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Introduction
Theobroma cacao L. has been cultivated in Central America since pre-Columbian times and has spread from the Amazon Basin of Guyana and the Upper Orinoco to southern Mexico and the tropics of Africa and Asia (Espinosa-García et al., 2015; Porto de Souza-Vandenberghe et al., 2022). The motivation for this crop today is to respond to the growing demand for cocoa, mainly in Europe, the United States and Asia, and to contribute to the economic and social development of the agricultural sector in more than 70 countries in the tropics, with approximately 5 million producers worldwide (Alonso-Báez et al., 2020; Gómez Hoyos et al., 2020; Porto de Souza-Vandenberghe et al., 2022). The International Cocoa Organization reports that the world's major cocoa producers are Côte d'Ivoire, Ghana and Ecuador. In the 2022 harvest, 5.24 million tons of cocoa were produced, with African countries contributing about 75 % of the world's annual production, with Côte d'Ivoire being the most representative with about 44 % (Paracchini et al., 2022). Mexico is the thirteenth largest cocoa producing country in the world, with Chiapas and Tabasco being the states with the highest production in the country with just over 20 000 tons of cocoa per year, representing 0.4 % of world production (Alonso-Báez et al., 2020).
The cocoa fruit is composed of approximately 10 % of the seed, as this is the product that is marketed, the other 90 % of the fruit remains as residual biomass, which is composed of the cocoa been shell (CBS), mucilage or pulp, placenta, and cocoa pod husk (CPH). The CBS is the covering that covers the cocoa seed, representing between 10 and 20 % of the total weight of the seed; the CPH is the covering of the ripe fruit, once the seed is removed, the pulp and placenta represent about 5 % of the weight of the fruit. These parts represent the largest volume of residual biomass, between 70 and 80 % of the weight of the fruit, so it is estimated that more than 16 million tons of CPH per year around the world (Sánchez et al., 2023; Vásquez et al., 2019).
CPH is an undervalued resource; it tends to remain in growing areas and accumulates in large piles, so its decomposition can cause environmental and phytosanitary problems, such as plant diseases that directly affect crop yields (Nii et al., 2021). This shell is composed of epicarp, mesocarp, a sclerotic lamina, and endocarp, whose chemical composition is cellulose, hemicellulose, lignin, pectins, and ash (Campos-Vega et al., 2018; Grob et al., 2021). Cellulose is a linear polymer of D-glucose units linked by β-1,4 bonds. Hemicellulose is a branched polysaccharide composed of pentoses and hexoses. Lignin is an aromatic heteropolymer composed of phenylpropane units (p-coumarilic, sinapyl, and coniferyl alcohols) (Zheng et al., 2017). These compounds can be depolymerized into molecules that serve as the basis for obtaining value-added chemical products; for example, the fermentation of cellulose and hemicellulose sugars could yield biofuels or sweeteners; from lignin, pesticide dispersants, emulsifiers, or their incorporation into polyurethanes and polyesters (Asiedu et al., 2019; Chávez-Sifontes & Domine, 2013; Porto de Souza-Vandenberghe et al., 2022). Likewise, its pectin content, composed mainly of uronic acid, has potential as a food additive (Paz-Cedeno et al., 2022). Finally, phenolics and flavonoids with oxide-reducing or antioxidant effects could be used in functional foods (Valadez-Carmona et al., 2017). CPH is considered a potential, renewable, low-cost, and readily available resource, attractive to the industrial sector and of interest to the scientific community, which is seeking alternatives for valorization and the development of technologies and patents for their use. This could bring additional benefits to the cocoa production chain (Porto de Souza-Vandenberghe et al., 2022).
