Stability of tea polyphenols solution with different pH at different temperatures

ABSTRACT

Although tea polyphenols possess a variety of biological activities, the oxidative stability of tea polyphenols limits its application in the diet as preventive medicine. To enlarge biological activity of tea polyphenols, we investigated changes of tea polyphenols with different pH (3, 4, 5, 6, and 7) at different temperatures (4, 25, and 100°C). Changes in transmittance, deterioration, color values, and contents of catechins were evaluated. The results showed that tea polyphenols with a pH of 3–6 remained stable at 4 and 25°C. With increase of temperature, the tea polyphenol solutions became darker and less green, but deeper yellow in color. When the heating temperature was 100°C, a significant reduction in both total catechins and transmittance was observed. Individual catechins undergo epimerization in this process. Not only temperature and pH, but also heating time influenced the epimerization. Total contents of catechins incubated in pH 3, 4, 5, 6, and 7 citrate buffer solutions for 24 h declined by 15, 24, 41, 57, and 96% at 100°C, respectively. Therefore, tea polyphenols were pH-sensitive: the lower the pH, the more stable the tea polyphenols during storage. Storing at low temperature and acidic pH conditions did not significantly affect the characteristics of tea polyphenols.

KEYWORDS:

• Tea polyphenols
• Stability
• Tea drinks
• Temperature
• pH

Introduction

Tea is one of the most highly consumed beverages in the world.^[1–3] Tea polyphenols (TP) are the major nutraceutical components in tea and account for 30–42% of the dry weight of the solids in brewed green tea.^[4,5] Tea catechins account for 70% of total TP. The major tea catechins are (−)-epigallocatechin-3-gallate (EGCG), (−)-epicatechin gallate (ECG), (−)-epigallocatechin (EGC), and (−)-epicatechin (EC). These epicatechins can change to their epimers those are non-epicatechins, i.e., (−)-gallocatechingallate (GCG), (−)-catechin gallate (CG), (−)-gallocatechin (GC), and (−)-catechin (C). The polyphenolic constituents have drawn wide attention to some extent due to their natural abundance and biological activities, which include antioxidant,^[6,7] anti-mutagenic,^[8] anti-cancer,^[9] anti-allergic,^[10] and anti-ultraviolet (UV) activities.^[11] Therefore, it is important to minimize changes of TP in tea beverages, tea foods, instant tea powder, and cosmetics during production, distribution, and storage. Figure 1b showed that the transmittance of TP solutions with a pH of 3–6 had almost no significant change and maintained a stable state in 24 h at 25°C. Also, the transmittance of TP solutions had a significant decline at the first hour at 25°C and declined gradually in the following hours. The transmittance of TP solutions with pH 7 was bigger at 4°C than that at 25°C after 24 h. Figure 2b showed that the absorbance values of TP solutions with a pH of 3–6 had almost no significant change and maintained a stable state in 24 h at 25°C. Also, the absorbance values of TP solutions increased gradually with time at 25°C. These results were consistent with the result reported by Wang, Kim, and Lee.^[21] The absorbance of TP solutions had great difference at different pH. The absorbance values of TP solutions with pH 7 was bigger at 25°C than that at 4°C after 24 h, suggesting that the absorbance values of TP solutions had great difference at different temperatures.

The stability of TP is dependent on both temperature and pH. It was noted that TP degraded faster with the increase of either pH, oxygen concentration, or temperature.^[12] Hong^[13] reported that several factors, including pH, concentration of proteins, antioxidant levels, and the presence of metal ions, could affect the stability of EGCG, of which pH is probably the most critical. TP solutions were very unstable in neutral and alkaline solutions and decomposed in a few minutes, whereas, they were relatively stable under acidic conditions.^[14] Tea catechins were found to be unstable in sodium phosphate buffer (pH 7.4), conditions under which 80% of them were lost in only 3 h;^[15] and 40% of them were lost in 3 h in boiling water.^[15] It was also shown that tea catechins degraded by at least 50% during the first month of storage in commercial soft drinks and even in acidic ones.^[15] TP solution degraded irreversibly to a yellowish-brown solution of the deterioration products, mainly through oxidation and dimer formation.^[16] This browning is undesired, e.g., in bottled green tea beverages and also declines TP biological activities.

To date, in spite of the numerous potential health benefits revealed, TP are scarcely added to food or drink products, and in the few cases it is the amounts added, and moreover, the residual amounts left by the time of consumption, are apparently insufficient to produce beneficial effects. It is important to understand the stability of TP in foods or drinks during processing and storage in order to gain the optimum health benefits from them. Previous studies evaluated the stability of TP only from change of the content of catechins, and do not comprehensively demonstrate the changing trends of TP from sensory evaluation, including transmittance, deterioration, color value, and nutritional quality. Hence, the objective of this work was to characterize the degradation of TP under diverse conditions, and to provide improved ways for storing TP and TP products such as tea beverages, tea foods, instant tea powder, and cosmetics.

Materials and methods

Chemicals

TP (caffeine < 0.5%) were purchased from Changsha Huacheng Biotech, Inc. (Changsha, China), which were extracted from tea. Acetic acid (high-performance liquid chromatography [HPLC] grade) and methanol (HPLC grade) were purchased from Chengdu Changzheng Huabo Co., Ltd. (Chengdu, China). Chemical standards for EGC, EGCG, EC, ECG, C, and GCG were purchased from Sigma Aldrich Chemical CO., Ltd. (USA). All other chemicals used in this study were analytical grade.

Sample preparation

To examine the stability of TP with different pH (3, 4, 5, 6, and 7) at different temperatures (4, 25, and 100°C), 50 mg of TP were maintained in 100 mL of 100 mM citrate buffer solutions at different temperatures for 24 h. The appropriate volume of water was periodically added to compensate for loss due to evaporation.

