Cocoa is widely consumed in the form of chocolates, and the consumption rate is rising, because of the increasing popularity of chocolate confectioneries worldwide. Cocoa can also be found in beverages, cosmetics, pharmaceuticals and toiletries [1
]. Cocoa beans begin as seeds in fruit pods of the tree Theobroma cacao
L., where each fruit pod contains 30 to 40 beans embedded in a mucilaginous pulp. Raw cocoa has an astringent, unpleasant taste and flavor. To obtain the characteristic cocoa flavor and taste, raw cocoa must be fermented, dried and roasted [2
]. The fresh cocoa bean is approximately composed of 32%–39% water, 30%–32% fat, 8%–10% protein, 5%–6% polyphenols, 4%–6% starch, 4%–6% pentosans, 2%–3% cellulose, 2%–3% sucrose, 1%–2% theobromine, 1% acids and 1% caffeine [3
Commercial cocoa is obtained from beans that originated as seeds from the ripe pods of the plant, Theobroma cacao
, which is cultivated in plantations in tropical regions throughout the world [4
]. The three major cocoa growing regions are West Africa, Southeast Asia and South America. Indonesia produces about 15% of the world’s cocoa beans and ranks third in terms of international production. Indonesia has expanded its cocoa production and is now producing almost as much as Ghana. Indonesian cocoa production has the potential to substantially increase, but such increase would depend on local political and economic factors. As a member of the International Cocoa Organization (ICCO) and a signatory to the 2001 International Cocoa Agreement, Papua New Guinea is obliged to promote a sustainable cocoa economy. Cocoa trees may be grown in China, particularly in the southern region of Hainan province. The quality of the cocoa beans from that region was said to be similar to that of Indonesian cocoa beans [5
The processing of cocoa beans comprises two major steps, namely, fermentation and drying. At cocoa plantations, fermentation and drying may primarily be looked upon as a curing process to stabilize the fresh beans through the microbial degradation of the firmly adhering, perishable pulp and through drying [6
Genotype, soil, climate and harvest conditions, as well as processes, such as fermentation, drying and roasting, have important effects on the characteristics of cocoa. Unfermented and partly fermented cocoa beans are the beans of the Theobroma cacao
L., which are dried without previously being fermented or partly fermented or by using improper procedures. Research has shown that these beans do not develop any chocolate flavor when roasted and are excessively astringent and bitter [7
]. Malaysia is currently making use of these types of beans, which are imported from Indonesia, especially for cocoa liquor, powder and cocoa butter production. Cocoa manufacturers usually blend the unfermented and partly fermented beans with fully fermented beans to obtain the desired flavor characteristics and to reduce the excessive astringency and bitterness.
So far, comprehensive studies have yet to be conducted on the total content variation of phenol and flavonoid and of gallic acid and epicatechin. Moreover, no study has explored the content and composition of free amino acids in commercially fermented and dried cocoa from different origins. To address this gap in the literature, the present study is conducted by showcasing the variations in fermented, dried cocoa samples from different countries. This paper also reports on the content and composition of total and free amino acids of cocoa samples originating from three countries. The aim of the present study is to assess the influence of unfermented, fermented and dried cocoa beans from box fermentations performed by different countries in the last two years.
2. Experimental Section
Gallic acid, epicatechin and Folin-Ciocalteu phenol reagent were obtained from Sigma Chemical Co., Ltd. (St. Louis, MO, USA). Methanol and acetonitrile (HPLC grade) were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). All other chemicals used were of analytical grade and were obtained from Sinopharm Co., Ltd. (Shanghai, China).
Theobroma cacao beans of different geographic origins (Papua New Guinea, Indonesia and China) and of the Trinitario (hybrids of Criollo and Forastero) type were used in this study and were supplied by the Spice and Beverage Research Institute (Hainan, China) and Huadong Cocoa Co. Ltd. (Wuxi, China), and the cacao beans from Papua New Guinea and Indonesia were gotten from the main producing region and were the varieties cultivated popularly there. Fermented cocoa beans from Papua New Guinea were named C1, and fermented and unfermented cocoa beans from Indonesia were named C2 and C3, respectively. Fully ripe Hainan cocoa pods (Theobroma cacao L.) were handily harvested in 2010 and 2011 and were fermented in a box for six days. The fermentation mass was turned manually every day by transferring the coca pods from one box to another. Cocoa beans that underwent six-day fermentation were collected and sundried until a 7% moisture content was reached. The beans (named C4 and C5) were then stored in a refrigerator for later use. The five sundried samples, in turn, were roasted in an oven at 160 °C for 30 min in the laboratory and were milled using a Retsch blender (Restch, Haan, Germany) to obtain cocoa liquor. The five samples were marked as CR1, CR2, CR3, CR4 and CR5, respectively.
