Industrial Crops and Products 24 (2006) 8794

Effect of pulp preconditioning on the content of polyphenols in cocoa beans (Theobroma Cacao) during fermentation
R. Nazaruddin , L.K. Seng, O. Hassan, M. Said
School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia Received 17 February 2004; accepted 8 March 2006

Abstract Polyphenol components were extracted from Malaysian mix-hybrid cocoa beans from different treatments of fermentation namely; post harvest pod storage, bean spreading and pressing. The polyphenol compounds were analyzed using high pressure liquid chromatography. The ()-epicatechin, (+)-catechin, theobromine and caffeine of control treatment at 5 days pod storages were 11.87, 4.31, 21.08 and 3.85 mg/g, respectively. Pre-fermentation treatments were found to be signicant in affecting the changes in acidity, degree of fermentation and the polyphenol content of cocoa bean. During fermentation, all pre-treated samples showed decreased levels of ()-epicatechin and (+)-catechin but the rate of decrease were found to be different. It was also found that the ()-epicatechin and (+)-catechin content before and after fermentation was affected by the type of pre-fermentation treatment used. Percentage lost for ()-epicatechin and (+)-catechin during fermentation at different degree of fermentation ranged from 6 to 17% and 0.95 to 1.62%, respectively. It was observed that pod storage treatments until 15 days have signicant effect in the reduction of acidity, fermentation index (FI) and polyphenol contents compared to spreading and pressing methods. Recoveries (Rv) ranged from 90.90 to 93.92% with % coefcient of variances (CVs) of 6.51, 8.57, 2.42 and 6.37 for ()-epicatechin, (+)-catechin, theobromine and caffeine, respectively. This study indicates that 15 days pulp preconditioning is the optimum conditions for the degradation of ()-epicatechin and (+)-catechin, theobromine and caffeine. 2006 Elsevier B.V. All rights reserved.
Keywords: Acidity; Cocoa beans; Fermentation; Pod storage; Polyphenols content

1. Introduction Recently, polyphenols have gained much more attention, owing to their antioxidant capacity (free radical scavenging and metal chelating) and their possible benecial implications in human health, such as in the treatment and prevention of cancer, cardiovascular disease, anti-microbial and other pathologies (Agarwal and Mukhtar, 1996). Cocoa is rich in polyphenols particu-

Corresponding author. Tel.: +60 3 89213817; fax: +60 3 89213232. E-mail address: naza@ukm.my (R. Nazaruddin).

larly in catechins (avan-3-ols) and procyanidins. The polyphenols in cocoa beans are stored in the pigment cells of the cotyledons and cocoa leaves (Osman et al., 2004). Depending on the amount of anthocyanins those pigment cells, also called polyphenol-storage cells, are white to deep purple. Polyphenols in green and black tea, grape seeds, grapes and red wine have raised much attention but chocolate has not been investigated intensively 1998). up to now (ZumbeA, Polyphenol and alkaloids, which comprise approximately 1420% of the whole bean weight, are very important in affecting the quality of cocoa beans (Kim and Keeney, 1983). Three groups of polyphenols can

