Search Results (35)

Although beer fermentation has traditionally been carried out with Saccharomyces, the boom in craft brewing has led to the use of non-conventional yeast species for beer production. This group also includes non-Saccharomyces starters, which are commonly used in winemaking and which have different technological characteristics compared to standard representatives of the Saccharomyces genus. One of the important characteristics of the non-Saccharomyces group is the richer enzyme profile, which leads to the production of beverages with different taste and aroma profiles. The aim of this study was to investigate sweet and hopped wort fermentation with seven strains of active dry non-conventional yeasts of Lachancea spp., Metschnikowia spp., Torulaspora spp. and a mixed culture of Saccharomyces cerevisiae and Torulaspora delbrueckii. One ale and one lager active dry yeast strain were used as control strains. The extract consumption, ethanol production, degree of fermentation, pH drop, as well as the yeast secondary metabolites formed by the yeast (higher alcohols, esters and aldehydes) in sweet and hopped wort were investigated. The results indicated that all of the studied types of non-conventional yeasts have serious potential for use in beer production in order to obtain new beer styles. For the purposes of this study, statistical methods, principle component analysis (PCA) and correlation analysis were used, thus establishing the difference in the fermentation kinetics of the growth in the studied species in sweet and hopped wort. It was found that hopping had a significant influence on the fermentation kinetics of some of the species, which was probably due to the inhibitory effect of the iso-alpha-acids of hops. Directions for future research with the studied yeast species in beer production are presented. Full article

Winemaking involves a microbial ecosystem where yeast diversity, shaped by terroir and winemaking conditions, determines wine characteristics. Understanding the microbial diversity of vineyards and spontaneous fermentation is crucial for explaining a winery’s typical wine profile. Studying and inoculating indigenous strains make it possible to produce high quality wines, reflecting the production environment. This study analyzes the yeast species involved in 16 spontaneous fermentations (8 in 2022 and 8 in 2023) from grapes of four distinct vineyards under two sets of winemaking conditions. A total of 1100 yeast colonies were identified by MALDI-TOF and DNA sequencing techniques. Saccharomyces (S.) cerevisiae and Hanseniaspora uvarum were the most prevalent species, alongside significant populations of non-Saccharomyces yeasts such as Lachancea thermotolerans and Metchnikowia pulcherrima, which were the most abundant ones. Minor yeast species, including Aureobasidium pullulans, Starmerella bacillaris, Kazachstania servazzi, and other Hanseniaspora spp., were also detected. The results demonstrated that yeast diversity in spontaneous fermentations varied according to vineyard origin and winemaking conditions. Differences between the two vintages studied indicated that annual climatic conditions significantly influenced yeast diversity, especially among non-Saccharomyces species. This substantial diversity represents a valuable source of indigenous yeasts for preserving the typicity of a winery’s wines under controlled conditions. Full article

CRISPR technology, which is derived from the bacterial adaptive immune system, has transformed traditional genetic engineering techniques, made strain engineering significantly easier, and become a very versatile genome editing system that allows for precise, programmable modifications to a wide range of microbial genomes. The economies of fermentation-based manufacturing are changing because of its quick acceptance in both academic and industry labs. CRISPR processes have been used to modify industrially significant bacteria, including the lactic acid producers, Clostridium spp., Escherichia coli, and Corynebacterium glutamicum, in order to increase the yields of bioethanol, butanol, succinic acid, acetone, and polyhydroxyalkanoate precursors. CRISPR-mediated promoter engineering and single-step multiplex editing have improved inhibitor tolerance, raised ethanol titers, and allowed for the de novo synthesis of terpenoids, flavonoids, and recombinant vaccines in yeasts, especially Saccharomyces cerevisiae and emerging non-conventional species. While enzyme and biopharmaceutical manufacturing use CRISPR for quick strain optimization and glyco-engineering, food and beverage fermentations benefit from starter-culture customization for aroma, texture, and probiotic functionality. Off-target effects, cytotoxicity linked to Cas9, inefficient delivery in specific microorganisms, and regulatory ambiguities in commercial fermentation settings are some of the main challenges. This review provides an industry-specific summary of CRISPR–Cas9 applications in microbial fermentation and highlights technical developments, persisting challenges, and industrial advancements. Full article

