Effect of huitlacoche (Ustilago maydis DC Corda) paste addition on functional, chemical and textural properties of tortilla chips

AMADOR-RODRÍGUEZ, Karla Yuritzi; MARTÍNEZ-BUSTOS, Fernando; PÉREZCABRERA, Laura Eugenia; POSADAS-DEL-RÍO, Francisco Aníbal; CHÁVEZ-VELA, Norma Angélica; SANDOVAL-CARDOSO, Ma. Lorena; GUEVARA-LARA, Fidel Effect of huitlacoche (Ustilago maydis DC Corda) paste addition on functional, chemical and textural properties of tortilla chips Ciência e Tecnologia de Alimentos, vol. 35, núm. 3, julio-septiembre, 2015, pp. 452-459 Sociedade Brasileira de Ciência e Tecnologia de Alimentos Campinas, Brasil


Introduction
Busy lifestyles and the increasing demand from consumers for meals and snacks that are quick sources of good nutrition have prompted the food industry to develop foods meeting the requirements (Reis & Abu-Ghannam, 2014), and tortilla chips made from blue corn are a viable option (Sánchez-Madrigal et al., 2014).Tortilla chips (TC) are defined as the snacks made from milled alkaline-treated (nixtamalized) corn or alkaline-treated corn flours that are formatted, cooked, and partially dried and fried or baked (McDonough et al., 2001;Mehta, 2001).Pigmented corn contains anthocyanins and phenolic compounds which are phytochemicals synthesized in the plant by secondary metabolism; although these compounds are considered nonnutritive, interest in antioxidant and bioactive properties has increased due to their potential health benefits (Rice-Evans et al., 1996;Heinonen et al., 1998;Setchell & Cassidy, 1999;Salinas-Moreno et al., 2003;Del Pozo-Insfran et al., 2006;He & Giusti, 2010).
Huitlacoche (Ustilago maydis DC Corda) (also known as cuitlacoche) is a mushroom consumed traditionally in México; this dimorphic fungus is responsible for the formation of corn smut, characterized by galls or tumors principally in ears, but also in stems, leaves and tassels of the plant host (Zea mays L.) (Bölker, 2001).Huitlacoche is a good source of protein (10-25% db), with a high content of lysine; it also has a high content of dietary fiber (54-64% db) and essential fatty acids such as linoleic and linolenic acids (Valverde et al., 1995;Beas et al., 2011).Huitlacoche has been remarked as a functional food that produces bioactive substances, the latter of which can be used to create fortified food products (Valverde et al., 1995;Ruiz-Herrera & Martínez-Espinoza, 1998;Valdez-Morales et al., 2010;Beas et al., 2011;Juárez-Montiel et al., 2011).Traditionally it is consumed with nixtamalized corn products, is highly edible and has no diversity processing.The objective of this research was to study the effect of huitlacoche paste addition on functional, chemical and textural properties of tortilla chips.

Practical Application:
As obesity and diseases related to bad eating habits rise, developing sources of good nutrition and health benefits has become a major research focus in recent years.The addition of a new and functional ingredient for tortilla chips proposes to enhance the nutritional and bioactive compounds of nixtamalized corn products.Huitlacoche is an important source of fiber, lysine, essential fatty acids and bioactive compounds.Corn-based snacks increased over the last 30 years because they were included in main meals as well as in between-meal nourishments.corn flour (TM1) (MINSA, Estado de México, México) and commercial blue baked tortilla chips (TM2) (Salmas, Sanissimo, Monterrey, N.L., México) were purchased in a local market of Aguascalientes (Aguascalientes, México); huitlacoche corn cobs were purchased in Irapuato (Guanajuato, México).Huitlacoche galls were separated from the corn cobs and cooked (stir-fried in an electric pan with no additives) for 15 min at 95 °C.After cooling, galls were processed into a uniform huitlacoche paste (HP) with a food processor (Quick N´ Easy Processor, Black & Decker, Shelton, CT, USA) and stored in a -15 °C freezer in one-liter air-tight plastic containers.

