ABSTRACT

The objectives of the present study were to determine the effects of the brewing method on the amount of the major catechins, methylxanthines, total polyphenols and antioxidant capacity in green and black teas, and to correlate the chemical composition of tea infusions to their sensorial properties by the combined use of the electronic nose and tongue. For this purpose, tea infusions were prepared from 7 green teas and 6 black teas using different extraction times (3 - 5 and 10 min). The extraction of total polyphenols and catechins was increased by prolonging the infusion time from 3 - 5 to 10 min, while the caffeine amounts did not increase. The antioxidant activity of teas was well correlated with polyphenol content. Electronic tongue and electronic nose were able to discriminate green teas from black teas on the basis of their sensorial properties. Considering the taste, green teas were characterized by astringency and aftertaste-astringency and this sensation was increased by prolonging the infusion time; black teas were perceived as more bitter, sour, salty and the extraction time affected the astringent sensation. The aroma of green and black teas was discriminated by specific sensors and the increase of the extraction time produced more richly flavoured infusions. This work adds information about correlation between sensorial properties, antioxidant capacity and chemical composition of green and black teas.

1. Introduction

Tea is the most widely consumed beverage throughout the world, appreciated because of its aroma and taste characteristics as well as beneficial health effects, including antioxidant activity, anticarcinogenic and antihypertensive effects [1]. All teas originate from a single evergreen plant, the Camellia sinensis var. sinensis and assamica, and diverse processing methods produce the various types of tea.

The major distinctive element of the different types of tea is the degree of oxidation, the leaf’s enzyme reaction to oxygen, a process that is improperly referred to as fermentation. The characteristics of raw material and the fermentation process greatly influence the chemical composition of teas [2], which are generally classified into three major categories: unfermented green tea, partially fermented oolong tea, and fully fermented black tea. In western countries, other than black tea, the consumption of green tea is an increasing and relatively recent trend and the market is continuously growing. The nature and quantity of chemicals contained in a cup of tea, as well as its sensory properties, are related to numerous factors, such as the starting material (variety and cultivar), the environmental conditions where the plant was grown, the period of year when the leaves were picked, the type of leaves used to produce the tea and the processing methods employed. Moreover, the chemical components are also influenced by the preparation method (extraction/ brewing conditions), that differs according to the varying cultures and traditions (type of water, use of teabag or loose-leaf, quantity of tea in relation to the amount of water, temperature of the water, steeping time). These compositional differences affect the health-promoting properties of tea, which are mainly due to its polyphenolic composition and content. Polyphenols in tea infusions are mainly represented by catechins and polymeric flavonoids derived by enzymatic oxidation of catechins (theaflavins and thearubigins). Flavonol glycosides, gallic and chlorogenic acids have also been detected in tea infusions [3]. Teas also contain methylxanthines: caffeine, one of the major components in tea extracts, and theobromine [4,5]. Water temperature can influence the extraction of tea components; tea infused at higher temperature had higher levels of catechins, especially epigallocatechin gallate and epigallocatechin, and caffeine than those extracted at lower temperature [6-8]. Another important brewing factor is the infusion time. It has been reported that the amount of catechins and caffeine increases with increasing duration of infusion [4,7]. Reference [9] reported that the total flavonoid content from loose leaf and bagged green tea increased with prolonged extraction time till 10 and 15 min, whereas longer extraction time can lead to catechin degradation. These results were in accordance with those reported by [10], who observed that catechins tend to degrade during prolonged extraction time.

Various studies investigated the effects of the type of tea and the brewing conditions (mainly water temperature, agitation, tea:water ratio and infusion time) in order to optimize the extraction of polyphenolic components and therefore maximize the nutraceutical characteristics of the beverage [1,3,4,7]. However, very few works take into account the effects of the varying conditions of tea preparation on the sensory attributes of the resulting beverage [5,11], though the sensory quality of teas is the primary aspect for the consumers to express their preference and choice. The sensory quality of food is generally determined by a panel of tasters, which is an expensive method in terms of time and labor, and sometimes gives inaccurate or subjective results. The electronic nose (enose) and electronic tongue (e-tongue) are fast, simple and non-destructive technologies applied successfully in the food field. There are some literature references about the e-nose application for the discrimination of different types of tea [12], for tea quality evaluation [13,14] and also for the detection of the optimum fermentation time in black tea manufacturing process [15,16]. E-tongue has been successfully used to discriminate different types of teas [17,18] and also to evaluate the geographical origin and quality level [19]. A recent research has evaluated black tea quality by using e-nose and e-tongue, showing that a better classification can be obtained when these two devices are used in combination [20]. To our knowledge, there are no works combining e-nose and e-tongue in order to relate the sensorial characteristics with the chemical composition of green and black teas.

