Effect of the Nixtamalization Process on the Dietary Fiber Content ...

Effect of the Nixtamalization Process on the Dietary Fiber Content, Starch Digestibility, and Antioxidant Capacity of Blue Maize Tortilla Luis A. Bello-Pérez,1,† Pamela C. Flores-Silva,1 Gustavo A. Camelo-Méndez,1 Octavio Paredes-López,2 and Juan de Dios Figueroa-Cárdenas3 ABSTRACT

Cereal Chem. 92(3):265–270

Nixtamalization is an ancient process developed by the Mesoamerican cultures. Initially, volcanic ashes were used and then calcium hydroxide in commercial production, and more recently nixtamalization with calcium salts (NCS) has been proposed. The aim of this study was to evaluate the effect of NCS on carbohydrate digestibility and antioxidant capacity in the elaboration of blue maize tortillas. NCS in blue tortillas showed a high amount of total dietary fiber (14.27 g/100 g), the main fraction being insoluble dietary fiber. The contents of resistant starch and slowly digestible starch did not change

with the nixtamalization process. The predicted glycemic index value was lower in blue tortillas with the NCS process (58) than with the traditional nixtamalization process (71). In general, NCS in blue tortillas presented a higher antioxidant capacity than traditional tortillas (ferric reducing antioxidant power method), indicating that phenolics present in blue maize maintain their activity after cooking. It can be concluded that the nutraceutical features (high dietary fiber content and antioxidant capacity) of blue maize tortillas are enhanced when they are elaborated with the NCS process.

The development of functional foods is a growing area in the food industry, and traditional products such as tortillas are not excluded from it. To enhance the nutritional features of tortillas, diverse raw materials such as soy, nopal cladodes, amaranth, chia, flaxseed, unripe banana, cassava, and bean, as well as iron and other micronutrients, have been added (Rendón-Villalobos et al. 2002; AgamaAcevedo et al. 2004, 2005; Sayago-Ayerdi et al. 2005; Bello-Pérez et al. 2006; Islas-Hernandez et al. 2006; Hernandez-Uribe et al. 2007; Dunn et al. 2008; Richins et al. 2008; Osorio-Diaz et al. 2011; Grajales-Garcia et al. 2012; Aparicio-Saguilan et al. 2013). However, the addition of these other ingredients modifies the tortilla characteristics for the consumer, who is accustomed to traditional tortillas that present a specific taste, flavor, and texture. Eating a healthier food at the expense of sensory characteristics is too much of a compromise, and consequently the preference for tortillas with other ingredients is low and the market is small. The search for functional foods acceptable to consumers is therefore a challenge for the food industry. In addition to the erosion of nutritional properties, the traditional process to produce tortillas is highly polluting, and an alternative ecological nixtamalization process has been developed (Campechano Carrera et al. 2012). This alternative process uses calcium salts (calcium sulfate, calcium carbonate, and calcium chloride) in the nixtamalization of maize and produces only minor amounts of contaminant solids because the pericarp is not removed. The calcium salts are mixed with maize grains (1% w/w of maize grain), and the blend is heated to the boiling point, cooled down, and then steeped for 16 h. Afterward, the maize grains are washed to eliminate excess salt and are ground to obtain masa, which is then dried to produce nixtamalized maize flour (Mexican patent 289339, Figueroa et al. 2011). The nixtamalized maize flour keeps the majority of maize components, among them the nonstarch polysaccharides present in the pericarp, which increases the nixtamalized maize flour yield. Tortillas and other products (snacks, tostadas, and tamales) made with nixtamalized maize flour generally have higher dietary fiber (DF) content than products made with the traditional nixtamalization process. However, because changes in the maize nixtamalization process with the nixtamalization with calcium salts

