Search Results (31)

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

Vin Santo is a passito wine produced mainly in Tuscany. In the traditional production of Vin Santo, fermentation occurs naturally. Only a few reports have explored the microbial ecology of Vin Santo. Therefore, the present study aimed to investigate the microbial ecology and its impact on the fermentative kinetics in traditional processes of Vin Santo carried out in two different Tuscan wineries. Despite the different systems used for drying the grapes, both wineries showed similar microbial ecology. Non-Saccharomyces yeasts were the dominant microbial population during grape drying in different succession, even though in the end, the dominant species (at different percentages) in both were Metschnikowia pulcherrima, Kloeckera apiculata, and Starmerella bacillaris. The spontaneous fermentations were instead both dominated by Saccharomyces cerevisiae, however in different concentration throughout the process, leading to a different ethanol content—12% (v/v) and 10.8% (v/v) in winery A and B, respectively. In both wineries, acetic bacteria and moulds did not grow. Considering the intraspecific biodiversity of S. cerevisiae populations, the vinifications of both wineries displayed very similar biodiversity indices. No single strain of S. cerevisiae dominated the entire fermentation process. The analysis identified 30 distinct genetic patterns in the fermentations of winery A and 23 in the fermentations of winery B. The work provided an insight into the microbial communities and their metabolomic interactions during Vin Santo production which could improve the management and control of the process. Full article

This paper explores the broad probiotic and functional properties of two non-Saccharomyces strains (MI120 and MI125) and one Saccharomyces cerevisiae BB06 strain (as a reference probiotic). Torulaspora delbrueckii MI120 and Starmerella bacillaris MI125 were identified via 5.8S rDNA sequencing. All the strains survived well in simulated gastrointestinal conditions and had strong antioxidant activity (>68%). S. bacillaris MI125 excelled in antimicrobial activity against Gram-positive and Gram-negative bacteria. S. bacillaris MI125 and Sacch. cerevisiae BB06 resisted all the tested antibiotics. No strain displayed hemolytic behavior. The freeze-dried yeast strains achieved survival rates between 76.62% and 93.38%. Based on our physiological characterization analysis (carbon assimilation, ethanol tolerance, acetic acid and H₂S production, temperature and low pH tolerance, enzymatic pattern, and killer phenotype), all the strains showed interesting attributes, with Sacch. cerevisiae BB06 fermenting vigorously in malt extract medium. Beer fermented with T. delbrueckii MI120 had the highest phenolic content (96.02 μg GAE·mL⁻¹) and antioxidant activity (90.43%), matching commercial Sacch. cerevisiae US-05 in sensory traits such as taste and aroma. However, after two months in bottled beer, the yeast viability decreased to 2–3 log CFU·mL⁻¹. The pilot brewing and the assessments of the strains’ technological, physico-chemical, and sensorial properties confirmed their suitability for industrial brewing. Overall, T. delbrueckii MI120 emerges as a promising brewing strain, and S. bacillaris MI125 is a potential probiotic. 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

One approach towards maintaining healthy microbiota in the human gastrointestinal tract is through the consumption of probiotics. Until now, the majority of probiotic research has focused on probiotic bacteria, but over the last few years more and more studies have demonstrated the probiotic properties of yeast, and also of species besides the well-studied Saccharomyces cerevisiae var. boulardii. Probiotic strains have to present the ability to survive in harsh conditions of the host body, like the digestive tract. Must fermentation might be an example of a similar harsh environment. In the presented study, we examined the probiotic potential of 44 yeast strains isolated from Polish wines. The tested isolates belonged to six species: Hanseniaspora uvarum, Pichia kluyveri, Metschnikowia pulcherrima, Metschnikowia ziziphicola, Saccharomyces cerevisiae and Starmerella bacillaris. The tested strains were subjected to an assessment of probiotic properties, their safety and their other properties, such as enzymatic activity or antioxidant properties, in order to assess their potential usefulness as probiotic yeast candidates. Within the most promising strains were representatives of three species: H. uvarum, M. pulcherrima and S. cerevisiae. H. uvarum strains 15 and 16, as well as S. cerevisiae strain 37, showed, among other features, survivability in gastrointestinal tract conditions exceeding 100%, high hydrophobicity and autoaggregation, had no hemolytic activity and did not produce biogenic amines. The obtained results show that Polish wines might be a source of potential probiotic yeast candidates with perspectives for further research. Full article

