*3.5. Chelating Activity on Fe2*<sup>+</sup>

The changes in ferrous ion chelating activity of HMW fractions, caused by roasting of beans of all tested cocoa groups, are presented in Figure 3. All HMW isolates obtained from raw and roasted cocoa beans were able to chelate ferrous ion (Fe2<sup>+</sup>), being the most powerful pro-oxidants among various species of metal ions. The ferrous ion in the Fenton reaction can catalyze the generation of potentially toxic reactive oxygen species (ROS), such as hydroxyl radicals (•OH) that initiate lipid peroxidation [28]. It was also found that, similar to the WAAC, the HMW fractions from raw Criollo beans showed the higher chelating activity (55.16%) than those derived from raw Forastero and Trinitario beans (53.31% and 43.39%, respectively). To the best of our knowledge, this is the first study showing the interplay between roasting conditions and the iron chelating activity of HMW fractions isolated from different groups of cocoa beans. Two-way ANOVA revealed that the effect of cocoa variety, roasting conditions,

and their interactions was highly significant (*p* < 0.001). The results of this study demonstrate that the chelating activity of HMW fractions isolated from beans of the three cocoa types was increased by roasting at temperatures in the range of 110 to 150 ◦C, which may be ascribed to structural changes in melanoidins and phenolic compounds. Moreover, the changes in the iron binding ability greatly depended on the roasting air humidity (0.3–5.0%) and were considerably smaller when the RH was elevated. The chelating activity of the HMW isolates obtained from roasted samples varied from 45.21% to 92.07%. The increase in the metal-chelating activity was more pronounced in HMW isolates from Forastero and Trinitario beans, while the lowest changes were observed in the Criollo samples. Irrespective of the cocoa type, the greatest increase in the iron chelating ability occurred when cocoa beans were roasted at 150 ◦C and the lower relative humidity of air (RH of 0.3%) whereas the lowest rise was caused by roasting at 110 ◦C with humid air (RH of 5.0%). These results are consistent with the described above differences in the free radical scavenging activities and the reducing properties of HMW cocoa. However, unlike to the WAAC, the melanoidin fraction of Forastero beans roasted at higher temperatures exhibited the highest ferrous ion chelating potential, compared to other cocoa groups (even in the relation to Criollo samples).

**Figure 3.** Total phenolics content of HMW fractions isolated from raw and roasted, at different at different temperatures and relative air humidities, cocoa beans of different groups. Results are presented as means ± SD from triplicate assays. Bars with the same lowercase letter (a–h) within each variety do not differ significantly according to Tukey's HSD test at *p* < 0.05.

Such differences could be explained by variations in the composition of studied HMW melanoidin fractions that are strongly determined by cocoa variety and roasting conditions [19,20]. In addition, this phenomenon suggests that Maillard reactions that took place during roasting of cocoa beans at the higher temperatures resulted in formation of high molecular weight brown melanoidins, exhibiting the high iron chelating activity. Our findings confirm recently reported observation that MRPs with metal-chelating ability are generated in model systems due to heat treatment. Gu et al. [24] found that high molecular weight MRPs had higher metal-chelating potential than low molecular weight MRPs. They also suggest that the metal-chelating activity of MRPs is possibly affected by the presence of the hydroxyl or pyrrole groups in their structures. This was a clear evidence that the studied HMW cocoa melanoidins may prevent against oxidative damage by sequestering Fe (II) ions that participate in transition metal ions-catalyzed hydrogen peroxide decomposition with generation of hydroxyl radicals.

#### *3.6. Characterization of Cocoa Melanoidins by Fourier Transform Infrared Spectroscopy*

To provide a comprehensive explanation about the antioxidant properties of HMW cocoa fractions, it was necessary to precisely characterize the chemical structure of these materials. We report here for the first time the ATR-FTIR spectroscopic characterization of HMW cocoa melanoidins. The FTIR spectra of HMW isolates obtained from raw and roasted cocoa beans were collected with FTIR spectrometer equipped with an ATR sample accessory. The ATR-FTIR spectra (within the 400 cm−<sup>1</sup> to 4000 cm−<sup>1</sup> wavenumber region) of the cocoa melanoidins are given in Figure 4.

