Original papers
Changes in the Content of Pectic Substances in Tea Leaves (Camellia sinensis L.) during Steam Processing of Green Tea
2014 Volume 20 Issue 4 Pages 859-865
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2014 Volume 20 Issue 4 Pages 859-865
We examined the change in the content of pectic substances in tea leaves during the steaming of green tea. In four cultivars of tea leaves, water-soluble pectin (WSP) content increased with steaming time, whereas the dilute hydrochloric acid soluble pectin (HSP) content remained almost constant. A β-elimination reaction proceeds throughout the steaming process, so it was postulated that some water-insoluble forms of pectin in HSP convert to a water-soluble form via β-elimination degradation with increasing steaming time. When tea leaves were steamed, pectin fractions with a molecular weight of 1,100 × 103 were degraded from water-insoluble HSP and eluted as WSP. Additionally, as steaming time increased, the fraction of smaller molecular weight pectin increased.
It has been elucidated that the bitter or astringent taste of catechins and the umami taste of amino acids such as theanine are important factors in the taste of green tea (Nakagawa et al., 1981). The taste of green tea changes depending not only on the conditions used to brew the tea, but also on how the tea leaves are processed. When raw tea leaves are processed differently, the tea products differ in quality. As an example, fukamushi tea steamed for a longer time tastes less astringent than asamushi tea steamed for a shorter time. The amounts of several components, such as catechins and amino acids, have typically been considered to be indices of the quality of the tea. However, the amounts of these components do not change during the course of tea processing (Takayanagi and Anan, 1986). In contrast, the amount of water-soluble pectin (WSP) increases during the steaming process with increased steaming time (Takayanagi et al., 1987). It was therefore assumed that the increase in WSP masked the astringent taste of catechins, as fukamushi tea tastes less astringent (Horie and Kohata, 1999). Recently, Hayashi et al. (2005) used a taste sensor system and 1H-NMR spectroscopy to reveal that gallate-type catechins form complexes with pectin, and that this complexation is a factor in reducing catechin astringency. These findings showed that WSP plays an important role in the quality of green tea.
Pectic substances are complex polysaccharides principally composed of poly-galacturonic acids present in the primary cell walls and middle lamella of plant cells. Pectic substances are degraded and solubilized during the maturing or post-harvest processing of plants. This is caused by both enzyme-catalyzed and non-enzymatic mechanisms. In tea processing, enzymes are inactivated by an initial heat treatment. During thermal processing, pectin undergoes non-enzymatic β-elimination degradation (Albersheim et al., 1960). In this paper, we examined the change in the content of pectic substances in tea leaves during the steaming process by varying cultivars, harvesting time, and maturity of the tea leaves. Additionally, we investigated the extent of β-elimination degradation and the molecular weight distribution of WSP during the steaming process.
Preparation of tea leaves Tea leaves were cultivated on the plantation of the National Institute of Vegetable and Tea Science in Kanaya-Shishidoi, Shimada, Shizuoka, Japan. Four cultivars (‘Yabukita’, ‘Saemidori’, ‘Fuushun’, ‘Okumidori’) of tea leaves were plucked in 2012 by season: first crop (May 2012), second crop (June 2012), and fall-winter crop (October 2012). Leaves from the first crop season were also plucked by their degree of maturity: younger, optimum, and over-matured. On average, the interval between these three levels of maturity was 2 days. About 100 g of plucked tea leaves (raw leaves) were immediately frozen and the rest were steamed. About 100 g of tea leaves were steamed for 30, 60, 120, and 300 s for the ‘Yabukita’ cultivar, and for 60 and 300 s for the other three cultivars. The steamed leaves were then frozen. All frozen tea leaves were lyophilized and powdered using a cyclone sample mill.
Preparation of alcohol-insoluble substances (AIS) Powdered tea leaves (1.0 g) were suspended in 200 mL of 70% ethanol and stirred overnight at room temperature. The supernatant was isolated by filtration. The residue was re-suspended in 70% ethanol and filtered again; this was repeated until the remaining free sugars were below 5 µg/mL (for measurements of neutral and acidic polysaccharides; Figure 4 and Table 4) or 20 µg/mL (for all other experiments) of D-glucose, determined using the phenol-sulfuric acid method (Dubois et al., 1956). The residue was washed with 99% ethanol, then with acetone, dried, weighed and milled using a vibrating sample mill.
Changes in WSP and WSN content in ‘Yabukita’ tea leaves during steaming by crop season. 1: first; 2: second; 3: fall-winter
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Extraction of pectic substances 0.1 g of milled AIS was suspended in 40 mL of water and stirred for 24 h at 25°C. The supernatant was filtered using a glass fiber filter (Advantec GS-25, Advantec, Dublin, CA, USA). The filtrate was labeled ‘WSP’. The residue on the glass fiber filter was then extracted with 50 mL of dilute hydrochloric acid (0.05 N) for 1 h in a boiling water bath, then filtered (Advantec No. 5B). This filtrate was added to the WSP and labeled ‘dilute hydrochloric acid soluble pectin’ (HSP).
