Appl Microbiol Biotechnol (2010) 86:721–729 DOI 10.1007/s00253-009-2352-6
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Inhibitory effect of hydroxycinnamic acids on Dekkera spp. Victoria Harris & Vladimir Jiranek & Christopher M. Ford & Paul R. Grbin
Received: 2 September 2009 / Revised: 12 October 2009 / Accepted: 9 November 2009 / Published online: 2 December 2009 # Springer-Verlag 2009
Abstract Simple phenolic components of wine, hydroxycinnamic acids (HCAs) are known to have antimicrobial properties. This study sought to determine the potential of ferulic acid as an antifungal agent for the control of Dekkera. Growth was inhibited by all HCAs examined in this study, with ferulic acid being the most potent at all concentrations. In the presence of ethanol, the inhibitory effects of ferulic acid were amplified. Scanning electron microscopy images reveal cellular damage upon exposure to ferulic acid. Thus, manipulation of ferulic acid concentrations could be of industrial significance for control of Dekkera and may be the basis for differences in susceptibility of wines to Dekkera spoilage. Keywords Antimicrobial . Brettanomyces . Spoilage . Scanning electron microscopy . Phenolic acids . Wine
Introduction Dekkera yeasts are often considered undesirable organisms. Dekkera bruxellensis is of particular concern within the wine industry, due to the variety and potency of the negative impacts it imparts (Grbin and Henschke 2000; Grbin et al. 2007; Loureiro and Malfeito-Ferreira 2003; Silva et al. 2004; van der Walt and van Kerken 1959). In addition, the yeast displays tolerance to low pH and high concentrations of ethanol and sulphur dioxide, which are usually sufficient to V. Harris (*) : V. Jiranek : C. M. Ford : P. R. Grbin School of Agriculture, Food and Wine, The University of Adelaide, PMB1, Glen Osmond 5064, Adelaide, South Australia, Australia e-mail:
[email protected]
control unwanted microorganisms in wine (Barata et al. 2007; du Toit et al. 2005; Silva et al. 2004). Strategies for the control and prevention of Dekkera contamination of wines are important for the maintenance of wine quality. Thus, equipment that comes into contact with juice, must, and wine is ideally subjected to routine and rigorous sanitisation practices. Maintaining appropriate wine parameters such as pH and sulphur dioxide can minimise Dekkera growth. Where these measures fail and the presence of the organism becomes evident, further additions of sulphur dioxide can be made; however, they are not always successful (Barata et al. 2007). Further concerns over the detrimental health effects associated with sulphur dioxide have led to reduced usage of this preservative by winemakers. The development of alternate and ideally natural antimicrobials is therefore warranted to combat the potential for increasing susceptibility of wines to microbial spoilage (García-Ruiz et al. 2008). Hydroxycinnamic acids (HCAs) are endogenous components of grapes and are considered natural food preservatives (Ou and Kwok 2004; Smid and Gorris 1999). Of these HCAs, ferulic acid has in fact been used to prevent spoilage of foods since 1975 (Ou and Kwok 2004). HCAs have been reported to inhibit growth of a variety of organisms including plants, fungi, and bacteria (Campos et al. 2003; Ravn et al. 1989; Stead 1993; Van Sumere et al. 1971; Walker et al. 2003). In particular, growth of a number of yeast species is inhibited by HCAs in concentrations upwards of 1 mM, with ferulic and p-coumaric acids generally being found to be most inhibitory (Baranowski et al. 1980; Baranowski and Nagel 1983; Edlin et al. 1995; Harris et al. 2008; Ravn et al. 1989; Stead 1995; Walker et al. 2003). HCAs are also known to have health-giving properties. They are anti-inflammatory compounds that may decrease the risk of cancer, diabetes, cardiovascular
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Appl Microbiol Biotechnol (2010) 86:721–729
and neurodegenerative diseases (Ogiwara et al. 2002; Sakai et al. 1999; Srinivasan et al. 2007). Given these purported health benefits and their antimicrobial properties against wine-related yeast and bacteria, HCAs may have a future role in the control of Dekkera. Some microbes detoxify HCAs present in wine by decarboxylating them to vinylphenols, which at low concentrations smell smokey, sweet and fruity (Heresztyn 1986; Maga 1978; van Beek and Priest 2000), but at higher concentrations they may have undesirable characteristics (Chatonnet et al. 1993). Vinylphenols can be further metabolised, predominantly by Dekkera, into ethylphenol derivatives (Chatonnet et al. 1992, 1995). In this way pcoumaric acid is converted into 4-ethylphenol that has an aroma reminiscent of Bandaid® and barnyards. Ferulic acid is metabolised into 4-ethylguaiacol which smells spicy and smokey (Chatonnet et al. 1992). Therefore, the addition of HCAs as a strategy to control unwanted organisms must occur at a fungicidal concentration so as to eliminate the risk that undesirable volatile phenols are produced. At present, insufficient data exists to define these fungicidal concentrations. In this study, growth of Dekkera species was examined in the presence of a range of single and multiple HCAs. The additional influence of pH and ethanol were also considered. The objective of this work was to evaluate the extent of inhibition and the potential of ferulic acid as a novel antimicrobial against Dekkera.
