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Oct 19, 2016 - immune system, VAD increases infant mortality rates and the incidence and severity of infectious diseases.2 In Mexico, the high prevale...
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Effect of Traditional and Extrusion Nixtamalization on Carotenoid Retention in Tortillas Made from Provitamin A Biofortified Maize (Zea mays L.) Aldo Rosales,†,‡ Edith Agama-Acevedo,† Luis Arturo Bello-Pérez,† Roberto Gutiérrez-Dorado,§ and Natalia Palacios-Rojas*,‡ †

Centro de Desarrollo de Productos Bióticos del Instituto Politécnico Nacional, Kilometer 8.5 Carretera Yautepec-Jojutla, Colonia San Isidro, Apartado Postal 24, 62731 Yautepec, Morelos, Mexico ‡ Global Maize Program, International Maize and Wheat Improvement Center (CIMMYT), CIMMYT Research Station, Kilometer 45 Carretera Mexico-Veracruz, El Batan, Texcoco, CP 56237, Estado de México, Mexico § Programa Regional del Noroeste para el Doctorado en Biotecnología, Facultad de Ciencias Químico-Biológicas, Universidad Autónoma de Sinaloa, Culiacán, Sinaloa 80010, Mexico S Supporting Information *

ABSTRACT: Provitamin A (proVA) enhanced maize was developed to help alleviate vitamin A deficiency in maize-consuming populations. Nixtamalization (lime-cooking process) is the most commonly used maize-preparation method in Mexico and Central America. In this study, the effect of traditional nixtamalization (TN) and nixtamalized extrusion (NE) on proVA retention was evaluated. Kernel conversion to TN dough led to high proVA apparent retention (>100%), while kernel conversion to NE flour led to lower retention (85%). However, TN tortilla proVA carotenoid concentration was similar to the kernels’ original concentration and slightly higher in NE tortillas. Genotypic variation has a strong effect on proVA retention in TN dough and NE flour, but no such variation in proVA retention was observed in tortillas. Tortillas prepared with proVAenhanced maize, using either TN or NE, are a good source of proVA carotenoids. Also, dough made using TN and proVAenhanced maize is a high proVA-content ingredient for other food products. KEYWORDS: biofortification, lime-cooking, provitamin A retention, tortillas

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In 2012, the first proVA-enriched maize hybrids developed by conventional breeding were released in Zambia and Nigeria. To date proVA hybrids have been also released in Zimbabwe and Malawi.5 Several hundred food products are derived from maize worldwide; but in Africa in particular, maize is mainly eaten boiled, roasted, as thin porridge and thick porridge (nshima, tza-tza, sadza), or as cooked grits (samp), all prepared mainly with white maize.6 However, consumer acceptance studies of products made from orange maize have suggested that acceptability is not an issue for preschool and school children and that nutritional benefit is a significant driver for consumers, especially women and parents. Therefore, testing proVA retention in vastly consumed food products is important for further dissemination and promotion of proVA enriched maize.7 In Mexico, maize consumption is around 253 kg per capita per year and the most common preparation process is limecooking, also called nixtamalization.8 This process involves cooking (35−50 min, 80−95 °C) and steeping maize kernels (8−16 h, room temperature) in calcium hydroxide. More than

INTRODUCTION Vitamin A deficiency (VAD) continues to be a widespread public health problem despite the relative success of supplementation/fortification programs. An estimated 250 million preschool children have VAD worldwide.1 VAD has been linked to adverse health outcomes including night blindness, corneal scarring, and blindness. By weakening the immune system, VAD increases infant mortality rates and the incidence and severity of infectious diseases.2 In Mexico, the high prevalence of subclinical VAD in children is of great concern for public nutrition. Recent studies in specific areas of the country found low levels of retinol in serum of preschool children, and there is a risk that these reserves may become even lower due to the high prevalence of infectious diseases such as giardiasis.3,4 All yellow maize genotypes contain carotenoids, although the content of carotenoids with provitamin A activity (proVA) (βcarotene, α-carotene, and β-cryptoxanthin) is relatively low (2 μg g−1, on average).5 ProVA biofortified maize, with content up to 8 μg g−1 of proVA, has been developed to help alleviate VAD in maize-consuming populations worldwide. ProVA-enriched maize breeding is led by the International Maize and Wheat Improvement Center (CIMMYT) and the International Institute of Tropical Agriculture (IITA) in collaboration with public and private research partners in Southern Africa, and is supported by the HarvestPlus Program (www.harvestplus.org). © 2016 American Chemical Society

