Citation: Rojas-Candelas, L.E.;

Díaz-Ramírez, M.; Rayas-Amor, A.A.;

Cruz-Monterrosa, R.G.;

Méndez-Méndez, J.V.;

Villanueva-Carvajal, A.;

Cortés-Sánchez, A.d.J.

Nanomechanical, Structural and

Antioxidant Characterization of

Nixtamalized Popcorn Pericarp. Appl.

Sci. 2022, 12, 6789. https://doi.org/

10.3390/app12136789

Academic Editor: Francisco

Artés-Hernández

Received: 8 June 2022

Accepted: 23 June 2022

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applied sciences

Article

Nanomechanical, Structural and Antioxidant Characterizationof Nixtamalized Popcorn PericarpLiliana Edith Rojas-Candelas 1 , Mayra Díaz-Ramírez 1,* , Adolfo Armando Rayas-Amor 1,Rosy Gabriela Cruz-Monterrosa 1, Juan Vicente Méndez-Méndez 2 , Adriana Villanueva-Carvajal 3

and Alejandro de Jesús Cortés-Sánchez 4

1 Departamento de Ciencias de la Alimentación, División de Ciencias Biológicas y de la Salud,Universidad Autónoma Metropolitana Unidad Lerma, Av. de las Garzas No. 10, Col. El Panteón,Lerma de Villada C.P. 52005, Estado de México, Mexico; dralilianaerojascandelas@gmail.com (L.E.R.-C.);a.rayas@correo.ler.uam.mx (A.A.R.-A.); r.cruz@correo.ler.uam.mx (R.G.C.-M.)

2 Centro de Nanociencias y Micro y Nanotecnologías, Instituto Politécnico Nacional, Luis Enrique Erro s/n,Zacatenco, Gustavo A. Madero, Ciudad Mexico C.P. 07738, Mexico; najuvi@hotmail.com

3 Facultad de Ciencias Agrícolas, Universidad Autónoma del Estado de México, Campus Universitario“El Cerrillo” A.P. 435, Toluca C.P. 50200, Estado de México, Mexico; adrcarvajal@gmail.com

4 Consejo Nacional de Ciencia y Tecnología (CONACYT), Unidad Nayarit del Centro de InvestigacionesBiológicas del Noroeste (UNCIBNOR+), Calle Dos No. 23. Cd. del Conocimiento, Av. Emilio M. González, Cd.Industrial, Tepic C.P. 63173, Nayarit, Mexico; alecortes_1@hotmail.com

* Correspondence: m.diaz@correo.ler.uam.mx

Abstract: Expanded popcorn grain is widely consumed as a healthy snack all around the world;however, the study of the behavior of its components by processes such as nixtamalization is scarce.Therefore, the aim of this work was to characterize the nanomechanical, structural, and antioxidantproperties of nixtamalized popcorn grain pericarp. FT-IR results showed that the secondary structureof proteins of the nixtamalized pericarp was α-helix with 42.10%, the turn was 21.5% and 36.33%β-sheet, and proteins of the pericarp did not present the random coil structure. Pericarp showedantioxidant activity, as their values were 550.1 ± 2.9 and 44.2 ± 1.6 (TE)/mL for ABTS and DPPH,respectively; total phenols content was 0.21 ± 0.008 (TE)/mL; reducing power values were around 29to 31%; hydroxyl radical scavenging ranged from 36 to 55% and iron chelation around 115 to 140%compared to the standard acids. Thickness values of the nixtamalized pericarp by SEM image analysiswere 0.15 ± 0.1 mm near the pedicel inferior tip, 0.07 ± 0.01 mm at middle, and 0.03 ± 0.02 mm atupper of the grain. Young’s modulus value was 261.72 ± 23.58 MPa with a Gaussian function fitting atthe distribution of all values. This research provides novel and valuable information for understandingthe nanomechanical and protein arrangement, as well as and the antioxidant activity of nixtamalizedpopcorn grain pericarp in order to promote other processes and uses for this kind of pericarp maize.

Keywords: popcorn pericarp; FT-IR and SEM structure; Young’s module; antioxidant activity

1. Introduction

Popcorn grain is composed by endosperm (83%), germ (11%), pedicel (1%), andpericarp (3.4 to 9.5%). The pericarp contains hemicellulose (67%), cellulose (23%), starch(7%), proteins (1.4%), lipids (1%), lignin (0.1%), and sugars (0.5%) [1]. In addition, itcontains phenolic acids such as ferulic acid. It is recognized that the pericarp of popcorngrain is strongly linked to the expansion process, the nutritional quality, and the sensoryproperties of popcorn kernels [2]; and the antioxidant activity varies significantly with thecolor of the pericarp and the expansion process [2,3]. Pericarp is a semipermeable barriercovering the endosperm and germ [4] that influences the drying rates [5] and diffusion ofcalcium and water into the inner kernel during nixtamalization [1].