2-phenylethanol (2-PE) has been widely utilized as an aromatic additive in various industries, including cosmetics, beer, olive oil, tea, and coffee, due to its rose-honey-like aroma. However, no reports have investigated the production of 2-PE by Starmerella bacillaris. Here, S. bacillaris (syn., Candida zemplinina, and named strain R5) was identified by analysis of morphology, physiology and biochemistry, and 26S rRNA and ITS gene sequence. Then, based on the analysis of whole-genome sequencing and comparison with the KEGG database, it was inferred that strain R5 could synthesize 2-PE from L-phe or glucose through the Ehrlich pathway or shikimate pathway. For further verification of the 2-PE synthesis pathway, strain R5 was cultured in M3 (NH₄⁺), M3 (NH₄⁺ + Phe), and M3 (Phe) medium. In M3 (Phe) medium, the maximum concentration of 2-PE reached 1.28 g/L, which was 16-fold and 2.29-fold higher than that in M3 (NH₄⁺) and M3 (Phe + NH₄⁺) media, respectively. These results indicated that 2-PE could be synthesized by strain R5 through the shikimate pathway or Ehrlich pathway, and the biotransformation from L-phe to 2-PE was more efficient than that from glucose. The qRT-PCR results suggested that compared to M3 (Phe + NH₄⁺) medium, the mRNA expression levels of YAT were 124-fold and 86-fold higher in M3 (Phe) and M3 (NH₄⁺) media, respectively, indicating that the transport of L-phe was inhibited when both NH₄⁺ and Phe were present in the medium. In the M3 (Phe) and M3 (Phe + NH₄⁺) media, the mRNA expression level of ADH5 was higher than PDC, hisC, GOT1, and YAT, and it was 2.6 times higher and 2.48 times higher, respectively, compared to the M3 (NH₄⁺) medium, revealing that the key gene catalyzing the dehydrogenation of benzaldehyde to 2-PE is ADH5. Furthermore, strain R5 exhibits tolerance to high concentrations of 2-PE, reaching 3 g/L, which conferred an ideal tolerance to 2-PE. In summary, the synthesis pathway of 2-PE, mainly for the Ehrlich pathway, was proved for the first time in S. bacillaris, which had not been previously explored and provided a basis for non-Saccharomyces yeast-producing 2-PE and its applications. Full article

In this study, six fermentation trials were carried out: co-inoculation and sequential inoculation of Saccharomyces cerevisiae and Starmerella bacillaris in the presence and absence of oak chips. Moreover, Starm. bacillaris strain was attached to the oak chips and co-inoculated or sequentially inoculated with S. cerevisiae. Wines fermented with Starm. bacillaris adhered to oak chips showed a higher concentration of glycerol (more than 6 g/L) than the others (about 5 g/L). These wines also showed a higher content of polyphenols (more than 300 g/L) than the others (about 200 g/L). The addition of oak chips induced an increase of yellow color (b* value of about 3). Oak-treated wines were characterized by a higher concentration of higher alcohols, esters and terpenes. Aldehydes, phenols and lactones were detected only in these wines, independently from the inoculation strategy. Significant differences (p < 0.05) were also observed in the sensory profiles. The fruity, toasty, astringency, and vanilla sensations were perceived as more intense in wines treated with oak chips. The white flower descriptor showed a higher score in wines fermented without chips. Oak surface-adhered Starm. bacillaris cells could be a good strategy to improve the volatile and sensory profile of Trebbiano d’Abruzzo wines. Full article

