Screening tea hybrid with abundant anthocyanins and investigating the effect of tea processing on foliar anthocyanins in tea

Anthocyanins, among the polyphenolic constituents, are water-soluble pigments produced as secondary plant metabolites that belong to the flavonoid family of bioactive compounds that give their characteristic red, blue and purple colours to fruits, vegetables and flowers (Saeed et al., 2018; Szpadzik et al., 2019; Gundesli et al., 2019; Gundesli, 2020). Anthocyanidins are widely used as human dietary supplements owing to their bioactivities, such as antimicrobial (Bendokas et al., 2018), antioxidant (Skrzyński et al., 2016), anti-hyperlipidaemia (Qin et al., 2009), anti-hypertension (Naruszewicz et al., 2007), alleviating postprandial inflammation (Jokioja et al., 2020) and anti-metastatic activities (Paramanantham et al., 2020). Anthocyanins are of particular interest to the food industry because of their bioactivity and ability to impart vibrant colours to a variety of food products, serving as natural dyes in cyanic colours ranging from salmon pink to red and violet to dark blue. A growing number of clinical evidence suggest that the intake of anthocyanins including their precursor's anthocyanidins at levels equivalent to those present in the recommended daily amounts of fruits and vegetables (i.e., 30–35 mg · d⁻¹) may decrease the risk of several age- and obesity-related chronic diseases (Pojer et al., 2013; Cassidy et al., 2016; Yang et al., 2017). However, consumption of anthocyanins is still low (Wallace and Giusti, 2019). It was estimated that the daily intake of anthocyanins was 11 mg, with about one-third of the population having no intake (Wallace and Giusti, 2019). A finely ground tea powder prepared using tea leaves is now widely used as an antioxidant ingredient in food processing (Sakurai et al., 2017). The tea products rich in anthocyanins will be a potential dietary supplement of anthocyanins. The breeding of tea cultivars with abundant foliar anthocyanins is significant.

The normal tea leaves in green colour contain a low level of anthocyanins. Tea cultivars with purple leaves, owing to their high content of anthocyanins, are increasingly attracting the attention of tea growers and tea consumers (He et al., 2018; Shen et al., 2018; Kilel et al., 2018). However, the available cultivars of purple leaf tea are limited. ‘Zijuan’ (ZJ) is a tea cultivar with purple leaf from Yunnan province in China where the weather is hot and humid (Bao et al., 2008; He et al., 2018). However, the ‘ZJ’ has two defects, that is, first, susceptible to freezing winter, resulting in severe freezing-induced injury in winter at high latitude areas; second, late sprouting time in spring, leading to late harvesting of spring tea.

Catechins and amino acids are important indicators for determining the quality potential of a tea cultivar (Jang et al., 2008). Cultivars producing quality black tea usually have a high level of catechins and a higher ratio of catechins to amino acids than those producing quality green tea. During black tea processing, tea catechins are significantly reduced due to oxidation, but information on anthocyanin changes during tea processing has not been available (Luo, 2014). In the current work, a hybridisation using a purple leaf cultivar ‘ZJ’ as female parent and early sprouting and winter resistant cultivar ‘Wuniuzao’ (WNZ) as male parent was carried out. In the hybrid progenies, four green leaf hybrids and four purple leaf hybrids were obtained. In the obtained purple leaf hybrids, the phenological phase, freezing resistance, leaf yield potential and tea quality-related chemical compositions including anthocyanins, catechins, amino acids and caffeine were determined. Moreover, the changes in foliar anthocyanins during black tea and green tea processing were also investigated. This study aims to screen a winter-resistant and early sprouting hybrid containing a high level of anthocyanins and to investigate the effect of tea processing on foliar anthocyanins in tea leaves.

There was a great difference in the content of foliar anthocyanins between tea cultivars. Breeding tea cultivars with abundant foliar anthocyanins is interesting for developing a novel source of anthocyanins dietary supplements. Tea cultivar ‘Mooma 1’ had 0.5918 μg · g⁻¹ DW) of foliar anthocyanins (He et al., 2018). Li et al. (2020) reported that a purple leaf tea cultivar ‘Ziyan’ had total content of anthocyanins up to 1,079.8 μg · g⁻¹ (DW). Purple shoots of cultivar ‘Zixin’ contained 35 μmol · g⁻¹ anthocyanins (equivalent 20,860 μg · g⁻¹ pelargonidin 3,5-di-o-glucoside) (Shen et al., 2018). Tea hybrid ‘B-2’ obtained in the present study had total content of anthocyanins 4,298.55 μg · g⁻¹ (FW), being 28.8% higher than ‘ZJ’ (Table 3). Fresh tea shoots with three leaves and a bud usually contain about 75% moisture (Luo, 2014). Assuming the fresh shoots of ‘B-2’ contained 75% moisture, its total content of anthocyanins would be above 17,000 μg · g⁻¹ (DW). Exactly, Figure 6 shows that fresh leaves of ‘B-2’ contained 16,076.4 μg · g⁻¹ (DW) of total anthocyanins. Hybrid ‘B-2’ showed better performance in anthocyanins content, winter resistance and sprouting time than ‘ZJ’, and so it will be a potential candidate for producing anthocyanins rich tea or tea products.

