# Analytical Methods to Assess Polyphenols, Tannin Concentration, and Astringency in Hard Apple Cider
## 1 Introduction  
Tannins are an essential group of components in many foods and beverages and are reported to influence visual appearance [1] and texture [2], as well as the flavor profile [3; 4; 5]. These relationships have been thoroughly described in beverages such as wine [3] and beer [6] but other fermented products such as hard cider are lacking that level of understanding. While tannins can have unwanted effects in a mostly colorless product, such as cider, ranging from haziness [7] to undesired browning [8], the positive contributions to texture, mouthfeel, and perceived volume of the beverage [3] are worth mentioning. In the United States, hard ciders are fermented wine-like apple beverages with ethanol concentrations commonly exceeding 7% by volume. Since most commercial hard apple ciders are blends of different cultivars, tannins can be considered important blending quality indicators in addition to sugar, acidity, and pH.  
The overall concentration of tannin material in cider depends on apple cultivar [9], culturing techniques [10], and processing conditions [11], as well as fermentation and aging treatments [12]. Interestingly, all apple tissue, skin, pulp, and seeds, contain condensedtannins with the highest concentration in the skin. Tannin material in apple cider differs from tannins found in grape wine, mainly due to differences in the average degree of polymerization (aDP) and constituent composition [13]. [PERSON] et al. (2006) found that tannins in cider are generally less polymerized with an aDP between 1.61 and 3.69, compared to wine with an aDP up to 13. Cider tannins are also fairly uniform, containing mostly epicatechin and some catechin in terminal positions [9]. These specific characteristics may impact perceived bitterness and astringency, since it was demonstrated that a lower aDP favors bitterness over astringency [5].  
Some phenolic components in apples, for example hydroxycinnamic acids, can have a similar sensory effect [14] but are easier to describe analytically than tannins. Small polyphenols have been analyzed in apples and can be used to characterize and predict bitterness [15]. Tannins or procyanidins on the other hand include a wide range of molecule sizes, which makes them difficult to characterize and quantify accurately. Analyzing the amount of final tannins in cider presents the additional challenge that most of the photometric assays were developed for wine and validated for that specific matrix [3, 16, 17, 18, 19]. Apple cider usually demonstrates higher concentrations of polysaccharides [20] that have been shown to react with tannin material, making reactions less predictable [21] and potentially the assay less accurate [22].  
A variety of tannin assays were specifically developed for red wines and include the observation of polymeric pigments in the visible light range, and others focus on absorbance in the ultraviolet (UV) spectrum. All of those assays, however, use a fairly broad approach to analyze this diverse group of molecules by either precipitation (Methylcellulose Precipitation Assay [23], Astringency Index [24], or Harbertson Adams Assay [16]) or non-specific color reactions (Folin Ciocalteu method [25, 26] or Iron Reactive Phenolics [18]). Reaction conditions, standards, and assay time can vary significantly and yield different information that might be more or less adaptable to other beverages such as apple cider.  
The goal of this study was to demonstrate the use of spectrophotometric assays for polyphenols and tannins in a wide variety of hard apple ciders and suggest interpretation and modification approaches for that matrix compared to wine made from grapes.