UHPLC–MS Characterization, and Antioxidant and Nutritional Analysis of Cocoa Waste Flours from the Peruvian Amazon

Cocoa (Theobroma cacao) is a food product used worldwide and a key raw material for chocolate manufacturing. Cocoa possesses bioactive compounds such as methylxanthines, flavonoids, procyanidins, and related molecules with medicinal or health-promoting properties. Cocoa shell and pod husk have been proposed as a by-product with several interesting bioactivities, and the gummy residue or glue (a sticky, gluey by-product known as “mucilage” in Spanish) is used to produce liquors and is eaten as a food in Perú. However, little is known about the chemical composition and bioactivity of flours made from Peruvian cocoa ecotype wastes such as those from the vein and pod husk of the fruits. This study aimed to characterize the in vitro antioxidant properties and nutritional values of flours made from the waste from a special ecotype of cocoa (CCN-51). The chemical fingerprinting was performed using UHPLC–HESI orbitrap mass spectrometry and allowed the detection of 51 compounds. GC-FID was used for the determination of individual fatty acid contents, and the antioxidant activity was assessed by several assays (DPPH, FRAP, and ABTS). The flours obtained were composed of a good amount of dietary fiber, carbohydrates, and minerals, as well as several bioactive polyphenolic compounds, fatty acids, and amino acids with nutraceutical properties, making the flours a rich and promising food as well as a good source for the preparation of functional foods or nutraceuticals.


Introduction
In recent years, fruit and vegetable by-products or food wastes have been shown to be a good source of bioactive compounds that can be extracted and reintroduced into the food chain or in food matrices as natural food additives for the production of functional foods or nutraceuticals [1]. In addition, the reduction of food waste is growing as an important process of environmental and economical welfare. Cocoa by-products, primarily cocoa pod husks, are produced in vast quantities around the world, representing 70-80% of the dry weight of the fruit [2]. They are usually thrown away as a production waste, which has a detrimental environmental impact [3]. Cocoa beans are mainly used to produce cocoa powder and chocolate. It is estimated that a total of around 4-5 million tons of cocoa is produced worldwide per year [4] and 700,000 tons of cocoa bean shells are produced as waste every year [1], being a good source of proteins, fatty acids, and reported polyphenolic produced as waste every year [1], being a good source of proteins, fatty acids, and reported polyphenolic compounds [5]. Cocoa-rich products can have beneficial effects on human health due to their beneficial antioxidant components. For instance, it has been proposed that cocoa products can prevent degenerative diseases, metabolic disorders, and cancer, acting as antiobesogenic, antidiabetic, and antihypertensive factors that are associated mainly with the number of phenolic compounds accounting for approximately 8% of cocoa beans [6][7][8]. The stem bark of cocoa has also shown anti-inflammatory properties [9]. Regarding cocoa bean shells, several revalorizations have been proposed for food, livestock feed, or industrial uses, and several health properties have been reported, including anticarcinogenic, antibacterial, anti-inflammatory, antidiabetic, antiviral, neuroprotective, and cardioprotective effects [5]. There are three main varieties of cocoa cultivated in the Amazon region, with the "forastero" variety being the variety that receives the greatest degree of exploitation. This variety is responsible for 96% of worldwide production due to its resistance to pests [10,11]. The main phenolic compounds in cocoa are the group of flavonoids, which include anthocyanins, flavonols, and flavanols. Other phenolic compounds found in cocoa products are amino acid and phenolic acid conjugates (N phenol amino acids, NPAs), stilbenes, and phenolic acids [12]. Finally, cocoa products contain a good quantity of alkaloids known as methylxanthines [13]. The purine alkaloids theobromine (3,7-dimethylxanthine), caffeine (1,3,7-trimethylxanthine), and theophylline (1,3-dimethylxanthine) are the most common methylxanthines found in cocoa [14]. They are biologically active alkaloids responsible for the bitter taste of cocoa. These alkaloids also possess desirable pharmacological effects, e.g., gastric secretion, diuresis, bronchodilation stimulation of the central nervous system, and stimulation of skeletal muscles in high doses. To date, there is little research conducted about nutritional properties and chemical fingerprinting of the metabolites from cocoa waste products; however, cocoa pod husk is considered a cheap and good source of pectin [2]. In the present study, we present the phenolic composition of two flours made from waste (vein and pod husk) from cocoa CCN-51 variety and their antioxidant potential together with nutritional properties, the content of fatty acids, methylxanthines, proximal composition, and mineral content. Therefore, this study aims to describe the chemical fingerprinting by UHPLC-MS analysis and nutritional properties of cocoa waste products, namely, flours made from vein and pod husk ( Figure 1) of a selected Peruvian ecotype of cocoa, evaluation of the antioxidant activity, and their potential as a food or food product.

