Exploring The Relative Astringency of Tea Catechins and Distinct Astringent Sensation of Catechins and Flavonol Glycosides via an In Vitro Assay Composed of Artificial Oil Bodies

Abstract

Artificial oil bodies covered by a recombinant surface protein, caleosin fused with histatin 3 (a major human salivary peptide), were employed to explore the relative astringency of eight tea catechins. The results showed that gallate-type catechins were more astringent than non-gallate-type catechins, with an astringency order of epicatechin gallate > epigallocatechin gallate > gallocatechin gallate > catechin gallate > epigallocatechin > epicatechin > gallocatechin > catechin. As expected, the extension of brewing time led to an increase in catechin content in the tea infusion, thus elevating tea astringency. Detailed analysis showed that the enhanced proportion of gallate-type catechins was significantly higher than that of non-gallate-type catechins, indicating that tea astringency was elevated exponentially, rather than proportionally, when brewing time was extended. Rough surfaces were observed on artificial oil bodies when they were complexed with epigallocatechin gallate (a catechin), while a smooth surface was observed on those complexed with rutin (a flavonol glycoside) under an atomic force microscope and a scanning electron microscope. The results indicate that catechins and flavonol glycosides induce the sensation of rough (puckering) and smooth (velvety) astringency in tea, respectively.

Keywords: artificial oil bodies; astringency; caleosin; catechins; histatin 3.

Figure 1

(A) Flotation of aggregated artificial oil bodies interacting with eight catechins in tea: ECG, EGCG, GCG, CG, EGC, EC, GC and C. Aggregated artificial oil bodies formed a visible emulsion layer on upper portion of the cuvette. (B) Light microscopy of aggregated artificial oil bodies interacting with eight catechins. Artificial oil bodies were allowed to interact with each catechin at room temperature for 60 min before taking the photos. Bars represent 40 μm. (C) The relative astringency of eight catechins. The relative astringency was calculated by measuring the thickness of emulsion layer in each catechin interaction, normalized with the thickness of emulsion in ECG interaction set as 100%. Statistical significance was processed by one-way ANOVA with Tukey’s test posttest (n = 3). * indicates p < 0.05, ** indicates p < 0.01.

Figure 2

(A) Flotation of aggregated artificial oil bodies in tea infusion prepared with extended brewing time. A visible emulsion layer on the upper portion of the cuvette was observed for artificial oil bodies mixed with a tea infusion prepared via brewing for 10, 20, 30 or 40 min. (B) Light microscopy of artificial oil bodies in tea infusion. Aggregation of artificial oil bodies in the four tea infusions was observed at room temperature for 60 min before taking the photos. Bars represent 40 μm. (C) Relative astringency of the four tea infusions. The relative astringency was calculated by measuring the thickness of emulsion layer and normalized by setting the thickness of emulsion in 10 min-brewing tea infusion set as 100%. Statistical significance: * indicates p < 0.05, *** indicates p < 0.001 (n = 3).

Figure 3

(A) HPLC profiles (OD 280 nm) of the four tea infusions prepared with different brewing time. The contents of four major catechins in infusion prepared by brewing the same tea for 10, 20, 30 or 40 min were analyzed and compared. The peaks of caffeine and the four major catechins, EGCG, EGC, ECG and EC, were indicated in the profile of tea infusion when they were brewed for 40 min. (B) Relative contents of EGCG, EGC, ECG or EC in the four tea infusions. The relative contents of EGCG, EGC, ECG and EC were estimated by calculating their peak areas in the HPLC profiles in (A). The content of each catechin in 10 min-brewing tea infusion was set as 100%.

Figure 4

Topography images of artificial oil bodies alone (A) or complexed with EGCG (B) or rutin (C) in atomic force microscopy. Artificial oil bodies were mixed with or without 5 mM of EGCG or rutin and observed under the atomic force microscope.

Figure 5

Relative surface roughness of artificial oil bodies (AOB) alone or complexed with EGCG or rutin. Surface roughness was analyzed by NanoScope Analysis (Ver. 7.4). Relative surface roughness was calculated by sampling (n = 5) images in Figure 5, and the average value of surface roughness of AOB alone was set as 100%. Statistical significance: *** indicates p < 0.001.

Figure 6

Topography images of artificial oil bodies alone (A) or complexed with EGCG (B) or rutin (C) in scanning electron microscopy. Artificial oil bodies were mixed with or without 5 mM of EGCG or rutin and observed under the scanning electron microscope (scale bar = 2 μm).

Figure 7

SDS-PAGE of the recombinant caleosin–histatin 3 overexpressed in E. coli. Total proteins of E. coli cells with caleosin–histatin 3 overexpressed after isopropyl β-D-1-thiogalactopyranoside (IPTG) induction were resolved in SDS-PAGE. Soluble (sup.) and insoluble (ppt) proteins extracted from E. coli cells were also analyzed. Labels on the left indicate the molecular masses of commercial marker proteins (Genemark, Taichung, Taiwan).