The effect of hydroxycinnamic acids on growth and H+-ATPase activity of the wine spoilage yeast, Dekkera bruxellensis

Abstract

Hydroxycinnamic acids are lipophilic compounds naturally present in grape must, and proposed to have antimicrobial properties. Consequently, microorganisms that grow in media containing these acids must have efficient adaptation mechanisms. In Saccharomyces cerevisiae hydroxycinnamic acids enter into the cell where they are deprotonated causing a decrease in internal pH, this variation in the intracellular pH is counteracted by an increase in the activity of the H + -ATPase pump Pma1p. Dekkera bruxellensis however, is able to transform hydroxycinnamic acids into volatile-less toxic derivates, a mechanism used by few yeast species. Nonetheless, D. bruxellensis could also have an adaptation mechanism similar to that of S. cerevisiae. Our results showed that hydroxycinnamic acids caused a longer lag phase during D. bruxellensis growth, particularly when supplementing media with ferulic acid. Additionally, extracellular pH decreased while Pma1p activity increased during lag phase in media supplemented with p-coumaric acid. These results suggest the existence of a complementary mechanism of resistance to hydroxycinammic acids in D. bruxellensis which involves the H + -ATPase pump Pma1p.

Comparative transcriptome assembly and genome-guided profiling for Brettanomyces bruxellensis LAMAP2480 during p-coumaric acid stress

“…2 ). This is similar to what has been previously reported for B. bruxellensis LAMAP1359 when grown in the presence of p -coumaric acid 14 . In the presence of weak acids, the role of Pma1p is vital to restore homeostasis 20 42 43 , which exerts a high energy demand, consuming until 60% of the total cellular ATP 20 , causing ATP levels to reduce to a point where growth rate declines 20 44 45 .…”

“…P-coumaric effect on B. bruxellensis LAMAP2480 growth was evaluated according to previous protocols developed in our laboratory with slight modifications 14 . The kinetic parameters evaluated were: specific growth rate (μ) 93 , Lag phase duration 94 , and efficiency was defined as area under curve (AUC) and expressed as a percentage using as 100% the control condition.…”

“…In order to describe the response of B. bruxellensis LAMAP2480 to p -coumaric stress, we evaluated seven physiological parameters: extracelullar pH, p -coumaric acid, 4-vinylphenol, 4-ethylphenol, acetic acid and glucose levels, and Pma1 enzyme activity. Extracellular pH was evaluated using previously described methods 22 , with adaptations to our local conditions and culture media 14 . p -coumaric acid, 4-vinylphenol, 4-ethylphenol, acetic acid and glucose were quantified by high performance liquid chromatography (HPLC) (Shimadzu Scientific Instruments, Colombia, MD, USA) using a Shimazdu Shim-Pack VP-ODS column 96 for p -coumaric acid, 4-vinylphenol and 4-ethylphenol, and a Bio-Rad HPX87H column 97 for acetic acid and glucose.…”

“…p -coumaric acid, 4-vinylphenol, 4-ethylphenol, acetic acid and glucose were quantified by high performance liquid chromatography (HPLC) (Shimadzu Scientific Instruments, Colombia, MD, USA) using a Shimazdu Shim-Pack VP-ODS column 96 for p -coumaric acid, 4-vinylphenol and 4-ethylphenol, and a Bio-Rad HPX87H column 97 for acetic acid and glucose. Finally, plasma membrane ATPase Pma1p activity was estimated by calculating the rate of phosphate production after ATP hydrolysis 98 , as previously described 14 .…”

“…The presence of hydroxycinnamic acids in the culture medium can inhibit the growth of a wide range of microorganisms. However, this inhibition depends on the acid concentration 5 9 10 11 12 and the type of molecule, with p -coumaric and ferulic acids showing the greater degree of inhibition 5 6 9 13 14 . The concentrations that can inhibit B. bruxellensis growth have been previously studied 6 15 , as well as the mechanisms involved 16 17 18 .…”