Capsaicinoids content and proximate composition of Mexican chili peppers (Capsicum spp.) cultivated in the State of Chihuahua

Abstract

Capsaicinoids content and proximate composition of Mexican chili peppers, widely used in the State of Chihuahua, México, were determined. Capsaicinoids content was determined by high-performance liquid chromatography (HPLC) technique. Proximate composition was determined by official AOAC methods. Scoville Heat Units (SHU) were also calculated. Capsaicin content ranged between 22.8 mg/kg and 9097.3 mg/kg dry matter. Dihydrocapsaicin content ranged between 36.8 mg/kg and 4023.6 mg/kg dry matter. Scoville Heat Units ranged between 961 and 211,248. Moisture content was determined with differences (p < 0.05) among chili pepper types and cultivars ranging from 54.8 g/kg dry matter to 130.1 g/kg dry matter. Ash content (p < 0.05) ranged from 58.1 g/kg dry matter to 96.1 g/kg dry matter showing statistical differences. Protein content ranged from 120.5 g/kg dry matter to 152.2 g/kg dry matter showing no statistical differences. Fat content (p < 0.05) ranged from 22.6 g/kg dry matter to 137.6 g/kg dry matter showing statistical differences.

Se determinó el contenido de capsaicinoides y la composición proximal de los chiles más utilizados en el estado de Chihuahua, México. El contenido de capsaicinoides de determinó mediante la técnica de cromatografía líquida de alta resolución. La composición proximal se determinó mediante métodos oficiales de la AOAC. Las unidades Scoville (SHU) también fueron calculadas. El contenido de capsaicina estuvo en un rango de 22,8 mg/kg–9097,3 mg/kg masa seca y dihidrocapsaicina en un rango de 36,8 mg/kg–4023,6 mg/kg masa seca. El rango de unidades Scoville (SHU) fue de 961–211248. El contenido de humedad presentó diferencias (P < 0,05) entre los chiles y fue de 54,8 g/kg a 130,1 g/kg masa seca así como también el contenido de ceniza presentó diferencias (P < 0,05) con un rango de 58,1 g/kg a 96,1 g/kg masa seca. El contenido de proteína no presento diferencias (120,5 g/kg a 152,2 g/kg masa seca). El contenido de grasa también presentó diferencias (P < 0,05) y estuvo en un rango de 22,6 g/kg a 137,6 g/kg masa seca. El contenido de capsaicinoides y la composición proximal de chiles mexicanos que no han sido estudiados es información esencial para investigación futura.

Keywords:

• capsaicinoids
• capsaicin
• capsicum
• chili pepper
• dihydrocapsaicin
• proximate composition
• Scoville Heat Units

Palabras clave:

• capsaicinoides
• capsaicina
• capsicum
• chiles
• dihidrocapsaicina
• composición proximal
• unidades Scoville (SHU)

Introduction

Chili peppers have been used in Mexico as food and condiment for at least the past eight centuries and are an integral part of the Mexican diet and culture (Contreras-Padilla & Yahia, 1998). In the United States, there has been a great increase in the demand of this fruit because of the increased multiculturalism, popularity of ethnic restaurants, and increased use of “flavored” vegetables in the diet to avoid fats and cholesterol (Lawless, 1989). Furthermore, chili peppers and by-products such as “paprika” are among the most consumed spices throughout the world mainly because of their sensory attributes such as aroma, color, and pungency (Garces-Claver, Arnedo-Andres, Abadia, Gil-Ortega, & Alvarez-Fernandez, 2006).

The pungency of this fruit is due to a group of alkaloids called capsaicinoids. These compounds are secondary metabolites produced in the placenta of the fruit (Minamiyama, Kinoshita, Inaba, & Inoue, 2005). The most important members of this group are capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin (Williams, Raghavan, Orsat, & Dai, 2004). Capsaicin and dihydrocapsaicin constitute approximately 90% of total capsaicinoids content of chili peppers, capsaicin being the most abundant (Govindarajan, 1986; Iwai, Suzuki, & Fujiwake, 1979; Kawada, Watanare, Katsura, Takami, & Iwai, 1985; Kosuge & Furuta, 1970). The capsaicin content in chili peppers is variable and ranges from 0.1% to 1% of the fruit weight, approximately, but the amount can vary depending on the temperature at which the plant is grown, the age of the fruit, and the light (Al Othman, Ahmed, Habila, & Ghafar, 2011; Tucker, 2001).