The objectives of the present study were: (A) to determine the effects of the infusion time on the amount of the major catechins (epicatechin, epigallocatechin, epigallocatechin gallate, and epicatechin gallate) and methylxanthines (caffeine and theobromine), total polyphenols and antioxidant capacity in green and black teas; (B) to evaluate how the chemical composition of green and black tea infusions affects their sensorial properties, assessed by the combined use of e-nose and e-tongue.

2. Materials and Methods

2.1. Materials

Seven green teas and six black teas in loose leaf form were used; tea samples were prepared using the brewing method typically applied in Western Europe for home preparation: 2.5 g of tea leaves were infused in 250 mL of low mineral content water heated to boiling temperature. The dipping time was 3 min and 5 min for green and black teas, respectively, as usually suggested for these products. All teas were also infused for 10 min, in order to evaluate the enhancement of the extraction rates and the relevant effect on tea sensorial properties. Detailed information about the samples and brewing conditions are reported in Table 1.

Low mineral content water was used for tea brewing (S. Anna, Vinadio, Cuneo; fixed residue 42.8 mg/l; sodium 0.0001%; hardness 3.1 French degrees).

(−)-Epigallocatechin (EGC), (−)-epicatechin (EC) and gallic acid (GA) were purchased from Sigma (Sigma Aldrich Italia). Caffeine (CA), theobromine (THEO), (−)- epigallocatechin gallate (EGCG) and (−)-epicatechin gallate (ECG) were purchased from Fluka (Sigma Aldrich Italia).

Acetonitrile and methanol were HPLC grade; all other reagents were analytical grade.

2.2. HPLC Analysis of Catechins and Methylxanthines

The analyses were carried out with a HP 1100 apparatus (Chemstations, Agilent Technologies) using ZORBAX Eclipse XDB-C18 (5 µm, 4.6 mm × 150 mm) column with a specific precolumn. The temperature was maintained at 35˚C. The flow rate was 1 mL/min and the injection volume was 20 mL. Peaks were detected at 210 nm and 280 nm.

The mobile phase and gradient conditions were adapted from [21]. A gradient elution was performed using eluent A (acetonitrile) and eluent B (0.1% orthophosphoric acid in water) as follows: 10% A and 90% B was used in the first 3 min, then 35% A was reached in 9 min and maintained from 12 to 15 min. After 15 min, the mixing was programmed to 10% A and 90% B and the

column was conditioned with the initial eluent for 10 min.

Six concentrations of the standard compounds (gallic acid, the four catechins and the two methylxanthines) were prepared and injected. Methylxanthines and catechins in tea infusions were identified by comparison of retention time and adsorption spectra with reference standards. Tea infusions were analyzed in triplicate.

2.3. Determination of Total Polyphenols and Antioxidant Activity

Total polyphenols were determined by the Folin-Ciocalteau method [22]. 0.5 mL suitably diluted samples were mixed with 2.5 mL water, 0.5 mL Folin-Ciocalteau reagent and, after 3 - 5 min, 2 mL 10% Na₂CO₃. Water was added to total 10 mL and samples were stored for 90 min in the dark. The absorbance at 750 nm was read against a blank (by Jasco Uvidec 650 spectrophotometer) and total polyphenols were quantified by a calibration curve built with gallic acid. All samples were analysed in triplicate and data are expressed as mg/L gallic acid equivalents (GAE).

The antioxidant activity of tea infusions was determined by the DPPH* (2,2-diphenyl-1-picrylhydrazyl) assay as previously reported [23]. Dose-response curves were built for each sample and the amount of antioxidant corresponding to 50% inactivation of the DPPH* radical (I50), was calculated. Since I50 is an inverse index (the higher the antioxidant activity, the lower the I50 value), data were converted into trolox equivalents (milligram of trolox per 100 mL of tea infusion) using a calibration curve built with trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid). All determinations were carried out in triplicate and data are expressed as mmol/L Trolox equivalents (TE).

2.4. Electronic Nose

A Portable e-nose (PEN2) from Win Muster Airsense (WMA) Analytics Inc. (Schwerin, Germany) was used. It consists of a sampling apparatus, a detector unit containing the sensor array, and a pattern recognition software (Win Muster v.1.6) for data recording and elaboration. The sensor array is composed of 10 Metal Oxide Semiconductor (MOS) type chemical sensors: W1C (aromatic) W5S (broadrange) W3C (aromatic) W6S (hydrogen) W5C (arom-aliph) W1S (broad-methane) W1W (sulphur-organic) W2S (broad-alcohol) W2W (sulph-chlor) W3S (methane-aliph). The sensor response is expressed as resistivity (Ohm).