The frozen cocoa bean samples were lyophilized (Labconco, Kansas, USA) until dry or until the beans became brittle and easily broken. The lyophilized beans were deshelled, degermed and ground using a Retsch blender (Restch, Haan, Germany). Small pieces of solid carbon dioxide were added occasionally to prevent cocoa lipids from melting, due to frictional heat caused by grinding.
2.2. Measurement of Average Bean Weight and Fat
The cocoa samples were placed in a ventilated oven at 60 °C until the constant dry weight of the bean components (beans and nibs) was achieved. The dry weights of the samples were then recorded. The results were shown as the gross weight and net weight of one cocoa bean.
The lyophilized dry cotyledons were crushed, and 5.0 g portions were extracted repeatedly in a Soxhlet apparatus (Soxtec 2050, Foss, Hoganas, Sweden) with 500 mL of petroleum ether (b.p. 40–70 °C). Fat content was determined and expressed as a percentage of weight [9
2.3. Measurement of Color and Absorbance at 420 nm
A 5.0 g portion of ground cotyledons was boiled in 45 mL of water and homogenized at 5000 rpm for 45 s. The homogenate was filtered and then centrifuged (1500× g
, 15 min). The supernatant was determined at 420 nm using a UV-Vis Shimadzu UV-1601 spectrophotometer (Tokyo, Japan) [10
Color was measured using an X-rite colorimeter (Xrite Inc., Grand Rapids, MI, USA). The results were expressed in the L
* colorimetric system, according to the International Commission of Illumination, in which a color can be defined conventionally by three numerical parameters: sample luminance L
* (quantity of reflected light), chromatic coordinated a
* (red-green axis) and b
* (yellow-blue axis) [11
2.4. Measurement of Total Phenolic Content and Total Flavonoid Content
The defatted residue (cocoa powder) was air dried and stored at −20 °C before being extracted in boiling water for 1 h at a concentration of 20 mg/mL−1. After cooling at room temperature, the samples were centrifuged, yielding the final extract for analysis.
The total phenolic phytochemical concentration was measured using the Folin-Ciocalteu method reported by [12
]. Afterwards, 1 mL of appropriately diluted samples and a standard solution of gallic acid were added to a 25 mL volumetric flask containing 9 mL of ddH2O. A reagent blank using ddH2O was prepared. One milliliter of Folin-Ciocalteu phenol reagent was added to the mixture and was then shaken. After 5 min, 10 mL of 7% Na2
solution was added prior to mixing. The solution was then immediately diluted to a volume of 25 mL with ddH2O and then mixed thoroughly. After incubation for 90 min at 23 °C, the absorbance relative to that of a prepared blank at 750 nm was measured using a spectrophotometer (Shimadzu UV-1601, SHIMADZU, Tokyo, Japan). The total phenolic contents of the samples were expressed in milligrams per serving of gallic acid equivalents (GAE). All samples were prepared in five replications.
The total flavonoid concentration was measured using a colorimetric assay developed by [13
]. One milliliter of appropriately diluted sample was added to a 10 mL volumetric flask containing 4 mL of ddH2O. At time zero, 0.3 mL of 5% NaNO2
was added to each volumetric flask; at 5 min, 0.3 mL of 10% AlCl3
was added; and at 6 min, 2 mL of 1 M NaOH was added. Each reaction flask was then immediately diluted with 2.4 mL of ddH2O and then mixed. The absorbances of the mixtures upon the development of pink color were determined at 510 nm relative to a prepared blank. The total flavonoid contents of the samples were expressed in milligrams per serving of epicatechin equivalents (ECE). All samples were prepared in five replications.
2.5. Measurement of Gallic Acid (GA) and Epicatechin (EC) Content
The (−)-epicatechin and gallic acid content were determined and quantified using the modified method of [14
]. Dried cocoa samples were ground in a blender. Pieces of dry ice were added to the beans to prevent the cocoa lipids from melting, due to frictional heat caused by grinding. After grinding, the powder was sieved through a 710 μm screen. Powdered samples were defatted for 16 to 18 h using petroleum ether (b.p. 40–70 °C) as solvent. The samples were dried in a vacuum oven at 65 °C for 5 min and then stored in the dark inside a desiccator over silica gel prior to the extraction procedures.
Concentrations of epicatechin and gallic acid were determined through High Performance Liquid Chromatography (HPLC, Agilent 1260 Infinity, Agilent, Waldbronn, Germany) and ultraviolet (UV, Agilent 1260 Infinity, Agilent, Waldbronn, Germany) detection (280 nm). Before analysis, defatted cocoa (4 g/L) was dissolved in 90% (v/v) water with 2% (v/v) acetic acid (pH 2.5) and 10% (v/v) acetonitrile, placed in an ultrasonic bath for 10 min and then filtered using a 0.45 μm cellulose filter. The mobile phase, at a flow rate of 1.0 mL/min, consisted of water plus acetic acid (pH 2.5, eluent A) and acetonitrile (eluent B) with the following gradients: 0.0 min, 90% A and 10% B; 20.0 min, 85% A and 15% B. Quantification was performed through external calibration with standard solutions of epicatechin and gallic acid. Results were expressed in milligram components per 10 g of cocoa product.