Determination of galacturonic acid and total neutral sugar content The content of galacturonic acid in WSP and HSP was determined according to the 3,5-dimethylphenol method (Scott, 1979) and expressed as anhydrogalacturonic acid. WSP and HSP (0.25 mL) were individually mixed with 0.25 mL of 2% (w/v) sodium chloride in a reaction tube, then 4 mL of sulfuric acid was added and the tube contents were immediately mixed. The solution was heated in a water bath at 70°C for 10 min and then cooled to room temperature. The solution was then mixed with 0.20 mL of glacial acetic acid containing 0.1% (w/v) 3,5-dimethylphenol. The optical density was measured at 450 – 400 nm using a spectrophotometer (Beckman DU7500, Beckman Coulter, Brea, USA) at the time between 10 and 40 min from the addition of the 3,5-dimethylphenol solution. D-Galacturonic acid solution (0 – 80 µg/mL) was used to construct a standard curve for the determination of galacturonic acid content. The content of total neutral sugars (D-glucose equivalent) in the water extract from AIS was determined by the phenol-sulfuric acid method after correction for interference from galacturonic acid.
Determination of the extent of the β-elimination reaction The periodate-thiobarbituric acid test (Weissbach and Hurwitz, 1959; Okamoto et al., 1964) was used to detect unsaturated uronide products from the β-elimination reaction. WSP (0.4 mL) was added to 0.5 mL of a 0.05 N solution of sodium periodate in 0.125 N sulfuric acid and heated in a water bath at 80°C for 15 min. After cooling to room temperature, the solution was mixed with 1 mL of a 1% solution (w/v) of sodium arsenite in 0.5 N hydrochloric acid and 4 mL of 0.3% (w/v) thiobarbituric acid in water. The mixture was stirred and heated in a boiling water bath for 10 min. After cooling to room temperature, the optical density was measured at 548 nm in a spectrophotometer.
Determination of the degree of methylation (DM) of pectic substances DM was determined by following the titration method described by Gee et al. (1958). AIS was treated with acidic ethanol (75% ethanol and 5% concentrated hydrochloric acid). After 15 min stirring, the sample was filtered and washed with 70% ethanol until a negative reaction of chlorine with silver nitrate was observed. The acidic ethanol-treated AIS was suspended in 9% aqueous ethanol containing 1.8% sodium chloride, then the free acidity was determined by titration with 0.01 N sodium hydroxide (a mL) using a phenol red indicator (Saeed et al., 1975). After saponification with 0.5 N sodium hydroxide and neutralization with 0.5 N hydrochloric acid, the sample was titrated again with 0.01 N sodium hydroxide (b mL) using a phenol red indicator. The DM was calculated as follows: DM (%) = 100b/ (a + b).
Determination of molecular weights The WSP was injected onto a HiPrep 26/60 Sephacryl S-400 HR column (GE Healthcare, Little Chalfont, England). The sample was chromatographed on the size exclusion column at a flow rate of 1.0 mL/min, using a mixture of 0.1 M sodium acetate and 0.15 M aqueous sodium chloride as eluent. 3.0 mL fractions were collected and analyzed for galacturonic acid content by the 3,5-dimethylphenol method. Dextran (MW 2,000,000, 670,000, 150,000 and 80,000, Sigma-Aldrich, St. Louis, MO, USA) was used as the standard.
Data analysis All colorimetric analyses were conducted twice independently; the measurements shown are average values. The effects of cultivar, steaming time, degree of maturity and crop season on the content of WSP, HSP and water-soluble neutral polysaccharide (WSN) were evaluated by using two-way or three-way analysis of variance (ANOVA; add-in Excel software 2012, SSRI, Tokyo, Japan)
Figure 1 shows changes in the WSP and HSP content in tea leaves of four cultivars during steaming. The leaves were harvested during the first crop season and picked at optimum maturity. Table 1(a) and (b) are ANOVA tables showing the WSP and HSP content in tea leaves of four cultivars during steaming for 0, 60, and 300 s. The samples were picked at optimum maturity during the first crop season. WSP content increased with increasing steaming time, but there was no significant difference in the content of WSP and HSP among the four cultivars. Figure 2 shows changes in WSP and HSP content during steaming in ‘Yabukita’ tea leaves harvested during the first crop season by the degree of maturity. Table 2(a) and (b) are ANOVA tables showing the WSP and HSP content in tea leaves of the four cultivars during steaming for 0, 60, and 300 s; the leaves were harvested during the first crop season at three degrees of maturity. The WSP content, which increased with increasing steaming time, was the same for all cultivars and degrees of maturity. Conversely, the HSP content was essentially constant for all steaming times, except for 0 s (raw leaves). The HSP content differed significantly among the cultivars and the degree of maturity. Figure 3 shows the changes in the WSP and HSP content in ‘Yabukita’ tea leaves during steaming by crop season. Table 3(a) and (b) are ANOVA tables showing the WSP and HSP content in tea leaves of four cultivars during steaming by crop season (steaming time was 0, 60, and 300 s). The WSP content increased with increasing steaming time, but the WSP content in raw leaves and the degree of increase by steaming in leaves harvested during the fall-winter crop season were lower than in leaves harvested during the first and second crop seasons. The crop season and the steaming time had an enormous impact on WSP content. There was also a significant difference among cultivars in the content of WSP, although to a much lesser degree. Conversely, the HSP content did not change with steaming in the second or fall-winter crop seasons, unlike the first crop season (Table 2). There were significant differences among the cultivars and crop seasons. Overall, the WSP content increased as steaming time increased, whereas the HSP content was essentially constant, regardless of the steaming time. It was postulated that a water-insoluble form of HSP converted to a water-soluble form as steaming time increased.
