Brettanomyces Bruxellensis Strains Research Articles

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.

What kind of sanitation should be applied to remove <em>Brettanomyces bruxellensis</em> biofilms?

The capacity for five Brettanomyces bruxellensis strains to form biofilm on stainless steel was confirmed, and the sanitation of these biofilms was tested using a solution of lactic acid and a reference method (a solution of foaming caustic soda and peroxide at 5 %). Different responses were observed depending on the strain: lactic acid solution induced a slight reduction in cell population, while the reference method resulted in the elimination of the adhered cells for three strains, but generated VBNC states for two others. The effects of sanitation on the biofilm formed is strain-dependent.

Biodiversity among Brettanomyces bruxellensis Strains Isolated from Different Wine Regions of Chile: Key Factors Revealed about its Tolerance to Sulphite.

Brettanomyces bruxellensis is regarded as the main spoilage microorganism in the wine industry, owing to its production of off-flavours. It is difficult to eradicate owing to its high tolerance of adverse environmental conditions, such as low nutrient availability, low pH, and high levels of ethanol and SO2. In this study, the production of volatile phenols and the growth kinetics of isolates from various regions of Chile were evaluated under stressful conditions. Through randomly amplified polymorphic DNA (RAPD) analysis, 15 strains were identified. These were grown in the presence of p-coumaric acid, a natural antimicrobial and the main precursor of off-flavours, and molecular sulfur dioxide (mSO2), an antimicrobial synthetic used in the wine industry. When both compounds were used simultaneously, there were clear signs of an improvement in the fitness of most of the isolates, which showed an antagonistic interaction in which p-coumaric acid mitigates the effects of SO2. Fourteen strains were able to produce 4-vinylphenol, which showed signs of phenylacrylic acid decarboxylase activity, and most of them produced 4-ethylphenol as a result of active vinylphenol reductase. These results demonstrate for the first time the serious implications of using p-coumaric acid, not only for the production of off-flavours, but also for its protective action against the toxic effects of SO2.

Simultaneous Detection of Multiple Wine-Spoilage Organisms Using a PCR-Based DNA Dipstick Assay.

Background: The presence of microbial contaminants such as Brettanomyces in wine can lead to undesirable wine. Therefore, monitoring for the presence of these spoilage organisms is critical for winemakers to ensure the quality of their end product. Objective: To address this problem, Molecular Epidemiology, Inc. (MEI, Seattle, WA) has developed a wine-spoilage organism detection kit consisting of a multiplex PCR DNA dipstick that simultaneously detects these organisms. Methods: Wine samples obtained from local wineries that tested negative by routine microbiological culture were spiked with the target microorganisms, while samples that were designated as spoiled by the wineries were used as-is without spiking for assessing the performance characteristics of the DNA dipstick assay. Microbial enumeration was performed following standard microbiological plating methods. Samples spiked with low cell numbers (<5 cells per 100 mL) were enriched using wine enrichment media (WSE; optional component of the kit) prior to analysis using the DNA dipstick assay. Suitability of WSE medium to support the growth of wine-spoilage microorganisms was compared with standard microbiological media. Results: Testing of 92 diverse bacterial and yeast strains commonly found in winery and food operations and 50 various strains of spoilage organisms isolated from wineries indicated that the dipstick assay can exclusively detect the target wine-spoilage microorganisms. All target spoilage organisms in samples containing low cell numbers (<5 cells per 100 mL) were detected by dipstick assay 48 h postenrichment in WSE, except for a few strains of Brettanomyces bruxellensis that required longer incubation times. Conclusions: The wine-spoilage organism detection kit has a detection limit of 10 cells/mL. Highlights: The kit can be used at different stages of the wine-making process to detect multiple spoilage-causing microorganisms in a single assay, thus offering a convenient test system for winemakers interested in monitoring the quality of their product.

Sulfur dioxide response of Brettanomyces bruxellensis strains isolated from Greek wine

Brettanomyces bruxellensis is the most common spoilage wine yeast which can provoke great economic damage to the wine industry due to the production of undesirable odors. The capacity of the species to adapt in various environmental conditions offers a selective advantage that is reflected by intraspecific variability at genotypic and phenotypic level. In this study, microsatellite analysis of 22 strains isolated from Greek wine revealed the existence of distinct genetic subgroups that are correlated with their geographical origin. The response of these strains to increasing levels of sulfur dioxide confirmed the presence of both sensitive and tolerant strains, which belong to distinguished genetic clusters. The genetic categorization of B. bruxellensis strains could be used by the winemakers as a diagnostic tool regarding sulfur dioxide sensitivity.

