Andrea CARIDI
Unit of Microbiology, Department of Scienze e Tecnologie Agro-Forestali e Ambientali (DiSTAfA),
Faculty of Agricultural Sciences, "Mediterranea" University of Reggio Calabria, Via Feo di Vito,
I-89122 Reggio Calabria, Italy, tel 0965.801256, fax 0965.312603, e-mail acaridi@unirc.it
VALIDATION OF A METHOD FOR ASSESSING THE ABILITY OF WINE YEASTS TO ADSORB GRAPE PIGMENTS
The cell walls of yeasts used in winemaking adsorb the grape pigments which affect the colour of the wine. This adsorption is a strain-dependent trait, with some wine yeasts adsorbing more pigments than others. The colour of wine, in particular red wine, can generally be controlled by selecting strains of wine yeasts that not adsorb grape pigments and by macerating grape solids during winemaking for extended time (1-3). Wine yeasts affect the colour of wine in three principal ways: (a) by producing the enzyme anthocyanin-β-D-glucosidase (4,5) or the reactive metabolites pyruvic acid (6,7) and acetaldehyde (8,9); (b) by releasing mannoproteins and different polysaccharides (10,11); and (c) by retaining pigments on their cell walls (12-14). This last process has important consequences for wine quality because it removes a large amount of grape pigments. To assess the amount of grape pigments that wine yeasts can adsorb on mannoproteins in the outermost layer of their cell walls, a consistent analytical method was needed. Recently, a simple but consistent method was developed (15-17) that enables the colour of yeasts grown on chromogenic grape media in Petri dishes to be photographically processed, by measuring their red-green-blue (RGB) parameters using Photoshop and statistically analyzing the colorimetric data. In order to confirm biomass colour results, microvinification trials using red must were performed, and the obtained wines were analysed. As expected, the relationship among the RGB components of biomass colour and the analytical parameters of wines was strong but not complete, i.e. not for all the strains, because, as already stated, colour and phenolic content of wines are influenced by a variety of yeast factors, other than grape pigment adsorption on cell walls.
AIM OF THE WORK
The purpose of the present research was to confirm the results and validate the latter method by determining the total polyphenol content of the yeast biomass by Folin-Ciocalteu index (FCI) analysis and studying its correlation to the colorimetric data. Yeast colour depends on yeast ability to adsorb grape pigments: white is related to low adsorption, dark brown to high adsorption.
INTRODUCTION
REFERENCES
MATERIALS AND METHODS
RESULTS
Table 1 - Means and significant differences (P<0.05) among 20 wine yeasts for the RGB parameters of yeasts grown on Grape Skin Agar prepared using grape skins from the cultivars Greco Nero (GN), Magliocco (Ma), and Nero d’Avola (NA) as measured using Photoshop. The values included in the first and in the last homogeneous group, according to LSD analysis, are bold-typed; a, b…m indicate the significance of the comparison in the same column.
Strains YPD Agar
Red component Green component Blue component
1042 192c 180cd 152bc 12233 183b 159b 151b BM45 196c 186de 161cd ICV D254 233j 223i 210i Sc226 212fg 200g 165de Sc560 205de 189ef 168de Sc708 225i 216h 189h Sc1304 226ij 179cd 150b Sc1483 209def 201g 179fg Sc1661 183b 174c 155bc Sc1766 211efg 200g 166de Sc1864 253l 254j 239j Sc2489 166a 141a 129a Sc2621 203d 186de 161cd Sc2640 220hi 201g 169de Sc2659 210efg 200g 170e Sc2717 216gh 212h 182gh TT77 245k 225i 191h TT173 210defg 195fg 172ef TT254 194c 181d 167de Average 210 195 172 CV1 10 13 14 Range 166-253 141-254 129-239 1
Table 2 - Means and significant differences (P<0.05) among 20 wine yeasts for the RGB parameters of yeasts grown on YPD Agar as measured using Photoshop. The values included in the first and in the last homogeneous group, according to LSD analysis, are bold-typed; a, b…l indicate the significance of the comparison in the same column.
1 Coefficient of variation
GraSkiGN FCI_GraSkiGN GraSkiMa FCI_GraSkiMa GraSkiNA FCI_GraSkiNA Red -0.76180.0001 Red -0.75180.0001 Red -0.73890.0002
Green -0.68690.0008 Green -0.64900.0020 Green -0.64130.0023
Blue -0.75610.0001 Blue -0.71370.0004 Blue -0.69770.0006
YPD FCI_YPD
Red -0.24420.2994 Green -0.20480.3864 Blue -0.13900.5589 1
Table 3 - Correlation coefficients and their statistical significance (superscript-typed) between Folin-Ciocalteu index (FCI) of yeasts grown on YPD Agar and Grape Skin Agar (GraSki) - prepared using grape skins from the cultivars Greco Nero (GraSkiGN), Magliocco (GraSkiMa), and Nero d’Avola (GraSkiNA) - and the related RGB parameters of the yeasts as measured using Photoshop. The pairs of variables having P-values below 0.05 are significantly correlated and therefore values are bold-typed.