Received: Revised: Accepted: Published: 8289

June 30, 2016 October 18, 2016 October 19, 2016 October 19, 2016 DOI: 10.1021/acs.jafc.6b02951 J. Agric. Food Chem. 2016, 64, 8289−8295

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Journal of Agricultural and Food Chemistry

Traditional Nixtamalization. Sample 350 g lots of maize were cooked for 35 min at 80 °C in 1.5 L of 1.2% calcium hydroxide and steeped for 16 h in the dark at room temperature. The cooking solution (nejayote) was discarded, and the resulting nixtamal was washed three times with tap water to remove the pericarp and excess lime, as done traditionally. Nixtamal was ground into dough using a commercial stone grinder. One hundred grams of dough of each genotype were collected in plastic containers and stored at −80 °C until carotenoid analysis was performed. Nixtamalized Extrusion. Extrusion was performed as described by Milán-Carrillo et al. (2006).13 Briefly, 500 g lots of kernels were broken up to obtain grits (1 to 2 mm). The grits were then mixed with lime powder (0.5 g of lime per 500 g of maize), and enough water was added to reach a moisture content of 28%. Extrusion was carried out using a single-screw laboratory extruder (Brabender model 20 DN, South Hackensack, NJ, USA) (19 mm screw diameter, 20:1 length-todiameter ratio, 2:1 nominal compression ratio, and 2.4 mm die opening). Extrusion was performed at 80 °C and 240 rpm screw velocity. The feed rate was 70 g min−1. Extrudates were cooled, dried at room temperature in darkness for 16 h, milled in a hammer-type cyclone mill (Lab Mill Perten 3100), and passed through a 30-mesh sieve (0.5 mm). They were then packed in aluminum bags and stored at −80 °C until carotenoid analysis was performed. Tortilla Cooking. Dough was prepared by mixing the extruded flour with sufficient water to reach the desired consistency. Dough (from traditional processing and extrusion) was rounded and shaped into flat disks using a manual machine. The dough disks were cooked on a hot griddle at 250 ± 5 °C for 30 s on one side, followed by 30 s on the other side, and then turned back on the first side until puffing. Tortillas were left to cool and then packed in polyethylene bags, after which samples were stored at −80 °C for further analysis. Each process (TN and NE) was done twice, and replicate samples were taken at each stage of the process. Physical Kernel Analysis. Grain physical characteristics were determined using standard procedures as described in Galicia et al. (2012).19 Flotation index (FI) and hectolitric weight (HW) (Method 84-10 from AACC, 2000)20 are indirect measurements of kernel hardness. Hundred kernel weight (HKW) was determined as a measure of kernel size (AACC 2000, Method 55-10).20 Carotenoid Analysis. Carotenoid extraction was performed as described by Galicia et al. (2012).19 Briefly, 600 mg samples (fine powder of maize kernels, dough, flour, and tortillas) in 6 mL of ethanol (with 0.1% butylated hydroxytoluene) underwent 5 min precipitation in an 85 °C water bath before being subjected to 10 min saponification with 500 μL of 80% (w/v) KOH in water. After saponification, samples were immediately placed in ice to which 3 mL of cold deionized water was added. Two hundred microliters of the internal standard (β-apo-8′-carotenal) was added, and samples were vortexed. Carotenoids were extracted 3 times with 3 mL of hexane by centrifugation at 800g, and the hexane fraction was extracted. The combined extracted hexane layers were dried under nitrogen and reconstituted in 500 μL of 50:50 methanol:dichloroethane (v/v). All carotenoid extraction procedures and analysis were conducted under yellow light. Two microliters of the sample was injected into Acquity UPLC Water equipment. Separation was performed using an Acquity UPLC BEH C18 1.7 μm, 2.1 × 100 mm column and an Acquity column in-line filter. LUT, ZEA, BCX, and BC were identified based on their characteristic spectra and by comparing their retention times with known standard solutions. 9-cis-β-Carotene and 13-cis-β-carotene were identified and quantified based on BC standard. ProVA was computed as all-trans-BC + (1/2)(13-cis-BC) + (1/2)(9-cis-BC) + (1/ 2)(BCX). Apparent Carotenoid Retention. Apparent retention (AR) was calculated as follows:

300 food products commonly consumed in Mexico alone are derived from nixtamalized maize;9,10 this provides an opportunity for innovative markets to promote biofortified maize consumption both in the country and worldwide. Nixtamalization also improves niacin bioavailability, increases calcium and resistant starch content in the final products, reduces phytate content (important mineral antinutrient), and significantly reduces mycotoxins in raw kernels.10 The most common maize-preparation method is traditional nixtamalization (TN), where lime-cooked kernels are ground into dough (masa) from which various products are made, tortillas being the most common, with a consumption of 78.49 kg per capita per year in Mexico.8 The second most common maize preparation method is the use of nixtamalized flour (NF), which has the advantage that it only needs to be rehydrated to prepare tortillas.8,10 The use of nixtamalized flour reduces labor costs and problems associated with maize acquisition and storage prior to processing, as well as providing greater opportunities for automatization.11 Nixtamalized extrusion (NE) is a process where maize grits are mixed with lime and water to reach a moisture content of about 35%. The uncooked blend is continuously fed into an extruder. Extrusion cooking (about 7 min at 80 °C, followed by 16 h at room temperature) can produce dough suitable for making tortillas having similar characteristics as those produced using the TN and NE processes.12 Extrusion offers advantages such as energy saving, less waste production, better quality control, and different product shapes, and compared to NF, the basic equipment needed is less expensive.13 Previous studies have shown that the three processes (TN, NF, and NE) can lead to the loss of nutraceutical compounds such as phenolics,14 anthocyanins,15 and total carotenoids;16−18 however, these studies were conducted with a limited number of normal yellow genotypes, and little is known about the effect of nixtamalization and tortilla cooking on the levels of provitamin A carotenoids16by traditional and extrusion nixtamalization. Based on these considerations, the objectives of this research were to compare the effects of TN and NE on proVA carotenoid retention and study the genotypic effects on proVA retention throughout the TN and NE processes.

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MATERIALS AND METHODS

Chemicals and Reagents. All chemicals for UPLC analyses were HPLC-grade and purchased from Merck (Darmstadt, Germany) and Sigma (St. Louis, MO, USA). Ultrapure water was used for UPLC and carotenoid extraction. The standards for β-carotene (BC) were purchased from Sigma, while those for lutein (LUT), zeaxanthin (ZEA), and β-cryptoxanthin (BCX) were purchased from Carotenature (Switzerland). Food grade lime (Nixtacal), commercially available in Mexico, was used for the nixtamalization. Plant Materials. Seven experimental proVA-enriched hybrids were used in this study; these hybrids, called biofortified experimental hybrids (BEH), were numbered from 1 to 7. Two commercial yellow maize hybrids (CYH) were used as control (CYH1, CYH2). The genotypes were chosen based on maturity (intermediate), kernel hardness (medium and hard), and proVA content (between 1.9 and 12.7 μg g−1 DW). Genotypes BEH2, 3, 4, 5, and 7 were used for extrusion experiments only. Ear moisture content at harvest was about 18%, and ears were dried at room temperature (24 °C) in the shade. Sample Preparation. Kernel moisture was brought down to about 10%. Then samples were divided for TN and NE. Prior to grinding, dough and tortilla samples were lyophilized in darkness for 6 days at −80 °C using a VirTis Benchtop 2KBTXL freeze-dryer. Kernel, dough, and tortilla samples were ground in a Cyclotech 1093 laboratory mill with a 0.5 mm sieve.