0926-6690/$ see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2006.03.013

88

R. Nazaruddin et al. / Industrial Crops and Products 24 (2006) 8794

be distinguished: catechins or avan-3-ols (ca. 37%), anthocyanins (ca. 4%) and proanthocyanidins (ca. 58%). The main catechin is ()-epicatechin with up to 35% of polyphenol content. In one study (Kim and Keeney, 1984), the ()-epicatechin contents ranged from 34.65 to 43.27 mg/g of defatted sample in freshly harvested Catongo and Forastero cocoa beans. In smaller amounts (+)-catechin as well as traces of (+)-gallocatechin and ()-epigallocatechin have been found. Nazaruddin et al. (2001) also report the total polyphenols ranged from 45 to 52 mg/g in cocoa liquor, 34 to 60 mg/g cocoa beans and 20 to 62 mg/g in cocoa powder. For ()-epicatechin contents, the avarage are 3.81 mg/g in cocoa powder, 2.53 mg/g in cocoa liquor and 4.61 mg/g in cocoa beans. The anthocyanin fraction consists mainly of cyanidin3--l-arabinosid and cyanidin-3--d-galactosid. Procyanidins are mostly avan-3,4-diols, that are 48 or 46 bound to condensed dimers, trimers or oligomers with epicatechin as the main extension sub-unit (Romanczyk et al., 1997). The total amount of soluble polyphenols in the dried fat-free mass of fresh cocoa beans is 1520% (equals approximately 6% in air dried cocoa beans, containing 54% fat and 6% water), in fermented beans approximately 5% (10% and more being considered a sign of a bad fermentation). These values are valid for Forastero beans, Criollo cocoa beans have approximately 2/3 of the amount of polyphenols, anthocyanins have not been found (Lange and Fincke, 1970). Reaction of polyphenol along with sugar and amino acids are responsible for the production of avor and color of the roasted cocoa beans, while alkaloids contribute bitterness to the fermented beans. It is an accepted fact that proper fermentation is a prerequisite for the formation of avor precursors (Lehrian and Patterson, 1983). Methods of fermentation determine the quality of products produced especially the avor. Previous studies on post harvest pod storage and bean spreading had shown marked improvement in chocolate avor and reduction in sourness, bitterness and astringency (Meyer et al., 1989; Biehl et al., 1990). In commercial production, similar effect was obtained through a combination of pod storage, pressing and air-blasting (Said et al., 1990). Variations in the condition during fermentation such as pod storage and duration will affect the pH, titrable acidity (TA) and temperature during fermentation, thus inuence the enzymes activities and avor development (Biehl et al., 1990). The free and condensed cocoa polyphenol are valuable antioxidants but the presence of these compounds and the xanthine alkaloids have been implicated for astringency and bitterness in cocoa and cocoa products (Kim and Keeney, 1983). Very little

researches have been done on the changes in polyphenol compounds and xanthine alkaloids during fermentation especially on pulp preconditioning methods. The presence of polyphenols in plants is dependent on several factors including degree of ripeness, variety, processing and storage (Porter et al., 1991; Hammerstone et al., 1999). This study will attempt to evaluate the effect of pulp preconditioning on the content of polyphenol compounds, ()-epicatechin and (+)-catechin as well as xanthine alkaloids (theobromine and caffeine) during fermentation. The information obtained from this study is essential in understanding and solving the problems associated with the quality of Malaysian cocoa beans. 2. Materials and methods 2.1. Samples Ripe cocoa pods of mixed-hybrids were obtained from Malaysian Cocoa Board, Lower Perak, Malaysia. Pod storage, bean spreading and pressing were carried out as reported earlier (Meyer et al., 1989). The fermentations varied according to the pod storage time, weight of beans used, the duration of the fermentation, the number turns and the timing of these turns. The pod storage time is the time that the pods were stored after harvesting but before splitting. Samples for analysis were taken at predetermined time and the pulp was removed by rubbing the beans on rough gunnysack. The beans were sun-dried until they reached moisture content of about 7%. Cocoa beans were stored in an air-conditioned room (2223 C) until used. All the chemical standards such as ()-epicatechin, (+)-catechin, theobromin and caffeine (purity: 99100%) were purchased from the Sigma Chemical Co. (St. Louis, MO). Milli-Q (Millipore, Bedford, MA, USA) double-distilled water (resistance = 18 mX) was used. All solvents and reagents were of American Chemical Society (ACS) grade or better. 2.2. Titrable acidity (TA) and pH TA and pH of the nibs were determined according to section 42.1.04, AOAC 16th ed., 1995. Five grams samples of nibs were homogenized for 30 s in 100 ml of hot distilled water and then vacuum ltered through Whatman lter paper #4. A 25 ml aliquot was pipetted into a beaker and the pH was measured using a pH meter (model MP230 Mettler Toledo MP230, Switzerland). A further 25 ml aliquot was titrated to an end point pH of 8.1 with 0.01N NaOH. The values reported as mmoles of sodium hydroxide per 100 g dry nibs.

