Comparing the Glories, Iland and Bovine serum albumin when measuring phenolic compounds and whether there is a correlation between grape and wine content?

by | Feb 1, 2017 | Article

Characterising grape and wine phenolic composition in red wines has  become very important in the South African wine industry. Phenolic
compounds play a critical role in colour, astringency, mouth feel and general quality of a wine [1, 2]. The phenolic compounds in red grapes might give an indication of phenolic and colour compositions of the resulting wines. It is therefore important for the winemaker to optimise the extraction of phenolic compounds to suit the specific  style of wine to be made.

A prediction of the amount of phenolics found in a wine by measuring the phenolics in the grape, would aid the winemaker to  better determine the best??a suitable harvest date. Determining the extractability of certain phenolics would also be helpful when planning grape processing. In order to be able to do this, a greater understanding of phenolic compounds, their extraction from grapes and the methods used to measure the phenolic content in the grape and wine, is needed. The aim of this study was to assess the anthocyanins, tannin and total phenolic composition of different South African red musts using various methods. Measurements were also done in the corresponding wines (including after malolactic fermentation) to investigate the correlation from must to wine. When considering a method to determine phenolic compounds, a few factors should be kept in mind such as the appropriateness of the method, accuracy, price, time, difficulty level, equipment available and labour. It is very important that the method be quick and easy to perform during harvest time, to be able to acquire fast and accurate answers to any harvest date enquiries.

Various methods exist to conduct these measurements in grapes.  The bovine serum albumin precipitation method (BSA)[3] measures the condensed tannin concentration of grapes and wine, using a spectrophotometer. The principle of this method is based on the precipitation of condensed tannins by the BSA. Other methods used for measurements in grapes are the Iland[4] and Glories[5] methods. The Iland method is designed to measure the total phenols and anthocyanins. This is done by acidifying grape pulp after which 50% alcohol is added. The Glories method measures the extractable
anthocyanins, as well as the total anthocyanins by acidifying the pulp to different degrees without any alcohol additions. It can also measure seed tannins, skin tannins and total phenolics. The Glories method also allows the calculation of the extractability index of anthocyanins and measures the contribution of seed tannins to the phenols.



FIGURE 1. Biplot of grape phenolic data obtained from different cultivars. M: Merlot, S: Shiraz, P: Pinotage, C: Cabernet Sauvignon SeTG: Seed tannins (Glories), Mp%G: Contribution of seeds to phenols (Glories), TPIG: Total phenols (Glories), A1G: Total anthocyanins (Glories), SkTG: Skin tannins (Glories), A3.2G: Extractable anthocyanins (Glories), AI: Anthocyanins (Iland). (Du Toit and Visagie, 2012).

FIGURE 3a. PCA plot of wine samples after malolactic fermentation. M: Merlot, S: Shiraz, P: Pinotage, C: Cabernet Sauvignon. (Du Toit and Visagie, 2012).
FIGURE 2b. Score plot of wine samples after alcoholic fermentation. Tanna: Tannins, SO2a: SO2 resistant pigments, TPa: Total phenols, MCDa: Modified colour density, CDa: Colour density, CPa: Co-pigmented anthocyanins, Aa: Anthocyanins, TRPa: Total red pigments. (Du Toit and Visagie, 2012).
FIGURE 3b. Score plot of wine samples after malolactic fermentation. Tannb: Tannins, SO2b: SO2 resistant pigments, TPb: Total phenols, MCDb: Modified colour density, CDb: Colour density, CPb: Co-pigmented anthocyanins, Ab: Anthocyanins, TRPb: Total red pigments. (Du Toit and Visagie, 2012).

Materials and methods 

Four red cultivars, Pinotage, Merlot, Shiraz, and Cabernet Sauvignon  were chosen from the 2010 vintage. Vineyard lo cations varied from Stellenbosch, Robertson, Rawsonville, Durbanville, Somerset-West, Franschhoek and Hermanus. Grapes were picked at commercial harvest date after which they were processed. The must was inoculated with NT116 (Anchor Yeast Biotechnologies) one hour after crushing and destemming and punch downs were performed every day to facilitate phenolic and colour extraction. Fermentation was performed at 23°C until dry. The skins were pressed after alcoholic
fermentation in an open basket to a pressure of 0.5 bar after which it was inoculated with Viniflora Oenos (CHR-Hansen) to start the malolactic fermentation (MLF).

Analyses of the berry samples using the Glories, Iland and BSA methods were done and full descriptions of the methods were  reported in published scientific articles[6]. BSA analyses were also done on the wines as well as different spectrophotometer analyses to measure colour density, modified wine colour density, total red pigments, total phenolics, estimate of SO2 resistant pigments and copigmented anthocyanins[4, 7]. Other wine analyses included total anthocyanins and tannin concentrations[3, 5].

Results and Discussion
Colour and phenolic composition of grapes There were no significant differences between the cultivars for most of the phenolic and anthocyanins analyses of the grapes (Table 1). The extractability index of the anthocyanins seemed to be higher for the Cabernet Sauvignon grapes, this means the colour was easier to extract for this cultivar. Merlot exhibited a higher seed tannin content, which could be undesirable as it increased the astringency and could associate with proteins and polysaccharides instead of stabilising anthocyanins[8]. There was no clear grouping of the cultivars in the
constructed biplot (Figure 1) pointing to other factors such as the origin of the grapes, terroir, viticultural practices and ripeness levels being the predominant influences on the phenolic composition of the grapes[9]. A strong, positive correlation between the total anthocyanins, extractable anthocyanins and skin tannins, all measures using the Glories method, as well as anthocyanins measured using the Iland method could be seen. In a positive correlation, as the values of one of the variables increases, the values of the other variable will also increase. These groups of compounds were extracted under similar conditions[5] and would thus make sense to be correlated.