Plant Material
The cocoa fruits (Theobroma cacao), CCN-51 variety, were collected in October 2020 directly from the cocoa farm of Mr. Américo Hernández, located in the Pajonal zone, Pardo Miguel district, Rioja province, San Martín region, Peru (05 • 43'22.5" S and 77 • 29'20" W, 800 m altitude). The selected fresh cocoa fruits were taken to the laboratory in dark bags where they were washed, brushed, disinfected using a 200-ppm sodium hypochlorite solution for 30 min, and rinsed with water. Then, the pulp was removed, and the fruit opened, wherein the veins were removed, and the pod husks were separated. Then, thermal drying for both waste parts of cocoa fruits was carried out at 70 • C for 48 h. Finally, a blade mill was used (Grindomix GM 200), and the flours were milled using each part of waste and stored. The extract for HPLC analysis and biological activity was prepared by extracting the flour (500 mg), with 10 mL of HPLC-grade methanol with 1% formic acid for 10 min with sonication (3 times). Extractions were combined, the solvent was evaporated under vacuo and filtered thorough Whatman paper number 1, and the yellow gummy residue was obtained (19.5 mg for the vein extract (3.9%) and 12.7 mg for the pod (2.54%) and stored at -20 • C.

HPLC-DAD Analysis of Catechins and Methylxanthines
Catechins and methylxanthines by HPLC were carried out by means of a chromatographic analysis according to the protocol of Oliviero et al. [15] and Brunetto et al. [16], with some modifications. A chromatograph (Hitachi LaCrhom Elite ® , Technologies America, Inc., Clarksburg, MD, USA) equipped with a vacuum degasser, a quaternary pump, and a diode-array detector (DAD), calibrated at 280 nm, was employed. The separation of (+)catechin and (-)-epicatechin was carried out in an RP-18E column whose dimensions were 5 µm in particle size, 250 mm in length, and 4.6 mm in diameter. As mobile phase, methanol (A) acidified with 0.1% formic acid (B) was used, with elution gradients of 0.01 min 60% of A; 5-12 min 80% A; 13-14 min 60% of A. The flow rate of the mobile phase was 1.0 mL/min. The identification of the peaks was carried out by comparing with standards of (+)-catechin and (-)-epicatechin (Sigma-Aldrich ® , St. Louis, MO, USA) and theobromine and caffeine standards (Sigma-Aldrich ® , St. Louis, MO, USA). The theobromine and caffeine separation were carried out with the same column as above.

Determination of Proximate Composition
AOAC procedures were used in all determinations [17,18]. The water content was determined by oven-drying the sample up to a constant weight, the crude protein content by the Kjeldahl method (N × 6.25), the fiber content by gravimetric method after acidic hydrolysis of the samples, the total lipid extracted in a Soxhlet apparatus using petroleum ether as solvent, and the ash content by incineration in a muffle furnace at 550 ± 15 • C. Total carbohydrates were calculated as difference: 100 − (g water + g protein + g fiber + g fat + g ash).
Results were expressed in grams per 100 g fresh weight (g/100 g fw). The experiments were carried out in triplicate.

Mineral Analysis
For the mineral analysis, the flours were dried to ash at 550 • C [18]. The ash in each case was boiled with 10 mL of 20% hydrochloric acid in a beaker, and then filtered into a 100 mL standard flask and made up to 100 mL with distilled deionized water. Levels of minerals, potassium (K), calcium (Ca), magnesium (Mg), sodium (Na), zinc (Zn), manganese (Mn), copper (Cu), and iron (Fe) were determined from the resulting solution using atomic absorption spectroscopy (Varian AA240). The values obtained for each parameter are averages of three determinations for a given food sample.