Preparation of nixtamalized corn flour (NF) and tortilla chips (TC)
An alkaline-cooking method was used (Salinas-Moreno & Arellano, 1989).Briefly, maize kernels (24-kg lots) were cooked for 30 min in lime solution [0.8% (w/v) Ca(DH) 2 in distilled water] at 85 °C using a 1:2 (w/v) ratio of grain to cooking medium, followed by a steeping time of 14 h.The cooking liquor was drained and discarded and the nixtamal (alkaline-cooked maize) washed with running tap water for 40 s.The cooked grain was ground into masa in a stone mill (M100, Fumasa, Puebla, México), and finally dehydrated using a flash type dryer (M2000, CINVESTAV-GAV, Querétaro, Qro., México).
For tortilla chip processing, formulations were rehydrated to 55% water content and mixed using speed two for 10 min (KitchenAid® NSF Certified® Commercial Series 8-Qt Bowl Lift Stand Mixer, Benton Harbor, MI, USA).Formulations were processed in a semi-industrial tortilladora (Villamex V-100, Guadalajara, Jalisco, México;Grupo Villamex, 2003).Briefly, the masa was shaped and cut into thin triangles (1 mm thickness) and the pieces were cooked on an iron hot band (270 °C±10 °C) for 45 s each side.After cooling, the pieces were baked in a semi-industrial convection oven at 180 °C (Zucchelli Forni, 260493, Verona, Italy) for 15 min, after which they were cooled and stored in air-tight packaging until evaluations and labeled: tortilla chips from NCF as CTC (conic tortilla chips); from NXF as XTC (creole tortilla chips); from NMF as MTC [MINSA (commercial flour) tortilla chips].

Physical and proximate analyses
The physical properties of tortilla chips were analyzed by approved AACC International methods: moisture content (MC) by method 44-15.02,crude protein (%N × 6.25) by method 46-10.01,crude fiber and fat (ether extract) were determined according to methods 32-10.01 and 30-10.01,respectively (American Association of Cereal Chemists, 1999).Total dietary fiber (TDF) was analyzed using the Total Dietary Fiber Assay Kits TDF-100A and TDF-C10 (Sigma Chemical Company, St. Louis, MD, USA).Three repetitions were made for each analysis.

Color determinations
Color changes were determined using a MiniScan XE colorimeter (model 45/0-L, Hunter Associates Laboratory, Reston, VA, USA).Total color differences (ΔE) at the different periods of time were calculated from the determined CIELAB L* a* b* values according to Equation 1.

Total soluble phenolics contents
Total soluble phenolics of samples was determined using a colorimetric method (Singleton et al., 1999).A calibration curve of gallic acid using 30% (v/v) methanol as solvent was used.Sample extracts were prepared accordingly using 500 mg of sample homogenized with 10 mL of 30% (v/v) methanol and agitated for 1.5 h.The extracts were centrifuged at 3200 × g (Thermo IEC model CL3-R, USA) for 30 min and decanted.Thirty microliters of extract, 3 mL of deionized water, and 200 μL of Folin-Ciocalteu's phenol reagent were mixed and allowed to stand for 10 min at room temperature.The reaction was neutralized with 600 μL of a 20% (w/v) sodium carbonate solution.The mixture was incubated for 20 min.The absorbance was measured at 757 nm using a spectrophotometer (BioMate 3, Thermo Fisher Technologies, Wisconsin, USA), and a reagent blank was used.The results were expressed as milligrams of gallic acid equivalents per 100 g of sample (mg GAE/100 g).This determination was performed in triplicate for each extract.

