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.

Contributions of Hanseniaspora species to Pinot Noir microbial terroir in Oregon's Willamette Valley wine region.

The apiculate yeast genus Hanseniaspora has appeared frequently in enological research for more than 100 years, mostly focused upon the species H. uvarum due to its notable capacity to cause spoilage. Recently, there has been increased research into the potential benefits of other Hanseniaspora species, such as H. vineae, in producing more complex wines. Furthermore, large-scale DNA sequencing-based (metabarcoding) vineyard ecology studies have suggested that Hanseniaspora species may not be evenly distributed. To address potential differences across geographical areas in Oregon, we sampled extensively from 12 vineyards within the Willamette Valley American Viticultural Area (AVA), across 2 sub-AVAs (Eola-Amity Hills and Yamhill-Carlton). Metabarcoding was then used to assess the contribution of Hanseniaspora to the grape berry fungal community and the impact of wine processing on diversity. While 6 of the 23 recognized Hanseniaspora species were present on Pinot Noir grapes in the Willamette Valley AVA, differences between vineyards were driven by the abundance of H. uvarum. Significant positive correlations between the amount of H. uvarum present in must and at cold soak, and then cold soak to early ferment were observed. While intuitive, it is worth noting that no prior studies have observed this across such a large number of grape samples from different vineyards. Our results provide clear evidence that the abundance of H. uvarum on grapes may be an important predictor of potential impacts on wine quality, particularly if performing cold soak, which acts as an enrichment step. IMPORTANCE: Hanseniaspora yeasts are frequently found in uninoculated wine fermentations, and depending upon the species present, their contributions to the wine may be positive or negative. We found that in Oregon's Willamette Valley, the most common species of Hanseniaspora in Pinot Noir vineyards was the known spoilage organism, H. uvarum. This species was one of the strongest contributors to differences in fungal communities between different vineyards and was enriched during typical Pinot Noir processing. These results support Hanseniaspora as an integral and functional component of vineyard "microbial terroir" within Oregon.

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.

Effect of Hanseniaspora vineae and Saccharomyces cerevisiae co-fermentations on aroma compound production in beer.

In recent years, the boom of the craft beer industry refocused the biotech interest from ethanol production to diversification of beer aroma profiles. This study analyses the fermentative phenotype of a collection of non-conventional yeasts and examines their role in creating new flavours, particularly through co-fermentation with industrial Saccharomyces cerevisiae. High-throughput solid and liquid media fitness screening compared the ability of eight Saccharomyces and four non-Saccharomyces yeast strains to grow in wort. We determined the volatile profile of these yeast strains and found that Hanseniaspora vineae displayed a particularly high production of the desirable aroma compounds ethyl acetate and 2-phenethyl acetate. Given that H. vineae on its own can't ferment maltose and maltotriose, we carried out mixed wort co-fermentations with a S. cerevisiae brewing strain at different ratios. The two yeast strains were able to co-exist throughout the experiment, regardless of their initial inoculum, and the increase in the production of the esters observed in the H. vineae monoculture was maintained, alongside with a high ethanol production. Moreover, different inoculum ratios yielded different aroma profiles: the 50/50 S. cerevisiae/H. vineae ratio produced a more balanced profile, while the 10/90 ratio generated stronger floral aromas. Our findings show the potential of using different yeasts and different inoculum combinations to tailor the final aroma, thus offering new possibilities for a broader range of beer flavours and styles.

Comparison of volatile compounds and sensory profiles of low-alcohol pear beverages fermented with Saccharomyces cerevisiae and different non-Saccharomyces cerevisiae.

This study aimed to assess the impact of Saccharomyces cerevisiae and different non-Saccharomyces cerevisiae (Zygosaccharomyces bailii, Hanseniaspora opuntiae and Zygosaccharomyces rouxii) on the volatile compounds and sensory properties of low-alcohol pear beverages fermented from three varieties of pear juices (Korla, Laiyang and Binzhou). Results showed that all three pear juices were favorable matrices for yeasts growth. Non-Saccharomyces cerevisiae exhibited a higher capacity for acetate ester production compared to Saccharomyces cerevisiae, resulting in a significant enhancement in sensory complexity of the beverages. PCA and sensory analysis demonstrated that pear varieties exerted a stronger influence on the crucial volatile components and aroma characteristics of the fermented beverages compared to the yeast species. CA results showed different yeast strains exhibited suitability for the fermentation of specific pear juice varieties.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Genome sequencing, annotation and exploration of the SO2-tolerant non-conventional yeast Saccharomycodes ludwigii.

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 ß-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.

Variable Spontaneous Mutation and Loss of Heterozygosity among Heterozygous Genomes in Yeast.

Mutation and recombination are the primary sources of genetic variation. To better understand the evolution of genetic variation, it is crucial to comprehensively investigate the processes involving mutation accumulation and recombination. In this study, we performed mutation accumulation experiments on four heterozygous diploid yeast species in the Saccharomycodaceae family to determine spontaneous mutation rates, mutation spectra, and losses of heterozygosity (LOH). We observed substantial variation in mutation rates and mutation spectra. We also observed high LOH rates (1.65-11.07×10-6 events per heterozygous site per cell division). Biases in spontaneous mutation and LOH together with selection ultimately shape the variable genome-wide nucleotide landscape in yeast species.

