Food Science and Technology Research
Online ISSN : 1881-3984
Print ISSN : 1344-6606
ISSN-L : 1344-6606
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Dynamics of Water-soluble Pectin in the Roasting Process during Green Tea Manufacturing
Hisako HironoYuzo Mizukami
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2018 Volume 24 Issue 1 Pages 177-181

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Abstract

We examined changes in water-soluble pectin (WSP) and dilute hydrochloric acid soluble pectin (HSP) contents of tea leaves, and pectin content of tea infusions in the roasting process during refined tea manufacturing by crop season. In all crop seasons, WSP content in tea leaves increased with roasting temperature. HSP content was almost constant or increased with roasting temperature by crop season. Changes in pectin content of tea infusions by roasting temperature were proportional to or lagged slightly behind WSP content changes of tea leaves in all crop seasons. In all crop seasons, the molecular weight (MW) distribution of both WSP in tea leaves and pectin in tea infusions was biased toward the lower-MW range with increasing roasting temperature. In parallel, major peaks in the MW distribution of pectin in tea infusions shifted to the lower-MW range in all crop seasons. It was postulated that β-elimination degradation was involved in WSP production from water-insoluble forms of pectin in HSP and the degradation of WSP itself during the roasting process.

Introduction

Green tea is made from raw tea leaves picked at the appropriate time during the first, second, third, and fall-winter crop seasons. The harvested tea leaves are immediately steamed to inactivate oxidizing enzymes, and then dried and shaped by rolling, thereby reducing the moisture content to about 5%. This semi-finished green tea product is called unrefined tea. Unrefined tea does not yet have the value of a finished product, since unrefined tea leaves are characterized by an uneven shape, a moisture content that is still high and raw odors. Consequently, the tea requires secondary processing before it can be sold as a finished product, known as refined tea. Secondary processing involves sorting, cutting and shaping, roasting and blending. In this refined tea manufacturing process, the unrefined teas are roasted to reduce the water content of tea leaves, thereby improving storage behavior and adding flavor by attenuating raw odors (Kubota and Hara, 1970). The roasting of unrefined tea is conducted under diverse conditions; tea leaves harvested in the fall-winter crop season are roasted more heavily to impart a stronger flavor, while leaves harvested in the first crop season are roasted more lightly for a subtle flavor, representing a feature of the tea itself.

Pectins are complex polysaccharides, principally composed of poly-galacturonic acids, present in the primary cell walls and middle lamella of plant cells, and greatly affect the rheological behavior of many plant-derived food products. Generally, heating results in decreased pectin polymer size and reduced viscosity in fluid products. In addition, Hayashi et al. (2005) revealed that gallate-type catechins, which are main components of green tea, form complexes with pectin, and this complexation is a factor in reducing catechin astringency.

During unrefined tea manufacturing processes, it has been observed that the water-soluble pectin (WSP) content in tea leaves increases during the steaming process, which is in the initial stages of tea processing, via non-enzymatic β-elimination degradation, and steaming time proportionally influences the increase of WSP content in tea leaves and pectin content in tea infusions (Takayanagi et al., 1987a; Takayanagi et al., 1987b; Matsuo et al., 2012; Hirono and Uesugi, 2014; Hirono, 2016). During green tea manufacturing, pectin might be degraded by heating during the roasting process in refined tea manufacturing as is degraded during the steaming process in unrefined tea manufacturing. In fact, it has been reported that heavily roasted tea, ‘houji cha’, has the highest WSP content among the various categories of green tea (Nakagawa, 1976; Maeda and Nakagawa, 1977). Nakagawa (1976) reported that roasting of unrefined tea leaves resulted in increased WSP content, but a decrease in the molecular weight (MW) of WSP in tea leaves. The rate of β-elimination degradation is accelerated with increasing temperature (Albersheim et al., 1960), and the conformation of WSP, which varies according to heating conditions, might be an explanatory factor for variations in the astringency and viscosity of roasted teas. Further, WSP dynamics in the roasting process following unrefined tea manufacturing, which is the final process in refined tea manufacturing, need to be precisely examined in order to assess the diverse roasting conditions. In this paper, we examined the changes in WSP content, dilute hydrochloric acid soluble pectin (HSP) content as a reference for water-insoluble pectin in tea leaves, and pectin content in tea infusions, based on the temperature of tea leaves during roasting by crop season. In addition, we investigated the changes in the MW distribution of both WSP in tea leaves and pectin in tea infusions, and the extent of β-elimination degradation during the roasting process.