The bioconversion of environmental wastes into energy is gaining much interest in most developing and developed countries. The current study is concerned with the proper exploitation of some industrial wastes. Cellulosic fiber waste was selected as a raw material for producing bioethanol as an alternative energy source. A combination of physical, chemical, and enzymatic hydrolysis treatments was applied to maximize the concentration of glucose that could be fermented with yeast into bioethanol. The results showed that the maximum production of 13.9 mg/mL of glucose was achieved when 5% cellulosic fiber waste was treated with 40% HCl, autoclaved, and followed with enzymatic hydrolysis. Using SEM and FTIR analysis, the instrumental characterization of the waste fiber treatment confirmed the effectiveness of the degradation by turning the long threads of the fibers into small pieces, in addition to the appearance of new functional groups and peak shifting. A potent yeast strain isolated from rotten grapes was identified as Starmerella bacillaris STDF-G4 (accession number OP872748), which was used to ferment the obtained glucose units into bioethanol under optimized conditions. The maximum production of 3.16 mg/mL of bioethanol was recorded when 7% of the yeast strain was anaerobically incubated at 30 °C in a broth culture with the pH adjusted to 5. The optimized conditions were scaled up from flasks to a fermentation bioreactor to maximize the bioethanol concentration. The obtained data showed the ability of the yeast strain to produce 4.13 mg/mL of bioethanol after the first 6 h of incubation and double the amount after 36 h of incubation to reach 8.6 mg/mL, indicating the efficiency of the bioreactor in reducing the time and significantly increasing the product. Full article

Biogeography is a key concept associated with microbial terroir, which is responsible for the differentiation and uniqueness of wines. One of the factors influencing this microbial terroir is the vegetation, which in turn is influenced by climate, soil, and cultural practices. Remote sensing instruments can provide useful information about vegetation. This study analyses the relationship between NDVI, calculated using Sentinel-2 and Landsat-8 satellite images of different veraison dates, and microbial data obtained in 2015 from 14 commercial (organic and conventional) vineyards belonging to four Designations of Origin (DOs) from Galicia (northwest Spain). Microbial populations in grapes and musts were identified using PCR techniques and confirmed by sequencing. Statistical analyses were made using PCA, CCA, TB-PLS, and correlation analyses. This study confirms that the NDVI is positively correlated with the diversity of yeasts, both in grapes’ surface and must samples. Moreover, the results of this study show: (i) Sentinel-2 images, as well as Landsat-8 images, can establish differences in NDVI related to yeast terroir in grapes and musts, as it is the most relevant DO factor, (ii) Sentinel-2 NDVI and yeast biogeography are moderately to strongly correlated, (iii) Sentinel-2 achieved a better delimitation of the DOs than Landsat-8 and can establish more accurate differences in NDVI–yeast terroir correlations, and (iv) a higher NDVI was associated with the yeast biogeographical patterns of the DOs with higher species richness (S) consisting of weakly fermenting yeasts (Hanseniaspora uvarum, Pichia spp., Starmerella bacillaris, and Zygosaccharomyces spp). However, NDVI values did not correlate well with biogeographic patterns of yeasts previously studied at frequency level (proportion or percentage of each species) in each particular DO. This study suggests that satellite imagery has the potential to be a valuable tool for wine quality management and a decision-making instrument for DO regulators and winegrowers. 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

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. Full article

Starmerella bacillaris is a non-Saccharomyces yeast proposed for must fermentation together with Saccharomyces cerevisiae because of its high glycerol and moderate volatile acidity production. Furthermore, it was demonstrated that the same S. bacillaris strains that possess interesting technological properties exhibited antifungal activity against Botrytis cinerea, suggesting the release of this yeast in the vineyard. To obtain a positive effect during the following winemaking process, the maintenance of suitable concentrations of S. bacillaris is essential. Therefore, to obtain information on the survival of S. bacillaris, a small-scale field trial was performed. One week before the harvest, two different concentrations of S. bacillaris (10⁶ and 10⁷ cells/mL) were sprayed on Pinot grigio bunches, and the strain concentration was monitored by means of qPCR during the subsequent fermentation process. In addition, the combined effect of different winemaking techniques was evaluated, i.e., the vinification of juice, juice with marc and cryomaceration treatment. Results demonstrated that, under the tested conditions, S. bacillaris released in the vineyard remained viable for one week on grape bunches and increased glycerol content during the subsequent fermentation process. Different vinification protocols influenced cell concentrations. In particular, the cryomaceration treatment, due to the use of low temperature, supported S. bacillaris growth due to its cryotolerant aptitude. The collected data open new perspectives on the control of alcoholic fermentation, involving both vineyard and cellar management. Full article