R. Nazaruddin et al. / Industrial Crops and Products 24 (2006) 8794

89

2.3. Fermentation index (FI) FI were determined according to Gourieva and Tserrevitinov (1979). 0.5 g ground cocoa nibs was extracted with 50 ml of 97:3 mixture of methanol:HCl. The homogenate was allowed to stand in refrigerator (8 C) for 1619 h and then vacuum ltered. The ltrate was made up to volume in 50 ml volumetric ask. The fermentation index of the sample was obtained by calculating the ratio of absorbance at 460 nm to the absorbance at 530 nm (LKB Biochrom Novaspec II UV Spectrometer, UK). Three replicate readings were obtained for each sample. 2.4. Determination and quantication of standard ()-epicatechin, (+)-catechin, theobromine and caffeine The analysis was performed according to the method of Kim and Keeney (1983), using a Waters Symmetry C18 column (4 m particle, 3.8250 mm), with the eluent comprising of 87% water:8% methanol:5% acetic acid, owing at 0.8 ml/min (Jasco 980 HPLC Pump) and detected using a Waters 484 Variable wavelength detector, set at 280 nm. All solvents (HPLC Grade) and samples were ltered through a nylon 0.45l membrane. Quantitative analysis was carried out by using the individual standard curves for each type of catechins and methylxanthines. Recovery (Rv) studies were carried out on a cocoa extracts containing specic amounts of standards. Analyses were carried out in duplicate before and after the addition of standards. 2.5. Statistical analysis Statistical signicance was determined using analysis of variance (ANOVA) and were compared by the Duncans multiple range test using SAS version 6.12 (SAS Institute, Cary, North Carolina). Signicance was accepted at 5% level (p 0.05). 3. Results and discussion 3.1. Reproducibility and recovery for determination of theobromine and caffeine compounds The HPLC method used was capable of resolving all compounds of interest. Fig. 1 showed the chromatogram for theobromine and caffeine compound that were separated in the mobile phase. The result showed both types of methylxanthines analyzed gave a good separation resolution. Chromatogram I is the result of

cocoa beans sample extraction analysis. Co elution peak a with standard theobromine can be seen clearly in chromatogram III. Peak b was identied as caffeine through the spiking test in chromatogram II. The reproducibility of the extraction step was calculated by measuring the peak height on six different extracts of the same cocoa samples, compared to the internal standard. Table 1 shows the reproducibility and recovery of the extraction method in determining theobromine caffeine, ()-epicatechin and (+)-catechin. Recoveries ranged from 89.11 to 94.65% with %CVs of 6.51, 8.57, 2.42 and 6.37 for ()-epicatechin, (+)-catechin, theobromine and caffeine, respectively. This is an indication of good reproducibility of the extraction method used. As for the recovery, the method used also gave a relatively high percentage of recoveries (>90%). Overall, the data collected from this study show the extraction method by Kim and Keeney (1984) is reliable. 3.2. Regression equations and coefcient of determinations (r2 ) The regression equations and r2 s for the standards curves of different methyxanthine compounds are presented in Table 2. The r2 -values were very good for caffeine (0.9951) and ()-epicatechin (0.9917) standards. However, the r2 for theobromine, (+)-catechin and total polyphenol were only 0.980, 0.979 and 0.981, respectively. 3.3. pH, Titrable acidity and fermentation index (FI) Table 3 and Fig. 2 showed the pH and titrable acidity of cocoa nib from different days of pod storage fermentation. As seen in Table 3, the pH at 0 day was extremely higher which is 6.5 then slowly decreased for 34 days, until the pH reached at 4.4. This is due to during fermentation processes, the adhering pulp becomes liquid and under anaerobic conditions micro-organisms will produce acetic acid and ethanol. These processes will inhibit germination of seeds and contribute to structural changes in fermented beans such as the removal of enzymes and substrates. Cell liquids move across cell walls and are spread all over the cocoa nib. This occurs generally after 2448 h of bean fermentation. By the third day, the mass of beans will have been fairly evenly heated to 45 C and will remain between this temperature and 50 C until the fermentation is complete. Thus, it is necessary to mix the beans occasionally for aeration and to ensure that those being initially in the outside of the heap are exposed to the temperature in the interior (Kim and Keeney, 1984; Lange and Fincke, 1970).