Spoilage potential of brettanomyces bruxellensis strains isolated from Italian wines

Brettanomyces bruxellensis is an important wine spoilage agent. In this study a population of Brettanomyces strains isolated from Italian wines was thoroughly investigated to evaluate adaptability to wine conditions and spoilage potential. The presumptive isolates of Brettanomyces were identified at species level with 26S rRNA gene sequencing and species-specific PCR, and subsequently subjected to analysis of intra-species variability through the study of intron splice sites (ISS-PCR). Although, some strains were tracked in wines from different regions, extensive genetic biodiversity was observed within the B. bruxellensis population investigated. All strains were evaluated for their growth ability in the presence of ethanol, high sugar content, low pH, different temperatures and sulphur dioxide, using optical density and flow cytometry measurement. The ability of yeasts to produce ethyl phenols in red wines with different chemical compositions was evaluated by means of high performance liquid chromatography with electrochemical detection (HPLC-ECD). The results highlighted wide variability in B. bruxellensis in response to wine limiting factors and in terms of the accumulation of ethyl phenols. As regards this last aspect, the differences found among strains were closely related to chemical composition of wine and strain resistance to environmental stress factors, making a priori evaluation of risk of wine alteration quite difficult. These results suggest that strategies for the control of Brettanomyces should be tailored on the basis of strain distribution and wine characteristics.

Hydroxycinnamoyl Glucose and Tartrate Esters and Their Role in the Formation of Ethylphenols in Wine.

Synthesized p-coumaroyl and feruloyl l-tartrate esters were submitted to Brettanomyces bruxellensis strains AWRI 1499, AWRI 1608, and AWRI 1613 to assess their role as precursors to ethylphenols in wine. No evolution of ethylphenols was observed. Additionally, p-coumaroyl and feruloyl glucose were synthesized and submitted to B.bruxellensis AWRI 1499, which yielded both 4-ethylphenol and 4-ethylguaiacol. Unexpected chemical transformations of the hydroxycinnamoyl glucose esters during preparation were investigated to prevent these in subsequent synthetic attempts. Photoisomerization gave an isomeric mixture containing the trans-esters and undesired cis-esters, and acyl migration resulted in a mixture of the desired 1-O-β-ester and two additional migrated forms, the 2-O-α- and 6-O-α-esters. Theoretical studies indicated that the photoisomerization was facilitated by deprotonation of the phenol, and acyl migration is favored during acidic, nonaqueous handling. Preliminary LC-MS/MS studies observed the migrated hydroxycinnamoyl glucose esters in wine and allowed for identification of feruloyl glucose in red wine for the first time.

Influence of Oenococcus oeni and Brettanomyces bruxellensis on Hydroxycinnamic Acids and Volatile Phenols of Aged Wine

The effect of two Oenococcus oeni strains, Viniflora® CiNe™ and Viniflora® CH11, with and without cinnamoyl esterase activity, respectively, on the contents of the hydroxycinnamic acids (HCAs) p-coumaric and ferulic acid, and their respective volatile phenols, 4-ethylphenol and 4-ethylguaiacol, was investigated over six months in Cabernet Sauvignon wines inoculated with two different Brettanomyces bruxellensis strains, CBS 73 and CBS 2499. Both CiNe™ and CH11 showed growth and malolactic fermentation in the wines. There were no clear growth interactions between B. bruxellensis and O. oeni. Furthermore, B. bruxellensis did not inhibit MLF by CiNe™ or CH11. The HCA concentrations in all wines increased until 114 days of fermentation, after which they decreased from day 114 to day 180. Wines inoculated with CiNe™ had the highest concentrations of HCAs throughout the fermentations. While CiNe™ degraded more of the tartaric ester bound forms of HCAs into free HCAs, there was no significant difference in the production of volatile phenols as compared with wines inoculated with CH11. However, there was a significant difference between wines with the two strains of B. bruxellensis in the level of volatile phenols and HCAs on day 180. Thus, the level of volatile phenols in wine depends more on strain differences of B. bruxellensis than on cinnamoyl esterase activity of O. oeni.

