Study of the fortification with bean and amaranth flours in nixtamalized maize tortilla

Abstract

The aim of this study was to compare the physicochemical, rheological and textural tortillas of nixtamalized maize flour fortified with common bean (Phaseolus vulgaris) and amaranth (Amaranthus spp.) flours in three different proportions (3F7A, 5F5A and 7F3A) with respect to commercial maize flour (TR). Also, their effect on growth was assessed by a bioassay of two generations with Wistar rats. Treatments obtained similar efficiencies to TR, plus a significant increase in the protein. They showed a similar behavior in water absorption capacity (WAC) and rollability, crucial factors to the proper development of the dough and its acceptation, respectively. Furthermore, the treatments presented a significant difference (p < 0.05) in bioassay, showing 3F7A similar behavior between casein control. The formulations developed in this research, primarily 3F7A, are a viable option for bioavailable protein-fortified tortillas, rich in lysine, that show textural and rheological properties analogous to regular tortilla.

El objetivo de este estudio fue comparar fisicoquímica, reológica y texturalmente tortillas de harina de maíz nixtamalizado fortificadas con frijol (Phaseolus vulgaris) y amaranto (Amaranthus spp.) en tres proporciones diferentes (3F7A, 5F5A, 7F3A) con respecto a la harina de maíz comercial (TR). Además, se evaluó su efecto sobre el crecimiento mediante un bioensayo de dos generaciones con ratas Wistar. Los tratamientos mostraron rendimiento similar a TR, además de aumentar significativamente la proteína. Éstos revelaron un comportamiento similar en capacidad de absorción de agua (CAA) y rollabilidad, factores cruciales para el correcto desarrollo de la masa y aceptación, respectivamente. Además, los tratamientos presentaron diferencia significativa (p < 0,05) en el bioensayo, mostrando un comportamiento similar entre 3F7A caseína control. Las formulaciones desarrolladas en la investigación, principalmente 3F7A, son una opción viable como tortillas fortificadas con proteína biodisponible rica en lisina y propiedades de texturales y reológicas análogas a la tortilla regular.

Keywords:

• maize tortilla
• fortification
• development

Palabras clave:

• tortilla
• fortificación
• desarrollo

Introduction

The maize tortilla is considered to be an excellent source of calories due to its high content of starch, and it is an excellent choice for people with gluten intolerance. Unfortunately, the maize tortilla lacks a good quality protein (Serna-Saldivar & Amaya-Guerra, 2008). Mexico produces over 230,000 tons of nixtamalized maize flour every month, equivalent to 370,000 tons of tortillas and a monthly consumption of 4.1 kg of tortilla/person (Serna-Saldivar & Amaya-Guerra, 2008). The tortillas and other maize products are the most consumed by the Mexican population. In rural areas, this cereal provides about 70% of calories and 50% of total protein (Pérez-Herrera, Esquivel-Esquivel, Rosales-Serna, & Acosta-Gallegos, 2002). Since the early 1970s, fortification plans of maize tortilla have been developed with different protein sources such as common bean (Phaseolus vulgaris) or soybean (Glycine max) but without reaching the general population, due to lack of financial resources to purchase these foods (Serna-Saldivar & Amaya-Guerra, 2008). In addition, these products have different sensory characteristics to the traditional tortilla, texture being one of the most important factors for the approval of certain foods by consumers as well as its rheology (Rodríguez-Sandoval, Fernández Quintero, & Ayala Aponte, 2005). Both textural and rheological properties are critical in the research and development of new products because they are intrinsically related to the physicochemical and functional properties of the constituents of the food system, as well as operating variables that apply at different stages of the process (Rodríguez-Sandoval et al., 2005). Maize tortillas, fortified or not, have particular physical characteristics and rheological properties due to high concentrations of starch in their systems. The starch owes much of this functionality to two major high-molecular-weight carbohydrate components: amylose and amylopectin, as well as to the physical organization of these macromolecules into the granular structure (French, 1984). It is of great importance to understand the physicochemical characteristics of starches so as to predict the functional properties of starchy food products that improve the acceptability of them (Méndez-Montealvo, Sánchez-Rivera, Paredes-López, & Bello-Pérez, 2006). The aim of this study was to compare the physicochemical and rheological properties of the resulting doughs and tortillas of nixtamalized maize flour fortified with bean and amaranth flours in different proportions, and their biological evaluation, to commercial maize flour.

