Comparison of Glories Iland and Hand-Crushed Methods for Assessing Grape Phenolics

Introduction

It is well established that grape seeds contribute significantly to the phenolic composition of red wine. A higher proportion of seeds in the ferment therefore results in increased extraction of seed-derived tannins, which are generally more astringent and bitter than tannins originating from grape skins.

The phenolic composition of grapes can be analysed to provide winemakers with valuable information for decision-making. Such analyses can assist in determining optimal harvest timing, predicting the phenolic profile of the resulting wines, and guiding maceration and pressing strategies.

Numerous laboratory methods for grape phenolic analysis have been published. Ideally, such methods should provide reliable results within a short time frame. This is often achieved by homogenising whole red grapes and adding ethanol to promote rapid extraction of phenolic compounds, which can subsequently be quantified using spectrophotometric techniques. One such method, commonly referred to as the Glories method, estimates the potential contribution of grape seeds to the final tannin concentration.

In this study, three extraction methods were evaluated to assess phenolic compounds in red grapes with differing seed levels. These methods included the Glories method, the Iland method, and the recently developed hand-crushed method. Unlike the Glories and Iland methods, which rely on grape homogenisation, the hand-crushed method involves manual crushing of the berries, while avoiding damage to the seeds.

Materials and methods

The effect of seeds on phenolic composition in grape extracts was studied using three extraction methods: Iland, Glories and machine-crushed. These methods differ in solvent, time, pH and grape preparation. Two crates (approximately 18 kg each) of Shiraz and Merlot grapes were collected from two vineyards in Stellenbosch and immediately frozen at −20°C. For each cultivar, 2 500 frozen berries were randomly taken from the crates and thoroughly mixed to ensure a uniform sample. From this mixed sample, 2 000 berries per cultivar were manually deseeded using a surgical blade. For grape phenolic analysis, a sub-sample of 500 berries per cultivar was taken from the same mixed batch. Of these, 200 berries were used for the homogenate extraction methods and 300 berries for the machine-crushed extraction method. All berries in this sub-sample were also manually deseeded following the same procedure.

The separated skins and seeds were weighed and recombined with the relevant volume of juice to create three seed-to-skin ratio treatments, each prepared in triplicate: seedless (SL), normal seed-to-skin ratio (1× seeds), and double seed-to-skin ratio (2× seeds). These treatments were used to evaluate the effect of seed content on phenolic extraction.

Three extraction methods were applied to each treatment. The Iland method involved a one-hour extraction of grape homogenate in 50% ethanol. The Glories method consisted of a four-hour extraction of grape homogenate in aqueous solutions at pH 1.0 and pH 3.2. The third method was an in-house machine-crushed protocol, in which grapes were mechanically crushed, heated using a microwave, and extracted for three hours in 50% ethanol. This was done to ensure better repeatability between grape batches and the operator than the hand-crushed method.

All extraction procedures were carried out using the same conditions for both cultivars and for all seed-to-skin ratio treatments. The crushed grapes were treated using a custom-made small-scale crusher, as shown in Figure 1. In Figure 2, the grape homogenate can be compared to the machine-crushed grapes.

FIGURE 1. A custom-made small-scale crusher was used in this work.

FIGURE 2. Grape berries being fed into the machine crusher (left), machine crushed berries (middle) and berry homogenate (right).

Results

Iland method

Using the Iland method (Table 1), anthocyanin concentration and colour density showed no significant differences across treatments for either cultivar. However, tannin concentrations and total phenolic index varied. An increase in seed number led to higher tannin concentrations, while total phenolics increased significantly from the seedless to the other treatments in Shiraz and from 1× seeds to 2× seeds in Merlot.

Machine-crushed method

This method used crushed berries with intact seeds and microwave assistance (Table 2). Using this method, the colour density, anthocyanins and total phenolics did not differ significantly. Only in the Merlot were the seedless and 2×-seed treatments significantly different.

Overall, both the Iland and machine-crushed methods showed similar trends in anthocyanin levels and colour density, which were unaffected by seed presence. This suggests that seeds do not influence anthocyanin extraction, consistent with other authors (2014), who reported that the absence of seeds does not enhance anthocyanin release from skins.

Glories method

Widely used for analysing grape phenolics, the Glories method provides extractable anthocyanin index (EA%) and seed tannin contribution (MP%). Interestingly, the seedless treatments showed higher potential anthocyanins (A pH 1). In Shiraz, 1× seeds had higher EA% than 2× seeds. In Merlot, EA% did not differ significantly across treatments. Surprisingly, seedless treatments still showed tannins attributed to seeds (MP%), despite the absence of seeds. For 1× and 2× seeds, MP% differences were not significant, though an upward trend was noted in both cultivars.

This anomaly raises questions about the validity of the Glories method for seed tannin contribution. MP% is calculated by subtracting estimated skin tannins (derived from anthocyanin absorbance at 520 nm multiplied by a constant factor of 40) from total phenols (280 nm absorbance). In seedless treatments, this calculation may falsely attribute tannins to seeds, suggesting methodological limitations.

Comparative insights

Across methods, tannin concentrations were influenced by seed presence: treatments with seeds generally produced higher tannin levels. However, in machine-crushed Shiraz extracts, this trend was absent, likely because seeds remained intact. In contrast, homogenisation in the Iland and Glories methods breaks seeds, enhancing tannin extraction.

Overall, more seed leads to higher tannin extraction into grape extract, but not to higher anthocyanin levels. The validity of the Glories index to estimate the contribution of seeds to wine tannin concentrations should also be reassessed. Grape phenolic analyses can also be performed for the industry using the PhenoLAB platform at the South African Grape and Wine Research Institute. The PhenoLAB platform uses the Iland method for tannin analysis, as it correlates well with wine astringency and, unlike the Glories method, does not appear to produce false-positive contributions from seed tannins in the absence of seeds.

In part two of this work, wines made from these different seed levels will be presented.

(This article has been written with the assistance of Copilot.)

For more information, contact Wessel du Toit at [email protected].

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