**4. Discussion**

The advantage of our study is that four active constituents of tea were measured using HPLC in one tea type (oolong) and changes relative to GABA-enrichment were documented. Further, our method of sample preparation was appropriate for determining constituent content in a cup of tea prepared according to usual consumption practices and in our studies of effects on a student cohort. In future, testing these constituents across a range of commercially available products would be valuable for a wider comparison of different tea types. Choice of oolong tea for the current study was based on mid-range fermentation. Analysis of constituents of GABA-enriched green or black teas will likely yield differing results.

Previous studies using HPLC have determined GABA to be present to varying extents in commercially prepared GABA-enriched teas, including 1560 mg/100 g [9]; 19 mg/100 g [6] and 272 mg/100 g [4]. The type of tea according to the degree of oxidation (e.g., oolong, black) was not specified in these studies. On the other hand, GABA concentration in freshly prepared GABA tea has been measured at between 30 and 700 mg/100 g (average 275 mg/100 g) using HPLC, with concentrations dependent on the method of preparation (aerobic vs anaerobic cycling, or both), and plant

*GABA-enriched Oolong Tea: Reducing Stress in a Student Cohort May Involve More than Just… DOI: http://dx.doi.org/10.5772/intechopen.106896*

#### **Figure 3.**

*Representative reverse-phase HPLC chromatograms of caffeine and EGCG in (A) regular oolong, and (B) GABA-enriched oolong teas.*

part used (leaves, buds or stem) [5, 7, 8, 23, 24]. These reported results compare with our finding of 40.2 mg GABA/100 g GABA-enriched oolong tea.

In non-GABA enriched (i.e., regular) teas, GABA quantities have been measured using HPLC, Zhao et al. [9] found the concentration of GABA in teas differed according to tea type. Relative concentrations from greatest to least were white (50.5 mg/100 g) > black (31.1–41.5 mg/100 g) > green (13.8–33.9 mg/100 g) > oolong (14.8–20.7 mg/100 g). On the other hand, Syu and colleagues [6] found the GABA content of different teas to be green (19.6–105 mg/100 g) equal to oolong (10–101 mg/100 g) > black (34–55 mg/100 g). In contrast to our findings of 5 mg GABA/100 g regular oolong tea, others have shown, on average, higher GABA concentrations in commercially prepared regular oolong teas.

The differences between GABA content determined in our study and findings from previous investigations may be explained by the fact that our teas were prepared according to manufacturer's instructions using simple aqueous extraction, as in making a cup of tea, without repeated extractions using different solvents and drying down to increase yield. In this way, we provide a realistic estimation of the concentration of GABA that one may encounter in a commonly prepared cup of tea.

Like GABA, theanine content measured across a range of commercial tea types is found to vary in concentration, and even within the different tea types, there is significant variation [6, 25, 26]. For example, commercially prepared white teas measured between 53 and 3337 mg theanine/100 g w/w [25] while oolong teas ranged from 85 to 282 mg/100 g [25], averaging 101 mg/100 g [6]. Our findings of 163.2 mg/100 g of theanine in a cup of regular oolong tea are in keeping with these previous studies. One study that prepared tea for analysis similar to the method used in the present investigation (brewing for a specified period of time in 200 mL water) found a standard (200 mL) cup of black tea contained 24.2 mg L-theanine, white tea contained 11.5 mg, while a cup of green tea contained the least theanine at 7.9 mg [27]. These values are lower than those that we report and may be related to the type of tea tested. Oolong tea was not investigated by Keenan and colleagues [27].

Syu and colleagues [6] measured theanine content in GABA-enriched tea and found it occurred in a higher concentration (198 mg/100 g) than regular teas on average. This contrasts with our finding that GABA-enriched tea measured half the theanine concentration (82.4 mg/100 g) of the regular oolong tea. On the other hand, Wang et al., [8] and Tsushida and Murai [7] found that GABA enrichment did not substantially alter theanine concentration compared to non-enriched teas. Moreover, theanine content in black teas was shown to be equivalent to green and oolong teas [25–27], therefore it appears to remain unaffected by oxidation.