Fatty Acid Profile
Fats were cold extracted using the Bligh and Dyer method [19]. Fatty acid profiles were obtained by gas chromatography of the fatty acid methyl ester derivatives. Derivatives were obtained by esterification with KOH in methanol (2 M). The fatty acid derivatives were extracted with hexane and analyzed through a Varian-450 gas chromatograph (Varian Inc., Palo Alto, CA, USA). The chromatograph was equipped with a VF-WAXms (60 m × 0.25 mm) capillary column and flame ionization detector (FID). Helium was used as the carrier gas, and the temperature program was as follows: 3 min at 130 • C, gradual heating to 220 • C for 9 min, 35 min at 220 • C, cooling to 130 • C, and 130 • C for 5 min. Individual peaks were identified by referring to a fatty acid methyl ester standard solution and analyzed under the same operation conditions [17].
2.8. Antioxidant Activity 2.8.1. DPPH Scavenging Activity DPPH scavenging activity was determined by the method developed by Brand-Williams et al. [20]. To 3.9 mL of a solution of the DPPH• radical (100 µM) dissolved in 80% methanol, 0.1 mL of the extract (2 mg/mL), previously filtered on a membrane filter (0.45 µm), was added, and the mixture was stirred vigorously and set in the dark for 30 min at 25 • C. After that time, the absorbance at 517 nm was read in a UV-visible Cary60 spectrophotometer. The concentration of DPPH• in the reaction medium was obtained from a calibration curve by linear regression. The control consisted of 0.1 mL of 80% aqueous methanol and 3.9 mL of DPPH • solution (100 µM). The results are expressed in TEAC, that is, antioxidant activity equivalent to Trolox (µmol Trolox/g of extract). The reference synthetic antioxidant Trolox, at a concentration of 5-30 µM in 80% methanol solution, was tested under the same conditions.

ABTS Bleaching Capacity
ABTS bleaching capacity was determined by the method developed by Re et al. [21]. The reaction was started with the addition of 1500 µL of an ABTS•+ solution in PBS buffer (0.70 ± 0.02 at λ = 734 nm) to 500 µL of the extract (2 mg/mL) in a cuvette kept at 30 • C. It was homogenized and allowed to react for 7 min, and then the absorbance reading was made at a wavelength of 734 nm, using a Cary60 UV-visible spectrophotometer. The results are expressed in TEAC (µmol Trolox/g of extract). The calibration curve for TEAC was constructed using different concentrations of Trolox (4-14 µM) in PBS buffer solution under the same conditions.

Ferric-Reducing Antioxidant Power Assay (FRAP)
The ferric-reducing antioxidant power (FRAP) was determined according to the Benzie and Strain method [22]. A volume of 10 µL of the extract (2 mg/mL) was mixed with 90 µL of distilled water and 900 µL of the FRAP reagent (2.5 mL of the 2,4,6-tripyridyl-s-triazine solution at a concentration of 10 µM in HCl 40 mM; 2.5 mL of FeCl 3 20 µM and 25 mL of acetate buffer 0.3 µM at a pH of 3.6). The absorbancy was read at 593 nm after 7 min in a UV-visible Cary60 spectrophotometer. The results are expressed in TEAC, that is, antioxidant activity equivalent to Trolox (µmol Trolox/g of extract).

Total Phenolic (TP) Content
The content of total phenols was estimated by a colorimetric method based on the procedures described by Velioglu et al. [23] with some modifications. In essence, 100 µL of the extract (2 mg/mL) was mixed with 750 µL of the Folin-Ciocalteu reagent diluted in a 1/10 proportion of Milli-Q water. After 5 min in the dark, 750 µL of sodium bicarbonate (60 g/L) was added to the mixture. The tubes were kept in the dark for 90 min at 30 • C, then the absorbance was read at 725 nm, using a Cary60 UV-visible spectrophotometer. Gallic acid (10-100 µg) was used for the construction of the standard curve. The results are expressed as milligrams of gallic acid/g extract.

Statistical Analysis
All the experiments were repeated at least three times. The results were expressed as mean ± standard deviation (SD) using GraphPad Prism 8. Comparison of results was performed using one-way analysis of variance (ANOVA), followed by Tukey's HSD (honest significant difference) test (p < 0.05).