In Mexico, there is a vast quantity of chili pepper types and cultivars produced in the different regions within the country. In the State of Chihuahua, in the Mexican northwest region, fresh chili pepper types have a great demand. Some types, such as Jalapeno and Serrano, are mainly used to prepare sauces and guacamole (smashed avocado). Other chili pepper types, such as Piquin and De Arbol, are mainly used in dry state and ground. Piquin chili pepper is also consumed pickled. Chipotle peppers (smoked jalapenos) are also very popular because of their singular taste, a little bit sweet, and can be found canned and processed.

The majority of Mexican chili pepper types and cultivars have not been characterized. There are some studies reporting the capsaicinoid content of various different types and cultivars of chili pepper. These chili pepper types and cultivars include Habanero Orange and White, De Arbol, Piquin, Serrano, Jalapeno, Poblano, Yolo Wonder, Jupiter, Sincap, Dulce, Chawa, Sukurre, 730 F1, 1245 F1, Amazon F1, Serademre 8, Kusak 295 F1, Meiteimorok, Haomorok, Uchithi, Mashingkha, Umorok, and Chiengpi (Cisneros-Pineda et al., 2007; Contreras-Padilla & Yahia, 1998; Garces-Claver et al., 2006; Hernández, Ochoa, López, & García, 2009; Huffman, Schadle, Villalon, & Burns, 1978; Kurian & Starks, 2002; Marshall & Doperalski, 1981; Ornelas-Paz et al., 2010; Pino et al., 2007; Reilly, Crouch, & Yost, 2001; Sanatombi & Sharma, 2008; Topuz & Ozdemir, 2007). However, there is little investigation about the chili peppers included in this study regarding capsaicinoids content and proximate composition.

The objective of this study was to determine proximate composition and capsaicin and dihydrocapsaicin content in 15 types and cultivars of Mexican chili peppers, produced at the northwest region of the State of Chihuahua.

Materials and methods

Materials

The study was carried out on 15 different chili peppers produced in the state of Chihuahua: Guajillo, Ancho, Pasado, Pasilla, Puya, Tres Venas, Chipotle Meco, Jalapeno, Mirasol, Morita, Serrano, Chipotle, De Arbol (Capsicum annuum L. var. annuum), Piquin (Capsicum annuum L. var. aviculare), and Habanero (Capsicum chinense Jacq.). All these chili peppers were found at local markets and most of them were found on a dry state. The mature stage on these fruits was senescent red, and field-grown harvested. Habanero, Serrano and Jalapeno peppers were found on a fresh state, mature stage immature green and they were dehydrated following the method used by Kuzniar, Bowers, and Craig (1983) in an oven with air circulating at 60°C, for 4–5 days until reaching constant weight. These fruits were also field-grown harvested according to the sellers.

HPLC analysis of capsaicinoids

The capsaicinoids extraction method used in this study was an adaptation of the method reported by Cisneros-Pineda et al. (2007) using methanol instead of acetone (Kurian & Starks, 2002; Ornelas-Paz et al., 2010). Dried tissue of solid pepper samples were blended to a fine powder and 0.6 g of powdered chili pepper sample were mixed with 10 mL of methanol high-performance liquid chromatography (HPLC) grade. The mixture was kept for 2 h at 4°C. Samples were then centrifuged for 5 min at 11,000 × g and the supernatant was filtered through a polyethylene membrane of 0.45 μm of pore size previous to HPLC analysis. Three replicates were performed for every chili pepper type.

High-performance liquid chromatography separation and quantification of capsaicinoids was achieved following the method recommended by Ornelas-Paz et al. (2010). High-performance liquid chromatography set-up consisted of a Varian (Model 9012, Agilent Technologies, Inc., Santa Clara CA, USA) solvent delivery system and a Varian (Model 9050, Agilent Technologies, Inc., Santa Clara CA, USA) UV-VIS detector (set at a λ  = 236 nm). A Supelcosil LC-C₁₈ column (25 cm × 4.6 mm, 5 μm; Sigma-Aldrich, St. Louis, Missouri, USA) was used. Mobile phase consisted of acetonitrile/water/acetic acid at a ratio of 50:50:1 at a flow rate of 1 mL/min. Injection volume was 20 μL, run time 30 min and temperature 25°C.