Previous studies have shown the positive effects of non-conventional Metschnikowia spp. yeasts in mixed cultures with Saccharomyces cerevisiae on the properties of fruit wines. In this study, we investigated the effects of using conventional S. cerevisiae and non-conventional Metschnikowia pulcherrima yeasts as starter cultures in controlled mixed fermentations of honey wort. Other non-conventional yeasts were also tested for comparison, including Wickerhamomyces anomalus, Dekkera/Bretannomyces bruxellensis, and Wickerhamomyces anomalus. We evaluated the tolerance of the tested yeasts to high sugar content and analyzed the metabolic profiles of both monocultures and mixed systems. The M. pulcherrima strain showed the highest tolerance to 30% w/v glucose. The chemical complexity of fermented honey was improved using M. pulcherrima in co-starters with S. cerevisiae. The fermented honey samples were characterized by lower ethanol content, higher glycerol level, and rich volatilomes containing higher levels of both esters (ethyl acetate, 3-methylbutyl acetate, 2-methylpropyl acetate) and aliphatic alcohols (2-methylpropan-1-ol, 3-methylbutan-1-ol, and 2-methylbutan-1-ol). Similar characteristics were obtained using mixed populations of four strains: S. cerevisiae, M. pulcherrima, D. bruxellensis, and W. anomalus. Full article

This study employed shotgun metagenomics to investigate microbial dynamics, phage-bacteria interactions, and functional genes throughout a three-month apple vinegar fermentation process. A total of 5621 microbial species were identified, revealing three distinct phases: (i) Enterobacteria and non-Saccharomyces species dominated the initial substrate; (ii) S. cerevisiae and Leuconostoc pseudomesenteroides prevailed in the intermediate phase; and (iii) acetic acid bacteria (Acetobacter ghanesis and Gluconobacter spp.), alongside non-Saccharomyces species (Pichia kudriavzevii and Malassezia restricta), dominated the final stages. Bacteriophage analysis revealed the presence of phages targeting spoilage bacteria, such as Pseudomonas and Erwinia, suggesting a role in regulating microbial stability and enhancing fermentation control. Functional metagenomic analysis highlighted key pathways associated with microbial growth and metabolite production, including carbohydrate and amino acid metabolism, energy production, and glycan biosynthesis. Enzymes involved in stress adaptation and secondary metabolism, including oxidative phosphorylation and phenolic compound synthesis, demonstrated microbial resilience and their potential role in shaping the product’s sensory and functional properties. Moreover, Enterobacteriaceae species were associated with pectin degradation during the early stages, aiding substrate breakdown. These findings are crucial for microbial and phage management in fermentation technology, offering valuable insights for innovation in the vinegar industry. Full article

Yeast derivatives are additives commonly used in winemaking for different purposes. Their manufacturing process is not well standardized, being mostly based on thermal inactivation and enzyme-induced lysis; furthermore, the main strain currently authorized for their production belongs to Saccharomyces spp. In this study, Saccharomyces cerevisiae and Torulaspora delbrueckii were used as starting microorganisms, whereas ultrasounds and high hydrostatic pressure were performed to induce autolysis, with the aim to evaluate the possibility to use different strains and emerging technologies as alternatives to the traditional methods to produce yeast derivatives. The chemical composition of the products obtained as well as the volatile profile of wines aged on yeast derivatives were mostly affected by the treatments performed during the manufacturing process. T. delbrueckii showed a good aptitude as starting microorganism for producing derivatives, whereas emerging, non-thermal technologies could replace the traditional methods for inducing autolysis, allowing to obtain products with enhanced content of polysaccharides (up to 178 mg/g) and antioxidant compounds (up to 9 µmol/g), and with low odor impact. The possibility to manage the chemical composition of yeast derivatives for specific winemaking purposes may thus be possible, by using specific starting microorganism and by applying the most suitable treatment to induce autolysis. Full article

