- Research article
- Open Access
Impact of fermentation, drying, roasting and Dutch processing on flavan-3-ol stereochemistry in cacao beans and cocoa ingredients
© Hurst et al 2011
- Received: 25 April 2011
- Accepted: 14 September 2011
- Published: 14 September 2011
This paper reports a systematic study of the level of flavan-3-ol monomers during typical processing steps as cacao beans are dried, fermented and roasted and the results of Dutch-processing. Methods have been used that resolve the stereoisomers of epicatechin and catechin. In beans harvested from unripe and ripe cacao pods, we find only (-)-epicatechin and (+)-catechin with (-)-epicatechin being by far the predominant isomer. When beans are fermented there is a large loss of both (-)-epicatechin and (+)-catechin, but also the formation of (-)-catechin. We hypothesize that the heat of fermentation may, in part, be responsible for the formation of this enantiomer. When beans are progressively roasted at conditions described as low, medium and high roast conditions, there is a progressive loss of (-)-epicatechin and (+)-catechin and an increase in (-)-catechin with the higher roast levels. When natural and Dutch-processed cacao powders are analyzed, there is progressive loss of both (-)-epicatechin and (+)-catechin with lesser losses of (-)-catechin. We thus observe that in even lightly Dutch-processed powder, the level of (-)-catechin exceeds the level of (-)-epicatechin. The results indicate that much of the increase in the level of (-)-catechin observed during various processing steps may be the result of heat-related epimerization from (-)-epicatechin. These results are discussed with reference to the reported preferred order of absorption of (-)-epicatechin > (+)-catechin > (-)-catechin. These results are also discussed with respect to the balance that must be struck between the beneficial impact of fermentation and roasting on chocolate flavor and the healthful benefits of chocolate and cocoa powder that result in part from the flavan-3-ol monomers.
- Cocoa Bean
- Dark Chocolate
Dark chocolate and cocoa powders are associated with numerous health benefits, most notably to the improvement of factors impacting cardiovascular health. Evidence comes from epidemiological studies which associate decreased risk of stroke and cardiovascular disease with the long-term consumption of chocolate [1–3]. Further evidence comes from the notable improvements in endothelial function which lead to increased flow mediated dilation [4, 5] and increased flexibility of the vascular system, also known as augmentation [6, 7]. Studies also show that blood pressure decreases after short-term consumption of dark chocolate [8–10]. Additionally chocolate and/or cocoa have been shown to decrease platelet aggregation [11, 12] and decreases oxidation of low density lipoprotein . All of these responses are associated with improved health in the overall vascular system. When it comes to the heart itself, dark chocolate consumption has been associated with increased dilation  and improved blood flow  to the left anterior descending coronary artery. These benefits have been summarized in recent review articles [16, 17].
The flavan-3-ols in chocolate and cocoa have been implicated in these cardiovascular benefits. Dark chocolates are high in flavan-3-ols [18, 19] and natural, non-Dutch processed cocoa powders are richer still in these compounds [20, 21]. Research indicates that the monomeric flavanols, and especially (-)-epicatechin, are at least partly responsible for these effects. Evidence is based on uptake and conjugation of epicatechin and catechin in the blood, with their levels peaking at about 1 to 2 hours post-consumption [22–24]. The levels of epicatechin in blood are also co-incident with the peak in vasodilation seen in brachial arteries as measured by flow-mediated dilation. Bioavailability of flavanols appears to be highest for the monomers, (-)-epicatechin and (+)-catechin, with little of the dimeric oligomers and none of the trimers and higher oligomers appearing in the blood stream . Finally it has been shown that the absorption of (-)-epicatechin into the blood stream is greatest followed by (+)-catechin and then (-)-catechin .
