Search Results (57)

Kombucha fermentation is driven by a Symbiotic Culture of Bacteria and Yeast (SCOBY), a cellulose-rich biofilm that hosts a complex microbial consortium. While most kombucha studies focus on the liquid beverage, the SCOBY pellicle itself remains underexplored, particularly with respect to species-level microbial resolution and its intrinsic biological activities. In this study, a commercial kombucha SCOBY was characterized using full-length 16S rRNA gene and ITS amplicon sequencing based on Oxford Nanopore Technology, enabling species-level taxonomic resolution. In parallel, hydroalcoholic and aqueous extracts of dried SCOBY biomass were evaluated for in vitro antioxidant activity (DPPH and ABTS assays), antidiabetic-related enzyme inhibition (α-glucosidase and dipeptidyl peptidase-4, DPP4), and anti-aging-related enzyme inhibition (tyrosinase and elastase). The SCOBY bacterial community was strongly dominated by acetic acid bacteria, with Komagataeibacter saccharivorans and Acetobacter tropicalis accounting for more than 60% of total reads, reflecting a biofilm structure optimized for cellulose production and oxidative metabolism. The yeast community showed marked unevenness, with Brettanomyces bruxellensis representing over 80% of reads, consistent with its known role in ethanol production and stress tolerance within kombucha systems. In vitro assays revealed that hydroalcoholic SCOBY extracts consistently exhibited higher biological activity than aqueous extracts across all tested assays. However, both extracts showed substantially lower potency than purified reference compounds, indicating moderate but measurable bioactivity typical of complex fermented matrices. These findings support the potential valorization of SCOBY as a fermentation-derived biomaterial and functional ingredient while underscoring the need for further chemical characterization, mechanistic studies, and biological validation beyond enzyme-based assays. Full article

Kombucha tea fermentation is driven by microbial consortia composed of yeasts, acetic acid bacteria (AAB) and lactic acid bacteria (LAB), whose metabolic interactions determine the product’s functional and sensory characteristics. This study focused on the isolation and characterization of cultivable microorganisms from kombucha tea and the reassembly of four defined communities to evaluate their contribution to the chemical composition of the beverage based on the physicochemical parameters and multivariate analysis (PCA) of sugars, organic acids and ethanol. Microbial isolates, identified in this study, belonged to yeast (Saccharomyces cerevisiae and Brettanomyces bruxellensis), AAB (Novacetimonas hansenii, Komagataeibacter europaeus, Komagataeibacter intermedius and Acetobacter pasteurianus) and LAB (Liquorilactobacillus nagelii). Selected strains were combined to reassemble simplified communities. Fermentation trials demonstrated that community composition markedly influenced metabolite production and acidification (acetic acid and ethanol concentration ranged from 0.30 ± 0.08 and 2.29 ± 0.03 g/L, and from not determined to 27.31 ± 3.41 g/L, respectively). Consortia combining yeasts, AAB and LAB most closely reproduced the chemical composition of the original Kombucha tea, whereas simpler yeast–bacteria consortia produced chemically distinct beverages. Overall, these findings enhance our understanding of the ecological roles of kombucha-associated microorganisms and demonstrate that community composition is a key factor in shaping the chemical profile of the beverage. Moreover, the reassembly of defined microbial communities represents a promising strategy for selecting and applying functional microorganisms to valorize agri-food by-products through sustainable fermentation processes. Kombucha-derived communities, due to their ability to grow under acidic conditions, tolerate osmotic stress and metabolize complex sugar mixtures, could be versatile biofactories for the development of new fermented beverages or functional ingredients from low-value agri-food residues, contributing to circular bioeconomy strategies and waste reduction. Full article