Ergosterol is a key fungal sterol that is mainly found in the plasma membrane and is responsible for the proper membrane structure, rigidity, permeability and activity of membrane proteins. Ergosterol plays a crucial role in the ability of fungi to adapt to environmental stresses. The biosynthesis of ergosterol is also intimately connected with the antifungal resistance and virulence of pathogenic fungi. The most common etiological agents of life-threatening fungal infections are yeasts belonging to the genus Candida. The antifungal agents mostly used to treat Candida spp. infections are azoles, which act as competitive inhibitors of sterol demethylase, a key enzyme in the fungal ergosterol biosynthetic pathway. Although most studies on ergosterol biosynthesis, its regulation and the uptake of sterols are from the baker’s yeast Saccharomyces cerevisiae, the study of ergosterol biosynthesis and its relationship to antifungal drug resistance and virulence in pathogenic fungi is of utmost importance. The increasing antifungal drug resistance of Candida spp. and the limited armamentarium of antimycotics pose a challenge in the development of new therapeutic approaches. This review summarizes the available data on ergosterol biosynthesis and related phenomena in Candida albicans and non-albicans Candida species (Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida auris) with special emphasis on C. albicans and C. glabrata as the most common etiological agents of systemic candidiasis. Full article

The industry predominantly depends on synthetic or artificial additives, occasionally permitting the inclusion of natural molecules sourced from plants or replicated from their original counterparts. The production of bakery products increasingly uses sourdough to improve the quality of bread or to obtain “clean label” products (free of artificial additives). The additive production sector contributes to this concern through the synthesis of potentially harmful compounds, the utilization of hazardous chemicals and solvents, the management of resulting by-products, and reliance on non-renewable resources for manufacturing. One percent of the world’s population suffers from celiac disease. Celiac disease is treated by excluding gluten from the diet. Most gluten-free bakery products have low nutritional and sensory quality. Therefore, sourdough is being used to replace chemical yeast to improve the sensory and nutritional quality and increase the shelf life of gluten-free bakery products. Three gluten-free sourdoughs were prepared with different flours: brown rice, quinoa and amaranth, in order to compare them with traditional sourdough (wheat) and optimize the most suitable temperature for the conservation of sourdoughs. Physicochemical analysis (pH, titratable acidity and color), antioxidant activity (FRAP, ORAC and ABTS), total phenolic compound content (Folin–Ciocalteu), total aflatoxin content, lactic and acetic acid content and microbiological analysis (mold and yeast content and bacterial and fungal composition (microbiota composition)) were carried out during the elaboration process and at different storage temperatures. A higher microbiological quantity of molds and yeasts (7.97 log CFU/mL), non-Saccharomyces yeasts (7.78 log CFU/mL) and lactic acid bacteria (8.10 log CFU/mL) and fungal composition were observed in the amaranth sourdough. The wheat sourdough obtained a higher total content of phenolic compounds (33.03 mg GAE g⁻¹) and antioxidant capacity in ABTS and FRAP, but the quinoa sourdough had the highest ORAC content. In addition, it was observed that the adequate temperature for the conservation of the doughs is 25 °C, due to the predominance of Lactobacillus spp. and Pediococcus spp. bacteria in the sourdough. Therefore, pseudocereal sourdoughs (quinoa and amaranth) could be an alternative to incorporate into the preparation of gluten-free bread, since their microbial composition, physicochemical composition, antioxidant activity and total phenolic compounds would contribute to gluten-free bread and thus produce health benefits for people with celiac disease. Full article

Post-harvest decay of fresh table grapes causes considerable annual production losses. The main fungal agents of decay both in pre- and post-harvest are B. cinerea, Penicillium spp., Aspergillus spp., Alternaria spp., and Cladosporium spp. To date, the use of agrochemicals and SO₂ are the main methods to control grape molds in pre- and postharvest, respectively. Significant improvements, however, have already been made in to apply innovative and more environmentally sustainable control strategies, such as Biological Control Agents (BCAs), which can reduce disease severity in both pre- and post-harvest. In this study, 31 new non-Saccharomyces yeast strains, isolated from berries of native Apulian table grape genotypes, were tested for their in vivo effectiveness against grey mold of table grapes, resulting in two St. bacillaris (‘N22_I1’ and ‘S13_I3’), one S. diversa (‘N22_I3’), one A. pullulans (‘OLB_9.1_VL’) and one H. uvarum (‘OLB_9.1_BR’) yeast strains that were marked as efficient and good BCAs. Their mechanisms of action were characterized through in vitro assays, and additional characteristics were evaluated to assess the economic feasibility and viability for future technological employment. Their effectiveness was tested by reducing the working concentration, their antagonistic effect on a wide range of fungal pathogens, their ability to survive in formulations with long shelf life, and their safety to human health. Full article

