Effects of Chemical Components on the Amount of Green Tea Cream

Agricultural Sciences in China

June 2011

2011, 10(6): 969-974

Effects of Chemical Components on the Amount of Green Tea Cream XU Yong-quan1, CHEN Su-qin2, SHEN Dan-yu3 and YIN Jun-feng1 Key Laboratory of Machining and Quality Control of Tea and Beverage Plants, Ministry of Agriculture/National Engineering Technology Research Center for Tea Industry/Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310008, P.R.China 2 Shenzhen Shenbao Huacheng Foods Co. Ltd., Shenzhen 518020, P.P.China 3 Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang 311400, P.R.China 1

Abstract The relationship between the amount of tea cream and the chemical components contents in the original green tea infusion was investigated. The results showed that the amounts of tea cream in the green tea infusions obtained from different cultivars and different parts of new shoots were varied. There were many chemical components participating in the formation of green tea cream. However, there were only the contents of caffeine (Y=0.85, P<0.01) and polyphenols (Y=0.65, P<0.05) in the original green tea infusion highly correlated with the amount of green tea cream. Stepwise regression analysis of overall chemical components indicated that the contents of caffeine and gallated catechins in the original green tea infusion had a significant effect (P<0.01) on green tea cream levels. Cream (g L-1)=-172.071+ 0.129×Ccaffeine+0.024×Ggallated catechins (R2=0.936). The amount of green tea cream can be predicted by the contents of gallated catechins and caffeine in the original tea infusion. Principal component analysis also indicated that catechins, minerals, and polysaccharides were the important chemical components in the formation of green tea cream. Key words: green tea infusion, chemical components, amount of tea cream

INTRODUCTION Tea cream forms a hot aqueous tea infusion, which not only has an unattractive appearance but also damages both taste and color of tea. Cream not only presents in black tea infusion (Smith 1968; Sanderson 1972), but also in semi-fermented tea infusion (Chao and Chiang 1999a) and green tea infusion (Liang et al. 2002). The constituents of tea cream include polyphenols (including thearubigins, theaflavine, and catechins), caffeine, protein, free amino acids (including theanine, aspartic acid, glutamic acid, and so on), carbohydrates (including pectin and polysaccharides), organic acids, and minerals (Ca, Fe, Mg, K, and so on) (Luck et al. 1994; Chao and Chiang 1999a; Jobstl et al. 2005).

Most of them participate in the formation of green tea cream (Liang et al. 2002; Yin et al. 2009). The formation of tea cream is governed by various parameters including extracting temperature (Liang and Xu 2003), pH (Liang and Xu 2001), chemical composition (Smith 1989; Yin et al. 2009), and solid concentration (Bee et al. 1987). Polyphenols, carbohydrates, and caffeine are found to be the main components of green tea cream (Yin et al. 2009). However, there were no reports studying on relationship data model between the amount of tea cream and the chemical components in original green tea infusion. Main chemical compositions, including polyphenols, caffeine, amino acids, minerals, and so on, are varied in different tea cultivars (Chen and Zhou 2005). There are different chemical compositions in the different parts

Received 21 June, 2010 Accepted 5 October, 2010 Correspondence YIN Jun-feng, Professor, Tel: +86-571-86650031, E-mail: [email protected]

© 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(11)60083-7

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of new shoots. The contents of polyphenols, catechins, caffeine, free amino acids, protein, magnesium (Mg), zinc (Zn), and kalium (K) decrease gradually, while the contents of total sugar, flavones, starch, chlorophyll, calcium (Ca), manganese (Mn), and aluminium (Al) increase gradually, among shoots with one leaf and a bud, second leaf, third leaf, and fourth leaf (Selvendran et al. 1973; Chen 1982; Wan 2003). Consequently, green tea infusions processed from many different tea cultivars and different parts of new shoots demonstrated the effects of chemical components contents in green tea infusion on the amount of tea cream. This study was aimed to investigate the relationship between the amount of tea cream and the chemical components in the original green tea infusion.