Transmittance and deterioration studies by UV spectra measurement

Samples in citrate buffer solutions (pH 3–7) were stored at 4, 25, and 100°C. Aliquots of 2.5 mL of TP solution were transferred to 1 cm path length spectrophotometer cuvettes and covered with parafilm to prevent evaporation. The transmittance at 640 nm was observed for 24 h using an Ultrospec2450 spectrophotometer (Shimadzu Japan).^[17,18] The absorbance value at 425 nm was observed for 24 h using an Ultrospec2450 spectrophotometer (Shimadzu Japan) to study the deterioration of TP solutions.^[16]

Color of TP in diverse solutions

Samples were placed as a uniform layer (0.5 cm thick) on a 5 cm diameter Petri dish and a Tristimulus reflectance colorimeter (HunterLab, model D25) calibrated with a white standard tile (X = 82.45; Y = 84.46; Z = 101.44) were used. Color was recorded using the CIE-L* a* b* uniform color space (CIE-Lab), where L* indicates lightness, a* indicates hue on a green (–) to red (+) axis, and b* indicates hue on a blue (–) to yellow (+) axis.^[19]

HPLC analysis of tea catechins

The solution filtrated at 0.45 μm was done before injection. Briefly, an HPLC-20 (Shimadzu Japan), equipped with an auto injector, a C18 reversed-phase column (250 × 4.6 mm /5 um, Thermo) were used for the analysis of tea catechins. Mobile phases consisted of 2% glacial acetic acid in water (eluent A) and methanol (eluent B). A gradient system was adopted as follows: 0–25 min, linear gradient from 18 to 25% B; 25–30 min, linear gradient from 25 to 35% B; 30–32 min, linear gradient from 35 to 18% B; 32–37 min, 18% B. The sample injection volume was 10 μL. The flow rate was 0.9 mL/min. Tea catechins were detected at 278 nm.

Statistical analysis

All measurements were carried out in triplicate. Data were subjected to one-way analysis of variance (ANOVA) using the Compare Means Procedure of SPSS Statistics (19.0; SPSS Inc., Chicago, IL). The least significant difference (LSD) procedure was used to test for differences between means (differences were considered to be significant when p < 0.05).

Results and discussion

Transmittance change of TP solution with pH at different temperatures

The transmittance at 640 nm can describe the turbidity of the solution.^[17] The transmittance of TP solutions with different pH at different temperatures were monitored for 24 h (Fig. 1a–c). Figure 1a: Transmittance in the spectra at 640 nm change of TP solutions with a pH of 3–7 at 4°C; B: at 25°C; and C: at 100°C. Data are expressed as means ± standard deviation (SD) of n = 3 samples. The different letter in the same temperature indicated that the difference between the treatments is significantly through LSD test (p < 0.05)

Figure 1a showed that the transmittance of TP solutions with a pH of 3–6 had almost no significant change and maintained a stable state in 24 h at 4°C. The transmittance of TP solutions with pH 7 had a significant decline at the first 1 h and maintained a stable state in 10 h, and then had a significant decline after 24 h.

As shown in Fig. 1c, there were significant differences between pH 3 to pH 7 at 100°C. From Fig. 1c, we could see that the transmittances of TP solutions were bigger at low pH values. After boiling for 24 h, the transmittances of TP solutions with a pH of 3–7 were 85.87, 91.37, 84.70, 74.77, and 50.13, respectively, resulted in about 2, 7, 14, 24, and 49% reduction in transmittance. The transmittances of TP solutions at the same pH had great differences at the three different temperatures. The higher the temperature and pH was, the greater decline the transmittance. Perhaps catechins in liquid and solid environments might undergo degradation, oxidation, epimerization, and polymerization reactions forming new compounds,^[20] which resulted in the increase of the turbidity and the decline of the transmittance of the solution. The higher the temperature was, the more compounds would be formed.

Figure 1. Transmittance in the spectra at 640 nm change of TP solutions with pH 3–7 at A: 4°C; B: at 25°C; C: at 100°C. Data are expressed as means ± SD of n = 3 samples. The different letter in the same temperature indicated that the difference between the treatments is significantly through LSD test (p < 0.05).

Deterioration of TP solution with different pH at different temperatures

As the appearance of yellow color correlates with oxidation of TP,^[13,16] the absorbance values at 425 nm of solutions can be used to demonstrated the deterioration of TP solution. The absorbance values with different pH at different temperatures were observed for 24 h (Fig. 2a–c). Figure 2a: Absorbance in the spectra at 425 nm change of TP with a pH of 3–7 at 4°C; B: at 25°C; and C: at 100°C. Data are expressed as means ± SD of n = 3 samples. The different letter in the same temperature indicated that the difference between the treatments is significantly through LSD test (p < 0.05)

Figure 2a showed that the absorbance values of TP solutions with a pH of 3–6 had almost no significant change and maintained a stable state in 24 h at 4°C. The absorbance values of TP solutions with pH 7 had a significant increase at the first 1 h and maintained a stable state in 5 h, and then had a significant increase.

As shown in Fig. 2c, when the temperature was 100°C, the absorbance values had significant differences between pH 3 to pH 7. From Fig. 2c, we could see that the absorbance values of TP solutions were bigger at high pH values. The absorbance values increased gradually with time, as expected. There were great differences in the absorbance values of TP solutions at the same pH at the three temperatures. The higher the temperature was, the greater increase in the absorbance value.

Figure 2. Absorbance in the spectra at 425 nm change of TP with pH 3–7 at A: 4°C; B: at 25°C; C: at 100°C. Data are expressed as means ± SD of n = 3 samples. The different letter in the same temperature indicated that the difference between the treatments is significantly through LSD test (p < 0.05).

Color change of TP solution with different pH at different temperatures

Color measurements enabled us to distinguish the influence of the temperature and pH on some quality characteristics of TP. Changes in color of TP solutions during heating and storage were expressed as L*, a*, and b* values. Table 1a–c showed that the color difference indicator L* values decreased while indicators a* and b* values increased with the elevation of temperature and pH, indicating the development of a brown color. The TP solutions, which contained anthocyanins, a* and b* values were positive, indicating a red and yellow color. The L* value is the indicator of lightness–darkness, and the higher it is, the lighter is the liquor.^[22]

Table 1a. Color change of TP solution with pH 3–7 at 4°C.