Three types of cocoa are recognized, two morpho-geographical groups known as Criollo and Forastero cocoa, and a third derived from the natural cross between Criollo and Forastero, called Trinitario. Research has primarily focused on the Forastero and Trinitario, while little is known about the Mexican Criollo (Ricaño-Rodríguez et al., 2018). In Mexico, there is a variety called ‘Carmelo’, recognized under Breeder's Right 1036, granted to producer C. Carlos Hernández Echeverría, who through a breeding process obtained this variety, considered homogeneous, distinct, and stable. This was published in the Official Gazette of the Federation (DOF: 29/09/2015) and established by article 4o. fraction I, of the Federal Law on Plant Varieties, Carlos Hernández Echevarria’s Right to be recognized as Breeder of the Cocoa Plant Variety (Theobroma cacao L.), named ‘Carmelo’ (Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación [SAGARPA], 2015). The Carmelo variety has its genetic origin in Criollo (Alonso-Báez et al., 2020). In this regard, the limited information available on Mexican Criollo cocoa is mainly related to cocoa seeds, so even less is known about by-products compared to vegetative varieties, and much less is known about the Carmelo variety, as due to the breeder's right, valid until 2031, this variety has been little propagated and studied.
Therefore, the objective of this research was to determine the antioxidant activity and chemical characterization of the main components of the shell of the pod of T. cacao variety Carmelo. To contribute to the knowledge of this variety and present elements of valorization of a highly available residue generated by the cocoa agroindustry.
Materials and methods
Raw material and study area
The raw material was the cocoa pod husk (CPH) of T. cacao variety Carmelo, which consists of the exocarp, mesocarp, sclerotic layer, and endocarp. The samples were collected from two locations: ranchería Río Seco in the municipality of Cunduacán (latitude: 18° 7' 55.90" N, longitude: 93° 18' 4.49" W) and the farm Jesús María in the municipality of Comalcalco (latitude: 18° 11' 0.22" N, longitude: 93° 14' 28.02" W), both located in the state of Tabasco, Mexico. The collection sites were at an elevation of 10 and 13 m (Figure 1). The plant material was provided by the C. Carlos Hernández Echeverría.
Reagents
Reagents: 96 % sulfuric acid (H2SO4), 99 % glacial acetic acid (CH₃COOH), 80-85 % sodium chlorite (NaClO2), 99 % sodium hydroxide (NaOH), 99.5 % sodium carbonate (Na2CO3), Folin-Ciocalteu phenol reagent, diammonium salt (ABTS) 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) 98 %, and (±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, also known as Trolox, were purchased from SUPELCO (Merck KGaA, Saint Louis, Missouri, USA).
Cocoa pod husk preparation
The cocoa pod husk (CPH) were dried at room temperature with partial sun exposure for 18 days. Subsequently, they were ground in a laboratory mill (Thomas Wiley 4, Pennsylvania, USA). The ground material was then sieved using U. S. STD 45 and 60 mesh sieves (W. S. Tyler, Ohio, USA) to obtain particle size classifications of 355 μm and 250 μm, respectively. The meal retained on the 250 μm particle size 60 mesh was used for all chemical determinations. Finally, the samples were weighed as appropriate for each determination and dried to a constant weight in a conventional oven at 102 °C.
Main chemical components
The determination of lignocellulose was based on the use of two standards from the Technical Association of the Pulp and Paper Industry (TAPPI), one standard from the American Society for Testing and Materials (ASTM), and a widely used chlorite-acetic method for the disintegration of lignocellulosic material (Rowell, 2012). TAPPI standard 204 cm-97 was used with modifications for sample preparation, solvent extraction, and quantification of extracts (TAPPI, 1997). The solvents used in this standard were replaced by hexane, dichloromethane, and methanol to eliminate the use of benzene, which is highly toxic and carcinogenic, as specified in the standard. For the determination of lignin, TAPPI standard 222 om-02 was used (TAPPI, 2002a); for ash quantification, TAPPI standard 211 om-02 (TAPPI, 2002b); and for the contents of holocellulose and cellulose ASTM standard D 1103-77 (ASTM, 1985). Finally, the hemicellulose content was quantified by weight difference. All determinations were performed in quadruplicate and the results were expressed as mean percentages ± standard deviation.
