Abstract: Background and Objective: Cocoa bean shell (CBS) has phenolic content and potential as cheaper source of antioxidants. The objective of this study was to evaluate the effects of cocoa bean fermentation on total phenolic content and antioxidant activity of cocoa bean shell. Materials and Methods: Cocoa beans were placed in fermentation box following spontaneous fermentation for 24, 48, 60, 72, 96 and 120 h. The CBS was separated from the cocoa beans and extracted with acetone-water (70:30 v/v). Folin-Ciocalteu method, 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and Fourier Transform Infrared Spectroscopy (FTIR) analysis were used to determine total phenolic contents, antioxidant activity and functional group of the samples, respectively. Results: For total phenolic contents, absorbance of various samples concentrations was plotted against gallic acid standard curve and the value indicated that CBS from partially fermented cocoa beans (24 h) contained the highest amount of phenolic content. Various concentrations of samples were also used for antioxidant activity measurement. Highest scavenging activity of 88.67±1.12% was obtained by 100 μg mL–1 CBS from partially fermented cocoa beans (24 h), while the lowest of 44.60±2.48% was obtained by CBS from 120 h fermented cocoa beans. A high correlation was observed between phenolic content and antioxidant activity. The FTIR analysis shows that CBS from 24 h fermented cocoa beans had more functional group compare to other samples. Conclusion: Fermentation of cocoa bean can affect total phenolic content and antioxidant activity of the CBS. Fermentation also causes functional group shift of CBS. The results of the study showed that CBS is a good source of antioxidants and phenolic compounds. Further study on the structural elucidation of the CBS individual phenolic compounds and evaluation of their mechanisms of action is recommended.
INTRODUCTION
Cocoa bean shell (CBS) is cocoa industry by-product with low economic value. The CBS is part of the cocoa bean that separated from the cotyledons. The CBS makes about 15% of the weight of the cocoa bean1. Indonesia processed 425.000 t of cocoa beans in 2014, generating approximately 63.750 t of shells2.
Cocoa beans are reportedly contain high level of polyphenols with antioxidant activity, mainly flavan-3-ol (monomeric epicatechin and catechin, as well as their oligomers from dimers to decamers, the procyanidins), with small amounts of anthocyanin (mainly cyanidin glycosides) and flavonols (quercetin glycosides)3,4. Their polyphenolic structure has one or more aromatic ring and at least has two hydroxyl groups5, with ability to inhibit free radicals3. The chemical structure of cocoa flavanols and procyanidins determine their antioxidant activity play as radical scavenging and metal chelating agent6.
However, most of studies are mainly concentrated on cocoa bean with far less attention on the CBS, despite its potential as cheaper source of antioxidants. It has beneficial effects such as antioxidants7-10, antimicrobial11 and anti-caries1,12,13. Nsor-Atindana et al.11 reported that total polyphenolic and total flavonoid content of CBS were 41.82 mg GAE g–1 and 5.49 mg CE g–1, respectively. Its polyphenolic compounds are procyanidins, epicatechin, p-hydroxybenzoic acid, anthocyanin, proanthocyanin and clovamide7,14,15.
Cocoa beans fermentation is a critical step in cocoa processing for the development of the chocolate flavour and taste due to aroma precursors generation and affect phenolic content of cocoa beans. Fermented cocoa beans had much lower epicatechin content than the partially fermented16. Moreover, polyphenol content of CBS from fermented cocoa beans is higher than CBS from partially fermented cocoa beans. Cocoa beans fermentation lead in death and cell permeability damaged of seed, resulting diffusion of polyphenol compounds from pigment cells to all parts of the cotyledons. During fermentation, the migration of epicatechin from cocoa beans into CBS has occurred16,17. Coinciding with the decreased of epicatechin in the bean, epicatechin in the shell increased, during the second and third day of fermentation. At the end of fermentation (6 days), epicatechin content of both cocoa beans and shells decreased16. Although, polyphenolic compounds usually accumulate in the outer parts of plants such as shells, skins, etc.18, there is limited information about the effect of cocoa beans fermentation to the antioxidant compound of CBS. The objectives of this study were to evaluate the effects of cocoa beans fermentation on phenolic content, antioxidant activity and to determine the functional groups of CBS extract using FTIR analysis.