90

R. Nazaruddin et al. / Industrial Crops and Products 24 (2006) 8794

Fig. 1. HPLC chromatogram of cocoa bean extracts (chromatogram I), chromatogram II: standard of caffeine (b), chromatogram III: standard of theobromine (a).

This trend corresponds well with the one noted in TA (Fig. 2). Changes in TA follow a decreasing of pH trend for all pod storage treatments. Overall, 15 days pod storage produced cocoa beans with high pH 6.0. The value of pH was found to be greater than the standard Malaysian estate beans, which is 4.44.7. TA, which is a better indicator of acidity than pH, showed an expected trend of steadily decreasing over time in all the treatments. Fresh beans (0 day fermentation) for pod storage were found to be high in its TA, conrming to current Malaysian cocoa industry problems of high acidity in beans. After 1015

day pod storage, the beans were seen to have less acidity than other treatments. Besides the decreasing of TA during fermentation period, other polyphenol compounds such as anthocyanins will hydrolysed to anthocyanidins. The latter compounds will polymerise along with simple catechins to form complex tannins. Anthocyanins usually disappear rapidly during fermentation process. Thus, anthocyanin content has been considered as a good index for determination of the degree of cocoa bean fermentation (Lange and Fincke, 1970). Data from FI can be used

Table 1 Effect of defatting treatment on the recoveries (Rv) and coefcient of variance (CV) of cocoa beans Sample Theobromine Rv (%) Cocoa bean 94.65 CV (%) 2.42 Caffeine Rv (%) 93.54 CV (%) 6.37 ()-Epicatechin Rv (%) 90.96 CV (%) 6.51 (+)-Catechin Rv (%) 89.11 CV (%) 6.57

R. Nazaruddin et al. / Industrial Crops and Products 24 (2006) 8794 Table 2 Regression equations and coefcient of determinations (r2 ) Concentration of standards (ppm) Standards of methylxanthin compounds (% peak area) Theobromine 5 10 20 30 40 50 60 80 100 120 200 300 400 500 y r2 153.74 190.97 300.13 343.35 422.72 0.2839x 0.1117 0.98 Caffeine 32.69 60.45 88.02 127.86 152.45 0.6488x + 0.1426 0.9951 (+)-Catechin 20.08 28.14 42.22 63.93 78.26 1.2861x + 0.1618 0.9785

91

()-Epicatechin 79.02 145.74 200.73 304.03 38.48 1.2926x + 0.8864 0.9917

to monitor the degree of fermentation of cocoa beans. Fig. 3 showed means data of FI from different days of pod storage fermentation. It has been suggested that fermented beans with FI values of 1 and below, indicate under fermentation (Gourieva and Tserrevitinov, 1979). As fermentation progressed, more and more condensation products of anthocyanins, such as cyanidin3--d-galactosid and cyanidin-3--l-arabinosid were formed, as shown by increasing absorbance at 460 nm but decrease absorbance at 530 nm (Kim and Keeney, 1983). In term of pH, the decreasing trend was noted in all the different treatments. Fig. 3 also shows a rapid change in FI was observed during the rst 3 days of fermentation, which subsequently slowed down since the condensation product became less soluble. Overall, pod storage of more than 1015 days were seen to have FI

value of more than 1. Compared to other treatments, this treatment is capable of producing well-fermented beans. 3.4. Polyphenols content in cocoa beans during fermentation The effects of pulp preconditioning on the content of polyphenolic compounds and xanthine alkaloids are shown in Table 4 (catechin) and Fig. 4 (epicathechin), Fig. 5 (theobromine) and Fig. 6 (caffeine). Generally, except for (+)-catechin and caffeine, the pulp preconditioning caused reduction in the content of polyphenolic compounds and xanthine alkaloids in the beans. However, the effect of 5, 10 and 15 days pod storage on the content of (+)-catechin and theobromine was not

