Comparison of Quality Characteristic and Antioxidant Potential of Cultivated Pu-erh and Gushu Pu-erh Tea Extracts at Two Temperatures

Pu-erh, a type of post-fermented dark tea, has attracted much attention of food scientists because of its health-promoting effects. pH, titratable acidity, color value, antioxidant potential and free amino acid contents of cultivated Pu-erh and Gushu Pu-erh teas extracted at 90 and 100°C were compared in this study. The pH of Gushu Pu-erh tea extract was slightly acidic than cultivated Pu-erh. Gushu Pu-erh contained higher antioxidant potentials and free amino acid content at the both extraction temperatures than the cultivated Pu-erh. The antioxidant potentials and free amino acid content were high in the tea extracted at 100°C. The results of this study showed that Gushu Pu-erh tea extracted at 100°C for 3 min with 30 s of shaking yields high amounts of phenolics and free amino acids.

Tea is one of the most popular ancient beverages in the world and is prepared in various forms as green tea (non-fermented), oolong tea (semi-fermented), black tea (fully fermented by oxidising enzymes) and dark tea (post-fermented by microbes). Pu-erh tea, originally produced in the Yunnan Province of China, is categorized as a type of post-fermented dark tea as opposed to the fermented tea, such as black tea. Pu-erh teas are typically prepared via two methods. Firstly, the raw pu-erh tea is produced by pressing large and unoxidized tea leaves which are then fermented for several years at room temperature. Secondly, the pu-erh teas are ripened for several months using microbes under optimum conditions before subjected for being pressed 1 . Pu-erh tea has got reddish to brownish red or gray appearance, thick and bright red infusion color, bittersweet taste and a unique moldy odor which becomes more prominent with the fermentation and the leaves aging 2 .
Recently, Pu-erh tea has attracted much attention because of its health benefits. Nowadays, in addition to being a favorite drink in China and other Southeast Asian countries, it has also been popular in Japan, USA, Britain and other countries. Studies have shown a multiple health-promoting effects of Pu-erh tea as a functional beverage, including anti-oxidative [3][4][5] , anti-mutagenic 6 , antibacterial 6,7 , antiviral 8,9 , antitumor 10 , cholesterol-lowering 11 , anti-obesity 12,13 , hypoglycemic 14 and anti-allergic 15 activities.
Pu-erh tea made from 'Gushu', meaning ancient tea trees in Chinese, is highly demanded and expensive as well. Gushu Pu-erh are prepared from the tea leaves obtained from trees of several hundred years old. Pu-erh teas are also prepared from cultivated tea trees, which are, in this study, referred to as Pu-erh cultivated. The market value of Pu-erh teas greatly varies with the age of the tea tree which the leaves were obtained from. Although there have various reports on Pu-erh tea been published, a comparative study considering the effect of extracting temperatures and types of Pu-erh tea is not found documented. Considering the effect of extracting temperature 16 and age of tea tree 17 on their quality and functional properties, the objective of this study was to compare the quality characteristics and antioxidant properties of Gushu Pu-erh and cultivated Pu-erh teas. This study will provide an insight into the effect of extraction temperature and source of tea leave on the quality and functional properties of Pu-erh teas.

Chemicals and materials
Folin-Ciocalteu phenol reagent and DPPH were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents used were analytical grade.
Two commercial tea samples, cultivated Pu-erh and Gushu Pu-erh manufactured in Yunnan province of China were used in this study.

Preparation of tea extracts
Two kinds of extract were prepared, one at 90°C and the other at 100°C with the two tea samples and were named as follows.
CP-90: a 1.5-g of dried sample of cultivated Pu-erh tea was extracted with 150 mL of boiling water and incubated at 90°C for 3 min with shaking for 30 s; GP-90: a 1.5-g of dried sample of Gushu Pu-erh was extracted with 150 mL of boiling water and incubated at 90°C for 3 min with shaking for 30 s; CP-100: a dried sample of cultivated Puerh tea (1.5 g) extracted with boiling water (150 mL) and incubated at 100°C for 3 min with shaking for 30 s; GP-100: a dried sample of Gushu Pu-erh tea (1.5 g) extracted with boiling water (150 mL) and incubated at 100°C for 3 min with shaking for 30 s. The extraction conditions were designed to get a close similarity to an actual tea brewing. determination of ph and titratable acidity A pH Meter (Model 250; Beckman Coulter, Inc., Fullerton, CA, USA) was used to measure the pH value of tea extracts. Titratable acidity (lactic acid in g/L) was measured by mixing 5 mL of the extracts and 125 mL of deionized water, followed by titration with 0.1 N sodium hydroxide to an endpoint pH of 8.2.