Color change	pH	0 h	1 h	3 h	5 h	10 h	24 h
L*	3	92.00 ± 1.0	92.13 ± 0.5	92.10 ± 0.4	92.01 ± 0.4	92.31 ± 0.4	91.93 ± 0.2
		0a	1a	7a	1a	9a	2a
	4	92.00 ± 1.0	91.74 ± 0.4	91.77 ± 0.3	91.80 ± 0.5	92.11 ± 0.7	91.83 ± 0.3
		0a	8a	5a	8a	3a	2a
	5	92.00 ± 1.0	91.56 ± 0.1	91.67 ± 0.4	91.51 ± 0.5	91.90 ± 0.3	91.49 ± 0.1
		0a	7a	5a	1a	3a	2a
	6	92.00 ± 1.0	90.98 ± 0.1	91.21 ± 0.3	91.31 ± 0.4	91.50 ± 0.3	91.23 ± 0.1
		0a	1b	2ab	3ab	8ab	0ab
	7	92.00 ± 1.0	89.18 ± 0.1	88.77 ± 0.7	88.61 ± 0.5	89.02 ± 0.3	88.56 ± 0.5
		0a	5b	0b	4b	2b	1b
a*	3	1.73 ± 0.15	1.89 ± 0.14	1.93 ± 0.10	1.95 ± 0.09	1.93 ± 0.16	1.78 ± 0.27
		a	a	a	a	a	a
	4	1.73 ± 0.15	2.13 ± 0.40	2.04 ± 0.21	2.10 ± 0.27	2.10 ± 0.31	1.95 ± 0.23
		a	a	a	a	a	a
	5	1.73 ± 0.15	2.31 ± 0.09	2.22 ± 0.10	2.18 ± 0.12	2.19 ± 0.09	1.97 ± 0.25
		b	a	ab	ab	ab	b
	6	1.73 ± 0.15	2.82 ± 0.16	2.66 ± 0.14	2.53 ± 0.12	2.55 ± 0.08	2.27 ± 0.29
		c	a	a	ab	ab	b
	7	1.73 ± 0.15	4.12 ± 0.21	4.16 ± 0.06	4.24 ± 0.05	4.41 ± 0.16	4.11 ± 0.18
		c	b	ab	ab	a	b
b*	3	11.17 ± 1.2	12.24 ± 0.2	12.08 ± 0.1	12.09 ± 0.2	11.91 ± 0.4	11.81 ± 0.2
		6b	9a	9ab	9ab	2ab	1ab
	4	11.17 ± 1.2	11.73 ± 0.7	11.82 ± 1.0	11.57 ± 0.2	11.26 ± 0.2	11.39 ± 0.0
		6a	1a	5a	0a	0a	9a
	5	11.17 ± 1.2	11.66 ± 0.7	11.61 ± 0.6	11.82 ± 0.7	11.18 ± 0.7	11.44 ± 0.4
		6a	5a	7a	2a	9a	7a
	6	11.17 ± 1.2	10.78 ± 0.6	10.78 ± 0.6	10.71 ± 0.2	10.64 ± 0.3	11.02 ± 0.1
		6a	5a	5a	7a	2a	0a
	7	11.17 ± 1.2	11.19 ± 0.3	11.19 ± 0.3	11.93 ± 0.0	12.81 ± 0.1	13.14 ± 0.3
		6b	2b	2b	7b	5ab	9a

Table 1a showed that the L* and b* values had no significant change at 4°C in 24 h at a pH of 3–6. When the pH was 7, the L* values decreased and b* values increased with time in 24 h, and resulted in about 3.74% reduction and 17.6% increase. The a* values had no significant change in 24 h at a pH of 3–4 and a significant increase at a pH of 5–7.

Table 1b showed that the L* values and b* values had no significant change at 25°C in 24 h at a pH of 3–6. When the pH was 7, the L* values decreased and b* values increased with time in 24 h, and resulted in about 13% reduction and 160% increase. L* values of the 25°C samples were significantly lower than for the 4°C reference ones, indicating a lower lightness during 25°C storage. The a* values had no significant change in 24 h at a pH of 3–4 and a significant increase at a pH of 5–7. The a* and b* values increased with pH, suggesting that the TP solution became deeper yellow. There were no statistically significant differences in the three indicators between 4 and 25°C treatments at a pH of 3–4.

Table 1b. Color change of TP solution with pH 3–7 at 25°C.

Color change	pH	0 h	1 h	3 h	5 h	10 h	24 h
L*	3	92.00 ± 1.0	92.23 ± 0.5	92.19 ± 0.4	92.11 ± 0.5	92.28 ± 0.3	91.88 ± 0.2
		0a	2a	6a	4a	5a	8a
	4	92.00 ± 1.0	91.91 ± 0.6	91.86 ± 0.5	91.85 ± 0.4	92.12 ± 0.5	91.78 ± 0.3
		0a	8a	0a	8a	5a	0a
	5	92.00 ± 1.0	91.79 ± 0.3	91.66 ± 0.4	91.77 ± 0.3	91.93 ± 0.2	91.58 ± 0.1
		0a	5a	0a	5a	7a	7a
	6	92.00 ± 1.0	91.30 ± 0.4	91.33 ± 0.3	91.14 ± 0.2	91.47 ± 0.3	91.14 ± 0.2
		0a	0a	9a	3a	6a	5a
	7	92.00 ± 1.0	89.16 ± 0.5	88.63 ± 0.6	87.92 ± 0.7	84.41 ± 0.6	79.97 ± 1.2
		0a	8b	0b	0b	6c	6d
a*	3	1.73 ± 0.15	1.68 ± 0.12	1.77 ± 0.07	1.88 ± 0.17	1.79 ± 0.10	1.82 ± 0.23
		a	a	a	a	a	a
	4	1.73 ± 0.15	1.97 ± 0.38	1.96 ± 0.13	1.88 ± 0.13	1.76 ± 0.10	1.71 ± 0.18
		a	a	a	a	a	a
	5	1.73 ± 0.15	2.06 ± 0.05	1.99 ± 0.07	1.95 ± 0.13	1.86 ± 0.16	1.73 ± 0.22
		b	a	a	ab	ab	b
	6	1.73 ± 0.15	2.61 ± 0.14	2.42 ± 0.09	2.35 ± 0.09	2.14 ± 0.08	1.98 ± 0.15
		d	a	ab	b	bc	c
	7	1.73 ± 0.15	4.51 ± 0.22	4.49 ± 0.15	4.55 ± 0.23	6.00 ± 0.48	8.20 ± 0.87
		d	c	c	c	b	a
b*	3	11.17 ± 1.2	12.10 ± 0.3	12.09 ± 0.1	11.99 ± 0.3	12.17 ± 0.2	12.11 ± 0.5
		6a	2a	7a	3a	0a	6a
	4	11.17 ± 1.2	11.40 ± 0.2	11.39 ± 0.1	11.61 ± 0.1	11.74 ± 0.3	11.86 ± 0.5
		6a	4a	9a	5a	9a	0a
	5	11.17 ± 1.2	11.24 ± 0.8	11.33 ± 0.6	11.44 ± 0.7	11.64 ± 0.7	11.92 ± 0.5
		6a	2a	3a	6a	2a	7a
	6	11.17 ± 1.2	10.43 ± 0.2	10.73 ± 0.1	11.26 ± 0.5	11.74 ± 0.5	12.34 ± 0.7
		6ab	7b	7b	5ab	7ab	3a
	7	11.17 ± 1.2	11.82 ± 0.3	13.25 ± 0.3	15.19 ± 0.9	21.68 ± 1.4	28.99 ± 2.4
		6d	0d	6cd	7c	2b	2a

As shown in Table 1c, when the temperature was 100°C, the L* values had a significant decline after 1 h and declined gradually over time, and the a* and b* values increased with time. However, there was a large difference in yellowness between 100 and 25°C. Storing at 100°C showed an increase in b* value from 11 to 47 in 24 h, whereas storing at 25°C increased its b* value from 11 to only 29. TP solutions stored at 100°C were more prone to color changes. These results suggested that the TP solution darkened and became deeper yellow as the temperature was raised. What is more, the higher the pH was, the greater decline in the L* values. The results suggested that the solution darkened as the pH raised. These results might be related to the oxidation and degradation of TP under hot conditions.