Starmerella bacillaris is often isolated from environments associated with grape and winemaking. S. bacillaris has many beneficial properties, including the ability to improve the flavor of wine, the production of beneficial metabolites, and the ability to biocontrol. S. bacillaris CC-PT4 (CGMCC No. 23573) was isolated from grape and can inhibit methicillin-resistant Staphylococcus aureus and adaptability to harsh environments. In this paper, the whole genome of S. bacillaris CC-PT4 was sequenced and bioinformatics analyses were performed. The S. bacillaris CC-PT4 genome was finally assembled into five scaffolds with a genome size of 9.45 Mb and a GC content of 39.5%. It was predicted that the strain contained 4150 protein-coding genes, of which two genes encoded killer toxin and one gene encoded lysostaphin. It also contains genes encoding F1F0-ATPases, Na(+)/H(+) antiporter, cation/H(+) antiporter, ATP-dependent bile acid permease, major facilitator superfamily (MFS) antiporters, and stress response protein, which help S. bacillaris CC-PT4 adapt to bile, acid, and other stressful environments. Proteins related to flocculation and adhesion have also been identified in the S. bacillaris CC-PT4 genome. Predicted by antiSMASH, two secondary metabolite biosynthesis gene clusters were found, and the synthesized metabolites may have antimicrobial effects. Furthermore, S. bacillaris CC-PT4 carried genes associated with pathogenicity and drug resistance. Overall, the whole genome sequencing and analysis of S. bacillaris CC-PT4 in this study provide valuable information for understanding the biological characteristics and further development of this strain. Full article

Wine quality is determined by the particular yeast strains prevailing at various stages of fermentation. Therefore, the ability to make an easy, fast, and unambiguous discrimination of yeasts at the strain level is of great importance. Here, the tandem repeat-tRNA (TRtRNA) method with the 5GAC or ISSR-MB primer sets and random amplified polymorphic DNA (RAPD) analysis with (GTG)3, R5, and RF2 oligonucleotides were tested on various non-Saccharomyces wine yeast species. The TRtRNA-PCR employing ISSR-MB showed the highest capacity in discriminating Lachancea thermotolerans and Metschnikowia pulcherrima isolates. RAPD with RF2 was the most efficient method in resolving Starmerella bacillaris isolates, although it produced few polymorphic bands. RAPD with R5 showed the highest capacity to discriminate among the Issatchenkia orientalis, Hanseniaspora guilliermondii, and Pichia anomala isolates. RAPD with either R5 or RF2 exhibited the highest ability to discriminate among the Torulaspora delbrueckii isolates. RAPD with (GTG)3 was the most discriminating method for the H. uvarum isolates. Here we concluded that both TRtRNA-PCR and RAPD-PCR offer rapid means for typing non-Saccharomyces species. However, each method performs better for a given species when paired with a particular primer set. The present results can be useful in wine research for the fast fingerprinting of non-Saccharomyces yeasts. Full article

Haze can appear in white wines as a result of the denaturation and subsequent aggregation of grape pathogenesis-related (PR) proteins. Yeast cell-wall polysaccharides, particularly mannoproteins, represent a promising strategy to reduce the incidence of this phenomenon. The aim of this study was to evaluate the effects of 13 Starmerella bacillaris strains, in sequential fermentation with Saccharomyces cerevisiae, on wine protein stability of three white wines (Sauvignon blanc, Pinot grigio, and Manzoni bianco). The resulting wines were characterized in terms of their chemical composition, content of PR proteins and polysaccharides, and heat stability. In addition, the mannoprotein fraction was purified from six wines, five produced with S. bacillaris and one with S. cerevisiae EC1118 used as control. Generally, wines produced with S. bacillaris strains were more heat-stable, despite generally containing higher amounts of PR proteins. The increased heat stability of Starmerella wines was attributed to the stabilizing effect resulting from their higher concentrations of both total polysaccharides and mannoprotein fractions. In particular, for the most heat unstable wine (Manzoni bianco), the low MW mannoprotein fraction resulted to be the most involved in wine stability. The ability to produce wines with different heat stability was demonstrated to be strain-dependent and was more evident in the most unstable wines. By reducing fining waste, the use of S. bacillaris as an enological starter can be proposed as a new tool to manage wine protein stability for a more sustainable winemaking. Full article