Table 3 Effect of pod storage fermentation on the pH and titrable acidity of cocoa beans Days of fermentation Days of pod storagea 0b pH 0 1 2 3 4 5
a b

5b TA 0.01b 0.01a 0.01c 0.08c 0.02c 0.08b,c 0.03 0.04 0.20 0.32 0.33 0.11 0.00a 0.01a 0.01a 0.00a 0.01a 0.01a pH 6.64 5.78 5.53 4.76 4.51 5.28 0.00a 0.06c 0.08a 0.01b 0.04b 0.11c TA 0.02 0.05 0.07 0.16 0.23 0.09 0.00a 0.01a 0.00c 0.00b 0.01b 0.01b

10b pH 6.67 5.89 5.58 4.61 4.68 5.44 0.04a 0.07b 0.04a 0.10b 0.05b 0.23b TA 0.03 0.05 0.08 0.23 0.17 0.09 0.00a 0.01a 0.01c 0.04b 0.00b 0.01b

15b pH 6.66 6.01 5.40 5.56 5.70 6.03 0.01a 0.01a 0.21b 0.24a 0.02a 0.09a TA 0.02 0.05 0.10 0.09 0.08 0.05 0.00a 0.00a 0.01b 0.01c 0.00c 0.00c

6.54 6.08 4.73 4.33 4.35 5.31

Values are expressed as mean standard deviation of two determinations. Means in the same column with the same letter are not signicantly different (p 0.05).

92

R. Nazaruddin et al. / Industrial Crops and Products 24 (2006) 8794

Fig. 2. Relationships between pod storage fermentation and the total TA in cocoa beans. TA of cocoa beans were measured by titration of 0.01 N NaOH: ( ) 0 day pod storage (control); ( ) 5 days pod storage; ( ) 10 days pod storage; ( ) 15 days pod storage.

signicant (Table 4 and Fig. 5). Earlier study on the effect of pod storage on the content of ()-epicatechin, theobromine and caffeine from different clones also demonstrated almost the same pattern (Clapperton et al., 1992). This observation suggested that the xanthine alkaloids are not involved in the physiological changes in the beans during fermentation. The slightly lower content of ()-epicatechin, (+)-catechin and theobromine in pressed beans as compared to spread beans may be due to loss as exudates during pressing. During fermentation of cocoa beans, polyphenols diffuse with cell liquids from their storage cells and undergo oxidation to condensed high molecular mostly insoluble tannins. These reactions are both non-enzymatic and catalysed by the enzyme polyphenol oxidase, even though this enzyme is strongly inactivated during the rst days of fermentation. The occurrence of condensation reactions is conrmed by the sharp decrease of epicatechin content between

Fig. 3. Interaction between pod storage fermentation and the index of fermentation in cocoa beans. The fermentation index of the sample was obtained by calculating the ratio of absorbance at 460 nm to the absorbance at 530 nm: ( ) 0 day pod storage (control); ( ) 5 days pod storage; ( ) 10 days pod storage; ( ) 15 days pod storage.

the second and fth day of fermentation (Fig. 5). Epicatechin and catechin content, respectively, is reduced to approximately 1070% during fermentation. This is not only due to the oxidation process but also caused by diffusing of polyphenols into fermentation sweatings (Kim and Keeney, 1984). The changes in (+)-catechin during fermentation are shown in Table 4. The reduction in pulp volume might facilitate the oxidation and polymerization of ()-epicatechin and its oxidation products as clearly demonstrated by the beans from 15-day pod storage. The reduction in this catechin will lower the level of astringency in the cocoa beans. Previous work by other

