Wine Microorganisms Research Articles

UV-C and Nanomaterial-Based Approaches for Sulfite-Free Wine Preservation: Effects on Polyphenol Profile and Microbiological Quality

Controlling the microorganisms employed in vinification is a critical factor for successful wine production. Novel methods aimed at lowering sulfites used for wine stabilization are sought. UV-C irradiation has been proposed as an alternative for reducing the viable cell count of microorganisms in wine and grape juice. Nevertheless, UV-C treatment poses the risk of altering the chemical properties of wine. Therefore, this study aimed to test and implement iron oxide–silica core–shell nanomaterial functionalized with TiO2 in UV-C treatment of white and red wines. Material for the study consisted of the synthesized nanocomposite, Saccharomyces cerevisiae as a model yeast, and Muscaris and Cabernet Cortis wines. The viability of yeasts under treatment, the physiochemical properties of wine, and polyphenol content were tested. Studies have shown that nanomaterial can modulate the effects of UV-C treatment regarding yeast viability and polyphenol content, and the effectiveness of the treatment depends on the wine type. These results open up discussion on the possible use of the proposed hurdle technology in winemaking to control the polyphenol composition and alcohol reduction.

Microorganisms in Fermented Foods and Their Effects on the Human Body

In recent years, microorganisms in fermented foods have shown an important role in regulating human health. Studies have shown that probiotics have significant health benefits in fermented foods, such as improving gut health, stimulating immune function, and promoting metabolism, and mental health. Specifically, lactic acid bacteria in yogurt can increase the number of beneficial bacteria in the intestines and reduce the growth of pathogenic bacteria, and yeasts such as Saccharomyces cerevae have multiple health effects on intestinal microbiota protection and anti-inflammation. In addition, various enzymes and metabolites produced by Aspergillus during fermentation process can improve the nutritional value of food. However, although lots of research has been done, the understanding of the microbial mechanism under different fermentation conditions is still insufficient. In this article, the types and influencing factors of microorganisms in yogurt, wine and seasoner were reviewed. The effects of temperature, pH value, fermentation time and other conditions on microbial activity and product quality were analyzed. The results showed that suitable fermentation conditions could significantly increase microbial activity, improve product flavor and health function, and provide a theoretical basis for optimizing the production process of fermented food. The article provides a theoretical basis for improving the fermented food production process, thus helping to improve product quality and market competitiveness. However, this article has some limitations like the lack of understanding of interactions between different microorganisms. Future research should focus on examining the collaborative relationship between these microbes to improve the health benefits and production efficiency of fermented foods.

Impact of <i>Pediococcus parvulus</i> and <i>Saccharomyces cerevisiae</i> on <i>Brettanomyces bruxellensis</i> mousy compound production

The presence of mousy off-flavours in wine, attributed to the production of N-heterocycles by lactic acid bacteria or Brettanomyces bruxellensis, poses a significant challenge in the winemaking industry. This study is the first one to investigate the impact of co-cultures focusing on B. bruxellensis with two common wine microorganisms, Pediococcus parvulus and Saccharomyces cerevisiae on the formation of mousy compounds. The screening was conducted in a synthetic medium under controlled conditions. It reveals the nuanced effects of the microorganism combinations. Depending on the P. parvulus or S. cerevisiae strain co-inoculated with B. bruxellensis a synergistic effect is observed on the 2-acetyltetrahydropyridine (ATHP) production, while an inhibitory effect on 2-acetylpyrroline (APY) production by P. parvulus is highlighted when co-inoculated with B. bruxellensis. These findings shed light on the potential impact of the alcoholic fermentation yeast (S. cerevisiae) on the production of mousy off-flavours. Finally, a correlation between ATHP and 2-ethyltetrahydropyridine (ETHP) appears to be confirmed with a ratio of 1:10 between ETHP and ATHP produced in the N-heterocycles assay medium.

