Search Results (104)

Yeast biosensors represent a promising biotechnological innovation for ensuring the safety and quality of fermented beverages such as beer, wine, and kombucha. These biosensors employ genetically engineered yeast strains to detect specific contaminants, spoilage organisms, or hazardous compounds during fermentation or the final product. By integrating synthetic biology tools, researchers have developed yeast strains that can sense and respond to the presence of heavy metals (e.g., lead or arsenic), mycotoxins, ethanol levels, or unwanted microbial metabolites. When a target compound is detected, the biosensor yeast activates a reporter system, such as fluorescence, color change, or electrical signal, providing a rapid, visible, and cost-effective means of monitoring safety parameters. These biosensors offer several advantages: they can operate in real time, are relatively low-cost compared to conventional chemical analysis methods, and can be integrated directly into the fermentation system. Furthermore, as Saccharomyces cerevisiae is generally recognized as safe (GRAS), its use as a sensing platform aligns well with existing practices in beverage production. Yeast biosensors are being investigated for the early detection of contamination by spoilage microbes, such as Brettanomyces and lactic acid bacteria. These contaminants can alter the flavor profile and shorten the product’s shelf life. By providing timely feedback, these biosensor systems allow producers to intervene early, thereby reducing waste and enhancing consumer safety. In this work, we review the development and application of yeast-based biosensors as potential safeguards in fermented beverage production, with the overarching goal of contributing to the manufacture of safer and higher-quality products. Nevertheless, despite their substantial conceptual promise and encouraging experimental results, yeast biosensors remain confined mainly to laboratory-scale studies. A clear gap persists between their demonstrated potential and widespread industrial implementation, underscoring the need for further research focused on robustness, scalability, and regulatory integration. Full article

The spoilage bacterium Acetobacter aceti poses a major threat to wine quality by causing acetification, driving the need for effective control methods. This study investigated the inactivation of A. aceti using pulsed electric field (PEF) and elucidated the multi-target mechanisms. The results demonstrated that PEF efficacy was highly dependent on the electric field intensity. PEF treatment at 30 kV/cm achieved a >3-log reduction in viable cell counts, with a Weibull model analysis indicating a critical inactivation threshold of 21.64 kV/cm. Mechanistic investigations revealed that PEF induced irreversible damage to the cell membrane, evidenced by morphological rupture (SEM) and a 4-fold increased permeability (flow cytometry), which led to a massive leakage of intracellular contents. Critically, this physical damage was correlated with profound physiological disruption, including the inactivation of key spoilage enzymes alcohol dehydrogenase (ADH, 80.0% relative activity loss) and aldehyde dehydrogenase (ALDH, 93.1% relative activity loss). Furthermore, PEF induced severe oxidative stress (4.25-fold increase in ROS and 3.30-fold increase in MDA) and a collapse in energy metabolism. Collectively, these findings reveal a synergistic inactivation mechanism, which establishes a strong theoretical foundation for the potential development of PEF as a non-thermal strategy to control acetic spoilage in winemaking. Full article

Sulfiting agents are common preservatives in the food and beverage industry to inhibit spoilage microorganisms. Sulfite produced by the dissolution of sulfur dioxide (SO₂) in water is used as a microbial inhibitor and antioxidant during winemaking. Thus, sulfite resistance is a desirable trait for wine yeasts. However, consumer health concerns regarding SO₂ exposure require a better understanding of the molecular basis of sulfite resistance/response. In this study, we have developed a highly SO₂-stress-resistant Saccharomyces cerevisiae strain (F3) using evolutionary engineering by repeated batch selection at gradually increased potassium metabisulfite (K₂S₂O₅) levels. F3 was resistant to 1.1 mM K₂S₂O₅ stress, which was strongly inhibitory to the reference strain, and cross-resistant to oxidative, heat, and freeze–thaw stresses. F3 also had enhanced cell wall integrity and altered carbon metabolism, indicating its potential for industrial applications, including winemaking. Comparative whole genome sequencing revealed point mutations in SSU1 and FZF1 that are related to SO₂ transport; ATG14, related to autophagy; and other genes involved in vacuolar protein sorting. Comparative transcriptomic analysis showed significant upregulation of SSU1 and differential expression of genes related to transport and carbohydrate metabolism. These findings may shed light on the molecular mechanisms contributing to SO₂ resistance and industrial robustness in S. cerevisiae. Full article

Natural wines represent a new trend in winemaking without the use of preservatives and starter cultures, revealing the unique quality traits of grapes, wine, and terroir, but are susceptible to spoilage or undesirable fermentations. This study investigated the microbial populations associated with organic grapes, must, and natural wines of the Limniona red grape variety, focusing on different production stages and fermentation vessels. Samples included immature and ripe grapes, initial and fermenting must, filtered and unfiltered wines, and final bottled and filtered wines. These were analyzed in order to enumerate key groups of microorganisms and identify beneficial yeasts and bacteria of alcoholic and malolactic fermentation, respectively, as well as potential markers of off-flavors. Culture-dependent methods were used to enumerate yeasts and bacteria, while Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and 16S rRNA sequencing provided taxonomic resolution. Beneficial fermentation microorganisms (especially Saccharomyces yeasts) were scarce in initial grapes, where other contaminants or wild yeasts were present. Gradually, as fermentation progressed, there was a prevalence of Saccharomyces cerevisiae strains of increased diversity in matured wine, as well as several lactic acid bacteria (LAB) of malolactic fermentation. Most LAB were identified as Lactobacillus and Oenococcus species. Other bacteria from environmental sources, irrelevant to alcoholic/malolactic fermentation or spoilage, like Burkholderia, were also present during the vinification process. The type of vessel affected the type of LAB that prevail, with an abundance of Oenococcus in clay vessels versus Lactobacillus species in stainless-steel vessels. Notably, Lentilactobacillus parafarraginis can be linked to off-flavors if they represent a high percentage of the wine microbiota. These findings highlight the importance of understanding, monitoring and controlling microbial succession during production stages in order to prevent sensory faults and ensure the stable quality of natural wines. 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

