Ó Springer 2005
Biotechnology Letters (2005) 27: 1565–1570 DOI 10.1007/s10529-005-1795-9
Retention of browning compounds by yeasts involved in the winemaking of sherry type wines Julieta Merida1, Azahara Lopez-Toledano1, Trinidad Marquez2, Carmen Millan2, Jose M. Ortega2 & Manuel Medina1,* 1
Department of Agricultural Chemistry, University of Cordoba, Edificio Marie Curie, Campus de Rabanales, E-14014 Cordoba, Spain 2 Department of Microbiology, University of Cordoba, Edificio Severo Ochoa, Campus de Rabanales, E-14014 Cordoba, Spain *Author for correspondence (Fax: +34 957 212 146; E-mail:
[email protected]) Received 3 June 2005; Revisions requested 1 July 2005; Revisions received 27 July 2005; Accepted 28 July 2005
Key words: acetaldehyde, browning, catechin, Saccharomyces, white wine, yeasts
Abstract Wine model solutions were used to study the ability of dehydrated yeasts to retain the brown products formed in the reaction between (+)-catechin and acetaldehyde. Saccharomyces cerevisiae races capensis and bayanus, two typical flor yeasts involved in the biological aging of sherry wines, had a higher capacity to retain coloured compounds than S. cerevisiae fermentative yeast. Of the flor yeasts, capensis exhibited a higher colour reduction capacity than bayanus. Such differences may account for the different rate at which browning compounds are removed at different times of year during the biological aging of wines.
Introduction Fino wines, which are typical of southern Spain (denominations of origin of sherry and MontillaMoriles), are characteristically of a very pale yellow bright, an almond flavour and a pungent odour. These wines are obtained by biological ageing in wooden casks filled to 5/6 of their capacity. Their sensory properties are due to flor yeasts that grow on the wine surface during the ageing process developing an aerobic metabolism. In the sherry zone, the major yeast species involved in the process include Saccharomyces beticus, S. cheresiensis and S. montuliensis (Casas 1985, Martı´ nez de la Ossa et al. 1997), which are synonymous with S. cerevisiae race capensis, S. cerevisiae race bayanus and S. cerevisiae race aceti in the Montilla-Moriles zone (Guijo et al. 1986). The former is the major yeast in flor films at the earliest stages of ageing, while the latter two are more frequent at later stages of the
process, probably as a result of their increased tolerance to the high acetaldehyde concentrations typical of this type of ageing (up to 400 mg/l). Fino wines retain their very pale colour throughout their long biological aging period (5–7 years). This non-browning has traditionally been ascribed to the isolation of the wine from atmospheric O2 exerted by flor yeasts growing on the wine surface (Martı´ nez de la Ossa et al. 1987, Domecq 1989). This protection may be the result of both restricted diffusion of atmospheric O2 to the wine by the yeasts acting as a protective layer and of competition between yeasts and phenols, the compounds responsible of browning reactions, for dissolved O2 in the wine. This latter phenomenon was observed by Salmon et al. (2002) in model solutions of phenolic compounds into contact with yeast lees. Nevertheless, this argument may be incomplete because the protective effect of flor yeasts is not exerted uniformly throughout the year as a result of the strict
1566 temperature conditions required for flor yeast growth (Ibeas et al. 1997). Therefore, flor yeasts grow strongly in spring and autumn, bringing the wine partially in contact with air in the summer and winter. As a result, the wine browns to some extent during the latter two seasons and regains its original colour later, when flor yeasts have recovered its surface, at a variable rate depending on the ambient conditions prevailing in the cellar. This suggests that flor yeasts not only exert a protective effect but also retain browning products thereby avoiding a progressive increase in their concentration and, as a result, the browning of wine occurs during all its biological aging (Baron et al. 1997). Indeed, some authors (Bonilla et al. 2001) have used baker’s yeasts as fining agents for colour correction of white wines. Basically, wine can brown by three different chemical pathways involving phenols, it being the flavans the most widely studied compounds in this respect. One pathway (catalysed by metals such as Fe and Cu), involves the oxidation of phenols that condense to polymerized quinones (Es-Safi et al. 2003). An other way involves the condensation of flavans with glyoxylic acid, this being formed by oxidation of tartaric acid of the wine (Es-Safi et al. 2000). The third pathway involves the direct condensation of phenols with acetaldehyde (Fulcrand et al. 1996). Whatever is the cause, the wine increases in its yellow–browness. The former two pathways involve oxidation reactions and require O2 to be in contact with the wine. The third, however, is a sole condensation reaction so it may reasonably develop to a greater extent in spite of the above-described protective effect of flor yeasts. On the other hand, this pathway is favoured because biological aging increases the content in acetaldehyde by oxidation of ethanol by flor yeasts, which increases the concentration of this compound by two or three times. Because fino wine exhibits little browning during the aging process, at least partially, one can reasonably ascribe its colour stability to the retention of brown compounds by flor yeasts. In this work, the ability of dehydrated yeasts to retain compounds formed in the reaction between (+)-catechin and acetaldehyde was examined with a view to compare the efficiency of various types of yeasts in preserving the pale colour of fino sherry type wines.