Experimental

Formulation of flours and doughs

We used commercial nixtamalized maize flour for different formulations. Amaranth flour was obtained from previously puffed grains. The bean flour was obtained from cooked and grinded seeds (Cárdenas-Quintana, Gómez-Bravo, Díaz-Novoa, & Camarena-Mayta, 2000). The samples were sieved through a mesh (# 40) and then mixed in different proportions to obtain three treatments (Table 1). The flours of each treatment were mixed with water until the doughs were smooth and the humidity was around 55%. Moisture was analyzed by gravimetric method (10.136 AOAC) and protein by Kjeldhal method (930.29 AOAC) (Helrich, 1990), lysine by derivatization in high performance liquid chromatography (Kamp, 1991) and tryptophan by a florescence assay kit (Table 1).

Table 1. Formulations of flour fortified with different proportions of amaranth and bean flours.
Tabla 1. Formulaciones de harinas fortificadas con diferentes proporciones de harina de amaranto y frijol.

Treatment	Formulation
3F7A	900 g/kg maize, 30 g/kg bean, 70 g/kg amaranth
5F5F	900 g/kg maize, 50 g/kg bean 50 g/kg amaranth
7F3A	900 g/kg maize, 70 g/kg bean, 30 g/kg amaranth
TR	1000 g/kg nixtamalized maize

Color

The color measurements were made by colorimetric tristimulus reflectance method. Instrument conditions were: an opening diameter of 65 mm and a viewing angle of 10°, calibrated with a standard plate Master Color Data Hunter brand. The color was measured according to the technique used by Martínez et al. (2001). For the determination of color in the tables, this was placed inside a black box to keep out light and the parameters a*, b* and L* were determined in triplicate, both in flour and tortillas.

Dough yield

Tortillas were made using a manual tortilla machine (Lenin, Mexico), where the prepared dough was obtained in the form of wafers of 0.1 mm wide and 12.5 ± 0.1 cm in diameter. Testing of the tortillas was made 30 min after processing (ambient temperature = 25°C).

Texture

The adhesiveness and cohesiveness of the doughs were determined by a texture profile analysis using a texture analyzer TA XT2 (Texture Analyzer plus, UK). The results obtained from the determination were analyzed using Microcal Origin 6.0 software. The adhesiveness and cohesiveness tests were carried out at a speed of 2 mm/s (length = 4 mm with a count = 4). The shear stress was performed under conditions of 2 mm/s speed, 6 mm length and with count 6. The tensile test was carried out with a speed of 2 mm/s, 15 mm longitude and count 1. The rollability proof of the tortillas was made subjectively (Martínez et al., 2001).

Absorption rate and water solubility

These methods consist of calculating the amount of dissolved material the proportion of water absorbed after stirring a suspension at room temperature. They were determined according to the methodology described by Anderson, Conway and Griffin (1969). The water absorption index (WAI) was expressed as the ratio between the weight of the residue after centrifugation and the dry weight of the sample, from which was subtracted the weight of the residue from evaporation of the supernatant. The water solubility index (WSI) was expressed as the ratio of the residue from evaporation and the dry weight of the sample. Both were measured three times per treatment and calculated as follows:

Gelatinization enthalpy

The determination of temperatures and enthalpies of gelatinization was made using a thermogravimetric technique, specifically, differential scanning calorimetry (DSC) (Arambula, Gutierrez Arias & Moreno Martinez, 2006). We used a DSC 288e (Mettler, Toledo, Spain). The program consisted of a temperature sweep of 30 to 100°C using a temperature ramp of 5°C/min and an empty pan as reference. The trays used were sealed aluminum pans, Tzero of 40 microliter. The thermograms were analyzed using universal analysis software.

Bioassay

We conducted a two-generation bioassay to assess the effect of protein fortification in growth. Wistar rats were around one month of age, and were fed for 10 days. These rats were reproduced for a second generation. Pregnant and lactating mothers were fed the same diet as their offspring, until one month of age. Each rat was housed in individual cages under standard conditions (12:12 h light/darkness, 50% relative humidity and 21°C ± 2). They were fed and water ad libitum. The assay had a control with 10% casein (Amaya Guera, Alanis Guzman, & Serna Saldivar, 2004). Nitrogen was determined in urine and faeces collected from Wistar rats during a period of 10 days (Amaya Guerra, Alanis Guzman, & Serna Saldivar, 2004). The digestibility and nitrogen retention value was calculated using the following formulas:

Results and discussion

The results shown by the fortification with bean and amaranth flour revealed a significant difference (p < 0.05) between TR (100% maize formulation) and the different treatments, namely that the 5F5A formulation has a higher protein value, increasing 23.28%, with respect to TR (Table 2). In addition, the fortification with both flours contribute an important increase of lysine and tryptophan, limit amino acids in maize products, presenting an increase of 48.07% for lysine and 40% for tryptophan in 3F7A treatment. Similar results were obtained in a nixtamalized process of maize–bean tortillas, where the research showed 9.71% protein, lysine 30 g/kg protein and 4.26 tryptophan g/kg protein with 95:5 maize:bean blend (Cuevas-Martínez, Moreno-Ramos, Martínez-Manrique, Moreno-Martínez, & Méndez-Albores, 2010).