Table 1c. Color change of TP solution with pH 3–7 at 100°C.

Color change	pH	0 h	1 h	3 h	5 h	10 h	24 h
L*	3	92.00 ± 1.	91.23 ± 0.2	90.77 ± 0.2	90.35 ± 0.1	90.43 ± 1.0	88.43 ± 0.1
		00a	0a	5b	1b	2b	9c
	4	92.00 ± 1.	91.22 ± 0.2	90.61 ± 0.1	90.23 ± 0.2	89.96 ± 0.6	86.56 ± 0.1
		00a	5ab	9b	8b	8b	1c
	5	92.00 ± 1.	89.56 ± 0.3	88.43 ± 0.6	87.80 ± 0.5	88.19 ± 1.4	84.47 ± 1.5
		00a	5b	9b	0b	1b	4c
	6	92.00 ± 1.	88.22 ± 0.5	86.71 ± 1.1	85.92 ± 0.7	85.30 ± 2.1	77.48 ± 0.7
		00a	3b	4bc	6c	8c	9d
	7	92.00 ± 1.	85.30 ± 0.2	81.49 ± 0.4	73.22 ± 1.2	59.54 ± 0.8	43.11 ± 6.5
		00a	8b	7b	1c	6d	5e
a*	3	1.73 ± 0.1	1.93 ± 0.19	1.91 ± 0.08	2.18 ± 0.09	2.38 ± 0.32	3.39 ± 0.18
		5c	c	c	bc	b	a
	4	1.73 ± 0.1	1.76 ± 0.11	1.59 ± 0.07	1.78 ± 0.07	2.02 ± 0.35	3.82 ± 0.12
		5bc	bc	c	bc	b	a
	5	1.73 ± 0.1	2.29 ± 0.14	2.46 ± 0.16	2.80 ± 0.19	2.76 ± 0.64	4.66 ± 0.89
		5c	bc	b	b	b	a
	6	1.73 ± 0.1	2.91 ± 0.51	3.54 ± 0.50	4.18 ± 0.32	4.91 ± 1.38	10.18 ± 0.6
		5d	cd	c	bc	b	1a
	7	1.73 ± 0.1	5.34 ± 0.52	10.33 ± 0.3	21.07 ± 1.0	36.40 ± 0.8	37.56 ± 2.2
		5d	d	2c	5b	8a	7a
b*	3	11.17 ± 1.	14.42 ± 0.1	15.95 ± 0.5	17.83 ± 0.4	19.49 ± 2.1	26.28 ± 0.9
		26d	9c	1c	2bc	0b	1a
	4	11.17 ± 1.	14.32 ± 0.4	16.28 ± 0.0	17.38 ± 0.4	18.69 ± 1.2	25.57 ± 0.6
		26e	9d	8c	7bc	4b	2a
	5	11.17 ± 1.	17.77 ± 0.4	19.99 ± 1.0	21.38 ± 1.0	21.20 ± 1.7	26.50 ± 2.7
		26d	0c	1bc	2b	4b	1a
	6	11.17 ± 1.	19.81 ± 1.0	22.35 ± 1.6	24.19 ± 1.2	25.41 ± 3.6	35.59 ± 1.0
		26d	2c	3bc	6b	2b	6a
	7	11.17 ± 1.	23.37 ± 0.4	25.36 ± 0.3	31.19 ± 1.5	42.17 ± 1.6	46.83 ± 2.7
		26e	7d	8d	5c	8b	7a

Values are expressed as means ± standard deviations of triplicate measurements.

Different superscript lowercase letters (a, b, c, d) in same row indicate that the difference between the treatments is significantly through LSD test (p < 0.05).

T = temperatures.

Change in contents of catechins of TP solution with different pH at different temperatures

The effect of pH on stability of catechins was best illustrated when the pH value of citrate buffer solutions were set between 3 and 7. Thermal stability of tea catechins with different pH was examined at different temperatures (4, 25, and 100°C) for 24 h in the present study (Table 2a–c). The HPLC analysis showed that the yield of total catechins in TP solution was 388 mg/L (78% of TP). Among six catechins of TP, EGCG was most abundant (>70% of total catechins), followed by ECG (>15%), GCG, EGC, EC, and C. Table 2a–c visually illustrated the decreases in the values of both individual and total catechins levels of the solution at different temperatures.

Table 2a. Change in contents of catechins of TP solution with pH 3–7 at 4°C.