Standards of capsaicin and dihydrocapsaicin (Sigma-Aldrich, St. Louis, Missouri, USA) were used to identify and quantify capsaicinoids. High-performance liquid chromatography grade methanol (Avantor Performance Materials, Center Valley, PA, USA) was used to prepare the solution for standards curves for capsaicin and dihydrocapsaicin. Standard curves were prepared using concentrations of 5–1000 μg/mL. Two replicates were performed with a correlation coefficient of 0.996.

Proximate composition

Moisture and ash were determined gravimetrically by desiccation at 105°C and by incineration at 550°C, respectively, following AOAC method 934.01 for moisture and AOAC method 942.05 for ash. Crude protein was obtained by Kjeldahl method using AOAC method 988.05. Protein content was obtained by calculation of the Kjeldahl nitrogen × 6.25. Fat was extracted from chili pepper using the method described by Soxhlet (1879). Carbohydrates were determined by difference.

Scoville Heat Units determination

Scoville Heat Units (SHU) were estimated by the method of Todd, Bensinger, and Biftu (1977). The determination of pungency on SHU was accomplished by multiplying the individual capsaicinoid contents by the corresponding value of threshold pungency.

Statistical analysis

Experimental data are shown as the mean ± standard error of assays run in triplicate for capsaicinoids content and proximate composition. Results were statistically compared by Tukey's test at 5% after a one-way ANOVA was conducted. Data were processed in the SAS software (SAS Institute, Inc. 1999).

Results and discussion

Capsaicinoids of chili peppers

Capsaicin and dihydrocapsaicin concentrations were determined for the 15 chili pepper types and cultivars. These two capsaicinoids are good predictors of the pungency of chili peppers since they constitute about 90% of the total capsaicinoid content (Reilly et al., 2001). Capsaicin and dihydrocapsaicin contents for each chili pepper type and cultivars are shown in Table 1. As a general trend, capsaicin and dihydrocapsaicin content of the studied chili peppers correlated well (Figure 1), showing an average relative proportion of about 2:1, supporting previous research (Govindarajan & Sathyanarayana, 1991; Zachariah, Bhageerathy, & Parthasarathy, 2008). However, some chili peppers of the variety Capsicum annuum L. var. annuum showed an average relative proportion of 1:1, while chili peppers of the variety Capsicum chinense Jacq., and Capsicum annuum L. var. aviculare showed a ratio of 2:1 (Hayman & Kam, 2008). These findings were previously documented by Neumann (1966).

Table 1. Capsaicinoids content of 15 commercial types and cultivars of dry chili peppers produced in the Mexican northwest region in Chihuahua State (mg/kg dry matter)¹.
Tabla 1. Contenido de capsaicinoides de 15 diferentes tipos y cultivares de chile comerciales producidos en la región noroeste del estado de Chihuahua (mg/kg masa seca)¹.

Chili pepper	Capsaicin	Dihydrocapsaicin	SHU^2
Guajillo	22.85 ± 0.06 a	36.85 ± 0.43 a	961.13 ± 7.91
Ancho	42.82 ± 1.97 a,b	42.19 ± 1.31a	1368.60 ± 52.67 a
Pasado	38.34 ± 3.83 a,b	58.38 ± 4.46 a	1557.06 ± 133.36 a
Pasilla	49.21 ± 1.51 a,b	68.76 ± 0.89 a	1899.34 ± 38.70 a
Puya	53.88 ± 1.03 a,b,c	67.40 ± 1.32 a	1952.61 ± 37.92 a
M. Tres Venas	66.87 ± 0.11 a,b,c,	77.12 ± 2.73 a	2318.25 ± 45.80 a,b
Chipotle Meco	163.82 ± 1.96 a,b,c,d	126.29 ± 0.39 a,b	4670.86 ± 25.27 b
Jalapeno	373.51 ± 29.11 d,e	210.40 ± 12.41 a,b,c	9400.83 ± 668.52 c
Mirasol	353.77 ± 5.66 c,d,e	231.70 ± 3.54 a,b,c	9426.09 ± 148.21 c
Morita	338.25 ± 5.17 b,c,d,e	334.94 ± 0.59 b,c	10838.24 ± 73.70 c
Serrano	627.48 ± 17.03 e,f	399.77 ± 5.48 c,d	16538.78 ± 185.92 d
Chipotle	883.04 ± 24.40 f	552.65 ± 6.90 d,e	23114.68 ± 281.72 e
De Arbol	1293.36 ± 16.24 g	641.74 ± 5.79 e f	31155.10 ± 354.68
Piquin	2656.74 ± 81.46 h	1031.57 ± 1.35 f	59381.77 ± 1289.64 g
Habanero	9097.35 ± 183.33 i	4023.63 ± 142.55 g	211247.65 ± 656.47 h
Note: ^1Each value is an average of three samples ± standard error of the mean. The means followed by different letters in the same column are significantly different (p < 0.05) by Tukey's test. ^2SHU: Scoville Heat Units.
Nota: ^1Cada valor es el promedio de tres muestras ± el error estándar de la media. Las medias seguidas de letras diferentes en la misma columna son significativamente diferentes (P < 0,05) mediante la prueba de Tukey. ^2 SHU: Unidades Scoville