Growth and volatile phenol production by Brettanomyces bruxellensis in different grapevine varieties during fermentation and in finished wine

Three red grapevine varieties, Touriga Nacional, Syrah and Cabernet Sauvignon, were evaluated for their susceptibility to spoilage by two strains of Brettanomyces bruxellensis during grape juice fermentation and in finished wine. B. bruxellensis ISA 2211 survived well during both alcoholic and malolactic fermentations. The 4-ethylphenol concentration at the end of malolactic fermentation was 176 µg L−1 for T. Nacional, 190 µg L−1 for C. Sauvignon and 149 µg L−1 for Syrah. These levels were slightly increased during 3 months of incubation, even though no culturable cells of B. bruxellensis could be detected after malolactic fermentation. However, a viable but non-culturable (VBNC) population of 3–4 log cells mL−1 was observed in all grape varieties when analyzed by flow cytometry. On the other hand, commercial varietal wines, inoculated with of B. bruxellensis ISA 1791, could attain levels of 6–8 log CFU mL−1 and 4-ethylphenol reached 1750, 1512 and 2707 µg L−1 in T. Nacional, C. Sauvignon and Syrah, respectively. Overall, the production of 4-ethylphenol was mostly dependent on the existence of growing B. bruxellensis populations, regardless of the grape variety. In finished wines, 4-ethylphenol was always produced in concentrations far above its preference threshold, indicating that T. Nacional, Syrah and C. Sauvignon are equally susceptible to the “horse sweat” taint.

Comparative phenomics and targeted use of genomics reveals variation in carbon and nitrogen assimilation among different Brettanomyces bruxellensis strains

Recent studies have suggested a correlation between genotype groups of Brettanomyces bruxellensis and their source of isolation. To further explore this relationship, the objective of this study was to assess metabolic differences in carbon and nitrogen assimilation between different B. bruxellensis strains from three beverages, including beer, wine, and soft drink, using Biolog Phenotype Microarrays. While some similarities of physiology were noted, many traits were variable among strains. Interestingly, some phenotypes were found that could be linked to strain origin, especially for the assimilation of particular α- and β-glycosides as well as α- and β-substituted monosaccharides. Based upon gene presence or absence, an α-glucosidase and β-glucosidase were found explaining the observed phenotypes. Further, using a PCR screen on a large number of isolates, we have been able to specifically link a genomic deletion to the beer strains, suggesting that this region may have a fitness cost for B. bruxellensis in certain fermentation systems such as brewing. More specifically, none of the beer strains were found to contain a β-glucosidase, which may have direct impacts on the ability for these strains to compete with other microbes or on flavor production.

Brettanomyces bruxellensisAroma-Active Compounds Determined by SPME GC-MS Olfactory Analysis

A survey of 95 Brettanomyces strains was undertaken to identify strains that consistently give positive aroma characteristics (e.g., spicy, fruity, and floral). These strains were grown in a defined medium, and five human evaluators rated each strain according to aroma quality, and their ratings were coupled with a solid phase micro-extraction with gas chromatography (SPME GC-MS) analysis. None of the strains yielded universally positive aromas for the evaluators. A further characterization of nine of these strains grown with both aromatic amino acids (phenylalanine, tryptophan, and tyrosine) and hydroxycinnamic acids (caffeic, p-coumaric, and ferulic acids) indicated that low levels of compounds that were most important in differentiating the strains may contribute to a positive sensorial perception of Brettanomyces strain aromas under these conditions. To define the components associated with positive Brettanomyces character, the volatile aroma compounds produced by five Brettanomyces bruxellensis strains were analyzed. Compound detecting in SPME GC-MS was coupled with olfactory analysis (SPME GC-MS-O), using nine individual evaluators to identify compounds most associated with Brettanomyces character and to assess the breadth of descriptive terms used by different individuals for the same compound. Twenty-two compounds were identified as having an impact on aroma, including the well-known ethylphenols and vinylphenols, as well as several fatty acids, alcohols, esters, terpenes, and an aldehyde.