Compounds	pH	0 h	1 h	3 h	5 h	10 h	24 h
C	3		3.06 ± 0.58	2.97 ± 0.86	2.57 ± 0.53
		2.97 ± 0.55a	a	a	a	2.75 ± 0.51a	2.24 ± 0.66a
	4		2.72 ± 0.57	3.16 ± 0.12	3.13 ± 0.18
		2.97 ± 0.55a	a	a	a	3.24 ± 0.26a	3.01 ± 0.33a
	5		3.39 ± 0.21	3.15 ± 0.22	3.33 ± 0.18
		2.97 ± 0.55a	a	a	ab	3.29 ± 0.06ab	3.06 ± 0.07b
	6		3.14 ± 0.11	2.96 ± 0.39	3.13 ± 0.11
		2.97 ± 0.55a	a	a	a	3.04 ± 0.21a	3.10 ± 0.15a
	7		3.14 ± 0.47	3.21 ± 0.52	3.32 ± 0.34
		2.97 ± 0.55a	a	a	a	3.15 ± 0.88a	2.99 ± 0.80a
EC	3	10.17 ± 1.04	10.64 ± 0.1	9.93 ± 0.97	10.34 ± 0.7		10.39 ± 0.39
		a	7a	a	7a	10.39 ± 0.51a	a
	4	10.17 ± 1.04	9.12 ± 2.04	10.26 ± 0.4	10.12 ± 0.4
		a	a	4a	6a	9.70 ± 0.65a	9.98 ± 0.92a
	5	10.17 ± 1.04	9.90 ± 1.01	9.72 ± 0.93	9.78 ± 0.73
		a	a	a	a	8.87 ± 1.36a	8.91 ± 1.19a
	6	10.17 ± 1.04	10.71 ± 0.9	10.50 ± 0.9	10.37 ± 0.9		10.39 ± 0.82
		a	6a	3a	2a	10.44 ± 0.75a	a
	7	10.17 ± 1.04	9.74 ± 0.45	9.79 ± 0.46	9.61 ± 0.36
		a	a	a	a	9.82 ± 0.65a	9.06 ± 0.64a
EGC	3	17.00 ± 1.00	18.10 ± 0.9	18.20 ± 0.6	17.10 ± 0.7		16.71 ± 1.32
		a	6a	0a	2a	17.40 ± 0.69a	a
	4	17.00 ± 1.00	17.10 ± 0.4	16.83 ± 1.4	16.92 ± 1.4		17.03 ± 0.79
		a	5a	6a	1a	17.20 ± 1.88a	a
	5	17.00 ± 1.00	17.87 ± 0.5	17.58 ± 0.5	18.47 ± 0.3		18.69 ± 1.07
		b	9ab	9ab	8a	18.89 ± 0.91a	a
	6	17.00 ± 1.00	17.31 ± 1.0	17.25 ± 1.8	17.75 ± 0.9		18.10 ± 0.94
		a	3a	0a	0a	17.70 ± 0.69a	a
	7	17.00 ± 1.00	14.19 ± 0.3	14.28 ± 0.4	13.06 ± 0.4	12.43 ± 0.71c	11.56 ± 0.42
		a	2b	4b	0c	d	d
ECG	3	60.07 ± 4.90	59.71 ± 4.7	59.92 ± 4.1	60.62 ± 4.3		61.61 ± 4.75
		a	5a	0a	6a	60.50 ± 4.05a	a
	4	60.07 ± 4.90	58.96 ± 5.0	59.45 ± 4.3	59.80 ± 4.3		61.29 ± 2.59
		a	2a	4a	7a	61.48 ± 3.78a	a
	5	60.07 ± 4.90	61.42 ± 4.4	60.29 ± 4.7	57.90 ± 1.9		59.31 ± 1.73
		a	5a	8a	7a	57.84 ± 1.69a	a
	6	60.07 ± 4.90	57.63 ± 6.3	56.44 ± 3.6	56.37 ± 3.8		59.52 ± 0.94
		a	6a	1a	6a	59.25 ± 1.34a	a
	7	60.07 ± 4.90	53.52 ± 1.3	54.44 ± 1.9	54.22 ± 1.4		54.85 ± 3.34
		a	2b	5b	0b	53.44 ± 1.86b	b
GCG	3	18.57 ± 1.50	19.37 ± 1.6	18.08 ± 1.2	19.48 ± 1.2		19.65 ± 0.81
		a	4a	3a	3a	18.80 ± 1.33a	a
	4	18.57 ± 1.50	17.85 ± 2.3	20.49 ± 2.1	20.44 ± 1.8		19.42 ± 2.25
		a	7a	9a	9a	20.87 ± 1.40a	a
	5	18.57 ± 1.50	18.98 ± 1.0	18.60 ± 0.9	19.06 ± 1.1		20.21 ± 0.88
		a	3a	3a	4a	19.36 ± 1.44a	a
	6	18.57 ± 1.50	18.62 ± 1.7	18.68 ± 1.9	18.74 ± 1.8		19.82 ± 0.62
		a	8a	2a	9a	19.48 ± 0.71a	a
	7	18.57 ± 1.50	17.25 ± 0.9	17.40 ± 0.7	17.02 ± 0.8	17.65 ± 0.61a	16.27 ± 1.30
		a	3ab	1ab	9ab	b	b
EGCG	3	279.67 ± 10.50a	284.57 ± 7.50a	282.34 ± 9.22a	280.16 ± 7.27a	275.71 ± 6.14a	281.50 ± 2.96a
	4	279.67 ± 10.	281.08 ± 4.22a	280.81 ± 4.09a	281.95 ± 3.	286.08 ± 10.57a	274.66 ± 11.63a
		50a			43a
	6
	5	279.67 ± 10.50a	275.20 ± 2.96ab	268.61 ± 3.99b	273.29 ± 2.65ab	276.69 ± 0.92ab	277.27 ± 1.74ab
	6	279.67 ± 10.50a	270.58 ± 15.87a	276.17 ± 6.56a	277.28 ± 8.96a	275.84 ± 8.32a	275.96 ± 7.99a
	7	279.67 ± 10.	242.69 ± 11	242.45 ± 10	234.30 ± 13	230.32 ± 15.4	210.49 ± 9.8
		50a	.54b	.57b	.54b	2bc	4c
Catechins	3	388.43 ± 16.85a	395.45 ± 12.36a	391.43 ± 10.42a	390.27 ± 14.12a	385.54 ± 9.91a	392.10 ± 5.93a
	4	388.43 ± 16.85a	386.84 ± 2.44a	391.02 ± 5.51a	392.37 ± 3.88a	398.57 ± 13.49a	385.39 ± 13.24a
		388.43 ± 16.	386.77 ± 1.	377.96 ± 8.	381.83 ± 5.	384.93 ± 3.06	387.46 ± 3.3
		85a	45a	90a	60a	a	8a
	5
	6	388.43 ± 16.	377.98 ± 25	382.01 ± 14	383.62 ± 16	385.75 ± 10.1	386.89 ± 8.0
		85a	.51a	.78a	.55a	9a	0a
	7	388.43 ± 16.	340.53 ± 12	341.57 ± 10	331.53 ± 14	326.82 ± 15.9	305.21 ± 9.9
		85a	.84b	.77b	.49b	8bc	9c

As shown in Table 2a, total contents of catechins incubated in pH 3, 4, 5, and 6 citrate buffer solutions for 24 h had almost no significant change and maintained a stable state at 4°C. Catechins degraded readily at pH 7, 21% of total catechins were lost after 24 h. Moreover, when TP with pH 7 were stored at 4°C for 1 h, a 12% loss of catechins were observed. Storing for an additional 9 h led to only an additional 3% loss of catechins. Maybe the initial oxygen concentration was higher in the TP solution so that oxidation of catechins was extensive.^[12] Additional storing would decrease the oxygen concentration in the solution and reduce the reaction between TP and oxygen.

During 4°C storage, all of the six catechins with pH 3–6 had no significant change. When the pH was 7, the contents of C and EC had no significant change at 4°C. The content of EGC, ECG, GCG, and EGCG decreased by 32, 9, 12, and 25% in the pH 7 citrate buffer, respectively. EGCG was the most unstable among all the kinds of catechins when it was dissolved in the pH 7 citrate buffer for 24 h. The ionization state of EGCG, an indication of its proton-donating ability, maybe a factor contributing to degradation of EGCG.^[23]

Table 2b showed that total contents of catechins incubated in pH 3, 4, 5, and 6 citrate buffer solutions for 24 h had almost no significant change and maintained a stable state at 25°C. When TP were dissolved in pH 7 buffer, degradation of the catechins was extensive: up to more than 67%. But, only 21% of total catechins were lost at 4°C after 24 h. So, it had been generally considered that green tea catechins were stable under low temperature and acidic pH conditions.^[24]

Table 2b. Change in contents of catechins of TP solution with pH 3–7 at 25°C.