Materials and methods Reagents (+)-Catechin was from Sigma–Aldrich; and acetaldehyde, ethanol and acetic acid were purchased from Merck (Darmstadt, Germany). Yeasts The yeasts studied were: (a) Industrially dehydrated Saccharomyces cerevisiae baker’s yeast supplied by Mauripan (Fleischmann, Canada), which was used as the reference yeast. (b) S. cerevisiae fermentative yeast (ATCC, MYA425). (c) S. cerevisiae races capensis and bayanus flor yeasts (ATCC, MYA2451 and ATCC 90919, respectively). The fermentative yeast and the two flor yeasts were isolated in the Montilla-Moriles winemaking region (southern Spain) and cultured on YPD medium (1% yeast extract, 2% peptone, 2% dextrose), collected by centrifugation, washed with distilled water twice and dried in an air stream at increasing temperatures from 30 to 45 °C. An initial catechin/yeast ratio of 1:2 (w/w) was used in all the experiments. Total cell numbers were determined by direct counting under a light microscope in a Thoma chamber. Reactions (+)-Catechin (14.6 g/l in 12% (v/v) ethanol, 50.3 mM) was supplied with a 21.2 mM acetaldehyde. The pH was adjusted to 2.2 with acetic acid (this acid was used instead of tartaric acid to avoid oxidation of the latter to glyoxylic acid, which could develop a browning pathway different from that of interest in this work). The resulting solution was incubated at 20±1 °C for 48 h. Spectrophotometric measurements, HPLC and MS analyses Samples were passed through membrane filters (0.45 lm pore size) and their absorbance at 420 nm measured. Also after filtration through
1567 16
3
(+)-catechin
2.5 12 2 10 8
1.5
6 1 4
HP expressed as mg/l of (+)-catechin
HP
14
(+)-catechin (mg/l)
filters of 0.45 lm, samples were analysed by HPLC equipped with a diode array detector [250 mm 4.6 mm i.d., 5 lm particle size C18 column; flow-rate, 1 ml/min; solvent A, water/ formic acid (98:2 v/v); solvent B, acetonitrile/water/formic acid (80:18:2, by vol); elution from 5% to 30% B in 35 min, from 30% to 100% B in 5 min]. Eluted compounds were quantified as (+)-catechin at 280 nm. The compounds formed in the reaction were identified by MS. Mass data were acquired in two ways: in the scan mode (by scanning the m/z range 150–1066 using a 1.2 step size) and in the multiple ion mode (using mass ranges around specific m/z values). The chromatographic and mass spectrometry conditions have been described by Lopez-Toledano et al. (2004).
0.5 2 0
0 0
6
12
18
24 Time (h)
30
36
42
48
Results and discussion
Fig. 1. Changes in the concentration of (+)-catechin and polymers of high molecular weight (HP) during the reaction between (+)-catechin and acetaldehyde.
Based on the mechanism proposed by Fulcrand et al. (1996), the reaction between (+)-catechin and acetaldehyde takes place through successive condensations involving C6 or C8 carbons of (+)-catechin so a total of four dimers (C6–C6, C8–C8, C6–C8 and C8–C6) are possible, although not all of them are detected in the practice. Each dimer in turn can yield several trimers and so on. All oligomers consisting of more than three units of flavan were eluted in the HPLC at similar retention times, and could not be resolved. These peaks were considered to be a group and were named HP (highly polymerized) materials as they corresponded to oligomers with a high degree of polymerization. Figure 1 shows the variation of the (+)-catechin and HP contents in the model solution over 48 h. As can be seen, the monomer concentration decreased gradually with increasing reaction time (50% after 48 h), as a result the increase in HP content and consequently in the brown colour was quite uniform throughout the studied period. In order to examine the interaction between the yeasts and the reaction products, dehydrated baker’s yeast (24 g) were added to a litre of the solution of (+)-catechin and acetaldehyde after 24 h of reaction. This yeast was used as reference on account of its ability to interact with browning compounds in white wines (Bonilla et al.
