Comparison of the antioxidant capacities of teas
The results of our studies show that EPR method based on a new semi-empirical mathematical model is an adequate to analyze the antioxidant activity of food products.
Based on the mathematical model, the total antioxidant capacity TEAC was determined (Table 2) for each of the studied teas.
No total antioxidant capacity TEAC values determined by EPR spectroscopy were found in the literature. However, TEAC values obtained in this work are congruent with the values determined through the use of other research methods [1, 10, 23]. TEAC values found in literature varied between 143 and 3100 (µmol Trolox/100 mL) for black tea [1, 10, 16, 21, 24], between 480 and 6200 (µmol TE/100 mL) for green tea [16, 17, 21] and between 728 and 1205 (µmol Trolox/100 mL) for earl grey tea [17]. All TEAC values obtained in the present study, for all tea types, are located within the lower limits of values given in the literature. Results of the total antioxidant capacity TEAC values obtained for black, green and earl grey teas from individual manufacturers were analyzed (Table 2). This analysis allowed to determine differences in the antioxidant properties of teas depending on their production method. Green tea was the best DPPH free radical scavenger, with average TEAC value amounting to 495 ± 157 (μmol/1 g). Black tea was characterized by lower antioxidant activity, with average TEAC value equaling 335 ± 34 (μmol/1 g), and the lowest was exhibited by earl grey tea, with an average TEAC value of 248 ± 59 (μmol/1 g) (Table 2). Only for manufacturer P6, a higher TEAC value was noted for black tea as compared with green and earl grey tea (Table 2).
In general, the obtained results confirm the higher TEAC value of unfermented green tea than of fermented black tea, which is in line with data found in literature [12, 13, 25], although some studies indicate a reverse trend [15] or lack of differences in the antioxidant properties of teas manufactured via different technological processes [1, 23]. In case of earl grey tea, earlier studies indicated a smaller polyphenol content compared to green and black tea [23], which can be explained by a lower total antioxidant potential of earl grey tea, despite the bergamot oil content, renowned for its antioxidant properties [26].
Significant variances in the TEAC values obtained in individual groups (green, black and earl grey tea) depending on the manufacturer are also worth mentioning. In case of green tea, this variance amounts up to 417.1 (μmol/1 g). Such large discrepancies in the TEAC values indicate that not only the production method (fermentation or its absence), but also individual technological processes of eliminating the natural moisture from the tea leaves (so-called wilting), twisting the leaves, duration and method of conducting fermentation, duration and temperature of drying, utilized by different manufacturers, as well as the origins of the tea itself, can determine the antioxidant properties of the product which reaches the consumer.
NMR spectroscopy
Catechins, which are most abundant in green teas, are believed to present the greatest impact on the antioxidant activity of tea infusions. Along with an increase in the catechin content comes an increase in the antioxidant activity [16, 17]. The significantly higher content of catechins in green tea, as compared to black tea, was confirmed by NMR studies, which were conducted for each tea studied. Individual signals were assigned based on data from literature [27, 28]. The differences are apparent especially in the aromatic proton range, where in the 1H NMR spectra obtained for green teas, signals were observed at 6–6.1 ppm, 6.5–6.6 ppm and 6.8–6.9 ppm, confirming the presence of EGC, EGCG, ECG and EC in green tea; whereas in the black tea and earl grey tea spectrum, the signals in this area have significantly lower intensity and can originate from theaflavins (TF), formed from catechins at the fermentation stage during black tea manufacturing (Fig. 2). Moreover, the signal at 7.1 ppm, which is evident in black and earl grey tea’s spectra, indicates enriching the tea with gallic acid during the fermentation process.
The obtained NMR spectra thus confirm that the fermentation process changes the antioxidant content of tea both in terms of quantity as well as quality. There are known studies which show that the proton content in aromatic groups correlates with the antioxidant capacity of food products [3, 6]. An analysis of the NMR spectra of the samples tested in this study also confirms this thesis. Green tea, characterized by the highest average TEAC value, also provided the highest H
ar content values (Table 2). The observed decrease in H
ar aromatic protons occurred in the following order: green tea > black tea > earl grey tea. The correlation factor between the aromatic proton content and the antioxidant capacity value is 0.92 for TEAC measured in (μmol/100 mL of infusion) and 0.89 for TEAC measured in (μmol/1 g of tea), which indicates a strong positive correlation between these quantities (Fig. 3).
Influence of additives on the antioxidant properties of tea
In order to determine the influence on the tea of commonly used additives, antioxidant properties were tested for black tea B1 with addition of: honey, lemon juice, milk, and sugar. The additives themselves—lemon juice and honey—were also tested. These additives were used in quantities that are normally used while drinking tea. Due to the reduction of ascorbic acid in high temperatures, examining the impact of lemon juice on the antioxidant properties of tea was conducted after adding lemon juice to both hot and cold infusions. The TEAC results obtained for the tea with additives were within the range from 515.7 (μmol/100 mL of infusion) for hot tea with added lemon juice, to 668.6 (μmol/100 mL of infusion) for tea with added honey (Table 3).
Table 3 The TEAC values obtained for B1 tea with additives and TEAC values for honey and lemon juice
Among the additives tested, only honey significantly impacted the total antioxidant capacity TEAC values. The other additives contributed to only a slight decrease in the TEAC values, within the margin of error. The lack of influence of sugar on the antioxidant properties of tea is not surprising, as white sugar does not display any antioxidant properties. This is confirmed by both the results of the tests conducted in this study, as well as of studies conducted using the volumetric method [29]. Individual reports show an insignificant effect of sugar on antioxidant values and demonstrate insignificant antioxidant amounts in some varieties of refined and unrefined sugar [29].
Lemon juice, which is known for its antioxidant properties, as well as the ascorbic acid found in lemons, are one of the most popular antioxidants [30]. The decrease in the antioxidant properties of tea after adding lemon juice may therefore seem surprising. The TEAC for lemon juice is 452.3 (μmol/100 mL) (Table 3), which explains a slight decrease in the antioxidant properties of tea after adding lemon juice. It thus turned out that a black tea infusion is a better antioxidant than lemon juice. It is worth noticing that the TEAC value for the infusion with added lemon juice was higher after cooling than the TEAC value of the infusion where lemon juice was added while it was still hot. This is most likely caused by the process of thermal degradation of ascorbic acid.
Tea is often consumed with addition of milk, so an analysis of its influence on antioxidant values appears interesting. The addition of milk, similarly to sugar and lemon juice, did not have a significant influence on the antioxidant properties of tea. It has been determined that there are non-covalent interactions between polyphenols contained in tea infusions and polar proteins found in milk. These interactions may influence the antioxidant potential of the polyphenols found in black tea. The results of the research conducted to date are ambiguous. Some studies indicate a decrease in antioxidant potential of tea after adding milk [31], others signal an increase in the antioxidant value [32]. There are also studies known where no change in the antioxidant properties of tea was observed after adding milk [33].
The only studied additive to significantly impact the antioxidant properties of tea (Table 3) was honey. The antioxidant properties of honey are mainly determined by flavonoids: (chrysin, pinocembrin, pinobanksin, quercetin, kaempferol, luteolin, galangin, apigenin, hesperetin, myricetin), phenolic acids (caffeic, coumaric, ferrulic, ellagic, chlorogenic), ascorbic acid, catalase, peroxidase, carotenoids and products of the Maillard reaction contained in honey [34, 35]. The amount of these components varies considerably depending on the type of flower and the geographical origin of honey [34, 36]. Moreover, the processing and storage methods may affect the composition of honey, and subsequently its antioxidant properties [35].