Physical–chemical characterization and antioxidant properties of extruded products made from mixtures composed of corn grits and red potato flour (Oxalis tuberosa)

ABSTRACT Some physicochemical characteristics and antioxidant activity of extrudates made from corn grits and red potato flour were evaluated. The inclusion of red potato flour altered values of water absorption index, water solubility index, expansion index and fracturability of extrudates. Color analysis of extrudates showed a reduction of luminosity from 44.37 ± 0.87 to 25.06 ± 1.74. Likewise, extrudates made with red potato flour at 100% presented higher content of total phenols compared to the control extrudates (0.44 ± 0.02 and 2.88 ± 0.03 mg GA/g dry sample, respectively); consequently, an increase in antioxidant capacity was observed using DPPH, ABTS and FRAP assays. The images obtained by fluorescence microscopy provided information on the protein content. The proximal chemical analysis indicated that extrudates substituted with red potato flour at 10% showed higher protein content (7.78 g/100 g), compared to T5. This study concludes that when adding red potato flour, is possible to develop extrudates with good physicochemical characteristics and antioxidant properties.


Introduction
Snacks are one of the most popular foods around the world, they are eaten between regular meals and include a variety of products of different textures. In this group of foods, extruded products play an important role due to its high stability and durability (Hirth, Leiter, Beck, & Schuchmann, 2014;Kolniak-Ostek et al., 2017). Extrusion is a process by which snack products are made and has become a very important method due to the ease of operation and ability to produce a variety of sizes, shapes and textures (Chalermchaiwat, Jangchud, Jangchud, Charunuch, & Prinyawiwatkul, 2015). Generally, cereals are the most used ingredients in extrusion processes due to their ability to provide texture, structure, volume and many other characteristics desired in finished products (Seth, Badwaik, & Ganapathy, 2015). However, in recent years different studies have been carried out to evaluate the effect of different fruits, vegetables and starches for making food products that contain high nutritional value compounds, characteristics which promote healthy living (Morales et al., 2015). Red potato (Oxalis tuberosa) is a tuber which is considered a good source of starch, has antibacterial, antifungal properties and antioxidant properties (Chirinos et al., 2009). Also, there are studies on the nutritional value of these tubers that indicate their potential as an alternative ingredient for use in the food industry (Condori et al., 2008). Therefore, the objective of the present study was to investigate the suitability of mixing corn grits and red potato flour to produce extrudates as well as evaluating the physicochemical characteristics and functional properties of the final product.

Materials
To prepare red potato flour, the tubers were cut into slices of 2 to 3 mm in length and dehydrated in a drying oven (Muebles Inoxidables Luckie SA, Mexico City, Mexico) at an average temperature of 55°C for 6 h. Subsequently, they were crushed and passed through a sieve (425 μm mesh) to homogenize the particle size. Yellow corn grits (21-22 µm) were purchased at a local store.

Preparation of extrudates
Before preparing the extrudates, the corn grits and red potato flour were mixed in the following ratios of: (90:10) T1, (80:20) T2, (70:30) T3, (60:40) T4 and (50:50%) T5, respectively. Both the mixtures as red potato flour and corn grits were hydrated with distilled water until an average of 30% of moisture. They were then packed in airtight bags and refrigerated for 12 h to balance the water distribution.
The extrusion process was carried out in a twin screw extruder (PRISM USALAB 16 Thermo, Electron Corporation, Newington, NH, USA). The diameter of the cylinder and its length/diameter ratio (L/D) was 16 mm and 25:1, respectively. It was equipped with six temperature zones and a round die of 3 mm in diameter. The extruder was operated at a screw speed of 130 rpm and fed at a rate of 28 g/min. The extrusion temperatures were 98-102, 135-130 and 100-90°C for the treatments (T1, T2, T3, T4, T5 and control); for red potato flour they were 85-100, 115-110 and 93-82°C.

Chemical composition of extrudates
AOAC standard methods were used to determine moisture, lipid, ash, total fiber content and nitrogen (methods 925.10, 920.39, 923.03, 985.29 and 920.87, respectively, AOAC, 2002). The carbohydrates content were quantified by difference.