Total anthocyanins
Total anthocyanins were assayed according to Abdel-Aal & Hucl (1999) by measuring the absorbance of ethanolic extracts.Tortilla chip samples (1.5 g) were homogenized with 10 mL of an acidified ethanol solution (ethanol and 1 N HCl, 85:15, v/v).The solution was mixed and adjusted to pH 1 with 4 N HCl, agitated for 30 min, and then centrifuged at 3200 × g (Sorvall RC5C, Sorvall Instruments, Dupont, Wilmington, DE, USA).The supernatant was poured into a 25 mL volumetric flask and brought to volume with acidified ethanol.The absorbance was measured at 535 nm using a spectrophotometer (BioMate 3, Thermo Fisher Technologies, Wisconsin, USA), and a reagent blank was used.The total anthocyanin content was expressed as milligrams of cyanidin 3-glucoside per kilogram of sample (mg C3G/kg) and was calculated using Equation 2: Where C is the concentration of total anthocyanin expressed as milligrams of cyanidin-3-glucoside (mg C3G) per kilogram of sample; ε is the molar absorptivity for cyanidin-3-glucoside (25965 cm -1 mol -1 ) and MW is the molecular weight of cyanidin-3-glucoside, 449 Da.

Trolox equivalent antioxidant capacity (TEAC)
Antioxidant capacity was evaluated following the TEAC-DPPH method (Brand-Williams et al., 1995;Fukumoto & Mazza, 2000).A calibration curve of 1 mM Trolox was used.The extracts were prepared in darkness using a procedure similar to that described for the determination of phenolics content, except that absolute methanol was used.An aliquot of 600 μL of DPPH in methanol was added to 100 μL of extract.The mixture was allowed to stand at room temperature in the dark for 20 min, after which absorbance at 515 nm was measured using a spectrophotometer (BioMate 3, Thermo Fisher Technologies, Wisconsin, USA).Results were expressed as micromoles of Trolox equivalents per gram of sample (μmol TE/g).This determination was performed in triplicate for each extract.

Breaking force
Tortilla chip texture was evaluated using a texture analyzer with a Kramer shear cell.A 454-gram load cell was used.The probe of the cell traveled at 5 cm/min.Dne piece of tortilla chip was placed in the Kramer shear cell for testing.Breaking forces until peak values were obtained from time-deformation curves.

Statistical analyses
All results were expressed as means ± standard deviation values (n=3).Statistical comparisons between two groups were made by using Student´s t test.With several groups, one-way analysis of variance was used; Tukey´s analyses (p<0.05) were applied for means with significant differences, and Pearson correlation was calculated.The data were analyzed using the GraphPad Prism Version 5.0 software package (GraphPad Software, Inc., San Diego, CA, USA).(Salinas-Moreno et al., 2003).The frying or baking processes cause darkening due to the Maillard reaction or caramelization (Maga & Liu, 1993;Buttery & Ling, 1995).

Breaking force
Fracture forces for baked chips were significantly different among the different blue corn flours used (P<0.05).CTC showed the highest resistance to fracture or hardness (22.84-27.54kgf); and XTC formulations showed less resistance to fracture (13.10-20.91kgf); this difference can be attributed to the formation of complexes of starch with calcium resulting in a more compact molecule and therefore harder tortilla chips (Gomez et al., 1991;Zazueta-Morales et al., 2002).TM1 had the lowest value (8.72-16.25 kgf) and this may be due to the additives (carboxymethyl cellulose and/ or guar gum) declared in the label.Fracture force was inversely correlated to protein content (r = -0.879)and directly to oil content (r = 0.952).Singh et al. (1996) reported that the force required to compress the product increased in direct proportion to the soya flour in the dough.The texture properties of tortilla chips is related to the removal of moisture during the baking process at high temperatures, which leads to the formation of larger air cells and cracks in the structure resulting in low fracture force values (Sánchez-Madrigal et al., 2014).This is because at high temperatures, large cracks are formed due to the quick vaporization and diffusion of moisture through the pores (Kayacier & Singh, 2003).High peak load force values (50 to 140 kgf) were reported in baked tortilla chips made from commercial nixtamalized instant-masa flours (not blue color) (Kayacier & Singh, 2003).Plascencia-González et al. (1999) produced corn tortilla chips from lime-cooked blue maize with good sensory characteristics (color, flavor and texture).
The texture properties of tortilla chips is related to the removal of moisture during the baking process at high temperatures, which leads to the formation of larger air cells and cracks in the structure resulting in low fracture force values (Sánchez-Madrigal et al., 2014).This is because at high temperatures, large cracks are formed due to the quick vaporization and diffusion of moisture through the pores (Kayacier & Singh 2003).
Huitlacoche addition influences the breaking force as the addition increases, the high content of dietary fiber (54-64% db) of huitlacoche (Valverde et al., 1995;Beas et al., 2011) may influence the water absorption index.The boiling temperature of water inside the sample increased quickly at high temperature and high pressure causing larger cracks (Kayacier & Singh, 2003).