Minerals Profile
To profile and quantify the minerals present in CPH, 0.5 g of CPH was placed in a microwave Teflon cell (Xpress Plus CEM), followed by the addition of 10 mL of 69 % HNO3. The sample was then introduced into a CEM microwave instrument, model Mars 6, using the U. S. EPA 3051 method for sample digestion (U. S. Environmental Protection Agency, 2007). The resulting digestion was volumized to 25 mL with deionized water to facilitate the identification and quantification of the concentration of each metal. Subsequently, an iCAP Q ICP-MS instrument (Thermo Scientific, Bremen, Germany) was used to identify and quantify the concentration of each metal (µg∙g-1), employing a calibration curve of standard solutions. Mineral determinations were conducted in triplicate, and the results were expressed as the mean ± standard deviation.
Methanolic extract
An extract was prepared with 0.5 g dry base of CPH and 25 mL of 80 % methanol, adjusted to pH 3 with 0.1 N HCl. This mixture was subjected to vortex agitation at 3 000 rpm for 3 min, followed by sonication for 15 min, then placed in a rotary incubator at 30 °C and 150 rpm for 30 min, finally it was centrifuged at 3 000 rpm for 20 min, the excess was transferred to a volumetric flask and made up to 25 mL with 80 % methanol. This extract was used to determine the antioxidant properties of the CPH. The method used was reported by Hernández-Rodríguez et al. (2019), who indicate that, since phenolic compounds are polar substances, they can be extracted using organic solvents that have been acidified to promote protonation.
Phenolic content
The total phenolic content of CPH was determined by the method reported by Hernández-Rodríguez et al. (2016). Aliquots of 25 μg of methanolic extract of CPH were prepared, to which 25 μL of distilled water and 20 μL of Folin-Ciocalteau reagent were added, then 30 μL of 20 % Na2CO3 solution was added and allowed to react for 30 min in the absence of light, the absorbance was measured at a wavelength of 760 nm in a μQuant microplate reader (Biotek, Instruments Inc., USA). The result was expressed as milligram equivalents of gallic acid per gram of cocoa pod dry weight (mg GAE∙g-1).
Flavonoid content
The total flavonoid content was determined according to the method of of Hernández-Rodríguez et al. (2019), 0.5 mL of CPH methanolic extract was mixed with 2.5 mL of distilled water and 0.15 mL of 5 % NaNO2 solution. The mixture was allowed to stand for 6 min, after which 0.3 mL of 10 % AlCl3 6H2O was added. Following a 5-minute rest, 1 mL of 5 % NaOH was added and the solution was vortexed at 3 000 rpm for 3 min. Subsequently, 200 µL of the aliquot was added to the microplate in quadruplicate, and absorbances were measured at 510 nm using the μQuant microplate reader (Biotek Instruments Inc., USA). The result was expressed as milligram equivalents of catechin per gram of cocoa pod shell dry matter (mg CE∙g-1).
ABTS (2,2′-azino-bis[3ethylbenzothiazoline-6-sulfonic acid]) assay
The assay was carried out using the method described by Arzeta-Ríos et al. (2020). First, solution 1 was prepared by combining 7.4 mM ABTS and 2.6 mM sodium persulfate in equal volumes of 10 mL, and then incubating the mixture for 16 h at room temperature in the absence of light. Next, 600 µL of solution 1 was taken and topped up to 10 mL with absolute methanol. In a microplate reader μQuant (Biotek, Instruments Inc., USA), 20 µL of CPH extract, 20 µL of Trolox solution in concentrations ranging from 5 to 60 µL, and 180 µL of solution 1 were placed. The absorbances were then read at 734 nm after 10 minutes, and the data from the calibration curve were used to determine the results, which are expressed in µmol of Trolox equivalents per gram of CPH dry base (µmol TE∙g-1) required to trap the ABTS radical.
