The role of non-Saccharomyces species in releasing glycosidic bound fraction of grape aroma components — a preliminary study
Mendes Faia Food Science Department, ICETA – Universidade de Trás-os-Montes e Alto Douro, PO Box 202, 5001 Vila Real, Portugal (e-mail: email@example.com).
Aims: The purpose of the study was to evaluate the effect of β-glycosidase activity in wine yeasts in releasing terpene glycosides from grape juice.
Methods and Results: Glycosidase activity was screened in 160 yeasts by testing their ability to hydrolyse arbutine on agar plates. Only non-Saccharomyces species exhibited β-glycosidase activity. Enzyme activity, based on hydrolytic activity on p-nitrophenyl-β-glycoside, was mainly located in the whole cell fraction, with smaller amounts in permeabilized cells being released into the growth medium. The hydrolysis of glycosides was determined by HRGC-MS, confirming the role of yeast in the liberation of monoterpenols, especially linalool and geraniol.
Conclusions: The results indicate the potential of microbial β-glycosidases for releasing flavour compounds from glycosidically-bound, non-volatile precursors, with significant implications for wines made from less aromatic grapes.
Significance and Impact of the Study: This study confirms the role of non-Saccharomyces species in enhancing wine aroma and flavour, suggesting that the future lies with controlled use of mixed cultures in winemaking.
Non-Saccharomyces yeasts are an ecologically and biochemically diverse group which can alter the fermentation dynamics, composition and flavour of wine (Soden et al. 1998). The non-Saccharomyces yeasts usually dominate in mature grape berries, accounting for 50–75% of the total yeast population (Fleet and Heard 1993). Despite their inability to achieve complete fermentation, they persist during the early stages. The level of their contribution to alcoholic fermentation depends on the extent of damage caused by moulds, or by any mechanical action, the temperature of fermentation, the degree of clarification of the grape juice, the addition of sulphur dioxide and inoculation with selected yeasts. The supporters of natural fermentation have claimed that non-Saccharomyces yeasts enhance wine aroma and flavour. The aroma compounds in wine are mainly the terpenes, which are metabolites derived from mevalonic acid, and are characterized by multiples of branched, five-carbon units resembling isoprene (Boulton et al. 1995). The most important group from an oenological point of view are the monoterpenes (10-carbon compounds) because of their volatility and odour when present in a free form (Williams et al. 1982). They are responsible for the varietal character of many grapes (Gunata 1994). In recent years, it has been found that a considerable portion of these compounds occurs in bound forms, particularly glycosides, which do not seem to contribute to the aroma unless they are hydrolysed and released. The glycosidically-bound forms can be converted into the free odourous forms (linalool, nerol, geraniol, α-terpineol and citronellol), by hydrolysis with glycosidases (Bayonove et al. 1992). Wine yeasts have been extensively studied to find extracellular activities, mainly β-glycosidase (Rosi et al. 1994; Miklósy and Pölös 1995; Mateo and Di Stefano 1997), in order to correlate enzyme activity with the aroma imparted to the wines. It has been found that wine-related yeasts, particularly non-Saccharomyces species, can hydrolyse part of the bound monoterpenes, thus modifying the aromatic profile (Gunata 1994) or enhancing the varietal character of wines (Laffort et al. 1989). Nevertheless, there are few data available regarding β-glycosidase activity in apiculate yeasts. Moreover, no data are available on their effects on monoterpenol glycosides of grape juice. The aim of this work was to evaluate the β-glycosidase activity of indigenous wine yeasts, and to determine their role in the aroma and character of wines.
MATERIALS AND METHODS
Isolation and identification of yeasts
The yeasts were isolated by plating the sample of must or wine, after appropriate dilution, into Yeast Malt Extract agar. The identification of the isolates was carried out by performing physiological and morphological tests, according to the keys proposed by Barnett et al. (1990) and Yarrow (1999), supported by the Barnett et al. (1987) computer programme.
Screening β-glycosidase activity
Screening of β-glycosidase activity was carried out in 160 isolates from musts and wines from the Douro Region on agar plates containing arbutine as carbon source. The composition of the medium was (g l–1): yeast nitrogen base (Difco), 6·7; arbutine (Sigma), 5; Agar (Difco), 20. The pH was adjusted to 5·0 prior to sterilization by heating to 121°C for 15 min. Immediately after sterilization, 2 ml ferric ammonium citrate solution (1% w/v) were added to 100 ml medium. After 2, 4, 6 and 8 days of incubation at 25°C, β-glycosidase activity was observed by the appearance of a dark brown colour in the colonies. An uninoculated plate of arbutine agar was used as control.
To characterize enzymatic activity, the isolates showing β-glycosidase activity were inoculated into a liquid culture medium in which arbutine was substituted by 20 g glucose, according to the methodology described by Rosi et al. (1994). The inoculum was prepared earlier by inoculating each strain into Yeast Malt Extract medium and incubating at 25°C for 48 h. The effect of aeration was evaluated in two separate experiments: for anaerobic conditions, 2% inoculum was added to 250 ml screw-capped flasks filled to 80% of their volume and incubated without shaking at 25°C for 3 days; for aerobic conditions, 250 ml screw-capped flasks filled to 20% of their volume were inoculated and incubated in an orbital shaker at 150 rev min–1 at 25°C for 24 h. Microbial growth was monitored by periodic readings of the absorbance at 600 nm. Before the enzyme assay, the samples were diluted with uninoculated medium to uniform absorbance.
