Comparison of antioxidant activity and flavanol content of cacao beans processed by modern and traditional Mesoamerican methods
© Chin et al.; licensee Chemistry Central Ltd. 2013
Received: 4 February 2013
Accepted: 18 March 2013
Published: 5 April 2013
The use of cacao, in its familiar food and beverage form, dates back about 3,800 years. By the time of the discovery of America by Europeans, the Aztecs and Mayans had developed methods for drying, roasting and grinding cacao beans for use in beverages and foods. Today, the same processes are used, but have been adapted to modern machinery. The current study was conducted to compare the traditional Mesoamerican processing and modern processing methods and their impact on the antioxidant activity and flavan-3-ol content of the resulting processed cocoa mass. Two cocoa bean types were used: unfermented cocoa beans commonly produced and consumed today by the native peoples of Mesoamerica, and fermented Ivory Coast cocoa beans representing the most common country of origin for cocoa in the world today. Both bean types were processed by roasting and grinding using traditional and modern processing methods. The traditional method consisted of roasting whole beans on an earthenware comal heated with wood charcoal, de-shelling the beans, then grinding the beans to a paste using a stone metate and mano. The modern method consisted of roasting cocoa beans with pilot-scale equipment that simulates large-scale chocolate manufacturing using whole bean drum roasters, deshelling the beans followed by mechanical grinding using a ball mill producing a chocolate paste. The antioxidant capacity and flavanol content of these chocolate pastes were then determined. The data show that the antioxidant capacity and flavanol content of unprocessed Mexican Lavado cocoa beans were significantly higher than that of the unprocessed, fermented Ivory Coast cocoa beans. In unfermented Mexican Lavado cocoa beans, the traditional and modern processing revealed no clear advantage with respect to the antioxidant and flavanol contents. However, for fermented Ivory Coast beans, there were some differences in the level of flavanols between traditionally processed and modern processed fermented beans. These differences, while significant for some, but not all of the flavanol measures, were attributed to final roast temperatures achieved and to the possible lack of uniformity of bean fermentation.
Cacao originated and evolved in the tropical rainforests of the upper Amazon and Orinoco Rivers in South America . When fresh fruit of cacao, also known as cocoa or cacao pods, are harvested, the hard woody pod is broken open and the cacao seeds, better known as cocoa beans, with their surrounding white, sweet pulp, are revealed. Fresh cacao beans at this stage taste bitter and contain little or no cacao flavor [2–4]. The archeological evidence dating back to about 1900 to 1400 BC, indicates that cacao juice expressed from the sweet pulp was the first product of the cacao seed to be used . The archeological records suggest that the first consistent use of the brown, roasted cacao paste that was made into cocoa drinks, sauces and solid cocoa-containing foods dates back to about 1,000 BC . The forms of cocoa that we are familiar with today have these brown cocoa solids —chocolate liquor and cocoa powder—which are the ingredients of chocolate candy, cakes, cookies and hot cocoa beverages.
Cacao is known to contain very high levels of flavan-3-ols (flavanols) that occur both as monomers of epicatechin and catechin and as polymerized flavanols, or procyanidins [9–13]. The flavanols are commonly associated with cardiovascular health benefits which include lower rates of morbidity and mortality due to stroke and heart attack, improved vascular endothelial function, reduction in blood pressure and increases in HDL cholesterol [14, 15].
Studies have shown that flavanols are lost during transformation of cocoa beans into chocolate. Therefore, one can ask the compelling question regarding the level of these flavanols: Do traditional processing methods for roasting cacao beans have an advantage over the modern methods used today to produce roasted cocoa beans for making chocolate, perhaps because the process is more natural or not mechanized? The objective of this study is to compare the level of antioxidant capacity, flavanol monomers as well as oligomers and total flavanols in cacao beans processed by traditional Mesoamerican methods to the levels found in cocoa beans processed by modern methods. Here we have studied two types of cacao beans: unfermented cacao beans from southern Mexico, locally known as Lavado beans (lavar, in Spanish, meaning to wash) and conventionally fermented Ivory Coast beans. Beans of each type then were processed in a modern pilot plant to simulate industrial processing and were compared to beans prepared by the traditional Mesoamerican method which included comal roasting, hand winnowing and stone grinding on a metate.