The scope of this work is the study of the evolution of Brettanomyces bruxellensis, the main wine spoilage yeast, during bottle fermentation of sparkling wines. Lambrusco (Emilia, Italy) was considered as a model wine, for its high concentration of precursors for B. bruxellensis activity, especially cinnamic acids. Five Lambrusco base wines furnished by a cooperative winery were inoculated with a 3-log concentration of B. bruxellensis and then underwent secondary fermentation in the bottle. Two strategies of contrast to B. bruxellensis, already successfully applied in red winemaking, were tested here for the first time in bottle fermentation: chitosan and a yeast proposed as a biocontrol agent. Bottle fermentation was monitored from a chemical and microbiological perspective. The resulting sparkling wines were analyzed by GC and HPLC–MS/MS to verify the presence of the key molecules indicating B. bruxellensis activity—biogenic amines, volatile phenols, and pyridines. Sensory analysis was also performed to establish the effects of the treatments on the overall wine profile. The results demonstrate that B. bruxellensis is capable of growing up to 5-log units, causing severe alterations of the wines, both from a chemical and sensorial point of view. The addition of chitosan at the beginning of bottle fermentation effectively mitigated the effects of B. bruxellensis, resulting in the wines being similar to the uncontaminated control. The effectiveness of the biocontrol agent under these conditions was lower and requires further investigation. Full article

Brettanomyces bruxellensis is a yeast that causes spoilage in red wines due to its ability to produce volatile phenols, compounds associated with major sensory defects. Specific monitoring of low populations of this species in complex ecosystems such as wine during fermentation or aging often relies on plating onto selective media supplemented with cycloheximide. However, the variability of B. bruxellensis sensitivity to this antibiotic needs to be better characterized. A collection of 175 B. bruxellensis strains was, thus, grown on YPD medium supplemented with increasing concentrations of cycloheximide (0 to 0.5 g.L⁻¹), and yeast development was monitored for 20 days by image analysis. This study revealed significant inter-strain variability, with some strains showing very late or even no growth at high cycloheximide concentrations. The cycloheximide inhibitory effect was also dose- and population-dependent. In addition, colony size was frequently reduced at high doses. Additional tests were conducted on a subset of strains grown in wines with either low pH or high alcohol content or containing sulfur dioxide and then plated in the presence of increasing concentrations of cycloheximide. This revealed a cumulative effect of wine and cycloheximide stresses that resulted in an even higher delay in yeast detection. The results confirm the huge phenotypic diversity of the species and highlight the need to adapt the plates’ incubation time, particularly when the selectivity and the doses of cycloheximide needed are high (samples taken in pre-fermentation phases or during fermentation) or in case of stressful wine analysis, to minimize the risk of false negatives. Full article

The application of ultraviolet-C (UV-C) light has emerged as a promising non-thermal alternative to chemical preservatives in winemaking. This study investigates the efficacy of UV-C treatment on the microbial stability of Pinot noir wine during a 12-week storage period at 20 °C, with a focus on the spoilage yeast Brettanomyces bruxellensis. Microbiological analysis demonstrated complete and sustained inactivation of 10⁵ CFU/mL Brettanomyces bruxellensis after UV-C treatment with no detectable regrowth during the 12-week storage period. Untreated wine showed 1-log increase in Brettanomyces bruxellensis during the 12-week storage period and significant production of volatile esters and 4-ethylguaiacol. At the same time, a significant reduction in coumaric acid concentration was determined and attributed to Brettanomyces bruxellensis metabolism. UV-C-treated wine showed marginal increases in 4-ethylguaiacol attributed to the residual activity of Brettanomyces bruxellensis after UV-C treatment. Volatile esters did not significantly change during the 12-week storage period. The findings of this study demonstrate that UV-C treatment can ensure the microbiological storage stability of red wine. Full article

