J Sci Food Agric 199569,467-474

The Tea

Phenolic - . Liquors- Pigment -Part I :

Composition of Black Predicting Quality*

Ian McDowell, Sarah Taylor? and Clifton Gay Natural Resources Institute, Central Avenue, Chatham Maritime, Chatham, Kent ME4 4TB, UK (Received 16 December 1994; revised version received 28 April 1995; accepted 4 July 1995)

Abstract: The phenolic constituents of 95 black teas, representing nine countries, were analysed and assessed by HPLC. The procedure resolved 38 coloured phe- nolic components which were classified as theaflavins (4). thearubigins (23) and flavonol glycosides (1 1). The relative levels of these constituents were monitored by HPLC. One of the thearubigin constituents was unresolved chromato- graphically, but nevertheless quantified as a hump. The data set was statistically analysed using principal component analysis (PCA) and multiple regression. PCA indicated that three of the quality attributes, quality/j?auour, brightness/ briskness and quality with milk, were strongly correlated with each other. The fourth attribute, colour/strength, was found to be the most effective in discrimi- nating between teas, particularly between high strength teas from Assam and Tanzania and low strength teas from Sri Lanka and Malawi. Multiple regression was used to investigate the relationship between tasters’ scores and the chemical constituents. The model established gives an R2 of 63% based on six com- ponents; five resolved thearubigins and the unresolved thearubigin hump. These results indicate the potential to develop objective chemical assessment pro- cedures for determining the quality of teas and is the first report of the flavour impact of individual thearubigins constituents.

Key words: black tea, quality, phenolic,

INTRODUCTION

Tea has a complex and unique phenolic composition which is responsible for many aspects of its flavour and value. Black tea is produced from the fresh shoots of the tea bush, followed by a series of processing stages to produce the familiar dried leaf (Robertson and Owuor 1993). The most important stage in terms of the phenol- ic composition is the fermentation. This is more accu- rately described as a complex of enzymically stimulated oxidation reactions in which the most important is the conversion of fresh leaf catechins to theaflavins (TF) and thearubigins (TR) (Roberts 1962; Sanderson 1972; McDowell and Taylor 1993). These substances ate only formed in black tea manufacture. The analysis of these products remains a formidable analytical challenge, not least, because the TR appear to be chemically diverse and remain substantially uncharacterised.

* Part of this paper was presented at the SCI Food Commod- ities & Ingredients Group symposium Recent Advances in the Chemistry of Tea held at the University of Surrey on 24 March 1994.

To whom correspondence should be addressed.

thearubigin, HPLC, PLA

Tea producers require objective procedures to enable them to assess quality in a systematic way. This would enable information to be obtained which would allow producers to improve tea quality and value systemati- cally, rather than relying on intuitive approaches and ad hoc solutions to quality problems. Objective methods have to be based on an assessment of the chemical com- position of tea and this study attempts to address the two major problems associated with devising suitable methods. The first is that the unique flavour character of tea relies substantially on the phenolic composition of the leaf which has greater complexity and diversity than any other beverage. The second is that the market assessment and value of tea is carried out by highly trained tasters, who, over the years, have developed a specialised vocabulary for tea flavour not readily acces- sible to untrained assessors.

The phenolic composition of tea remains a formida- ble analytical challenge. However, this report utilises a HPLC separation procedure (Bailey et a1 1991) devel- oped to resolve as many of the phenolic components of tea as possible. Bailey et a1 (1991) separated 29 TR and devised a classification system based on their UV-Vis

467 J Sci Food Agric 0022-5142/95/%09.00 0 1995 SCI. Printed in Great Britain

468 I McDowell, S Taylor, C Gay

TABLE 1 Summary of origins of the 95 teas analysed

~~

Kenya Tanzania Assam Malawi Sri Lanka Bangladesh Zimbabwe South Africa Uganda

Total

20 7

27 5 7

18 1 2 8

95

spectra. This procedure is used here to aid the charac- terisation of any TR which are particularly important for quality. In addition, all the teas have been assessed by professional tasters and the results quantified. This allows comparisons between the chemical composition of the teas and the qualities considered to be of particu-

lar importance by the trade. It was decided to use multi- variate statistical procedures due to the success of this approach in discovering relationships in large date sets.