Isolation, selection and characterisation of microorganisms with potential to form unique consortium for wine production

This paper focuses on a selection of microorganisms that should eventually form a Defined Consortium of Wine Microorganisms. The consortium might serve as a sophisticated oenological product for the production of wine with attractive organoleptic features. The Defined Consortium of Wine Microorganisms was obtained from non-saccharomyces and saccharomyces yeasts and lactic acid bacteria isolated from places where autochthonous microflora can be expected to occur (grape berries, wine lees or cellar spaces). A total of 42 microorganisms were obtained by surface smears, fallouts from the air or isolation from fermented must. All isolates were then tested and the best strains were selected on the basis of technological and phenotypic characterization using standard microbiological techniques. The main criteria for the selection of strains were the ability to ferment and the production of organoleptically active compounds. Sulphur dioxide or ethanol tolerance, β-glucosidase activity, a taxonomic identification, the tendency to produce sulphane or the ability of lactic acid bacteria to perform malolactic fermentation were also considered. According to these results, the non-saccharomyces yeast Starmerella bacillaris, the saccharomyces yeast Saccharomyces cerevisiae and the lactic acid bacterium Levilactobacillus brevis were selected as part of the Defined Consortium of Wine Microorganisms.

Correlation between changes in flavor compounds and microbial community ecological succession in the liquid fermentation of rice wine.

Microorganisms play an important role in regulating flavor compounds in rice wine, whereas we often don't understand how did they affect flavor compounds. Here, the relations between flavor compounds and microbial community ecological succession were investigated by monitoring flavor compounds and microbial community throughout the fermentation stage of rice wine. The composition of microbial community showed a dynamic change, but 13 dominant bacterial genera and 4 dominant fungal genera were detected throughout the fermentation stages. Saccharomyces presented a strong negative correlation with fungi genera but had positive associations with bacteria genera. Similarly, flavor compounds in rice wine were also showed the dynamic change, and 112 volatile compounds and 17 free amino acids were identified in the whole stages. The alcohol-ester ratio was decreased in the LTF stage, indicating that low temperature boosts ester formation. The potential correlation between flavor compounds and microbial community indicated that Delftia, Chryseobacterium, Rhizopus and Wickerhamomyces were the core functional microorganisms in rice wine. These findings clarified the correlation between changes in flavor compounds and in microbial community in the liquid fermentation of rice wine, and these results have some reference value for the quality improvement and technological optimization in liquid fermentation of rice wine.

Analysis of the effect of rice wine koji on the fermentation quality of rice wine based on high-depth sequencing

Rice wine koji is made from “ChengQu” as the parent species and is prepared by enriching microorganisms in the environment, which plays a decisive role in the formation of quality rice wine. This study explored the microbiome of 18 rice wine koji collected from Xiaogan, Fangxian, Hubei, Dazhou, and Sichuan and analyzed the sensory quality and microbiome of rice wine made with rice koji as a fermentation agent. Although our results showed that there were large differences in microbial communities and functions in rice wine koji, samples from the same region had a higher similarity, and the closer the distance, the higher the similarity. Five suspected new species (Lactococcus sp., Weissella sp., Lactobacillus sp., and Acetobacter sp.) were discovered by ultra-high-depth metagenomic sequencing; their functional maps and phase correlation analysis showed that they contributed positively to the aroma of rice wine. The sensory quality and microbial community structure of rice wine fermented with different rice wine koji have obvious differences, consistent with the structural difference of microorganisms in rice wine koji in different regions. These findings indicate that rice wine koji directly determines the structure of microorganisms in rice wine and directly or indirectly affects the sensory quality of rice wine.

Physiochemical properties and Enzymatic profile of indigenous yeast isolated from palm wine in Nigeria

This study was aimed at investigating the physiochemical properties of fresh palm wine and enzymatic activities of indigenous yeast strains isolated from palm wine sample (Elaeis guineensis). The physiochemical properties was evaluated after 1h of tapping giving the following values 7.29a±0.02%, 2.25a±0.03%, 476.58d±0.04 mg/L and 5.2c±0.06 for ethanol, TTA, reducing sugar and pH respectively. The yeast strains was isolated and characterized based on their morphological, physiological, and biochemical characteristics. Two yeast cells were identified as Saccharomyces cerevisiae and Geotrichum candidum. They were screened for their ability to synthesize amylase and cellulase. Results revealed significant variation in the enzymatic profiles of the indigenous yeast strains. Cellulase activity was present suggesting the ability to break down cellulose; and a starch degrading ability indicated by amylase activity. All the yeast stains had good cellulase and amylase enzyme profile but YT3 which was identified as Geotrichum candidum exhibited the highest enzyme activity of 0.719U/ml (amylase) and 0.182U/ml (cellulase), suggesting their potential for efficient sugar utilization during fermentation. The findings from this study proved that indigenous microorganisms in palm wine can act as a source of enzymes especially cellulase and amylase; and the enzyme yield will most likely improve after optimization. The identification of yeast strains with enhanced enzymatic potential offers opportunities for the large-scale and industrial production of the enzyme.