Colour and phenolic composition of the wine
The principle component analyses (PCA) (Figure 2a) on the wine data just after fermentation did not show clear trends, or groupings in terms of cultivar. Loadings (Figure 2b) did, however, show positive correlations between total red pigments, anthocyanins, colour density, modified colour density and total phenols which were negatively correlated with tannins. This grouping was not surprising as the former contained all the colour associated compounds. After MLF, some grouping occurred with Merlot positioning more on the left of PC1 (Figure 3a). No other grouping tendencies were seen for the other cultivars. Loadings (Figure 3b) showed that the total phenol, total red pigments, anthocyanin and colour density were significantly lower for the Merlot wines when compared to the other cultivars. In general, the colour and phenolic concentration was lower after MLF. This confirmed the general belief that MLF could affect colour and phenolic characteristics of a wine[10, 11].

Correlations from grape to wine
The BSA method proved to be well equipped to assess grape tannin levels and how they reflected in the wine. Significant positive correlations (data not shown) between grape and wine phenolic characteristics were observed when analysed with both the Glories and Iland methods. Strong correlations were observed between anthocyanins (Iland), colour density (after alcoholic fermentation), modified colour density (after alcoholic fermentation) and modified colour density (after MLF). The total anthocyanin content (Glories) and the extractable anthocyanins were also positively and significantly correlated to the above mentioned wine parameters. It would seem as if the correlations delivered by the Iland method might be slightly better
to predict wine colour. The use of ethanol during the Iland procedure might explain this observation. The importance of assessing correlations not only after alcoholic fermentation but also after the completion of MLF was evident. In the future, studies should also investigate the effect of barrel ageing
on the phenolic composition. Using the extractability index (EA) The extractability index is an indicator of the extractability of anthocyanins,
by taking into account the total anthocyanins and anthocyanins extracted under winemaking conditions (both measured using the Glories method). It would thus be expected that the EA would correlate with the anthocyanins and colour levels in wine. This, however, was not the case in this study. There was no significant correlation between EA and most colour characteristics of the wine. This raises the question of the efficiency of the EA to predict total
extractability of anthocyanins from ripe grapes. However, it could still provide information on how quickly the anthocyanins can be extracted during winemaking operations particularly with regard to skin contact and fermentation.

Which method to use: Glories or Iland?
The question arises, which of these methods to use when attempting  to predict the phenolics and colour indicative compounds in grapes that would be convertedto wine? This is especially important for the commercial cellars as equipment such as the HPLC is normally not available. According to the findings in this study, the Iland method ight be slightly more suitable, as it yielded slightly better correlations with most of the wine data. The Glories method, however, had an advantage by delivering additional information such as skin tannins and the contribution of seed tannins to the total phenolic profile of the wine. Of course other factors such as equipment available, chemicals used, waste management and time should be borne in mind
when making this decision. This work might give wine producers, as well as wine analyses laboratories, with valuable information regarding the suitability of these methods to characterise the phenolic composition of South African red grapes and their resulting wines.

The author would like to thank Winetech, Thrip and the NRF for financial support, the vineyards and cellars which donated grapes for this project, Lorraine Geldenhuys, Hanneli van der Merwe and Andy Roediger for technical support and Professor Martin Kidd for the statistical analyses.
1. M. Rossouw, J. Marais, South African Journal of Enology and Viticulture. 2012, 25, 94.
2. W. J. Du Toit, K. Lisjak, J. Marais, M. du Toit, South African Journal of
Enology and Viticulture. 2006, 27, 57.
3. J. F. Habertson, R. E. Hodgins, L. N. Thurston, L. J. Schaffer, M. S.
Reid, J. L. Landon, C. F. Ross, D. O. Adams, American Journal of
Enology and Viticulture. 2008, 59, 210.
4. P. Iland, Chemical analysis of grapes and wine: techniques and concepts,
Patrick Iland Wine promotions, Campbelltown, Australia, 2004.
5. P. Ribéreau-Gayon, Y. Glories, A. Maujean, D. Dubourdieu, Handbook
of Enology, Vol. 2, 2 ed., John Wiley & Sons Ltd, Chichester, 2006.
6. W. J. Du Toit, M. Visagie, South African Journal of Enology and
Viticulture. 2012, 33, 33.
7. R. B. Boulton, American Journal of Enology and Viticulture. 2001, 52,
8. G. González-Neves, D. Charamelo, J. Balado, L. Barreiro, R. Bochiccio,
G. Gatto, G. Gil, A. Tessore, A. Carbonneau, M. Moutounet, Analytica
Chimica Acta. 2004, 513, 191.
9. D. De Beer, E. Joubert, J. Marais, M. Manley, D. Van Schalkwyk, South
African Journal of Enology and Viticulture. 2006, 27, 151.
10. L. Geldenhuys, University of Stellenbsoch (Stellenbosch), 2009.
11. Z. Guadaluoe, B. Ayestaran, Eur. Food Res. Technol. 2008, 228, 29.

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