It has long been known that heavy flavanol losses occur when cocoa beans are fermented [26–28]. Losses also occur during roasting , Dutch processing [20, 21] and baking with alkali-leavening agents . Fresh cacao beans contain about 12.8 to 43.2 mg/g of (-)-epicatechin, depending on variety [28–31]. Fresh beans also contain about 20 to 30-times less (+)-catechin . A recent paper comprehensively summarized the importance of fermentation, drying, roasting and Dutch processing on the levels of epicatechin and catechin in cacao beans and cocoa ingredients. This report found that epicatechin, compared to catechin, is as much as 30-times greater in fresh and dried cocoa beans, but as conventional processing occurs, there is a loss in epicatechin and at times an increase in catechin .
Two reports have shown that roasting or Dutch processing can result in the formation of a new stereoisomer, (-)-catechin, an enantiomer not found in fresh cacao beans [32, 33]. But the first report by Gotti et al.  only had limited quantitative data for unripe and ripe beans and the second report by Kofink et al.  only measured ratios of stereoisomers, not their absolute levels. These, and other reports [20, 31–34], speculate that the (-)-catechin is a product of epimerization coming from (-)-epicatechin. One paper did not measure the levels of the stereoisomers of either epicatechin or catechin, only measuring total epicatechin and total catechin . However a recent report  concluded that (-)-epicatechin epimerizes to (+)-catechin after roasting and Dutch processing. Obviously there is need to quantify the levels of these flavan-3-ol stereoisomers after typical processing steps common to chocolate and cocoa powder manufacture. Additionally it is important to resolve the disparity in the literature regarding epimerization of (-)-epicatechin to either (-)-catechin or to (+)-catechin and to resolve this controversy in the literature.
This manuscript describes the first comprehensive study of the impact of fermentation, drying, roasting and alkali-(Dutch) processing of cacao beans and cocoa ingredients by measuring the absolute levels of stereoisomers of epicatechin and catechin. The methods sufficiently resolve the (+) and (-) isomers of epicatechin and catechin, enabling us to show that processing with heat and with alkali result in significant loss of both (-)-epicatechin and (+)-catechin as well as the de novo appearance of (-)-catechin, not found in fresh plant material, but formed in fermented as well as roasted and Dutch-processed cocoa beans and ingredients.
HPLC-grade standards of (-)-epicatechin, (-)-catechin and (+)-catechin hydrate were purchased from Sigma.
Preparation of the chocolate samples followed a procedure, which was modified from the method used by Donovan et al. in 2006 to observe the bioavailability in chocolate samples . Powdered samples (2 g) were extracted with 3 × 4.5 mL hexanes and the residue was dried under an argon stream overnight. The defatted and dry solid was further extracted with 3 × 2.5 mL acetone:H2O:AcOH (70:28:2, v:v:v). The combined extracts were concentrated to approximately 2 mL under a stream of argon over 36 h. The solution was diluted to exactly 5 mL with a mixture of acetone:H2O:CH3CN (2:2:1, v:v:v).
Fresh cacao pods, both unripe and ripe, of the Forestaro type were obtained from the U.S.D.A. Field Station, Mayaguez, Puerto Rico. Unripe pods were green and the pulp was a spongy white foam typical of unripe pods. Ripe pods were yellow and/or orange and the pulp was moist and watery, typical of ripe pods.
Dried Cacao Beans
Unfermented washed and dried beans, sometimes referred to as cacao lavado, were commercially obtained from AMSA in Mexico. Cacao beans with various degrees of fermentation from Sulawesi, Ivory Coast and Papua New Guinea (PNG) were commercially available, fair to average quality beans typical of the origin. Commercial beans are nominally ≤ 8% moisture.
Natural and Dutch-processed (alkalized) cocoa powders were obtained from J.B. Cocoa. Natural cocoa powder represents no Dutch processing with an average extractable pH of 5.76 while low, medium, and heavy Dutch processed samples were characterized by increasing pH values of 7.13, 7.36, and 7.92 respectively as described in Miller et al. 2008 .