Brettanomyces bruxellensis has been described as the main spoilage microorganism in wines due to its ability to produce volatile phenols, which negatively impact the final product’s organoleptic properties. This yeast can grow and survive in environments that are too nutritionally poor and stressful for other microorganisms, and one of the stressful conditions it can endure is the high alcohol content in wine. In this study, cell wall morphology and the expression of some genes related to its composition were characterized under increasing ethanol concentrations to establish a possible ethanol resistance mechanism. B. bruxellensis LAMAP2480 showed greater resistance to β-1,3-glucanase activity when grown in media supplemented with 5% or 10% ethanol compared with the control assay (without ethanol). Transmission electron microscopy showed no significant differences in cell wall thickness during the different adaptation stages. However, the amount of wall polysaccharides and chitin briefly increased at 1% ethanol but returned to baseline at 5% and 10%. The amount of wall-associated protein increased progressively with each increment in ethanol concentration. In addition, overexpression of the ROM2 and KNR4/SMI1 genes was observed at 10% ethanol. These results suggest that the integrity of the cell wall might play an important role in the adaptation of B. bruxellensis to an ethanol-containing medium. Full article

Bioprotection in winemaking refers to the use of naturally occurring microorganisms—mainly non-Saccharomyces yeasts—to inhibit the growth of spoilage microbes and reduce the need for chemical preservatives like sulfur dioxide (SO₂). Numerous studies have demonstrated the benefits of non-Saccharomyces as bioprotectants. However, the use of Saccharomyces cerevisiae as a bioprotectant has been studied very little. Furthermore, it can offer many advantages for the production of sulfite-free wines. To test if S. cerevisiae could be used in bioprotection, we compared the ability of different strains to inhibit the growth of Brettanomyces bruxellensis and Hanseniaspora uvarum. Among the strains tested, the S. cerevisiae Sc54 strain isolated from the vineyard of the Bekaa plain was selected. To investigate its mechanisms of action, we analyzed its metabolite production, including acetic acid and ethanol. Taking into account the low levels of these metabolites and the lack of similar inhibition patterns in media supplemented with acetic acid and ethanol, it appears that other factors contribute to its antagonistic properties. Nutrient competition was ruled out as a factor, as the growth inhibition of B. bruxellensis and H. uvarum occurred rapidly within the first 24 h of co-culture. In this study, we explored the role of the S. cerevisiae killer toxin (Sc54Kt) as a bioprotective agent against H. uvarum and B. bruxellensis spoilage yeasts. Purification procedures with ethanol allowed the extraction of Sc54Kt, yielding two concentrations (0.185 and 0.5 mg/mL). Remarkably, semi-purified Sc54Kt exhibited inhibitory effects at both concentrations under winemaking conditions, effectively controlling the growth and metabolic activity of the target spoilage yeasts. Overall, these findings demonstrate that S. cerevisiae Sc54 not only exerts a strong bioprotective effect but also contributes to improving the quality of wine. The results suggest that S. cerevisiae Sc54 is a promising bioprotective agent for mitigating spoilage yeasts in winemaking, offering a natural and effective alternative to conventional antimicrobial strategies. Full article

The spoilage of wine caused by Brettanomyces bruxellensis and Hanseniaspora uvarum poses a significant challenge for winemakers, necessitating the development of effective and reliable strategies to control the growth of these yeasts, such as grape must bioprotection. Despite evidence that certain microorganisms can inhibit the growth of Brettanomyces bruxellensis and Hanseniaspora uvarum, the specific mechanisms driving this inhibition remain unclear. The primary objective of this study is to elucidate the underlying mechanisms responsible for this inhibitory effect. We analyzed one Metschnikowia pulcherrima (Mp2) and two Lachancea thermotolerans (Lt29 and Lt45) strains, all of which demonstrated significant killing and inhibitory effects on Brettanomyces bruxellensis (B1 and B250) and Hanseniaspora uvarum (Hu3137) in synthetic must at pH 3.5 and 22 °C. The effectiveness of these two strains exhibited varying inhibition kinetics. The strains were monitored for growth and metabolite production (L-lactic acid, ethanol, and acetic acid) in both single and co-cultures. The low levels of these metabolites did not account for the observed bioprotective effect, indicating a different mechanism at play, especially given the different growth profiles observed with added L-lactic acid and ethanol compared to direct bioprotectant addition. Following the production, purification, and quantification of killer toxins, different concentrations of toxins were tested, showing that the semi-purified Mp2Kt, Lt29Kt, and Lt45Kt toxins controlled the growth of both spoilage yeasts in a dose-dependent manner. These bioprotectant strains also showed compatibility with Saccharomyces cerevisiae in co-cultures, suggesting their potential use alongside commercial starter cultures. Full article