(NCS) method are few, the sensory characteristics of tortillas are similar to those made with the traditional method. In Mexico, diverse varieties of maize are grown, including pigmented varieties with colors such as violet, red, black, and blue. These pigmented varieties are used to produce tortillas at local and regional levels. The color of the pigmented maize varieties is because of water-soluble dietary phytochemicals (anthocyanins) that are classified in the flavonols group. Anthocyanins may be present in the pericarp, the aleurone layer, or both grain structures. The potential health benefits of anthocyanins are well known, principally their ability to act as antioxidants and as radical scavengers. More recently, anthocyanins have been found to interact and bind noncovalently to macronutrients in foods such as protein, lipids, and carbohydrates (Bordenave et al. 2014). It has been reported that flavonols interfere with enzymes that hydrolyze starch in the gut (Lo Piparo et al. 2008) and with glucose transporters present in the intestinal brush border (Hanhineva et al. 2010). Both phenomena impact starch digestion and postprandial glucose response. The use of the NCS method with white maize produced tortillas with a higher DF content and also restricted starch hydrolysis owing to the arrangement of nonstarch polysaccharides in the food matrix (Bello-Pérez et al. 2014). In theory, blue tortillas made with the NCS process should give tortillas with better nutraceutical features owing to the anthocyanins that are present in the grain pericarp. The aim of this study was therefore to evaluate the effects of the NCS process on the DF content, starch digestibility, and antioxidant capacity of blue maize tortillas.

† Corresponding

author. Phone: +52 735 3942020. E-mail: [email protected]

1 Instituto

Politécnico Nacional, CEPROBI, 62731 Yautepec, Morelos, Mexico. Unidad Irapuato, Querétaro, Qro. C.P. 76230, Mexico. 3 CINVESTAV-IPN Unidad Quer´ etaro, Querétaro, Qro. C.P. 76230. Mexico. 2 CINVESTAV-IPN

http://dx.doi.org/10.1094/CCHEM-06-14-0139-R © 2015 AACC International, Inc.

MATERIALS AND METHODS Traditional Nixtamalization Process. To obtain flour, maize grain was processed by the traditional method: 10 kg was cooked with 20 L of water and 1.0% (w/w) of calcium hydroxide to 90!C for 23 min, and the cooked grains were steeped for 16 h at room temperature. The cooking liquor (or nejayote) was collected, and the nixtamal of the traditional process was rinsed. The nixtamal was ground in a volcanic stone mill to obtain fresh masa. The masa was passed through a flash dryer at 260!C for 4 s to obtain dehydrated flour. Subsequently, this flour was ground in a mill (model 200, Pulvex, Mexico D.F., Mexico) with a hammer head and a 0.5 mm mesh screen (Campechano Carrera et al. 2012). NCS Process. Flour was obtained with the new method described by Figueroa et al. (2011). Calcium salts to obtain whole grain nixtamalized flour replaced the lime. Maize (10 kg) was cooked for 23 min with 20 L of water and 1% (w/w) of calcium carbonate salt; the cooked grains were steeped for 16 h at room temperature. The Vol. 92, No. 3, 2015