Microbial and Commercial Enzymes Applied in the Beverage Production Process

Enzymes are highly effective biocatalysts used in various industrial processes, playing a key role in winemaking and in other fermented beverages. Many of the enzymes used in fermentation processes have their origin in fruits, in the indigenous microbiota of the fruit, and in the microorganisms present during beverage processing. Besides naturally occurring enzymes, commercial preparations that usually blend different activities are used (glucosidases, glucanases, pectinases, and proteases, among others). Over the years, remarkable progress has been made in enhancing enzyme performance under operating conditions. The winemaking industry has observed a significant improvement in production levels, stimulating the introduction of technological innovations that aim to enhance efficiency and wine quality. Enzymes have traditionally been used in the beverage industry; however, others have been introduced more recently, with numerous studies aimed at optimizing their performance under processing conditions, including the use of immobilized enzymes. Therefore, one major goal of the current review is to give a detailed overview of the endogenous enzyme potential of wine microorganisms, as well as of enzymes obtained from grapes or even commercial preparations, studied and already in use in the beverage industry, and to present the future trends in enzyme production and application.

UV-C treatment: A non-thermal inactivation method for microbiological stabilisation of must and wine

UV-C treatment is discussed as an effective and efficient method to inactivate harmful microorganisms in wine and other viticultural products. In comparison to other stabilisation techniques, the application of UV-C is thought to be beneficial to reduce energy costs and to minimize SO2 addition. The object of this work was to determine the lethal UV-C dose for harmful microorganisms such as Brettanomyces bruxellensis and Acetobacter aceti. The concept of 5-log inactivation was applied and the Weibull model was used to compare different microbial and wine parameters. Microbial relevant UV-C doses and 2-fold overdose treatments and how they affected chemical and sensory changes of wine were investigated. Riesling and Pinot noir wine, which have different absorbance at 254 nm, were individually inoculated with microorganisms at different inoculation numbers. The results showed that the Weibull model is appropriate to predict the lethal UV-C dose. Already at microbially relevant doses, UV-C treatment can lead to significant changes in the colour and concentration of aroma compounds in white wine. Higher concentrations of 2-aminoacetophenone were found with increasing UV-C doses. Hence, UV-C overdosing can cause the “atypical ageing” off-flavour in wine. However, microbially relevant UV-C doses change the sensory properties of wine more towards a typical ageing character.

Evaluating the influence of operational parameters of pulsed light on wine related yeasts: focus on inter- and intra-specific variability sensitivity

In oenology, there is a growing demand by consumers for wines produced with less inputs (such as sulphite, frequently used for microbial control). Emerging control methods for managing microorganisms in wine are widely studied. In this study, the efficiency of pulsed light (PL) treatment was investigated. A drop-platted system was used to evaluate the impact of three PL operational parameters: the fluence per flash, the total fluence and the flash frequency. Fluence per flash appeared to be a key parameter prior to total fluence, thus demonstrating the importance of the effect of peak voltage during PL treatments. The efficiency of PL treatment was assessed on 198 strains distributed amongst fourteen yeast species related to wine environment, and an important variability in PL response was observed. Brettanomyces bruxellensis strains were strongly sensitive to PL, with intraspecific variation. PL was then applied to red wines inoculated with 9 strains of B. bruxellensis, Saccharomyces cerevisiae and Lachancea thermotolerans. Results confirmed interspecific response variability and a higher sensitivity of B. bruxellensis species to PL. Wine treatments with a total fluence of 22.8 J cm−2 resulted in more than 6 log reduction for different B. bruxellensis strains. These results highlight the potential of PL for wine microbial stabilization.

