Prebiotic potential of green and black tea phenolics: Metabolite and microbiota profiling, and green biorefinery for sustainable production

Tea is the world’s most commonly consumed beverage after water. Besides its widely appreciated flavour and taste, its popularity is also due to various health-promoting effects, which are mainly attributed to its high content of phenolic compounds. Therefore, sustainable production and high-value application of tea phenolics is of great industrial interest. Implementation of green biorefinery strategies aiming at sustainable production of tea phenolics can be facilitated by the valorisation of agricultural waste streams and the utilization of emerging energy-efficient extraction technologies. The first aim of this thesis was to evaluate the potential of using fresh old tea leaves as a renewable source of tea phenolics, and to explore the use of pulsed electric field assisted extraction as a novel sustainable method of obtaining phenolics from tea leaves. As a second aim, the prebiotic potential of tea phenolics will be investigated. Comprehensive understanding of the metabolic fates of tea phenolics upon fermentation by human gut microbiota, and the gut microbiota modulatory effects of tea phenolics is essential for the further application of tea phenolics as prebiotics in food and pharmaceutical industries. With regards to this, the reciprocal interactions between monomeric and dimeric flavan-3-ols, and human gut microbiota were investigated in this thesis as well. 

In Chapter 1, the current knowledge on the phenolic compounds in green and black tea, the green biorefinery strategy for tea phenolics extraction, and their prebiotic effect were summarised. Green tea contains green tea catechins (GTCs), which are mainly monomeric flavan-3-ols, whereas black tea mainly contains oxidatively coupled black tea phenolics (BTPs), which includes dimeric, oligomeric, and polymeric compounds. Furthermore, the differences in gut microbial metabolism and gut microbiota modulatory effects between GTCs and BTPs were compared. To simplify this comparison, epigallocatechin-3-gallate (EGCG) was selected as the representative compound of GTCs, whereas theaflavin-3,3′-digallate (TFDG) and theasinensin A (TSA) were selected as the representative compounds of BTPs. 

The detailed profiling of the phenolic composition of old tea leaves (OTL) is an essential first step to enable the valorisation of this waste stream. In Chapter 2, the phenolic composition of OTL was studied and compared with that of young tea leaves (YTL), by using untargeted UHPLC-Q-Orbitrap-MS analyses. Principal component analysis illustrated distinct differences in overall phenolic profiles between OTL and YTL. Of all the detected phenolic compounds, 14 phenolic compounds, including rutin, epigallocatechin, epicatechin, EGCG, epicatechin gallate, and epiafzelechin gallate, underwent the most extensive quantitative changes upon leaf aging. Degalloylation of flavan-3-ols commonly occurred during leaf aging. Overall, after aging of the leaves, we observed a 1.7- and 3.0-fold decrease in flavanols and phenolic acids, respectively, and a 1.5-fold increase in flavonols. 

Conventionally, a thermal dehydration procedure, which is a time and energy intensive process, is performed prior to phenolics extraction from fresh tea leaves. In Chapter 3, we proposed to utilize pulsed electric field (PEF) to replace the conventional thermal dehydration procedure as pre-treatment before extraction. The optimized PEF conditions required an energy input of 22 kJ/kg and induced a temperature increase of 1.5 °C. Compared to oven drying, PEF pre-treatment increased the extraction rate by approximately 2 times, without significantly altering the phenolic profiles of the extracts. Scanning electron microscopy imaging revealed that PEF pre-treatment induced the formation of inhomogeneously distributed pores and protuberances on the surface of leaf tissues, which might facilitate the penetration of extraction solvent and allow the migration of phenolics out of the cell.  

Due to the poor absorption of tea phenolics in the small intestine, it has been suggested that the interactions of tea phenolics and gut microbiota may play a crucial role in health benefits of tea phenolics. In Chapter 4, metabolic fate of EGCG and its impact on gut microbiota were integrally investigated via in vitro fermentation. As revealed by UHPLC-Q-Orbitrap-MS, EGCG was promptly degraded into a series of (hydroxylated) phenylcarboxylic acids, through consecutive ester hydrolysis, C-ring opening, A-ring fission, dehydroxylation, and aliphatic chain shortening. Microbiome profiling indicated that EGCG stimulated beneficial bacteria Bacteroides, Christensenellaceae, and Bifidobacterium, and inhibited the pathogenic bacteria Fusobacterium varium, Bilophila, and Enterobacteriaceae. Furthermore, changes in concentrations of metabolites including 4-phenylbutyric acid and phenylacetic acid, were strongly correlated with changes in abundance of specific gut microbiota.  

TFDG, which is characterized by its benzotropolone moiety, is a representative black tea phenolic. In Chapter 5, we investigated the microbial metabolic fate of TFDG and its gut microbiota modulatory effect in comparison with EGCG. Despite the similar flavan-3-ol skeletons, TFDG was more slowly degraded and yielded a distinctively different metabolic profile. Theanaphthoquinone was discovered to be the main microbial metabolite of TFDG. Additionally, a number of (hydroxylated) phenylcarboxylic acids were formed, albeit at low concentrations compared to EGCG metabolism. Despite the distinctively different metabolite profiles found for EGCG and TFDG, microbiome profiling demonstrated several similarities in gut microbiota modulatory effects. This included growth promoting effects on Bacteroides, Faecalibacterium, Parabacteroides, and Bifidobacterium, and inhibitory effects on Prevotella and Fusobacterium.  

TSA, a dimer of EGCG with a biphenylhexaol moiety, is also a representative black tea phenolic. In Chapter 6, the microbial metabolism of TSA by human gut microbiota was investigated. Purified TSA was incubated with human faecal microbiota, and EGCG and procyanidin B2 (PCB2) were used for comparison. After degalloylation, the core biphenylhexaol structure of TSA remained intact during fermentation. Conversely, EGCG and PCB2 were promptly degraded into a series of (hydroxylated) phenylcarboxylic acids. Computational analyses comparing TSA and PCB2 revealed that TSA’s stronger interflavanic bond and more compact stereo-configuration might underlie its lower fermentability.  

In Chapter 7, the results obtained in previous chapters were put into perspective in an integrated discussion. Firstly, the sustainable production of tea phenolics from old tea leaf biomass was evaluated. For further valorisation of OTL, tea phenolic extraction should be followed up with extraction of proteins, carbohydrates, and lignocellulose. To this end, a green biorefinery framework was proposed. Secondly, we discussed the differences in metabolic fates of pure tea phenolic compounds of EGCG, TFDG and TSA. Additionally, the microbial metabolism of crude extracts of green tea and black tea was compared. Overall, our findings indicated that dimeric tea phenolics had lower fermentability by human gut microbiota, resulting in reduced degradation rate and lower yield of downstream metabolites of (hydroxylated) phenylcarboxylic acids. Thirdly, we discussed the prebiotic potential of GTCs and BTPs, and based on the results of Chapter 5, we concluded that both GTCs and BTPs possess prebiotic properties. Moreover, we argued that tea phenolics fit well within the recently updated definition of prebiotics, due to their reciprocal interactions with gut microbiota. The higher fermentability of GTCs suggests that green tea possesses more pronounced prebiotic properties than black tea and may, thereby, also have superior health benefits.  

In conclusion, we proposed an integrated green biorefinery strategy for tea phenolic production, and illustrated their potential application as novel prebiotics.