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pericarp-free water was separated from the grain, and the clean liquor (nejayote) was eliminated. The nixtamal was milled in a stone mill to obtain the masa. The masa was dehydrated in a flash dryer at 260!C and ground in a mill (model 200, Pulvex) with a hammer head and a 0.5 mm mesh screen (Campechano Carrera et al. 2012). Tortilla Processing. Flours were hydrated to obtain masa, which was molded by pressure and extruded in an MOT-G model commercial tortilladora (Tortilladoras González, Naucalpan, Mexico) into thin circles to obtain 2 mm thick tortillas. Tortillas were cooked on a hot griddle for 1 min per side at an approximate temperature of 250 ± 5!C. The analyses were conducted on powders obtained after freeze drying, except for the chewing/dialysis test. Chemical Composition. The moisture content was determined by drying 2.00 ± 0.01 g of sample for 1 h at 130!C, according to AACC International Approved Method 44-15.02. Ash, protein (N × 5.85), and fat were assessed according to AACCI Approved Methods 08-01.01, 46-13.01, and 30-25.01, respectively. Soluble, Insoluble, and Total DF. The insoluble and total DF contents were determined with AACCI Approved Method 32-05.01. Soluble DF was calculated by difference: total DF – insoluble DF. Starch Digestibility Tests. Total starch was measured according to AACCI Approved Method 76-13.01. Resistant starch (RS) was measured by AACCI Approved Method 32-40.01. Available starch was calculated as the difference between total starch and RS. Rapidly Digestible (RDS) and Slowly Digestible Starch (SDS) Fractions. The RDS and SDS fractions were determined with the procedure proposed by Englyst et al. (1992). Starch Hydrolysis Index (HI) of Products “as Eaten” (Chewing/Dialysis Test). The in vitro rate of starch hydrolysis was assessed with the protocol developed by Granfeldt et al. (1992) and reported in tortillas by Sayago-Ayerdi et al. (2005). Samples of tortillas containing 1 g of total starch were tested. Data were plotted as degree of hydrolysis versus time curves, and the HI was calculated as the area under the curve (0–180 min) for the test product expressed as a percentage of the corresponding area for commercial white bread chewed by the same person. The average HI was calculated from the six digestion replicates run for each sample, and means were compared by the Wilcoxon matched-pair signed-rank test, with each person being his or her own control. The predicted glycemic index (pGI) was calculated from HI values, using the empirical formula proposed by Granfeldt et al. (1992): pGI = 0.862(HI) + 8.198, for which the correlation coefficient (r) is 0.806 (P < 0.00001). Extraction Procedure for Polyphenols. Extraction was performed by the method used by Ovando-Martinez et al. (2009) with some modifications. Samples were extracted by shaking at room temperature with methanol/water acidified with HCl (70:30 v/v, pH 2, 20 mL/g sample, 60 min, room temperature, 28 g) and acetone/ water (70:30 v/v, 20 mL/g sample, 60 min, room temperature, 28 g). After centrifugation (10 min, 25!C, 3,000 × g), supernatants were combined and used to determine extractable polyphenol contents and antioxidant capacity. Determination of Total Anthocyanin and Polyphenol Contents. Total anthocyanin content was evaluated with methanol/water extraction and was determined spectrophotometrically with the differential pH method described by Zhao et al. (2008) with some modifications (Camelo-Méndez et al. 2013). Results were expressed as milligrams of cyanidin 3-glucoside/100 g of dry matter. Extractable polyphenols were determined by the Folin–Ciocalteu procedure (Singleton and Rossi 1965) with some modifications (Ivanova et al. 2011). Sample (0.1 mL) was mixed with 0.5 mL of Folin–Ciocalteu reagent and swirled. After 3 min, 1.5 mL of sodium carbonate solution (7%) was added and mixed, and then it was filled to a total of 10 mL with distilled water. Determination was performed at a wavelength of 765 nm in a spectrophotometer, and total polyphenol content was expressed as milligrams of gallic acid equivalents per gram of dry matter by using a calibration curve. 266