Figure 1. Capsaicin and dihydrocapsaicin correlation for studied chili peppers.

Figura 1. Correlación de capsaicina y dihidrocapsaicina para los chiles estudiados.

Dihydrocapsaicin was more abundant than capsaicin in Guajillo, Pasado, Pasilla, Puya and Mirasol Tres Venas, which are found on a dry state. These peppers showed an atypical capsaicinoids' profile and were the least pungent. In these varieties, capsaicin concentration ranged between 22 mg/kg and 66.87 mg/kg, and dihydrocapsaicin concentration between 36 mg/kg and 77.12 mg/kg. These results support the findings by Cisneros-Pineda et al. (2007), and Garces-Claver et al. (2006) who found that some C. annuum cultivars contain higher concentrations of dihydrocapsaicin than the amounts found in C. chinense (Habanero). Ancho pepper showed a low capsaicinoids' concentration with a similar content of capsaicin and dihydrocapsaicin (42.82 mg/kg and 42.19 mg/kg, respectively). Morita pepper had this characteristic as well with the difference that this pepper is about 10 times more pungent than Ancho (338 mg/kg capsaicin and 334 mg/kg dihydrocapsaicin). Capsaicin was more abundant than dihydrocapsaicin in Ancho, Chipotle Meco, Jalapeno, Mirasol, Morita, Serrano, Chipotle, De Arbol, Piquin and Habanero.

Habanero chili pepper is known for its high capsaicinoids' concentration and it is the least used in the Mexican northwest region. Habanero pepper was the richest in capsaicinoids. Our findings of capsaicin and dihydrocapsaicin contents in Habanero pepper, 9097 mg/kg and 4023 mg/kg, respectively, were similar to those previously reported by Kurian and Starks (2002) and slightly higher than those reported by Garces-Claver et al. (2006) and Ornelas-Paz et al. (2010). Our results showed higher levels of capsaicin and dihydrocapsaicin on Habanero than those of Cazares-Sanchez et al. (2005) due, probably, to the fact that the fruits were cultivated in a greenhouse and the conditions were not optimal than that of its area or origin, such as altitude. It is also possible that the variety of this pepper has a naturally low concentration of capsaicinoids. Research by Cisneros-Pineda et al. (2007) showed higher results, compared to ours, in part because of genetic factors to each cultivar and environmental conditions.