The wide versatility of the nixtamalization process for detaching the pericarp allowsfor increasing the soluble fiber, decreasing the lipid content and promoting higher protein

Appl. Sci. 2022, 12, 6789. https://doi.org/10.3390/app12136789 https://www.mdpi.com/journal/applsci

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content in the derived product, such as tortillas. In addition, the nixtamalization increasesthe solubility and availability of amino acids with major nutritional value, such as gluteninand tryptophan, and the calcium is accumulated in the pericarp [6]. Therefore, the useof nixtamalization could modify the nutrimental quality of the pericarp; however, theinformation about it is scarce.

Regarding the studies on popcorn pericarp, technological development could improvethe knowledge of its properties with the use of several analytical tools such as sensorialand physicochemical analyses, microscopy, spectroscopy techniques, and antioxidantassays [2,3,7–9]. The published studies are about the effect of the expansion process onits biochemical composition [10,11] and the effect of pericarp thickness on the expansionprocess [12] and genetic traits [13], but the studies about the nanomechanical and structuralproperties, which explains, in part, the macro-scale material changes and properties, are scarce.

Regarding the nutrimental composition and technological functions of the pericarp,this work aimed to perform a physicochemical, protein structure, antioxidant activity, andnanomechanical characterization of nixtamalized popcorn using microscopy techniques,image analysis, nanoindentation, antioxidant assays, and FT-IR. Consequently, this workprovides valuable and unpublished information on nixtamalized popcorn pericarp regard-ing its cellular architecture, physicochemical, nanomechanical, antioxidant power, andstructural properties. Based on the information obtained, it is proposed that this pericarp maybe an alternative source of antioxidants and others compounds to apply in different industriesand could provide guidelines for the characterization of another type of corn pericarp.

2. Materials and Methods2.1. Sample

The pericarp was obtained from the popcorn grains (Great Value, Wal-Mart, Inc.,Bentonville, AR, USA) after the nixtamalization process. The nixtamalization process wasperformed according to the Mexican Norm, NMX-FF-034/1-SCFI-2002, where 200 g ofpopcorn grains were added to 300 mL of NaOH 2N solution, and it was put into a waterbath at 50 ◦C for 15 min. After cooling, the pericarp was manually removed from the grain,and it was stored in hermetic bags at 25 ◦C until its analysis.

2.2. Proximate Chemical Composition

The moisture of nixtamalized popcorn grain pericarp was determined accordingto the AOAC 942.05 methodology using a thermobalance (OHAUS, MB45, Parsippany,NJ, USA). Lipid content was determined by the method of [14], while ash content wasanalyzed following the AOAC 942.05 methodology. Protein total content of the pericarpwas determined using an automated Kjeltec 8200 (FOSS, Minneapolis, MN USA) [15],and soluble protein was determined using the Bradford method [16]. The quantification ofreducing sugars of pericarp popcorn was assessed in a spectrophotometer (Thermo Scientific-Genesys Uv, Carlsbad, CA, USA) at 540 nm according to the methodology reported by [17].The total sugar was determined by adding 0.6 mL of 5% phenol to 10 uL of the 0.05% sample,kept in the dark, and stirred using a spectrophotometer (Thermo Scientific-Genesys Uv,Mexico) at 490 nm [18]. The measurements were expressed as the average of three replicatesvalues ± standard deviation.

2.3. Fourier-Transform Infrared Spectroscopy (FT-IR)

The study of pericarp structure was carried out by FT-IR spectroscopy (Agilent Carymodel 630, Santa Clara, CA, USA), according to [19]. Furthermore, FT-IR determined thesecondary structure of proteins in the pericarp. The deconvolution method was used to eval-uate the region of a protein, band amide 1. This band extends from 1610 to 1694 cm−1 [20],and the assigned structures were turns: 1662–1684 cm−1, random coil: 1640–1650 cm−1,α-helix: 1650–1658 cm−1, and β-sheet: 1610–1639 and 1685–1669 cm−1.

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2.4. Total Phenolic Content

Total phenolic content was determined according to the methodology reported by [21],with modifications for the measurement in the microplate reader applied by [22].

2.5. Antioxidant Activity2.5.1. ABTS and DPPH

DPPH radical scavenging activity was determined by the methodology of Bobo-García et al. [22], while ABTS radical scavenging activity was determined according toLeite et al. [23]. The results are expressed in mM Trolox equivalents (TE)/mL using a Troloxcalibration curve and as a percentage of scavenging of each radical compared to threecontrol solutions: coumaric acid, caffeic acid, and ferulic acid (0.1 M). A microplate readerwas used (Multiskan Go, Thermo Fisher Scientific, Waltham, MA, USA).

2.5.2. Reducing Power

The reducing power of the sample was determined according to [24]. The resultsare presented as a percentage reduction in absorbance compared to four control solutions:ferulic acid, caffeic acid, Trolox, and coumaric acid (0.1 M). A microplate reader was used(Multiskan Go, Thermo Fisher Scientific, Waltham, MA, USA).

2.5.3. Hydroxyl Radical

The methodology of the hydroxyl radical reported by [25] was used in this research.The results are presented as a percentage reduction in absorbance compared to four controlsolutions: ferulic acid, caffeic acid, Trolox, and coumaric acid (0.1 M). A microplate readerwas used (Multiskan Go, Thermo Fisher Scientific, Waltham, USA).