Cacao beans from either unripe or ripe pods were removed within three days of harvest, hand dissected using a scalpel, and freed from the surrounding, foamy pulp (in the case of unripe beans) or watery pulp (in the case of ripe beans). The seed coat was cut away leaving the cotyledons, hypocotyl axis and root radicle. Beans from unripe and ripe pods were freeze-dried to moisture levels at or below 5%.
Commercial grade cacao beans from Ivory Coast were subjected to increasing degrees of roasting using a Blodget Oven. Three hundred grams (300 g) of shell-free cacao nibs were placed on wire mesh trays and roasted in the oven maintained at 163°C. Low, medium, and high roasted nibs were roasted at 13, 20, and 25 minutes respectively.
Degree of Fermentation
The degree of fermentation of cacao beans was evaluated by acidic methanol extraction and colorimetric determination using a ratio of 460/530 nm based on method of Gourieva and Tserevitinov . The calculated fermentation index values of the three bean samples were: Mexican Lavado (0.5), Sulawesi (0.79), Ivory Coast (0.96), and PNG (1.38).
Tabular summary of results
Comparison of data for determination of flavanol stereo-isomer levels of triplicate samples of beans and powder analyzed in this study versus data from an earlier study where only catechin and epicatechin were determined
Data on stereoisomer concentrations
Data on individual compounds 
Unprocessed Raw Beans
9.77 ± 0.80
0.57 ± 0.05
12.8 ± 0.06
0.39 ± 0.01
13.35 ± 2.42
0.58 ± 0.10
12.4 ± 4.05
0.46 ± 0.17
Unfermented Dried Beans
- Mexican lavado
4.88 ± 0.87
0.19 ± 0.05
0.17 ± 0.03
Fermented Dried Beans
- Light - Sulawesi
1.87 ± 0.32
0.12 ± 0.01
0.10 ± 0.01
- Medium - Ivory Coast
0.94 ± 0.22
0.08 ± 0.02
0.08 ± 0.00
1.69 ± 0.10
0.08 ± 0.00
- Heavy-Pap. New Guin
0.56 ± 0.02
0.07 ± 0.00
0.09 ± 0.02
0.78 ± 0.04
0.05 ± 0.00
- Ivory Coast
0.66 ± 0.19
0.08 ± 0.01
0.42 ± 0.10
0.50 ± 0.03
0.52 ± 0.02
Dutch processed Powder
0.16 ± 0.09
0.06 ± 0.00
0.48 ± 0.01
0.26 ± 0.29
0.36 ± 0.34
Comparison of data to other literature results
Also in Table 1 for comparative purposes, data from Payne et al.  is shown in which the method of Nelson and Singletary  was used to determine total epicatechin and total catechin in unripe, ripe dried, fermented cacao beans and in Dutch processed cocoa powder. The levels of total epicatechin in closely, but not perfectly, correspond to the levels of (-)-epicatechin seen in this study. This difference probably represents the natural variation of cacao beans and powder samples rather than a systematic error in either of the analytical procedures. The same can be said for the amounts of total catechin icompared to the amount of (+)- and (-)-catechin summed as total catechin. We conclude that, within the natural variation of cacao ingredients surveyed and sample to sample differences, the findings we have reported for the resolved isomers of (-)-epicatechin, (+)-catechin and (-)-catechin closely agree with the results found previously for total epicatechin and total catechin in similarly processed samples . Our results for the amounts of catechin isomers are also in agreement with the amounts found in Venezuelan cocoa beans from unripe pods by Gotti et al.  who measured 15.3 ± 0.09 mg/g (-)-epicatechin and (+)-0.45 ± 0.01 mg of (+)-catechin.