Comprehensive yeast strain characterization is an important issue for the wine industry as market demands require controlled production of distinctive high-quality wines. Glycosides form an important reservoir of varietal grape wine aroma, and their hydrolysis into olfactory-active compounds essentially depends on the fermenting yeast genera and strains. Among the 14 Metschnikowia, Pichia, Torulaspora and 18 Saccharomyces spp., rapid screenings by agar plate and activity assay, including the substrates arbutin, cellobiose and p-nitrophenol-β-D-glucopyranoside, revealed the most glycosidase-active strains. In the novel co-fermentation setups, five selected non-Saccharomyces and a Saccharomyces strain were separated by a 14 kDa cut-off membrane, allowing respective viable cell counts but facilitating metabolite transfer. Chemical analysis focused on aroma glycosides, with extensive quantification by GC-MS with SIDA on the extracted and hydrolyzed compounds. Olfactory profiles obtained for the non-Saccharomyces wines demonstrated a significant impact of these yeasts, albeit mainly correlated with increased hydrolysis of monoterpene glycosides, and surpassed by a technical Aspergillus niger enzyme. While screenings of non-Saccharomyces strains indicated enhanced glucosidase activity under winemaking conditions, their effect was lower than expected and dominated by ester formation. Interestingly, Saccharomyces yeast cell vitality was increased via in co-fermentation, and non-Saccharomyces strains displayed extended viabilities with high ethanol tolerances. Full article

Madeira wine is produced via spontaneous alcoholic fermentation arrested by ethanol addition. The increasing demand of the wine market has led to the need to standardize the winemaking process. This study focuses on identifying the microbiota of indigenous yeasts present during Madeira wine fermentation and then evaluates the impact of selected indigenous non-Saccharomyces as pure starter culture (Hanseniaspora uvarum, Starmerella bacillaris, Pichia terricola, Pichia fermentans, and Pichia kluyveri) in the chemical and phenolic characterization of Madeira wine production. Results showed that the polyphenol content of the wines was influenced by yeast species, with higher levels found in wines produced by Pichia spp. (ranging from 356.85 to 367.68 mg GAE/L in total polyphenols and 50.52 to 51.50 mg/L in total individual polyphenols through HPLC methods). Antioxidant potential was higher in wines produced with Hanseniaspora uvarum (133.60 mg Trolox/L) and Starmerella bacillaris (137.61 mg Trolox/L). Additionally, Starmerella bacillaris stands out due to its sugar consumption during fermentation (the totality of fructose and 43% of glucose) and 15.80 g/L of total organic acids compared to 9.23 g/L (on average) for the other yeasts. This knowledge can be advantageous to standardizing the winemaking process and increasing the bioactive compounds, resulting in the production of high-quality wines. Full article

The topic of microbial interactions is of notable relevance in oenology, being connected with their impact on microbial biodiversity and wine quality. The interactions among different couples of microorganisms, in particular yeasts and lactic acid bacteria representative of the must/wine microbial consortium, have been tested in this study. This interaction’s screening has been implemented by means of plate assays, using culture medium, grape juice, and wine agar as substrates. Different antagonistic phenomena have been detected, belonging to the following interaction categories: yeast-yeast, yeast-bacteria, bacteria-yeast, and bacteria-bacteria. In general, the inhibitory activity has been observed in all three media agar used as substrates, resulting in more frequent on culture medium, followed by grape juice and, finally, wine. Specifically, the work is one of the first reports demonstrating the reciprocal interactions between non-Saccharomyces yeasts (NSY) and malolactic bacteria. The findings shed new light on the co-inoculation of the yeast starter culture with malolactic bacteria, as well as the biocontrol potential of Lactic Acid Bacteria (LAB) strains. Highlighted microbial interactions are relevant for the management of alcoholic fermentation, malolactic fermentation, and the development of distinctive aroma profiles, control of spoilage yeasts, and the selection of tailored mixed starter cultures. In addition, the plate assay method could be a fast, cheap, and suitable method to exclude negative interactions among Saccharomyces spp., NSY, and malolactic bacteria during trials from regional spontaneous fermentations with the aim to select tailored mixed starter cultures. Full article