Zuo [28] found caffeine concentrations were fairly stable across different commercial teas tested, including green (up to 99 mg/ 100 g), oolong (37–121 mg/100 g) and black (43 mg/100 g) teas. Roughly equivalent amounts of caffeine were also found across different teas [29–31]. Caffeine constituted 3.62% w/w of white tea, ~2.40% w/w of green teas tested (0.77–3.35% w/w), 2.77% w/w of oolong tea, and ~2.90% w/w of black teas (2.41–3.69% w/w) [29–31].

Compared with previous studies, we show higher concentrations of caffeine in the teas we tested: 267 mg/100 g dried tea leaf in regular oolong tea and 402 mg/100 g in GABA-enriched oolong tea. These differences in concentration may be explained by the method of tea sample preparation for HPLC analysis. Zuo [28] extracted tea samples in methanol and HCl then further diluted samples in water, focusing on efficient extraction. Wang and colleagues [8] dried samples, infused them in 80°C water for 20 minutes, while we infused tea samples directly in 90°C water for 10 minutes in accordance with manufacturer's instructions for consumption.

Caffeine content has previously been shown to be relatively unaffected by the cycling fermentation process involved in creating GABA-enriched tea, with no difference between freshly prepared green (3.2 mg/100 g) and GABA-enriched (3.3 mg/100 g) [8] However, we found variation in caffeine content in GABA-enriched compared with regular oolong tea.

Our findings show a lower concentration of EGCG (356 mg/100 g) in regular oolong tea compared to some previous studies, and levels near the lower limit of detection in GABA-enriched oolong tea (2.6 mg/100 g). For example, Tang and colleagues [32] found that EGCG varied between 2070 mg/100 g and 3670 mg/100 g across commercially prepared oolong teas tested. This may be due to sample preparation as well as tea source. Tang et al., [32] extracted samples with 10 mL tetrahydrofuran at 30 °C for 30 minutes, followed by 10 mL methanol:acetic acid:water

## *GABA-enriched Oolong Tea: Reducing Stress in a Student Cohort May Involve More than Just… DOI: http://dx.doi.org/10.5772/intechopen.106896*

(50:3.7:46.3, *v/v/v*) mixture at 30 °C for 30 minutes to obtain all soluble components. While this sample preparation technique may be more rigorous in extraction of different constituents, it does not represent common consumption practices. Thus, our findings provide a more realistic estimate of EGCG concentrations consumed in a cup of tea. The significant reduction in EGCG content in GABA-enriched compared with regular oolong tea observed in our study likely arose through the additional fermentation process required to increase GABA content in the tea.

There are many ways of increasing the levels of GABA in food and beverages. There is a wide range of GABA-enriched fermented food products [33], lactic acid fermented green tea being an example [34]. The GABA content of mulberry leave powder as a potential functional food ingredient has been increased by sodium glutamate immersion, cold shock and anoxia [35]. A different approach is being used in tomatoes, with CRISPR/Cas9 gene editing technology used to selectively increase GABA levels by deleting the autoinhibitory domain of the enzyme that converts glutamate to GABA [36]. GABA levels in such gene-edited tomatoes increased by 11 to 18-fold accompanied by a drastic reduction in glutamate and aspartate levels. Sales of these tomatoes were launched onto the Japanese market in 2021[37]. Such gene editing technology can be applied to annual crops like tomatoes but would be difficult to readily apply to tea.

In conclusion, teas contain a range of bioactive constituents including GABA, theanine, EGCG and caffeine. GABA-enriched oolong tea was shown to have 8 times more GABA than regular oolong tea, although the quantity may not be sufficient alone to account for the stress-reducing effects of this GABA-enriched tea. It is very likely that additional constituents measured here contribute to the purported relaxant effects of GABA-enriched tea, possibly via interaction with the GABAergic system. The differences in GABA, theanine, caffeine and EGCG content between GABAenriched and regular oolong teas demonstrated here may arise through the additional fermentation process required to enrich GABA content in the tea. In particular, the increase in the amount of caffeine may be significant.