Analysis of unripe, ripe cacao beans
In Table 1 is shown the results for the analysis of flavan-3-ols stereoisomers in cacao beans that were unripe and ripe and were subsequently freeze dried. Fresh ripe cacao beans have the highest levels of (-)-epicatechin (13.35 ± 2.24 mg/g) with 24-times less (+)-catechin (0.56 ± 0.05 mg/g) and no (-)-catechin. Unripe beans also have high levels of (-)-epicatechin (9.77 ± 0.80 mg/g) but somewhat lower than that of ripe beans. The (+)-catechin level (0.57 ± 0.05 mg/g) of unripe beans is the same as ripe beans.
Effect of cacao bean fermentation
The results for lightly fermented cacao beans, as represented by commercially available Sulawesi cacao beans, are compared to medium fermentation, as represented by Ivory Coast beans, and heavy fermentation, as represented by Papua New Guinea beans, are also shown in Figure 4. Sulawesi has a tradition of either not fermenting or fermenting for a short period, Ivory Coast has a tradition of fermenting for four to five days and Papua New Guinea has a tradition of fermenting for six or more days, although in the beans that were analyzed the absolute length of fermentation was not truly known to us. Compared to dried, unfermented beans, the light fermented Sulawesi beans had an average of 1.87 ± 0.32 mg/g of (-)-epicatechin, 0.12 ± 0.01 mg/g (+)-catechin and 0.10 ± 0.01 mg/g of (-)-catechin. The Ivory Coast beans had an average of 0.94 ± 0.22 mg/g of (-)-epicatechin, 0.08 ± 0.01 mg/g (+)-catechin and 0.08 ± 0.00 mg/g of (-)-catechin. The heaviest fermented beans, the Papua New Guinea beans, had the least (-)-epicatechin (0.56 ± 0.02 mg/g), (+)-catechin (0.07 ± 0.00 mg/g) and (-)-catechin (0.09 ± 0.00 mg/g). Extending the fermentation time decreased the level of (-)-epicatechin and (+)-catechin from levels found in ripe and dried beans but also caused a relative increase in (-)-catechin, compared to the other stereoisomers. We interpret this to mean that the temperature of the fermentation mass could be responsible for the appearance of (-)-catechin during fermentation since it has been observed that fermentation temperatures as high as 60°C for as long as two days can occur .
Effect of roasting on flavan-3-ol stereoisomers
Balance between chocolate palatability and healthfulness
As pointed out in the introduction, the association of the cardiovascular benefits of chocolate and cocoa with the flavanols is supported by an ever growing body of literature. So, in order to maximize the impact of cocoa or dark chocolate on health, why not make these products out of unfermented beans? Indeed this concept is the basis of several unique ingredients or chocolates [38, 39]. But there are good reasons why fermented cocoa beans are used, which mostly concern taste and palatability. One of the quality measures used to determine the final price of cocoa beans is the "cut test". This is a measure of the oxidation of the anthocyanins in the cocoa beans; grey and purple beans being unfermented and brown beans being fully fermented [40, 41]. Chocolate makers know that optimally fermented cocoa beans will be less astringent, presumably due to the reduction of flavanols, and these beans also will have more cacao flavor, presumably due to chocolate flavor precursors that, when roasted, produce cacao flavor. So a balance needs to be struck between retaining the health benefits associated with the flavanol antioxidants, especially (-)-epicatechin, and the beneficial impact that fermentation has on roasted flavor and to mitigate excessive astringency. Similar logic holds for roasting. Roasting develops chocolate flavor and aroma, a fact well known to the Mayans who roasted on traditional charcoal heated comals and to anyone who has roasted cocoa beans, whether it is done industrially or in a skillet [40, 41]. In addition to flavor development, roasting also loosens the cocoa shell, making it easier to remove from the cotyledons of the cocoa bean. Roasting also is a critical pasteurization or even a sterilization step that increases the microbiological safety of the resulting roasted beans, chocolate liquor or cocoa powder. A balance needs to be struck between preserving the healthful benefits associated with the flavanol antioxidants, especially (-)-epicatechin, and the loss that occurs during roasting and flavor development that may be key to certain chocolate brand identity.