Changes in pectic acid content in tea leaves of four cultivars during steaming. The leaves were harvested at optimum maturity during the first crop season. 1: Yabukita; 2: Saemidori; 3: Fuushun; 4: Okumidori
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Changes in pectic acid content in ‘Yabukita’ tea leaves during steaming by degree of maturity. The leaves were harvested during the first crop season. 1: younger; 2: optimum; 3: over-matured
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Changes in pectic acid content in ‘Yabukita’ tea leaves during steaming by crop season. 1: first; 2: second; 3: fall-winter
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Polysaccharide conjugates from green tea have attracted much attention due to their biological activities, especially as antioxidants. These conjugates are composed of neutral sugars as well as acidic sugars (Chen et al., 2008). We therefore also examined the change in the WSN content during steaming. Figure 4 shows the change in the WSP and WSN content in ‘Yabukita’ tea leaves during steaming by crop season. Table 4(a) and (b) are ANOVA tables showing the WSP and WSN content in tea leaves of four cultivars during steaming by crop season (steaming time was 0, 60, and 300 s). In contrast to the WSP content, which increased with increasing steaming time, WSN did not increase with steaming for any crop season. Therefore, it was the unique nature of the acidic polysaccharide, pectin, which was changed by steaming.
Figure 5(a) shows changes in the relative content of unsaturated uronides, products of the β-elimination reaction, during steaming based on raw leaves harvested during each crop season. Figure 5(b) shows changes in the relative content of WSP during steaming based on raw leaves at each crop season. There was a high correlation between the change in unsaturated uronide content and WSP content associated with steaming time. Therefore, the results show that the β-elimination reaction was involved in increasing the WSP content with increasing steaming time. It is known that the higher the methyl ester content, the greater the degradation of pectic substances by the β-elimination reaction (Sajjaanantakul et al., 1989). We reasoned that because of a decrease in esterification rates of pectic substances in tea leaves harvested during the fall-winter crop season, the degree of increase in the WSP content by steaming for that crop season was lower than for the first and second crop seasons. We measured the esterification rate of pectic substances in raw tea leaves at each crop season using AIS. The results showed that there was no significant difference in esterification rate among leaves harvested during the various crop seasons for any of the cultivars (about 60% to 70%, data not shown). Swamylingappa et al. (1994) reported that the breakdown of pectins by β-elimination is more extensive in Danvers carrots than in B9304 carrots, but there was no varietal difference in overall pectin ester content. We believe that difficulties in degrading pectic substances in tea leaves harvested during the fall-winter crop season are due to something other than the esterification rate of pectic substances.
Changes in relative content of unsaturated uronides, products of the β-elimination reaction, (a) and relative content of WSP (b) in ‘Yabukita’ tea leaves during steaming based on raw leaves according to crop season. 1: first; 2: second; 3: fall-winter
Figure 6 shows the molecular weight distribution of WSP in ‘Yabukita’ tea leaves from the first crop season by steaming time. Each absorbance value is shown as a relative value divided by WSP content at each steaming time. In the steamed leaves, a strong peak is seen around MW 1,100 × 103. This means that pectin fractions with a molecular weight of 1,100 × 103 are degraded from water-insoluble forms of HSP and eluted as WSP when the tea leaves are steamed. Additionally, as steaming time increases, the smaller molecular weight fractions increase. This means that the longer the steaming time, the greater the degradation of WSP. This shows that as the steaming time increased, the WSP content and the amount of higher molecular weight fractions eluted increased; in addition, the degradation of the eluted WSP also changed the molecular weight distribution of WSP. It seems that it is not just the WSP content, but also the molecular weight of the WSP, that is critical to the elution behavior of WSP comprising various molecular weight pectins. We plan to investigate the dynamics of WSP and the characteristics of WSP elution during green tea processing after the steaming process.
Molecular weight distribution of WSP in ‘Yabukita’ tea leaves harvested during the first crop season by steaming time. Each absorbance value is shown as a relative value divided by the WSP content at each steaming time.
Acknowledgments This research was partly supported by a Research Grant from the National Agriculture and Food Research Organization Gender Equality Program. The authors thank Masako Ishikawa for research assistance.
