Inactivation of polyphenol oxidase in Camellia sinensis for the production of high quality instant green tea

Abstract

A concerning situation has developed over the past few years where several tea estates had to close down due to high labour costs and low profitability. Solutions are desperately required to save these estates from further regression and to prevent others from joining their ranks. One solution is to redirect the tea factories from the current production of black tea to producing a value added commodity such as a high quality green tea extract with an increased market value. The aim of this study was to find an economically viable PPO inactivation method that can be implemented in existing tea factories for the production of high quality instant green tea. Further enhancement of quality may then be achieved by high throughput cultivar screening where those with a higher natural catechin to caffeine content will be favourable. Six different PPO inactivation methods (steaming, blanching, fluid bed drying, panfrying, grilling over direct heat and grilling over indirect heat) were explored. Four independent experiments were performed in duplicate with these six methods using fresh tea leaves donated by a tea estate in Tzaneen, South Africa. All samples were dried in a fluid bed drier and milled after PPO inactivation. Biochemical analysis of specific quality parameters followed where extractions from these green tea leaf samples were tested for theaflavin content, caffeine content, flavan-3-ol content, total free amino acid content, colour and taste. The six PPO inactivation methods were compared by data evaluation of the individual quality parameters where certain quality parameters carried a higher weight than others. For the purpose of this project, the catechin to caffeine ratio was the most important quality determinant to yield a high value IGT. Also, to prove effectiveness of the PPO inactivation method, low TF content was compulsory. All PPO inactivation methods explored proved successful in rapidly inactivating PPO enzyme. As hypothesised, a significant difference in biochemical composition is brought about between green teas produced by employing different PPO inactivation methods. Blanching was found to be the most efficient PPO inactivation method as well as the method resulting in the highest catechin to caffeine ratio (16.67:1 for 4 min blanch vs. 5.72:1 for 17 min FBD as determined by HPLC analysis). A freeze dried extract from a 1.5 minute blanched sample (IGT) was compared with foreign IGTs originating from Sri-Lanka, Kenya, China and India by HPLC analysis. A more than two fold greater catechin to caffeine ratio was obtained for the sample originating from the blanching method (9.08:1 vs. 2.81-5.02:1). A high quality, naturally low caffeine, instant green tea can therefore be produced by utilizing the blanching method of PPO inactivation. To allow for inter and intra cultivar screening, tentative identification of novel catechins (digallated catechins) and their HPLC retention times was done using HPLC-ESI-MS/MS. Potential HPLC retention times for EC-digallate were detected at tR 62.0 ± 0.2, 70.7 ± 0.1 and 76.7 ± 0.2 minutes while tR 64.6 ± 0.1 and 65.8 ± 0.1 minutes were detected for EGC-digallate. With this information at hand, cultivars of a higher quality, hence increased economical potential, can be identified upon confirmation by NMR. HPLC-ESI-MS/MS screening coupled with NMR confirmation is to be continued to detect several other novel flavan-3-ols that could not be detected in the 4 IGTs of different origin used in this study. This study gives an overview of the biochemical differences between green tea leaves prepared using six different PPO inactivation methods. The aim of this study was met by identification of the significant increase in quality brought about by PPO inactivation using the blanching method, which is also economical for use in Africa. Blanching of tea leaves caused a significant decrease in caffeine. Therefore, the 1st hypothesis, stating that the six polyphenol oxidase inactivation methods investigated will produce instant green teas with different catechin to caffeine ratios, is accepted. Also, a means to perform large scale screening of individual tea trees in Africa for their novel flavan-3-ol content was provided by tentative identification of these novel catechins by LC-MS. Thus, the 2nd hypothesis, stating that application of LC-MS will aid in the identification of HPLC retention times of compounds (novel catechins) from a crude extract, is also accepted.