Material and methods
Cocoa bean samples
Traditional processing method
Cocoa beans were roasted using methods that resembled traditional, ancient processing and typical of those still used today in Central America for local use. A 50-cm diameter earthenware comal was obtained from a Mexican market and was supported by several bricks. The comal was heated from beneath with charcoal (Figure 1). Once the center of the comal reached 250–300°C surface temperature as measured with a RayTek Mini-Temp Model MT6 laser thermometer (Fisher Scientific), 250 g of whole, dried cocoa beans were placed on the comal and heated. The beans were stirred occasionally with a spatula to avoid burning the outer shell. When beans developed a characteristic cacao aroma, they were heated for another three to four minutes, removed from the comal and cooled. Final external bean temperatures were 125–130°C as measured by a RayTek laser thermometer. The shells of roasted beans were fractured into small pieces using a stone mano and removed by hand or winnowed by sifting and blowing away the lighter shells from the heavier cocoa nibs. Shell-free nibs were ground using a stone metate and mano purchased from Melissa Guerra Cookware (Figure 2; bottom). The metate was kept slightly warm by charcoal underneath. The nibs were ground several times to produce a smooth, melted paste with a glossy appearance. Several batches were processed, combined and three aliquots were taken for analysis.
Modern processing method
Modern industrial roasting was accomplished on a pilot plant scale using 1362 g of whole, dried cocoa beans in a drum-style US Roaster. Burner temperature was set at 150°C and 2% water was added at the beginning of the roast. The beans were roasted to a final moisture content of less than 1% with a bean temperature of about 155°C. Shell-free nibs were obtained using a bench-top BLT cracker and winnower. Nibs were made into liquor with a bench-top Netsch Ball Mill and ground to a 25 μm particle size. Several batches were processed, combined, and three aliquots were taken for analysis.
Composite samples from each type of roasting were analyzed, to avoid measuring batch to batch variability but rather focusing on the impact of the different processing methods. In all cases, unroasted and roasted cocoa beans by both traditional and modern processing methods had their shells removed and were ground to a consistent particle size. Prior to analysis for the three flavanol tests described below, the ground mass was defatted by extracting three times with hexane . Final results are reported on per gram of original cocoa bean material.
Antioxidant capacity measurement
Total antioxidant activity of unroasted and roasted cocoa bean samples was measured by the ORAC assay. ORAC is a widely used fluorescent method for assessing antioxidant capacity in food and biological samples. It is based on the inhibition of a peroxy radical induced oxidation initiated by the thermally based decomposition of 2, 2'- azobis- 2- methyl- propanimidamide, using fluorescein as a fluorescent probe and Trolox as a standard substrate. The results are expressed as μmol Trolox equivalent (TE) per gram [17, 18].
Flavanol monomer determination
The flavanol monomers (±)-catechin and (±)-epicatechin were measured by the HPLC method of Nelson and Sharpless  standardized with (+)-catechin hydrate and (−)-epicatechin using the method recently validated using a five-laboratory ring test . We define and report measures of (±)-epicatechin as epicatechin and (±)-catechin as catechin.
Flavanol monomer to decamer determination
The flavanol oligomers with degree of polymerization (DP) ranging from DP 1 to 10 were measured by the HPLC method of Robbins et al. with each sample extract standardized using individual purified oligomeric standards isolated and characterized as described by Hurst et al.. These standards were obtained from Planta Analytica (Danbury, CT). The data are reported as Flavanols DP 1–10.
Total flavanol determination
Total flavanols were measured by the DMAC method which has been validated by a three laboratory ring-test described by Payne et al.. This method is based on the specific reaction of p-dimethylaminocinnamaldehyde with the flavanol structure. The test was standardized using procyanidin B2 purchased from Planta Analytica (Danbury, CT). The method is specific to monomeric and polymeric flavan-3-ols as well as to related compounds such as epigallocatechin gallate and epicatechin-3-gallate. Monomers, i.e., epicatechin and catechin, react more strongly than higher MW oligomers with DMAC. Thus mg/g may not change, but the various proportions of monomers to DP2 and higher may be altered with proces sing. These results are reported as Total Flavanols.
The data presented in this paper represent analysis of three representative samples for each sample type. Values for antioxidant activity and flavanol contents are expressed as averages and standard deviations. Due to the small sample populations, p-values were calculated on the basis of a significant difference. Therefore, at a 95% confidence level, p-values ≤0.05 represent a significant difference between two sets of data.