Compounds	pH	0 h	1 h	3 h	5 h	10 h	24 h
C	3	2.97 ± 0.55	2.82 ± 0.42	2.67 ± 0.62	2.57 ± 0.47	2.99 ± 1.08
		a	a	a	a	a	2.66 ± 0.49a
	4	2.97 ± 0.55	2.61 ± 0.53	3.13 ± 0.09	3.08 ± 0.17	3.12 ± 0.21
		a	a	a	a	a	2.51 ± 0.78a
	5	2.97 ± 0.55	3.09 ± 0.35	2.81 ± 0.48	3.25 ± 0.15	3.16 ± 0.31
		a	a	a	a	a	2.98 ± 0.11a
	6	2.97 ± 0.55	3.22 ± 0.10	2.69 ± 0.99	3.10 ± 0.29	2.84 ± 0.08
		a	a	a	a	a	2.77 ± 0.37a
	7	2.97 ± 0.55	4.23 ± 0.86	3.83 ± 0.62	4.74 ± 0.27	4.53 ± 0.12
		a	a	a	a	ab	5.71 ± 0.51a
EC	3	10.17 ± 1.0	10.61 ± 0.0	10.00 ± 0.9	10.28 ± 0.7	9.70 ± 0.50	10.01 ± 0.75
		4a	9a	6a	0a	a	a
	4	10.17 ± 1.0	8.84 ± 1.93	10.11 ± 0.4	9.52 ± 0.06	9.17 ± 0.67
		4a	a	5a	a	a	9.48 ± 1.34a
	5	10.17 ± 1.0	9.80 ± 0.96	9.00 ± 0.12	9.32 ± 0.64	8.32 ± 0.80
		4a	ab	ab	ab	b	8.42 ± 0.76b
	6	10.17 ± 1.0	10.27 ± 1.0	9.85 ± 0.74	9.24 ± 0.92	9.35 ± 0.31
		4a	1a	a	b	b	8.35 ± 0.75b
	7	10.17 ± 1.0	9.78 ± 0.50	9.22 ± 0.28	8.95 ± 0.28	8.99 ± 0.45
		4a	ab	ab	b	b	8.08 ± 0.84b
EGC	3	17.00 ± 1.0	17.45 ± 0.7	17.38 ± 0.9	17.09 ± 0.6	17.71 ± 2.0	17.63 ± 1.95
		0a	1a	9a	2a	7a	a
	4	17.00 ± 1.0	16.87 ± 1.2	16.92 ± 1.5	16.83 ± 1.4	16.90 ± 1.6	17.05 ± 1.16
		0a	3a	5a	0a	8a	a
	5	17.00 ± 1.0	17.11 ± 0.4	16.73 ± 1.2	18.27 ± 0.2	18.38 ± 1.2	18.20 ± 1.67
		0a	6a	6a	2a	6a	a
	6	17.00 ± 1.0	16.97 ± 1.0	16.95 ± 1.2	17.43 ± 1.1	16.56 ± 0.0	15.84 ± 2.00
		0a	5a	8a	7a	3a	a
	7	17.00 ± 1.0	14.12 ± 0.8	10.25 ± 0.7	15.63 ± 0.5	13.80 ± 1.5
		0a	4b	9c	9ab	1b	9.22 ± 0.87c
ECG	3	60.07 ± 4.9	61.16 ± 4.6	61.07 ± 4.3	60.22 ± 4.3	61.67 ± 4.7	61.00 ± 4.24
		0a	2a	7a	0a	0a	a
	4	60.07 ± 4.9	59.38 ± 4.5	59.36 ± 4.4	59.98 ± 4.2	60.37 ± 3.4	59.34 ± 3.38
		0a	8a	4a	3a	5a	a
	5	60.07 ± 4.9	61.47 ± 5.0	60.00 ± 3.0	58.48 ± 1.3	58.23 ± 1.1	61.00 ± 2.56
		0a	3a	9a	4a	2a	a
	6	60.07 ± 4.9	56.77 ± 3.6	56.20 ± 3.6	56.50 ± 3.0	59.93 ± 3.5	60.57 ± 1.27
		0a	0a	1a	9a	2a	a
	7	60.07 ± 4.9	58.56 ± 4.1	53.20 ± 1.2	52.66 ± 1.8	48.34 ± 2.4	47.49 ± 2.88
		0a	4ab	9b	7b	1b	c
GCG	3	18.57 ± 1.5	19.32 ± 1.6	17.95 ± 1.1	19.67 ± 1.0	19.19 ± 0.4	19.89 ± 0.69
		0a	5a	6a	4a	4a	a
	4	18.57 ± 1.5	17.41 ± 2.0	20.43 ± 2.2	20.69 ± 1.6	20.18 ± 1.6	20.07 ± 1.57
		0ab	7b	4ab	8a	4ab	ab
	5	18.57 ± 1.5	19.07 ± 1.0	18.73 ± 0.4	19.37 ± 1.0	19.45 ± 1.3	20.49 ± 0.71
		0b	7ab	8ab	0ab	2ab	a
	6	18.57 ± 1.5	20.09 ± 2.2	18.58 ± 1.9	19.55 ± 2.3	20.15 ± 1.9	19.51 ± 0.83
		0a	4a	6a	8a	6a	a
	7	18.57 ± 1.5	17.29 ± 0.6	16.03 ± 0.1	15.63 ± 1.2	14.32 ± 1.2	9.00 ± 0.63c
		0a	1ab	1b	6b	3b
EGCG	3	279.67 ± 10.50a	283.32 ± 8.56a	283.05 ± 7.51a	280.60 ± 6.38a	275.65 ± 4.05a	276.89 ± 5.79a
	4	279.67 ± 10	278.83 ± 1.	280.24 ± 2.	282.41 ± 2.	283.38 ± 4.	280.17 ± 3.6
		.50a	47a	65a	69a	31a	7a
	5	279.67 ± 10	282.18 ± 3.	273.47 ± 6.	282.18 ± 7.	282.02 ± 1.	279.58 ± 2.7
		.50a	48a	78a	18a	85a	1a
	6	279.67 ± 10.50a	267.28 ± 6.83a	268.12 ± 7.33a	266.11 ± 9.31a	266.88 ± 7.23a	266.51 ± 10.87a
	7	279.67 ± 10	240.09 ± 10	196.91 ± 13	166.75 ± 15	146.60 ± 10	47.34 ± 2.25
		.50a	.48b	.50c	.85d	.32e	f