UHPLC-MS Analysis of Cocoa Extract
The fingerprinting of the flour from cocoa pod husk and vein were created and investigated by means of UHPLC-high-resolution MS with DAD analysis. The negative mode was used for the identification of phenolic compounds, while the positive mode was used for anthocyanins and methylxanthines. Some of the metabolites identified are reported for the first time in flours made from waste from this species. In total, 51 metabolites were detected and tentatively identified including phenolic acids, amino acids, anthocyanins, flavonoids, alkaloids, terpenes, and fatty acids (See Figures 2 and 3 and Table 1). A detailed analysis is provided below.

Chemical Composition and Nutritional Properties of Flours
The chemical composition and nutritional properties of flours were performed according to previous methodologies [15][16][17]19]. This included proximal analysis (Table 2), mineral content (Table 3), and content of fatty acids (Table 4). Table 2 shows proximal composition such as the humidity, ashes, protein, lipids, carbohydrates, and fiber, while Table 3 shows the mineral contents of two flours for cocoa waste material. The results of physicochemical properties showed that the proximal composition and the caloric value of these flours made of cocoa waste had a high fiber content that is good for use as a supplement (35.48 ± 1.47 and 7.26 ± 0.17% in pod husk and vein flour, respectively) and carbohydrate content (41.89 and 57.96%, respectively), which make them two highly caloric flours. The composition showed differences with other flours derived from foods, such as banana flour (Musa paradisiacal L.), which showed total starch of 73.36% and dietary fiber of 14.52% [24], while in buckwheat, for instance, in the flour, these values were dietary fiber: 6.77%, ashes: 1.82%, starch: about 78.4%, protein: 10%, and lipid content: 2%, but in its bran, starch: 40.7%, ashes: 4%, protein: above 21%, and lipid content: around 7% [25]. Soy flours collected at Cedar Rapids showed protein at 52%, fat at 0.8%, and ashes at 6.31% [26], while a group of flours obtained from the species of Prosopis showed protein: 7.17-11.2%, crude fiber: 2.7-3.4%, and carbohydrates: all around 8.2% [27]. The composition of cocoa pod husk in the literature showed great variability; however, our results are in accordance with those reported by Mariatti et al. [28]. Briefly, ashes: 5.9-13.0 (8.62%), lipids: 0.6-4.7 (0.51%), proteins: 2.9-9.1 (8.50%), fiber: 18.3-59.0 (35.48%), and carbohydrates: 17.4-47.0 (41.89%) ( Table 2). The flours were analyzed for mineral content (Ca, Na, K, Mg, Cu, Mn, Zn, and Fe) and were high in K (112.04 and 54.03 mg K/100 g for pod husk and vein, respectively) and low in sodium (0.47 and 0.09 mg Na/100 g for pod husk and vein, respectively), which is also good for hypertensive people. The pod husk had the highest contents of Mg, Ca, Mn, Zn, and Fe ( Table 3). The content of Ca makes the flour a good supplement for bones. The cocoa pod husks have been studied for use as fertilizers due to their high mineral content [2]. The fatty acid profile of the flours was investigated using a standardized protocol [19]. Saturated fatty acids were 44.99% and 39.40% in vein and pod husk flours, respectively (Table 4). While the pod husk showed more polyunsaturated fatty acids (52.54%), the vein showed more monounsaturated fatty acids (37.11%), and thus the pod husk flour has more healthy fatty acids. Indeed, the pod husk is richest in dietary poly-UFAs, and it is more suitable as a food or food supplement for its nutritional values. Because of its high values in proteins, crude fats, fibers, and mineral levels, cocoa pod husk has been widely explored as feed for poultry and/or animals [3]. Moreover, the main methylxanthine alkaloid compounds: theobromine, caffeine, and the two main catechins were quantified, and the results are shown in Table 5. The pod husk showed the highest content of theobromine (10.21 µg/g), while the vein showed the highest content of caffeine, epicatechin, and catechin (1.11 µg/g, 3.40 µg/g, and 3.09 mg/g, respectively; Table 5).