MATERIALS AND METHODS Tea leaves and preparation of the tea infusion New shoots of seven different cultivars of tea plant [Camellia sinensis (L.) O. Kuntze] were harvested in August 2007 from Hangzhou Tea Respository, National Germplasm located in the Tea Research Institute of Chinese Academy of Agricultural Sciences for this study. Among the seven cultivars of new shoots, there were six different cultivars with two leaves and a bud, which were named as: GPCS1953, GPCS2409, GPCS2407, GPCS1317, GPCS0022, and GPCS2532, and Zhenghe dabaicha ( PCS0034) with four leaves and a bud. Every new tea shoot of GPCS0034 was separated into four parts which were the bud and first leaf, the second leaf and its implicative stem, the third leaf and its implicative stem, and the fourth leaf and its implicative stem, which were named as: GPCS0034 (1), GPCS0034 (2), GPCS0034 (3), and GPCS0034 (4). There were 10 tea samples in total, which were ranged by the amount of tea cream in the infusions as shown in Table 1. The fresh tea leaves were then processed into dry tea leaves. After withering for 3 h, the tea leaves were panned at 130°C for 8 min, rolled for 5 min, first dried at 95°C for 30 min and second dried at 80°C to a final moisture content of about 4%. The dry tea leaves were stored in a freezer at -20°C to maintain their quality. The ground tea leaves (20-40 mesh) were extracted

XU Yong-quan et al.

Table 1 The list of tea cultivars for green tea samples used in the present study1) Number

Tea cultivar

1 2 3 4 5 6 7 8 9 10

GPCS1953 GPCS2407 GPCS2409 GPCS0034 (1) GPCS0022 GPCS0034 (2) GPCS1317 GPCS0034 (4) GPCS0034 (3) GPCS2532

1)

Amount of tea cream (g L-1) 2.01 a 1.78 b 1.25 c 1.23 c 0.59 d 0.52 e 0.49 e 0.47 e 0.43 f 0.35 g

Each value is the mean for three replicates. Means within a column followed by the same letter were not significantly different at the P=0.05 level.

with distilled water with 1:20 (w/w) leaf/water ratios at 70°C for 10 min. The extract was filtered through 300 mesh filterstuff and then quickly cooled down to 25°C or lower with a glass condenser using water (15°C) as a cooling medium. The cooled extract was then centrifuged at 10 000×g at 10°C for 15 min to obtain a clarified infusion, which was heated to 90°C, and every 45 mL was quickly infused into a transparent bottle. The bottles were 45 mL PET bottles, which were sterilized with 100°C water for 15 min before filling. Tea infusion in the bottles, which was the original tea infusion, was cooled down to 25°C, and then stored at 10°C for 20 d to observe tea cream formation.

Analysis of the amount of tea cream The amount of tea cream in tea infusions was determined according to the method described by Nagalashmi et al. (1984). The tea liquor in the bottles was centrifuged at 10 000×g at 10°C for 15 min. The supernatant was discarded. The precipitated tea cream was obtained and the bottle was washed twice with aliquots of 5 mL distilled water in a weighed glass dish having a diameter of 9 cm and dried at 80°C for 48 h.

Analysis of free amino acids and tea polyphenols The content of free amino acids in the tea infusion was determined with a spectrophotometer [UV-2550, Shimadzu (Suzhou) Instruments Manufacturing, Co., Ltd., Suzhou, China] by ninhydrin dying method as described by Yao et al. (2006). The content of tea polyphenols in the tea infusion was determined by the spectrophotometric method with FeSO4, 3.5×10-3 mol L-1

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Effects of Chemical Components on the Amount of Green Tea Cream

potassium sodium tartrate buffer (The First Research Institute of China Standards Publisher 2003).

Analysis of protein and flavones The protein content in the tea infusion was determined by bicinchoninic acid (BCA) method (Genmed Scientifics Inc., Boston, USA). The flavone content in the tea infusions was determined at 420 nm by the spectrophotometric method with 1% AlCl3 as described by Zhong (1989).