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Condensed Tannins. Residues from the methanol/acetone/ water extraction were treated with 10 mL/L HCl in butanol for 3 h at 100!C (Reed et al. 1982) for condensed tannins determination. Condensed tannins were calculated from the absorbance at 550 nm of the anthocyanidin solutions. Condensed tannins from Mediterranean carob pod (Ceratonia siliqua L.) supplied by Nestlé S.A. were treated under the same conditions to obtain standard curves. Hydrolyzable Polyphenols. Hydrolyzable polyphenols comprise hydrolyzable tannins, phenolic acids, and hydroxycinnamic acids that are released from the food matrix by strong acidic hydrolysis. They were determined by a methanol/H2SO4 9:1 (v/v) hydrolysis at 85!C for 20 h on the residues of the methanol/acetone/water extraction (Hartzfeld et al. 2002). After centrifugation (10 min, 25!C, 3,000 × g), supernatants were combined and used to determinate the hydrolyzable polyphenols by the Folin–Ciocalteu method (Montreau 1972). The results were expressed as gallic acid equivalents. Antioxidant Capacity. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Method. Inhibition of free radical DPPH was evaluated by the method proposed by Brand-Williams et al. (1995) with some modifications; it was carried out measuring the absorbance at 0 and 16 min at 515 nm, and a standard curve was produced in mM Trolox. 2,29-Azino-bis(3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS) Method. Inhibition of ABTS free radical was performed by the methodology proposed by Re et al. (1999). It was carried out measuring the absorbance at 0 and 4 min at 593 nm. Results were expressed in milligram equivalent of Trolox per gram of dry weight by using a calibration curve for Trolox. Ferric Reducing Antioxidant Power (FRAP) Method. The FRAP method was performed by the methodology proposed by Benzie and Strain (1996); it was carried out measuring the absorbance at 0 and 6 min at 593 nm. Results were expressed in milligram equivalent of Trolox per gram of dry weight by using a calibration curve for Trolox. Statistical Analysis. Results were expressed as means ± standard deviations. All experiments were randomized, and analysis of variance and Pearson correlations were conducted with Statgraphics Plus version 5.1 software (Manugistics, Rockville, MD, U.S.A.) and mean separation with the LSD test (P < 0.05). RESULTS AND DISCUSSION Chemical Composition of Flour and Tortillas. The proximal analysis of nixtamalized flours obtained with the two processes (traditional and NCS) is presented in Table I. A difference in moisture content was found in the flours obtained with the nixtamalization methods. This behavior is related to the components of the maize grain that are preserved after nixtamalization. The moisture content of NCS tortillas (along with the respective flour) was lower than for traditional tortillas (Table II), an issue that could be important during tortilla storage. There was no difference in protein content, but the ash and lipid contents showed slight differences between the two flours (Table I). This difference suggests that lipid saponification was achieved in the NCS method but even more in the commercial tortilla, which had 18–24 h of steeping. The main component of tortillas is carbohydrate, in particular starch (Table II). The carbohydrate content was higher in NCS tortillas than traditional tortillas because nonstarch polysaccharides present in the pericarp of the grain were maintained in the NCS, as was observed by the insoluble DF content. Different proximal composition has been reported in maize tortillas, depending on the maize variety used (Agama-Acevedo et al. 2004, 2005; Mora-Avilés et al. 2007; Grajales-Garcia et al. 2012). NCS tortillas presented higher total DF than the traditional and commercial tortillas (Table II). This nutritional feature is important because tortilla consumption has been related to overweight and obesity (Rodr´ıguez-Ram´ırez et al. 2011). However, the total DF is an important fraction of tortillas that is not hydrolyzed by digestive enzymes and is higher in tortillas obtained with the NCS process.

Tortillas also have a higher amount of insoluble DF than soluble DF, and the NCS tortillas showed the highest insoluble DF content. The insoluble DF is important because it adds bulk to the stool and appears to help food pass more quickly through the gut. Also, in vivo and in vitro studies have demonstrated the ability of insoluble DF to absorb carcinogens (Steinmetz and Potter 1991; Slavin 2001). Starch Digestibility. The main component of nixtamalized flours and tortillas is starch, which is highly available (Table III). A difference in RS content was found between the traditional and NCS tortillas, but NCS tortillas showed a lower available starch content. The difference between blue tortillas (made with both nixtamalization methods) and commercial tortillas was owing to maize variety. In general, pigmented maize has soft endosperm, whereas crystalline endosperm is present in the reference white maize used commercially to make white tortillas that are widely consumed. The RS content of white tortillas is low because the nixtamalization and baking processes result in an almost complete starch gelatinization. The RS fraction present in tortillas can be related to the food matrix, which restricts the enzyme that hydrolyzes starch (Singh et al. 2010), and to the production of indigestible glucans by pyrodextrinization at a high temperature (Laurentin et al. 2003). The nixtamalization process did not modify the enzymatic hydrolysis rate of starch in blue tortillas, because both tortillas presented similar contents of RDS and SDS (Table IV). The results from this test suggest that the starch present in tortillas is digested TABLE I Proximal Analysis, Fiber Contents, and Starch Contents of Flours Prepared with the Different Nixtamalization Procedures (g/100 g)w Component

Traditional Flour

Moisturex

5.49 9.08 8.48 1.63 80.81 10.13 6.92 3.21 74.88 2.17 72.71

Protein Lipid Ash Carbohydratesy Total DF Insoluble DF Soluble DFy Total starchz Resistant starchz Available starchy