2.6. Measurement of Total Amino Acids
The defatted residue or cocoa powder was hydrolyzed with 6 M hydrochloric acid at 110 °C for 24 h under vacuum. The hydrolysate was submitted to an automated online derivatization with O-phthalaldehyde and reversed phase high performance liquid chromatography (RP-HPLC) analysis in an Agilent 1100 (Agilent Technology, Palo Alto, CA, USA) assembly system using a Zorbax 80A C18 column (4.6 i.d. × 180 mm) running at 0.5 mL/min. The results acquired were analyzed with the aid of ChemStation for LC 3D software (Agilent Technology, Palo Alto, CA, USA).
2.7. Measurement of Free Amino Acids
Determination of free amino acids was carried out using the extraction method of [15
], with slight modifications. Only l
-amino acids were quantified as cocoa flavor precursors. Seven hundred milligrams of defatted powder and 1.4 g of polyvinylpyrrolidone (PVP) were homogenized for 5 min at 0 °C in 15 mL distilled water and then adjusted to pH 2.5 using glacial acetic acid. The mixture was then centrifuged at 13,000× g
for 15 min and then filtered through Whatman No. 4 filter paper. The filtrate was made to reach 50 mL using distilled water. Twelve milliliters of acetone and dl
-butyric acid (AABA, internal standard) were added to 3 mL of the filtrate. The mixture was then mixed thoroughly using a Polytron homogenizer, kept at room temperature for 30 min and centrifuged at 13,000× g
for 15 min. Acetone was then removed by streaming with nitrogen gas. The amino acids were converted into phenylthiocarbamyl (PTC) amino acids using phenyl isothiocyanate (PITC). Twenty microliters of sample extract were used. The free amino acids were separated using RP-HPLC with gradient elution at a flow rate of 0.8 mL/min. Free amino acids were detected at 254 nm. Solvent A of the gradient elution was acetate buffer at pH 5.7, and solvent B was acetonitrile:deionized water (60:40). The gradient elutions were as follows: 0 min, 100% A, 0% B; 5 min, 75% A, 25% B; 13 min, 52% A, 48% B; 13.5 min, 0% A, 100% B; 16.5 min, 0% A, 100% B; 17 min, 100% A, 0% B; and 22 min, 100% A, 0% B. A Waters Pico-Tag Free Amino Acids Column (3.9 mm × 300 mm i.d., Waters, Millipore Corporation, Milford, MA, USA) was used for the analysis, which was employed at a temperature of 37 °C.
2.8. Measurement of Flavor with Electronic Nose (E-Nose)
The e-nose (model Gemine, Alpha M.O.S., Toulouse, France) with MOS chambers equipped with six sensors (LY/AA, LY/gCT, T30/1, P30/2, T70/2, PA/2) was connected to an auto sampler (CTC 100). A change in mass of a chemical compound caused a change in electrical resistance, as indicated by each sensor.
A 3.0 g mashed sample was placed into a glass vial. Using a crimping tool, each sample vial was sealed with a fitted cap and septum. Each vial was sealed tightly to prevent leaks and uncharacteristic decrease in sensor signal. The sample vials were then placed in the instrumental tray for further analysis. In this study, the sample was incubated at 50 °C for 10 min. The data collected using the e-nose were analyzed using principal component analysis (PCA) to differentiate the cocoa beans.
2.9. Statistical Analysis
One-way single-factor analysis of variance (ANOVA) was performed using SPSS software (version 16.0, SPSS Inc., Chicago, IL, USA). The F ratio was used to determine statistical significance at p < 0.05. A multiple-comparison test using Fisher’s least significance difference (LSD) was conducted. PCA was employed to describe the variability of sensory data.
The varieties, growing conditions and fermentation of cocoa beans seem to influence their average bean weight, level of polyphenols and amino acids. Such observation held true, as the present study found varying values in the cocoa bean samples used. Although the Hainan cocoa beans were lighter than the other samples, their total polyphenolic and flavonoid content were higher or equal to the cocoa beans from Papua New Guinea and Indonesia. Aside from the interest in the quality standards and economic specification of raw material, manufacturers today pay great attention to declaring their products as functional food. Thus, our data provide additional knowledge to be considered in the promotion of cocoa plantations in China.
The results obtained from this study are essential in understanding and solving the problems associated with the quality of raw cocoa beans. Further research is needed to determine the effect of the growing conditions, storage time and fermentation on the physico-chemical and flavor quality attributes of industrial raw cocoa material. Such a direction is intended for improving the quality of raw cocoa beans sourced from China, Indonesia and Papua New Guinea.