MATERIALS AND METHODS
Materials: The CBS used in the experiments were obtained from fermented cocoa bean (24, 48, 72, 96 and 120 h of fermentation) and CBS from unfermented cocoa bean used for comparison. Forastero cocoa was collected in June 2015 from a local farmer in Yogyakarta, Indonesia. Hexanes, methanol and gallic acid were purchased from Merck Millipore (Germany), acetone was purchased from Mallinckrodt (Ireland). The DPPH and Folin-Ciocalteu were purchased from Sigma-Aldrich (Germany). All other chemicals and solvents were of analytical grade.
Fermentation: Cocoa pods were sorted based on visual appearance and those with physical damage or disease symptoms were dismissed. Selected pods were manually opened and the beans were placed in boxes following spontaneous fermentation for 120 h. Cocoa mass was manually mixed every 48 h in a wooden box with approximately 100 kg of cocoa raw beans stored per unit. Nibs were manually sampled, daily and in the center of the fermentation mass, about 5 kg each sampling. Samples were washed to stop the fermentation, then dried for a week using combination of solar and cabinet drying. The end of drying was determined when cocoa beans moisture content was down to 7%.
Extraction procedure: The CBS was separated from the cocoa beans and subjected to the procedure described in previous method with some modifications1,19. Dried samples were ground in a blade grinder. Approximately 20 g of the ground CBS was defatted with 40 mL of hexane for 30 min in a magnetic stirrer and followed by centrifugation at 2.054 g for 20 min. This defatting procedure was repeated twice. Afterwards, defatted CBS material was extracted with a 400 mL of acetone-water (70:30), stirred for 4 h and centrifuged at 2.054 g for 30 min. This extraction procedure was repeated three times. Acetone was evaporated at 40°C and under vacuum. Concentrated extracts were dried with freeze dryer.
Total phenolic compounds: Total phenolic content of the extracts was determined by a colorimetric Folin-Ciocalteu reagent assay20. Briefly, 0.1 mL of extract was added to test tube, followed by the addition of 0.5 mL of the Folin Ciocalteu reagent and 6 mL of distilled water. After mixing and resting for 3 min, 20% sodium carbonate was added (w/v, 1.5 mL) and the test tube was adjusted with distilled water to 10 mL. After 2 h incubation at room temperature, absorbance was determined spectrophotometrically at 760 nm. Distilled water was used as a blank or control. Gallic acid (25-500 μM) was used as standard and the results were expressed as mg gallic acid equivalents per gram of dried weight (mg GAE g–1 DW). Analyses were carried out in triplicates.
DPPH radical scavenging activity: Scavenging free radical potentials were tested in the ethanolic solution of DPPH with a slight modification21. From aqueous extracts at different concentrations, solution was prepared by adding 1 mL of the extract and 2 mL of the DPPH solution. To determine the antioxidant activity, ethanol solution of 0.1 mM DPPH was prepared. The absorbance was measured at 517 nm. A reference sample was prepared with 1 mL methanol. The BHT was used as positive control. The antiradical activity was calculated as a percentage of DPPH decoloration using the following equation:
FTIR analysis: FTIR spectra of all samples were recorded between 4000 and 400 cm–1 using Shimadzu IR Prestige 21 spectrometer for all the samples. The discs were prepared by first mixing 1 mg of dried sample with 100 mg of potassium bromide in an agate mortar and then pressing the resulting mixture at 10 t cm-2.
Statistical analysis: Data were reported as Means±Standard Deviation. Statistical comparisons were evaluated by analysis of variance (ANOVA) using SPSS (version 16). Duncan MRT Post-ANOVA test was used to compare treatment means. The difference between treatment means was judged at 5% significance level.