EXPERIMENTAL

Samples

Ninety-five commercial teas (Table l), representing nine countries, were obtained from a London tea broker (George White & Co; now Thompson Lloyd & Ewart Ltd) after tasting and scoring by the company. The quality attributes selected, in discussion with the brokers, were colourlstrength, brightnesslbriskness, qualitylflaoour and quality with milk. These are the main attributes used by the international tea trade to assess quality and hence the value of black teas.

The scoring system was on a scale of 1-10 for each of three at tributes (brisknesslbrightness, colourlstrength and Javourlquality) and on a scale of 1-20 for quality with milk, giving a total possible score of 50.

3

E a

10 2 0 3 0 40 T i m e ( m i n . )

l c3 2 0 3u 4 0 T l m e ( m i n . )

Fig 1. HPLC chromatograms of an Assam tea. (a) Chromatogram at 380 nm with the 38 phenolic substances annotated. TF, theaflavin; TR, thearubigin, and FG, flavonol glycoside. (b) Chromatogram at 460 nm with annotation of the theaflavins and the

thearubigins which have most significance for quality.

Phenolic pigment composition o j black tea liquors-Part I 469

TABLE 2 Classification of thearubigin substances’

Component Retention Classijicat ion time (min)

TR 1 TR2 TR3 TR4 TR5 TR6 TR7 TR8 TR9 TRlO TR11 TR12 TR13 TR14 TR15 TR16 TR17 TR18 TR19 TR20 TR2 1 TR22

5.26 7.43 8.06 8.78 9.75 9.90

10.47 11.81 13.77 15.14 15.73 16.22 2560 27.10 30.74 32.27 33.52 34.10 31.49 39.18 40.00 41.35

Type I1 Type I Type II Type I Type 11 Type II Chlorogenic acid Type 11 Type 11 Type I1 Type 11

Type 11

Type I

Type I1

Theaflavic acid Theaflavic acid

Theaflavin Theaflavic acid Theaflavin Theaflavic acid Type 11 Type II

a Based on the UV-Vis spectra classification of Bailey et a1 (1991).

a m p l e preparation

Boiling water (100 ml) was poured over 4 g of tea and infused for 10 min at 80°C. The infusion was vacuum filtered through Whatman No 5 filter paper and the

TABLE 3 Identification of theaflavins

Component Retention Classification time (min)

TF 1 41.04 Theaflavin TF2 43.68 Theaflavin-3-gallate TF3 45.25 Theaflavin-3’-gallate TF4 45.57 Theaflavin-3,3’digallate

extract was placed in a refrigerator at 6°C for 5 min before injection into the HPLC system.

HPLC equipment and conditions

The equipment consisted of an ACS 350/04 Ternary pump (ACS, Macclesfield, UK), a Rheodyne 7010 injec- tor (Jones Chromatography, Cardiff, UK) fitted with a 20 p1 loop and a Pye Unicam PU 4020 UV-Vis detec- tor (Philips Analytical, Cambridge, UK) set at 380 nm. The column and mobile phase conditions are described elsewhere (Bailey et a1 1991).

Statistical analysis

Statistical analysis was carried out on the integrated peak areas of the 38 selected phenolic components and the taster’s scores using a Statgraphics software package (Statistical Graphics Corporation, Rockville, USA). Stepwise multiple regression analysis was undertaken using an F ratio of 4.0 as an exclusion-inclusion cri- terion for all potential regressors.

TABLE 4 Probable identification of flavonol glycosides”

Component Comment Retention time (min)

Classijication

FG 1 18.30 Myricetin rhamnosylglucoside FG2 Merged 18.92 Myricetin galactoside FG2 Merged 18.92 Myricetin glucoside FG3 19.86 Quercetin glucosylrhamnosylgalactoside FG4 FG FG5 FG6 FG7 FG8 FG9 FGlO FG FG11

20.37 Not determined 21.56

23.05 23.71 24.25 2544 27.69 27.98

Not determined 28.60 29.42

Quercetin glucosylrhamnosylglucoside Kaempferol glucosylrhamnosylgalactoside Quercetin rhamnosylgalactoside Quercetin galactoside Quercetin glucoside Kaempferol glucosylrhamnosylglucoside Kaempferol galactoside Kaempferol rhamnosylglucoside Quercetin rhamnoside Kaempferol glucoside

’ Based on the HPLC retention time order of Engelhardt et al(l992).

470

2.3

1.3

0.3

Component 2

-0.7

I McDowell, S Taylor , C G a y

-

-

- .