Analysis of carbohydrates and polysaccharides The carbohydrate content was determined by the anthrone-sulphuric acid reaction, using glucose as a standard (Fu et al. 2001). To prepare polysaccharides, 5 mL of tea infusion was precipitated with 10 mL of 100% (v/v) ethanol at 10°C for 24 h, and centrifuged (10 min, 5000×g) to get the sediment (Chen et al. 2005). Polysaccharides were dissolved, the volume was made to 25 mL with distilled water, and then analyzed according to the method used for carbohydrate analysis.

Analysis of tea catechins and caffeine Analysis of tea catechins and caffeine was carried out by HPLC method (Liang et al. 2002). The tea infusion was filtered through a 0.2 μm Millipore filter before injection (Model Shimadzu LC-2010A, Shimadzu Corporation, Kyoto, Japan). The HPLC conditions were as follows: injection volume, 5 μL; column, 5 μmDiamonsilTM C18 (4.6 mm×250 mm); temperature, 40°C; mobile phase A, acetonitrile/acetic acid/water (6:1:193); mobile phase B, acetonitrile/acetic acid/water (60:1:139); gradient, 100% mobile phase A to 100% mobile phase B by linear gradient during the early 45 min and then 100% mobile phase B up to 60 min; flow rate, 1 mL min-1; detector, Shimadzu SPD ultraviolet detector (Shimadzu Corporation, Kyoto, Japan) at 280 nm.

Analysis of minerals The minerals in the tea infusions were determined by ICP-OES (IRIS/AP, Thermo Jarrell Ash Corp., USA).

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Analytical conditions were as follows: Detector CID, low wavelength max intergration time: 15 s; high wavelength max intergration time: 5 s; nebulizer pressure: 28 psi; pump speed: 100 r/min; auxiliary gas: medium (1.0 L min-1); RF power: 1 150 W.

Statistical analysis Results are presented as mean value (at least three replicates). Analysis of variance and significant differences among means, bivariate correlations, stepwise regression, and principal component analysis were worked out by one-way ANOVA using SPSS (ver. 13.0, SPSS Inc., Chicago, USA). Stepwise regression analysis was performed on the data obtained from the ten green tea samples to test the contribution of chemical components to the formation of green tea cream. Principal component analysis was performed to detect the most important factors of variability and describe the relationship between variables and the amount of tea cream.

RESULTS AND DISCUSSION Bivariate correlations analysis of the amount of tea cream and the contents of chemical components in the original green tea infusions There were different amounts of tea cream in the green tea infusions processed from 10 tea samples (Table 1), which might be caused by different contents of the chemical constituents in the original tea infusion. In the previous study (Yin et al. 2009), polyphenols, carbohydrates, and caffeine were found the main components of green tea cream, while catechins [mainly including (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), (-)-epicatechin (EC), and (-)-epicatechin gallate (ECG)] and seven minerals (including Ca2+, K+, Mg2+, Mn2+, Na+, Zn2+, and Ni+) were also found in the green tea cream. Both caffeine-polyphenols and polyphenols-protein were considered as the main hypothetic complexation of Paochung tea cream (Chao and Chiang 1999a). Eight main minerals constituents, including Al3+, Ca2+, K+, Mg2+, Na+, Mn2+ Zn2+, and Ni+ were found in the original green tea infusions, and most

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of them participated in the formation of green tea cream. The correlation between the amount of tea cream and the contents of overall chemical components in the original infusion (Table 2) was studied. It was found that only caffeine (Y=0.85, P<0.01) and polyphenols (Y=0.65, P<0.05) were highly correlated with the amount of green

tea cream. The contents of polyphenols and caffeine in green tea cream also had a positive correlation with the amount of tea cream (Yin et al. 2009). Polyphenols and caffeine are both found to be the predominant in black tea (Smith 1968; Rutter and Stainsby 1975) and in semi-fermented tea (Chao and Chiang 1999a).