Cocoa powder and dark chocolate are appreciated, first and foremost, for their flavor and texture. By attempting to make cocoa ingredients healthier by shortening fermentation time or minimizing roasting for similar, we would expect that the result would be a bitter, astringent ingredient with lower than expected cocoa flavor. So as with many things, a balance needs to be struck between different benefits; in this case flavor versus healthfulness.
The project was funded by the Hershey Center for Health & Nutrition
- Buijsse B, Feskens EJ, Kok FJ, Kromhout D: Cocoa intake, blood pressure and cardiovascular mortality: The Zutphen elderly study. Arch Intern Med. 2006, 166: 411-417. 10.1001/.411.Google Scholar
- Mink PJ, Scrafford CG, Barraj LM, Harnack L, Hong CP, Nettleton JA, Jacobs DRJ: Flavonoid intake and cardiovascular disease mortality: a prospective study in post menopausal women. Am J Clin Nutr. 2007, 85: 895-909.Google Scholar
- Buijsse B, Weikert C, Drogan D, Bergmann M, Boeing H: Chocolate consumption in relation to blood pressure and risk of cardiovascular disease in German adults. Eur Heart J. 2010, 10: 1093-1101.Google Scholar
- Heiss C, Dejam A, Kleinbongard P, Schewe T, Seis H, Kelm M: Vascular effects of cocoa rich in flavan-3-ols. J Amer Med Assoc. 2003, 290: 1030-1031. 10.1001/jama.290.8.1030.View ArticleGoogle Scholar
- Engler MB, Engler MM, Chen CY, Malloy MJ, Browne A, Elisa BS, Chiu Y, Kwak H-K, Milbury P, Paul SM, Blumberg J, Mietus-Snyder ML: Flavonoid rich dark chocolate improves endothelial function and increases epicatechin concentrations in healthy adults. J Am Coll Nutr. 2004, 23: 197-204.View ArticleGoogle Scholar
- Fischer ND, Hughes M, Gerhard-Herman M, Hollenberg NK: Flavanol-rich cocoa induces nitric oxide-dependent vasodilation in healthy humans. J Hypertension. 2003, 21: 2281-2286. 10.1097/00004872-200312000-00016.View ArticleGoogle Scholar
- Vlachopoulos C, Aznaouridis K, Alexopoulos N, Economou E, Andreadou I, Stefanadis C: Effect of dark chocolate on arterial function in healthy individuals. Am J Hypertension. 2005, 18: 785-791. 10.1016/j.amjhyper.2004.12.008.View ArticleGoogle Scholar
- Taubert D, Berkels R, Roesen R, Klaus W: Chocolate and blood pressure in elderly individuals with isolated systolic hypertension. J Amer Med Assoc. 2003, 290: 1029-1030. 10.1001/jama.290.8.1029.View ArticleGoogle Scholar
- Grassi D, Necozione S, Lippi C, Croce G, Valeri L, Pasqualetti P, Desideri G, Blumberg J, Ferri C: Cocoa reduces blood pressure and insulin resistance and improves endothelial-dependent vasodilation in hypertensives. Hypertension. 2005, 46: 398-405. 10.1161/01.HYP.0000174990.46027.70.View ArticleGoogle Scholar
- Taubert D, Roesen R, Lehmann C, Jung N, Schomig E: Effects of low habitual cocoa intake on blood pressure and bioactive nitric oxide: A randomized controlled trial. J Amer Med Assoc. 2007, 298: 49-60. 10.1001/jama.298.1.49.View ArticleGoogle Scholar
- Heiss C, Dejam A, Kleinbongard P, Schewe T, Seis H, Kelm M: Vascular effects of cocoa rich in flavan-3-ols. J Amer Med Assoc. 2003, 290: 1030-1031. 10.1001/jama.290.8.1030.View ArticleGoogle Scholar