Expansion index
The expansion index was calculated by dividing the diameter of the extrudate by the diameter of the die used (Philipp, Oey, Silcock, Beck, & Buckow, 2017). The measurement of both diameters was carried out using a vernier calipers (Fowler, model 0-6-0-150, Mexico), and the data used were the average of 10 replicas.

Water absorption index and water solubility index
The determination of the water absorption index (WAI) and water solubility index (WSI) was carried out according to the technique described by Fiorda, Soares, Silva, Moura, and Grosmann (2015) with some modifications. A 1-g sample (previously ground) was dissolved in distilled water at room temperature (25 ± 1°C), the obtained slurry was vortexed for 30 min and centrifuged at 1500 rpm. The WAI is the weight of gel obtained after removal of the supernatant per unit weight of original dry solids, and the WSI is the weight of dry solids in the supernatant expressed as a percentage of the original weight of the sample.

Fracture tests
The maximum breaking force by-product compression was determined in a universal machine for mechanical tests (Universal Testing System, Instron model 4411), using a 4900 N load cell (Pérez-Navarrete, Cruz-Estrada, Chel-Guerrero, & Betancur-Ancona, 2006).

Color analysis
The color parameters in the extrudates were evaluated with a colorimeter (Minolta, CM-508D, Osaka, Japan). The data were provided as CIE L*a*b* coordinates, which define the color in a three-dimensional space (Peksa et al., 2016).

Differential scanning calorimetry (DSC)
The DSC analysis for the flour and extrudates were carried out according to the technique described by Moreira, Chenlo, and Arufe (2015). The previously ground sample (15-30 mg) was introduced into an aluminum basket and hermetically sealed and the test consisted of equilibrating the sample (without adding water) at 30°C and then heating it up to 250°C at a constant heating rate of 10°C/min. The thermal properties were determined using a differential scanning calorimeter (Star System DSC1, Mettler Toledo Ltd., Victoria, Australia). All temperatures (beginning, maximum and end) were obtained by an endothermic curve.

Total phenolic content
Aqueous extracts were obtained by weighing 0.025 to 0.2 g of dry sample (DS) (extrudates previously ground), and were diluted in 10 ml of distilled water, followed by vortexing for 1 min and centrifuged at 4000 rpm for 10 min. Total phenolic content was determined using the Folin Ciocalteu colorimetric technique as described by Ondo and Ryu (2013). Aliquots of 1.58 mL of extract were oxidized with the Folin-Ciocalteu reagent, and after 8 min the reaction was neutralized with 20% sodium carbonate. Immediately, the mixture was incubated for 15 min at 50°C and the absorbance was read at 765 nm against the blank. The results were expressed as mg GA/g DS.

Evaluation of antioxidant activity
The antioxidant activity was determined by the DPPH and ABTS free radical scavenging capacities. In the case of DPPH, 0.5 mL of extract was reacted with 1.95 mL of DPPH methanol solution (0.1 mM). A reaction kinetics was then plotted where the percentage inhibition was determined at 517 nm (Balestra, Cocci, Pinnavaia, & Romani, 2011). The results were expressed as a percentage inhibition. The ABTS test was carried out according to the technique described by Rotta, Haminiuk, Maldaner, and Visentainer (2017), with some modifications: 50 μL of extract was diluted with 1450 μL of ABTS, 30 min after was measured the absorbance to 732 nm.
The FRAP test was carried out as described by Rotta et al. (2017), the absorbance was measured at 593 nm and the results were expressed in terms of μmol of Trolox/g DS. FRAP reagent was prepared following the method described by Safdar et al. (2017).

Fluorescence microscopy
The microstructure analysis was carried out according to Peighambardoust, Dadpour, and Dokouhaki (2010). Samples of pre-ground extrudates were dispersed on a slide to be stained with sodium fluorescein and rhodamine B (10% solution). The stained samples were visualized in a fluorescence microscope (Nikon Eclipse Ti-U, Japan), with fluorescent light emitters, attached to a digital camera and a 3-position RGB filter disc (red, green and blue). Observation of the protein-carbohydrate interaction was performed with the red-green RGB using the 10X objective. The analysis of the RGB fluorescent micrographs was carried out using ImageJ software v.1.42q (Bethesda, USA). For quantitative fluorescence analysis, 15 images with higher fluorescence were selected, from which regions of interest (ROI) of 80 × 80 μm were taken. Subsequently, the ROI was converted to gray scale, to quantify the values of protein and carbohydrate fluorescence; from these ROIs, the average value was extracted using the ImageJ histogram.