Table 2 Main chemical components of the original green tea infusions1) Chemical components Polyphenols (g L -1) Amino acids (g L-1) Caffeine (g L-1) Protein (g L-1) Carbohydrates (g L-1) Polysaccharides (g L-1) Flavones (g L-1) GC (g L-1) C (g L-1) EC (g L-1) EGCG (g L-1) EGC (g L-1) ECG (g L-1) Gallated catechins (g L-1) Total catechins (g L-1) Al3+ (mg L-1) Ca2+ (mg L-1) Mg2+ (mg L-1) K+ (mg L-1) Mn2+ (mg L-1) Na+ (mg L-1) Zn2+ (mg L-1) Ni+ (mg L-1)

Sample no. 1 10.19 0.69 1.4 0.18 4.27 0.42 0.46 0.69 0.37 0.51 2.45 1.33 0.63 3.18 6.08 7.7 16.6 46.0 574.5 24.6 16.8 2.7 0.2

2 7.83 0.94 1.47 0.16 3.44 0.32 0.25 0.43 0.21 0.4 2.09 1.36 0.42 2.56 4.96 3.8 14.9 63.0 662.5 50.8 16.7 3.2 0.3

3 9.7 0.81 1.08 0.11 2.41 0.34 0.26 0.19 1.05 0.45 2.04 0.5 1.2 3.3 5.48 5.7 12.2 37.1 500.0 10.3 16.6 3.0 0.1

4 9.73 0.7 1.13 0.12 4.33 0.37 0.29 0.21 0.17 0.53 2.87 1.37 0.79 3.72 6 4.3 14.5 35.6 486.9 23.9 14.4 2.8 0.2

5

6

7.16 0.74 1.09 0.1 4.57 0.21 0.29 0.28 0.11 0.39 1.91 1.43 0.38 2.38 4.59 3.5 18.5 30.0 464.2 22.4 15.0 2.8 0.1

7

8.65 0.89 1.02 0.06 4.76 0.35 0.41 0.26 0.11 0.62 2.34 1.72 0.61 3.03 5.75 9.2 19.2 40.6 521.5 32.2 14.9 2.5 0.2

8.06 0.8 1.07 0.13 5.11 0.33 0.57 0.39 0.1 0.53 1.94 1.76 0.44 2.5 5.28 5.9 17.3 46.2 573.2 25.3 16.1 2.9 0.2

8 8.2 0.95 0.92 0.15 5.83 0.4 0.44 0.26 0.08 0.54 1.88 1.81 0.42 2.76 5.06 19.8 20.5 43.4 560.2 33.7 15.9 2.6 0.2

9

10

7.51 0.97 1 0.16 5.6 0.37 0.43 0.32 0.09 0.6 2.16 1.94 0.51 2.37 5.71 14.7 24.0 38.1 582.3 35.2 16.0 2.7 0.2

7.21 0.81 1.14 0.15 3.98 0.3 0.46 0.39 0.12 0.51 1.48 1.71 0.38 1.92 4.64 4.6 14.8 48.1 608.0 20.8 16.8 3.0 0.2

Correlation 2) 0.65* -0.35 0.85 ** 0.38 -0.56 0.32 -0.43 0.52 0.45 -0.43 0.51 -0.59 0.37 0.53 0.40 -0.36 -0.52 0.39 0.19 0.14 0.30 0.32 0.28

Each value is the mean for three replicates. The correlation coefficient of the amount of tea cream and the contents of chemical components in original green tea infusion. * P<0.05, ** P<0.01. 1) 2)

Stepwise regression analysis of overall chemical components in the original green tea infusions With the dependent variable of the amount of green tea cream, stepwise regression analysis of overall chemical components in the original tea infusions was carried out. Stepwise regression analysis indicated that caffeine (C) and gallated catechins (G) had a significant effect (P<0.01) on green tea cream levels (Tables 3 and 4). Cream (g L -1 )=-172.071+0.129×C+0.024×G (R 2 =0.936) In the former research, it was observed that polyphenols, caffeine, EGCG, and ECG were the principal constituents of green tea cream formation (Yin et al. 2009). Polyphenol-caffeine complex and polyphe-

Table 3 Model summary of stepwise regression analysis1) Model 1 2 1)

R 0.848 0.968 3) 2)

R2

Adjusted R 2

SE

0.719 0.936

0.684 0.918

15.45263 7.85450

Stepwise regression (criteria: probability-of-F-to-enter 0.50, probability-

of-F-to-remove 0.100). Dependent variable: green tea cream. 2) Predictors: (constant), caffeine. 3) Predictors: (constant), caffeine, gallated catechins.

nol-polyphenol interactions were the primary interactions in tea cream formation, and the numbers of gallate and hydroxyl groups of the polyphenols are the important factors (Rutter 1971; Collier et al. 1972; Rutter and Stainsby 1975). The gallated catechins have been identified to be active sites for a hydrogen bond, and a much greater tendency to precipitate with protein and caffeine than the non-ester-form catechins (Chao and Chiang 1999b).