Inactivation of Brettanomyces bruxellensis by High Hydrostatic Pressure technology

High Hydrostatic Pressure (HHP) technology was tested on two strains of Brettanomyces bruxellensis isolated from Rioja red wines for analyzing the microbial inactivation reached in different oenological conditions. With this purpose, these strains were inoculated in synthetic wine with different pH and ethanol content. The inactivations reached with 100 MPa, 200 MPa and 300 MPa applied from one to seven minutes were tested just after the treatment and after a week.Current consumers demand quality, free-additive wines. This has made oenological industry search alternative non-thermal technologies. The present results supported that high pH and high ethanol wines shortly treated at 100 MPa made B. bruxellensis undetectable. Moreover, low pH and low ethanol wines content should be at least treated at 200 MPa, and wines with intermediate ethanol content should be carefully analyzed in terms of strain composition before applying HHP. Finally, treatments at 300 MPa managed a complete inactivation regardless of oenological conditions.

REMOVAL OFBRETTANOMYCES BRUXELLENSISFROM RED WINE USING MEMBRANE FILTRATION

ABSTRACT While sulfites help limit growth of the spoilage yeast, Brettanomyces, SO2 has been reported to decrease cell size, thereby potentially decreasing the porosities of filtration membranes required for removal. Brettanomyces bruxellensis strains B1b and F3 were inoculated into red wines and after 12 days, half of the wines received ≈0.5 mg/L molecular SO2 prior to filtration on day 20. Strain B1b was retained by filtration through 1.2 µm membranes from wines with or without SO2. In contrast, two of three filtration replicates of wines inoculated with F3 exhibited growth approximately 40–50 days after filtration through the same porosity. However, this strain was removed from wines using 0.8 µm membranes. Results indicate that Brettanomyces may enter a “viable-but-not-culturable” physiological state upon exposure to SO2. While culturability and morphology of B. bruxellensis was affected by SO2 exposure, this factor did not impact removal of this yeast by membrane filtration. PRACTICAL APPLICATIONS Cells of B. bruxellensis have been observed to decrease in size upon exposure to sulfites (SO2), thereby possibly affecting filtration recommendations for its removal from red wines. Though culturability on plate count agar and microscopic morphology was influenced by SO2, use of membrane filters with porosities ≤0.8 µm removed the spoilage yeast from red wines. In those situations where a larger membrane porosity is desired, additional SO2 must be added to curtail potential spoilage by cells of Brettanomyces that pass through the filtration medium.

Preliminary evaluation of infrared spectroscopy for the differentiation of Brettanomyces bruxellensis strains isolated from red wines

The objective of this study was to evaluate different infrared spectroscopy methods in combination with chemometrics for the differentiation between Brettanomyces bruxellensis strains. These methods of discrimination were applied to intact yeast cells of B. bruxellensis strains and on wines spoiled by the same strains. Eleven wine isolates of B. bruxellensis were evaluated for volatile phenol production in red wine and their genetic diversity was determined by Restriction Endonuclease Analysis–Pulsed Field Gel Electrophoresis (REA-PFGE). Fourier transform mid-infrared (FTMIR) spectroscopy was used to obtain spectral fingerprints of the spoiled wines. Attenuated total reflectance (ATR) was used to obtain spectral fingerprints from the intact cells of the 11 B. bruxellensis strains. The groupings from the genetic fingerprints obtained with REA–PFGE were used as reference firstly for comparison with the groupings observed with the FTMIR spectral fingerprint of the wines and secondly for the FTIR-ATR spectral fingerprints from the whole cells. Results indicated that ATR-IR spectra obtained by scanning whole cells of B. bruxellensis could be useful for rapid strain typing in comparison or complementary to molecular techniques and FTMIR spectra from wines provide a useful resource for the discrimination between B. bruxellensis contaminated wines.

Sulphur dioxide affects culturability and volatile phenol production by Brettanomyces/ Dekkera bruxellensis

The effect of different sulphur dioxide concentrations on culturability and viability of seven strains of Brettanomyces bruxellensis was tested in a synthetic wine medium (SWM) and a different response to molecular SO 2 among strains was detected. Sulphur dioxide induced a viable but non culturable (VBNC) state in all the strains. The greater percentage of VBNC cells were identified for five strains at molecular SO 2 concentrations of 0.2 mg/L and for two strains at the concentration of 0.4 mg/L. Vinyl phenols were detected in media containing VBNC or not viable B. bruxellensis, suggesting that its spoilage metabolism could be maintained during wine storage. Overall, this study indicates that SO 2 is a chemical stressor inducing VBNC state in B. bruxellensis grown in synthetic wine medium . Further studies are needed to evaluate the effects of SO 2 on the metabolism of this yeast in wine spoilage.