FRAP (ferric reducing antioxidant power) assay
The FRAP assay was carried out following the method described by Hernandez et al. (2019), FRAP reagent was prepared using a mixture of sodium acetate buffer (300 mM, pH 3.6), 10 mM 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) dilution (in 40 mM HCl), and 20 mM ferric chloride dilution in a 10:1:1 ratio. An aliquot (20 μL) of the extract was mixed with 180 μL of the FRAP dilution and 60 μL of distilled water. The absorbance was measured at 595 nm using a μQuant microplate reader (Biotek, Instruments Inc., USA). The results were expressed as µmol Trolox equivalents per gram of CPH dry weight (µmol∙g-1).
The determinations of total phenolic content, flavonoid content, the ABTS and FRAP assay were performed in quadruplicate, and the results were expressed as the mean ± standard deviation.
Results and Discussion
The Carmelo variety is characterized by its elongated fruit with an absent to weak basal constriction, an obtuse apex shape, medium length, a large diameter, a moderately elongated length-to-diameter ratio, an orange exocarp, a moderately rough surface, medium depth of furrows, medium thickness, light cream flesh, a low number of white seeds, and a sweet taste.
The determination of extractables in polar solvents, lignocellulosic constituents, mineral profile and content, quantification of phenolics, flavonoids, and antioxidant activity of T. cacao variety Carmelo shells is shown in Table 1.
Table 1 Composition of the major chemical constituents of the cocoa pod husk of Theobroma cacao L. variety Carmelo.
| Chemical Constituent | Average ± σ This study | Average Other research | Reference | |
|---|---|---|---|---|
| Lignocellulosic (%) | Hexane extractables | 0.06 ± 0.01 | ND | This work |
| Dichloromethane extractables | 0.32 ± 0.05 | ND | This work | |
| Methanol extractables | 19.01 ± 1.29 | ND | This work | |
| Total extractables | 19.39 ± 1.35 | 23.66 | Titiloye et al. (2013) | |
| Lignin | 23.28 ± 0.44 | 24.16 27.90 14.70 |
Sandesh et al. (2020)
Redgwell et al. (2003) Daud et al. (2014) |
|
| Cellulose | 30.52 ± 1.03 | 30.41 28.25 35.40 35.00 |
Titiloye et al. (2013)
Sandesh et al. (2020) Daud et al. (2014) Campos-Vega et al. (2018) |
|
| Hemicellulose | 19.09 ± 1.44 | 16.75 22.40 |
Sandesh et al. (2020)
Redgwell et al. (2003) |
|
| Ashes | 7.75 ± 0.69 | 8.32 / 8.42 6.70 / 10.02 6.40 / 8.40 6.70 / 13.00 |
Martínez et al. (2012)
Campos-Vega et al. (2018) Lu et al. (2018) Vásquez et al. (2019) |
|
| Minerals (µg∙g-1) | Al | 22.79 ± 2.46 | ND | This work |
| V | 0.06 ± 0.00 | ND | This work | |
| Cr | 0.37 ± 0.01 | ND | This work | |
| Fe | 164.44 ± 1.79 | 58 466 |
Vriesmann et al. (2011)
Aregheore (2002) |
|
| Co | 0.15 ± 0.01 | ND | This work | |
| Ni | 10.70 ± 0.30 | 3.10 / 6.27 | Barraza et al. (2021) | |
| Cu | 6.91 ± 0.24 | 0.55 6.18 |
Moyin-Jesu (2007)
Vriesmann et al. (2011) |
|
| As | 0.04 ± 0.00 | ND | This work | |
| Cd | 1.00 ± 0.02 | 0.60 / 0.80 0.16 / 0.27 | Gramlich et al. (2016) | |
| Ba | 63.58 ± 0.36 | 98 | Barraza et al. (2021) | |
| Zn | 63.44 ± 0.86 | 41.50 / 47.00 42.90 / 45.80 90 39 |
Barraza et al. (2021)
Gramlich et al. (2016) Aregheore (2002) Vriesmann et al. (2011) |
|
| Hg | 0.03 ± 0.02 | ND | This work | |
| Pb | 0.18 ± 0.01 | ND | This work | |
