INTRODUmIONThe maceration of green tea shoots during black tea manufacture results in the enzymic oxidation of catechins and their subsequent conversion to theaflavin (TF) and thearubigin (TR) compounds (Roberts 1958; Roberts and Myers 1959). Together these represent up to 30% of the dry weight of black tea, and their colour and astringency are major factors affecting the brightness, depth of colour, mouthfeel and overall quality of plain black teas (Hilton and Ellis 1972).

Reverse-phase HPLC of liquors prepared from a Lattakari Assam black tea was used to assess the contribution of black tea phenolic pigments to 'tea cream ', a precipitate which forms routinely following caffeine complexation of the phenols when a strong infusion of black tea cools. In this study caffeine was added back to tea liquor fractions and reconstituted decaffeinated whole tea liquor, and the extent to which the theaflavins, thearubigins and flavonol glycosides were removed from solution in the form of insoluble complexes was monitored by HPLC. The poorly characterised thearubigins are shown to be the principal ( % 86 %) pigmented phenolic components of 'tea cream'. Theaflavins ( % 12 YO) and flavonol glycosides ( x 2%) were also present. Evidence is presented for a synergistic interaction between theaflavins and thearubigins during cream formation. The use of caffeine precipitation is proposed as an aid to the fractionation and quantification of phenols including thearubigins in black tea liquors.

An enzymic in-vitro model tea fermentation system has been used in combination with gradient elution reversed-phase HPLC to study the oxidative breakdown of black tea theaflavins in the presence and absence of ( -)-epicatechin. It was found that theaflavin, theaflavin-3-monogallate, theaflavin-3'-monogallate and theaflavin-3,3'-digallate were not substrates for tea polyphenol oxidase (EC 1 . 14.18.1) whereas (-)-epicatechin alone, or mixtures of (-)-epicatechin and a theaflavin were rapidly converted to a complex mixture of thearubigin-like substances. While the chromatographic profiles varied with the identity of the theaflavin precursor each resembled the equivalent profile obtained with a whole black tea liquor. It is concluded that theaflavins are degraded by a coupled oxidation with ( -)-epicatechin-quinone. It is suggested that thearubigins may form via these pathways during black tea manufacture.

An enzymic in-vitro model tea fermentation system has been used in combination with gradient elution reversed-phase high-performance liquid chromatography to study the formation from individual flavan-3-01s of resolvable and unresolvable thearubigin-like substances. It has been found that in 30 min at pH 5.6 tea polyphenol oxidase (EC 1.14.18.1) produces a distinctive range of resolvable thearubigin-like products from each flavan-3-01 substrate examined (( +)-catechin, ( -)-epicatechin, (-)-epigallocatechin, ( -)-epicatechin gallate and (-)-epigallocatechin gallate). However, (-)-epicatechin, and to a much lesser extent ( +)-catechin, were the only flavan-3-01s converted to an unresolvable thearubigin-like hump under these conditions. In addition, it was demonstrated that varying the pH of incubation in the range 5.0-6.0 had little effect on the profile of products obtained from the simple catechins, but a pronounced effect in the case of the gallocatechins. It is concluded that the combination of (-)-epicatechin and polyphenol oxidase is the simplest model of tea fermentation that can be used to generate in quantity thearubigin-like materials for structure elucidation.

A previously published method for the HPLC analysis of the anthocyanidin pigments produced by the autoxidative depolymerization of proanthocyanidins by heating in a solution of n-butanol/HCl (95/5 v/v) and iron(II1) salts (Porter's reagents) has been refined. The pigments are detected by monitoring at 530 nm, but by monitoring the eluate also at 280 nm and 380 nm a wider variety of products resulting from the same autoxidation and/or hydrolysis/ transesterification reactions can be detected. Such products have been shown to include aglycones (particularly flavonols from flavonol glycosides) and n-butyl gallate (from galloyl esters). The advantages of monitoring the wider range of transformation products are illustrated by applying the method to the characterisation of two heterogeneous thearubigin fractions isolated from black tea liquor. The caffeine-precipitatable thearubigin fraction contained 181 g proanthocyanidins kg-' (as procyanidin dimer equivalents), a trace ( 2 2 g kg-') of flavonols (as aglycones), 74 g kg-' galloyl ester and 5.6 g kg-' elemental N (determined by microanalysis): caffeine was not detectable and some 74% of the fraction remains uncharacterised. The theafulvin fraction contained 105 g proanthocyanidins kg-' (as procyanidin dimer equivalents), c 1 g kg-' quercetin and 71 g kg-' galloyl ester, but neither caffeine nor elemental N was detectable; some 82% of the fraction remains uncharacterised. The proanthocyanidins of both hump fractions contained not only prodelphinidins and procyanidins but also propelargonidins which have not previously been reported in tea.