Three mL of tea infusions were placed in 10 mL Pyrex^â vials fitted with a pierceable Silicon/Teflon disk in the cap. After 10 min equilibration at 50˚C ± 1˚C, the measurement started. The headspace was pumped over the sensor surfaces for 60 s (injection time) at a flow rate of 300 mL/min, during this time the sensor signals were recorded. After sample analysis the system was purged for 180 s with filtered air prior to the next sample injection to allow reestablishment of the instrument baseline. The sensor drift was evaluated by using a standard solution of 1% ethanol included in each measurement cycle. For all the experimental period (4 weeks max) no sensor drift was experienced. All samples were analyzed in triplicate.

2.5. Electronic Tongue

Analyses were performed with the Taste-Sensing System SA 402B (Intelligent Sensor Technology Co. Ltd., Japan). The detecting part of the system consists of detecting sensors whose surface is combined with artificial lipid membranes having different response properties to chemical substances on the basis of their taste. The detecting sensors used in this work were: CT0 specific for saltiness, CA0 for sourness, C00 for bitterness and aftertaste bitterness, AE1 for astringency and aftertaste stringency. The measurement principle of the e-tongue is based on the capability of tasty substances to change the potential of the detecting sensors through electrostatic or hydrophobic interaction with the hydrophilic and hydrophobic groups of the lipid membranes. The detecting sensors were first dipped into the reference solution (30 mmol/L potassium chloride and 0.3 mmol/L tartaric acid) and the electric potential measured for each sensor was defined as Vr. Then the sensors were dipped for 30 s into 60 mL room temperature tea infusion and for each sensor the measured potential was defined as Vs. The “Relative value” (Rv) was represented by the differences between the potentials of the sample and the reference solution (Vs-Vr). Sensors were rinsed with fresh reference solution for 6 s and then dipped into the reference solution again. The new potential for the reference solution was defined as Vr’. The difference Vr’-Vr between the potentials of the reference solution after and before sample measurement is the “Change of Membrane Potential caused by Absorption value” (CPAv) and corresponds to the “aftertaste”. Before a new cycle measurement, electrodes were rinsed for 90 s with a washing solution (ethanol 30%) and then for 180 s with the reference solution. Each sample was evaluated in duplicate and the averages of the sensor outputs were converted to taste information. The “taste values” were calculated by multiplying the Rv and CPAv of the sensors for appropriate coefficients based on Weber-Fechner law, which gives the intensity of sensation considering the sensor properties for tastes [24].

2.6. Statistical Treatment of Data

E-nose and e-tongue data were standardized and analyzed by means of Principal Component Analysis (PCA) using the SCAN software for chemometric analysis (MINITAB Inc., PA, USA, 1998). Chemical data were subjected to one-way analysis of variance (ANOVA) and comparison among means was determined according to Fisher’s least significant difference (LSD) test, at 95% confidence level.

3. Results and Discussion

Table 2 reports the amounts of individual methyllxanthines and catechins in the black and green teas analysed. Concerning methylxanthines, all teas contained relatively low concentration of THEO, higher in black than in green teas. Pettiagalla (B1-B2) and Thowra (B7-B8), which are assamic varieties, showed the highest THEO content. Also in the case of CA, the highest amounts were measured in black teas; this is in agreement with literature data [4]. Green teas Chun Mee (G3-G4), Sencha Tokumushi (G5-G6) and Sencha Ariake (G11-G12) showed CA content similar to that found in black teas; lower CA levels were measured in Sencha Fuji-yama (G9-G10) and Bancha Yanagicha (G13-G14). Bancha is a Japanese green tea, considered naturally low in caffeine because it is made from more mature leaves, which contain less caffeine. In the case of methylxanthines, significant differences in the two infusion times were observed only for Lung Ching (G7-G8) and Thowra (B7-B8). Concerning catechins, for all investigated teas EGC and EGCG were the prevailing compounds, with values ranging from 2.33 mg/g to 26.65 mg/g for EGC and from 2.08 mg/g to 27.81 mg/g for EGCG; the content of EC and ECG was lower, ranging from 0.48 mg/g to 10.96 mg/g. It can be noted that Bancha Tostato Hojicha (G1-G2) had the lowest catechin content and Sencha Ariake (G11-G12) had the highest content. The prolonged infusion time not always led to significantly higher levels of extracted catechins. In the case of black teas, differences for all catechins were significant in Margaret’s Hope (B11-B12). For green teas, the effect of prolonged infusion was significant for Lung Ching (G7-G8), Sencha Fuji-yama (G9-G10) and Bancha Yanagicha (G13-G14).