Electrophoresis with sodium dodecyl sulfate and polyacrylamide (SDS-PAGE)
The SDS-PAGE analysis was carried out according to the process as described by Mena, Moreno-Gordaliza, Moraleja, Cañas, and Gómez-Gómez (2011) with some modifications. Proteins from the dried and ground samples (0.1 mg) were extracted using a sample buffer (Tris-HCl pH 6.8, SDS 2% and bromophenol blue 0.03%). Subsequently, they were incubated for 5 min at 100°C and centrifuged at 6000 rpm for 10 min. Protein separation was performed in a Mini-Protean 3 vertical gel system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using an 8% polyacrylamide gel at 120 V for 1 h. After electrophoresis, the gel was stained with Coomassie blue for 12 h, then the reaction was stopped using 7% acetic acid and 30% ethanol in a distilled water solution. Molecular weights were estimated using widerange molecular weight markers.

Statistical analysis
The effect of mixing red potato flour with corn grits on the physicochemical and functional properties was determined by an analysis of variance, and the results were expressed as mean ± SD (standard deviation). To determine the difference between treatments, the Tukey´s test at the level of significance (p < 0.05) with the NCSS 10 statistical package (NCSS 10 Data Analysis, USA) was conducted. All the experiments were carried out in triplicate.

Selection of the best formulations
Extrudates processed with different concentrations of red potato flour and corn grits were analyzed through physical tests. An increase WSI, IE and fracturability of extrudates were found, while there was a decrease in WAI values. While a decrease in L* was observed showing that extrudates with a higher percentage of red potato flour had a darker color (T5:25.06 ± 1.74) compared with the extrudates control (44.37 ± 0.87). Also, it was observed that high concentrations of red potato flour led to an increase in phenolic compounds. Additionally, a significant increase in antioxidant capacity (DPPH, ABTS and FRAP) with regard to the control was recorded (11.91%, 148.38 and 80.81 μmol Trolox/g DS, respectively), since extrudates substituted with up to 50% red potato flour presented values of antioxidant capacity of 45.44%, 515.61 and 208.87 μmol Trolox/g DS, respectively. Based on the above and to simplify subsequent analyzes such as proximal chemical analysis, DSC, protein profile and epifluorescence microscopy, it was concluded that T1 and T5 were the best treatments, since T1 did not show a significant difference with respect to control in terms of physical characteristics. While T5 presented higher content of total phenols and high antioxidant activity with respect to the other treatments.

Physical characterization of extrudates
The transformation of starch during extrusion is responsible for the physicochemical properties of extruded products such as the WAI, WSI, degree of expansion, product density and structure or texture (Peksa et al., 2016). Table 1 shows the results of WAI, in which no significant differences were found with regard to the control (p < 0.05), presenting a value of 3.40 ± 0.22, and it was observed that this parameter is inversely proportional to the concentration of red potato flour. This behavior could be due to the higher content of soluble polysaccharides released from the starch polymer chains after the extrusion process, since the number of available hydroxyl groups to form hydrogen bonds with water depends on the degree of gelatinization, and this affects the decrease or increase of the values of WAI (Becker, Eifert, Soares, Tavares, & Carvalho, 2014;Peksa et al., 2016). Regarding WSI (Table 1) all the treatments presented significant differences with respect to the control (6.18 ± 0.30%), and extrudates made with red potato flour showed the highest WSI value (20.19 ± 0.72%). Rodríguez-Sandoval, Lascano, and Sandoval Table 1. Effect of the extrusion process on the physical and antioxidant properties of extrudates made from mixtures of corn grits and red potato flour.
Tabla 1. Efecto del proceso de extrusión sobre las propiedades físicas y antioxidantes de botanas elaborados a partir de mezclas de grits de maíz y harina de papa roja. T1  T2  T3  T4  T5  RPF  WAI 3.40 ± 0.22 a 3.34 ± 0.21 a 3.22 ± 0.14 a 3.22 ± 0.14 a 3.11 ± 0.51 a 3.04 ± 0.18 a 3.  (2012) reported higher WSI for potato starch compared to wheat flour (7.45 ± 0.72%, and 2.09 ± 0.26%, respectively); one reason for this behavior could be starch degradation from the increased exposure of the product to high temperatures within the barrel, a higher shearing action of the mixture or the amount of polysaccharides released from the starch granules after the addition of excess water to the starch, as higher humidity contents (higher than 18%) help the polysaccharides to dissolve easily in the food matrix, thus increasing the WSI (Seth et al., 2015). Table 1 also shows the results of expansion index, observing a higher expansion index in extrudates made from red potato flour (4.21 ± 0.32) compared with the control (3.02 ± 0.04). Rodríguez-Miranda et al. (2011) reported that increasing the concentration of taro meal in mixtures, there was an increase in the total carbohydrate content in the feed material, which led to further expansion. Seth et al. (2015) reported that higher percentages of protein and fiber in corn affected the expansion of the extrudates, a possible reason being that a higher concentration of protein can result in fluctuations in pressure and temperature as the material moves to through the extrusion barrel, which negatively affects the expansion (Sumargo, Gulati, Weier, Clarke, & Rose, 2016).