**Figure 4.** Fourier transform infrared spectroscopy (FTIR) spectra of HMW fractions isolated from raw and roasted, at different at different temperatures and relative air humidities, cocoa beans of different groups.

The heterogenic nature of the cocoa melanoidins has been confirmed by Fourier transform infrared spectra of HMW fractions isolated from different types of *T. cacao* beans, which exhibit prominent absorption bands in the broad region 400–4000 cm<sup>−</sup>1, the characteristic for various classes of compounds. Common features as well as particular vibrations, specific to from phenolic compounds and its derivatives, polysaccharides, and proteins, are found in the spectra. The ATR-FTIR spectra of all HMW cocoa melanoidin fractions contains a wide band at 3350 cm−<sup>1</sup> that belongs to the H-bond stretching vibrations of O–H hydroxyl groups, strong stretching band at about 1650 cm−<sup>1</sup> assigned to the double bond stretching of carbonyl (C=O), C=C, or C=N [42] and characteristic absorption bands appeared between 900–600 cm−<sup>1</sup> due to the stretching vibrations of the entire anhydroglucose ring. All mentioned bands confirms the presence of a phenol group sensitive to hydrogen bonding [25]. Moving forward, it confirms that more phenolic compounds bound to melanoidins might also contribute to the observed strong antioxidant capability of HMW cocoa fractions. The antioxidant activity of phenolic compounds is attributed to its molecular structures, particularly the number and positions of the hydroxyl groups, and the nature of substitutions on the aromatic rings [43]. Therefpre, more active antioxidant compound possesses more hydroxyl groups. The changes in the spectra gradually occur, depending on the cocoa type and roasting conditions. Our results showed that thermal treatment temperatures between 110 and 150 ◦C generally caused an increase in intense of the peak of HMW fractions isolated from cocoa beans (except for Trinitario samples). The peaks at 2922 and 2850 cm−<sup>1</sup> in the FTIR spectra were attributed to the stretch of the C–H of aromatic ring and would be due to stretching vibrations of CH2 and CH3 groups that can originate from fatty acids present in the cocoa melanoidin fractions [25]. A igher peak in the region 2920 cm−<sup>1</sup> clearly reflects adsorption of aliphatic compounds in the melanoidins structure. Some reports revelated that interactions between reactive carbonyl compounds arising from lipid oxidation reactions and amino acids or proteins might play an important role in the formation of brown HMW macromolecular compounds upon high-temperature processing of cocoa beans [44,45]. Bands in the range of 1616–1690 cm−<sup>1</sup> were ascribed to N–H bending vibrations from amine or amide groups, and C=O stretching vibrations from flavonoids, phenolic acids and its derivatives, quinones, and lipids [45,46]. The band centered at about 1650 cm−<sup>1</sup> mainly corresponds to the C=O stretching vibrational mode of the different structures of the protein backbone. The band at 1515 cm−<sup>1</sup> was attributed to C=C stretching vibrations from aromatic rings of phenolic compounds [25,26,45]. Mot [46] noticed that the intensity of the peak at 1630 cm−<sup>1</sup> is closely correlated with the antioxidant activity of plant extracts. In this study, in the FTIR spectrum of HMW fractions of Trinitario beans, the intensity of the 1630 cm−<sup>1</sup> peak is significantly lower compared to the Criollo and Forastero melanoidins. This behavior corroborates the results obtained in DPPH• and ABTS scavenging assays and could be a consequence of the higher number of phenolic groups in the HMW melanoidin fractions of Criollo and Forastero groups compared to those of Trinitario type. Moreover, the relative intensity of this peak increases during roasting of almost all cocoa beans, which may be the result of adsorption of phenolic compounds and/or its derivatives in the melanoidin structures. This was a clear evidence that the studied HMW melanoidin fractions possessed residues (OH or NH2 groups) that could act simultaneously as hydrogen and electron donors. Our previous study on UHPLC-DAD-ESI-HR-MSn analysis of HMW melanoidin fractions derived from two different types of cocoa beans revealed that both free and bound phenolic compounds, including three flavan-3-ols, seven phenolic acids, one phenolic aldehyde, and four n-phenylpropenoyl-L-amino acids (NPAs), are present in these fraction [20]. Most of the phenolics in all the HMW melanoidin fractions were present in the bound form. It was also found that HMW fractions obtained from roasted cocoa beans had the higher content of bound phenolics than those from unroasted cocoa beans. This observation is consistent with the results of other authors who showed that phenolic compounds can be incorporated into the melanoidin skeleton during coffee roasting [15]. The spectral region from 1400 to 650 cm−<sup>1</sup> is called the fingerprint region. This region of the infrared spectrum contains vibrations that are specific for the large number of infrared bands, including C–O, C–C, and C–N single bond stretches, C–H bending vibrations, and some bands due to benzene rings [25,26]. The peaks in the range of 1020–1060 cm−<sup>1</sup> corresponded to C–O–C and C–O stretching vibrations of the glycoside linkage and C–O bond stretching vibration in glycerol. According to the literature, the occurrence of this band is an evidence of polysaccharide and lipids in samples [25,26].