Ozonized Oleic Acid as a New Viticultural Treatment? Study of the Effect of LIQUENSO® Oxygenate on the Carpoplane Microbial Community and Wine Microorganisms Combining Metabarcoding and In Vitro Assays

In this study, an amplicon metagenomic approach was used to determine the effect of repeated treatments with ozonized oleic acid on the microbial community of grapevine carpoplane. Differences in community composition of treated vineyards were compared to non-treated and conventionally treated samples regarding the prokaryotic and eukaryotic microbiome at two developmental stages (BBCH 83, BBCH 87). The results showed effects both on occurrence and on abundance of microorganisms and the community assembly. Wine-relevant genera such as Acetobacter and members of the former genus Lactobacillus could be identified as part of the natural microbiota. The impact of the new viticultural treatment on these organisms was assessed in liquid culture-based microtiter assays. Therefore, we investigated an array of two acetic acid bacteria (AAB), four lactic acid bacteria (LAB) and nine saccharomyces and non-saccharomyces yeasts. Brettanomyces bruxellensis, Saccharomyces cerevisiae, Pediococcus sp. and Acetobacter aceti revealed the highest sensitivities against ozonized oleic acid (LIQUENSO® Oxygenat). Culture growth of these organisms was significantly reduced at an ozonide concentration of 0.25% (v/v), which corresponded to a quarter of the concentration used in the vineyard. The metabarcoding approach in combination with complementary in vitro assays allow new insights into treatment effects on the community and species scale.

Wine Microbial Consortium: Seasonal Sources and Vectors Linking Vineyard and Winery Environments

Winemaking involves a wide diversity of microorganisms with different roles in the process. The wine microbial consortium (WMC) includes yeasts, lactic acid bacteria and acetic acid bacteria with different implications regarding wine quality. Despite this technological importance, their origin, prevalence, and routes of dissemination from the environment into the winery have not yet been fully unraveled. Therefore, this study aimed to evaluate the WMC diversity and incidence associated with vineyard environments to understand how wine microorganisms overwinter and enter the winery during harvest. Soils, tree and vine barks, insects, vine leaves, grapes, grape musts, and winery equipment were sampled along four seasons. The isolation protocol included: (a) culture-dependent microbial recovery; (b) phenotypical screening to select fermenting yeasts, lactic acid, and acetic acid bacteria; and (c) molecular identification. The results showed that during all seasons, only 11.4% of the 1424 isolates presumably belonged to the WMC. The increase in WMC recovery along the year was mostly due to an increase in the number of sampled sources. Acetic acid bacteria (Acetobacter spp., Gluconobacter spp., Gluconoacetobacter spp.) were mostly recovered from soils during winter while spoilage lactic acid bacteria (Leuconostoc mesenteroides and Lactobacillus kunkeii) were only recovered from insects during véraison and harvest. The fermenting yeast Saccharomyces cerevisiae was only isolated from fermented juice and winery equipment. The spoilage yeast Zygosaccharomyces bailii was only recovered from fermented juice. The single species bridging both vineyard and winery environments was the yeast Hanseniaspora uvarum, isolated from insects, rot grapes and grape juice during harvest. Therefore, this species appears to be the best surrogate to study the dissemination of the WMC from vineyard into the winery. Moreover, the obtained results do not evidence the hypothesis of a perennial terroir-dependent WMC given the scarcity of their constituents in the vineyard environment along the year and the importance of insect dissemination.

Silicon carbide (SiC) membranes in œnology

In a winery, clarification and stabilization steps by crossflow microfiltration are limited by fouling phenomenon which reduces production flows and can alter the aromatic potential of wines, forcing professionals to use several successive filtration steps. The objectives of this industrial-scale study are to (i) introduce SiC membranes at different vinification steps (ii) evaluate the hydraulic and retention performances of SiC membranes and (iii) carry out a comparative study between SiC membranes and polymeric hollow fibers commonly used in the wine industry. It appears that SiC membranes are able to filter diverse matrices with very satisfactory production flow and retention efficiency. This particular process was optimized by developing an efficient regeneration protocol. The clarification and stabilization of the matrices by SiC membranes allowed (i) the production of clear and brilliant wines, (ii) the retention of wine microorganisms, (iii) higher production flows than hollow fibers, while conserving wines interest compounds, (iv) and the filtration of matrices that cannot be processed by hollow fibers.

Discovering the Influence of Microorganisms on Wine Color.

