Withering timings affect the total free amino acids and mineral contents of tea leaves during black tea manufacturing

2.1 Sample preparation

Fresh tea leaves were collected from National Tea Research Institute (NTRI) Mansehra, Pakistan. The leaves were then spread out on wire trays and withered by passing fresh air over them at about 25 °C for 21, 22, 23, 24 h (Emdadi et al., 2009). The control samples were analyzed as fresh i.e. 0 h withered. Three replicates were used for each treatment. The humidity of the air at the site of the tea leaves collection and during the withering process was 55–78% and 70–80%, respectively. Moisture was lost during the withering process along with biochemical changes in the leaves. All the samples were analyzed for the following parameters in triplicate.

2.2 Moisture content

The fresh tea leaves (i.e. non-withered) were dried in oven at 70 °C for 24 h, and the withered tea samples were dried at 105 °C for 3 h. The percentage of moisture contents of fresh and withered tea leaves was calculated when the samples reached constant weights (AOAC, 2005). The dried samples were then grounded to powder, and preserved in air tight packets for further analysis.

2.3 Total free amino acids

The method used for the determination of total amino acid in tea solution was based on international standards (ISO 3103, 1980) with modification (Yao et al., 1993, 2006). Briefly, ground tea leaves samples (1 g) each of fresh and withered leaves were weighed into separate beakers. Hot distilled water (100 ml) was added to each beaker and the leaves were allowed to infuse for 10 min. The infusions were filtered through Whatman filter paper No. 1 prior to analysis. The total free amino acids were determined by taking 1 ml of tea infusion, 0.5 ml of phosphate buffer solution (pH 8.0) and 0.5 ml of 2% ninhydrin solutions containing 0.8 mg/ml of tin chloride in 25 ml volumetric flask. The mixture was heated for 15 min in boiling water bath and cooled to room temperature. It was then diluted to 25 ml with distilled water and laid aside for 10–15 min. The optical density of the solution was checked at 570 nm by spectrophotometer (UV 1700 Shimadzu, Japan) with 15 mm colorimetric cup, using distilled water as a blank. Using theanine as a standard, standard curve was prepared by plotting the absorbance of a series of working standards against their respective concentrations. Total amino acid contents were calculated by using the formula reported by Yao et al. (2006).

2.4 Ash content

Accurately weighed 2 g of finely powdered samples from each treatment was taken in pre-weighed china dish. The samples were ignited with the help of blow pipe. The ignited samples in crucibles were placed in a muffle furnace at 600 °C for 3 h. The crucibles were taken out, cooled in desiccator for 30 min and reweighed. From the difference in weights, the percent ash was calculated (Ferrara et al., 2001).

2.5 Minerals analysis

The dried and powdered samples (2 g) each of fresh and withered tea leaves were taken in 100 ml digestion flasks. Nitric acid (10 ml) was added to each flask and left over night. After 24 h, 4 ml perchloric acid (HClO₄) was added to each flask. The flasks were heated gently at first and then vigorously until colorless solutions were obtained. Heating was discontinued when volumes were reduced to 3 ml. The contents were cooled and diluted with distilled water up to 100 ml. The solutions were used for analysis of selected minerals. Na and K were determined by flame photometer (PFP7 Jenway, UK), P by spectrophotometer (UV 1700 Shimadzu, Japan) and Mg, Fe, Cu, Zn, Mn, Al, Ni, Cd and Pb by Atomic Absorption Spectrophotometer (Analyst 200 Perkin Elmer, USA) using their respective standard curves. All the mineral concentrations were expressed in mg/kg.

2.6 Statistical analysis

Experimental data were analyzed using the statistical package MStatC (Michigan State University, USA). A one-way ANOVA procedure followed by Least Significant Difference (LSD) test was used to determine the significant difference (P < 0.05) between treatment means (Steel and Torrie, 1997). Each mean was calculated from triplicate values.

2.7 Method validation

Calibration was performed for each mineral element using its aqueous standard solution. The detection limits were calculated according to IUPAC rule (Long and Winefordner, 1983) in order to evaluate the analytical characteristics of the method for each element.

The precision of method was evaluated in eight replicate determinations on each of four different randomly chosen samples. The reliability of the method was confirmed by using a standard reference material i.e. Standard Reference Material 1571 Orchard Leaves from National Bureau of Standards.

3.1 Moisture and ash contents

Significant (P < 0.05) variations were observed in the moisture contents of tea leaves during withering (Table 1). Maximum average moisture content (76.4%) was examined in fresh leaves (0 h withered) followed by 21 h withered tea leaves (68.5%). The lowest amount of moisture (63.8%) was noted in 24 h withered tea samples (Fig. 1). Water was continuously lost during the withering process that ultimately resulted in the flaccid leaves. As such with the moisture contents, the ash contents were significantly affected by the withering timing (P < 0.05). The average ash content of fresh tea leaves was 3.74% lower than the ash contents of the withered leaves (Fig. 3, Table 1). The highest amount of ash content i.e. 5.85% was examined in 23 h withered tea samples that decreased to 5.75% after 24 h withering.