2.5.4. Iron Chelation

The chelating activity of the Fe +2 ion was determined by the methodology of [26] witha few modifications. The results are presented as a percentage reduction in absorbance com-pared to four control solutions: ferulic acid, caffeic acid, Trolox, and coumaric acid (0.1 M).A microplate reader was used (Multiskan Go, Thermo Fisher Scientific, Waltham, USA).

2.6. Scanning Electron Microscopy (SEM)

Dried samples were coated with gold using a sputter coater (SPI supplies, West Chester,PA, USA) and observed with a scanning electron microscope (Hitachi, SU3500 I, SantaClara, CA, USA) at 10.0 kV. The pericarp thicknesses were obtained from SEM images. Allimage analyses were performed in the software ImageJ (v. 1.46, National Institute of Health,Bethesda, MD, USA).

2.7. Nanoindentation and Imaging with Atomic Force Microscopy (AFM)

The dried pericarp was observed using AFM (Bruker, Bioscope Catalyts ScanAsyst, SantaBarbara, CA, USA), and it was placed on a fixation glass slide system. ScanAsyst mode wasused, and thus scans of 2 × 2 µm2 and 1 × 1 µm2 taken, producing five images. The studywas carried out in ambient conditions, and the cantilevers used in this study were (DNP-10A) silicon cantilevers of spring constant 0.540 Nm-1 and resonant frequency 1 kHz. Usingmatrices, Young’s modulus (YM) is indented with the point and shot method (NanosScope 1.4,Bruker, Santa Barbara, CA, USA). They also used a maximum indentation force of 50 nNand a ramp rate of 1 Hz indentation depth. Three hundred force curves were obtained frompericarp using the Hertz–Sneddon model to find YM values in NanoScope software [27].

2.8. Statistical Analysis

The measurements were performed at least in triplicate and are expressed as averagevalues and deviation standards. Data were compared using the ANOVA-Tukey test and sig-nificant differences (p < 0.05). Statistical analyses were carried out in SigmaPlot software, v.12

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(Systat Software Inc., Palo Alto, CA, USA). Frequency histograms of Young’s modulus valueswere performed to fit the data with the best model to describe the data distribution

3. Results and Discussion3.1. Physicochemical Parameters

The pericarp is a film that covers the maize endosperm; it is around 5% of all kernels [1].The ash is the content of minerals in the sample, and in this research (Table 1), we foundhigher values (67.77 ± 3.13%) in the pericarp compared with those of the literature (1.50to 0.88%) [2], probably because our corn underwent a nixtamalization process to detachthe pericarp from the grain, and therefore, this process provided more minerals to thepericarp. The reducing sugar content of pericarp showed a higher value (3.93 ± 0.14%) thanother research values in popcorn where the whole kernel was occupied (0.07 to 0.23% [7]),probably due to the sugar alkaline process by alkaline-high temperature treatment; butalso, complex polysaccharides are maintained because the value of non-reducing sugars is6.99 ± 0.23%. The value of lipid content in pericarp (6.76 ± 0.13%) is similar to [2], whichworked with popcorn kernels of the red pericarp. They reported values of 5.53 ± 0.97%when popcorn was unexpanded. Moisture (5.71 ± 0.53%) and protein (7.2 ± 0.33%) contentvalues of popcorn pericarp were lower than the reported (10.46 to 10.76 and 13.42 to 9.76%,respectively) [2]. Furthermore, Table 1 shows that the pericarp has more insoluble proteinthan soluble due to the fact that insoluble protein comes from the alkaline hydrolysis of thepericarp, while the soluble protein comes from the germ and even endosperm.

Table 1. Physicochemical parameters of nixtamalized popcorn pericarp.

Parameter

Ash (%) 67.77 ± 3.13Lipid (%) 6.76 ± 0.13

Total sugars (%) 10.93 ± 0.13Reducing sugars (%) 3.93 ± 0.14

Non-reducing sugars (%) 6.99 ± 0.23Moisture (%) 5.71 ± 0.53Nitrogen (%) 1.15 ± 0.04

Crude protein total (%) 7.2 ± 0.33Crude protein soluble (%) 0.11 ± 0.005

Crude protein insoluble (%) 7.10 ± 0.30Results expressed as mean value ± standard deviation.

3.2. FTIR Spectra of Pericarp

The pericarp contains polysaccharides such as cellulose (23–40%) and hemicellulose(50–67%); in the FT-IR spectrum (Figure 1), these polysaccharides are observed at 1160 cm−1,and polysaccharide skeletons with arabinose residues in bands 1079 and 1100 cm−1 [28].The presence of these residues is probably due to ferulic acid [29] (Figure 1, spectrum at1519 cm−1) since it has been observed that this acid can modify the structure of arabinoseto bind it to cellulose and hemicellulose [30]. The pericarp also contains lipids (3050 cm−1)and proteins (presence of aromatic molecules around 1609, 1608, 1516, and 1517 cm−1) [31].These results are according to Table 1.