- \ t \

-1 .7

-2.7

-3.7

-2.8 -0.8 1.2 3.2 5.2

Component 1

Fig 2. Plot of the loadings of the tasters' attributes on the first two principal components, explaining 91 % of the variability.

RESULTS AND DISCUSSION

Phenolic classification

A total of 38 coloured black tea phenolic constituents were resolved and monitored by HPLC using the pro- cedure of Bailey et a1 (1991). A typical chromatogram at 380 nm is shown in Fig 1 with peak annotation as T F (TF1 to 4), resolved TR (TR1 to 22) and flavonol glyco- sides (FG1 to 11). The retention times and identity or

classification of these peaks are listed in Tables 2-4. The chromatographic conditions used in this study (Bailey e t al 1991) have resulted in a marked improvement in resolution of the substances of interest when compared to the results used in a previous statistical study (McDowell et a1 1991). In the previous study, TR could not be confidently allocated to unknown peaks, in con- trast to this study, where 22 chromatographically resolved TR have been classified. Resolved TR can be further classified on the basis of spectra using the scheme devised by Bailey et a1 (1991) and the results are reported in Table 2. In addition, quantification of a polymeric TR fraction (Bailey et a1 1991, 1992) has been carried out. Black teas generally show a convex broad band or 'hump' underneath the resolved peaks. This was quantified by finding the total area under both the hump and resolved peaks, and subtracting this value from the sum of the resolved peaks integrated on a valley to valley basis. This is similar to the procedure used by Powell et a1 (1992).

McDowell et a1 ( 1 990), Bailey e t a1 ( 1 990) and Cattell and Nursten (1977) have demonstrated that many of the constituents assumed to be TR are FG. On this basis, some of the peaks of the chromatograms (Fig 1) were assigned as FG in addition to the T F and TR. The assignments were based on the UV-Vis spectra gener- ated by a diode array HPLC detector (Bailey et al 1990, 1991) and the retention time order (Engelhardt et a1 1992).

Statistical analysis

Principal component analysis ( P C A ) PCA was carried out on the four tasters' attributes to determine whether relationships exist between tasting

TABLE 5 The highest correlation coellicients ( r ) between individual substances and tasters' attributes significant at the 5% level

~ ~ ~ ~ _ _ _ _ _ _ _ ~

Total score Brisknesslbrightness Colourlstrength Qualityljavour Quality with milk

TR2 TR12 TF2 TR Hump TF4 TR15 TR21 TF3 TR 14 TR18 TR19 TR16 FG4 TR8 TR17 TR5 TR9

0.65 0.58 0.57 0.50 0.49 0.47 0.47 0.45 0.42 0.40 0.40 0.39 0.34 0.33 0.33 0.32 0.32

TR2 TR12 TF2 TF4 TR21 TR Hump TR19 TR15 FG4 TR6 TR 14 TR5 TR17 TR9 TR18 TR8 FG7

0.57 TR2 0.56 TR12 0.47 TF4 0.42 TR15 0.40 TR21 0.40 TF2 0.39 TR14 0.37 TR18 0.37 TR19 0.33 TF3 0.33 TR Hump 0.3 1 TR5 0.3 1 TR4 0.30 TR16 0.30 TR7 0.29 TR13 0.27 TR11

0.59 TR2 0.51 TR2 0.59 TR12 0.51 TF2 0.57 TR15 0.42 TF3 0.57 TF2 0.41 TR Hump 0.56 TR Hump 0.41 TR12 0.55 TR9 0.39 TR17 0.52 TF4 0.38 TR16 0.49 TR16 0.34 TR21 0.48 TR19 0.34 TF4 0.45 TR8 0.31 TFl 0.45 FG4 0.31 TR15 0.43 TR18 0.30 TR8 0.42 FG2 0.30 TR9 0.38 TR6 0.29 TR14 0.33 TR14 0.29 TR18 0.31 FGS 0.29 FG7 0.30 FGl 0.28 TR3

0.54 0.5 1 0.42 0.42 0.40 0.38 0.36 0.36 0.35 0.32 0.3 1 0.30 0.30 0.30 0.30 0.29 0.27