Brettanomyces bruxellensis is recognized as the main spoilage yeast in red wines, producing volatile phenols that negatively impact wine quality. However, few studies have investigated strain diversity within wineries. Understanding the diversity and distribution of B. bruxellensis strains in different wines can provide insights into the origin and timing of contamination. This study aimed to evaluate the presence and diversity of B. bruxellensis biotypes during the production of four red wines in the same winery and to identify critical contamination stages. The analysis covered the entire process, from grape to six months of aging. B. bruxellensis yeasts were isolated and identified, and representative strains were typified by RAPD analysis. The results suggest that B. bruxellensis contamination did not originate from a single source. The grapes harbored low levels of B. bruxellensis, yet all wines were positive before barrel filling. This study demonstrates that winery equipment can serve as a vector for Brettanomyces introduction. Two critical contamination stages were identified: the shared use of equipment during fermentation, facilitating strain dissemination across wines, and the reuse of barrels, introducing new strains during aging. Additionally, some winery practices further promote B. bruxellensis spread and proliferation. Full article

The biochemistry and physiology of raw material, the metabolism of microorganisms, and the methods used for processing and storage can affect the stability of wines. Due to the antimicrobial action of sulfur dioxide and dimethyl dicarbonate, the aim of this study is to determine the optimal treatment protocol to maintain the physico–chemical and microbiological stability of white wines with high residual sugar. Thus, the present research focuses on analyzing the influence of both treatments, combined or separate, on 45 wine samples obtained from a blend of Muscat Ottonel and Fetească Regală grape varieties, where different doses of 6% aqueous SO₂ solution (40, 80, and 160 mg/L) and dimethyl bicarbonate (0, 100, and 200 mg/L) were used. In order to assess the ability of dimethyl dicarbonate to suppress microorganisms, varying concentrations of Brettanomyces bruxellensis and Schizosaccharomyces pombe yeasts were inoculated (0, 30, 100 CFU/mL wine). The results indicate that, while sulfur dioxide cannot be entirely substituted in wines, both treatments can effectively lower or inhibit the activity of spoilage microorganisms. For the wines’ physico–chemical and microbiological stability, the treatment that used the synergistic force of sulfur dioxide (160 mg/L) and dimethyl dicarbonate (200 mg/L wine) performed the best. Full article

In recent years, the wine industry has been researching how to improve wine quality along the production value chain. In this scenario, we present here a new tool, MicroVi, a cost-effective chip-sized microscopy solution to detect and count yeast cells in wine samples. We demonstrate that this novel microscopy setup is able to measure the same type of samples as an optical microscopy system, but with smaller size equipment and with automated cell count configuration. The technology relies on the top of state-of-the-art computer vision pipelines to post-process the images and count the cells. A typical pipeline consists of normalization, feature extraction (i.e., SIFT), image composition (to increase both resolution and scanning area), holographic reconstruction and particle count (i.e., Hough transform). MicroVi achieved a 2.19 µm resolution by properly resolving the G7.6 features from the USAF Resolving Power Test Target 1951. Additionally, we aimed for a successful calibration of cell counts for Saccharomyces cerevisiae. We compared our direct results with our current optical setup, achieving a linear calibration for measurements ranging from 0.5 to 50 million cells per milliliter. Furthermore, other yeast cells were qualitatively resolved with our MicroVi microscope, such as, Brettanomyces bruxellensis, or bacteria, like, Lactobacillus plantarum, thus confirming the system’s reliability for consistent microbial assessment. Full article

