**1. Introduction**

In recent years, a number of biological properties and potential health benefits of consuming cocoa-derived products have been investigated, e.g., antioxidant, anti-inflammatory, anti-carcinogenic, and antifungal properties. Much research has focused on the cocoa phenolic compounds, such as flavonoids (procyanidins, anthocyanins, flavonols, etc.), as a potential health-promoting compounds due to its antioxidant capacity and abundance in the cocoa beans. However, the bioactive compounds composition of cocoa beans is influenced by many factors including variety, climatic and agronomic conditions, post-harvest practices, and storage conditions [1–3]. Moreover, thermal processing may also cause a change in the level of phytochemicals in cocoa beans and their antioxidant capacity. Many studies have shown that phenolic compounds, which have been suspected as primarily factors responsible for the antioxidant properties of cocoa beans, are susceptible to degradation and oxidative

condensation during thermal processing [2,4–7]. A recent study showed that, beside loss of phenolic compounds, roasting of cocoa beans induced only negligible changes in the total antioxidant capacity, probably due to the higher extractability of the cellular matrix compounds and/or formation of new antioxidants trough Maillard reactions, such as reductones and melanoidins [5,6,8]. Melanoidins are colored, nitrogen-containing, and polymeric compounds that form as a result of the final stage of the Maillard reactions. These compounds occur widely in many treated processed foods, such as coffee, bakery products, cooked potatoes, cocoa, roasted barley, and beef [9]. One of the most important properties that melanoidins contribute to foodstuffs is brown color. In addition, these macromolecules have considerable structural variability that result in diverse biological effects. The interest in the physiological role of melanoidins present in many heat-treated foods has increased dramatically over the last decade, particularly in relation to human health [2,9,10]. The biological effects exerted by melanoidins on the human body are thought to be strongly related to their ability the chelate metal cations, the capacity of scavenging superoxide anions and hydroxyl radicals, and the decomposing ability of hydrogen peroxide [10,11], which may be responsible for the antioxidant, anticancer, and antimicrobial properties [12]. Food melanoidins can also act as dietary fiber in the gastrointestinal tract and promote the growth of beneficial *Bifidobacteria* in the gut [9,11]. Current studies have shown that these biological functions are thought to be associated, at least in part, with the presence of phenolic compounds in the melanoidin structure [11,13]. Phenolic compounds, especially phenolic acids, are considered to contribute more to the healthful effects than the other constituents of melanoidins [13,14]. However, the exact mechanism of melanoidins antioxidant activity has not fully been elucidated to date. Moreover, despite numerous studies evaluating the biological and molecular properties of melanoidins obtained from both model systems and real foodstuffs, such as coffee, bread, honey, heated potato fiber, and malt [9,11,15], only very few authors have attempted to determine the biological activities of melanoidins isolated from cocoa beans and cocoa-derived products. Summa et al. [16,17] investigated the antibacterial, mutagenic, and radical-scavenging effects of four molecular weight fractions (>30, 30–10, 10–5, and <5 kDa) isolated by ultrafiltration from raw, pre-roasted (80–90 ◦C for 10 min), and roasted (130–160 ◦C for 15–20 min) cocoa beans. In those experiments, all high-molecular weight (HMW) fractions showed activity against the pathogenic bacteria *Enterobacter* and *Escherichia*. More recent studies have reported that LMW (<10 kDa), intermediate molecular weight (10–30 kDa), and HMW (>30 kDa) fractions isolated from cocoa powder by ultrafiltration have a dose-dependent inhibitory activity against α-glucosidase [18]. This activity was attributed to the presence of compounds in the low, intermediate, and high molecular weight fractions, including brown melanoidins, proteins, phenolic compounds, as well as polysaccharides, whether or not they were bound to the melanoidin skeleton or to unknown MRPs [18]. In our previous study, we investigated the effect of different roasting conditions, including temperature (110, 120, 135, or 150 ◦C) and relative air humidity (0.3% or 5.0%) on the physicochemical properties and the profiles of free phenolics and bound phenolics of high molecular weight Maillard reaction products isolated from Criollo, Forastero, and Trinitario beans from different regions of Africa [19,20]. In our previous work, we also demonstrated that the of the cocoa melanoidins are good sources of bound phenolics [19]. In that respect, it is noteworthy that the relation between the structure of these compounds and their health-related properties should be comprehensively investigated. Especially when, for some groups of people, cocoa beans represent a significant source of these compounds in their diet, the accurate assessment of the contribution of the chemical and structural properties of melanoidins to their antioxidant activity should be carried out.

In continuation with our previous study, this work was designed to carry out a comparative investigation of total phenolic content, antioxidant activity, and structural characteristic of HMW melanoidin fractions isolated from cocoa beans, both raw and roasted at different temperatures (110, 120, 135, and 150 ◦C) and relative air humidity levels (RH 0.3% and 5.0%) of three *Theobroma cacao* L. types. To the best of our knowledge, this is the first report concerning the determination of in vitro antioxidant activity and ATR-FTIR structural characterization of isolated HMW melanoidin fractions of Criollo, Forastero, and Trinitario beans from different regions of Africa.

#### **2. Materials and Methods**

#### *2.1. Materials and Chemicals*

Gallic acid, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), sodium acetate, ferric chloride hexahydrate, ferrozine, and ammonium acetate were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade and were purchased from Chempur (Piekary Slaskie, Poland). Ultrapure water (resistivity 18.2 MΩ cm), obtained from a Milli-Q purification system (Millipore, Bedford, MA, USA), was used for all analyses.