Table 2. Results of moisture, protein, lysine and tryptophan (dry basis) of fortified nixtamalized maize flour with bean and amaranth flours in three different proportions.^1,2
Tabla 2. Resultados de humedad, proteína, lisina y triptófano (base seca) de harina de maíz nixtamalizado fortificada con harinas de frijol y amaranto en 3 diferentes proporciones.

Component	FAO requirement	TR	3F7A	5F5A	7F3A
Moisture (mL/kg)		98.60^a	103.10^a	101.50^a	101.80^a
Protein (g/kg)		85.90^a	91.66^b	105.90^c	100.20^c
Lysine^3	54.4	26.00^a	38.50^c	34.90^b	38.90^c
Tryptophan^3	11.0	7.00^a	9.80^d	8.50^b	9.00^c
Note: ^1Mean of three replicates. ^2Mean values with the different letter in the same line are significantly different (p < 0.05). ^3Amino acids are expressed in g/kg protein.
Nota: ^1Promedio de tres replicas. ^2Valores promedio con diferente letra en la misma línea son significativamente diferentes (p < 0,05). ^3Aminoácidos son expresados en g/kg de proteína.

Regarding color and texture, the analysis of doughs revealed significant differences (p < 0.05) in color, WAI, adhesiveness and cohesiveness of doughs (Table 3). The fortified doughs are notably more yellowish and less luminous by the addition of colored flours. Differences in the control behavior may be caused by diverse reasons, such as the use of commercial maize flour in assay, the presence of foreign starches and differences of amylase-amylopectin ratio.

Table 3. Color measurements, water absorption capacity (WAC), water absorption index (WAI) and water solubility index (WSI), adhesiveness and cohesiveness strengths of fortified maize flour doughs with bean and amaranth flours in three different proportions.^1,2
Tabla 3. Mediciones de color, capacidad de absorción de agua (CAA), índice de absorción (IAA) y de solubilidad de agua (ISA), fuerza de adhesión y cohesión de masas de maíz fortificadas con harinas de frijol y amaranto en 3 diferentes proporciones.

Treatment	L	a*	b*	WAC (%)	Mass efficiency (%)	WAI (g gel/g sample)	WSI (%)	Adhesiveness (g)	Cohesiveness (g)
TR	92.24^a	−0.81^a	0.62^a	1.32^a	2.31^a	5.45^a	4.24^a	196.47^b	36.16^b
3F7A	82.49^b	0.84^b	0.84^b	1.19^a	2.19^a	6.96^b	5.42^a	173.33^b	41.66^c
5F5A	82.95^b	0.77^b	12.62^b	1.28^a	2.28^a	7.01^b	5.10^a	152.66^a	36.62^b
7F3A	83.08^b	0.73^b	12.69^b	1.21^a	2.21^a	6.99^b	4.85^a	218.03^d	25.76^a
Note: ^1Mean of three replicates. ^2Mean values with different letter in the same column are significantly different (p < 0.05). L* = 0 yields black and L* = 100 indicates diffuse white. a*, negative values indicate green while positive values indicate magenta. b*, negative values indicate blue and positive values indicate yellow. % w/w (g/100 g).
Nota: ^1Promedio de tres replicas. ^2Valores promedio con diferente letra en la misma columna son significativamente diferentes (p < 0,05). L* = 0 produce negro, L* = 100 indica un blanco difuso. a*, valores negativos indican verde mientras valores positivos indican magenta. b*, valores negativos indican azul mientras valores positivos indican amarillo. % p/p (g/100g).

Some research had shown significant variability in commercial maize flours due to kernel type and storage and process conditions (Bedolla & Rooney, 1984; Flores Farías, Martínez Bustos, Salinas Moreno, & Ríos, 2002). Also, this maize flour type has guar gum, which affects the swelling behavior of starch during the blending and heating processes (Nagano, Tamaki, & Funami, 2008). Regarding the presence of foreign starches, amaranth and bean give shape and size heterogeneity of starch granules and variability to percentages of amylase and amylopectin, affecting retrogradation and viscosity (Paredes-López, Maza-Calviño, González-Castañeda, & Montes-Rivera, 1988; Paredes-López, Schevenin, Hernández-López, & Cárabez-Trejo, 1989), as well as the cohesiveness and adhesiveness of the dough (Serna-Saldivar, Canett, Vargas, Gonzales, & Bedolla, 2008).

