Development and application of a fast gas chromatographic method offer new insights into L-theanine production regulation in Camellia sinensis L

Authors: CHEN, YANNI - Hunan Agricultural University; LIU, SHUOQIAN - Hunan Agricultural University; Ferreira, Jorge; XIAO, LIZHENG - Hunan Agricultural University; GU, MEIYI - Hunan Agricultural University; LUO, YIPING - Hunan Agricultural University; ZHANG, TIANTIAN - Hunan Agricultural University; ZHANG, XIANGQIN - Hunan Agricultural University; LIU, ZHONGHUA - Hunan Agricultural University; HUANG, JIANAN - Hunan Agricultural University; TIAN, NA - Hunan Agricultural University

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/10/2021
Publication Date: 9/13/2021
Citation: Chen, Y., Liu, S., Ferreira, J.F., Xiao, L., Gu, M., Luo, Y., Zhang, T., Zhang, X., Liu, Z., Huang, J., Tian, N. 2021. Development and application of a fast gas chromatographic method offer new insights into L-theanine production regulation in Camellia sinensis L. Journal of Agricultural and Food Chemistry. 69(37):11142-11150. https://doi.org/10.1021/acs.jafc.1c04093.
DOI: https://doi.org/10.1021/acs.jafc.1c04093

Interpretive Summary: Theanine is only produced by tea plants and some mushrooms and is responsible for the umami flavor, anti-stress properties, and market quality of tea. However, without precise scientific tools it is difficult to accurately measure theanine in commercial teas and tea plants. Tea has been consumed for 4,500 years and is currently cultivated in approximately 60 countries and fast, affordable, and reliable analytical methods are needed to establish its chemical composition and theanine concentration. We have developed and validated a fast, affordable, and reliable analytical method to quantify theanine from tea plants (leaves and roots) and commercial teas (White, Green, Yellow, Oolong, Black, and Dark).Theanine extraction and quantification were completed in 40 minutes. Roots produced the most theanine, followed by leaves of White tea and Green tea. Teas that had undergone little to no fermentation (White and Green) had the most theanine while teas that had undergone extensive fermentation (Oolong, Black, and Dark tea) had the least amount of theanine. We report for the first time that an amorphous tissue, represented by a conglomerate of cells (callus), can produce theanine at the same concentrations as leaves of tea plants. Theanine production in callus indicates that roots or leaves are not necessary for theanine production and open the possibility for the large-scale industrial production of theanine in bioreactors without the need to cultivate plants in the field. Theanine has pharmacological effects on the nervous system and is sold as an over-the-counter additive in the USA as a sleep aid, sometimes added to melatonin. This knowledge is important for the industrial production of theanine for human consumption but without the customary extensive use of land, water, energy, labor, and fertilizers.

Technical Abstract: Tea is the most consumed beverage worldwide, and l-theanine in tea leaves significantly affects their flavor and market quality. We have developed and validated a fast and reliable gas chromatographic method with flame ionization detection (GC-FID) to quantify l-theanine after its extraction from Camellia sinensis (tea plant) and derivatization. The procedure was completed in 40 min, from extraction to chromatographic analysis, with a recovery rate of more than 93% and allowing a high sample throughput. The GC-FID intraday precision was within 0.57–2.28%, while the interday precision ranged from 1.57 to 13.48%. The intraday accuracy ranged from -6.84 to 5.26%, while the interday accuracy ranged from -1.08 to 3.12%. The limit of detection was 2.28 µg/mL, and the limit of quantification was 6.47 µg/mL. The GC-FID method was validated by high-performance liquid chromatography with UV detection (HPLC–UV) and was used to investigate the biosynthesis and regulation of l-theanine in tea plants. We found that plants fed with ethylamine significantly increased l-theanine concentrations in roots, while exogenous supplementation of glutamic acid, carbamide, and glutamine did not significantly affect the l-theanine level in roots. Our results also indicated that roots were not indispensable for the biosynthesis of l-theanine, which was detected in undifferentiated embryonic calluses in concentrations (g/100 g dry weight) as high as in leaves of whole plants (1.67 and 1.57%, respectively) and without any exogenous theanine precursor supplementation.