± ± ± ± ± ± ± ± ± ± ±

during the 20 min after ingesting the tortilla. However, it is important to keep in mind that a fine powder is used in the test, which increases the enzyme accessibility to the substrate (starch). In our opinion, this test is an interesting approach to starch digestion. The SDS content of tortillas is an important issue because of the physiological effects (e.g., satiety) associated with the liberation and adsorption of glucose along the small intestine (Zhang and Hamaker 2009; Lee et al. 2013). Semi–In Vitro Rate of Hydrolysis and Predicted Glycemic Index (pGI). The course of the hydrolysis of blue tortillas (Fig. 1) differed between those elaborated with the traditional and NCS processes, and a similar pattern was found between traditional blue and commercial tortillas. The NCS blue tortillas showed the slowest hydrolysis. This semi–in vitro method has been shown to correlate well with the postprandial glycemic response in vivo, because a portion of the tortilla is chewed, leaving small pieces to TABLE III Total Starch (TS), Resistant Starch (RS), and Available Starch (AS) Contents of Blue Maize Tortillas (g/100 g)y Tortilla Traditional NCS Commercialz y

z

y z

RS

AS

2.13 ± 0.09b 1.54 ± 0.06c 2.58 ± 0.34a

71.00 ± 2.71a 67.55 ± 1.44b 63.92 ± 0.86b

Values are means of three replicates ± SD. Dry matter basis. Means in a column not sharing the same letter are significantly different (P < 0.05). NCS = nixtamalized calcium salt. TS and RS were measured with AACCI Approved Methods 76-13.01 and 32-40.01, respectively; AS was calculated by difference. White maize tortilla.

NCS Flour

0.09a 0.09a 0.08a 0.04a 0.09b 0.83b 1.64b 0.93a 0.58a 0.07a 0.56a

2.51 8.98 6.49 1.48 83.04 14.11 11.93 2.18 68.23 1.88 66.35

± ± ± ± ± ± ± ± ± ± ±

0.05b 0.09a 0.30b 0.03b 0.08a 1.42a 2.31a 1.28a 1.17b 0.07b 1.17b

are means of three replicates ± SD. Dry matter basis. Means in a row not sharing the same letter are significantly different (P < 0.05). NCS = nixtamalized calcium salt, and DF = dietary fiber. As is basis. Calculated by difference. Measured with AACCI Approved Methods 76-13.01 (total starch) and 32-40.01 (resistant starch).

TABLE IV In Vitro Starch Digestibility of Blue Maize Tortillas with the Englyst Method (g/100 g)y Tortilla Traditional NCS Commercialz y

w Values

x

TS 73.13 ± 2.62a 69.08 ± 1.50b 66.50 ± 0.72b

z

RDS

SDS

57.74 ± 4.29a 59.58 ± 3.15a 52.55 ± 5.31b

2.19 ± 2.12a 3.85 ± 3.96a 3.60 ± 5.74a

Values are means of three replicates ± SD. Dry matter basis. Means in a column not sharing the same letter are significantly different (P < 0.05). Values were adjusted according to the total starch content of tortillas determined by AACCI Approved Method 76-13.01. RDS = rapidly digestible starch; SDS = slowly digestible starch; and NCS = nixtamalized calcium salt. White maize tortilla.

TABLE II Proximal Analysis and Fiber Contents of Tortillas Prepared with the Different Nixtamalization Procedures (g/100 g)w Component Moisturey Protein Lipid Ash Carbohydratesz Total DF Insoluble DF Soluble DFz

Traditional 42.67 9.83 8.48 1.76 79.93 11.16 8.27 2.89

± ± ± ± ± ± ± ±

0.93b 0.25a 0.37a 0.03a 0.23c 1.10a 1.65b 0.56a

NCS 39.11 9.27 6.65 1.69 82.39 14.27 13.30 0.97

± ± ± ± ± ± ± ±

1.19c 0.10b 0.08b 0.02a 0.08b 0.42a 1.31a 0.91b

Commercialx 45.88 7.73 3.41 1.30 87.56 12.60 11.61 0.99

± ± ± ± ± ± ± ±

0.40a 0.06c 0.21c 0.06b 0.06a 1.73a 2.89a 1.24b

are means of three replicates ± SD. Dry matter basis. Means in a row not sharing the same letter are significantly different (P < 0.05). NCS = nixtamalized calcium salt, and DF = dietary fiber. White maize tortilla. As is basis. Calculated by difference.