Materials and Methods

Preparation of tea leaves    Tea leaves were cultivated at the National Institute of Fruit Tree and Tea Science plantation in Kanaya-Shishidoi, Shimada, Shizuoka, Japan. Tea leaves (‘Yabukita’ cultivar) were harvested during the first crop season, in May 2012 and April 2013, and during the second crop season, in June 2015. Harvested tea leaves were immediately steamed for 25–45 s and were then manufactured into unrefined teas. After the initial steaming process, unrefined tea processing proceeded sequentially from primary drying, tea rolling and secondary drying, to the final rolling. As for the fall-winter crop season, tea leaves (‘Yabukita’ cultivar) were cultivated at a tea plantation in Hatsukura, Shimada, Shizuoka, Japan and were similarly manufactured into unrefined teas. These unrefined teas, made in the first (equally mixed 2012 and 2013), the second and the fall-winter crop seasons, were respectively used as the raw materials for roasting, after sorting by sieving, cutting and eliminating stems of teas and powdered teas using an optical tea stem separator and a pneumatic separator. Raw tea leaves (1200, 1000 and 800 g), from the first, second and fall-winter crop seasons, respectively, were roasted using a reciprocating sieve (BAISEN; Yamamasu Ltd., Shizuoka, Japan) at a rotation speed of 12 rpm and air speed of 0.2 ms−1. Tea leaves were removed from the reciprocating sieve at 3 s after the surface temperature of tea leaves reached 90–140°C for the first season, and 90–180°C for the second and fall-winter crop seasons. Roasting at each temperature setting was repeated three times.

Determination of WSP and HSP contents in tea leaves    Unroasted and roasted tea leaves were powdered using a ZM200 cyclone sample mill (Retsch GmbH, Haan, Germany). Preparation of Alcohol-Insoluble Substances (AIS), WSP and HSP extraction from AIS, and determination of WSP and HSP contents by the colorimetric method were conducted using the 3,5-dimethylphenol method as described previously (Hirono and Uesugi, 2014; Hirono 2016). The extraction method is briefly described as follows: AIS was prepared by washing powdered tea leaves using 70% ethanol until the remaining free sugars were below 20 µg/mL of D-glucose. AIS was suspended in water and stirred for 24 h at 25°C. The supernatant was filtered, and the filtrate was labeled WSP. The residue on the filter was extracted with dilute hydrochloric acid (0.05 N) for 1 h in a boiling water bath, and then filtered. This filtrate was added to the WSP and labeled HSP. WSP and HSP contents are given as average analysis values of three independent roasted tea samples.

Determination of pectin content in tea infusions    Six grams of unroasted and roasted tea leaves (not powdered) were infused with 600 mL of hot water, immediately after boiling, using a GV-3 teapot (Celec Co., Ltd., Tajimi, Japan) and left at room temperature for 5 min. One hundred milliliters of the infusion without tea leaves was freeze-dried, and the lyophilized concentrate was then dissolved in distilled water (DW). A precipitate was formed by the addition of a final concentration of 70% ethanol, dissolved in DW, and then subjected to colorimetric determination of pectin content. The pectin content in tea infusions was calculated per 100 g of dried tea leaves. Pectin contents are given as average analysis values of three independent roasted tea samples.