Our results demonstrate that ATR-FTIR spectroscopy may be used as a direct and nondestructive method for the rapid investigation of the structural characteristics and functional properties of the HMW melanoidin fractions. According to our findings, the presence of different types of compounds in HMW melanoidin fractions is responsible for their bioactive properties (e.g., reducing power, antioxidant capacity, chelating activity) and its different mechanisms of action.

#### **4. Conclusions**

In conclusion, the present study, to the best of our knowledge, was the first time a comprehensive study was carried out on the total phenolic content and antioxidant activities, as well as structure–activity relationships of HMW cocoa melanoidin fractions isolated from raw and roasted, under different temperature and relative air humidity conditions, cocoa beans of different *T. cacao* groups. The results showed that the cocoa type and roasting conditions affect the total phenolic content and antioxidant properties of HMW melanoidin fraction isolated from the studied beans. The ATR-FTIR analysis revealed the presence of different bioactive compounds with various mechanism of action in HMW cocoa melanoidin fractions. We found that, both for TPC and in vitro antioxidant activity, HMW cocoa melanoidin fractions of Criollo beans showed significantly higher values than those of Fosrastero and Trinitario samples. Moreover, we observed that that roasting at higher temperatures with the lower relative humidity of air effectively enhances in vitro antioxidant potential of almost all HMW fractions isolated from cocoa beans. Consequently, irrespective of the cocoa type, the thermal processing at 150 ◦C and RH of 0.3% can be recommended to obtain HMW materials with the highest total phenolic content and strong in vitro antioxidant potential. This fact could indicate that the thermal processing of cocoa beans enhances the concentration of bioactive molecules in HMW fractions that contribute to the antioxidant response observed in in vitro tests. Our findings suggest, in general, that optimization of the roasting conditions and choosing an appropriate cocoa variety may provide functional advantages by enhancing in vitro antioxidant properties of HMW fractions of cocoa beans.

**Author Contributions:** Conceptualization, J.O.; formal analysis, J.O.; funding acquisition, J.O.; investigation, J.O. and D.Z.; methodology, J.O.; resources, J.O. and D.Z.; writing—original draft preparation, J.O.

**Funding:** This research was funded by NATIONAL SCIENCE CENTRE POLAND, grant number UMO-2012/ 05/N/NZ9/01399.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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