Results and discussion
Comparison of unprocessed Lavado and Ivorian beans
Antioxidant capacity (ORAC) and flavanol content comparison for unroasted, unfermented Lavado beans and unroasted fermented Ivory Coast beans used in this study
ORAC (μmol TE/g)
± − epicatechin (mg/g)
± − catechin (mg/g)
Flavanols DP 1–10 (mg/g)
Total flavanols (mg/g)
Washed, Unfermented Lavado
737 ± 32
6.53 ± 0.25
0.17 ± 0.02
37.18 ± 0.38
38.67 ± 1.15
Fermented Ivory Coast
420 ± 44
1.53 ± 0.06
0.09 ± 0.00
10.18 ± 0.09
14.33 ± 0.58
Since the focus of this study was a comparison of processing methods, the data presented in the following tables and graphs compare only the impact of traditional and modern bean processing on various antioxidant and flavanol measurements. A comparison will not be made to the unroasted starting material because the difference in moisture contents would create an unfair comparison.
Processing of Lavado (unfermented) beans
Antioxidant capacity and flavanol content comparison for unfermented Lavado cocoa beans roasted on a Mayan-style comal and ground on a metate (Traditional process) compared to the same beans roasted and ground using modern pilot plant equipment (Modern process)
Lavado beans, traditional process
Lavado beans, modern process
ORAC (μmol TE/g)
620 ± 17
613 ± 15
± − epicatechin (mg/g)
3.57 ± 0.15
3.73 ± 0.06
± − catechin (mg/g)
1.77 ± 0.15
2.53 ± 0.06
flavanols DP 1–10 (mg/g)
18.12 ± 0.72
18.90 ± 0.40
total flavanols (mg/g)
36.00 ± 1.00
38.67 ± 1.53
Our results with unfermented beans are different from those of Cervelotti et al. in 2008  who comparing the in vitro antioxidant activity and polyphenol content of finished chocolates made for standard, large scale manufactured products and for artisan manufacturing methods which used small batch roasting and stone, melangeur grinding. They concluded that artisan-made chocolate had higher antioxidant capacities when compared to mass produced chocolate as measured by Briggs-Rauscher reaction and by the Trolox Equivalent Antioxidant Capacity (TEAC) methods. They also found higher levels of polyphenols in the artisan chocolates. But their study made no attempt to measure the flavan-3-ol content of the chocolates, nor were they able to measure the antioxidant activities or the total polyphenol composition of the starting materials, as we have. Additionally, the Cervelotti study did not attempt to measure differences in their starting materials, nor differences in the processing methods used, since all of the chocolates produced would have used similar, but not identical, modern roasting and grinding techniques; the differences mainly being the scale of the manufacture. Our results with unfermented beans clearly show that there is a loss in antioxidant activity and flavanol content due to roasting but that there is no significant difference between the traditional process and the modern process in making chocolate mass.
Processing of Ivorian (fermented) beans
Antioxidant capacity, and flavanol content comparison for Ivory Coast beans roasted on a Mayan-style comal and ground on a metate (Traditional process) compared to the same beans roasted and ground using modern pilot plant equipment (Modern process)
Ivorian beans, traditional process
Ivorian beans, modern process
p-value * (if different)
ORAC (μmol TE/g)
383 ± 6
317 ± 15
± − epicatechin (mg/g)
1.60 ± 0.14
1.27 ± 0.06
± − catechin (mg/g)
0.54 ± 0.03
0.63 ± 0.05
flavanols DP 1–10 (mg/g)
8.30 ± 0.20
6.79 ± 0.30
total flavanols (mg/g)
17.67 ± 0.58
19.33 ± 1.15
When the Lavado bean processing data (Table 2) are compared to Ivory Coast processing data (Table 3), the same observation can be made that the antioxidant capacity and flavanol contents of roasted, unfermented Lavado beans are significantly higher than in the roasted, more-fermented Ivory Coast beans (Table 1). This also is consistent with data published by Payne, et al.  and others [10–12] where the flavanol monomer and oligomer content of roasted Lavado beans has been found to be significantly higher than roasted beans from fermented sources like Ivory Coast and Papua New Guinea.