Phenolic pigment composition of black tea liquors-Part I 47 1

TABLE 6 Between substance correlation coefficients (r) , at the 5% level,

of the best five substances taken from Table 5

T R 2 T R 1 2 T F 2 T R Hump T F 4

TR2 1.00 0.47 0.81 0.37 0.38 TR12 0.47 1.00 0.29 0.26 0.82 TF2 0.81 0.29 1.00 0-45 0.43 TR Hump 0.37 0.26 0.45 1 -00 0.36 TF4 0.38 0.82 0.43 0.36 1 .oo

terms. The results are presented in Fig 2 which is a plot of the loadings of the tasters attributes for the first two components. It is immediately apparent that three of the attributes (quality/flavour, quality with inilk and brisknesslbrightness) are strongly related attributes for the tasters’ used in this experiment as they make up the majority of component 1. Component 2 appears to be mainly made up of colourlstrength.

Correlation analysis Correlation analysis was carried out with all the sub- stances against the tasters’ attributes and the results for

the substances giving correlation coefficients of 0.3 and above are reported in Table 5. Looking at the total tasting scores, the highest correlations were found with the two TR substances, TR2 and TR12, which also have the best correlations for almost all individual attributes. TF2 and TF4 are also highly significant, having the third and fifth highest correlations for the total tasting scores. The TR Hump gives the fourth highest score. TR2 gave the highest correlation coefficient (r = 0.65) for a single substance, representing an R 2 of 42%, when compared to total score. The FG do not appear to be major contributors to quality as the highest correlation coefficients achieved are only r = 0.37 and 0-34 using FG4.

Table 6 shows the correlation relationships between the five substances discussed above. It can be seen that TR2 and TF2 give an r of 0.81 and TR12 and TF4 given an r of 0.82 indicating that these two pairs of sub- stances are strongly correlated to each other. Possible explanations are that their production during fermenta- tion maybe biochemically linked and/or their is flavour synergy between them.

The five substances were also analysed individually with the tasters attributes by PCA to ascertain how the chemical loadings aligned relative to the attributes in

TABLE 7 Models produced by stepwise multiple regression with adjusted R 2 values in bold

Total Brightness1 ColourJ Quality1 Quality with score briskness strength flavour milk

All 0.66 substances

TR2 TR4 TR16 FG6 TR19 TR Hump TR12

TR 063 TR2 TR4 TR16 TR19 TR Hump TR12

TF 0.44 TF 1 TF2 TF3 TF4

FG 0 1 1 FG4

055

TR4 FG7 TR12

055 TR 1 TR17 TR7 TR22 TR Hump TR12

029 TF2 TF3 TF4

013 FG4

065

TR2 TR16 TR20 TR2 1 TR13

065 TR2 TR16 TR20 TR21 TR13

043 TF2 TF4

004 FG4

059

TR20 FGlO TRlO TR Hump TR12 FG 1 FG2

046 TR4 TR9 TR Hump TR12 TR13

031 TF1 TF3 TF4

020 FG2 FG3 FG4

0.44

TR2 FGll TR17 TR Hump FG2

0.45 TR2 FGl1 TR17 TR7 TR Hump

025 TF2

0.07 FG7

472 I McDowell, S Taylor, C Gay

the Fig 2 plot. All these components, including the TF, were more closely aligned with the colour/screngch vector than the other attributes.

Stepwise mulciple regression analysis Stepwise multiple regression analysis was then carried out in order to determine the combination of substances which best explain flavour quality. This method enables between substance interactions and synergy between substances to be taken into account. The results are shown in Table 7. Using all the substances, an adjusted R2 of 66% was obtained for the tasters' total score with a model containing seven substances (six TR and one FG). The other score relationship, with an adjusted R2 of above a%, is colourlstrength at 65% (the model con- tains five TR). This indicates that the colourlstrengrh attribute can be best explained by the TR confirming the interpretation discussed in the correlation section. When TR substances are analysed alone by stepwise multiple regression, high adjusted R 2 values of 63% for total score and 65% for colourlstrength are obtained. The regression plot for the former model is shown in Fig 3. The total score value of 63% is explained by the loss of the single flavonol glycoside (FG6) from the 66% obtained from the 'all substances' relationship. The same value of 65% obtained for colour/screngch with 'all substances' and with the TR is explained by both models using the same five TR.