Enzyme location in cells.
Enzymatic activity was evaluated in whole cells, in permeabilized cells and in the supernatant fluid. The whole cells were harvested by centrifuging 1 ml culture at 7012 g for 10 min at 4°C. After removing the supernatant fluid, the cells were washed twice with cold distilled water; the pellet was resuspended in 0·2 ml citrate-phosphate buffer (pH 5·0) and used for enzymatic assay. The permeabilized cells were obtained by centrifugation of 5 ml culture and washing the pellet several times with cold distilled water. The pellet was resuspended in 1 ml imidazole buffer (pH 7·5) and added rapidly to 50 μl glutathion (0·3 mol l–1), 10 μl Triton X–100 (10%) and 50 μl toluene/ethanol (1 : 4 v/v). Finally, the suspension was shaken vigorously for 5 min in a mechanical Vortex stirrer (Scientific Industries, Inc, Bohemia, NY, USA) and centrifuged at 7012 g for 10 min at 4°C. The yeast pellet was suspended in 5 ml cold distilled water from which 1 ml was removed, centrifuged and washed with cold water; the pellet was resuspended in 0·2 ml citrate-phosphate buffer.
Evaluation of enzymatic activity was carried out by determining the amount of p-nitrophenol (pNP) released from the p-nitrophenyl-β-D-glycoside (pNPG) used as substrate. To 0·2 ml of each suspension, 0·2 ml pNPG solution (5 mmol l–1 in citrate-phosphate buffer at pH 5·0) were added. The mixture was incubated for 1 h at 30°C and the reaction stopped by adding 1·2 ml carbonate buffer (pH 10·2). The amount of pNP released was determined in a spectrophotometer Shimadzu UV-2101 PC (Shimadzu Co, Kyoto, Japan) at 400 nm. For quantification, a standard curve was prepared with pNP ranging between 10 and 100 nmol ml–1. Enzymatic activity was expressed as nmoles of pNP produced per millilitre per hour. All the assays were performed in triplicate.
Evaluation of enzymatic activity in glycoside-bound terpenes in grapes
Extraction of glycosides from grape juice.
Muscat grape juice (200 ml) was eluted through a C18 column (Mega Bond Elut, 5 g Varian, Harbor City, CA, USA), followed by washing with 100 ml dichloromethane to remove the free fraction of terpenes. The bound fraction, the glycosides, was recovered by washing the column with 100 ml methanol (Rosi et al. 1995) and stored at −18°C.
Hydrolysis of grape glycoside extract.
Just before use, the extract was concentrated under vacuum, at 30°C, in a water-bath of a rotary evaporator. The whole yeast cells suspended in 10 ml citrate-phosphate buffer were added to this extract. As a control, 10 ml buffer without cells were used. The mixture was incubated at 30°C. After 48 h, the samples were centrifuged at 1753 g for 5 min and the released terpenes extracted in an ultrasound bath, using dichloromethane as solvent, according to the method described by Coccito et al. (1995).
Analysis by HRGC.
The gas chromatographic analysis was carried out in a Carlo Erba apparatus (Mega 5160) (Carlo Erba Strumentazione, Milan, Italy) equipped with a flame ionization detector (FID) and a fused silica capillary column of polyethylene glycol (DB-WAX of J & W Scientific, Folsom, CA, USA), 30 m, 0·32 mm ID, 0·5 μm film thickness. Operating conditions were as follows: injector and detector (250°C); carrier gas hydrogen (inlet pressure 68 948 Pa and split ratio 1:20); oven temperature programme: 5 min at 45°C, 3·5°C min–1 until 210°C, and 210°C for 20 min; volume injected 0·5 μl. All analyses were replicated. The components were identified by their relative retention times, which were determined by injection of standards and confirmed by GC-MS. Quantitative measurements were performed with 2-octanol as internal standard.
Analysis by GC-MS.
The analyses were performed in a GC-MS (Magnum, Finnigan Mat, San Jose, CA, USA) equipped with a fused silica capillary column of polyethylene glycol (DB-WAX of J & W Scientific), 30 m, 0·25 mm ID, 0·25 μm film thickness. Operating conditions were as follows: injector and interface temperature, 250°C; carrier gas helium (inlet pressure 82 737 Pa and split ratio 1 : 30); oven temperature programme: 5 min at 45°C, 3·5°C min–1 to 180°C, 180°C for 30 min; volume injected, 0·5 μl. Retention times and mass spectra of eluted peaks were compared with those of authentic standards, except 3-acetyl-1-propanol, 3-ethoxy-1-propanol, Ho-trienol, 3-methyl-thio-1-propanol, pyran linalool oxides, 3,7-dimethyl-1,5-octadien-3,7-diol and 3,7-dimethyl-1,7-octadien-3,6-diol, which were directly identified by the NIST library of MS equipment.
