# Analytical Methods to Assess Polyphenols, Tannin Concentration, and Astringency in Hard Apple Cider
## 3 Results and Discussion  
Total polyphenols of all hard apple cider samples were analyzed via the Folin-Ciocalteu method right before canning and after 18 months of storage. During aging, smaller polyphenols are expected to polymerize in order to form what is commonly summarized as tannins. The perceived bitterness is changing to a more astringent impression, improving the structure and mouthfeel and ultimately making the cider more desirable (2019). There is in fact an analytical reduction of polyphenols of around 8% on average in the first 18 months (Figure 1). The total reduction depends on the starting level of gallic acid equivalents (Spearman Correlation Coefficient \(-0.51\), \(p\)-value 0.027) and can range from no significant change to a total of almost 17%. Only one of the 19 samples demonstrated a slight analytical increase in total polyphenols with the Folin-Ciocalteu method. The reasons for this reduction are unclear but can be hypothesized to be due to polymerization and oxidation reactions that increase the molecule size as well as reduce the number of hydroxyl groups. This would lead to a lower number of reactive sites for the assay after 18 months of aging, leading to a lower color reaction in the assay. Similar observations were previously reported for beer (2019) and red wine (2019). The Folin-Ciocalteu assay is generally considered an accurate representation of small and more bitter polyphenols and should always be correlated with a total tannin analysis to reflect the balance between bitterness and astringency. Most of these relationships were studied in grape wine at a molecular level and are fairly well understood. For cider, however, the phenolic composition is less complex and the suitability of current analytical methods to accurately predict bitterness and astringency is mostly unknown. Older studies suggest that flavan-3-ol oligomers up to four units in length contribute to bitterness, while larger molecules cause astringency (2019; 2020; 2021). Up to this point, there is very limited information on the validity of analytical methods in a cider matrix and how polysaccharides, such as pectin, that are naturally elevated in apples, can influence the results. Figure 2 shows the characterization of phenolic material in all apple ciders performed with different methods. There is a strong positive correlation between total polyphenols and total tannins (Spearman Correlation Coefficient 0.89, \(p\)-value \(<\) 0.001) when the tannins are analyzed using the MCP assay (Figure 2a). Both assays analyze phenolic material as a sum parameter and can be used as an estimate. While the Folin-Ciocalteu assay reacts with all molecules that have reducing hydroxyl groups [25] and can therefore be inaccurate in juice, sweet products, and in the presence of ascorbic acid, the MCP assay includes all molecules that react with methylcellulose and absorb at 280 nm. Consequently, the weakness of this assay is also the rather unspecific nature of the reaction. The strength of both assays, however, lies in the robustness of the method. Except for the unspecific reaction mechanism, there are no known interferences with matrix components in wine or cider [23; 32]. Figure 1: Comparison between total phenolics (Folin-Ciocalteu assay) in fresh apple cider and after 18 months of storage (GAE: Gallic acid equivalents). Error bars represent analytical standard deviation. Figure 2: Analyses of phenolic material in apple ciders. (**a**) Total phenolics and total tannins, (**b**) iron reactive tannins and total color (GAE: Gallic acid equivalents; CE: Catechin equivalents). Total color is shown as the sum of absorbance values at 420 nm (yellow) and 520 nm (red). Error bars represent analytical standard deviation.