When the T F were analysed alone by stepwise multi- ple regression, their contribution appears considerably weaker than the TR, the highest R 2 values being 44% and 43% with total score and colourlstrength, respec- tively. But care must be taken as the correlation analysis indicates that TF2 and TF4 are highly corre- lated with TR2 and TR12 and there are only four T F for the stepwise multiple regression procedure to select from compared to 22 TR. The low R 2 value of 29% obtained with brightnesslbriskness is surprising as the T F are considered to be the main determinant of this unique characteristic of tea. The TR give a much better relationship with brightnesslbriskness, having a R2 value of 55%. This could be explained by some of the resolved T R having T F characteristics due to the presence of a benzotroplone ring structure. The theaflavic acids are well known, but their degree of contribution to brightnesslbriskness has not been ascertained. Table 2 indicates that four TR have theaflavic acid type spectra (TR 14, TR 15, TR 18 and TR20). However, in the selec- ted models of Table 7, only TR20 is identified as impor- tant for colourlstrength and quality/flaoour, the remaining three substances not appearing in the models. Two TR are 'theaflavin-like' (TR17 and TR19) due to the similarity of their UV-Vis spectra to the four main T F (TFl, TF2, TF3 and TF4) (Bailey et a1 1991). In Table 7, TR17 appears to be important for brightness1 bri:kness and quality with milk and TR19 only appears

45 I- /

,/ 40

/

35

observed

30

25

20

15

15 20 25 30 35 40 45

predicted Fig 3. Stepwise multiple regression plot observed versus actual values using six thearubi in substances (TR2, TR4, TR12, TR16,

TR19 and TR Hump) with total score giving an adjusted R ! of 63%.

Phenolic pigment composition of black tea liquors-Part I 473

TABLE 8 Six thearubigins selected by the stepwise regression model

with the classification based on Bailey et a1 (1991)

Substance Classification

TR2 TR4 TR12 TR16 TR19 TR Hump

~

Type I Type I

Type I Type I1

Theaflavin

in the total score models. But neither substance was selected for the final model. The six substances selected for the best overall model by stepwise regression are presented in Table 8 with their classification. I t can be seen that three of the five resolved TR are type I pig- ments, the other two being a Type I1 and a ‘theaflavin- like’. The chromatogram in Fig l(b), monitored at 460 nm, is annotated with the TR selected by the model. The flavonol glycosides contribute very little to the above models.

The TR are produced during manufacture and are considered to be products of the oxidation of catechins during the fermentation stage. Their exact structures remain unknown but investigations are ongoing which may reveal structural details. In overall terms they are considered to contribute to body, strength and colour in tea liquors, but with their diversity, 23 TR monitored in this study, their flavour is likely to be more subtle.

CONCLUSIONS

These results indicate the potential to develop objective chemical assessment procedures for tea and is the first detailed report of the flavour impact of chromato- graphically resolved TR constituents. In a previous study (McDowell et a1 1991), the TR were not con- sidered due to analytical constraints. However, since then improvements in the separation of the TR were reported (Bailey et a1 1991) which allowed this study to resolve 23 TR and to assess their levels in a range of teas. Six TR were selected as a model which explained 63% (R2) of the relationship between the tasters’ scores and chemical composition. One of the six TR in the model was the chromatographically unresolved ‘hump’ which is considered to be relatively high molecular weight polymeric material (Bailey et a1 1992). This is the first report which determines this fraction in relation to quality. It was found to be important enough to be included in the six TR model developed in this study. The results indicate the TR have a greater impact on quality and flavour than the TF and FG. However, two of the resolved T R selected were found to be highly cor- related with two TF which indicates that the impact of

T F may be underestimated in this study and that these T F could be used in the model.

As the model has substantially reduced the number of substances that require analysis in order to predict quality, this could be used as a basis for future work to develop rapid methods of assessing quality which would be of practical use to the trade. TR2 and TR12, which are the individual substance with the highest corre- lations with quality could be targets for the develop- ment of rapid solid phase extraction/spectrophoto- metric methods, particularly as they are also correlated with two TF.

It is highly recommended that further work is carried out in order to confirm the results reported here as the results are dependent on the assessment of one firm of tasters. The variation between individual or groups of tasters has not been assessed under controlled condi- tions and possibly could be large. However, the results indicate that further progress could be made under con- trolled sensory assessment conditions to validate objec- tive procedures for using chemical composition for quality assessment.

ACKNOWLEDGEMENT

The authors wish to thank Mr David Panter of Thomp- son Lloyd & Ewart Ltd for obtaining and evaluating the teas used in this study.

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