- Murphy KJ, Chronopolos AK, Singh I, Francis MA, Moriarty H, Pike MJ, Turner AH, Mann NJ, Sinclair AJ: Dietary flavanols and procyanidin oligomers from cocoa (Theobroma cacao) inhibit platelet function. Am J Clin Nutr. 2003, 77: 1466-73.Google Scholar
- Kondo K, Hirano R, Matsumoto A, Igarashi O, Itakura H: Inhibition of LDL oxidantion by cocoa. Lancet. 1996, 348: 1514-View ArticleGoogle Scholar
- Flammer AJ, Hermann F, Sudano I, Spieker L, Hermann M, Cooper KA, Serafini M, Luscher TF, Ruschitzka F, Noll G, Corti R: Dark chocolate improves coronary vasomotion and reduces platelet reactivity. Circulation. 2007, 116: 2376-2382. 10.1161/CIRCULATIONAHA.107.713867.View ArticleGoogle Scholar
- Shiina Y, Funabashi N, Lee K, Murayama T, Nakamura K, Wakatsuki Y, Daimon M, Kumoro I: Acute effect of oral flavoniod-rich dark chocolate intake on coronary circulation, as compared with non-flavonoid white chocolate, by transthorasic Doppler echocardiography in healthy adults. Int J Cardiol. 2009, 131: 424-429. 10.1016/j.ijcard.2007.07.131.View ArticleGoogle Scholar
- Cooper KA, Donovan JL, Waterhouse AL, Williamson G: Cocoa and Health: A decade of Research. Brit J Nutr. 2008, 99: 1-11.View ArticleGoogle Scholar
- Corti R, Flammer AJ, Hollenberg NK, Luscher TF: Cocoa and Cardiovascular Health. Circulation. 2009, 119: 1433-41. 10.1161/CIRCULATIONAHA.108.827022.View ArticleGoogle Scholar
- Miller KB, Stuart DA, Smith NL, Lee CY, Mchale NL, Flanagan JA, Ou B, Hurst WJ: Antioxidant activity and polyphenol and procyanidins contents of selected commercially available cocoa-containing and chocolate products in the United States. J Agric Food Chem. 2006, 54: 4062-68. 10.1021/jf060290o.View ArticleGoogle Scholar
- Cooper KA, Campos-Gimenez E, Alverez DJ, Nagy K, Donovan JA, Williamson GA: Rapid reversed phase ultra-performance liquid chromatography analysis of the major cocoa polyphenols and interrelationships of their concentrations in chocolate. J Agric Food Chem. 2007, 56: 260-265.View ArticleGoogle Scholar
- Andres-Lacueva C, Monagas M, Khan N, Izquierdo-Pulido M, Urpi-Sarda M, Permanyer J, Lamuela-Raventtos RM: Flavanol and Flavonol contents of cocoa powder products: Influence of manufacturing process. J Agric Food Chem. 2008, 56: 3111-17. 10.1021/jf0728754.View ArticleGoogle Scholar
- Miller KB, Hurst WJ, Payne MJ, Stuart DA, Apgar J, Sweigart DS, Ou B: Impact of alkalization on the antioxidant and flavanol content of commercial cocoa powders. J Agric Food Chem. 2008, 56: 8527-8533. 10.1021/jf801670p.View ArticleGoogle Scholar
- Richelle M, Tavazzi I, Enslen M, Offord EA: Plasma kinetics in man of epicatechin from dark chocolate. Eur J Clin Nutr. 1999, 53: 22-26. 10.1038/sj.ejcn.1600673.View ArticleGoogle Scholar
- Schroeter H, Heiss C, Balzer J, Kleinbongard P, Keen CL, Hollenberg NK, Sies H, Kwik-Uribe C, Schmitz HH, Kelm M: (-)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans. P Nat Acad Sci USA. 2006, 103: 1024-1029. 10.1073/pnas.0510168103.View ArticleGoogle Scholar