Determination of molecular weight    The pectin solutions tested (determination of WSP content in tea leaves and pectin content in tea infusions) in triplicate (except for unroasted samples) were respectively combined according to roasting temperature and concentrated by lyophilization. Size exclusion chromatography using a HiPrep 26/60 Sephacryl S-500 HR column (GE Healthcare, Little Chalfont, UK) was performed with this concentrated pectin. The pectin samples were run at a flow rate of 0.8 mL/min, using a mixture of 0.1 M sodium acetate and 0.15 M sodium chloride aqueous solution (pH 5.6) as the eluent. Fractions of 3.0 mL were collected and analyzed for galacturonic acid content by the 3,5-dimethylphenol method. Dextrans (MW 670,000, 150,000 and 80,000; Sigma-Aldrich, St. Louis, MO) were used as standards. As for dextrans with MW of 1,000,000, the data from a certified analysis (GE Healthcare) was applied to the standard curve of MW.

Determination of the extent of β-elimination reaction    The periodate-thiobarbituric acid test, which detects unsaturated uronide products from the β-elimination reaction, was conducted as described previously (Hirono and Uesugi, 2014). The relative content was based on unrefined teas from each crop season, and is given as an average analysis value of three independent roasted tea samples.

Results and Discussion

Figure 1 shows the changes in WSP and HSP contents in tea leaves, and pectin content in tea infusions during the roasting of unrefined teas by crop season. In the first crop season, WSP content in tea leaves increased with roasting temperature. Conversely, HSP content was constant at all roasting temperatures. In the second crop season, WSP content in tea leaves increased with roasting temperature, and HSP content was almost constant at all roasting temperatures. In the fall-winter crop season, WSP and HSP contents in tea leaves increased with roasting temperature. Changes in pectin content of tea infusions by roasting temperature were proportional or lagged slightly behind changes in WSP content in tea leaves in all crop seasons. The pectin content of tea infusions increased significantly above 130, 155 and 135°C in the first, second and fall-winter crop seasons, respectively. In comparison, WSP and HSP contents in tea leaves and the pectin content in tea infusions of unrefined teas were 0.790, 2.101 and 0.274 g/100 g DW for the first crop season; 0.881, 2.022 and 0.241 g/100 g DW for the second crop season; and 0.414, 2.537 and 0.187 g/100 g DW for the fall-winter crop season.

Fig. 1.

Changes in WSP and HSP contents in tea leaves, and pectin contents in tea infusions during roasting of unrefined teas by crop season (i: first; ii: second; iii: fall-winter). Roasting time was 3 s after reaching each temperature.

Data are shown as an average value ± standard deviation. There were no significant differences between the same letters for each content (Tukey's test, p < 0.05).

; WSP content in tea leaves, ; HSP content in tea leaves, ; pectin content in tea infusions

We previously reported that WSP content in raw tea leaves and the degree of increase by steaming in tea leaves harvested during the fall-winter crop season were lower than in tea leaves harvested during the first and second crop seasons (Hirono and Uesugi, 2014). Although plantations producing raw tea leaves differed by crop season, the WSP content of unrefined tea leaves in the fall-winter crop season was also lower than that of other crop seasons. However, the increase in WSP content by roasting at 155 and 180°C in the fall-winter crop season was much higher than that in the second crop season.

Figure 2 shows changes in the MW distribution of WSP in tea leaves (a) and pectin in tea infusions (b) during roasting of unrefined teas. In all crop seasons, the MW distribution of WSP in tea leaves was biased toward the lower-MW range with increasing roasting temperature. In tea from the second and fall-winter crop seasons roasted at 180°C, the major peaks of the MW distribution of WSP were distinctly lower when compared to tea leaves roasted at below 155°C. As for tea infusions, the MW distribution of pectin was also biased toward the lower-MW range with increasing roasting temperature in all crop seasons. In parallel, the major peaks of the MW distribution of pectin in tea infusions shifted to the lower-MW range in all crop seasons. In tea infusions of unroasted leaves and leaves roasted at lower temperatures, higher-MW fractions of pectin were mainly present. In tea infusions of leaves roasted at higher temperatures (above 140, 155 and 155°C in the first, second and fall-winter crop seasons, respectively), the MW range of pectin corresponded to that of tea leaves, which were composed mainly of the lower-MW range.