During 25°C storage, all of the six catechin with a pH of 3–6 had no significant change. When the pH was 7, the contents of C had no significant change and EC had a significant decline. The order of stability of cis-catechins was EC > ECG > EGC > EGCG, and the content decreased by 20, 21, 46, and 83%, respectively, in the pH 7 citrate buffer after 24 h. This was thought to be that EGCG and EGC have three hydroxyl groups on their B rings, whereas ECG and EC only have two hydroxyl groups.^[21] During the oxidation process, catechins lose one hydrogen radical and form a semiquinone radical with an unpaired electron on the oxygen atom. It was suggested that the radical can form more freely on a ring possessing three hydroxyl groups than on a ring possessing two groups. This might explain why EGCG and EGC oxidized more rapidly than ECG and EC. No epimerization had been observed in the samples of strong infusions stored at 5 and 25°C.^[25] In neutral pH, EGCG is easily auto-oxidized. And, the two major oxidative products with dimeric structures formed in a time-dependent manner and were more hydrophobic or have higher molecular weights.^[13] EC and EGC are the precursors of theaflavin, one of the important oxidation derived components in black tea. So, the decline of EC and EGC thought to be due to oxidation to form dimers, or the intermediary products of theaflavin.^[26]

As shown in Table 2c, surprisingly, catechins with pH 3 exhibited a remarkable stability at 100°C. Boiling for 24 h resulted in about 15% reduction in total catechins. Total contents of catechins incubated in pH 4, 5, 6, and 7 citrate buffer solutions for 24 h declined by 24, 41, 57, and 96%, respectively. These observations were similar to that previously reported by Chen, Zhu, Tsang, and Huang,^[27] namely, that GTC was relatively stable at pH 3 and 4, but it degraded readily at pH 5 and 6. The higher the pH value of the medium, the greater the percentage of catechins that degraded. Furthermore, there was a declining trend in total catechins with increase of temperature, which suggested that some of the catechins were oxidized (besides the epimerization) and they might be responsible for the changes in the liquor color.^[22]

Table 2c. Change in contents of catechins of TP solution with pH 3–7 at 100°C.

Compounds	pH	0 h	1 h	3 h	5 h	10 h	24 h
C	3	2.97 ± 0.55	2.77 ± 0.12			6.23 ± 0.30	7.14 ± 0.60
		c	c	3.01 ± 0.24c	5.85 ± 0.60b	b	a
	4	2.97 ± 0.55	2.84 ± 0.14			5.98 ± 1.48	6.71 ± 0.23
		c	c	4.29 ± 0.43b	5.31 ± 0.50b	ab	a
	5	2.97 ± 0.55	3.31 ± 0.06			6.61 ± 1.12	7.53 ± 0.52
		d	cd	4.33 ± 0.67c	5.87 ± 0.37b	ab	a
	6	2.97 ± 0.55	5.31 ± 0.53			8.34 ± 1.05	9.44 ± 2.00
		c	b	7.76 ± 0.32a	8.71 ± 0.94a	a	a
	7	2.97 ± 0.55	10.54 ± 0.5	9.49 ± 2.18a		5.48 ± 0.96	3.43 ± 0.97
		b	1a	b	12.07 ± 5.32a	b	b
EC	3	10.17 ± 1.0	11.98 ± 0.8	12.20 ± 0.29	11.46 ± 0.32a	10.18 ± 1.1	9.32 ± 1.52
		4b	2a	a	b	8b	b
	4	10.17 ± 1.0	9.36 ± 0.12			9.85 ± 0.72	9.24 ± 0.55
		4a	a	9.45 ± 0.08a	9.90 ± 0.29a	a	a
	5	10.17 ± 1.0	8.67 ± 0.27			8.23 ± 0.48	5.70 ± 1.05
		4a	b	8.54 ± 0.26b	8.63 ± 0.32b	b	c
	6	10.17 ± 1.0	8.78 ± 0.82			4.51 ± 0.84	5.20 ± 2.45
		4a	a	6.06 ± 1.36b	5.55 ± 1.05b	b	b
	7	10.17 ± 1.0	5.33 ± 0.52	4.45 ± 0.50b		4.12 ± 0.17	1.81 ± 0.18
		4a	b	c	4.28 ± 0.67bc	c	d
EGC	3	17.00 ± 1.0	17.63 ± 0.7	16.33 ± 6.60		14.93 ± 2.5	14.46 ± 2.5
		0a	5a	a	14.69 ± 1.06a	2a	0a
	4	17.00 ± 1.0	16.43 ± 1.0	14.06 ± 0.58	14.46 ± 0.30	11.85 ± 0.6	10.75 ± 0.1
		0a	1a	b	b	0c	7c
	5	17.00 ± 1.0	14.72 ± 0.4	14.88 ± 0.54	14.67 ± 0.91	10.86 ± 0.9	5.84 ± 0.54
		0a	8b	b	b	8c	d
	6	17.00 ± 1.0	10.02 ± 1.1			6.10 ± 0.78	5.00 ± 0.76
		0a	5b	8.86 ± 0.13b	8.66 ± 0.33b	c	c
	7	17.00 ± 1.0	6.38 ± 0.70	6.05 ± 0.51c		5.21 ± 0.37	2.95 ± 0.49
		0a	c	d	6.99 ± 0.52b	d	e
ECG	3	60.07 ± 4.9	57.18 ± 5.6	56.61 ± 5.05	54.62 ± 4.56a	51.87 ± 3.4	47.32 ± 1.4
		0a	0ab	ab	b	5b	3b
	4	60.07 ± 4.9	59.48 ± 3.4	55.36 ± 0.67	53.56 ± 0.72	53.63 ± 3.4	49.14 ± 2.8
		0a	0a	ab	b	3b	7b
	5	60.07 ± 4.9	54.74 ± 2.0	50.11 ± 1.90		41.62 ± 1.6	30.44 ± 2.8
		0a	0b	bc	48.80 ± 2.38c	5d	0e
	6	60.07 ± 4.9	46.25 ± 0.7	27.87 ± 1.24		22.55 ± 1.5	22.45 ± 1.2
		0a	8b	c	28.23 ± 2.11c	9d	8d
	7	60.07 ± 4.9	24.13 ± 1.9	18.63 ± 2.87		8.62 ± 0.14	2.35 ± 1.44
		0a	2b	c	16.57 ± 2.77c	d	e
GCG	3	18.57 ± 1.5	17.54 ± 1.2	20.25 ± 1.75	20.52 ± 2.60	23.16 ± 1.2	29.99 ± 2.0
		0c	1c	bc	bc	7b	1a
	4	18.57 ± 1.5	20.95 ± 1.0	24.65 ± 0.93	29.78 ± 1.06	31.13 ± 0.4	37.36 ± 1.7
		0e	9d	c	b	8b	7a
	5	18.57 ± 1.5	32.80 ± 2.1	62.92 ± 3.95	71.46 ± 19.0	106.80 ± 8.	84.54 ± 2.3
		0d	5d	c	7bc	23a	3b
	6	18.57 ± 1.5	80.57 ± 5.0	130.02 ± 3.2	128.32 ± 6.3	115.08 ± 3.	64.15 ± 4.3
		0e	8c	7a	2a	02b	9d
	7	18.57 ± 1.5	99.16 ± 0.9	51.75 ± 2.34	36.66 ± 1.75c	4.21 ± 0.13
		0d	1a	b		e	0.46 ± 0.10f
EGCG	3	279.67 ± 10.50a	258.42 ± 6.81b	253.93 ± 4.98bc	241.35 ± 11.64c	234.89 ± 6.57c	220.07 ± 5.91d
	4	279.67 ± 10	275.76 ± 6.	254.69 ± 6.4	252.71 ± 7.7	231.87 ± 10	181.99 ± 9.
		.50a	30a	0b	5b	.96c	46d
	5	279.67 ± 10	251.99 ± 2.	212.43 ± 1.6	183.97 ± 3.9	147.68 ± 8.	94.91 ± 9.0
		.50a	27b	3c	0d	10e	4f
	6
		279.67 ± 10.50a	186.71 ± 8.11b	117.63 ± 9.57c	109.81 ± 7.07c	91.94 ± 5.44d	59.19 ± 4.85e
	7	279.67 ± 10	81.64 ± 2.6	55.72 ± 4.39	40.22 ± 3.34	8.71 ± 0.94	4.57 ± 0.70
		.50a	0b	c	d	e	e
Catechin s	3	388.43 ± 16	365.52 ± 14	362.33 ± 13.	348.49 ± 14.	341.26 ± 1.	328.30 ± 8.
		.85a	.34b	86bc	32bc	00c	78c
		388.43 ± 16.85a	384.81 ± 8.96ab	362.50 ± 6.47bc	365.71 ± 6.92b	344.30 ± 12.90c	295.20 ± 9.45d
	4
	5	388.43 ± 16	366.23 ± 4.	353.21 ± 5.7	333.40 ± 16.	321.79 ± 2.	228.96 ± 10
		.85a	74b	5b	59c	06c	.75d
	6	388.43 ± 16	337.64 ± 12	298.19 ± 7.6	289.28 ± 10.	248.51 ± 7.	165.42 ± 9.
		.85a	.06b	2c	43c	38d	82e
	7	388.43 ± 16	227.18 ± 5.	146.08 ± 6.3	116.80 ± 4.7	36.36 ± 2.3	15.58 ± 1.6
		.85a	68b	2c	9d	6e	6f