2001, Razmkhab et al. 2002). Figure 2 shows the decrease of different compounds in the solution as a result of their absorption by this yeast after 90 min of contact. As can be seen, except for dimer 3, the retention capability of the yeast increased with increasing molecular weight of the compound (10% for (+)-catechin versus 74% for HP). The increased retention of dimer 3 (52%) relative to the other two (14% for D1 and 22% for D2) can be ascribed to its low polarity. This hypothesis is supported by the fact that the former was eluted at a longer retention time than the trimers. Because the retention sequence (M < D1 < D2 < T2 < T1 < T3 < T4 < D3 < HP) was virtually identical with that of appearance of the chromatographic peaks, one can assume that the less polar compounds, which are most often also those with the higher molecular weights, are those more strongly retained by the yeasts. To compare the efficiency of the winemaking yeasts in retaining the browning compounds formed in the reaction, three types of yeasts were added to respective solutions of (+)-catechin –acetaldehyde under the same conditions as in the previous experiment. One of the yeasts (S. cerevisiae) was of the fermentative type and the other two (S. cerevisiae race bayanus and S. cerevisiae race capensis) were of the flor type. Figure 2 also shows the decrease of different
1568
Fig. 2. Decrease (after 90 min of contact with different yeasts) in the contents of the compounds formed after 24 h of reaction between (+)-catechin and acetaldehyde (M=monomer; D1, D2, D3=dimers; T1, T2, T3, T4=trimers; HP= polymers of high molecular weight).
compounds absorbed by the three winemaking yeasts studied. As can be seen, the baker’s yeast was that absorbing the largest amounts of all the compounds, followed by the flor yeasts and the fermentative yeast. By comparing between flor yeasts, bayanus was more effective than capensis. In spite of these quantitative differences, all yeast types exhibited the same selectivity for compounds of different nature. Thus, as noted earlier, the four yeast types absorbed the higher polymers (D3 excepted) in a more extent. Since this reaction can have a strong contribution on browning of white wines and the degree of this alteration is usually measured in the visible region at 420 nm, the A420 was recorded for all the solutions. Taking into account the high (+)-catechin concentration used, the initial solution was coloured (A420 = 0.182 u.a.). This colour increased as the reaction with acetaldehyde developed, it reaching 0.207 u.a. at 24 h, the time at which the four types of yeast were added. To compare the effectiveness in the absorption capability of the yeasts to coloured compounds, the cell numbers per gram of each yeast species was determined (Table 1). The dehydrated baker’s yeast exhibited the smallest
Table 1. Total cell numbers per gram of the dehydrated yeasts. Yeasts S. S. S. S.
cerevisiae cerevisiae cerevisiae cerevisiae
Total cells/g (dry wt) baker’s fermentative race capensis race bayanus
3.2 3.9 4.7 7.1
1010 1010 1010 1010
cell numbers per gram, so their cells were the largest of all. On the other hand, the flor yeasts exhibited the largest cell numbers per gram, particularly race bayanus, because their smaller cellular size. Figure 3 shows the decrease in A420 caused by a same number of cells (1012) of each yeast. Bayanus race exhibited a lower absorption capability per cell than capensis race, it being the capability of both in between those of the baker’s and fermentative yeasts. The baker’s yeast used as reference exhibited a much higher colour reduction capability than the others. Because this yeast was obtained by industrial dehydration and the remainder yeasts were dehydrated in the laboratory, the dehydration method may have influenced the ability of yeasts to retain browning
1569 and hence the rate at which the wine regains its original colour.
12
Decrease in A420 u.a./10 cells
0.16 0.14 0.12 0.10
Acknowledgements
0.08
The authors gratefully acknowledge financial support from the Spanish Government, Department of Science and Technology (AGL-2002-04154CO2-01 and 02) for the realization of this work.
0.06 0.04 0.02 0 Baker's
Bayanus
Capensis
Fermentative
Fig. 3. Decrease after 24 h of reaction in the absorbance at 420 nm caused by 1012 cells of the different yeasts kept in contact with the solution for 90 min.
compounds. The increased absorption capability of the flor yeasts relative to the fermentative yeast can be ascribed to the structure of their cell walls. Thus, the cell walls of the former contain molecules capable of binding among them to form flor films. This binding ability could additionally be used to retain coloured compounds, thereby increasing the colour absorption capability of flor yeasts relative to fermentative yeast. Likewise, this property may account for the difference in absorption capability between the two flor yeasts. According to Mauricio et al. (1997), capensis yeast forms a thick flor film, consisting of viable and non-viable cells, with a cellular density of 96.5107 cells/cm2; by contrast, bayanus yeast forms a very thin film of viable cells with a density of 8.2107 cells/cm2. This difference suggests a lower binding capability for bayanus cells, it leading to a decreased ability to bind coloured polymers formed in the browning of the wine. In conclusion, the protective effect to the increase of colour in white wines subjected to biological aging is more strongly exerted during the aging stage (flor yeasts) than alcoholic fermentation (fermentative yeasts). The differences between the two flor yeasts also seems to account for the different rate at which browning compounds are removed at different times of year. Thus, the distribution of yeast species forming the flor film can vary depending on the particular ambient conditions of the cellar, and it may affect the absorption of browning compounds
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