Flavor, composition and quality of wine are influenced by microorganisms present on the grapevine surface which are transferred to the must during vinification. The microbiota is highly variable with a prevalence of non-Saccharomyces yeasts, whereas Saccharomyces cerevisiae is present at low number. For wine production an essential step is the fermentation carried out by different starter cultures of S. cerevisiae alone or in mixed fermentation with non-Saccharomyces species that produce wines with significant differences in chemical composition. During vinification wine color can be influenced by yeasts interacting with anthocyanin. Yeasts can influence wine phenolic composition in different manners: direct interactions—cell wall adsorption or enzyme activities—and/or indirectly—production of primary and secondary metabolites and fermentation products. Some of these characteristics are heritable trait in yeast and/or can be strain dependent. For this reason, the stability, aroma, and color of wines depend on strain/strains used during must fermentation. Saccharomyces cerevisiae or non-Saccharomyces can produce metabolites reacting with anthocyanins and favor the formation of vitisin A and B type pyranoanthocyanins, contributing to color stability. In addition, yeasts affect the intensity and tonality of wine color by the action of β-glycosidase on anthocyanins or anthocyanidase enzymes or by the pigments adsorption on the yeast cell wall. These activities are strain dependent and are characterized by a great inter-species variability. Therefore, they should be considered a target for yeast strain selection and considered during the development of tailored mixed fermentations to improve wine production. In addition, some lactic acid bacteria seem to influence the color of red wines affecting anthocyanins’ profile. In fact, the increase of the pH or the ability to degrade pyruvic acid and acetaldehyde, as well as anthocyanin adsorption by bacterial cells are responsible for color loss during malolactic fermentation. Lactic acid bacteria show different adsorption capacity probably because of the variable composition of the cell walls. The aim of this review is to offer a critical overview of the roles played by wine microorganisms in the definition of intensity and tonality of wines’ color.

The inhibition of wine microorganisms by silver nanoparticles

The presented work aimed to study the inhibition using nanoparticles produced by the green synthesis in selected acetic acid and lactic acid bacteria, which are related to viticulture. The degree of ability to eliminate silver particles produced by green syntheses was determined using the plate method on Petri dishes. This is done using two different approaches - the method of direct application of the solution to the surface of the inoculated medium (determination of inhibition zones) and the method of application using nanoparticles to the inoculated medium. Gluconobacter oxydans (CCM 3618) and Acetobacter aceti (CCM 3620T) were studied from acet acetic bacteria. The lactic acid bacteria were Lactobacillus brevis (CCM 1815) and Pediococcus damnosus (CCM 2465). The application of silver nanoparticles was always in concentrations of 0, 0.0625, 0.125, 0.25, 0.5, and 1 g.L-1. All applied concentrations of silver nanoparticles showed an inhibitory effect on the monitored microorganisms. Silver particles could be used in wine technology for their antibacterial effects, mainly to inhibit microorganisms during vinification, as a substitute for sulfur dioxide.

Effects of ultrasound treatments on wine microorganisms

Ultrasound is one of the most promising non-thermal an emerging technique in food technology. The objective of the present work was to evaluate the effect of different ultrasonic treatments on the most important wine microbiota (Saccharomyces and non-Saccharomyces yeasts and lactic acid bacteria). Two stages were carried out: the assessment step, where six different ultrasonic treatments (with varying power, time, and pulses) were used on Saccharomyces cerevisiae, Brettanomyces spp., and Lactiplantibacillus plantarum; and the validation step, where two chosen ultrasonic treatments were used on Zigosaccharomyces bailli, Brettanomyces spp., Saccharomyces cerevisiae, Saccharomyces bayanus, Pichia membranifaciens, Schizosaccharomyces pombe, and Hanseniaspora osmophila. The most sensitive microorganism was Brettanomyces spp., and the most resistant was Lactiplantibacillus plantarum. Ultrasonic treatments had varying effects on vitality (delay of growth or maximum OD reduction) and on viability (reduction of microbial growth).

QTL mapping: an innovative method for investigating the genetic determinism of yeast-bacteria interactions in wine.

The two most commonly used wine microorganisms, Saccharomyces cerevisiae yeast and Oenococcus oeni bacteria, are responsible for completion of alcoholic and malolactic fermentation (MLF), respectively. For successful co-inoculation, S. cerevisiae and O. oeni must be able to complete fermentation; however, this relies on compatibility between yeast and bacterial strains. For the first time, quantitative trait loci (QTL) analysis was used to elucidate whether S. cerevisiae genetic makeup can play a role in the ability of O. oeni to complete MLF. Assessment of 67 progeny from a hybrid S. cerevisiae strain (SBxGN), co-inoculated with a single O. oeni strain, SB3, revealed a major QTL linked to MLF completion by O. oeni. This QTL encompassed a well-known translocation, XV-t-XVI, that results in increased SSU1 expression and is functionally linked with numerous phenotypes including lag phase duration and sulphite export and production. A reciprocal hemizygosity assay was performed to elucidate the effect of the gene SSU1 in the SBxGN background. Our results revealed a strong effect of SSU1 haploinsufficiency on O. oeni's ability to complete malolactic fermentation during co-inoculation and pave the way for the implementation of QTL mapping projects for deciphering the genetic bases of microbial interactions. KEY POINTS: • For the first time, QTL analysis has been used to study yeast-bacteria interactions. • A QTL encompassing a translocation, XV-t-XVI, was linked to MLF outcomes. • S. cerevisiae SSU1 haploinsufficiency positively impacted MLF by O. oeni.

