*3.8. In Vitro Determination of the Antioxidant Activity of the Samples* 3.8.1. Reducing Power (RP)

The RP of the conjugates was determined according to the method described by Oyaizu et al. [40]. It is based on the reduction of ferricyanide [Fe(CN)6] −3 to the ferrocyanide [Fe(CN)6] <sup>−</sup><sup>4</sup> anion. Owing to this reduction, Prussian blue was formed in the presence of Fe3+. PBS (0.2 M, pH = 6.6; 50 µL) and 50 µL of K3[Fe(CN)6] (1%) were added to 20 µL of the samples at the concentrations of 15, 7.5, 3.75, and 1.88 mg/mL in one 96-well microplate. The mixture was incubated at 40 ◦C for 20 min. Subsequently, 50 µL of TCA (10%) and 10 µL of FeCl<sup>3</sup> (0.1%) were added and incubated for 10 min at 40 ◦C. The absorbance was read at 700 nm using a microplate reader.

### 3.8.2. Hydroxyl Radical Scavenging Activity

The percentage hydroxyl radical (OH·) scavenging capacity of the conjugates was determined according to the method developed by Lin et al. [41]. In this procedure, OH· is generated using the Fenton reaction. Thus, 20 µL of each sample at the concentrations of 15, 7.5, 3.75, and 1.88 mg/mL were placed in a 96-well microplate. Subsequently, 50 µL of 1,10-phenanthroline and FeSO<sup>4</sup> (3 mM) were added. To start the reaction, 50 µL of H2O<sup>2</sup> were added and incubated at 37 ◦C for 60 min. The absorbance was measured using a microplate reader at 536 nm.

### *3.9. Statistical Analysis*

The statistical analysis of the data obtained during the determination of the extension of conjugation and the antioxidant activity tests was performed using GraphPad Prism v6.0, La Jolla, CA, USA. A one-way analysis of variance (ANOVA) and Tukey's test were performed for multiple comparisons. The statistical significance was set at *p* ≤ 0.05.

### **4. Conclusions**

The conjugation of peptide fractions from germinated soybeans to Fe3O<sup>4</sup> NPs was achieved for the first time using a multicomponent protocol. The U-4C reaction was an efficient and versatile procedure for the conjugation of bioactive peptide fractions to Fe3O<sup>4</sup> NPs. The experiments on the RP and hydroxyl radical scavenging activity demonstrated that the antioxidant capacity of the peptide fractions was maintained or increased after the conjugation. The only exception was the conjugation of the >10 kDa peptide fraction to the magnetite NPs coated with sodium citrate (conjugate 1). Its RP decreased slightly with respect to the nonconjugated peptide fraction. Fe3O4@APTES@ > 10 kDa showed the highest antioxidant capacity. The obtained results suggest a synergistic effect of the conjugation of peptide fractions from germinated soybeans to the coated Fe3O<sup>4</sup> NPs by the U-4C reaction.

**Author Contributions:** Conceptualization, H.Y.-M., R.M.-E., Y.E.A.-J. and C.D.-Á.; Methodology, Y.E.A.-J., M.G.-M., D.U.-R., E.C.-U. and D.N.-F.; Validation, H.Y.-M. and R.M.-E.; Formal analysis, Y.E.A.-J.; Investigation, Y.E.A.-J.; Resources, R.M.-E. and H.Y.-M.; Writing, Y.E.A.-J. and R.M.-E.; Supervision, R.M.-E., H.Y.-M., C.D.-Á. and E.C.-U.; Funding acquisition, R.M.-E. and H.Y.-M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by grants from Instituto Politécnico Nacional (SIP:20181718 project) and CONACyT (PY-SEP-CONACyT 242860 project). The work was also supported with a PhD scholarship to A.T.M. from CONACyT.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data of this research is available from the authors.

**Acknowledgments:** Y.E.A.J. acknowledges CONACyT for scholarship No. 622043 and H.Y.M. E.C.U. and R.M.E. acknowledge the economic support from EDI, COFAA, and SNI. The authors acknowledge CNMN-IPN and Liliana Alamilla-Beltrán (from ENCB-IPN) for the experimental

support provided. Y.E.A.J. is thankful to Eduardo Gonzalez-Martinez and Gerardo-Manuel Ojeda-Carralero for their valuable suggestions.

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

**Sample Availability:** Samples of the compounds Fe3O4@APTES@ > 10 kDa are available from the authors.

## **References**