In addition, stretching of OH and NH groups was observed around 3350 cm−1, andCH was at 2929 cm−1 [30]; this chemical structure influences the interactions with othercomponents and could modify the technological properties as foaming and emulsify-ing capacities of this system [32]. Finally, the protein of the pericarp was shown at the1610–1694 cm−1 band, concerning amide I and band amide II at 1500–1560 cm−1. Thesebands are the protein zone, and they allow us to know the secondary structure of proteins(Figure 2). The protein of the pericarp showed that the highest value was 42.10%, whichis an α-helix structure related to the emulsifier properties and force of the gel [32]. While21.56% and 36.33% are related to the β-sheet, the protein of the pericarp did not presentthe random coil structure. This behavior allows understanding that the proteins found in

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partially unfolded structures could react with other components of the pericarp because theactive sites are available and could react with other components or reactivate and changeproperties of the pericarp.

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(3050 cm−1) and proteins (presence of aromatic molecules around 1609, 1608, 1516, and 1517 cm−1) [31]. These results are according to Table 1.

Figure 1. FT-IR spectra of the pericarp.

In addition, stretching of OH and NH groups was observed around 3350 cm−1, and CH was at 2929 cm−1 [30]; this chemical structure influences the interactions with other components and could modify the technological properties as foaming and emulsifying capacities of this system [32]. Finally, the protein of the pericarp was shown at the 1610–1694 cm−1 band, concerning amide I and band amide II at 1500–1560 cm−1. These bands are the protein zone, and they allow us to know the secondary structure of proteins (Figure 2). The protein of the pericarp showed that the highest value was 42.10%, which is an α-helix structure related to the emulsifier properties and force of the gel [32]. While 21.56% and 36.33% are related to the β-sheet, the protein of the pericarp did not present the random coil structure. This behavior allows understanding that the proteins found in partially unfolded structures could react with other components of the pericarp because the active sites are available and could react with other components or reactivate and change properties of the pericarp.

Figure 1. FT-IR spectra of the pericarp.

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Figure 2. FT-IR spectra of pericarp for the amide band I region, which represents peak-fitting of the second derivative curves of the spectra.

3.3. Total Phenolic Content The obtained value of total phenolic content in the nixtamalized popcorn pericarp

(Table 2) (0.21 mgGAE/mL) is lower than the reported values in the literature, probably because of the nixtamalization process. In this regard, Zhang et al. [33] reported values of around 138.00 to 57.04 mg GAE/100 g for eight representative sweet corn varieties grown. Other research found 232.9 ± 3.6 mg GAE/g DM in the barley and spelt flour [34], while Ragaee et al. [35], who conducted a study on different grain cereals, found in hard wheat a value of 562 ± 28.8, barley 879 ± 24.0 and millet 1387 ± 13.3 (results were expressed as gallic acid equivalent (µg/g)), while Lopez-Martinez et al. [36] reported values in differ-ent corn with different colors: white 170.2 ± 4.5 mg/100 g, blue 343.2 ± 6.8 mg/100 g, red 465.3 ± 5.7 mg/100 g and, green 1760 ± 8.2 mg/100 g. The blue and red color of the pericarp is related to the anthocyanins content, which, in turn, positively affect the antioxidant activity of the grain [2].

Table 2. Antioxidant activity of pericarp (total phenolic content, ABTS and DPPH).

Sample Total

Phenolic Content ABTS DPPH

Pericarp 0.21 ± 0.008 550.1 ± 2.9 44.2 ± 1.6 The content of total phenols will be expressed in µg gallic acid equivalents/mL of sample (mg GAE/mL), ABTS and DPPH assays reported in mM Trolox equivalents (TE)/mL.

Figure 2. FT-IR spectra of pericarp for the amide band I region, which represents peak-fitting of thesecond derivative curves of the spectra.

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3.3. Total Phenolic Content

The obtained value of total phenolic content in the nixtamalized popcorn pericarp(Table 2) (0.21 mgGAE/mL) is lower than the reported values in the literature, probablybecause of the nixtamalization process. In this regard, Zhang et al. [33] reported values ofaround 138.00 to 57.04 mg GAE/100 g for eight representative sweet corn varieties grown.Other research found 232.9 ± 3.6 mg GAE/g DM in the barley and spelt flour [34], whileRagaee et al. [35], who conducted a study on different grain cereals, found in hard wheata value of 562 ± 28.8, barley 879 ± 24.0 and millet 1387 ± 13.3 (results were expressedas gallic acid equivalent (µg/g)), while Lopez-Martinez et al. [36] reported values indifferent corn with different colors: white 170.2 ± 4.5 mg/100 g, blue 343.2 ± 6.8 mg/100 g,red 465.3 ± 5.7 mg/100 g and, green 1760 ± 8.2 mg/100 g. The blue and red color ofthe pericarp is related to the anthocyanins content, which, in turn, positively affect theantioxidant activity of the grain [2].

Table 2. Antioxidant activity of pericarp (total phenolic content, ABTS and DPPH).

Sample TotalPhenolic Content ABTS DPPH

Pericarp 0.21 ± 0.008 550.1 ± 2.9 44.2 ± 1.6

The content of total phenols will be expressed in µg gallic acid equivalents/mL of sample (mg GAE/mL), ABTSand DPPH assays reported in mM Trolox equivalents (TE)/mL.