Genetic diversity and physiological traits of Brettanomyces bruxellensis strains isolated from Tuscan Sangiovese wines

Eighty four isolates of Brettanomyces bruxellensis, were collected during fermentation of Sangiovese grapes in several Tuscan wineries and characterized by restriction analysis of 5.8S-ITS and species-specific PCR. The isolates were subsequently analysed, at strain level, by the combined use of the RAPD-PCR assay with primer OPA-02 and the mtDNA restriction analysis with the HinfI endonuclease. This approach showed a high degree of polymorphism and allowed to identify seven haplotypes, one of them being the most represented and widely distributed (72 isolates, 85.7%). Physiological traits of the yeasts were investigated under a wine model condition. Haplotypes clustered into two groups according to their growth rates and kinetics of production of 4-ethylphenol and 4-ethylguaiacol. Hexylamine was the biogenic amine most produced (up to 3.92 mg l − 1 ), followed by putrescine and phenylethylamine. Formation of octapamine was detected by some haplotypes, for the first time.

Molecular identification of Brettanomyces bruxellensis strains isolated from red wines and volatile phenol production

The spoilage yeast Brettanomyces/Dekkera can persist throughout the winemaking process and has the potential to produce off-flavours that affect the sensory quality of wine. The main objective of this study was to select different strains of Brettanomyces bruxellensis isolated from red wines and to compare their volatile phenol production. From a collection of 63 strains, eight strains of B. bruxellensis were selected for volatile phenol production after the application of molecular techniques such as ISS–PCR, PCR–DGGE and REA–PFGE. All strains showed three large chromosomes of similar size with PFGE. However, unique restriction profiles of the chromosomes were visible after NotI digestion that clearly distinguished the strains. All strains were capable of producing large quantities of 4-ethylphenol and 4-ethylguaiacol from p-coumaric acid and ferulic acid, respectively in synthetic media. However, the diversity among strains for volatile phenol production differed between synthetic media and wine with regard to the maximum production levels of 4-ethylphenol and 4-ethylguaiacol. This study illustrated the diversity of B. bruxellensis strains that occur during winemaking.

Diversity in Spoilage Yeast Dekkera/Brettanomyces bruxellensis Isolated from French Red Wine. Assessment During Fermentation of Synthetic Wine Medium

Yeast Brettanomyces bruxellensis is a contaminant found worldwide and is responsible for red wine spoilage due to the development of animal and phenolic off-odours. During this study, 24 Brettanomyces bruxellenis isolates were obtained from red wine samples from two French wineries and these were discriminated as 23 strains. Nine strains coming from 2 wineries and 4 vintages were cultivated in synthetic wine medium for 1500 hours and they gave nine different behaviours. Four main growth patterns (with different growth steps and durations) and three main different sugar consumption profiles were obtained. Glucose and fructose were not limiting substrates for all strains. The production level of 4-ethylphenol was found to vary from strain to strain (from 0.350 to 2.773 mg L−1) and was independent of the biomass concentration. Some strains presented a coupled-to-growth production of volatile phenols, others did not. This study showed that different strains of Brettanomyces bruxellensis behaved differently, one from another, under the given conditions taking into consideration several aspects. The results thus demonstrate a large intraspecific diversity.

Development of a molecular method for the typing of Brettanomyces bruxellensis (Dekkera bruxellensis) at the strain level

In recent years, Brettanomyces/Dekkera bruxellensis has caused increasingly severe quality problems in the wine industry. A typing method at the strain level is needed for a better knowledge of the dispersion and the dynamics of these yeasts from grape to wine. Three molecular tools, namely random-amplified polymorphic DNA, PCR fingerprinting with microsatellite oligonucleotide primers and SAU-PCR, were explored for their relevance to typing strains of Brettanomyces bruxellensis. The results indicated that discrimination of each individual strain was not possible with a single PCR typing technique. We described a typing method for B. bruxellensis based on restriction enzyme analysis and pulse field gel electrophoresis (REA-PFGE). Results showed that electrophoretic profiles were reproducible and specific for each strain under study. Consequently, REA-PFGE should be considered for the discrimination of B. bruxellensis strains. This technique allowed a fine discrimination of B. bruxellensis, as strains were identified by a particular profile. This study constitutes a prerequisite for accurate and appropriate investigations on the diversity of strains throughout the winemaking and ageing process. Such studies will probably give clearer and more up-to-date information on the origin of the presence of Brettanomyces in wine after vinification when they are latent spoilage agents.