Secondary Aroma: Influence of Wine Microorganisms in Their Aroma Profile.

Aroma profile is one of the main features for the acceptance of wine. Yeasts and bacteria are the responsible organisms to carry out both, alcoholic and malolactic fermentation. Alcoholic fermentation is in turn, responsible for transforming grape juice into wine and providing secondary aromas. Secondary aroma can be influenced by different factors; however, the influence of the microorganisms is one of the main agents affecting final wine aroma profile. Saccharomyces cerevisiae has historically been the most used yeast for winemaking process for its specific characteristics: high fermentative metabolism and kinetics, low acetic acid production, resistance to high levels of sugar, ethanol, sulfur dioxide and also, the production of pleasant aromatic compounds. Nevertheless, in the last years, the use of non-saccharomyces yeasts has been progressively growing according to their capacity to enhance aroma complexity and interact with S. cerevisiae, especially in mixed cultures. Hence, this review article is aimed at associating the main secondary aroma compounds present in wine with the microorganisms involved in the spontaneous and guided fermentations, as well as an approach to the strain variability of species, the genetic modifications that can occur and their relevance to wine aroma construction.

Potential Use of Silica Nanoparticles for the Microbial Stabilisation of Wine: An In Vitro Study Using Oenococcus oeni as a Model.

The emerging trend towards the reduction of SO2 in winemaking has created a need to look for alternative methods to ensure the protection of wine against the growth of undesired species of microorganisms and to safely remove wine microorganisms. This study describes the possible application of silica nanospheres as a wine stabilisation agent, with Oenococcus oeni (DSM7008) as a model strain. The experiment was conducted firstly on model solutions of phosphate-buffered saline and 1% glucose. Their neutralising effect was tested under stirring with the addition of SiO2 (0.1, 0.25, and 0.5 mg/mL). Overall, the highest concentration of nanospheres under continuous stirring resulted in the greatest decrease in cell counts. Transmission electron microscope (TEM) and scanning electron microscopy (SEM) analyses showed extensive damage to the bacterial cells after stirring with silica nanomaterials. Then, the neutralising effect of 0.5 mg/mL SiO2 was tested in young red wine under stirring, where cell counts were reduced by over 50%. The obtained results suggest that silica nanospheres can serve as an alternative way to reduce or substitute the use of sulphur dioxide in the microbial stabilisation of wine. In addition, further aspects of following investigations should focus on the protection against enzymatic and chemical oxidation of wine.

A simple procedure for detecting Dekkera bruxellensis in wine environment by RNA-FISH using a novel probe

Dekkera bruxellensis, considered the major microbial contaminant in wine production, produces 4-ethylphenol, a cause of unpleasant odors. Thus, identification of this yeast before wine spoilage is crucial. Although challenging, it could be achieved using a simple technique: RNA-FISH. To reach it is necessary to design probes that allow specific detection/identification of D. bruxellensis among the wine microorganisms and in the wine environment and, if possible, using low formamide concentrations. Therefore, this study was focused on: a) designing a DNA-FISH probe to identify D. bruxellensis that matches these requirements and b) determining the applicability of the RNA-FISH procedure after the end of the alcoholic fermentation and in wine.A novel DNA-FISH D. bruxellensis probe with good performance and specificity was designed. The application of this probe using an in-suspension RNA-FISH protocol (applying only 5% of formamide) allowed the early detection/identification of D. bruxellensis at low cell densities (5 × 102 cell/mL). This was possible by flow cytometry independently of the growth stage of the target cells, both at the end of the alcoholic fermentation and in wine even in the presence of high S. cerevisiae cell densities.Thus, this study aims to contribute to facilitate the identification of D. bruxellensis before wine spoilage occurs, preventing economic losses to the wine industry.