| Total phenolic compounds (mg GAE∙g-1) | 24.59 ± 0.93 | 24.80 / 27.30 17.62 19.83 34.88 35.53 16.57 3.23 2.06 3.52 |
Nieto-Figueroa et al. (2020)
Jamaluddin et al. (2022) Jamaluddin et al. (2022) Jamaluddin et al. (2022) Castro-Vargas et al. (2019) Siqueira-Melo et al. (2011) Valadez-Carmona et al. (2017) Martínez et al. (2012) Martínez et al. (2012) |
|
| Flavonoids (mg CE∙g-1) | 2.35 ± 0.24 | 1.10 / 1.30 0.97 0.56 |
Nieto-Figueroa et al. (2020)
Valadez-Carmona et al. (2017) Castro-Vargas et al. (2019) |
|
| ABTS (µmol TE∙g-1) | 304.84 ± 57.18 | 227.70 / 147.00 30.60 24.13 42 / 24 |
Nieto-Figueroa et al. (2020)
Valadez-Carmona et al. (2017) Martínez et al. (2012) Lu et al. (2018) |
|
| FRAP (µmol TE∙g-1) | 145.80 ± 3.84 | 521.73 1.98 |
Jamaluddin et al. (2022)
Martínez et al. (2012) |
|
GAE: gallic acid equivalents (dry basis); CE: catechin equivalents (dry basis); TE: trolox Equivalent (dry basis). ABTS: 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid); FRAP: ferric reducing antioxidant power.
Extractables in organic solvents
In the determination of extractives from lignocellulosic materials using ethanol-benzene solvents, fats, waxes, phytosterols, low molecular weight carbohydrates, resins and salts can be obtained (Jani & Rushdan, 2014). The total content of cocoa pod shell extracts obtained in this study was 19.4 %, of which 19 % corresponds to those soluble in methanol, which could be low molecular weight carbohydrates and salts, 0.32 % corresponds to those soluble in dichloromethane, and a percentage of 0.068 % are those extracted in hexane, which according to the (TAPPI, 1997) standard could be waxes and fats. Despite the exclusion of benzene as a solvent of low polarity compounds, the content of extractables determined in this work is close to the 23.66 % reported by Titiloye et al. (2013) for CPH, who did not specify the composition of these extractables. In this sense, it can be observed that the substitution of benzene did not alter the release of CPH extractables for subsequent lignocellulosic determinations, in which it is necessary to have the material free of these extractables.
Lignin content
The lignin content in this study was 23.28 %, similar to the 24.16 % reported by Sandesh et al. (2020) and close to the 27.9 % reported by Redgwell et al. (2003) in cocoa pod using the Klason lignin method. On the other hand, Daud et al. (2014) determined 14.7 % lignin in CVC, a value much lower than that found in the present study and those cited above. According to Barhoum et al. (2020), the lignin content reported for other lignocellulosic materials such as wood is usually between 10 and 25 % depending on the species, some values reported for eucalyptus and pine are 21.5 % and 20 %, respectively, very similar to those reported in this study. Lignin has high value-added applications as a fertilizer, dispersant, chelating agent, and because of its high carbon content, it is a potential precursor for materials such as activated carbon and carbon electrodes. The paper industry generates 75 million tons of lignin per year in the pulp production process from wood fibers (Yao et al., 2022). However, this industry has been replacing wood fibers with recycled fibers as an environmental strategy (Jochem et al., 2021), so the production of lignin from CPH could be a viable option.