Implications for traditional processing and modern processing
People native to Mesoamerica and Northern South America have a tradition of consuming less-fermented or totally unfermented cocoa. This is due to the ease of processing and drying cocoa beans in humid climates, particularly during the rainy season, when the beans need to be quickly dried. Over the centuries these people have come to prefer the unfermented, Lavado beans. Regardless of the processing methods used, Lavado beans had the same level of flavanols and these levels were more than twice the level found in fermented beans after similar processing (compare Table 2 to Table 3). This may mean that people native to the Americas today and before the discovery of the New World by Columbus and Cortez, ate cocoa products made from cocoa mass (also called chocolate liquor) that contained about twice the level of flavanols found in today’s cocoa mass.
After Europeans began importing cocoa beans, first for use in beverages, and then for solid chocolate, hot cocoa beverages and later milk chocolate, there was increased demand for partially or fully fermented cocoa beans. The producers of cocoa in the Americas were asked to produce cocoa beans that had been fermented to suit European tastes and were paid a premium for these beans [3, 4]. As is shown in this study (Table 1), and by others [9–13], fermentation lowers the level of flavanols in the raw beans. The benefit to the European consumer was to have cocoa drinks or chocolate bars that were less bitter and had more cacao flavor. With the increased use of fermented beans and with the development of modern cocoa bean processing, there was a loss of flavanols from product, with the greatest loss of flavanols due to the preference for fermented beans. The results of the present study have shown that modern processing has little or no impact on flavanols (Table 3, Figure 5). Thus the health benefits of cocoa and chocolate flavanols and other beneficial bioactives need to be balanced with the flavor of the final product, for which fermentation of cocoa beans tends to increase cacao flavor impact and lessen bitterness. Despite the implementation of fermentation, dark chocolate and cocoa powder still have some of the highest concentrations of flavanols of any food in our diet . In fact a recent dose response study using conventionally processed cocoa powder in ever increasing amounts has found that as little as 5 g is sufficient to elicit significant vasodilation of the brachial artery, as measured by Flow Mediated Dilation .
The authors would like to thank the Hershey Center for Health and Nutrition for providing the samples and facilities for this research. We thank The Hershey Company for supplying support to E. Chin as part of her undergraduate summer internship. Thanks go to John Scharffenberger for his expert advice and direction for the ancient, traditional processing of cacao beans. Also, a special thanks to W. J. Hurst for the acquisition of an authentic clay comal from Latin America.
- Powis : Cacao and the San Lorenzo Olmec. Proc Natl Acad Sci. 2011, 108: 8595-8600. 10.1073/pnas.1100620108.View ArticleGoogle Scholar
- Coe SD, Coe MD: The True History of Chocolate. 1996, London, England: Thames and Hudson Ltd.Google Scholar
- Minifie BW: Chocolate, Cocoa and Confectionery. 1980, Westport, CT, USA: AVI Publishing CompanyGoogle Scholar
- Beckett ST: Industrial Chocolate Manufacture and Use. 1988, Blackie: Glasgow and London, 388-Google Scholar
- Henderson JS, Joyce RA, Hall GR, Hurst WJ, McGovern PE: Chemical and archaeological evidence for the earliest cacao beverages. Proc Natl Acad Sci USA. 2007, 104: 18937-18940. 10.1073/pnas.0708815104.View ArticleGoogle Scholar
- Hurst WJ, Tarka SM, Powis TG, Valdez F, Hester TR: Cacao usage by the earliest Maya civilization. Nature. 2002, 418: 289-290. 10.1038/418289a.View ArticleGoogle Scholar
- Edgar B: The power of chocolate: tracking the chemical signature of cacao across Mesoamerica. Archaeology. 2010, 63: Nov/Dec., 20–25Google Scholar
- Thompson SS, Miller KB, Lopez AS: Chapter 39, Cocoa and Coffee. Food Microbiology: Fundamentals and Frontiers, 3rd Ed. 2007, Washington DC: ASM PressGoogle Scholar