PERSPECTIVES
With the present study, a method to select
yeasts based on the adsorption ability of grape
pigments has been confirmed and validated.
The validated method can be helpful in yeast
selection because it represents a useful tool to
amplify the knowledge of wine yeast traits.
The simplicity of this method and its materials
suggest that it may be of particular interest to
makers of red wine, who are interested in a
low-cost, simple, and objective method of controlling
the colour of red wine.
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15.Caridi, A., Cufari, A., Ramondino, D. (2002). Isolation and clonal pre-selection of enological Saccharomyces. Journal of General and Applied Microbiology 48, 261─268.
16.Caridi, A., Sidari, R., Solieri, L., Cufari, A., Giudici, P. (2007). Wine colour adsorption phenotype: an inheritable quantitative trait loci of yeasts. Journal of Applied Microbiology 103, 735─742. 17.Sidari, R., Postorino, S., Caparello, A., Caridi, A. (2007). Evolution during wine aging of colour and tannin differences induced by wine starters. Annals of Microbiology 57, 197─201.
18.Rizzo, M., Ventrice, D., Varone, M.A., Sidari, R., Caridi, A. (2006). HPLC determination of phenolics adsorbed on yeasts. Journal of Pharmaceutical and Biomedical Analysis 42, 46─55. Twenty wine yeasts with different abilities to adsorb grape pigments were used: 16 strains of Saccharomyces
sensu stricto (DiSTAfA collection), two laboratory strains S. cerevisiae 1042 and S. bayanus 12233
(Department DIPROVAL collection, Coviolo, Reggio Emilia, Italy), and two commercial wine yeasts Lalvin BM45 and Lalvin ICV-D254 (Lallemand, Canada). The yeasts were grown at 28°C for 2 days on YPD medium (1% yeast extract, 1% peptone, 2% dextrose) solidified with 2% agar when required. The chromogenic medium Grape Skin Agar (GraSki) was prepared using grape skins from the black cultivars Greco Nero (GN), Magliocco (Ma), and
Nero d’Avola (NA). A control medium (YPD Agar) was also used. Grape skins were manually removed from
grapes, gently washed, dried at 55°C, finely ground by a blender and kept at room temperature in a closed plastic container until use.
Preparation of the chromogenic medium (pH 3.5): Dried grape skins 50 g l-1, peptone from casein 18.75 g l-1,
yeast extract 11.25 g l-1, glucose 50 g l-1, Na
2HPO4 62.5 g l-1, and citric acid monohydrate 125 g l-1 were
suspended in distilled water, treated at 100°C for 30 min, filtered through gauze, distributed into test tubes (4 ml/tube) and autoclaved at 121°C for 15 min. A solution of agar (33.33 g l-1) was prepared, distributed into
test tubes (6 ml/tube) and autoclaved at 121 °C for 15 min. One test tube containing the medium and one containing the agar solution were poured together into Petri dish (ø 60mm) and carefully mixed using a sterile L-shaped plastic spreader. After solidification, each dish was used to inoculate one yeast strain. After 10 days of incubation at 28°C, the biomass spread on a calibrated loop was processed for colour by RGB analysis. The colour assessment was performed on photographs of yeast (Photosmart 945: “macro” function, ISO-100 in automatic modality). The region of interest was set to 5x5 pixels taking four replicates for each strain. The intensity values ranged from 0 (black) to 255 (white) for each of the RGB components in a colour image. Accordingly, a low ability to adsorb grape pigments corresponded to high RGB values, i.e. yeast of a white colour; a high ability to adsorb grape pigments corresponded to low RGB values, i.e. yeast of a dark brown colour (Fig. 1). The results obtained by RGB analysis were confirmed and validated by FCI determined on the same yeast biomass (18).
All the analyses were performed in triplicate; data were subjected to statistical analysis using StatGraphics Centurion XV for Windows XP from StatPoint.
For the red component, yeasts grown on GraSki medium from the cultivar GN were distributed in 14 homogeneous groups, those grown on GraSki medium from the cultivar Ma in 13, those grown on GraSki medium from the cultivar NA in 12; for the green component, the strains were distributed in 14, 15, and 15 homogeneous groups, respectively; for the blue component, the strains were distributed in 11, 15, and 10 homogeneous groups, respectively. On the three GraSki media, the RGB component values had an extremely wide range: red from 2 to 210, green from 1 to 182, and blue from 6 to 178. The following correlation coefficients, all very highly significant (P <0.001), were observed: a) red component: GN x Ma=0.8687; GN x NA=0.9108; Ma x NA=0.7984; b) green component: GN x Ma=0.8122; GN x NA=0.8647; Ma x NA=0.7411; c) blue component: GN x Ma=0.8258; GN x NA=0.9027; Ma x NA=0.7544 (Tab. 1).
For the red component, the 20 strains grown on YPD agar were distributed in 16 homogeneous groups, for the green and blue components the strains were distributed in 12 homogeneous groups. The red component values ranged from 166 to 253, the green component values from 141 to 254 and the blue component values from 129 to 239 (Tab. 2).