Cellulose and hemicellulose content
The cellulose content was 30.52 %, which is similar to the 30.41 % reported by Titiloye et al. (2013) and close to the 28.25 % reported by Sandesh et al. (2020). Other cellulose contents of CPH are 35.4 % and 35 % reported by Daud et al. (2014) and Campos et al. (2018) respectively, using the chlorite method as in this work. The cellulose content of other lignocellulosic materials, such as pine and eucalyptus wood, is 40 and 54 %, respectively (Barhoum et al., 2020) which is much higher than that of CPH. The hemicellulose content was 19.09 %, which is between the values of 16.75 % and 22.4 % of cocoa pod hemicelluloses reported by Sandesh et al. (2020) and Redgwell et al. (2003), respectively. According to Barhoum et al. (2020), wood biomass has a hemicellulose content ranging from 11 % to 35.9 % for hardwoods and 24 % to 27 % for softwoods. Agricultural and forestry residues represent a viable option for the production of alternative fuels (Devi et al., 2021). CPH of the Carmelo variety have potential as a source of cellulose and hemicellulose, comparable to cocoa husks from Chiapas, Mexico, which were studied and reported by Hernández-Mendoza et al. (2021) as having potential for bioethanol production.
Ash content
The ash content is 7.75 %, similar to 8.42 and 8.32 % of different origins studied by Martinez et al. (2012), and between the ranges of 6.7 to 10.02 %, 6.4 to 8.4 %, and 6.7 to 13 % reported by Campos et al. (2018), Lu et al. (2018) and Vazquez et al. (2019), respectively. The ash content is an average value of the mineral salts that are part of the lignocellulosic composition of CPH and is approximately 25 times higher than the ash content that ranges from 0.26 to 0.52 % in highly available lignocellulosic materials such as pine wood of various species (Bernabé-Santiago et al., 2013). CPH ash has been studied as a fertilizer substitute in partial or total application (Campos-Vega et al., 2018). CVC has also been analyzed as compost and has been observed to increase soil pH and improve fertility by incorporating most of the essential elements; it also improves cocoa growth, offers a useful alternative to fungicides, and reduces the risk of black pod disease when properly managed and not just left as residue in cultivation areas (Doungous et al., 2018).
Mineral Profile
In relation to a high ash content, the CPH material has been widely recognized to be rich in mineral nutrients, with the following concentrations: K (2 768 - 112.04 mg∙100 g-1), Mg (110.9 - 21.23 mg∙100 g-1), Ca (254 - 6.15 mg∙100 g-1), Zn (39.74 - 7.22 mg∙100 g-1), Mn (35.72 - 7. 32 mg∙100 g-1), Na (10.5 - 0.47 mg∙100 g-1), Cu (8.55 - 6.18 mg∙100 g-1), Fe (5.8 - 5.04 mg∙100 g-1) and Se (0.01 mg∙100 g-1) (Vargas-Arana et al., 2022; Vriesmann et al., 2011). Minerals such as Mn, Cu, Zn, Ni and Mo are micronutrients required by plants, but there are others that are considered toxic to human health such as As, Cd, Cr and Pb (Barraza et al., 2021). Cd is known to accumulate in the cocoa bean, its derivatives and by-products, and other foods subproductos (Gramlich et al., 2016). Cd is a metal that is toxic to human health, has no biological function, occurs naturally in soil, and is increasing worldwide due to anthropogenic inputs (Gramlich et al., 2016).
CPH have been used locally in some West African countries for the production of ‘black’ soap. It has also been extensively used in composts and its potential as organic matter for soil replenishment, fertilization and biological control has been extensively studied (Doungous et al., 2018; Moyin-Jesu, 2007; Olubunmi-Kayode et al., 2018). However, the possibility of using it as an additive in the food industry has been raised, so it was important to measure some other minerals that have not been reported and could be considered toxic to human health, in addition to those already known.