- Harborne JB, Baxter H, Moss GP: Phytochemical Dictionary: a handbook of bioactive compounds from plants. 1999, London, United Kingdom: Taylor and Francis, 2Google Scholar
- Kim H, Keeney PG: (−)-Epicatechin content in fermented and unfermented cocoa beans. J Food Sci. 1984, 49: 1090-1092. 10.1111/j.1365-2621.1984.tb10400.x.View ArticleGoogle Scholar
- Porter LJ, Ma Z, Chen BC: Cocoa procyanidins: Major flavonoids and identification of some minor metabolites. Phytochemistry. 1991, 20: 1657-1663.View ArticleGoogle Scholar
- Lea PJ, Ford GD: Characterization of the polyphenols of cocoa. 1990, Glasgow: BioFlavors ConferenceGoogle Scholar
- Payne MJ, Hurst WJ, Miller K, Rank C, Stuart DA: Impact of fermentation, drying, roasting, and Dutch processing on epicatechin and catechin content of cacao beans and cocoa ingredients. J Ag Food Chem. 2010, 58: 10518-10527. 10.1021/jf102391q.View ArticleGoogle Scholar
- Hooper L, Kay C, Abdelhamid A, Kroon PA, Cohn JS, Rimm EB, Cassidy A: Effects of chocolate, cocoa and flavan-3-ols on cardiovascular health: a systematic review and meta-analysis of randomized trials. Am J Clin Nutr. 2012, 95: 740-751. 10.3945/ajcn.111.023457.View ArticleGoogle Scholar
- Reid K, Fakler TR, Frank OR, Stocks NP: Effect of cocoa on Blood Pressure (Review). The Cochrane Collaboration. 2012, John Wiley Press, 1-83.Google Scholar
- Association of Analytical Chemists, International: Cacao mass (fat-free) of chocolate liquor. Official Methods of the AOAC International, 16th Ed. AOAC Official Method. 1995, Gaithersburg, MD: AOAC International, 931.05Google Scholar
- Ou B, Hampsch-Woodill M, Prior R: Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. J Agric Food Chem. 2001, 49: 4619-4626. 10.1021/jf010586o.View ArticleGoogle Scholar
- Huang D, Ou B, Hampsch-Woodill M, Flanagan J, Deemer EK: Development and validation of oxygen radical absorbance capacity assay for lipophilic antioxidants using randomly methylated-cyclodextrin as a solubility enhancer. J Agric Food Chem. 2002, 50: 1815-1821. 10.1021/jf0113732.View ArticleGoogle Scholar
- Nelson BC, Sharpless KE: Quantification of the predominant monomeric catechins in baking chocolate standard reference material by LC/APCI-MS. J Agric Food Chem. 2003, 51: 531-537. 10.1021/jf0207474.View ArticleGoogle Scholar
- Shumow L, Bodor A: An industry consensus study on an HPLC fluorescence method for the determination of (±)-catechin and (±)-epicatechin in cocoa and chocolate products. Chemistry Central Journal. 2011, 5 (39): MS ID: 7184481134944588-Google Scholar
- Robbins RJ, Leonczak J, Johnson JC, Li J, Kwik-Uribe C, Prior RL, Gu L: Method performance and multi-laboratory assessment of a normal phase high pressure liquid chromatography-fluorescence detection method for the quantitation of flavanols and procyanidins in cocoa and chocolate containing samples. J Chromatorgr A. 2009, 1216: 4831-4840. 10.1016/j.chroma.2009.04.006.View ArticleGoogle Scholar
- Hurst WJ, Stanley B, Glinski JA, Davey M, Payne MJ, Stuart DA: Characterization of primary standards for use in the HPLC analysis of the procyanidin content of cocoa and chocolate containing products. Molecules. 2009, 14: 4136-4146. 10.3390/molecules14104136.View ArticleGoogle Scholar
- Payne MJ, Hurst WJ, Stuart DA, Ou B, Fan E, Ji H, Kou Y: The use of the DMAC assay for determination of total procyanidins in selected chocolate and confectionery products. J AOAC International. 2010, 93: 89-96.Google Scholar
- Cervelatti R, Greco E, Costa S, Guerra M, Speroni E: A comparison of antioxidant properties between artisan-made and factory-produced chocolate. International Journal of Food Science & Technology. 2008, 43: 1866-1870. 10.1111/j.1365-2621.2008.01765.x.View ArticleGoogle Scholar
- ICCO statistics for World Cocoa Production. 2009, http://www.icco.org/statistics/production-and-grindings/production.html,
- USDA Database for the Proanthocyanidin Content of Selected Foods. 2004, http://www.nal.usda.gov/fnic/foodcomp/Data/PA/PA.html,
- Monahan KD, Feehan RP, Kunselman AR, Preston AG, Miller DL, Lott MEJ: Dose dependent increases in flow-mediated dilation following acute cocoa ingestion in healthy older adults. J Appl Physiol. 2011, 111: 1568-1574. 10.1152/japplphysiol.00865.2011.View ArticleGoogle Scholar
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