Table 4 Effect of pod storage fermentation on the (+)-catechin content (mg/g) in cocoa beans Method of fermentation Control 5 days PS 10 days PS 15 days PS Spreading Pressing
a

Days of fermentationa 0 10.54 9.60 9.46 9.48 16.18 12.39 0.49 0.28 0.21 0.28 0.39 2.28 1 9.76 8.36 6.82 5.33 12.65 11.11 0.66 0.26 0.15 0.13 0.91 0.61 2 7.61 6.60 5.73 0.76 9.02 7.04 0.01 0.08 0.33 0.01 0.06 0.04 3 5.20 5.66 5.41 0.66 8.43 3.79 0.06 0.19 0.26 0.14 0.15 0.74 4 4.97 5.15 3.45 0.59 6.39 1.33 0.43 0.25 0.46 0.05 0.25 0.12 5 4.31 1.76 1.16 0.49 1.88 0.94 0.31 0.20 0.02 0.04 0.15 0.03

Values are expressed as mean standard deviation of two determinations.

R. Nazaruddin et al. / Industrial Crops and Products 24 (2006) 8794

93

Fig. 4. ()-Epicatechin content (mg/g) in cocoa beans during different type of fermentation. The analyses were carried out using a Waters Symmetry C18 column (4 m particle, 3.8250 mm), with the eluent comprising of 87% water:8% methanol:5% acetic acid, owing at 0.8 ml/min (Jasco 980 HPLC Pump) and detected using a Waters 484 Variable wavelength detector, set at 280 nm: ( ) 0 day pod storage (control); ( ) 5 days pod storage; ( ) 10 days pod storage; ( ) 15 days pod storage; () spreading; ( ) pressing.

Fig. 6. Caffein content (mg/g) in cocoa beans during different type of fermentation. The chromatographic conditions are shown in Fig. 3: ( ) 0 day pod storage (control); ( ) 5 days pod storage; ( ) 10 days pod storage; ( ) 15 days pod storage; () spreading; ( ) pressing.

researchers also show similar trend (Meyer et al., 1989; Biehl et al., 1990). A similar trend was observed for (+)catechin. The slower rate of degradation of (+)-catechin (2 days after fermentation) might be due to inactivation of polyphenoloxidase as a result of polymerization. The effect of pulp preconditioning on the changes of theobromine and caffeine was not very clear. This might be due to the fact that the loss of these compounds during fermentation was by exudation to the testa and not by chemical degradation.

4. Conclusions Pod storage caused a signicant reduction in the content of polyphenol compounds especially ()epicatechin, thus will reduce the astringency and bitterness in cocoa and cocoa products. The degradation of ()-epicatechin and (+)-catechin during fermentation was also inuenced by the pulp preconditioning. This study indicates that 15 days pulp preconditioning is the optimum conditions for the degradation of ()epicatechin and (+)-catechin, theobromine and caffeine. However, the effect on the content of xanthine alkaloids was not signicant, suggesting that the reduction during fermentation was mainly due to exudation.

Fig. 5. Theobromine content (mg/g) in cocoa beans during different type of fermentation. The chromatographic conditions are shown in Fig. 3: ( ) 0 day pod storage (control); ( ) 5 days pod storage; ( ) 10 days pod storage; ( ) 15 days pod storage; () spreading; ( ) pressing.