Accurately evaluating and quantifying microbial spoilage on wine grapes is a major challenge, especially in machine-harvested fruit that is no longer intact as a cluster when it arrives at the winery and cannot be visually inspected. The goal of the study was, using an infrared spectroscopy-based analytical system, to establish a unitless quantitative number that would reflect complex microbial spoilage and could be applied for all types of grapes with only one calibration model. Grapes (cultivars Riesling, Chenin Blanc, Chardonnay, Zinfandel and Petite Sirah) were hand-harvested in two consecutive vintages and separated in the vineyard into visually healthy clusters and infected grapes. Grapes were blended with increasing infection levels between 0 and 20% and models were created using known spoilage indicators. The resulting formula can be used to calculate a weighted index that reflects the microbial infection status of the grape material. Using a common spectroscopy instrument that is already present in larger wineries, the Grape Health Index allows for a quantitative quality assessment within two minutes at the test stand before the grape material is accepted. When visual inspection is not an option, this can help to make data-based quality and blending decisions at a very early stage. Full article

A study was carried out to identify the filamentous fungi and yeasts present in rotten wine grapes from two subzones of the Southern oasis of Mendoza winegrowing region, to assess the occurrence of tenuazonic acid (TA), a mycotoxin produced by the Alternaria genus, and to evaluate the wine spoilage potential of the associated yeasts in vitro and during microvinifications. The main fungal genera present were Alternaria (69.3%), followed by Aspergillus (16.8%), Penicillium (9.3%), and Cladosporium (4.6%), while the dominant yeast species Metschnikowia pulcherrima (23.1%), Aureobasidium pullulans (20.2%) and Hanseniaspora uvarum (13.0%) were followed by H. vineae (11.6%), Zygosaccharomyces bailii (10.4%), and H. guilliermondii (9.2%). Additionally, 94.1% of the rotten samples were contaminated with TA, with the highest level found in the Cabernet Sauvignon variety. No geographic association was found in the incidence of the different fungal genera or yeast species, nor in the occurrence of TA. Almost all of the tested yeasts produced H₂S, the majority of the Hanseniaspora strains produced acetic acid, and only one M. pulcherrima strain produced off-flavours in in vitro tests. Wines co-fermented with H. uvarum L144 and S. cerevisiae showed higher volatile acidity and lower fruity aroma and taste intensity. Therefore, processing bunch rot could pose a toxicological and microbiological risk to winemaking due to the high incidence of Alternaria and TA, as well as the potential of the associated yeasts to spoil wine. Full article

This study investigated the impact of berry and cluster thinning on the organoleptic and chemical quality of red wines produced with no-sulfites-added production, using ‘Monastrell’ grapes cultivated under organic viticulture. The experiment was conducted in a commercial vineyard in Murcia (Spain), applying three treatments: control, bunch reduction (BR), and berry thinning (BT). Grapes were vinified under identical conditions, and the resulting wines were analyzed after three months and five years of storage. Physicochemical parameters, volatile organic compounds (VOCs), and sensory profiles were evaluated. Thinning treatments significantly increased alcohol content, reducing sugars, polyphenol index, and the concentration of key aromatic compounds. Sensory analysis revealed that wines from thinned grapes exhibited more intense toasted, vegetal, and fruity notes, and presented greater color stability and fewer defects over time. Notably, only the control wine developed Brettanomyces-related off-flavors after five years. Consumer preference tests confirmed higher acceptance of BR and BT wines, based particularly on color, fruity aroma, and aftertaste. These findings suggested that thinning practices, especially bunch thinning, offer a cost-effective strategy to improve wine quality and stability in no-sulfites-added winemaking, reducing the risk of spoilage and enhancing consumer satisfaction. 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

Reusing agro-industrial by-products is a successful strategy that aligns with the 2030 Sustainable Development Goals. Red grape pomace poses a significant environmental challenge, particularly for wine-producing nations. Due to its high moisture content and seasonal availability, ensiling emerges as a potential method to prolong the nutritional value of red grape pomace, supporting the need for research into its application in animal nutrition. This study analyzed the bale ensiling process for red grape pomace by assessing its potential integration into ruminant diets and comparing its storage stability to untreated preservation methods. Baled silage units (approximately 300 kg each) were employed for this purpose. Analytical evaluations were conducted at 0, 7, 14, 35, 60, and 180 days of storage to monitor microbial and fermentation activity, nutritional composition, and bioactive attributes. Bale silage preserved the nutritional and microbial quality of red grape pomace for ruminant feed over a storage period of 180 days. The results demonstrated that bale silage successfully maintained the macro-composition, bioactive compounds, and antioxidant properties while reducing the fatty acid profile’s susceptibility to oxidation. By contrast, untreated storage led to significant spoilage. We concluded that bale ensiling is a suitable and effective technique that preserves red grapes for ruminant feed over a long period. 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