Brettanomyces bruxellensis yeasts cause wine spoilage by producing volatile phenol compounds with specific off-odors. Assessing the propagation of this species is challenging, especially for micro-wineries. In this study, wines produced in a micro-winery from the grapes of different varieties collected from three PGI regions of Moldova over three years were studied for the presence and infection level of Brettanomyces spoilage yeasts, using traditional microbiological and molecular methods. The results of Brettanomyces infection monitoring in mature wines might speak in favor of the hypothesis that grape berries can be a potential source of B. bruxellensis in wine. The contamination levels of mature wines with respective species fluctuated in accordance with the year of grape cultivation, being the highest during the 2023 vintage. This study shows the potential of applying sequencing analysis for tracking the source of Brettanomyces contamination in wineries. Full article

Background (1): The use of fungal chitosan as an antiseptic in wine appears as a promising alternative to sulfur dioxide for the elimination of Brettanomyces bruxellensis sensitive strains. Nevertheless, its utilization raises the question, “how are the treated wines different from the untreated ones?” Methods (2): Chitosan treatment residues were sought in the oligosaccharide and polysaccharide fractions and among 224 low MW ions (<1800 g·mol⁻¹) in several wines by using liquid chromatography (size exclusion HPLC or LC-MS) and GC-MS. Standard oenological parameters were also examined as well as possible sensory modifications by a panel of tasters composed of experts and non-experts. Results (3): None of these methods enabled the reproducible and reliable identification of a treated wine without comparing it to its untreated control. The fingerprints of treatment are not reliably detectable by the analytical methods used in this study. However, the treated wines seem permanently protected against the development of chitosan-sensitive strains of B. bruxellensis. Conclusions (4): If chitosan treatment modifies the wine, the associated changes were not identified by the liquid chromatography method mentioned above and they were not perceived by most people in our taster panel. However, the expected antimicrobial action of chitosan was observed on B. bruxellensis sensitive strains and persisted at least one year. Tolerant strains were less affected by these persistent effects. Full article

Gluconobacter oxydans (Go) and Brettanomyces bruxellensis (Bb) are detrimental micro-organisms compromising wine quality through the production of acetic acid and undesirable aromas. Non-Saccharomyces yeasts, like Metschnikowia species, offer a bioprotective approach to control spoilage micro-organisms growth. Antagonist effects of forty-six Metschnikowia strains in a co-culture with Go or Bb in commercial grape juice were assessed. Three profiles were observed against Go: no effect, complete growth inhibition, and intermediate bioprotection. In contrast, Metschnikowia strains exhibited two profiles against Bb: no effect and moderate inhibition. These findings indicate a stronger antagonistic capacity against Go compared to Bb. Four promising Metschnikowia strains were selected and their bioprotective impact was investigated at lower temperatures in Chardonnay must. The antagonistic effect against Go was stronger at 16 °C compared to 20 °C, while no significant impact on Bb growth was observed. The bioprotection impact on Saccharomyces cerevisiae fermentation has been assessed. Metschnikowia strains’ presence did not affect the fermentation time, but lowered the fermentation rate of S. cerevisiae. An analysis of central carbon metabolism and volatile organic compounds revealed a strain-dependent enhancement in the production of metabolites, including glycerol, acetate esters, medium-chain fatty acids, and ethyl esters. These findings suggest Metschnikowia species’ potential for bioprotection in winemaking and wine quality through targeted strain selection. Full article

The antimicrobial activity of an aqueous extract of the leaves of Schinus polygamus (cav.) Cabrera against microorganisms of food importance was evaluated. First, the leaf aqueous extract of Schinus polygamus was characterized, quantifying hydroxycinnamic acids and phenolic compounds. Then, a battery of strains was tested, including Escherichia coli ATCC 25922, Salmonella Typhimurium ATCC 14028, and Listeria monocytogenes ATCC 13932. Also, we tested wine spoilage yeasts such as Brettanomyces bruxellensis LAMAP2480, B. bruxellensis LAMAP1359, B. bruxellensis CECT1451, and Pichia guilliermondii NPCC1051. Tests were conducted using the kinetic curve of growth and cell viability counts. The results indicate that with 10% v/v of concentrated extract, it is possible to observe growth inhibition of all microorganisms studied, with statistically significant differences during the whole measurement time (70 h for bacteria and 145 h for yeast). Full article