w Values

x y z

Fig 1. Average in vitro starch hydrolysis curves of blue maize tortillas. u = Commercial (white tortilla); n = traditional; : = nixtamalized calcium salt; and d = white bread. Error bars represent SEM. Vol. 92, No. 3, 2015

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continue the enzymatic hydrolysis (similar to the consumption of tortillas by people). The HI calculated from the hydrolysis curves and the corresponding pGI are shown in Table V. The lowest HI was found in NCS blue tortillas. Traditional blue tortillas and commercial tortillas had similar HI values, which were similar to the HI reported in other commercial tortillas following the same method (HI = 77.6; Sayago-Ayerdi et al. 2005). The low HI found in NCS blue tortillas could be related to the presence of a higher level of nonstarch polysaccharide from the maize pericarp (e.g., arabinoxylans) that increase the viscosity of the bolus, decreasing the accessibility of the enzyme to the substrate. In general, the pGI of commercial tortillas and traditional blue tortillas, both elaborated with similar nixtamalization processes, indicates that consumption of tortillas provides an intermediate supply of glucose. TABLE V Hydrolysis Index (HI) and Predicted Glycemic Index (pGI) of Blue Maize Tortillasy Sample White bread reference Traditional tortilla NCS tortilla Commercial tortillaz y

z

HI

pGI

100 73.42 ± 10.01a 58.03 ± 5.50b 70.33 ± 7.28a

94 71a 58b 69a

Mean ± SD, n = 6. Values followed by the same letter within the same column do not differ significantly at P < 0.05. NCS = nixtamalized calcium salt. White maize tortilla.

However, the NCS method produces tortillas with a minor supply of glucose, which can be taken into account in the production of tortillas with low caloric impact. In blue tortillas, the different heat treatments from nixtamalization to tortillas produce changes in the anthocyanins that decrease its antioxidant capacity and the capacity to interact with carbohydrates (starch and nonstarch polysaccharides), as was suggested (Saura-Calixto 2011). Both white tortillas (data not shown) and blue tortillas obtained with the NCS method had similar pGI, indicating that interactions between starch and polyphenols are absent. However, in vivo tests can be performed to elucidate if interference with digestive enzymes and glucose transporters at the intestinal brush border by polyphenols in blue tortillas is involved. Polyphenol Content in Tortillas. The total anthocyanin content of fresh tortillas made by the traditional and NCS procedures did not show significant differences (P < 0.05) (Table VI). These values were lower than those reported by Del Pozo-Insfran et al. (2006) for nixtamal and nixtamal-acidified blue tortillas (0.10–0.18 mg/g of total anthocyanins) that used Mexican and American blue maize. Also, blue tortillas did not present significant differences (at P < 0.05) in the extractable polyphenols and condensed tannins (Table VI). However, the same trend was not seen with hydrolyzable polyphenols of tortillas made by the traditional and NCS methods. This pattern suggests that some strong interactions between polyphenols and other components present in the maize (nonstarch polysaccharides) are produced during the traditional nixtamalization process that decreased the amount of polyphenols liberated after the hydrolysis to obtain the hydrolyzable polyphenols. This is attributed to the different interactions between

TABLE VI Total Anthocyanin and Polyphenol Contents of Blue Maize Tortillas Made by the Traditional and Nixtamalized Calcium Salt (NCS) Methodsz Sample

Total Anthocyanin

Extractable Polyphenols

Hydrolyzable Polyphenols

Condensed Tannins

0.08 ± 0.00a 0.07 ± 0.06a

5.64 ± 0.28a 5.50 ± 0.34a

7.08 ± 2.58a 4.92 ± 1.58b

42.85 ± 7.21a 40.39 ± 5.59a

NCS tortilla Traditional tortilla z

Values are mean ± SD (n = 4), dry matter. Values ± SD followed by the same letter within the same column do not differ significantly (at P < 0.05) by tortilla type, LSD test. Total anthocyanin is expressed as mg of cyanidin 3-glucoside/100 g of dry matter, polyphenols as mg of gallic acid/g of dry matter, and condensed tannins as mg of condensed tannins/g of dry matter.

