# Analytical Methods to Assess Polyphenols, Tannin Concentration, and Astringency in Hard Apple Cider
## 3 Results and Discussion
Both total polyphenols and total tannins correlate significantly with the iron-reactive polyphenols shown in Figure 2b (Correlation coefficient 0.89, \(p\)-value < 0.001 for both correlations), indicating that the phenolic substances in apple cider are similar enough to those in grape wine, for which the original assays were developed to produce consistent results. This could be expected, since monomeric flavian-3-ols and hydroxycinnamic acids as well as polymeric polyphenols consisting of catechin and epicatechin can be found in both cider and grape wine [11; 34; 35]. Iron-reactive phenolics also show a strong positive correlation with cider color (Spearman Correlation Coefficient 0.76, \(p\)-value < 0.001), indicating that the brown and red color in apple cider is based on phenolic compounds. This is in agreement with previous studies conducted by [PERSON], et al. [33] and [PERSON] and [PERSON] [36]. The red color portion, as part of the total color, however, varies between cultivars. For the most part, only red flesh apples show significant concentrations of anthocyanins, which are also structurally different from anthocyanins in red grape wine [36]. Orange hues found in some apple ciders are caused by oxidized and polymerized polyphenols that are colorless in their reduced state [33]. The orange color, however, can only be quantified by adding the absorbance at 420 nm and 520 nm, even though the red portion might not originate from anthocyanins. One of the main objectives for analyzing tannins in beverages is the estimation of perceived astringency for the consumer. While astringency in the mouth is caused by the precipitation of lubricating salivary proteins by phenolic compounds [2], different tannin assays use different methods of precipitation. In order to mimic that tannin precipitable protein fraction, bovine serum albumin (BSA) is used as a model protein in some assays [16; 27]. The methylcellulose precipitation (MCP) assay, on the other hand, is performed under conditions that essentially salt-out all tannin material independent of the chemical composition. For red wines, this was demonstrated to correlate with perceived astringency [34; 35]. Figure 3 shows a comparison of the relationship between MCP and astringency data for red wines and ciders. While not perfect, the correlation between total tannins and the astringency index in red wines is good (n = 10, R\({}^{2}\) = 0.572). In ciders, the correlation does not exist (n = 19, R\({}^{2}\) = 0.003), even though some of the ciders are analytically clearly dominated by tannins. Figure 3: Correlation between tannin material and astringency in apple cider compared to red wine. The reasons for that inconsistency can be speculated to relate to higher polysaccharide concentrations in the hard cider compared to wine. Polysaccharides can interfere with the reaction between proteins and polyphenols [21; 22] and while reacting with methylcellulose, the interaction with BSA is limited. Consequently, it remains unclear if astringency can be accurately evaluated in apple cider analytically without the involvement of a trained sensory panel. The role of pectin in the sensory perception of astringency has been known for some time and is used commonly during sensory panels. Pectin-enriched water can be served in between red wine samples to cleanse the palate and prepare for the next sample [37; 38]. This strategy alludes to a known interaction between tannins and pectin, as well as between pectin and proteins in the saliva. It is also possible that the use of a different protein could solve the analytical problem, for example the application of saliva protein instead of BSA [35; 39]. Any cross-reaction between the protein and polysaccharides needs to be evaluated for the astringency index to be meaningful in high-polysaccharide environments. Figure 4 supports that hypothesis, with the direct comparison of tannin data produced by using methylcellulose and BSA as precipitation agents.