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Figure 1

Moisture content (%) of Camilla sinensis leaves at different withering times during black tea manufacturing. Fresh represents 0 h withering. The bars represent the standard errors of means.

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Figure 2

Total free amino acids (%) of Camilla sinensis leaves at different withering times during black tea manufacturing. Fresh represents 0 h withering. The bars represent the standard errors of mean.

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Figure 3

Ash content (%) of Camilla sinensis leaves at different withering times during black tea manufacturing. Fresh represents 0 h withering. The bars represent the standard errors of means.

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3.2 Total free amino acids

We found increase in the total free amino acids in tea infusion with the withering times. However, the increase was statistically non-significant (P > 0.05; Table 1). The lowest amount (1.60%) was examined in fresh leaves infusion whereas the highest amount i.e. 2.77% was observed in 23 h withered tea leaves infusion (Fig. 2). An increasing trend was noted up to 23 h after which a decline was observed in the total free amino acids of tea leaves.

3.3 Essential mineral elements

Analysis of variance for the essential mineral contents indicated that withering timings had a significant (P ⩽ 0.05) influence on Na, P, Mg, Cu, Zn and Mn contents whereas non-significant (P > 0.05) effect was observed for K and Fe contents of the tea leaves (Table 3). Sodium contents were highest (331.7 mg/kg) in fresh leaves whereas the lowest amount (151 mg/kg) was found in 23 h withered tea leaves (Table 2). The K contents ranged from 8588 mg/kg in 21 h withered to 8611 mg/kg in 24 h withered tea leaves. The highest amount of P (5932 mg/kg) was examined in 24 h and the lowest (4994 mg/kg) in 22 h withered leaves. A reversal of the process was observed for Mg where the highest concentration of magnesium (896 mg/kg) was found in 22 h withered leaves and the lowest (803 mg/kg) in 24 h withered leaves. The average Fe contents did not significantly change during withering and ranged from 110 mg/kg in fresh leaves to 114 mg/kg in 24 h withered leaves. Copper contents of tea leaves at different withering hours showed the highest concentration (12.6 mg/kg) at 24 h while the lowest (10.2 mg/kg) at 23 h. Similarly, the highest amount of Zn (25.6 mg/kg) was examined in 24 h withered leaves and the lowest amount (17.3 mg/kg) in fresh leaves. The Mn contents of tea leaves ranged from 158.6 (fresh) to 185 mg/kg (23 h withered).

3.4 Toxic mineral elements

Apart from the non-significant (P > 0.05) effect of withering timing on Cd contents (Table 5), we found significant (P < 0.05) effect of withering time on the toxic mineral elements of tea leaves (Al, Ni and Pb). The Al contents of the leaves ranged from 599 mg/kg in fresh to 624 mg/kg in 23 h withered leaves (Table 4). Similarly, the Ni contents showed lowest concentration (1.86 mg/kg) in fresh leaves and the highest (3.88 mg/kg) in 23 h withered tea leaves. The lowest amount of Pb (1.18 mg/kg) was also examined in fresh leaves whereas the highest concentration (2.45 mg/kg) was noted in 24 h withered tea leaves. Result regarding Cd contents of tea leaves withered for different intervals of time showed non-significant variations among the samples. The average Cd contents ranged from 0.09 mg/kg in 24 h withered tea leaves to 0.12 mg/kg in fresh leaves.

4.1 Temporal variations in the moisture, ash and total free amino acids with withering

This study investigated the impact of variation in withering time on the moisture, ash, total free amino acids and essential and toxic mineral contents of tea leaves during black tea manufacturing. The study revealed that significant biochemical changes took place during withering that might have profound influence on the quality of made tea. It was observed that the leaves started to lose moisture immediately after the start of withering process. As the degree of withering progressed, the leaves became more flaccid. This may be due to the fact that after detachment of leaves from the shoot, the permeability of cell membranes increases and that leads to the loss of water as vapors (Tomlins and Mashingaidze, 1997). The results of our findings are closely in agreement with Bhattacharyya and Ghosh, 1968 who reported moisture loss during withering of tea leaves. It should be noted that moisture contents play a key role in maintaining the quality of tea. High moisture contents enhance the growth of microorganisms; thereby the tea shelf life is reduced (Hall et al., 1988).

Free amino acid contents are considered as an important criterion for tea quality assurance and contribute to the overall quality in terms of taste and color (Yao et al., 2006). Theanine, a glutamic acid analog or amino acid derivative, has been shown to reduce mental and physical stress (Kimura et al., 2007) and improves cognition and mood in a synergistic manner with caffeine (Haskell et al., 2008). Our study suggests that the increased amount of amino acids in withered tea samples as compared to fresh tea leaves may be due to the breakdown of proteins into amino acids by peptidase (Yao et al., 2006). Apart from protein break down, sugar is also converted into amino acid during withering (DevChoudhary and Bajaj, 1980). Roberts and Sanderso, 1966 confirmed an increase in total free amino acids during the withering stage of tea manufacture. The rate of increase may be positively related to temperature up to the point where the tissues are killed, after which no further change takes place (Tomlins and Mashingaidze, 1997).