3.4. Antioxidant Activity3.4.1. ABTS and DPPH

Table 2 shows the values of ABTS (550.1 ± 2.9) and DPPH (44.2 ± 1.6) radical scav-enging activities of nixtamalized popcorn pericarp. Herrera-Balandrano et al. [37] workedwith nixtamalized maize bran and reported values from 29.49 to 31.69 µmol TE/g of DPPH,while Ragaee et al. [35] conducted a study on different grain cereals about their antioxidantactivity, and they found values of 4.33 ± 0.17 of DPPH and 8.8 ± 0.39 of ABTS in hardwheat, 21.00 ± 0.83 of DPPH and 14.9 ± 0.61 of ABTS in barley and 23.83 ± 0.67 of DPPHand 21.4 ± 0.43 of ABTS (results were expressed in µmol/g) in millet. These antioxidantactivities of grain cereals depend on environmental and genetic factors. Paraginski et al. [2]concluded that there are relationships between the bioactive compounds and the colorpericarp because the red pericarp grain had higher values of ABTS (13.99) than the others.The information on the total phenol content concluded that it is higher in whole kernel thanin the pericarp, and it also depends on the color, being higher when they are red and blueand when popcorn explodes due to the heat [2,8]. Furthermore, the total phenol contentand these antioxidant activities depend on the nixtamalization process.

3.4.2. Reducing Power

The results of reducing power are shown in Figure 3. The activity of the pericarp wascompared with the activity shown by Trolox, caffeic acid, ferulic acid, and coumaric acid(0.1 M), as the pericarp contains these last three acids. The pericarp exerts a reducing powerof 29% compared to the Trolox (0.1 M), 30% compared to caffeic acid (0.1 M), 33% comparedto ferulic acid, and 88% compared to coumaric acid (0.1 M), being the coumaric acid, thecontrol with a highest reducing power; high percentages are related to a bioactive componentperformance. The values of reducing power were not significantly different (p < 0.05) betweencaffeic acid and Trolox. Lopez-Martinez et al. [36] reported values of around 85% to 40% indifferent corn types, while Ivanišová et al. [34] studied reducing power in milling cereals suchas barley, and they obtained values of around 32–38% expressed as mg of Trolox equivalent.Another study found the lowest values in reducing power in yellow and white maize fromNigeria at 2.27 d ± 0.06 and 0.94 ± 0.03 (mg AAE g−1), respectively [38]. Therefore, weconcluded that the pericarp has an actual charge of reducing power.

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Figure 3. Percentage of reducing power of Pericarp comparison with Trolox, caffeic acid, ferulic acid, coumaric acid (0.1 M). Values reported as percentage (%). Values presented are the average of three replicates ± standard deviation. Different letters indicate that the values are significantly dif-ferent (p < 0.05).

3.4.3. Hydroxyl Radical The results of OH radicals are shown in Figure 4. The activity of the pericarp is

compared with the activity shown by Trolox, caffeic acid, ferulic acid, and coumaric acid (0.1M). The pericarp exerts OH radical scavenging activity of 51% compared to the Trolox (0.1 M), 36% compared to caffeic acid (0.1 M), 53% compared to coumaric acid (0.1 M), and 55% compared to ferulic acid. The activities showed by Trolox, caffeic acid, ferulic acid and coumaric acid are significantly different (p < 0.05). A high positive correlation (0.809) between total phenolic content and hydroxyl radical (OH) indicates that phenolic compounds catch radicals free [35]. Martínez-Tomé et al. [39] reported OH radical scav-enging activity values in cereal brands, wheat bran with 79.2% and oat bran with 41.3%, while in another study, protein hydrolyzates from corn gluten meal reported hydroxyl radicals of around 20 to 100% [40]. A study on bonito (kasuto) from Japan reported sim-ilar values of around 54 ± 1 with ascorbic acid and garlic (88 ± 4), and onion (89 ± 4) had higher values than popcorn pericarp [41]. These show that the pericarp has a significant OH radical scavenging activity and can be applied in different industries (food, nutraceutical and cosmetic) as a good source of antioxidant compounds.

Figure 3. Percentage of reducing power of Pericarp comparison with Trolox, caffeic acid, ferulic acid,coumaric acid (0.1 M). Values reported as percentage (%). Values presented are the average of threereplicates ± standard deviation. Different letters indicate that the values are significantly different(p < 0.05).