Proximate analyses
Some chemical characteristics of tortilla chips were also evaluated (Table 2).Crude fat was reduced as huitlacoche increased.Protein content of tortilla chips did not change significantly with the addition of huitlacoche (P<0.05),but it showed significant differences among raw materials.XTC showed the lowest amount of crude protein (6%) while commercial tortilla chips showed the highest.Total dietary fiber and crude fiber were incremented by the HP addition (P<0.05);total dietary fiber content was influenced by the flour used and by the addition of HP.Commercial tortilla chips (TM2) reported 5.5% of TDF in the label, while the treatments almost tripled this value, as CTC-9 shows.Levels of 2.9 g oil/100 g sample, 12.4 g protein/100 g and 54-64% total dietary fiber have been reported for raw huitlacoche (Beas et al., 2011), while those reported for blue corn were 4.2-4.8%oil and 9.5-10.4% protein, respectively (Salinas-Moreno et al., 2013).Crude fat was negatively correlated to anthocyanin content (r =-0.7430) because these pigments are water soluble (Wrolstad et al., 2005).

Total soluble phenolics, anthocyanin content and TEAC
The functional properties of raw materials were evaluated (Table 3).Nixtamalization did not result in significant differences (P<0.05) in total soluble phenolics contents; from grains to nixtamalized corn flours, these contents ranged from 84 to 92 mg GAE/100 g sample (db).Huitlacoche decreased its phenolics content from 538 to 393 mg GAE/100 g sample (db) as an effect of the cooking process.Previously reported values in raw huitlacoche include 390-640 mg GAE/100 g for total soluble phenolics (Beas et al., 2011).Compared to other foods, huitlacoche presented higher soluble phenolics content than some bean varieties (141-198 mg/100 g db) (Vinson et al., 2001).
The raw materials showed significant differences in antioxidant capacity (Table 3).Fresh huitlacoche and its paste had the highest TEAC values: 88.8 and 64.4 μmol TE/g sample, respectively.Among blue-corn grains and nixtamalized flours, creole grains (XG) and their nixtamalized flour (NXF) had the highest TEAC (16.3 and 17.0 μmol TE/g, respectively).
Total soluble phenolics contents in tortilla baked chips were significantly different among the various blue-corn masas analyzed (P<0.05; Figure 1).HP increased the total soluble phenolics content as it was increased from 0 to 9% (db) in the three nixtamalized corn flours used.CTC showed increasing total phenolics levels of 17.52, 20.46, 26.01 and 36.08 mg GAE/100 g sample as the HP was added at 0, 3, 6 and 9%, respectively; similar behaviors were shown by XTC and TM1.As a reference, the commercial tortilla chip (TM2) contained 20.5 mg GAE/100 g of sample.
Some studies have reported that the presence of polyphenolics such as catechin and free forms of ferulic acid in corn flours contribute to the antioxidant activity (De la Parra et al., 2007;López-Martínez et al., 2009).
Total anthocyanin contents in baked tortilla chips were significantly different among the various nixtamalized bluecorn flours (P<0.05; Figure 2).However, HP did not modify the total anthocyanin content as it was added from 0 to 9% (db) in the nixtamalized corn flours used.TM1 and TM2 showed the highest contents (49.48-55.10mg C3GE/kg).Figure 3 shows that TEAC values for baked tortilla chips were significantly different among the different formulations with HP (P<0.05).HP increased the antioxidant capacity of the tortilla chips as it was added at 3, 6 and 9% (db) to the three nixtamalized corn flours.The antioxidant capacity of CTC increased as the HP was added, showing values of 6.74, 7.72, 7.88 and 