Effect of lactic acid bacteria growth on Brettanomyces bruxellensis growth and production of ethylphenols

The yeast Brettanomyces bruxellensis is the most important wines spoilage yeast encountered during winemaking as it can survive in wine for long periods, requires minimal nutrients for growth, and can be difficult to control. Brettanomyces produces two major spoilage products, the volatile phenols 4-ethylphenol (Band-Aid, medicinal smell) and 4-ethylguaiacol (smoke, clove smell) by decarboxylation and subsequent reductions of the hydroxycinnamic acids p-coumaric acid and ferulic acid. Hydroxycinnamic acids are naturally present in grapes and wine and are also often present in the form of tartaric acid- hydroxycinnamic acid esters which B. bruxellensis cannot utilize. The first objective of this study was therefore to investigate the ability of other wine microorganisms to hydrolyze tartaric acid bound hydroxycinnamic acids and the impact this may have on volatile phenol production by Brettanomyces. Of the thirty five strains of wine microorganisms tested only one, the commercial strain O. oeni VFO, hydrolyzed tartaric acid bound hydroxycinnamic acids in wine and increased the concentrations of the free hydroxycinnamic acids p- coumaric acid and ferulic acid. Because of this, B. bruxellensis produced significantly higher concentrations of 4-ethylphenol and 4-ethylguiaciol when growing in wine where VFO had  conducted the malolactic fermentation. In contrast, wines that underwent MLF with O. oeni Alpha or VP41 contained similar 4-EP and 4-EG concentrations to the control. A subsequent study investigated interactions between the wine spoilage yeast Brettanomyces bruxellensis and wine lactic acid bacteria (LAB) and the impact on Brettanomyces growth and volatile phenol production. Studies in acidic grape juice (AGJ) broth (pH 3.50, 5% ethanol) demonstrated that growth of O. oeni could inhibit Brettanomyces growth in a strain dependent manner. Production of 4-ethyphenol was also delayed and reduced in a strain dependent manner. For example, in the control a maximum of 19 mg/L 4-EP was produced by B. bruxellensis UCD-2049 while during growth in media where VP41 had previously grown only 7.9 mg/L 4-EP was produced. When B. bruxellensis was inoculated into Pinot noir wine where malolactic fermentation (MLF) had been performed by O. oeni VFO, populations of B. bruxellensis quickly decreased below detectable levels and did not recover during the course of the experiment (50 days). In contrast, when B. bruxellensis UCD-2049 was inoculated into Pinot noir wine where Pediococcus damnosus OW2 had previously grown populations did not decrease and growth occurred in a similar manner to the control. The reason for this decrease in population was further explored by inoculating B. bruxellensis into AGJ broth (pH 3.50) where there had been previous growth of O. oeni Alpha or VP41. In half the treatments the O. oeni were removed by sterile filtration prior to Brettanomyces inoculation. Although there was delayed growth of B. bruxellensis in media where O. oeni had previously grown there was no significant difference between growth in sterile filtered and non-sterile filtered treatments. The relief of inhibition by sterile filtration suggests that neither nutrient depletion nor the production of an extracellular inhibitory compound by O. oeni were the causes of Brettanomyces inhibition, as sterile filtration would not have impacted these. Future work in this field should include screening a larger and more diverse group of wine microorganisms for the ability to hydrolyze tartaric ester bound hydroxycinnamic acids. This should include non-Saccharomyces yeast such as Kloeckera that are known to be present during early fermentation as well as spoilage bacteria such as Lactobacillus. In addition, the inhibition of B. bruxellensis should be explored further by testing O. oeni against a larger number of B. bruxellensis strains and additional mechanisms of inhibition should be investigated

Metabolism of Nonesterified and Esterified Hydroxycinnamic Acids in Red Wines by Brettanomyces bruxellensis

While Brettanomyces can metabolize nonesterified hydroxycinnamic acids found in grape musts/wines (caffeic, p-coumaric, and ferulic acids), it was not known whether this yeast could utilize the corresponding tartaric acid esters (caftaric, p-coutaric, and fertaric acids, respectively). Red wines from Washington and Oregon were inoculated with B. bruxellensis, while hydroxycinnamic acids were monitored by HPLC. Besides consuming p-coumaric and ferulic acids, strains I1a, B1b, and E1 isolated from Washington wines metabolized 40−50% of caffeic acid, a finding in contrast to strains obtained from California wines. Higher molar recoveries of 4-ethylphenol and 4-ethylguaiacol synthesized from p-coumaric and ferulic acids, respectively, were observed in Washington Cabernet Sauvignon and Syrah but not Merlot. This finding suggested that Brettanomyces either (a) utilized vinylphenols formed during processing of some wines or (b) metabolized other unidentified phenolic precursors. None of the strains of Brettanomyces studied metabolized caftaric or p-coutaric acids present in wines from Washington or Oregon.Keywords: Volatile phenols, Cinnamates, Hydroxycinnamic acids, Brettanomyces, Phenolic acidsKeywords: Volatile phenols, Cinnamates, Hydroxycinnamic acids, Brettanomyces, Phenolic acid