Values are expressed as means ± standard deviations of triplicate measurements.

Different superscript lowercase letters (a, b, c, d) in same row indicate that the difference between the treatments is significantly through LSD test (p < 0.05).

The four cis-configured catechins (EGCG, EGC, ECG, and EC) degraded steadily over time, whereas the two trans-configured catechins (GCG and C) increased during the first heating time and then decreased with time. These results suggested that epimerizations of catechins took place under the heating conditions. Courbat^[28] pointed out that a rapid epimerization of catechins occurred in alkaline solution. Nakagawa^[29] reported that the isomerization of catechins in the roasting process of green tea was not racemization but epimerization. In addition to oxidation products, EGCG could be degraded to GCG or EGCG dimers.^[30] We observed increase in GCG concurrent with EGCG reduction at the first 1 h. Therefore, it was concluded that the high temperature led to the epimerization. It had been recognized that catechins undergo epimerization at the C-2-position in hot aqueous solution and this epimerization could change the epi-structured catechins to non-epi-structured catechins.^[20] The relatively high amount of GCG found in some tea drinks was most likely the epimerization of EGCG during autoclaving.^[27] We also observed increase in C concurrent with EC. The degradation of EC and EGCG was obviously dependent on the pH of the solution. The reaction scarcely proceeded in slightly acid media at pH < 5; however, it was accelerated at pH > 6. So, production of trans-catechins (GCG, C) increased with increasing pH levels, suggesting that isomerization was more favored with increases in pH levels. Inferred from their stereochemical configuration, catechins with their “2,3-trans” structure are thermodynamically more stable than epicatechins, which are “2,3-cis,” so catechins isomerized at a lower rate than epicatechins.^[21]

Increased reaction products, GCG and C, finally decreased with prolonged heating process, which indicated that after the catechins reached the maximum level of epimerization, the predominant change become the degradation or oxidation of the catechins.^[26] The decline did not happen at a pH of 3–4 in 24 h. And, it happened after 1, 3, and 10 h at a pH of 7, 6 and 5, respectively. Furthermore, it was thought that not only temperature and pH, but also heating time influenced the epimerization. These results were similar to that previously reported by Komatsu,^[25] namely, that a dominant reaction of EC in slightly acidic media was isomerization as far as the reaction fitted an apparent first order kinetics, and once other reactions, such as oxidation and/or polymerization, proceeded, the first order kinetics was disturbed. In this process, there are several competing reactions occurring, including epimerization, oxidation, and degradation making prediction of the reaction of catechins more complex.

Conclusion

The results of this study highlighted the importance of temperature and pH in impacting the stability of TP. According to the stability testing, the present results suggested that TP were stable under low temperature and acidic pH conditions. The stability of TP were pH-dependent: the higher the pH, the more unstable the TP. Transmittance, deterioration, color, and contents of catechins were significantly affected at a pH of 7 or 100°C. These results were the initial step in understanding the behavior of TP in liquid systems, and the stability of TP should also be studied in solid systems, which could help us use TP in foods or drinks optimally. The results of the present study for the stability of TP suggest that the optimal pH tea drink should be below pH 7 and stored at 4 or 25°C against browning. We can protect TP against degradation and oxidization by adjusting pH and being stored at low temperature when tea drinking was obtained. Moreover, our laboratory will establish models of TP-caffeine, TP-tea protein, caffeine-protein, and TP-caffeine-protein, and comprehensively analyze the formation mechanism of green tea cream.

Funding

This work was supported by the National Natural Science Foundation of China (Grant no. 31100500), the Fundamental Research Funds for the Central Universities (XDJK2013B036).

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (Grant no. 31100500), the Fundamental Research Funds for the Central Universities (XDJK2013B036).