticides in laboratory assays against both summer- and winter-morph D. suzukii. Candida zemplininia or Hanseniaspora uvarum + C. zemplininia combined with lambda-cyhalothrin or cyantraniliprole, and H. uvarum combined with cyantraniliprole caused significantly higher mortality in winter- compared to summer-morph D. suzukii. Additionally, lambda-cyhalothrin combined with M. pulcherrima + H. uvarum resulted in greater mortality compared to single yeasts, H. uvarum for both summer- and winter-morphs and C. zemplininia for summer-morphs. M. pulcherrima + H. uvarum with spinosad significantly reduced the time-to-kill (50%) of summer-morphs compared to insecticide alone. Most yeast-based baits were comparable in terms of attract-and-kill efficacy to Combi-protec, a commercially available bait, although M. pulcherrima or H. uvarum + C. zemplininia in with cyantraniliprole were less effective. Our study suggests that yeast phagostimulants in attract-and-kill strategies should be adjusted for summer- and winter-morph D. suzukii for more effective control.

Yeast communities before and after spontaneous fermentation of wine grapes: a case study from Nova Scotia.

Wine fermentations are generally completed by the domestic yeast Saccharomyces cerevisiae, but many indigenous vineyard yeasts also influence wine flavour and aroma. Despite the flourishing wine industry in Nova Scotia, there has yet to be any systematic evaluation of these yeasts in Atlantic Canada. The yeast communities of pressed L'Acadie blanc grapes sampled from an organic vineyard in the Annapolis Valley in 2018 and 2019 were characterized before and after spontaneous fermentation by both Illumina and PacBio sequencing, to address and compare potential platform biases. Chemical and sensory evaluations were also conducted. Basidiomycete yeasts, including Vishniacozyma carnescens, Filobasidium globisporum, and Curvibasidium cygneicollum, dominated pre-fermentation diversity. Species of Saccharomyces made up ∼0.04% of sequences prior to fermentation, but 85%-100% after fermentation, with some replicates dominated by S. cerevisiae and some by S. uvarum. PacBio sequencing detected high proportions of Hanseniaspora uvarum, while Illumina sequencing did not. A better understanding of Nova Scotia vineyard yeast communities will allow local wine makers to make better use of non-traditional yeasts and spontaneous fermentations to produce high-quality wines unique to the region.

Brewing and probiotic potential activity of wild yeasts Hanseniaspora uvarum PIT001, Pichia kluyveri LAR001 and Candida intermedia ORQ001

Non-conventional yeasts can be isolated from a wide range of environmental sources and are often found in the beverage industry in mixed fermentations, in which the microbial community is usually not fully known. However, it is important to know the compositions of these starter cultures because in addition to enabling reproducibility during fermentation, other properties can be discovered. Thus, the objective of this work was to identify and characterize non-conventional yeasts isolated from the environment, evaluating their probiotic potential and possible use in beer brewing. Isolates were obtained from flowers, fruits, leaves and mixed-fermentation beers, with the species being identified by PCR. Yeasts with promising activity were evaluated regarding their growth under different pHs, temperature and the presence of organic acids. To explore probiotic potential, in vitro tests were performed for antimicrobial activity and co-aggregation with food-spoiling microorganisms, auto-aggregation and survival in simulated gastrointestinal tract conditions. In this study, Pichia kluyveri (LAR001), Hanseniaspora uvarum (PIT001) and Candida intermedia (ORQ001) were selected among 20 isolates for further study. P. kluyveri was the only strain that tolerated pH 2.5. Lactic acid was not inhibitory, but acetic acid and incubation at 37 °C had partially inhibitory effects on yeast growth. All yeasts tolerated α-acids from hops and up to 1% NaCl. Our results also suggest that these isolates are able to adhere to intestinal cells and positively influence the host to combat pathogens, as they showed auto-aggregation rates > 99% and antagonistic activity to pathogenic bacteria. The yeasts tolerated gastric environment conditions, but were more sensitive to pancreatic conditions. We conclude that these non-conventional yeasts have probiotic potential and promising application in beer fermentation.

Co-Fermentations of Kveik with Non-Conventional Yeasts for Targeted Aroma Modulation.

Kveik are consortia of yeast used for farmhouse ale production in Western Norway. Yeast strains derived from these mixtures are known, for example, for their high fermentation rate, thermotolerance, lack of phenolic off flavor production (POF-) and strong flocculation phenotype. In this study, we used five single cell yeast isolates from different Kveik yeasts, analyzed their fermentation and flavor production, and compared it with a typical yeast used in distilleries using 20 °C and 28 °C as the fermentation temperatures. One of the isolates, Kveik No 3, showed an impairment of maltotriose utilization and thus a reduced ethanol yield. Kveik fermentations for spirit production often harbor bacteria for flavor enrichment. We sought to improve Kveik fermentations with non-conventional yeasts (NCY). To this end we co-fermented Kveik isolates with Hanseniaspora uvarum, Meyerozyma guilliermondii and Pichia kudriavzevii using 5:1 ratios (Kveik vs. NCY) at 20 °C. The combinations of Kveik No 1 with P. kudriavzevii and Kveik No 1 with Hanseniaspora uvarum showed substantially increased amounts of specific volatile aroma compounds that were previously identified in the NCYs. Our results indicate that Kveik isolates appear to be suitable for co-fermentations with certain NCY to enhance beer or spirit fermentations, increasing the potential of these yeasts for beverage productions.