Capsaicin and dihydrocapsaicin contents (373 mg/kg and 210 mg/kg, respectively) in jalapeno pepper were very low compared to results reported by Contreras-Padilla and Yahia (1998) (17,093 mg/kg and 2180 mg/kg, respectively) and Ornelas-Paz et al. (2010) (2083 mg/kg and 1765 mg/kg, respectively). Research by García-Martínez, Miranda González, González González, & Nieto Pineda (2006), showed similar results compared to ours on capsaicin concentration (340 mg/kg) although dihydrocapsaicin was not estimated in this study. Capsaicin concentration reported by Huffman et al. (1978) was slightly lower (268 mg/kg) and dihydrocapsaicin concentration was not reported. Recent work by Topuz, Dincer, Ozdemir, Feng, & Kushad (2011) showed slightly higher results for capsaicin and dihydrocapsaicin (549 mg/kg and 597 mg/kg, respectively) in jalapeno pepper. They also studied the influence of drying methods on capsaicinoids composition of jalapeno pepper. They reported that oven drying method decreased capsaicinoids concentration compared to natural convective drying. We can assume that differences of capsaicinoid concentration in jalapeno pepper compared to other results in the literature may also be due to the drying method used in this study. Capsaicinoids content may be affected by the stage of maturation and cultivar, and also geographic location, growing, and processing conditions. Although these results were obtained using fresh jalapeño pepper, it has also been reported that peroxidases are involved in the degradation of capsaicinoids (Bernal et al., 1993). We can deduce that these different factors may be involved in the discrepancies found in the literature with our results. Serrano pepper concentrations for capsaicin and dihydrocapsaicin were higher than in Jalapeno (627 mg/kg and 399 mg/kg, respectively). Ornelas-Paz et al. (2010) also reported higher capsaicinoid content in Serrano than in Jalapeno. These discrepancies may be due to the vast quantity of different cultivars and types of Jalapeno and Serrano peppers existing in the market, growing conditions, genetic factors and probably the effect of peroxidase, as mentioned before. Also, the drying process used in this study for Serrano pepper may have influenced the differences in capsaicinoids' concentration compared to other results in the literature.

Total capsaicinoids (capsaicin and dihydrocapsaicin) concentration for De Arbol (80,520 mg/kg) and Piquin (53,710 mg/kg) reported by Contreras-Padilla and Yahia (1998) were also much higher than the values found in this study.

Results obtained in this study show that capsaicin and dihydrocapsaicin content in Chipotle (883 mg/kg and 552 mg/kg) is higher than that found in Chipotle Meco (163 mg/kg and 126 mg/kg, respectively). These findings validate research by Cisneros-Pineda et al. (2007) who found that capsaicinoids content is higher in ripe than in unripe peppers. The difference between the two is that Chipotle is smoked ripe (when the fruit reaches its red color) and Meco is smoked unripe (fresh state, or green). Fattorusso and Taglialatela-Scafati (2007) mention that differences in capsaicinoid accumulation may be due to environmental issues, such as temperature, light, moisture of the soil, and fertilization level.

Scoville Heat Units (SHU) were determined for the chili peppers studied. The SHU ranged between 961 and 21,4233 (Table 1). The fruits having higher capsaicin and dihydrocapsaicin contents result in higher SHU values. Guajillo, Ancho, Pasado, Pasilla, Puya, and Mirasol Tres Venas ranged between 961 and 2318 SHU, and showed no statistical differences among them. Jalapeno, Mirasol, and Morita ranged between 9401 and 10,838 SHU and showed no statistical differences among them. Habanero, Piquin, De Arbol, Chipotle and Serrano ranged between 16,539 and 211,248 and showed significant differences among them.

Proximate composition of chili peppers

The proximate composition of the 15 studied chili peppers is shown in Table 2. The moisture, fat and ash contents did show some differences among chili peppers (p > 0.05). Different protein contents were not observed among chili peppers (p > 0.05). There is little information about proximate composition for the chili pepper types and cultivars studied. Previous research by Tandon, Dravid and Siddappa (1964), on Indian chili peppers, showed similar results for moisture, ash, and protein on an average basis (8.94 g, 6.27 g, and 12.82 g, respectively). Research by Krishna De (2003) was slightly different (moisture 6.50 g, ash 8.00, protein 14.00 g, and fat 14.10 g) but there is no information on the origin of the types and cultivars studied for the proximate composition determination. Carbohydrate content was slightly higher comparing research by Krishna De (2003). Mean concentrations of carbohydrate ranged between 57.68% and 67.92% and Krishna De (2003) reports an average of 56.25%.

Table 2. Proximate chemical composition (g/kg) of 15 commercial types and cultivars of dry chili pepper used in the northwest region of the state of Chihuahua, Mexico¹.
Tabla 2. Composición proximal química (g/kg) de 15 tipos y cultivares comerciales de chile seco utilizados en la región noroeste del estado de Chihuahua, México¹.