Fig. 2.

Changes in molecular weight distribution of WSP in tea leaves (a) and pectin in tea infusions (b) during roasting of unrefined teas. Roasting time was 3 s after reaching each temperature. Crop seasons were first, second, and fall-winter (i, ii, and iii, respectively). Arrows in figures show molecular weight markers (dextrans).

Areas enclosed by a horizontal axis and individual curves were proportional to WSP content at each temperature.

Figure 3 shows changes in the relative content of unsaturated uronides, β-elimination reaction products, in WSP extracts from AIS in tea leaves during roasting of unrefined teas by crop season. β-Elimination reaction results in removal of the H atom at C-5 and the glycosidic residue at C-4 of galacturonic acid residues, leading to the formation of unsaturated uronides, which have a double bond between C-4 and C-5 (BeMiller and Kumari, 1972). The relative content was determined based on unrefined teas from each crop season. In all crop seasons, the relative content of unsaturated uronides increased with roasting temperature. There was a strong correlation between the roasting temperature-associated changes in unsaturated uronide content and WSP content in the first and fall-winter crop seasons. Therefore, the β-elimination reaction was involved in the roasting temperature-associated increases in WSP content, as previously reported for steam processing (Hirono and Uesugi, 2014). In light of the observation that WSP content in tea leaves increased and HSP content remained constant with increasing roasting temperature, except for the fall-winter crop season (Fig. 1), it was postulated that the water-insoluble form of HSP was converted to a water-soluble form with increasing roasting temperature. In the second crop season, the relative content of unsaturated uronides at 180°C was high for the low WSP content. It is proposed that the polymer is first depolymerized via β-elimination reaction, resulting in sufficiently small molecules so they are soluble in water, followed by partial fragmentation (Sila et al., 2009). Therefore, the high amount of unsaturated uronides at 180°C was attributed not only to the WSP production volume, degraded from the water-insoluble form of HSP, but also the degree of WSP degradation itself. In the fall-winter crop season, the increase in WSP content of unrefined tea leaves by roasting was much higher than the second crop season, and HSP content was also increased by roasting. This suggests that unrefined teas in the fall-winter crop season are susceptible to β-elimination reaction, and HSP was converted from the hydrochloric acid-insoluble form.

Fig. 3.

Changes in relative content of unsaturated uronides, products of the b-elimination reaction, during roasting of unrefined teas by crop season (i: first; ii: second; iii: fall-winter). Roasting time was 3 s after reaching each temperature.

Data are shown as an average value ± standard deviation. There were no significant differences between the same letters (Tukey's test, p < 0.05).

Okada and Furuya (1965) reported that the alcohol precipitation characteristics of tea extracts varied in structure among tea categories. ‘Sen cha’, which is usually roasted lightly, had continuous precipitates resembling silk floss, while ‘houji cha’, roasted heavily, had fragmentary precipitates. The shift to the lower-MW pectin range in unrefined tea infusions roasted at 180°C in the second and fall-winter crop seasons reflects this fragmentary structure. Differences between lower-MW pectin, present in ‘houji cha’, and higher-MW pectin, present in the more expensive ‘sen cha’, might be correlated with differences in the viscosity of these tea infusions.

It is known that the flavor component in ‘sen cha’ is affected by roasting (Mizukami, 2015). Moreover, from this study, it was revealed that roasting markedly changed WSP in green tea; with increasing roasting temperature, the WSP content increased and the MW distribution of WSP was biased toward a lower-MW range. Further examination of WSP changes and the presence of other components in tea infusions under various roasting conditions is necessary. We are now planning to investigate the effects of changes in the content and MW of pectin on the reduction of catechin astringency and viscosity of tea infusions.

Acknowledgments    This research was partly supported by a Research Grant from the National Agriculture and Food Research Organization Gender Equality Program. The authors would like to thank Masako Ishikawa for research assistance.

References
 
© 2018 by Japanese Society for Food Science and Technology
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