Antioxidant Activity and Total Polyphenol Content
The antioxidant capacity cannot be fully described by only one method; thus, for this study, we employed three different complementary antioxidant methods (ABTS, DPPH, and FRAP) that have been applied to the flours in addition to the phenolic content measured by spectrophotometry. It has been reported that the total phenolic content of cocoa varies on the basis of the growing region and extraction solvent technique [29]. Table 6 shows the antioxidant capacities of the three methods used in this study. The DPPH antiradical activity as well as the reducing power of the flours can serve as a significant indicator of their potential antioxidant activity. For this reason, the reducing power of ferric ions and total polyphenol content was also examined in the two cocoa waste flours ( Table 6). The FRAP of the flour from the vein exhibited a weak reducing power compared to the pod husk, but the total phenolic content (111.05 mg GAE/g flour) was higher than those reported for cocoa pod husk flour (CPHF) criollo variety (5.4 to 16.6 mg GAE/g flour) [30]. This difference may have been due to the location growth of cocoa, variety, and the solvent system used in the extraction of phenolics [29]. To show some comparison, antioxidant properties of refined and whole wheat flour by the oxygen radical absorbance capacity (ORAC) test for refined wheat flours ranged from 10.88 to 14.38 µmol TE/g (mean 12.52 µmol TE/g) while showing significantly lower values compared to their whole wheat flour counterparts, which ranged from 27.93 to 44.33 µmol TE/g (mean 35.74 µmol TE/g) from the same brand [31]. In addition the DPPH content reported for those wheat flours ranged among 4-5 µmol equivalent of Trolox/g [31], which is lower than our cocoa waste flours (46 and 87 µmol equivalent of Trolox/g for the vein and husk flours, respectively) ( Table 6). Each value represents the means ± SD of three replicates, n = 3, while different letters on the same column indicate significant difference using Tukey's test at 0.05 level of significance (p < 0.05).
Furthermore, our values for the TPC (Table 6) were higher than those reported for the TPC of cocoa shell hydroalcoholic extract (51.9 mg/g) [32] and lower than those obtained by Amin et al. (113 mg/g extract) who used ethanol as an extraction solvent [33]. Our DPPH activity was also higher (  [32] and is in accordance with that reported by Delgado-Ospina et al. (36 to 133 µmol Trolox/g) [30]. Furthermore, strong correlation was found between total phenolic and the three antioxidant assays DPPH (r = 0.9995, p < 0.001), ABTS (r = 0.9970, p < 0.0001), and FRAP (r = 0.9988, p < 0.0001).
In the flours, the antioxidant compound protocatechuic acid, PCA (3,4-dihydroxy benzoic acid), was detected; this is one of the main metabolites produced by anthocyanins and proanthocyanins and has been shown to possess antioxidant activity in vitro and in vivo [34]. In addition, catechin, epicatechin, and procyanidins detected in the flours are present in chocolate made of cocoa and considered the main phenolics in it (30%), being directly related to its antioxidant capacity [35]. The flavanols detected in cocoa food products (such as apigenin-7-O-glucuronide, quercetin 3-O-glucoside, and kaempferol 3-O-pentoside) are also responsible for the antioxidant activity of those products [36]. On the other hand, dietary-important unsaturated and saturated fatty acids were also detected, and saturated ones were measured by GC-FID (Table 4). In the cocoa flours, it was shown that chocolates showed a high concentration of saturated fatty acids, mainly stearic acid (18:0), and palmitic acid (16:0), followed by the unsaturated fatty acids, among which linoleic acid (18:6) and oleic acid (18:1n-9) were the more concentrated [37], having been very important in stopping the development of coronary diseases and high blood pressure [38].

Conclusions
In this study, two flours made of the vein and pod husk from a special ecotype of Peruvian cocoa (CCN-51) were investigated regarding their potential as a food or food supplement and their antioxidant capacities. Different compounds were detected in the two flours including methylxanthines, catechin, flavonoids, fatty acids, amino acids, phenolic acids, and other common acids. The principal methylxanthines and catechins were quantified, and the content of fatty acids was quantified individually for each waste material, making it a standardized food waste material. Proximal composition and mineral content were analyzed for the first time in these by-products, and the findings of physicochemical characteristics revealed that these flours generated from cocoa waste had a significant fiber and carbohydrate content, making it an energetic and healthy product. The mineral content (Ca, Na, K, Mg, Cu, Mn, Zn, and Fe) of these flours is shown to be a rich source of potassium and low in sodium, which is good for people suffering from high blood pressure. The pod husk had the highest contents of main dietary minerals, and therefore is better for this purpose. Flours made of cocoa waste have good nutritional properties and can be a good source of dietary phenolic compounds, which are essential for the preparation of nutraceuticals or food supplements. More biological tests and more analyses are necessary to test the health potential of these waste flours.