3.4.3. Hydroxyl Radical

The results of OH radicals are shown in Figure 4. The activity of the pericarp iscompared with the activity shown by Trolox, caffeic acid, ferulic acid, and coumaric acid(0.1M). The pericarp exerts OH radical scavenging activity of 51% compared to the Trolox(0.1 M), 36% compared to caffeic acid (0.1 M), 53% compared to coumaric acid (0.1 M), and55% compared to ferulic acid. The activities showed by Trolox, caffeic acid, ferulic acidand coumaric acid are significantly different (p < 0.05). A high positive correlation (0.809)between total phenolic content and hydroxyl radical (OH) indicates that phenolic com-pounds catch radicals free [35]. Martínez-Tomé et al. [39] reported OH radical scavengingactivity values in cereal brands, wheat bran with 79.2% and oat bran with 41.3%, while inanother study, protein hydrolyzates from corn gluten meal reported hydroxyl radicals ofaround 20 to 100% [40]. A study on bonito (kasuto) from Japan reported similar valuesof around 54 ± 1 with ascorbic acid and garlic (88 ± 4), and onion (89 ± 4) had highervalues than popcorn pericarp [41]. These show that the pericarp has a significant OHradical scavenging activity and can be applied in different industries (food, nutraceuticaland cosmetic) as a good source of antioxidant compounds.

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Figure 4. Percentage of hydroxyl radical of pericarp comparison with Trolox, caffeic acid, ferulic acid, coumaric acid (0.1 M). Values reported as percentage (%). Values presented are the average of three replicates ± standard deviation. Different letters indicate that the values are significantly dif-ferent (p < 0.05).

3.4.4. Iron Chelation The results of iron chelation are shown in Figure 5. The activity of the pericarp is

compared with the activity shown by Trolox, caffeic acid, ferulic acid, and coumaric acid (0.1 M). The pericarp exerts iron chelation of 134% compared to the Trolox (0.1 M), 116% compared to coumaric acid (0.1 M), 116% compared to ferulic acid (0.1 M), and 141% compared to caffeic acid. The activity showed no significant difference between ferulic acid and coumaric acid (p < 0.05). A study found values of iron chelation in six medicinal plant extracts, and Clinacanthus nutanshad had high values of around 88.2 ± 0.4 [42]. The values found in another study demonstrated that the pericarp has iron chelation signifi-cant enough to apply to the food, nutraceutical and cosmetic industries as a new source of antioxidant compounds [33].

Figure 4. Percentage of hydroxyl radical of pericarp comparison with Trolox, caffeic acid, ferulic acid,coumaric acid (0.1 M). Values reported as percentage (%). Values presented are the average of threereplicates ± standard deviation. Different letters indicate that the values are significantly different(p < 0.05).

3.4.4. Iron Chelation

The results of iron chelation are shown in Figure 5. The activity of the pericarp iscompared with the activity shown by Trolox, caffeic acid, ferulic acid, and coumaric acid(0.1 M). The pericarp exerts iron chelation of 134% compared to the Trolox (0.1 M), 116%compared to coumaric acid (0.1 M), 116% compared to ferulic acid (0.1 M), and 141%compared to caffeic acid. The activity showed no significant difference between ferulicacid and coumaric acid (p < 0.05). A study found values of iron chelation in six medicinalplant extracts, and Clinacanthus nutanshad had high values of around 88.2 ± 0.4 [42]. Thevalues found in another study demonstrated that the pericarp has iron chelation significantenough to apply to the food, nutraceutical and cosmetic industries as a new source ofantioxidant compounds [33].

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Figure 5. Percentage of iron chelation of pericarp comparison with Trolox, caffeic acid, ferulic acid, coumaric acid (0.1 M). Values reported as percentage (%). Values presented are the average of three replicates ± standard deviation. Different letters indicate that the values are significantly different (p < 0.05).

3.5. Scanning Electron Microscopy (SEM) SEM images (Figure 6) show the microstructure, thickness and fibers of the popcorn

pericarp; thickness values depend on the part of the grain, where the near pedicel tip is thicker than in the middle and end of grain (Figure 6(a1,b1,c1)) due to the fact that the pedicel is the principal structural medium that allows nutrient transportation and is re-lated to the vascular tissue development [43]. The middle and near pedicel fibers were more alienated than the final part of the grain. These fibers contain cellulose, hemicellu-lose, and lignin. Furthermore, the pericarp surface has a waxy layer appearance that al-lows mass transfer [1]. A close-up of pericarp images (Figure 6(a2,b2,c2) show the change in the fiber’s disposition and direction. The values of thickness at the end part of the popcorn pericarp (Figure 6(c1)) were similar to those reported in the Cacahuacintle sample from Mexico, the values of which were around 28 µm [44], while in the middle part of the pericarp (Figure 6(b1)), values of 70 ± 10 µm were found according to the values of Maíz Ancho and pozolero from Jalisco, Mexico [44]. Gutiérrez-Cortez et al. [1] reported values of white maize from Guanajuato, Mexico, and these authors found three zones with different thicknesses, which were around 75 to 87 µm. Finally, the thickness values of the near pedicel part were around 150 ± 10 µm—similar to the values of the study of Narváez-González et al. [9] in Popcorn, Cónico, Dulce, Arrocillo, and Nal-Tel.

Figure 5. Percentage of iron chelation of pericarp comparison with Trolox, caffeic acid, ferulic acid,coumaric acid (0.1 M). Values reported as percentage (%). Values presented are the average of threereplicates ± standard deviation. Different letters indicate that the values are significantly different(p < 0.05).