Control
The fracturability of the extrudates shows in Table 1, and it can be observed that treatments T4, T5 and red potato flour were the only samples that showed a significant difference (p < 0.05) with respect to the control, which presented the lowest fracturability value (201.58 N). Singh, Kaur, McCarthy, Moughan, and Singh (2009) reported that extruded snacks added with tuber flour (25% to 50%) resulted in an increase in the toughness of snacks and indicated that the differences in texture characteristics can be attributed to the variation in the dry matter and starch content, because the hardness of the snacks showed a positive correlation with the starch content of flours.
The effect of the addition of red potato flour on the color of the extruded products is shown in Table 1, observing a decrease in the values L*, a* and b* in all the treatments, and extrudates made with red potato flour at 100% showed the lowest values of L* = 25.65 ± 0.84, a* = 3.83 ± 0.64 and b* = 7.91 ± 1.58, compared to the control. Singh et al. (2009) prepared snacks made with 100% corn flour showed higher L* values, while the incorporation of potato flour (up to 25%) considerably reduced the L* values compared to the control. The color in the extrudates could be affected by the extrusion parameters, since the thermal processes contribute to the creation of complex brown polymeric compounds known as melanoidin pigments. As well as for the chemical composition of the raw material processed, the presence of greater amounts of sugars (main compounds involved in the Maillard reaction) may contribute to the darker shades of the final product, thus reducing the homogeneity of the color (Peksa et al., 2016).

Differential scanning calorimetry (DSC)
Gelatinization is an irreversible transition suffered by starch granules when subjected to heating in the presence of high moisture content. La Figure 1 shows the gelatinization temperatures of mixtures and extrudates, and no change was observed in the thermal transition temperature (118°C) for the control, while in start and end temperatures there were changes, being for the control (102.19 and 172.04, respectively), and for extruded control of 100.15 and 132.54, respectively. The extrusion also affected T1 since the raw material presented an initial, maximum and final temperature of 82.0, 100.2 and 137.6°C, respectively, these temperatures increased in the extrudates, showing temperatures of 142.0, 142.8 and 163.83°C, respectively. The extrusion process decreased the initial temperature (123.39 to 117.79°C) and the thermal transition temperature (133.12 to 130.05°C) for T5. The gelatinization temperatures obtained were high according to that reported in the literature, which may be due to the low moisture content of the samples analyzed (10-15%), since this transition is usually carried out in a temperature range between 60 and 75°C, at high moisture contents (>35%). The authors indicate that at this temperature, the low molecular weight polymers (amylose) begin to separate from the starch granule and when the temperature increases, the granules begin to collapse until the amorphous part is completely solubilized, while the crystalline part it is maintained in the aqueous solution. However, when the moisture content is restricted, gelatinization can be partially postponed to higher temperatures due to the fusion of the remaining amylopectin crystals (Alzate, Quintero, & Lucas, 2013;Moreira et al., 2015;Pineda-Gomez et al., 2011). The high temperatures of gelatinization could also be attributed to amylose-amylose, amylose-amylopectin interactions, as well as chemical bonds or interactions that occur during a thermal process. On the other hand, it is also important to determine the enthalpy of gelatinization since it indicates the amount of energy required to produce the disruption of the structure of the starch and depends on factors such as type of starch, humidity during its determination, among others (Pineda-Gómez et al., 2011). However, the study of starch transitions in cereal pastes such as doughs of corn flour is more complex than the study of starch isolated from different sources (Moreira et al., 2015).