Methods Dekkera strains
Table 1 Dekkera strains used during this investigation Species
Strain
Original source
Dekkera anomala
CBS 76 CBS 77 CBS 8139 CBS 2336 AWRI 1499 VH01a VH04a VH05a
Beer Stout Soft drink Wine Wine Wine Wine Wine
a
Investigation of the effect of ferulic acid on Dekkera growth in chemically defined medium at various pH values and ethanol concentrations Starter cultures were prepared from a single loop of freshly grown Dekkera (YPD agar) inoculated into 25 mL of YPD broth and grown at 30°C with agitation (180 rpm) to a density of 1×108 cells/mL. Chemically defined medium (CDM; Harris et al. 2008), 50 mL of medium in a 250-mL Erlenmeyer flask, was supplemented with 0–2 mM ferulic acid and inoculated to 5×106 cells/mL. Cultures were prepared in triplicate and incubated at 30°C with agitation. The influence of pH and ethanol on the growth of Dekkera isolates was examined with or without 0.5 mM of ferulic acid. Cultures were prepared as above but with media adjusted to pH values between 3, 3.8 (control), 4 and 5, or else containing ethanol at concentrations of 0, 5, 7, and 10% (v/v). Culture growth was determined by optical density measurements at 600 nm (OD600). Minimum inhibitory or non-culturable concentrations in CDM, wine medium and modified wine
Dekkera anomala and D. bruxellensis were obtained from the Centraalbureau voor Schimmelcultures (CBS), The Australian Wine Research Institute (AWRI) or isolated directly from wine (Table 1). Wine strains were isolated on
Dekkera bruxellensis
YPD agar (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose and 2% (w/v) bacteriological agar) supplemented with 0.01% (w/v) cycloheximide. Identity of wine isolates was confirmed by sequencing the internal transcribed spacer (ITS) region (Guillamón et al. 1998). Genomic DNA preparations were made (Adams et al. 1997), the ITS region was amplified and PCR products were sequenced at the Australian Genome Research Facility (Brisbane). Sequence alignment results had 99% to 100% homology to the D. bruxellensis ITS region (AF 043503 strain CBS 4914 NCBI-BLAST nucleotide; EMBL-EBI).
D. bruxellensis strains isolated from a wine as part of this study
The minimum inhibitory concentration (MIC) at which HCA(s) addition prevented the detectable growth of Dekkera was defined as that which resulted in an OD600 ≤ 0.05 units. Cultures subjected to the MIC and higher were subcultured (1 mL) into 9 mL of fresh CDM medium (HCA-free) to assess residual culturability. A lack of detectable growth (OD600 ≤0.05) after 2 weeks defined the minimum non-culturable concentration (MNC). Experiments were preformed in 10 mL of medium supplemented with ferulic, p-coumaric or caffeic acids added singularly or in combination. Supplementation rates for individual acids were 1, 2, 4, 8, 10 or 20 mM for CDM and wine medium (WM; Harris et al. 2008). The impact of ferulic acid with one or both p-coumaric and caffeic acids was also examined. In this case, a matrix approach was used to achieve a total combined HCA concentration of up to 4 (CBS 1499) or 8 mM (CBS 77), derived from every combination of individual HCAs being added at 1, 2 or 4 mM.
Appl Microbiol Biotechnol (2010) 86:721–729
Modified wine (Shiraz, 12.8% (v/v) ethanol,