Color measurement
The L* (lightness), a* (redness, + or greenness, "), and b* (yellowness, + or blueness, ") values of the extracts were determined using a Chroma Meter (CR-300; Minolta Corp., Osaka, Japan). A Minolta calibration plate (YCIE=94.5, XCIE=0.3160, YCIE=0.330) and a Hunter Lab standard plate (L*=97.51, a*= "0.18, b*= +1.67) were used to standardize the instrument using a D65 illuminant as described earlier 18 . dPPh radical scavenging activity DPPH radical scavenging activity was measured according to the methods described by Dhungana et al 19 with some modifications. A 0.8-mL of 0.2 mM DPPH ethanol solution was mixed with 0.2 mL of the tea extracts. The mixture was thoroughly mixed using a vortexer and left to stand for 30 min at room temperature under dark condition, and then the absorbance value was measured at 517 nm using a microplate spectrophotometer (Multiskan GO, Thermo Fisher Scientific, Vantaa, Finland).

determination of the total polyphenol content
The total polyphenol contents of tea varieties were estimated according to the Folin-Ciocalteau method 20 . A 250-µL of undiluted Folin-Ciocalteau reagent was added to 50 µL of the tea extracts. After 1 min, 750 µL of 20% (w/v) aqueous Na 2 CO 3 was added to the mixture and the volume was made up to 5.0 mL with distilled water. The mixture was incubated for 30 min at room temperature under dark condition and the absorbance value was measured at 760 nm using a microplate spectrophotometer (Multiskan GO, Thermo Fisher Scientific). Gallic acid was used to prepare a calibration curve and the total polyphenol contents were reported as gallic acid equivalents (µg GAE/mL extract).

aBts radical scavenging activity
T h e A B T S ( 2 , 2 ' -A z i n o -b i s ( 3ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity of tea extracts was analyzed according to the method described by Miller et al. [21]. The method is based on the ability of an antioxidant to reduce ABTS cation radical into its colorless form. The ABTS cation radical was generated by reacting 2.4 mM potassium persulfate with 7 mM ABTS solution. The reaction mixture was left in the dark at room temperature for 12 16 h. The reaction mixture was diluted with double distilled water to achieve an absorbance value of 0.7 ± 0.02 at 734 nm. The tea extracts and ABTS reagent were mixed and kept in dark for 30 min and the absorbance was measured at 734 nm using a microplate spectrophotometer (Multiskan GO, Thermo Fisher Scientific). The ABTS radical scavenging activity was calculated using the following equation.
where AC= absorbance of ABTS radical cation, AS= absorbance of a mixture of ABTS radical solution and tea extracts.

sod (superoxide dismutase)-like activity
The SOD-like activity was measured according to the method described by Debnath et al 22 . A reaction mixture prepared by adding 1.3 mL of Tris-HCl buffer (50 mM Tris, 10 mM EDTA, pH 8.5) and 100 µL of 7.2 mM pyrogallol was added to an aliquot (100 µL) of sample extracted and reacted at 25°C for 10 min. The reaction was stopped by adding 50 µL of 1 N HCl. The amount of pyrogallol oxidised during the reaction was measured at an absorbance 420 nm using a microplate spectrophotometer (Multiskan GO, Thermo Fisher Scientific). The SOD-like activity was determined using the following equation.

Flavonoid content
The flavonoid content was measured following the method described by Mohdaly et al 23 . A 100-µL of tea extract was mixed with 500 µL of methanol. A 50-µL of AlCl 3 , 50 µL of 1 M NaOH and 300 µL of double distilled water was added to the methanol-sample mixture. The reaction mixture was allowed to react in dark for 30 min and the absorbance value of the mixture was measured at 510 nm using a microplate spectrophotometer (Multiskan GO, Thermo Fisher Scientific). Quercetin was used to plot standard curve and flavonoid content was measured as quercetin equivalent (µg QE/mL extract).

Free amino acid composition
The free amino acid content of tea extracts was determined following the procedure described by Je et al 24 . An aliquot of the tea extract (1 mL) was hydrolyzed with 6 N HCl (10 mL) in a sealedvacuum ampoule at 110°C for 24 h. The HC1 was removed from the hydrolyzed sample using a rotary evaporator and a known volume (5 mL) of the reaction mixture was prepared with 0.2 M sodium citrate buffer (pH 2.2). The mixture was passed through a Sep-Pak C18 cartridge (Waters Co., Milford, MA, USA) and filtered through a 0.22-µm membrane filter (Millipore, Billerica, MA, USA). The amino acid profile was determined using an automatic amino acid analyzer (Biochrom-20, Pharacia Biotech Co., Stockholm, Sweden). All of the samples were run in duplicate and expressed as µg/mL of tea extract.

statistical analysis
All the data were subjected to analysis of variance (ANOVA) using SAS (SAS institute, Cary, NC, USA). A significant difference between means was determined at p<0.05 using Tukey test. A mean value of three replicates were reported, unless otherwise stated.

results and disCussion
General chemical characteristics pH and titratable acidity (TA) were considered to measure the general chemical composition of the tea extracts. The pH value of tea extracts was significantly varied with tea type and extraction temperature, whereas the difference in TA values was not significant ( Table 1). The tea extracts were slightly acidic and the highest pH value was found in CP-90 (5.72) and the lowest in GP-90 (5.01). The TA ranged from 0.09% (CP-90) to 0.12% (GP-100). The pH value of cultivated Puerh tea extracts was higher than that of the Gushu Pu-erh at both temperatures. TA is a predictor of the impact of acid content on flavor of food, whereas the pH indicates the ability of a microorganism to grow in a specific food 25 . The variation in pH can influence on flavor and shelf-life 26 .