The ash contents of the tea are affected by many factors such as processing methods, storage conditions, shooting periods and their interactions (Nas et al., 1991). The higher contents of ash in withered leaves, in our case, may be due to adulteration or unhygienic conditions during withering process. The ash content of tea is a highly valuable characteristic of its quality. Old and spurious leaves, as well as tea adulterated with mineral matter yield more ash as compared to fresh tender leaves.

4.2 Dynamics of essential mineral elements and withering time

We found that the Na contents of tea leaves were significantly reduced during withering. This may be due to the fact that during withering 3–4% solid matter in the leaf is lost through respiration and other biochemical processes (Hampton, 1992). Interestingly, unlike Na, K was not significantly affected by the withering process. The present values reported for Na and K in tea leaves are in agreement with those reported by Stagg and Millan (1975), Padmini et al. (2010) and Ozcan (2005). Na and K are of immense importance in regulating many body functions. Sodium regulates the fluid balance of body within and outside the cells whereas K plays a crucial role in fluid balance and nerve impulse transmission. The regular consumption of tea may contribute to the daily dietary requirements of several elements and the large amount of K in comparison with Na may be beneficial for hypertensive patients (Xie et al., 1998; Fernandez et al., 2002). Regarding P, an increasing trend was examined during the withering process. The possible reason for the increase in P contents may be the degradation of all phosphates esters and RNA contents during withering (Selvendran, 1969). Similarly, Mg content also increased up to 22 h withering but then decreased. There was no obvious reason for variation of Mg contents at different withering hours. However, Mg is an integral part of chlorophyll and during withering chlorophyll degrades which results in an increase or decrease of Mg contents (Tomlins and Mashingaidze, 1997).

The effect of withering was found non-significant on the Fe contents of tea leaves. The present values reported for Fe were quite higher than those compiled by Hussain et al. (2006) (0–2.75 mg kg⁻¹) for different commercial tea brands available in Pakistan. However, our results were reasonably in line with Ferrara et al. (2001) who reported a mean concentration of 146.9 mg Fe kg⁻¹ in Indian tea. Similarly, the average Cu content of tea leaves determined in the present study was fairly supported by the previous work done by Stagg and Millan (1975). They reported that tea contained 8–28 mg kg⁻¹ copper. Similarly, Gebretsadik and Chandravanshi (2010) reported that the Cu contents of tea ranged from 9.1 to 11.5 mg kg⁻¹. Cu is invaluable in plant metabolism and especially in tea it plays a functional role in polyphenol oxidase, an enzyme essential in fermentation during manufacturing of tea (Natesan and Ranganthan, 1990). Regarding Zn, the present results were comparable to those of Ashraf and Mian (2008) who examined that the Zn contents of tea ranged from 23.7 to 122.4 mg kg⁻¹. The progressive increase in Zn contents with withering time may be due to the gradual degradation of Zn containing enzymes, proteins and nucleic acid content of the leaves. Zn plays a vital role in the synthesis of nucleic acid and proteins and helps in utilization of phosphorus and nitrogen (Wardlaw and Hampl, 2006). An increasing trend was noted for Mn contents of tea during withering. The possible explanation for the increase may be the degradation of enzymes particularly mitochondrial superoxide dismutases and Mn-catalases. Mn containing enzymes participate in the urea cycle and carbohydrate metabolism (Wardlaw and Hampl, 2006).

4.3 Dynamics of toxic mineral elements and withering time

The toxic mineral elements such as Al, Ni, Pb were significantly affected by variation in withering time. However, withering time had no significant effect on the Cd content of tea leaves. Many factors contribute to metals accumulation in the tea leaves, such as soil, its organic matter contents, processing methods and environmental pollutions (Fernandez et al., 2002; Ozcan, 2005). We observed an increasing trend for Al, Ni and Pb contents, which may largely be attributed to the airborne elements and from dust blowing and pollution from the surrounding area (Salahinejad and Aflaki, 2010). It was concluded that field was not the only determinant of high toxic elements in the tea leaves. However, all the values were fairly in line with those reported in the literature (Stagg and Millan, 1975; Hussain et al., 2006; Salahinejad and Aflaki, 2010). High concentrations of toxic elements in beverages and food products have severe health implications for human. For example, increased amount of Ni can cause kidney failure, lung cancer and nasal sinus (Duda-Chodak and Blaszczyk, 2008). Pb can inhibit copper dependent enzymes needed for neurotransmitters, causing hyperactivity. Gout can arise from Pb toxicity raising uric acid level and impairing kidney functions (Hussain et al., 2006). Cd is a toxic metal having no obvious function in humans, animal or plants. Once accumulated in the kidney, it stays there and difficult to remove by excretion. Thus, long consumption of Cd contaminated tea is dangerous for health and can directly damage the nerves cells (Hussain et al., 2006).