- Holt RR, Lazarus SA, Sullards MC, Zhu QY, Schramm DD, Hammerstone JF, Frage CG, Schmitz HH, Keen CL: Procyanidin dimer B2 [epicatechin-4beta-8)epicatechin] in human plasma after consumption of a flavanol-rich cocoa. Am J Clin Nutr. 2002, 76: 798-804.Google Scholar
- Donovan JL, Crespy V, Oliveira M, Cooper KA, Gibson BB, Williamson G: (+)-Catechin is more bioavailable than (-)-catechin: Relevance to the bioavailability of catechin from cocoa. Free Rad Res. 2006, 40: 1029-1034. 10.1080/10715760600868545.View ArticleGoogle Scholar
- Roelofsen PA: Fermentation, drying and storage of cacao beans. Adv Food Res. 1958, 8: 225-296.View ArticleGoogle Scholar
- Bracco U, Graihle N, Rostagno W, Egli RH: Analytical evaluation of cocoa curing in the Ivory Coast. J Sci Food Agric. 1969, 20: 713-716. 10.1002/jsfa.2740201204.View ArticleGoogle 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
- Stahl LA, Miller KB, Apgar J, Sweigart DS, Stuart DA, Mchale N, Ou B, Kondo M, Hurst WJ: The preservation of cocoa antioxidant activity, total polyphenols, flavan-3-ols and procyanidin content in cooked and baked foods prepared with cocoa powder. J Food Sci. 2009, 74: 456-461. 10.1111/j.1750-3841.2009.01226.x.View ArticleGoogle Scholar
- Robinson T, Ranalli AW, Phillips AW: Changes in cocoa tannins during processing. J Agric Food Chem. 1961, 9: 295-298. 10.1021/jf60116a016.View ArticleGoogle Scholar
- Payne MJ, Hurst WJ, Miller KB, Rank C, Stuart DA: Impact of fermentation, drying, roasting and Dutch processing on epicatechin and catechin content of cacao beans and cocoa ingredients. J Agric Food Chem. 2010, 58: 10518-10527. 10.1021/jf102391q.View ArticleGoogle Scholar
- Gotti R, Furlanetto S, Pionzauti S, Cavrini V: Analysis of catechins in Theobroma cacao beans by cyclodextrin-modified micellar electrokinetic chromatography. J Chromatogr. 2006, 1112: 345-352. 10.1016/j.chroma.2005.11.058.View ArticleGoogle Scholar
- Kofink M, Papagiannopoulos M, Galensa R: (-)-Catechin in cocoa and chocolate: Occurrence and analysis of an atypical flavan-3-ol enantiomer. Molecules. 2007, 12: 1274-1288. 10.3390/12071274.View ArticleGoogle Scholar
- Caligiani A, Cirlini M, Palla G, Ravaglia R, Arlorio M: GC-MS detection of chiral markers of cocoa beans of different quality and geographic origin. Chirality. 2007, 19: 329-334. 10.1002/chir.20380.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
- Gourieva KB, Tserevitinov OB: Method of evaluating the degree of fermentation of cocoa beans. USSR Patent No. 646. 1979, 254:Google Scholar
- Thompson SS, Miller KB, Lopez AS: Cocoa and Coffee. Food Microbiology Fundamentals and Frontiers. Edited by: Doyle MP, Beuchar LR. 2007, American Society of Microbiology Press: Washington, DC, 837-850. 3Google Scholar
- MARS Incorporated. MARS Botanical: CocoaPro. 2010, [http://cirkuhealth.com/Cocoa-101/CocoaPro.aspx]
- Barry Callebaut Group. Acticoa. [http://www.acticoa.com/en]
- Beckett ST: Industrial chocolate manufacture and use. 1988, Blackie, London. van Nostrand Reinhold, NY, 388-Google Scholar
- Minifie BW: Chocolate, cocoa and confectionery: Science and technology. 1989, van Nostrand Reinhold, NY, 904-3Google Scholar