Color measurement
A color value of food plays a vital role in consumers' acceptability towards the food. Hunter's color values of the tea extracts were significantly different ( Table 2). The highest and lowest lightness values were found in CP-90 (48.07) and 46.53 (GP-100), respectively. In contrary the highest and lowest redness values were found in GP-100 (0.20)  Samples are defined in Materials and Methods (Preparation of tea extracts).

3)
Quoted values are means±SD of triplicate measurements. Values followed by different letters (a-d) in the same row are significantly different (p<0.05). and CP-90 (0.07), respectively. The higher redness value in CP-100 and GP-100 might be due to more extraction of coloring agents at 100°C than at 90°C. The yellowness value of cultivated Pu-erh (CP-90 and CP-100) was higher than that of Gushu Pu-erh tea extracts. The variation in color value among the sample extracts might be due to the effect of extracting temperature 16 and/or polyphenol Table  2 content 27 . antioxidant potential DPPH and ABTS radical scavenging activities, SOD-like activity, and total polyphenol and flavonoid contents were considered to evaluate the antioxidant potentials of tea extracts. Value of antioxidant potentials was high for the extraction at 100°C compared to 90°C and significantly different among the sample extracts (Table 3). Higher contents of phenolics in the tea extracted at higher temperatures were also observed in previous studies 28,29 . The results also showed that Gushu Pu-erh tea extract possessed high antioxidant potentials in terms of ABTS radical scavenging activity and total polyphenol and flavonoid contents compared to the cultivated Pu-erh ( Table 3). The higher antioxidant potential of Gushu Pu-erh tea extracts was possibly due to the tea leaves obtained from old tea trees. Ahmed et al 30 implied that the tea leaves harvested from older trees tastes much bitter, and the bitter taste of Pu-erh tea is more related to catechin, a group of polyphenol, content. It has been reported that the tea catechins demonstrate a range of cellular mechanisms that have antioxidative, anti-inflammatory, neuro-protective, anti-cancer, anti-microbial, and anti-atherosclerotic activities 31 .

Free amino acid composition
The content of individual free amino acids and their total amount in the four tea extracts are listed in Table 4. A total of 37 free amino acids were analyzed, out of which seven, seven and nine were detected at least in one sample extract as essential, non-essential, other free amino acids, respectively. Essential L-methionine and non-essential proline were not detected in any of the four tea extracts. Similarly, taurine, urea, L-á-amino asipic acid, L-á-amino-n-butyric acid, L-cystine, cystathionine, hydroxylysine, L-ornithine, 3-methyl-L-histidine, L-anserine, L-carnosine and hydroxy proline were the other free amino acids which were not detected in any sample extract. The highest amount of total free amino acids was found in GP-100 (691.56 ug/ mL) followed by CP-100 (566.68 ug/mL) and GP-90 (557.69 ug/mL). Gushu Pu-erh contained higher amount of total free amino acid than cultivated Pu-erh in the both extraction temperatures. L-phenylalanine was the most abundant essential and L-asparitic acid and L-glutamic acid were the most abundant non-essential amino acids detected in the tea extracts. ã-Amino-n-butyric acid (GABA) is primarily synthesized in plant tissues by decarboxylation of glutamic acid in the presence of glutamate decarboxylase 32 . GABA and glycine are related to learning and memory, stroke and neurodegenerative diseases; relieving anxiety, sedation, Table 4 anticonvulsant, and muscle relaxation function [33][34][35] .

ConClusion
This study compared the pH, titratable acidity, color value, antioxidant potential and free amino acid contents of cultivated Pu-erh and Gushu Pu-erh tea extracted at 90 and 100°C. The results of the present study showed that extraction temperature significantly affect the chemical and functional values of tea. Gushu Pu-erh showed lower pH but higher antioxidant potentials and free amino acid content at the both extraction temperatures. The antioxidant potentials and free amino acid content were high in the tea extracted at 100°C compared to at 90°C. Based on the nutritional and functional parameters investigated in the present study, Gushu Pu-erh tea extracted at 100°C for 3 min with 30 s of shaking provides high amounts of total polyphenol, flavonoid and free amino acid.