The content of photosynthetic pigments has genotypic features.

There is a direct relationship between the quality of raw materials and the content of flavonoids in tea.

There are significant losses of ascorbic acids during processing raw materials to finished tea, despite the equal conditions of collection.

The dynamics of GPOD activity in a 3-leaf sprout are variety-specific. There is a direct relationship between increased enzyme activity and hydrothermal factors.

The largest number of amino acids is synthesized in May, and in the future, AAs are included in the synthesis, the processes of transformations, and mutual conversions to other compounds.

The finished product of tea was made in the plant physiology and biochemistry laboratory of the Federal Research Centre the Subtropical Scientific Centre of the Russian Academy of Sciences using classical technologies.

Black tea (Figure 1) – in the production of black tea, the raw material passed the stages of withering, twisting (moisture loss up to 55%), fermentation (oxidation of polyphenols with the formation of theaflavins and thearubigins), drying (at a temperature of about 95 °C to a final water content of not more than 5 – 6%).

Green tea: steaming (temperature about 95 – 100 °C), twisting, drying (at a temperature of about 95 – 105 °C to the final water content of no more than 3 – 5%).

Ethanol (96% ethyl alcohol), U.S.P. – JSC LenReactiv (St. – Petersburg, Russia).

Potassium iodide (KJ), Reagent – JSC LenReactiv (St. – Petersburg, Russia).

Hydrochloric acid (HCl), U.S.P. - JSC LenReactiv (St. – Petersburg, Russia).

2,6-dichlorophenol indophenol (C12H6Cl2NNaO2xH2O), Reagent – LLC TD "Himmed” (LLC Sigma-Aldrich, USA.

Hydrogen peroxide (H2O2), N.F. - JSC Reachim (Moscow, Russia).

Guaiacol (C7H8O2), Reagent – LLC TD "Himmed” (LLC Sigma-Aldrich, USA).

Ninhydrin (C9H4O3*H2O), Reagent – JSC Interhim (St. – Petersburg, Russia).

The research began in 2016, the targets are samples from raw material (3-leaf fresh sprout) of cultivars Kolkhida and Sochi, and forms 3823, 582, 855, and 2264 (Sochi, Lazarevsky district, Krasnodar region, Russia) (Figure 2a, b).

We using a PE-5400VI spectrophotometer, manufactured by EKROSCHEM (Russia) and capillary electrophoresis system «Kapel 105-M», manufactured by LUMEX (Russia).

The main component determination of the antioxidant system was carried out in the plant physiology and biochemistry laboratory of Subtropical Scientific Centre. Contents of photosynthetic pigments using method invented by Shlyk and the calculated formulas of Smith and Benitez (Shlyk, 1971).

The determination of ascorbic acid was performed using the classical iodometric method (Vorobyov et al., 2013).

Flavonoid compounds with P-vitamin activity were determined by titration following the method of vitamin analysis (Voskresenskaya et al., 2006).

The content of free amino acids amount in samples was determined by the ninhydrin dyeing method and then use a high-performance liquid chromatography method (Speckman et al., 1958).

The activity of guaiacol peroxidase (GPOD) was determined by the spectrophotometric method (Vorobyov et al., 2013).

Statistical processing of the experimental data was carried out using the ANOVA package in STATGRAPHICS Centurion XV (version 15.1.02, StatPoint Technologies) and MS Excel 2007. Statistical analysis included univariate analysis of variance (method of comparing averages using variance analysis, t-test) and variance analysis (ANOVA). The significance of the difference between the means of the least significant difference (LSD) results with p <0.05 was considered statistically significant. All experiments were performed in triplicate and the values were expressed as mean ±SD. The differences between the samples were assessed using the unpaired t-test.

At the beginning of the active growing season (in May), the largest amount of total chlorophylls is synthesized (1.335 μg.g^-1), followed by a smooth drop by August (1.018 μg.g^-1). We found another pattern when studying the content of carotenoids and flavonoids (thearubigins and theaflavins). There is an increase in the content of these groups of pigments in June, a decline in July and, by August, and a slight increase again. The increase is due to unfavorable conditions – drought during these periods. Also, it was noted that the content of photosynthetic pigments shows genotypic features (Table 1).

The main photosynthetic pigment is chlorophyll a; carotenoids transfer additional energy to chlorophylls, performing a light-absorbing function, and divert excess energy from them, performing a light-protecting function (Endo et al., 1985; Nafissatou et al., 2011; Li et al., 2016). We noted that a significant accumulation of chlorophyll in the leaves is typical for the control cv. Kolchida. At that time, as the studied forms contain chlorophyll a is significantly lower (Table 1).

The content of chlorophyll b indicates the level of adaptation of plants to low light. For tea plants, this is not relevant, since the crop is grown in open spaces, and pruning the trellis stimulates the growth of leaves on the upper part of it. But often a tightly closed trellis restricts the space open to the sun's rays, and many of the leaves of the side surfaces are in the shade. In this case, the increased content of chlorophyll b is preferable for the photosynthetic activity of the leaves of this tier. We noted the same pattern as with chlorophyll a: more chlorophyll b is accumulated by the control variety; moreover, the differences are significant or on the border of materiality, as in the case of forms 3823 and 2264. It is important not only the content of a particular pigment but also their ratio. In all the tea plants studied, the a/b ratio ranges from 2.813 mg.g^-1 to 3.362 mg.g^-1, and the differences between the forms are insignificant (Table 1).