Total phenols and antioxidant activity
Extrudates elaborated from red potato flour had a higher total phenolic content (2.88 mg GA/g DS) compared to the control (0.44 mg GA/g DS), as shown in Table 1. Likewise, Nemś & Peksa (2018) when elaborating snacks with potato flour of three purple varieties (Salad Blue, Blue Congo and Valfi) and a red variety (Herbie 26), they observed that snacks made with Herbie 26 showed higher content of phenols with respect to the control (15.5 mg and 12.9 mg GAE 100 g DS, respectively). Regardless of the high temperatures of the extrusion process the content of phenolic compounds can be preserved because this process induces the hydrolysis of fibers and/or protein residues linked to polyphenols, changing the state of non-extractable polyphenols to extractable (Morales et al., 2015).
The antioxidant capacity based on free radical uptake activity was determined by DPPH and ABTS assays.
Regarding DPPH, Figure 2a shows the antioxidant capacity when 0.15 g of sample is used, observing a high percentage of inhibition in extrudates made with 100% red potato flour (55.49% inhibition), this result agrees with that obtained in the raw material (red potato flour), which presented 54.87% of inhibition against the DPPH radical. Similarly, in the kinetics obtained with 0.3 g of sample (Figure 2b), can be observed the increase in antioxidant activity in all treatments, being T5 presenting the highest percentage of inhibition (45.44%), with regarding the control (11.91%). Nemś et al. (2015) reported low antioxidant activity in snacks made with 50% of Blue Congo potato flour, Valfilos and products that were not substituted with purple potato flour. In contrast, a 50% increase in antioxidant capacity was observed after the addition of purple potato flour from the Salad Blue and Herbie 26 varieties.
Regarding the ABTS antioxidant activity evaluation, extrudates made with red potato flour at 100%, showed greater antioxidant activity (532.83 ± 0.009 μmol Trolox/g DS) against the ABTS radicals when compared to the other treatments (Table 1). A same behavior was observed in the antioxidant/ferric reducing power of the extracts (FRAP), because extrudates made with red potato flour presented high antioxidant capacity (412.57 ± 0.34 μmol Trolox/g DS), with respect to the other treatments. Nems et al. (2015) reported fried snacks enriched with colored potato flour had higher total polyphenol content than dough containing only industrial potato flour. Kolniak-Ostek et al. (2017) reported that both flour and extruded Jerusalem artichoke contained high levels of polyphenols, which resulted in increased antioxidant activity (260 μmol Trolox/g DS). This indicates that, independently of the addition of vegetable flour with a high content of antioxidant compounds, the antioxidant activity observed in extruded products as consequence of processing could be attributed to the release of phenolic compounds from cell walls, the interaction of phenols with proteins and the formation of Maillard reaction products due to high extrusion (Bisharat, Lazou, Panagiotou, Krokida, & Maroulis, 2015).