Spontaneous wine fermentation is a meaningful topic that cannot be disregarded among winemakers and consumers due to the peculiarity of the organoleptic profile that it confers to the wine. Nevertheless, in this process the activity of indigenous microorganisms might be a threat. We studied the evolution of the spontaneous fermentation process in a traditional Italian winery in order to understand the origin of spoilage microorganisms, and to characterize the peculiarity of the microbiota associated with spontaneous fermentation. Six Trebbiano and Montepulciano wine production chains were monitored by plate counts made by OIV methods and by Illumina MiSeq technique. Despite some compositional deficiencies, all grape musts were characterized by a highly concentrated microbial population. Non-Saccharomyces yeasts revealed an unexpected tolerance to ethanol, which has contributed to the evolution of alcoholic fermentation. Lactic bacteria were detectable from the very first steps of the winemaking process, with a prevalence of Leuconostoc spp. which is nowadays, rarely isolated in wine. The combination between culture-dependent and high-throughput sequencing (HTS) approaches allowed to estimate microbial diversity and growth dynamics in wine fermentations of different grape varieties and under different treatments; these results could be used by winemakers as a starting point to drive a more mindful, accurate and, controlled fermentation process and to set up the most suitable environmental conditions to enhance wine singularities. Full article

The type and quantity of precursor amino acids present in grape must that are used by wine yeasts affect the organoleptic and health properties of wine. The aim of this work was to conduct a preliminary screening among Saccharomyces and non-Saccharomyces indigenous strains, which were previously isolated from different Italian regional grape varieties. This was performed in order to evaluate their decarboxylase activity on certain important amino acids—such as arginine, proline, serine, and tyrosine—that are present in grape must. In particular, a qualitative test on 122 wine yeasts was performed on a decarboxylase medium using arginine, proline, serine, and tyrosine as precursor amino acids. Our results showed a considerable variability among the microbial species tested for this parameter. Indeed, Saccharomyces cerevisiae strains exhibited a high decarboxylase capability of the four amino acids tested; moreover, only 10% of the total (i.e., a total of 81) did not show this trait. A high recovery of decarboxylation ability for at least one amino acid was also found for Zygosaccharomyces bailii and Hanseniaspora spp. These findings can, therefore, promote the inclusion of decarboxylase activity as an additional characteristic in a wine yeast selection program in order to choose starter cultures that possess desirable technological traits; moreover, this also can contribute to the safeguarding of consumer health. Full article

Biological ageing is an essential process for obtaining some distinctive Sherry wines, such as Fino and Manzanilla. It occurs after the fermentation of the grape must due to the appearance of a biofilm on the surface of the wine called “veil of flor”. Yeasts belonging to the Saccharomyces cerevisiae species mainly comprise such biofilm. Although other species have also been found, these have been traditionally considered spoilage. Indeed, it has even been hypothesised that they may not be able to form biofilm on their own under such conditions. In the present work, four different non-Saccharomyces yeasts isolated from barrels in the Jerez area under biological ageing have been characterised through their physiological abilities, including extracellular enzymatic and biofilm-forming capabilities. Results showed not only a surprising ethanol tolerance, above 15.5% in all cases, but also a significant degree of extracellular enzyme production, highlighting the urease and proteolytic activities found in Pichia manshurica, as well as lipolytic activity in Pichia kudriavzevii, Pichia membranifaciens and Wicherhamomyces anomalus. As a conclusion, these non-Saccharomyces could be very interesting in the oenological field, beyond improving the organoleptic characteristics as well as technological features in these wines. Full article