Photosynthetic pigments are not only components of the antioxidant protection of the plant itself, but also are part of the biochemical components of the drink since the preparation of black and green tea pigments to some extent pass into the infusion (Lu et al., 2009; Li et al., 2018). Thermal processing induced the formation of degradation product: changes occur in their content: a significant part of the chlorophyll is destroyed during steam treatment, as well as during the drying process (Castillon et al., 1949; Nafissatou et al., 2011; Ošťádalová et al., 2014; Xu et al., 2018; Dini et al., 2019). Therefore, there is less chlorophyll in green tea than in raw materials, and there are even fewer green pigments in the production of black tea than in green tea. But this is good since the presence of chlorophyll in the finished tea affects its quality – the tea acquires an herbal smell and an uncharacteristic taste. The smallest amount of this pigment in green and black tea contains form 855 (0.33 mg.g^-1), which is preferred. Carotenoids provide not only the plant's resistance to exogenous stress but also its value as a source of antioxidants for humans and their content in ready-made (black and green) tea is a positive fact. However, we have not detected any changes in their content, and, as a rule, carotenoids are not taken into account in the qualitative assessment of tea.

Changes occur in the content of chlorophyll: during steam treatment, as well as during drying, a significant part of it is decayed. In the manufacture of black tea, green pigments remain even less than in green.

However, it should be noted that the presence of chlorophyll in the finished tea adversely affects the quality of the product. The smallest amount of this pigment in green and black tea contains form 855 (0.33 mg.g^-1), which is preferred. In respect of carotenoids, such profound changes are not observed by us, which is confirmed by the literature data, besides, carotenoids are not taken into account in the qualitative assessment of tea. However, they are an important component of the antioxidant system and they, therefore, required no less comprehensive study.

In the production of black tea, catechins are disintegrated by oxidation with the formation of flavonoids –TFs and TRs (Figure 3).

We were revealed that between the quality of raw materials and the content of flavonoids in tea there is a direct relationship (Table 2).

As we show, green tea TFs are slightly less than in sprouts and black tea. TFs are unstable compounds and during enzymatic oxidation, they easily pass into TRs, so in black tea, TRs are much more. In the varietal section, it can be noted that the raw materials of forms 855 and 582 (0.10 mg.g^-1 and 0.11 mg.g^-1 respectively), as well as black tea produced from form 582 (0.09 mg.g^-1), showed the highest values in the content of theaflavins, while the highest content of TRs was found in forms 582 and 3823 (1.33 and 1.17 mg.g^-1).

Currently, there is no single standard for the content of these pigments in the finished product. But, according to international rules, any blend of tea should have a ratio of TRs: TRs are not lower than 1:16 and in the highest quality tea 1:10. According to this indicator, tea made from raw materials collected from experimental plants meets international requirements (Kang et al., 2017; Li et al., 2018; Palanivel et al., 2020).

The least amount of AA is contained in the raw material of forms 2264 and 582 (319 and 320 mg.100g^-1, respectively) (Figure 4).

In black tea, the differences between varieties are insignificant, a slightly higher content of AA is noted in black tea made from forms 855 and 3823 (13.73 and 13.07 mg.100g^-1, respectively).

Vitamin C is a necessary nutrient in the production of collagen. Vitamin C deficiency results in the lost formation of collagen fibers, weakening vascular walls, and causing scurvy. Ascorbic acid is also being a tool in the biosynthesis of several secondary polyphenols such as theaflavins thearubigin (Sharangi, 2009; Palanivel et al., 2020). AA also acts as an antioxidant and is believed to play an important role in the prevention of lifestyle-related diseases such as cancer (Nafissatou et al., 2011; Palanivel et al., 2020). The native level of AA is very less in tea leaves and negligible in processed tea (Palanivel et al., 2020; Zimmermann and Gleichenhagen, 2011).

Our research show, that during processing raw materials (fresh sprouts) to finished tea (Figure 1), there are significant losses (up to 90%) of AA, despite the equal conditions of collection. In the production of black tea, due to the development of oxidative reactions during fermentation, disintegrated a significant part of this vitamin on average 96 – 97% (preserved only 3 – 4%).

In green tea, where fermentation is excluded, AA is decayed to a lesser extent. As a result, green tea is about 13% of the initial amount of AA in raw materials (sprout).

This issue requires additional research, which is planned by us in the future.

At the beginning of the growing season (May), the activity of the enzyme was low – in the range of 0.363 to 0.607 gunit in sec. In June, there is a decrease in activity, which, however, is not significant and is due to the biological characteristics of the tea culture.