In the case of amylose, the difference percentages of this starch component, 4.7–12.5% in amaranth (Kong, Bao, & Corke, 2009) and 34–39% in bean (Ovando-Martínez, Osorio-Díaz, Whitney, Bello-Pérez, & Simsek, 2011), possibly had an important role, because the amylose is correlated negatively to adhesiveness (Sahai, Buendía, & Jackson, 2001). Still, it is reported that water absorption capacity (WAC) has a greater effect on the tensile properties of the dough than the other factors mentioned above, as well the particle size of the starch granule (Pérez-Herrera et al., 2002).

Concerning the behavior about gelatinization of starch (Table 4) in the treatments; a significant difference (p < 0.05) was observed between treatments in Tonset values, as well not for Tp and ΔH. In previous research, it has been observed that with bigger Tonset values, the samples contained more protein and had been more physical and thermally processed (Sandoval Aldana, Rodríguez Sandoval, & Fernández Quintero, 2005; Wannenberger & Eliasson, 1993). These data are supported with the ΔH values, which suggest several process conditions; because some starches lost their birefringence and low energy is necessary for the phase transition (Méndez-Montealvo et al., 2006).

Table 4. Onset (Tonset) and peak (Tp) temperatures and gelatinization enthalpy (ΔH) by formulations of nixtamalized maize flour fortified with bean and amaranth flours in three different proportions.^1,2
Tabla 4. Temperaturas inicial (Tonset) y pico (Tp) y entalpía de gelatinización (ΔH) de formulaciones de harina de maíz nixtamalizado fortificadas con frijol y amaranto en 3 diferentes proporciones.

Treatment	Tonset (°C)	Tp (°C)	ΔH (mJ)
TR	64.43^a	71.11^a	6.30^a
3F7A	64.69^b	71.31^a	6.92^a
5F5A	65.29^b	71.83^a	6.35^a
7F3A	64.91^b	71.73^a	6.98^a
Note: ^1Mean of three replicates. ^2Mean values with different letter in the same column are significantly different (p < 0.05).
Nota: ^1Promedio de tres replicas. ^2Valores promedio con diferente letra en la misma columna son significativamente diferentes (p < 0,05).

In reference to the tortillas, the results show significant differences (p < 0.05) in color measurements, and cutting force. The tortillas obtained were less luminous and more yellow than the control but they presented similar values of rollability, tensile strength and breaking distance, which exposed a product with acceptable texture values, analogous to the control (Table 5). These results are similar to those reported for maize:bean blends (Cuevas-Martínez et al., 2010); showing 1.1N to tensile strength, 7.9N to cutting force and 1 to rollability. In that case, the color was not affected because the bean was a white bean variety. The decrease in cutting force values could be caused by the amaranth flour, as previous research has observed that inclusion of amaranth makes a softer and wetter dough and tortilla (Méndez-Albores et al., 2003).

Table 5. Color measurement, rollability, tensile strength, breaking distance and cutting force in fortified maize tortillas with bean and amaranth flours in 3 different proportions.^1,2
Tabla 5. Medición de color, rolabilidad, fuerza de extensión, distancia de ruptura y trabajo de corte en tortillas de maíz fortificadas con harinas de frijol y amaranto en 3 diferentes proporciones.

Treatment	L	a*	b*	Rollability^3	Tensile strength (N)	Breaking distance (mm)	Cutting force (N)
TR	74.12^a	1.20^a	19.46^a	1.00^a	1.65^a	6.03^b	6.66^d
3F7A	68.29^b	3.58^b	18.96^a	1.10^a	1.15^a	5.10^a	4.53^a
5F5A	68.12^b	3.49^b	19.33^a	1.12^a	1.08^a	5.33^a	5.22^b
7F3A	69.20^b	3.34^b	18.80^a	1.25^a	1.16^a	4.15^a	5.98^c
Note: ^1Mean of three replicates. ^2Mean values with different letter in the same column are significantly different (p < 0.05). ^3Tortilla rollability: 1 = tortillas with no breaking, 2 = a partial breaking at the center and edges of the tortilla and 3 = completely damaged tortillas. N = Newton, mm = millimeters.
Nota: ^1Promedio de tres replicas. ^2Valores promedio con diferente letra en la misma columna son significativamente diferentes (p < 0.05). ^3Tortilla rollability: 1 = tortilla sin rompimiento, 2 = parcial rompimiento en el centro y bordes de la tortilla y 3 = tortilla con daño completo. N = Newton, mm = milímetros.