RESULTS AND DISCUSSION
Extraction yields and total phenolic content: The yield of extracted materials and their total phenolic contents are reported in Table 1. This study used 70% acetone (70:30 acetone:water) as the most effective solvent for CBS antioxidants extraction10. The yield varies from 14.22% (extract from 48 h of fermentation) to 23.11% (extract from 72 h of fermentation).
| Table 1: | Yields and total phenolic contents of CBS extracts |
The data given are Mean±Standard Deviation for three different values of each sample. GAE: Gallic acid equivalents. Means within the same column with different letters differ significantly (p≤0.05) |
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Result in Table 1 showed that total phenolic content of CBS extract from unfermented and fermented cocoa bean tested varied, ranging from 19-39 mg GAE g–1 dry weight. The CBS extract from partially fermented cocoa bean (24 h) was found to have the highest total phenolic content (39.41 mg GAE g–1 dry weight). There was an increase in total phenolic content at 24 h of fermentation, but decreased with longer fermentation. Increasing polyphenols content due to diffused of polyphenols out of the cells through the CBS. Diffusion continues until 72 h of fermentation, no phenol was detected in cocoa bean cells22. Polyphenols diffuse along with cell liquids from their storage cells, oxidized and forming into high molecular mass, such as insoluble tannins. Polyphenol oxidase converts polyphenols into quinones. Polyphenols and quinones then form a complex with other polyphenols, proteins and peptides23. This might contribute to the increase of total phenolics or phenolics like complexes that contributed to higher absorbance readings.
After 24 h of fermentation, total phenolics were decreased until the end of fermentation (120 h). This could be attributed to the degradation or polymerization products of CBS polyphenols during fermentation. Mariani24 reported that total phenolic content of CBS from unfermented cocoa bean as 22.72 mg GAE g–1 dry weight and from fermented cocoa bean as 14.8 mg GAE g–1 dry weight. This were in line with other study reported that the total polyphenols in cocoa beans decreased by 10% during 6 days of fermentation25. During fermentation, polyphenol undergoes polymerization and form a complex with the protein26. In the cocoa bean, polyphenols content was reduced from 16.11% (unfermented) to a 6.01% in 6 days of fermentation27.
DPPH radical scavenging activity: In this study, free radical (DPPH) scavenging method was used to measure the total antioxidant activity of CBS extracts. Discoloration of violet color indicates that DPPH free radicals were quenched or reduced by the antioxidants of the sample.
| Fig. 1: | Correlation between free radical scavenging activity and total phenolic content of CBS extract. The CBS extract from fermented cocoa bean (24, 48, 72, 96 and 120 h of fermentation) and unfermented (0 h) cocoa bean |
| Table 2: | Antioxidant activity of CBS extracts at different concentration |
a-dEach value is presented as Mean±SD (n = 3). Means within the same column with different letters differ significantly (p≤0.05) |
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Total antioxidant activity shown in Table 2 was expressed as a percentage of inhibition.
Total antioxidant activity of CBS extracts were significantly affected by fermentation. In line with total phenolic content, CBS extract from partially fermented cocoa bean (24 h) was found to have the highest scavenging effect, while the lowest was found at 120 h of fermentation were consistent with the decreasing pattern of total phenolic. The order of total antioxidant activity obtained by different fermentation time was 24>48>96>72>0>120 h. Other study reported that antioxidant capacity (percentage of inhibition DPPH free radicals) of cocoa bean decrease from 96% in the first day of fermentation to 79% in the 6th day27. The results of CBS crude extract were still lower compare to those of BHT as pure compound. Total antioxidant activity of BHT at 10 and 100 μg mL–1 were 62.55 and 88.67%, respectively.
Antioxidant activity of phenolic as free radical scavenger associated with antioxidant capability such as a proton donor. Hydrogen protons donation is largely influenced by the number and position of the aromatic hydroxyl groups or hydroxyl from phenolics28,29.
Correlation between antioxidant compounds and antioxidant activity: The coefficient of correlation between the phenolic compound and antioxidant activity of the CBS is shown in Fig. 1.