Cultivar ‘ZJ’ in the present study had 3,336.82 μg · g⁻¹ (FW) (Table 3) or 13,380.81 μg · g⁻¹ (DW) (Figure 6) of total anthocyanins, which was lower than the results (29,140 μg · g⁻¹, DW) by Bao et al. (2008), but higher than that (377.13 μg · g⁻¹, DW) by Wang et al. (2017). These might be due to the differences in growing environments. Bao et al. (2008) sampled the ‘ZJ’ plants grown in Menghai of Yunnan Province (latitude 21.95°N, mean altitude 1,700 m), and Wang et al. (2017) sampled their ‘ZJ’ plants grown in Fuzhou of Fujian Province (latitude 26.05°N, mean altitude 10 m). The ‘ZJ’ plants in the present study are grown in Hangzhou of Zhejiang Province (latitude 30.27°N, mean altitude 20 m). Ultraviolet A and ultraviolet B upregulate the expression of structural genes such as F3’5 ’H encoding flavonoid 3’,5’-hydroxylase, DFR encoding dihydroflavonol 4-reductase and ANS encoding anthocyanidin synthase, as well as regulatory gene TT2 (TRANSPARENT TESTA 8) in the anthocyanin pathway, resulting in high accumulation of anthocyanins (Li et al., 2020). Ultraviolet radiation is usually stronger at high altitude areas than low altitude areas. This explains why the total content of anthocyanins of ‘ZJ’ in the present study was between those by Bao et al. (2008) and Wang et al. (2017).

Anthocyanins are one of the major contributors to the antioxidant activity of tea and tea products (Lu et al., 2015). The bioactivity of anthocyanins in tea varies with their composition and concentration. The most abundant component of anthocyanins in commercial tea prepared using cultivar ‘ZJ’ in Yunnan was delphinidin 3-O-beta-D-(6-(E)-p-coumaroyl) galactopyranoside (Jiang et al., 2013), while cyanidin 3-O-beta-D-(6-(E)-coumaroyl) glucopyranoside showed the highest in vitro antioxidant activity in the leaf of an Indian purple leaf tea cultivar (Joshi et al., 2017). When the same batch of fresh leaves plucked from plants of cultivar ‘ZJ’ were separated into two portions to be prepared into unfermented green tea and fermented black tea respectively, the black tea showed much lower antioxidant activity than the green tea (Lu et al., 2015), indicating tea processing has a great impact on the antioxidant activity of anthocyanins abundant tea leaf. The present study showed that green tea processing has a little effect on the content of anthocyanins, but black tea processing induced great loss in anthocyanins, especially in the rolling and fermentation stages (Figures 6–8). Black tea fermentation is a process of polyphenol oxidation, in which colourless catechins are oxidised and condensed into thearubigins and theaflavins in red and orange colours. Anthocyanins are a group of polyphenolic compounds. Oxidation of anthocyanins might be an important factor leading to the decrease in total content of anthocyanins during black tea processing. To maintain a high level of anthocyanins in the final tea products, it is considered that the anthocyanin abundant leaves harvested from the purple leaf tea cultivars should be prepared into unfermented green tea instead of fermented black tea.

The present study showed that hybrid ‘B-2’ had a higher content of anthocyanins than both its parents ‘ZJ’ and ‘WNZ’ (Table 3). The tea plant is an outcrossing species. This phenomenon suggests that the assortment of different parental alleles was favourable in ‘B-2’. However, total catechins content in ‘B-2’ was significantly lower than ‘ZJ’ and ‘WNZ’ (Table 4). The total content of anthocyanins in ‘A-1’ and ‘B-3’ was significantly higher than their male parent ‘WNZ’, but not significantly different from their female parent ‘ZJ’. Total catechins in ‘A-1’ were significantly higher than ‘WNZ’ but significantly lower than ‘ZJ’ (Tables 3 and 4). The biosynthesis of anthocyanins shares a common phenylpropanoid pathway with catechins (Liu et al., 2019; Wang et al., 2019), in which there may be a substrate competition between anthocyanins biosynthesis and catechins biosynthesis (Liu et al., 2019). It is considered that the high accumulation of anthocyanins in ‘B-2’ might be partially at the expense of catechins because of the substrate competition in the phenylpropanoid pathway.

Catechins and amino acids are important indicators to assess the quality of tea and the suitability of a cultivar for processing green tea or black tea. Usually, tea cultivars for processing black tea have a higher content of catechins and higher ratio of catechins to amino acids (C/A ratio) than those for processing green tea. Instead, green tea cultivars usually have a higher content of amino acids and lower C/A ratio than black tea cultivars (Liang et al., 1996; Jang et al., 2008). Nowadays, tea breeders use these indicators to predict the quality potential of a new tea cultivar for processing black tea or green tea (Liang et al., 1996). ‘ZJ’, a cultivar for processing black tea, had the highest level of catechins and the highest C/A ratio among the tested cultivars and hybrids. The C/A ratio of ‘A-1’, ‘B-2’, and ‘B-3’ was significantly lower than ‘ZJ’, among which ‘B-2’ was the least (Table 5), suggesting that ‘B-2’ is suitable for processing green tea, especially for anthocyanidins rich green tea.

An anthocyanins abundant, early sprouting and winter-resistant tea hybrid with purple leaf were obtained. Black tea processing has caused a significant loss of foliar anthocyanins and green tea processing has a little effect on foliar anthocyanins. To maintain a high level of foliar anthocyanins, fresh tea leaves with abundant anthocyanins from purple leaf cultivars should be processed into green tea instead of black tea.