In this study, 13 concentrations of minerals were determined in CPH of the Carmelo variety, presented in descending order of concentration: with the highest values between 164 and 63 µg∙g-1, Fe > Ba > Zn were found, followed by those with medium values between 22 and 1 µg∙g-1, Al > Ni > Cu > Cu > Cd and finally those with low values between 0.37 and 0.03 µg∙g-1, Cr > Pb > Co> V > As > Hg. Vriesmann et al. (2011) coincidentally reported higher Fe content in relation to Zn, with values of 58 and 39 µg∙g-1, respectively; Aregheore et al. (2002) agree with this trend, reporting 466 and 90 µg∙g-1 for Fe and Zn respectively, but with a value for Zn much higher than those reported by the generality. Barraza et al. (2021) reported Zn concentrations of 41.5 and 47 mg∙kg-1 and Ba concentrations of 98 mg∙kg-1 in cocoa beans in Ecuador, while Gramlich et al. (2016) reported Zn concentrations of 42.9 and 45.8 mg∙kg-1 in CPH of two varieties from Bolivia. Zn values are close to those reported in this study; however, those for Ba are much higher. Concerning Cu, concentrations of 0.55 mg∙kg-1 and 6.18 mg∙100 g-1 of CPH were reported by Moyin-Jesu (2007) and Vriesmann et al. (2011), respectively, which are similar to those reported in this study. Barraza et al. (2021) reported mean Ni concentrations of 3.10 and 6.27 mg∙kg-1 in two varieties of CPH from Ecuador, which are significantly lower than those found in this study for the Carmelo variety. On the other hand, Gramlich et al. (2016) reported Cd concentrations ranging from 0.6 to 0.8 and 0.16 to 0.27 mg∙kg-1 for CPH and seeds, respectively, observing that the concentration of Cd in the shells was significantly higher than that in the seeds. In this sense, the value reported in this study for Cd is relatively higher. Regarding the presence of Al in this lignocellulosic material. No previous reports were found on this matter; however, it is presumed that this element was bioadsorbed by the material, probably originating from the soil or water containing this type of contaminants. It is important to note that Al would not be suitable for food or pharmaceutical uses. Finally, for Cr, Pb, Co, V, As and Hg, there are no comparative references in CPH, but the concentrations determined by this study are low in relation to the rest of the minerals that make it up and in relation to Cd, as a reference of contaminant and toxic mineral. The concentrations of minerals in CPH will vary according to the origin, type of soil, type of crop, crop management and surrounding activity association (Barraza et al., 2021; Gramlich et al., 2016). However, if CPH material were to be considered for any food use, it would be necessary to verify that the volume contribution of contaminant minerals does not exceed the 2.5 µg∙kg-1 body weight tolerable monthly intake concentration established by the Joint FAO/WHO Expert Committee on Food Additives and the EFSA Panel on Contaminants in the Food Chain (European Food Safety Authority, 2012).
Total phenolic and flavonoid content
The total phenolic content for cacao nibs of variety Carmelo extracted with methanol-water was 24.59 mg GAE∙g-1 dry base sample, this result is comparable to the range of values reported by Nieto-Figueroa et al. (2020) from 24.8 to 27.3 mg GAE∙g-1 of cacao nibs from Tabasco Mexico dried by different methods. Moreover, the content is within the range reported by Jamaluddin et al. (2022) for phenolic contents of acetone (17.62 mg GAE∙g-1), ethanol (19.83 mg GAE∙g-1), and methanol (34.88 mg GAE∙g-1) extracts of CPH from Malaysia. These values are comparable to 35.53 mg GAE∙g-1 and 16.57 mg GAE∙g-1 of mango and grape peel reported by Castro-Vargas et al. (2019) and Siqueira et al. (2011), respectively. On the other hand, Valadez et al. (2017) reported 323.7 mg GAE∙100 g-1 of CPH from Chiapas, Mexico, which is similar to the phenolic content of ethanol (206.67-227 mg GAE∙100 g-1) and methanol-acetone (352.67-365.33 mg GAE∙100 g-1) extracts of CPH from two locations in Ecuador (Martínez et al., 2012). These values are significantly lower than those found in the present study and and those reported by other authors. However, in all cases the trend of higher phenolic content in methanol extracts is maintained.