The highest activity of GPOD during the entire vegetation period is characterized by the cultivar Sochi and form 582 (about 0.56 g-unit in sec) (Figure 5). The lowest activity was observed in forms 3823 and 2264, which indicates a low intensity of redox reactions in stressful situations, under the influence of changing environmental factors on plants.

Peroxidase is considered a marker of the physiological state of plants (Marinescu et al., 1999; Pandey et al., 2017). Several scientists note that during a period of stress, the isoenzyme system is reorganized, which ensures the plant's resistance to external factors and the regulation of homeostasis (McRae and Ferguson, 1985; Castillo, 1992; Sharangi, 2009). For example, researches have demonstrated that the expression level of peroxidases and superoxide dismutase increased after the heat treatment of creeping plants (Khan and Robinson, 1993; Huang et al., 2006).

We were shown that after May's surge in growth processes in tea plants, there is a period of rest, in which the metabolic processes are somewhat slowed down. As a rule, in the future, in our zone there is a period under a stressful hydrothermal condition, which affects the functional state of plants; in particular, we can note an acute water deficit. This leads to the manifestation of oxidative stress, which is expressed in an increase in the activity of GPOD, as a nonspecific reaction to a stress factor (Mittler, 2002; Thongsook and Barrett, 2005). However, the dynamics of enzyme activity in a 3-leaf sprout are variety-specific.

We performed a correlation analysis that showed a direct relationship between increased enzyme activity and hydrothermal factors. The most significant correlation was found between the activity of GPOD in freshly harvested 3-leaf tea sprouts and the amount of precipitation (r = 0.86).

Tea sprouts are used to prepare a drink whose nutritional value is made up of substances formed both in the process of photosynthetic reactions and in the process of processing raw materials, the basis of which is redox enzyme reactions (Kareska, 2009; Skhalyakhov et al., 2019). In the process of technological processing of raw materials to ready-made the oxidases that ensure the normal course of redox processes are important. Thus, peroxidase catalyzes the oxidation of polyphenols and certain aromatic amines using oxygen, hydrogen peroxide, or organic peroxides.

Given this fact, a lower level of activity of GPOD is a negative phenomenon, both for the stability of the plant itself and for the quality of the finished tea (Wright, 2005; Huang et al., 2006).

Studies have shown depending on the season of tea leaf collection, the content of ruthin in green tea ranges from 36 mg.100g^-1 to 41 mg.100g^-1, and in black tea-in the range of 17 mg.100g^-1 – 20 mg.100g^-1, which is lower than in green tea. Thus, a lower content of ruthin is characterized by the cv. Sochi (on average 10 mg.100g^-1 in black tea and 34 mg.100g^-1 in green), a higher-form 582 (about 23 mg.100g^-1 in black tea and 46 mg.100g^-1 in green).

Ruthin, namely tea catechin, is very significant for human health. In the absence of it, the permeability of capillary blood vessels increases, and their strength decreases (Vinson, 2000; Sinija, Mishra, 2008; Sharangi, 2009; Ferreira et al., 2020). When acting together with vitamin C (with which it is often found in food), the latter is better absorbed and retained in the body and more successfully prevents scurvy (Skotnicka et al., 2011). Ruthin is an equally important component of the antioxidant system of tea plants, it not only takes part in the main redox reactions but also increases the absorption of ascorbic acid (Gulati and Ravindranath, 1996). The content of ruthin in black and green tea changed as the content of ascorbic acid. The ruthin on average is 3 times more in green tea than in black (on average about 38.09 and 12.12 mg.100g^-1 respectively). At the same time, the variability of the indicator during the season is important: a more stable content of vitamin P in tea forms 2264, 3823, 582, and 855. A variation was revealed in the content of vitamin C and P depending on genotypic characteristics (Figure 4 and Figure 6).

We identified 11 AAs in Krasnodar tea, a large proportion of which is proline (30 – 70% of the total AAs content in the sample), valine (17 – 30%), and serine (about 10%) (Figure 7).

AAs are the component in tea that contributes full-bodied flavor and sweetness. Of these AAS, more than 60% is theanine, which is unique to tea. Theanine has a structure similar to that of glutamine, with its particular trait being a refined, rich flavor and sweetness (Karori et al., 2007). AAs other than theanine present in tea leaves include proline, asparagine, arginine, serine, and others (Kumar et al., 2015).

We were shown that the largest number of AAs is synthesized in May, and in the future, the processes of their transformations and mutual conversions to other compounds are probably included in the synthesis.

It is necessary to note a strong variability in the content of AAs, and only Pro is stable enough (V, % = 4 – 26% during vegetation). When processing raw materials into readymade tea, the number of AAs falls, which is due to oxidative deamination and their conversion into protein compounds that are involved in the formation of tea aroma.

Most of the AAs in the raw material are contained in the cv. Kolchida and Sochi. During processing, there are not only general changes – there are slightly fewer AAs in black than in green, which is understandable since it is during the fermentation of black tea that active processes of oxidative deamination take place. There are varietal differences in the metabolic reactions of converting AAs: in the black tea the AAs composition of the cv. Kolchida is more susceptible to changes, while in green tea, the composition of form 2264 changes a little bit.