Antioxidant activity as free radical scavenging was significantly correlated (p<0.05) to total phenolic content with R2 = 0.926 as shown in Fig. 1. Significant positive correlations indicated that free radical scavenging activity is mainly attributed to total phenolic content. In this study, total phenolic content was more likely to be responsible to scavenge most of free radicals in the studied. Therefore, it could be expected that phenolic compounds might have been the major contributor to antioxidant activity in the CBS.
Phenolic compounds activity largely depends on their molecular weight, structure and concentration of these compounds. The configuration and total number of hydroxyl groups in the flavonoid molecules influence their radical scavenging efficiency. The presence of the ortho-dihydroxy (catechol) structure in the B-ring, 2,3-double bond in conjugation with a 4-oxo function in the C-ring and hydroxyl groups at positions 3 and 5 enhances radical scavenging activity. Furthermore, the antioxidant activity of procyanidins also depends on their degree of polymerization30. Thus properties (hydrophilic or lipophilic) of certain compounds present in the cocoa samples may affect the results of antioxidant activity assays31.
FTIR analysis: The FTIR analysis to determine functional group of CBS extracts was carried out at wave number 4000-400 cm–1 The FTIR spectrum of CBS crude extract can be seen in Table 3. Vibration will occur when infrared radiation is absorbed by the bond in a molecule. The amount of absorption of a bond depends on the type of bond vibration. Therefore, different types of bonds will absorb infrared radiation at different wavelengths.
Table 3 shows that functional groups area between 4000-1400 cm–1 had identical spectrum, suggested that CBS polyphenols, either from unfermented or fermented, had identical functional groups. Changes in the CBS spectra from unfermented and fermented cocoa bean occur due to shift in the functional group during fermentation process.
Spectra at 3410-3394 cm–1 shows the absorption intensity was very strong and wide, indicating an O-H stretch. In the polyphenol molecule, the O-H bond is the most responsible of antioxidant activity as proton donor.
| Table 3: | FTIR spectrum band of CBS |
Changes in the number of OH due to fermentation determined the changes of antioxidant activity, thus lower number of O-H lead to smaller antioxidative activity. The antioxidative activity of polyphenols is also determined by O-H position in the molecule. The O-H bond at C3, C4 and C5 position in B-ring and O-H at C3 in C-ring play an important role. Loss of OH other than those positions did not affect antioxidative activity32.
The CBS obtained from 24 h of cocoa bean fermentation had more functional groups than others, thus had the highest antioxidant activity. Spectra at 1720 cm–1 showed a C=O stretch which came from carboxyl groups that are supported by very wide O-H absorption due to the bonding of hydrogen with its dimer. The CBS (24 h of fermentation) also has C-O-C symmetric (the glycoside bond) that are shown at 1103 cm–1.
CONCLUSION AND FUTURE RECOMMENDATIONS
The results of the study showed that CBS is a good source of antioxidants and phenolic compounds. It revealed that fermentation of cocoa bean can affect total phenolic content and antioxidant activity of the CBS. Fermentation also causes functional group shift of CBS. Further study on the structural elucidation of the CBS individual phenolic compounds and evaluation of their mechanisms of action is recommended.
SIGNIFICANCE STATEMENTS
The CBS is cocoa industry by-product with low economic value and contain polyphenolic compounds for example procyanidins, epicatechin and anthocyanin. However, most of studies are mainly concentrated on cocoa bean with far less attention on the CBS. Cocoa beans fermentation is a critical step in cocoa processing for the development of the chocolate flavor that can affect phenolic content of cocoa. In this study we evaluated the effects of cocoa beans fermentation on phenolic content, antioxidant activity and to determine the functional groups of CBS extract using FTIR analysis. This research is useful to prove that CBS has potential as cheap source of antioxidants.
ACKNOWLEDGMENT
This study was supported by The Pilot Program for Doctoral Degree Fund, Research and Development of Industry, Ministry of Industry with grant No. 50/BPKIMI/Kep/02/2015. The authors have no conflicts of interest to declare.