The phenolic content of cocoa nib extracts depends on the variety or genotype, the origin of the raw material, the extraction method (technology, conditions and solvents) and the pre-treatment. The time and method of drying have a significant influence, as dehydration of the raw material at low temperatures results in higher levels of phenolics, flavonoids and antioxidant activity (Sánchez et al., 2023; Valadez-Carmona et al., 2017). Regarding the flavonoid content of the CPH of the Carmelo variety, a value of 2.35 mg CE∙g-1 dry base sample was determined, which is close to the value of 1.1-1.3 mg CE∙g-1 reported by Nieto-Figueroa et al. (2020) and higher than the value of 0.097 g CE∙g-1 reported by Valadez et al. (2017) for cacao nibs from Chiapas, Mexico. CPH is mainly composed of catechin, quercetin, (-)-epicatechin, gallic acid, coumaric acid, and protocatechinic acid (Lu et al., 2018; Valadez-Carmona et al., 2017). The polyphenols present in CPH are bioactive compounds that represent a potential source for antioxidant-rich foods (Campos-Vega et al., 2018).
Antioxidant Activity
The value of CPH may be largely due to their high content of phenolics and antioxidants, which could benefit human health by being used as an additive in foods or other value-added products (Jamaluddin et al., 2022). The evaluation of antioxidant activity requires different test methods, as a single method can provide basic information, and the combination of several methods can describe the antioxidant properties in more detail. This is because each method measures the ability of a plant's antioxidants to scavenge specific radicals through different electron transfer pathways, either by inhibiting lipid peroxidation or by chelating metal ions (Cádiz-Gurrea et al., 2014; Martínez et al., 2012). In this study, ABTS and FRAP methods were used to evaluate the antioxidant capacity of CPH Carmelo variety. It was observed that ABTS was found to have a higher antioxidant capacity than FRAP, which is attributed to the low selectivity of the ABTS+ radical to reach with hydroxylated aromatic compounds, while FRAP is based on the ability of phenolic compounds to reduce Fe3+ to Fe2+ in the presence of 2,4,6-tripyridyl-s-triazine.
The antioxidant capacity values obtained in this study were 304.84 µM TE∙g-1 and 145.80 µM TE∙g-1 for ABTS and FRAP, respectively. These results are close to the ABTS antioxidant capacity (227.7-147 µM TE∙g-1) in cacao reported by Nieto et al. (2020) and to the ABTS antioxidant capacity of grape skin (240 µM TE∙g-1) reported by Siqueira et al. (2011) but are higher than the ABTS antioxidant capacity of carrot skin beta-carotene (79.15 µM TE∙g-1) reported by Jayesree et al. (2021). These values are higher than the ABTS antioxidant capacity of cocoa pod shell (30.6 µM ET∙g-1, 24.13 µM ET∙g-1 and 42-24 µM TE∙g-1) reported by Valadez et al. (2017), Martinez et al. (2012). and Lu et al. (2018), respectively. Regarding the FRAP assay, a wide range of values was found in the literature for the antioxidant capacity of CPH, ranging from 521.73 µM TE∙g-1 to 1.98 µM TE∙g-1 (Jamaluddin et al., 2022; Lu et al., 2018; Martínez et al., 2012). There is a linear correlation between phenolic compounds and the antioxidant capacity of a plant (Cádiz-Gurrea et al., 2014). According to Campos et al. (2018), the antioxidant activity of CPH has been studied in green chemistry as nanoparticles with larvicidal activity and against resistant bacteria, resulting in potentially viable products.
Conclusions
Lignocellulose content, antioxidant capacity and mineral profile of cocoa pod husk (CPH, Theobroma cacao L. Carmelo variety) were determined for the first time. With the data obtained in this study and those reported in the literature, it was found that the Carmelo variety does not present any peculiarities with respect to other varieties. CPH is a viable source of lignin, and the cellulose and hemicellulose contents have potential as a source of carbohydrates to produce biofuels; however, the high content of polyphenols and its antioxidant capacity characterize it as a potential source for the food and/or pharmaceutical industry. Although heavy metals were detected at trace levels in this study, the mineral profile should always be analyzed as it is determined by the area of origin. Contrary to cocoa bean, CPH has been little studied in terms of antioxidant capacity. Therefore, the present study contributes to the understanding of a by-product with valorization potential.