Tab. 3 shows the correlation coefficients and their statistical significance between FCI of the yeasts grown
on GraSki media and YPD Agar and the related RGB parameters measured using Photoshop on the yeasts. An inverse and highly (P <0.01) or very highly (P <0.001) significant correlation between FCI and RGB values was observed for all the three GraSki media utilised. On the contrary, data do not support any minimal correlation for the YPD Agar, so confirming the high accuracy, precision, and specificity of the proposed GraSki media.
Statistical distribution of the 20 strains in several homogeneous groups, based on their RGB behaviour on the four media, strongly validate the aim of the present study supporting the presence of strong differences in yeast ability to adsorb grape pigments.
Strains Red component Green component Blue component GN Ma NA GN Ma NA GN Ma NA 1042 153e 155h 152e 114f 120hi 118ef 131d 141hij 135d 12233 4a 2a 5a 1a 1a 1a 9a 12ab 13a BM45 171f 139f 161f 140h 100ef 117e 151f 118f 140de ICV D254 198k 164i 177ij 169l 131l 157kl 161gh 143hij 160g Sc226 192j 141f 166fg 161k 103fg 133gh 165h 128g 151f Sc560 173fg 147g 177ij 136h 109g 139hi 161gh 130g 163g Sc708 181hi 135f 149e 141hi 95e 109d 159g 117f 134d Sc1304 104c 26b 135d 79d 2a 103cd 96c 18b 116c Sc1483 104c 3a 114c 88e 3a 98c 97c 6a 111c Sc1661 175fg 140f 168gh 146i 107g 143ij 149ef 130g 149f Sc1766 111d 125e 165fg 69c 86d 126fg 95c 108e 152f Sc1864 183i 176k 180j 138h 129kl 136hi 161gh 153k 160g Sc2489 62b 68d 66b 58b 61c 53b 61b 69d 58b Sc2621 171fg 162i 194k 128g 124ijk 163lm 143e 145hijk 166gh Sc2640 181hi 167ij 189k 152j 134l 168m 160gh 149jk 172h Sc2659 176fgh 150gh 174hi 154j 115h 150jk 161gh 139h 162g Sc2717 190j 163i 180j 163k 123ij 157kl 158g 140hi 164g TT77 177gh 153gh 170gh 125g 89d 117e 152f 120f 147ef TT173 113d 56c 153e 76d 10b 117e 99c 43c 140de TT254 210l 171jk 189k 182m 129jkl 164lm 178i 147ijk 173h Average 152 123 153 121 89 124 132 108 138 CV1 34 47 30 38 53 32 32 46 29 Range 4-210 2-176 5-194 1-182 1-134 1-168 9-178 6-153 13-172 1 Coefficient of variation 1
Figure 1 - Biomass colour of the low adsorbing strain TT254 (on the top) and of the high adsorbing strain 12233 (at bottom) grown on Grape Skin Agar prepared using grape skins from the cultivars Greco Nero, Magliocco, and Nero d’Avola and on YPD Agar (from left to right: Greco Nero, Magliocco, Nero d’Avola, YPD Agar). The yeast biomass, after 10 days of anaerobic incubation on the above media, was spread on a calibrated loop and photographed. The image was processed to perform red-green-blue analysis (see Tables 1-2).
DISCUSSION
Statistical distribution of the 20 strains in several homogeneous groups, based on their RGB behaviour on the four media, strongly validate the aim of the present study and the previously obtained data, supporting the presence of strong differences in yeast ability to adsorb grape pigments. The employed methodology to get colour data on the yeast by RGB analysis allowed data to be subjected to statistical analysis. This analysis of the yeast distribution into homogeneous groups showed the presence of highly significant and correlated differences among the strains for GraSki media. Moreover, a high reproducibility of the analysis was observed when the utilised cultivar was changed. While, based on grape cultivar used for GraSki media, different colours were observed for yeasts; this variability did not modify, for the great majority of the yeasts, the homogeneous group assigned by statistical analysis to each strain. After growth on the three grape-skin-based media, the tested strains showed wide and significant differences for the Photoshop parameters; a highly significant correlation between these values and the corresponding Folin-Ciocalteu values was observed. It must, however, be restated that wine colour and phenolic content are also influenced by yeast factors apart from adsorption of grape pigments. Also, it is known that grape pigments, above all anthocyanins, are sensitive to pH changes. Furthermore, it has been observed that parietal mannoproteins vary from strain to strain, depending on the percentage of acidic oligosaccharides. Clearly, this influences parietal pH and consequently grape pigment colour. A further investigation into this phenomenon is desirable. Nevertheless, the present results exhibit a highly significant correlation with the corresponding Folin-Ciocalteu values, thus validating the use of the present method to screen and measure the ability of wine yeasts to adsorb grape pigments. One important implication of the present study is that GraSki media could be prepared for any grape cultivar required, thus allowing the selection of the most suitable yeast strain for each required wine. Yeast strain selection was demonstrated to be important in protecting the colour of wine during winemaking; consequently, strategies proposed to enhance the wine concentration of grape pigments via choice of yeast strain are appreciated.