The results of enzymatic activity experiments were analysed using the SAS® Procedures Guide (SAS Institute Inc, Cary, NC, USA). The Student’s t-test was used for paired comparisons.
Screening β-glycosidase activity
Screening of β-glycosidase activity was carried out in 160 isolates from musts and wines after 2, 4, 6 and 8 days of incubation at 25°C on agar plates containing arbutine as carbon source. Evidence of the presence of β-glycosidase was provided by the appearance of a dark brown colour in the colonies; this colour was intense in three, fair in eight and weak in 13 of the isolates. None of the other isolates exhibited the colour, indicating that they do not have the enzyme.
Location of enzyme activity
In three of those isolates which had the strongest reaction on the arbutine agar plates, identified as Kloeckera apiculata, Pichia anomala and Metschnikowia pulcherrima, enzyme activity was sought in whole cells (parietal activity), in permeabilized cells (endocellular activity) and directly in the medium (exocellular activity). A strain of Saccharomyces cerevisiae was used as control, even though the split of arbutine and the consequent browning of the colonies had been almost undetectable.
The K. apiculata strain showed the highest β-glycosidase activity (69·05 nmol pNP ml−1 h–1), followed by P. anomala (50·16 nmol pNP ml–1 h–1) and M. pulcherrima (9·83 nmol pNP ml–1 h–1). In S. cerevisiae, very weak enzymatic activity was observed (0·68 nmol pNP ml–1 h–1) (Table 1). The data obtained show that most enzyme activity was parietal, being significantly higher (P < 0·001) than endocellular and exocellular activities (Table 2).
Mean values of enzymatic activity in the species under study
Mean values of enzymatic activity in the species under study, independently of the growth conditions
Enzymatic activity increased significantly (P < 0·001) from 16·88 to 49·27 nmol pNP ml–1 h–1 under aerobic conditions (Table 3). However, the effect of aeration was different between species. In M. pulcherrima and K. apiculata, a 13- and 5·6-fold increase in β-glycosidase activity, respectively, was observed.
Effect of aeration on enzymatic activity in the species under study, independently of the location of the enzyme
Role of yeasts in releasing glycoside terpenols in an extract of a Muscat grape juice
As shown in Table 4, the K. apiculata strain exhibited the ability to release some monoterpenols from an extract of Muscat grape juice. A remarkable increase in linalool and geraniol was detected in the inoculated extract, 27·5 and 43·1 μg l–1, respectively. Amounts of 3,7-dimethyl-1,7-octadien-3,6-diol and 3,7-dimethyl-1,5-octadien-3,7-diol were two and almost three times higher in the presence of the yeast. A slight increase in the concentration of other terpenic compounds, such as Ho-trienol, nerol, trans-o-cimenol, α-terpineol and citronellol, was also observed.
Volatile compounds (μg l–1
of 2-octanol) released from an extract of Muscat grape juice incubated with Kloeckera apiculata
The role of β-glycosidase activity in wine yeasts has been extensively researched. Although Mateo and Di Stefano (1997) observed some enzyme activity in different strains of Saccharomycescerevisiae, most studies demonstrate that the higher enzyme producers are non-Saccharomyces species (Rosi et al. 1994; Miklósy and Pölös 1995). These findings were confirmed in the present study. It was also observed that enzyme activity, based on the hydrolytic activity of p-nitrophenyl-β-glycoside, was mainly cell-wall bound (parietal activity), as has been described for other species (McMahon et al. 1999). This activity was significantly (P < 0·001) increased by aerobic conditions of growth, as previously reported in Debaromyces hansenii by Rosi et al. (1994). On the other hand, exocellular activity, which was found only in K. apiculata and P. anomala, was significantly (P < 0·001) inhibited by aeration.
It has been assumed that yeasts, being responsible for hydrolysis of bound monoterpenes, can modify the aromatic profile of wines and even enhance their varietal character. The estimation of volatile components released from the extract of grape juice by HRGC/MS suggested that yeasts have the ability to increase free terpenols, especially in aromatic wine varieties such as Muscat. However, in two other Portuguese varieties (Códega and Malvasia Fina), no free terpenols were released, probably due to their neutral character. Only benzyl alcohol and 2-phenyl ethanol showed a remarkable increase in all assays independently of the grape variety (unpublished data). Therefore, non-Saccharomyces species have not been recommended for winemaking because most of them are, simultaneously, very weak producers of ethanol and strong producers of undesirable by-products, such as acetate and ethyl acetate. However, some species present in mature grape berries and at the beginning of alcoholic fermentation seem to play an important role in the aromatic profile of the wines, due to their high β-glycosidase activity. These findings have practical importance, suggesting the potential of a reliable mixed culture fermentation strategy for producing a greater flavour diversity in wine.
This work was carried out in the framework of the project PAMAF 6106. The authors thank A. Lage for his technical assistance and L. Mendes Ferreira for statistical analysis.