94

R. Nazaruddin et al. / Industrial Crops and Products 24 (2006) 8794 Kim, H., Keeney, P.G., 1983. Method of analysis for ()epicatechin content in cocoa beans by high performance liquid chromatography. J. Food Sci. 48, 548551. Kim, H., Keeney, P.G., 1984. ()Epicatechin content in fermented and unfermented cocoa beans. J. Food Sci. 49, 10901092. Lange, H., Fincke, A., 1970. Kakao und Schokolade. In: Acker, L., Bergner, K.-G., Diemair, W. (Eds.), Handbuch der Lebensmit` tel Band VI: Alkaloidhaltige Genussmittel, GewuErze, Kochsalz. Springer-Verlag, New York, Berlin, Heidelberg, pp. 210309. Lehrian, D.W., Patterson, G.R., 1983. Cacao fermentation. In: Rehm, H.J., Reed, G. (Eds.), Biotechnology, 5, p. 529. Meyer, B., Biehl, B., Said, M.B., Samarakoddy, R.J., 1989. Post harvest pod storage: a method of pulp preconditioning to impair strong nib acidication during cocoa fermentation in Malaysia. J. Sci. Food Agric. 48, 285304. Nazaruddin, R., Ayub, M.Y., Mamot, S., Heng, C.H., 2001. HPLC determination of methylxanthines and polyphenols levels in cocoa and chocolate products. Malays. J. Anal. Sci. 7 (2), 377386. Osman, H., Nazaruddin, R., Lee, S.L., 2004. Extracts of cocoa (Theobroma cacao L.) leaves and their antioxidation potential. Food Chem. 86, 4145. Porter, L.J., Ma, Z., Chan, B.G., 1991. Cacao procyanidins: major avonoids and identication of some minor metabolites. Phytochemistry 30 (5), 16571663. Romanczyk, L.J., Hammerstone, J.F., Buck, M.M., Post, L.S., Cipolla, G.G., Micceland, C.A., Mundt, J.A., Schmitz, H.H., 1997. Cocoa extract compounds and methods for making and using the same. Patent Cooperation Treaty (PCT) WO 97/36497, Mars incorporated, USA. Said, M.B., Jayawardena, M.P.G.S., Samarakoddy, R.J., Perera, W.T., 1990. Preconditioning of fresh cocoa beans prior to fermentation to improve quality: a commercial approach. Planter 66, 332345. A., 1998. Polyphenols in cocoa: are there health benets? ZumbeA, BNF Nutr. Bull. 23, 94102.

Acknowledgments The authors are very grateful to the Universiti Kebangsaan Malaysia and the research ofcers in Malaysian Cocoa Board, Lower Perak Station especially to Mr. Bakri Tukimon for the kind cooperation during this study. References
Agarwal, R., Mukhtar, H., 1996. Cancer chemoprevention by polyphenols in green tea and artichoke. In: Back, N., Cohen, I.R., Lajtha, D., Lajtha, A., Paoletti, R. (Eds.), Dietary Phytochemicals in Cancer Prevention and Treatment. Plenum Press, New York, pp. 3550. AOAC, 1995. pH of acidied foods. Section 42.1.04, method 981.12. In: Cunniff, P.A. (Ed.), Ofcial Methods of Analysis of AOAC International, 16th ed. AOAC International, Gaithersburg, MD, pp. 24. Biehl, B., Meyer, B., Said, M.B., Samarakoddy, R.J., 1990. Bean spreading: a method of pulp preconditioning to impair strong nib acidication during cocoa fermentation in Malaysian. J. Food Agric. 51, 3545. Clapperton, J., Hammerstone, J.F., Romanczyk, L.J., Yow, S., Chan, J., Lim, D., Lockwood, 1992. Polyphenols and cocoa avour. In: Proceedings of the Meeting of Group Polyphenol, JIEP 92, Lisbon. Gourieva, K.B., Tserrevitinov, O.B., 1979. Method of evaluating the degree of fermentation of cocoa beans. USSR Patent no. 646254. Hammerstone, J.F., Lazarus, S., Mitchell, A., Rucker, R., Schmitz, H., 1999. Identication of procyanidins in cocoa (Theobroma cacao) and chocolate using high-performance liquid chromatography/mass spectometry. J. Agric. Food Chem. 47, 490496.