3.5. Scanning Electron Microscopy (SEM)

SEM images (Figure 6) show the microstructure, thickness and fibers of the popcornpericarp; thickness values depend on the part of the grain, where the near pedicel tip isthicker than in the middle and end of grain (Figure 6(a1,b1,c1)) due to the fact that thepedicel is the principal structural medium that allows nutrient transportation and is relatedto the vascular tissue development [43]. The middle and near pedicel fibers were morealienated than the final part of the grain. These fibers contain cellulose, hemicellulose,and lignin. Furthermore, the pericarp surface has a waxy layer appearance that allowsmass transfer [1]. A close-up of pericarp images (Figure 6(a2,b2,c2)) show the changein the fiber’s disposition and direction. The values of thickness at the end part of thepopcorn pericarp (Figure 6(c1)) were similar to those reported in the Cacahuacintle samplefrom Mexico, the values of which were around 28 µm [44], while in the middle part ofthe pericarp (Figure 6(b1)), values of 70 ± 10 µm were found according to the values ofMaíz Ancho and pozolero from Jalisco, Mexico [44]. Gutiérrez-Cortez et al. [1] reportedvalues of white maize from Guanajuato, Mexico, and these authors found three zoneswith different thicknesses, which were around 75 to 87 µm. Finally, the thickness valuesof the near pedicel part were around 150 ± 10 µm—similar to the values of the study ofNarváez-González et al. [9] in Popcorn, Cónico, Dulce, Arrocillo, and Nal-Tel.

Appl. Sci. 2022, 12, 6789 10 of 14Appl. Sci. 2022, 12, x FOR PEER REVIEW 11 of 15

Figure 6. SEM micrographs of pericarp in different parts. Scales correspond to 1 mm. a1: The near pedicel part, b1: in middle part, c1: end part, and a2, b2, c2: close up of pericarp parts, images by fast Fourier transform (FFT).

3.6. Nanostructure and Nanoindentation Pericarp mechanics play a pivotal role in grain firmness and protection, but their

relationships with structure and stiffness remain unknown to a certain extent. For this reason, topography, stiffness, and the structure of the pericarp were studied using AFM. The surface of the popcorn pericarp is a heterogeneous topography with roughness val-ues (Ra) of around 4.32 ± 0.40 nm. The pericarp’s surface is associated with a wide dis-tribution of Young’s modulus (YM) values shown in the histogram of Figure 7 (261.72 ± 23.58 MPa). Furthermore, a Gaussian function was the most appropriate model for fitting the distribution of YM values according to the mathematical modeling optimization em-ployed. Similar topography was observed in corn kernels from Jilin province of China, where they had values of Ra 3.41 ± 0.89 nm [45]. Another study of eggshells reported similar values (5.30 to 8.81 nm) to pericarp, but they had different surfaces because the eggshell showed pores [46]. In contrast, Rojas-Candelas et al. [27] reported higher values of Ra around from 20.63 to 38.65 nm in apple tissues, but they have lower values of Young’s modulus compared with pericarp (0.90 ± 1.30 to 1.76 ± 1.01 MPa). Antoine et al. [47] reported values of YM in the pericarp of grain’s wheat (cv. Baroudeur and, cv. Scipion) where the values were 20.6 ± 2.8 and 28.9 ± 6.7 N/mm, respectively. Another study determined YM in bread wheat Buck-Amancay cultivar and ACA 315 cultivar from Argentina. They found values of an outer layer of around 2799 ± 659 and 1404 ± 121 MPa, respectively, with 10% moisture [48].

Mechanical characterization of pericarp allows knowing the physiological state of cells and the effect of individual biomolecules and assemblies on overall properties on the cell surfaces.

Figure 6. SEM micrographs of pericarp in different parts. Scales correspond to 1 mm. (a1): The nearpedicel part, (b1): in middle part, (c1): end part, and (a2,b2,c2): close up of pericarp parts, images byfast Fourier transform (FFT).

3.6. Nanostructure and Nanoindentation

Pericarp mechanics play a pivotal role in grain firmness and protection, but theirrelationships with structure and stiffness remain unknown to a certain extent. For thisreason, topography, stiffness, and the structure of the pericarp were studied using AFM.The surface of the popcorn pericarp is a heterogeneous topography with roughness values(Ra) of around 4.32 ± 0.40 nm. The pericarp’s surface is associated with a wide distributionof Young’s modulus (YM) values shown in the histogram of Figure 7 (261.72 ± 23.58 MPa).Furthermore, a Gaussian function was the most appropriate model for fitting the distri-bution of YM values according to the mathematical modeling optimization employed.Similar topography was observed in corn kernels from Jilin province of China, where theyhad values of Ra 3.41 ± 0.89 nm [45]. Another study of eggshells reported similar values(5.30 to 8.81 nm) to pericarp, but they had different surfaces because the eggshell showedpores [46]. In contrast, Rojas-Candelas et al. [27] reported higher values of Ra around from20.63 to 38.65 nm in apple tissues, but they have lower values of Young’s modulus comparedwith pericarp (0.90 ± 1.30 to 1.76 ± 1.01 MPa). Antoine et al. [47] reported values of YMin the pericarp of grain’s wheat (cv. Baroudeur and, cv. Scipion) where the values were20.6 ± 2.8 and 28.9 ± 6.7 N/mm, respectively. Another study determined YM in bread wheatBuck-Amancay cultivar and ACA 315 cultivar from Argentina. They found values of an outerlayer of around 2799 ± 659 and 1404 ± 121 MPa, respectively, with 10% moisture [48].