Fig 2. Antioxidant capacity (mM Trolox/g) measured by three different methods of blue maize tortillas made by traditional and nixtamalized calcium salt methods. Values are mean ± SD (n = 4), on a dry matter basis. The same lowercase letters indicate differences are not significant (at P < 0.05) by tortilla effect, according to the LSD test. EP = extractable polyphenols; HP = hydrolyzable polyphenols; CT = condensed tannins; DPPH = 1,1-diphenyl-2picrylhydrazyl; ABTS = 2,29-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); and FRAP = ferric reducing antioxidant power. 268

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polyphenols and carbohydrates, proteins, and lipids (Bordenave et al. 2014) and the stability characteristics of each compound (e.g., pH, polarity, number and position of methyl and hydroxyl radicals, double bonds, etc.). Also, previous investigations have determined that the specific occurrence and concentration of polyphenols present in anthocyanin-containing food matrices have an important effect on the stability of anthocyanins (Mazza and Miniati 1993; Bridle and Timberlake 1997; Del Pozo-Insfran et al. 2004, 2006). Although in this study individual anthocyanins were not identified, we suggest that cyanidin 3-glucoside is the main pigment in blue tortillas because it has been reported as the major anthocyanin in blue maize foods (Del Pozo-Insfran et al. 2006; Sánchez-Madrigal et al. 2014). Antioxidant Activity. The antioxidant activity of extractable and hydrolyzable polyphenols and condensed tannins of blue tortillas was measured by spectrophotometric methods: free radical inhibition (DPPH and ABTS) and reducing power (FRAP), and the results are presented in Figure 2. In general, the antioxidant activity of blue tortillas was in the following order: DPPH > FRAP > ABTS. Methodologies used in this study have been widely used for measuring antioxidant activity in food science. However, it is important to mention that they cannot be compared because each one measures the inhibition of different radicals and the reducing power of Fe3+. Therefore, correlations between the polyphenol and tannin contents with each antioxidant methodology were evaluated. Pearson coefficients indicate significant correlations (P < 0.05) between extractable polyphenols and DPPH inhibition (r = 0.93) and between condensed tannins and FRAP (r = 0.62), suggesting a direct relationship between polyphenol content and antioxidant activity. Differences in Pearson coefficients among the antioxidant methodologies used in this study are owing to the polarity of compounds and sensitivity characteristics of each technique. Also, it is important to note that this biological activity cannot only be attributed to the antioxidant characteristic of each molecule per se (double conjugate bonds, number and position of methyl and hydroxyl groups). Further, this activity can be attributed to the complex effects of the molecules (synergistic, additive, or antagonist) present in each extract and the affinity of each molecule to inhibit free radicals or reduce metals. The effect of the processing conditions did not affect the antioxidant activity represented by free radical inhibition as shown by the DPPH and ABTS methods. However, NCS tortillas showed a higher antioxidant capacity following the FRAP method. The methods used to determine antioxidant capacity are complementary, and their sensitivity depends on the different macromolecules and bioactive compounds present in the food. The highest total DF content in NCS tortillas could be responsible for this effect, according to the interactions between bioactive compounds and DF components previously reported (Saura-Calixto 2011). CONCLUSIONS The NCS process produced fresh blue tortillas with higher indigestible carbohydrate (total DF) and lower RS contents than its counterpart elaborated with the traditional nixtamalization process. Blue tortillas elaborated with both nixtamalization processes had similar RDS and SDS contents. However, NCS tortillas showed lower pGI and higher antioxidant capacity determined by FRAP, suggesting an effect of the bioactive compounds (extractable and unextractable polyphenols) present in blue tortillas on the starch digestibility. Blue tortillas elaborated with the NCS process could show some potential health benefits associated with consumption. ACKNOWLEDGMENTS The authors thank the support from CONACYT-Mexico (grant 203622), SIP-IPN, COFAA-IPN, and EDI-IPN. One of the authors (L. A. Bello-Pérez) also acknowledges the leave of study from COTEBAL-IPN and the grant from CONACYT-México (203622).

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[Received June 27, 2014. Accepted December 24, 2014.]

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