SDS-PAGE protein profile
The separation of proteins by SDS-PAGE resulted in different ranges of protein bands as shown in Figure 3. Regarding  protein the fractions of the control flour, three main protein groups with different molecular weights could be distinguished: 15 kDa, 18-25 kDa and from 37 to 60 kDa. In T1 the dominant proteins showed weights of 15 kDa, 20 kDa and 45-60 kDa, while for T5 only 2 main groups were observed, with bands of 18-20 kDa and 45-60 kDa. Santos et al. (2014) reported that bands corresponding to prolamins can be found in low molecular weight, represented by the monomers of α-zein (19 and 22 kDa), β-zein (14 kDa), δ-zein (10 kDa) and γ-zein bands (16 and 27 kDa). On the other hand, high molecular weight proteins were mainly observed in T5, which could indicate the presence of dimmers. As reported by Drochioiu et al. (2016), in corn flour they found a large characteristic band of α-zein dimmers visible below 50 kDa; it is important to mention that T1 and T5 also contain red potato flour (10% and 50%, respectively), so  some protein fractions present in these treatments could belong to this tuber, like ocatina which has an apparent molecular weight of 18 kDa (Monteghirfo & Yarleque-Chocas, 2007).
In the electrophoretic profile of extruded samples, no protein bands were identified in any of the treatments, which could be due to the high temperatures applied during the extrusion, since at temperatures above 100°C, some changes are involved, such as protein denaturation, association, dissociation and aggregation of subunits by covalent and non-covalent bonds. The results obtained agree with that reported by Chao and Aluko (2018) in samples of proteins isolate of peas, since in lanes corresponding to samples subjected to heating temperatures of 80°C, 90°C, and 100°C, they did not identify protein bands, and attribute it to the presence of protein aggregates induced by the heat treatment, because these aggregates were too large to enter the gel. The solubility of the proteins could also affect the identification of protein bands of the extrudates, since when increasing the extrusion temperature, protein aggregates insoluble in SDS are formed as a consequence of the crosslinking of proteins by disulfide bonds (Kristiawan et al., 2018), so it would have been convenient to use a writing agent such as mercaptoethanol for the extraction of proteins.

Epifluorescence light microscopy
Fluorescence microscopy depends on the autofluorescence of the components within a sample or the addition of selective fluorochromes to evaluate the presence, structural organization and spatial distribution of some specific components within a product (Corradini & McClements, 2017). The extrudates images obtained by fluorescence microscopy showed that the proteins and carbohydrates can be distinguished by different emission spectra, and from the fluorescence of proteins ( Figure 4a) and carbohydrates ( Figure 4b) was possible determine the relative location of these compounds, since there is no uniform dispersion of the compounds. The protein fluorescence was very intense for the control and T1, which is directly related to higher protein content, presenting fluorescence values of 99.89 ± 2.3 and 97.94 ± 2.3, respectively, while for T5 were 76.75 ± 2.7. Carbohydrate fluorescence was also lower in the control (77.89 ± 3.7) compared to T1 (88.48 ± 3.6) and T5 (87.47 ± 1.9). The lack of uniformity in the dispersion of these compounds could be attributed to the high extrusion temperatures, since in a study carried out by Hu, Wang, and Li (2017) reported that after treatment with superheated steam in wheat flour, protein aggregation occurred, and caused the starch granules to clump together into small lumps, thus showing interaction between the protein network and lumps of starch; therefore, applied fluorescein dye mixed with rhodamine B to indicate the starch and protein.

Proximal chemical analysis of extrudates
The chemical composition evaluation was carried out on extrudates treatments T1 and T5. The content of protein, fat, fiber, carbohydrate and ash for sample T1 were 7.78,    0.90, 2.18, 77.68 and 0.16 g/100 g, respectively. The composition varied for T5, with values of 6.0, 0.10, 1.51, 82.4 and 0.25g/100g, respectively. These values were comparable to those reported by Singh, Kaur, Shevkani, Singh, and Singh (2016) who found the following values in corn grits: protein, fat and ash content of 7.6, 1.1 and 1.7 g/100 g, respectively. Charoenkul, Uttapap, Pathipanawat, and Takeda (2011) reported that cassava flour has a protein content between 0.32-1.18, fat content from 0.10 to 0.72, ash content of 1.50-2.34 and fiber content of 1.53-2.48.

Conclusion
In this study, an effect of the mixture of corn grits and red potato flour on the physical, chemical and functional properties of extrudates was investigated, and it was observed that when adding red potato flour, extruded with a lower WAI, brown and less fracturability were obtained. However, there was an increase in WSI, expansion index and total phenolic content which were directly proportional to the concentration of red potato flour. Extrudates extracts also showed a high capacity to donate hydrogens and reduce ferrous ion, so it could be considered as a product with high antioxidant capacity. Based on these analyses, it is concluded that it is important to know the effect of the addition of unconventional flours for the processing of snacks, because both the quality and the structure of this type of product depend directly on the raw material used during its preparation.