Appl. Sci. 2022, 12, 6789 11 of 14

Appl. Sci. 2022, 12, x FOR PEER REVIEW 12 of 15

Figure 7. Frequency histogram of YM values, YM: Young’s modulus. The line indicates the fit with a Gaussian function (a). AFM height image (1 × 1 µm) of pericarp. Ra: roughness (b).

4. Conclusions The current study integrated different characterization techniques at the micro and

nanostructural level along with statistical tools and provided novel data to study the popcorn pericarp. The secondary structure of the pericarp protein through FT-IR pro-vided relevant information about the state that unfolds or folds the proteins. This study found that pericarp has significant antioxidant activities compared with other antioxi-dants, such as ferulic acid, caffeic acid, Trolox, and coumaric, and this behavior was re-lated to the nixtamalization process. The qualitative information obtained by SEM indi-cated that the thickness depends on the pericarp parts. Atomic force microscopy allowed us to examine the nanomechanical properties of the pericarp, which showed significant values of Young’s modulus that correspond to a film that covers the popcorn that cannot be stronger because it would not allow it to pop but it is necessary to protect the grain. Nixtamalization increased antioxidant activity.

Author Contributions: Conceptualization, L.E.R.-C., A.V.-C., and M.D.-R.; methodology, L.E.R.-C., A.V.-C., and M.D.-R.; software, L.E.R.-C.; validation, L.E.R.-C. A.d.J.C.-S., and M.D.-R.; formal analysis L.E.R.-C.; investigation L.E.R.-C. and J.V.M.-M.; resources L.E.R.-C., A.d.J.C.-S., and M.D.-R.; data curation L.E.R.-C.; writing—original draft preparation, L.E.R.-C.; writing—review and editing A.A.R.-A., and R.G.C.-M.; visualization, A.A.R.-A. and R.G.C.-M.; supervision, L.E.R.-C. and M.D.-R.; project administration, M.D.-R. funding acquisition, M.D.-R. All authors have read and agreed to the published version of the manuscript.

Funding: Not applicable.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The authors declare the transparency of data.

Acknowledgments: Liliana Edith Rojas Candelas wishes to thank COMECYT in Mexico State for her professorship and the financial support provided by COMECYT (CAT2021-0080), also Felipe Cervantes Sodi of Universidad Iberoamericana for the micrographs SEM.

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the man-uscript, or in the decision to publish the results.

Figure 7. Frequency histogram of YM values, YM: Young’s modulus. The line indicates the fit with aGaussian function (a). AFM height image (1 × 1 µm) of pericarp. Ra: roughness (b).

Mechanical characterization of pericarp allows knowing the physiological state of cells andthe effect of individual biomolecules and assemblies on overall properties on the cell surfaces.

4. Conclusions

The current study integrated different characterization techniques at the micro andnanostructural level along with statistical tools and provided novel data to study thepopcorn pericarp. The secondary structure of the pericarp protein through FT-IR providedrelevant information about the state that unfolds or folds the proteins. This study foundthat pericarp has significant antioxidant activities compared with other antioxidants, suchas ferulic acid, caffeic acid, Trolox, and coumaric, and this behavior was related to thenixtamalization process. The qualitative information obtained by SEM indicated that thethickness depends on the pericarp parts. Atomic force microscopy allowed us to examinethe nanomechanical properties of the pericarp, which showed significant values of Young’smodulus that correspond to a film that covers the popcorn that cannot be stronger because itwould not allow it to pop but it is necessary to protect the grain. Nixtamalization increasedantioxidant activity.

Author Contributions: Conceptualization, L.E.R.-C., A.V.-C. and M.D.-R.; methodology, L.E.R.-C.,A.V.-C. and M.D.-R.; software, L.E.R.-C.; validation, L.E.R.-C. A.d.J.C.-S. and M.D.-R.; formal analysisL.E.R.-C.; investigation L.E.R.-C. and J.V.M.-M.; resources L.E.R.-C., A.d.J.C.-S. and M.D.-R.; datacuration L.E.R.-C.; writing—original draft preparation, L.E.R.-C.; writing—review and editing,A.A.R.-A. and R.G.C.-M.; visualization, A.A.R.-A. and R.G.C.-M.; supervision, L.E.R.-C. and M.D.-R.;project administration, M.D.-R. funding acquisition, M.D.-R. All authors have read and agreed to thepublished version of the manuscript.

Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: The authors declare the transparency of data.

Acknowledgments: Liliana Edith Rojas Candelas wishes to thank COMECYT in Mexico State forher professorship and the financial support provided by COMECYT (CAT2021-0080), also FelipeCervantes Sodi of Universidad Iberoamericana for the micrographs SEM.

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the designof the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, orin the decision to publish the results.

Appl. Sci. 2022, 12, 6789 12 of 14

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