7.98 µmol TE/g sample at 0, 3, 6 and 9% of addition, respectively.XTC increased as well from 6.84 to 7.80 µmol TE/g sample.TM1 showed a different behavior, TM1-0 had 7.70 µmol TE/g sample, then the antioxidant capacity decreased in TM1-3 down to 7.02 µmol TE/g sample, and incrementing again from 7.44 µmol TE/g sample in TM1-6 up to 7.51 µmol TE/g sample in TM1-9.As a reference, the commercial tortilla chip (TM2) had 7.59 µmol TE/g sample.TM2 declared in the label: nixtamalized blue corn flours, salt (1%) and tocopherols (natural antioxidant).TEAC was negatively correlated to the L* parameter (r= -0.7382), while total soluble phenolics content was negatively correlated to L* (r=-0.7722)and positively to b* values (r=0.7038); the addition of HP and its black color influenced L*; on the other hand b* may have been influenced by blue corn.Sánchez-Madrigal et al. (2014) reported antioxidant capacities from 0.5 to 1.2 µmol TE/g in blue corn tortilla chips from extruded flours containing different sources of calcium.
Several works have also found that nixtamalization significantly reduces the total polyphenol content, antioxidant activity and anthocyanin content in corn and corn products, due to the thermal-alkaline process and by the leaching of these same compounds to the nejayote (Salinas-Moreno et al., 2003;Del Pozo-Insfran et al., 2006;De la Parra et al., 2007;Mora-Rochin et al., 2010).Meanwhile, López-Martínez et al. (2011) found a decrease in anthocyanins and phenolics due to the alkaline heat treatment in some varieties, and demonstrating that the loss of anthocyanins is associated with the variety of corn used; these authors also reported an increase in antioxidant capacity after cooking the tortilla dough preparation, due likely to an increase in soluble phenolics.Snacks produced with colored flour had 2-3 times higher antioxidant activities, 40% higher contents of polyphenols, attractive color and better expansion compared to control samples (Nemś et al., 2015).
Anthocyanin and phenolic contents have shown a high correlation with antioxidant activity and color of tortilla chips (Sánchez-Madrigal et al., 2014), however, in our work the commercial flour and tortilla chips showed no correlation among those parameters.Mexican official regulation (NDM-187-SSA1/ SCFI-2002;México, 2003) allows for the addition of a mixture of artificial colors; their sum should not exceed 500 mg/kg of product in tortillas; for tortilla chips, brilliant blue FCF1 is allowed (300 mg/kg maximum), and for flours no color addition is allowed.

Conclusions
The huitlacoche fungus is a valuable source of phenolic compounds and antioxidants.Its application as ingredient in the formulation of baked snacks resulted in an increase in the phenolic and in the antioxidant properties when compared to the controls without huitlacoche and to commercial tortilla chips.However, the heat treatment applied in precooking pieces and convection oven promoted loss of phenolic compounds, anthocyanins and antioxidant capacity.The functional, physicochemical and structural properties of tortilla chips changed significantly with the addition of huitlacoche (Ustilago maydis).The functional properties of tortilla chips were enhanced because of the huitlacoche phytochemical contents, which are related to health benefits.Thus, huitlacoche can be used as ingredient in the formulation of baked corn snacks generating products richer in antioxidants, phenolic compounds and fiber.

Table 2 .
Crude fat, protein, fiber and moisture contents of tortilla chips.

Table 3 .
Total soluble phenolics, anthocyanin content and Trolox equivalent antioxidant capacity of raw materials.