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

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

Impact of antagonistic yeasts from wine grapes on growth and mycotoxin production by Alternaria alternata.

AIMS: Alternaria alternata is a major contaminant of wine grapes, meaning a health risk for wine consumers due to the accumulation of toxic metabolites. To develop a successful biofungicide, the effectiveness of epiphytic wine grape yeasts against A. alternata growth and toxin production was assessed in vitro under temperature and aW conditions that simulate those present in the field. METHODS AND RESULTS: The effect of 14 antagonistic yeasts was evaluated on growth and alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) production by three A. alternata strains in a synthetic medium with composition similar to grape (SN) at three temperatures (15, 25 and 30°C). All Metschnikowia sp. yeast strains evaluated completely prevented A. alternata growth and mycotoxin production at all temperatures in SN medium. Meanwhile, the growth inhibition exerted by Starmerella bacillaris yeast strains was higher at 30°C, followed by 25 and 15°C, being able to show a stimulating or inhibiting effect. Hanseniaspora uvarum yeast strains showed a growth promoting activity higher at 15°C, followed by 25 and 30°C. Even at conditions where A. alternata growth was stimulated by the S. bacillaris and H. uvarum yeasts, high inhibitions of mycotoxin production (AOH, AME and TA) were observed, indicating a complex interaction between growth and mycotoxin production. CONCLUSION: There is a significant influence of temperature on the effectiveness of biocontrol against A. alternata growth and mycotoxin production. Metschnikowia sp. strains are good candidates to compose a biofungicide against A. alternata. SIGNIFICANCE AND IMPACT OF THE STUDY: Among the different antagonistic yeasts evaluated, only Metschnikowia sp. strains were equally effective reducing A. alternata growth and mycotoxin at different temperatures underlining the importance of considering environmental factors in the selection of the antagonists.

Integrating microbial metagenomics and physicochemical parameters and a new perspective on starter culture for fine cocoa fermentation.

Cocoa beans used for chocolate production are fermented seeds of Theobroma cacao obtained by a natural fermentation process. The flavors and chemical compounds produced during the fermentation process make this step one of the most important in fine chocolate production. Herein, an integrative analysis of the variation of microbial community structure, using a shotgun metagenomics approach and associated physicochemical features, was performed during fermentation of fine cocoa beans. Samples of Forastero variety (FOR) and a mixture of two hybrids (PS1319 and CCN51) (MIX) from Bahia, Brazil, were analyzed at 7 different times. In the beginning (0 h), the structures of microbial communities were very different between FOR and MIX, reflecting the original plant-associated microbiomes. The highest change in microbial community structures occurred at the first 24 h of fermentation, with a marked increase in temperature and acetic acid concentration, and pH decrease. At 24-48 h both microbial community structures were quite homogenous regarding temperature, acetic acid, succinic acid, pH, soluble proteins and total phenols. During 72-96 h, the community structure resembles an acidic and warmer environment, prevailing few acetic acid bacteria. Taxonomic richness and abundance at 72-144 h exhibited significant correlation with temperature, reducing sugars, succinic, and acetic acids. Finally, we recommend that dominant microbial species of spontaneous fine cocoa fermentations should be considered as inoculum in accordance with the farm/region and GMP to maintain a differential organoleptic feature for production of fine chocolate. In our study, a starter inoculum composed of Acetobacter pausterianus and Hanseniaspora opuntiae strains is indicated.

Non-conventional yeasts from fermented honey by-products: Focus on Hanseniaspora uvarum strains for craft beer production.

The increasing interest in novel beer productions focused on non-Saccharomyces yeasts in order to pursue their potential in generating groundbreaking sensory profiles. Traditional fermented beverages represent an important source of yeast strains which could express interesting features during brewing. A total of 404 yeasts were isolated from fermented honey by-products and identified as Saccharomyces cerevisiae, Wickerhamomyces anomalus, Zygosaccharomyces bailii, Zygosaccharomyces rouxii and Hanseniaspora uvarum. Five H. uvarum strains were screened for their brewing capability. Interestingly, Hanseniaspora uvarum strains showed growth in presence of ethanol and hop and a more rapid growth than the control strain S. cerevisiae US-05. Even though all strains showed a very low fermentation power, their concentrations ranged between 7 and 8 Log cycles during fermentation. The statistical analyses showed significant differences among the strains and underlined the ability of YGA2 and YGA34 to grow rapidly in presence of ethanol and hop. The strain YGA34 showed the best technological properties and was selected for beer production. Its presence in mixed- and sequential-culture fermentations with US-05 did not influence attenuation and ethanol concentration but had a significant impact on glycerol and acetic acid concentrations, with a higher sensory complexity and intensity, representing promising co-starters during craft beer production.