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Effects of Chemical Components on the Amount of Green Tea Cream

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Table 4 Coefficients of stepwise regression analysis1) Unstandardized coefficients B SE

Model 1 2

1)

(Constant) Caffeine (Constant) Caffeine Gallated catechins

-111.849 0.135 -172.071 0.129 0.024

Standardized coefficients β

34.166 0.030 21.282 0.015 0.005

t

Sig.

-3.274 4.522 -8.085 8.502 4.895

0.848 0.813 0.468

0.011 0.002 0.000 0.000 0.002

Dependent variable: green tea cream.

Principal component analysis (PCA) of overall chemical components of the original green tea infusions PCA was used to reduce the multivariate data to a few composite variables that would account for the most effect factor of variation in the formation of green tea cream. PCA on these attributes explained 69.85% of the variability in the data in the first three components, and they accounted for 28.84, 22.73, and 18.28% of the data variance (Table 5). Variables in the first component were grouped as the catechins component, indicating that set of variables related to general catechins. Variables in the second component were grouped as the mineral component, while the third component accounted for polysaccharides.

CONCLUSION Many chemical components including polyphenols, caffeine, protein, carbohydrates, polysaccharides, free amino acids, flavones, most catechins, and seven main minerals were found to participate in the formation of green tea cream. However, there were only the contents of caffeine (Y=0.85, P<0.01) and polyphenols (Y=0.65, P<0.05) in the original green tea infusion highly correlated with the amount of green tea cream. The stepwise regression analysis of overall chemical components indicated that the contents of caffeine and gallated catechins in the original green tea infusion had a significant effect (P<0.01) on the tea cream levels. Principal component analysis also indicated that catechins, minerals, and polysaccharides were important chemical components when green tea cream formed. The amount of green tea cream can be predicted by the contents of gallated catechins and caffeine in the original tea infusion.

Table 5 PCA of chemical components from ten green tea samples Component

Chemical components

1

Polyphenols Free amino acids Caffeine GC C EGC EGCG ECG EC Total catechins Gallated catechins Protein Carbohydrates Polysaccharides Flavones Al 3+ Ca2+ Mg2+ Ni + K+ Mn2+ Na+ Zn2+ Cumulative variance (%)

-0.718 0.578 -0.388 0.053 -0.829 0.947 -0.407 -0.832 0.434 -0.318 -0.672 0.127 0.857 -0.040 0.604 0.573 0.820 0.120 -0.370 0.359 0.499 -0.059 -0.391 28.838

2

3

-0.402 0.136 0.644 0.461 0.047 -0.108 -0.576 -0.360 -0.628 -0.605 -0.585 0.474 -0.417 -0.373 -0.091 -0.395 -0.365 0.756 0.191 0.728 0.306 0.730 0.826 51.572

0.518 0.028 0.455 0.676 -0.007 0.123 0.465 0.059 0.361 0.691 0.315 0.622 0.152 0.829 0.324 0.290 0.128 0.444 0.649 0.498 0.347 0.375 -0.199 69.849

Acknowledgements The authors thank Prof. Wang Huafu, Finlays Company, London, England, Prof. Chen Liang and Prof. Han Wenyan, Tea Research Institute, Chinese Academy of Agricultural Sciences, for their revision. This research was supported by the Special Scientific Research Funds for Commonweal Section of the Ministry of Agriculture, China (nyhyzx07-3-35), the Natural Science Foundation of Zhejiang Province, China (R3090394), and the Key Laboratory of Processing and Quality Control of Tea & Beverage Plants Products, Ministry of Agriculture, China (2010K1004).

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