Chili pepper	Moisture (g/kg)	Ash (g/kg)	Protein (g/kg)^2	Fat (g/kg)	Carbohydrates (g/kg)
Guajillo	91.1 ± 6.7 b,c	75.3 ± 0.2 a,b,c	128.9 ± 0.9 b,c	124.3 ± 1.5 a	580.0 ± 6.3 d,e
Ancho	101.1 ± 13.3 a,b,c	78.1 ± 9.1 a,b,c	120.5 ± 0.0 c	98.2 ± 1.2 b,c	602.1 ± 23.5 c,d,e
Pasado	89.2 ± 5.1 b,c	71.8 ± 12.2 b,c	126.1 ± 2.8 b,c	54.1 ± 1.5 e	661.8 ± 10.5 a,b
Pasilla	75.9 ± 2.5 c,d	58.5 ± 0.2 c	122.8 ± 8.5 c	137.6 ± 2.6 a	605.3 ± 8.1 c,d,e
Puya	70.6 ± 1.7 c,d	78.2 ± 0.8 a,b,c	132.5 ± 3.5 b,c	81.1 ± 7.4 c,d	637.6 ± 13.3 a,b,c
M. Tres Venas	90.5 ± 0.4 b,c	70.2 ± 1.8 b,c	132.8 ± 6.0 b,c	96.1 ± 1.4 b,c	610.5 ± 3.1 c,d,e
Chipotle Meco	84.9 ± 4.2 b,c	95.4 ± 2.4 a	152.2 ± 5.1 a	90.8 ± 0.2 b,c,d	576.8 ± 11.4 e
Jalapeno	101.3 ± 8.7 a,b,c	73.2 ± 1.6 b,c	143.6 ± 3.8 a,b	42.3 ± 2.0 e	639.7 ± 8.8 a,b,c
Mirasol	98.9 ± 2.0 b,c	96.1 ± 0.7 a	140.5 ± 4.0 a,b	74.9 ± 0.7 d	589.6 ± 4.6 c,d,e
Morita	107.8 ± 9.1 a,b	85.9 ± 14.3 a,b	141.2 ± 6.2 a,b	76.0 ± 1.8 d	589.1 ± 13.1 c,d,e
Serrano	112.3 ± 4.1 a,b	58.1 ± 0.7 c	127.8 ± 6.0 b,c	22.6 ± 1.3 f	679.3 ± 8.1 a
Chipotle	87.9 ± 2.3 b,c	69.2 ± 1.9 b,c	127.2 ± 0.1 b,c	86.6 ± 8.1 c,d	629.2 ± 12.3 b,c,d
De Arbol	56.5 ± 2.1 d	88.2 ± 6.3 a,b	127.5 ± 6.3 b,c	133.8 ± 10.7 a	594.1 ± 3.9 c,d,e
Piquin	54.8 ± 0.8 d	72.8 ± 0.2 b,c	137.2 ± 2.9 a,b,c	110.2 ± 9.7 b	625.0 ± 11.5 b,c,d,e
Habanero	130.1 ± 19.4 a	75.1 ± 0.3 a,b,c	135.2 ± 3.9 a,b,c	46.3 ± 1.8 e	613.3 ± 24.7 b,c,d,e
Note: ^1Each value is an average of three samples ± standard error of the mean. The means followed by different letters in the same column are significantly different (p < 0.05) by Tukey's test. ^2Kjeldahl nitrogen × 6.25. ^3Carbohydrates were determined by difference.
Nota: ^1Cada valor es el promedio de tres muestras ± el error estándar de la media. Las medias seguidas de letras diferentes en la misma columna son significativamente diferentes (P < 0,05) mediante la prueba de Tukey. ^2Nitrogeno Kjeldahl × 6,25. ^3 Cálculo de carbohidratos por diferencia.

Conclusions

Differences in capsaicinoids' concentration have been found for different chili peppers used in the State of Chihuahua, Mexico. It can be deducted from the current study that the varieties Capsicum chinense Jacq., and Capsicum annuum L. var. aviculare contain larger amounts of capsaicin and dihydrocapsaicin, having a ratio of 2:1, while some varieties of Capsicum annuum L. var. annuum showed an average proportion of 1:1. Variations in capsaicinoids quantity could be attributed to environment and genetic factors to each cultivar and the effect of the oven drying method used in this study for Serrano and Jalapeno peppers. It was found that chili peppers constitute a good source of protein compared to other vegetables regularly used in Mexico.

Acknowledgments

The authors would like to thank Veronica Gonzalez and Angel Esparza for their technical assistance and the Consejo Nacional de Ciencia y Tecnologia (CONACYT) for providing the funding for the graduate studies of author Orellana-Escobedo.