In the bioassay, the treatments showed similar behavior with respect to 1° and 2° generations of rats, indicating a significant difference (p < 0.05) for diets fortified with bean and amaranth flours with respect to TR. It is important to note that 3F7A, which has just 6.7% protein increase with respect to TR, showed a comparable behavior with the control, which contained 10% casein as nitrogen source (Table 6). It could be because this treatment showed an important increase in limiting the amino acids lysine and tryptophan, 48.07% and 40%, respectively. These values illustrate that fortification with bean and amaranth, particularly 3F7A, would increase weight in a comparable mode to the control, even with the presence of bean flour, which formerly was not recommended for fortification due the presence of antinutrimentals and the high cost of bean products (Serna-Saldivar, Canett, Vargas, Gonzales, & Bedolla, 1988).

Table 6. Comparison of growth of Wistar rats feed with diets based in fortified nixtamalized maize tortilla with bean and amaranth, in a two-generation bioassay.^1,2
Tabla 6. Comparación del crecimiento de ratas Wistar alimentadas con dietas basadas en tortillas de maíz nixtamalizado fortificadas con harina de frijol y amaranto en un bioensayo de 2 generaciones.^1,2

Diet	Protein (g/kg)	Wi (g)	Wf (g)	ΔW (g)	PER	APD	BV	NPU
1° Generation
Control	100.00^c	92.5^b	122^b	29.5^b	2.25^c	85.39^c	78.79^b	77.38^d
TR	85.90^a	87^a	111.5^a	24.5^a	1.67^a	76.64^a	70.94^a	54.37^a
7F3A	100.20^c	106.4^d	140.8^d	34.4^c	1.78^b	85.91^c	80.99^b	69.58^c
3F7A	91.66^b	87.75^a	129.75^c	42^d	2.13^c	85.36^c	79.40^b	67.78^c
5F5A	105.90^c	95.6^c	130.8^c	35.2^c	1.90^b	83.99^b	77.88^b	65.41^c
2° Generation
Control	100.00^c	61.5^b	84.5^c	23.0^c	2.22^d	95.38^c	92.81^c	93.50^d
TR	85.90^a	80.75^e	105^d	24.25^c	0.84^a	51.39^a	50.86^a	50.74^a
7F3A	100.20^c	57.5^a	68.17^a	10.67^a	1.52^b	92.86^b	90.62^b	84.15^b
3F7A	91.66^b	64^c	80.83^b	16.83^b	1.86^d	92.36^b	89.83^b	82.96^b
5F5A	105.90^c	71.5^d	82.67^c	11.17^a	1.63^c	94.68^c	93.15^c	88.20^c
Note: ^1Mean of six individuals. ^2Mean values with different letter in the same column are significantly different (p < 0.05). ^3Wi = initial weight, Wf = final weight, ΔW = Wf – Wi. ^4PER = protein efficiency ratio, APD = apparent protein digestibility, BV = biological value, NPU = net protein utilization.
Nota: ^1Promedio de seis individuos. ^2Valores promedio con diferente letra en la misma columna son significativamente diferentes (p < 0.05). ^3Wi = peso inicial, Wf = peso final, ΔW = Wf – Wi. ^4PER = rango de eficiencia proteica, APD = digestibilidad aparente de proteína, BV = valor biológico, NPU = utilización neta de proteína.

That the treatments increase the weight in Wistar rats significantly, even with a low percentage of protein in the diet, could be explained by the fact that it is known that low levels of protein consumption lead to a higher retention of nitrogen, even more when the type of protein fortification has balanced amino acid values (Chavez & Muñoz de Chavez, 2003; Rosado, López, Morales, Muñoz, G. & Allen, 1992).

Conclusions

After formulating maize:bean:amaranth blends, there was significant improvement in different fortified tortillas with regard to protein, lysine and tryptophan content, mainly with 3F7A, which has increases of up to 48% and 40% of these limiting amino acids, respectively. These formulations show physicochemical, rheological and textural properties similar to the commercial maize tortilla formulations, showing analogous results in WAC in the doughs, and the same rollability and tensile strength in tortilla. Therefore, as an alternative to resolve the problem of low protein quantity/quality of maize-based food products without variation in process or equipment, the fortification with amaranth and common bean, particularly with 3% bean and 7% amaranth, is suggested.

Acknowledgements

The authors thank the laboratory of Bioorganic Materials CINVESTAV IPN, Querétaro Unit, especially Dr. Juan de Dios Figueroa Cárdenas and his staff for the facilities provided to carry out this research.