#### *2.2. Plant Material*

Analyses and experiments were conducted on samples of fermented and dried cocoa beans of the three main *T. cacao* groups: Criollo and Trinitario originating from Madagascar, and Forastero from Ghana. All cocoa beans were harvested at their technological maturity in 2013 and purchased from commercial sources. Cocoa fruit reaches physiological maturity around 5 months after flowering and is harvested at 5–6 months. Cocoa pods harvested at 5–6 months are considered to be technologically matured as the beans have developed optimum quality [21]. The description of cocoa beans is presented in Table 1.

**Table 1.** Selected physical characteristics of different cocoa varieties used in the study.


Means sharing the different letters (a–c) are significantly different according to Tukey's HSD test at *p* < 0.05.

Raw cocoa beans of each group after removal of impurities and broken or chipped beans were convectively roasted in batches of 200 g in a tunnel with the forced air flow without circulation (adapted for either dry or humid air). Process air humidity was gradually increased using 4.0 MPa saturated steam from a generator. Roasting was performed at four temperatures (110, 120, 135, or 150 ◦C) and two relative air humidity levels (0.3% or 5.0%). The heat treatment parameters were chosen to obtain a range of roasted beans with acceptable physico-chemical and sensory properties. Usually, during roasting, the raw cocoa beans are exposed to temperatures that range from 135 to 150 ◦C, whereas the "fine or flavor" varieties require lower temperatures than the "bulk" ones [19]. It was terminated when bean moisture dropped to 2% as determined by drying until constant weight at 103 ± 2 ◦C. The time of thermal treatment was determined experimentally for each batch of cocoa beans, based on their initial water content and size. Roasting times at 150, 135, 120, and 110 ◦C were approximately 20, 40, 75, and 85 min, respectively. The application of higher relative air humidity prolonged the duration of thermal treatment. At the end of roasting, the beans were immediately cooled to approximately 20 ◦C for about 10 min. The roasted cocoa beans were kept in hermetically sealed plastic containers (500 g) and stored at −20 ◦C for subsequent analyses. The samples were analyzed within 6 to 12 h of storage. All roasting experiments were performed in duplicate for each cocoa type used.

#### *2.3. Extraction and Isolation of Cocoa Melanoidin*

The HMW melanoidin fraction was obtained from raw and roasted cocoa beans by dialysis according to our previously research [19]. Briefly, the cocoa beans were deshelled, ground, and defatted, and then extracted twice with 100 mL of water at 90 ◦C for 20 min in an orbital shaker (100× *g*). Subsequently, pooled extracts were cooled to room temperature and filtered through Whatman no. 4 filter paper to remove insolubles. An aliquot of the filtrate was dialyzed using a dialysis tubes (MW cut-off > 12.4 kDa, Sigma-Aldrich, Saint Louis, MO, USA) for 1 day under running tap water and for 2 days against 2000 mL of water at 4 ◦C with constant stirring. The water was changed 4–5 times until no further color was visible in the dialysate. The dialysate containing LMW compounds was removed. After dialysis, the retentate containing the HMW fraction was frozen at −20 ◦C and lyophilized (−50 ◦C, 0.9 MPa) using a DELTA 1-24LSC Christ freeze drier (Martin Christ, Osterode am Harz, Germany). All lyophilized HMW materials were then stored in plastic bags at −20 ◦C to prevent hydration until used.

#### *2.4. Determination of Total Phenolic Content*

Total phenolic contents (TPC) of HMW isolates from raw and roasted cocoa beans were determined using the Folin–Ciocalteu method, as described by Belšcak et al. [22]. Briefly, 100 μL of the suitably diluted lyophilized HMW cocoa sample with high-purity deionized water (1.5 mg/mL) or blank was mixed with 4 mL of high-purity deionized water and 0.5 mL of the Folin–Ciocalteu reagent. After 3 min, 1 mL of 20% (w/v) Na2CO3 solution was added to the mixture. The final volume was adjusted to 10 mL with high-purity deionized water. The solution was then mixed vigorously and allowed to stand at room temperature in the dark place for 60 min. The absorbance of reaction mixture was measured at 765 nm using a UV-1800 spectrophotometer (Shimadzu, Tokyo, Japan). For each sample, experiments were conducted in triplicate. The results were expressed as mg gallic acid equivalents (GAE) per gram of lyophilized HMW cocoa fraction dry weight (mg GAE/g dw).

## *2.5. Determination of the Free Radical-Scavenging Capacity*

The free radical-scavenging activity of the HMW isolates from raw and roasted cocoa beans was determined by the DPPH and ABTS assays as previously described by Oracz and Nebesny [8]. All analyses were carried out in triplicate, and the results obtained from the two tests were expressed as μmol Trolox equivalents per gram of lyophilized HMW cocoa fraction dry weight (μmol TE/g DW).

## *2.6. Determination of Ferric Reducing Antioxidant Power (FRAP)*

The FRAP assay was performed according to the protocol described by Pastoriza and Rufián-Henares [23] with slight modification. The fresh working FRAP solution was prepared by mixing 25 mL of 300 mmol/L acetate buffer (pH 3.6), 2.5 mL of 10 mmol/L TPTZ in 40 mmol/L HCl, and 2.5 mL of 20 mmol/L FeCl3·6H2O and then warmed at 37 ◦C for 30 min before using. Aliquots (0.1 mL) of the suitably diluted lyophilized HMW cocoa samples with high-purity deionized water (1.5 mg/mL) were mixed with 4 mL of fresh FRAP solution. The solution was then mixed vigorously and allowed to stand for 30 min at 37 ◦C. The absorbance of reaction mixture was measured at 593 nm using a UV-1800 spectrophotometer (Shimadzu, Tokyo, Japan). For each sample, experiments were conducted in triplicate. The results were expressed as μmol Trolox equivalents per gram of lyophilized HMW cocoa fraction dry weight (μmol TE/g dw).