% AR =

nutrient content per g of cooked food (dry basis) × 100 nutrient content per g of raw food (dry basis)

Statistical analysis. To identify and eliminate systemic variations between genotypes and to focus only on the carotenoid variation 8290

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0.05 0.13 0.26 0.29 0.09 0.19 0.06 0.04 0.33

proVA

± ± ± ± ± ± ± ± ± 0.00 0.02 0.06 0.04 0.02 0.01 0.02 0.01 0.14 ± ± ± ± ± ± ± ± ± 0.20 0.22 1.19 0.98 1.67 1.37 0.97 0.38 1.88 a b c c d e f a g 0.05 0.08 0.16 0.22 0.05 0.08 0.05 0.03 0.16 ± ± ± ± ± ± ± ± ±

all-trans-BC 8291

0.00 0.02 0.06 0.03 0.01 0.05 0.02 0.01 0.07 ± ± ± ± ± ± ± ± ± 0.00 0.07 0.08 0.07 0.06 0.15 0.01 0.01 0.13 ± ± ± ± ± ± ± ± ± ab a cd be fc e dg ef g 0.01 0.02 0.67 0.16 0.19 0.07 0.72 0.34 1.80 ± ± ± ± ± ± ± ± ± 0.65 0.32 9.16 3.20 6.91 3.96 11.53 4.47 12.56 0.42 0.87 0.56 0.36 0.53 0.15 0.63 0.55 0.40 ± ± ± ± ± ± ± ± ±

HKW (g)

30.20 36.49 25.42 28.27 29.32 29.65 28.52 25.93 30.80

hardness

hard intermediate soft hard hard very hard hard hard hard ± ± ± ± ± ± ± ± ±

a

25 56.5 71 25 23 5.5 19.5 24 19.5

1.41 3.53 3.13 1.41 2.83 2.12 3.53 4.76 3.54

FI (%)

CYH1 CYH2 BEH1 BEH2 BEH3 BEH4 BEH5 BEH6 BEH7

Table 1. Physical and Chemical Properties of Maize Kernelsa

LUT + ZEA

0.20 0.21 4.59 2.26 4.30 3.75 5.94 1.28 7.75

BCX

a a b c b d e f g

9-cis-BC

a a b c b d dc e f

μg g−1 DW

RESULTS AND DISCUSSION Physical and Chemical Properties of Maize Genotypes. Kernel hardness and size were the two physical parameters used to set nixtamalization conditions. Intermediate to hard kernels are preferred by traditional tortilla producers in Mexico, whereas hard to very hard kernels are used mostly by the nixtamalized flour industry.21 The genotypes were chosen based on kernel hardness. Nevertheless, once kernels in this experiment were harvested, their hardness was verified using the flotation index (FI) and test weight (TW) methodologies. As presented in Table 1, only BEH4 kernels were classified as very hard and BEH1 as soft. All genotypes had small kernels with hundred kernel weight (HKW) ranging between 25 and 30 g (Table 1). Therefore, for the TN process we used 1.2% calcium hydroxide and the same cooking time (35 min) at 80 °C for small and hard kernels. In general, harder and large kernels require a longer cooking time (up to 50 min) to ensure good hydration, softness, and high quality dough.10 The baseline kernel carotenoid content before any cooking was also monitored. As expected, all the BEH had higher proVA carotenoid content than the CYH1 and CYH2 (Table 1). Among the different BEH genotypes, LUT + ZEA accounted for 39.56% (ranging from 24.67 to 59.00%) of total carotenoids. BCX and all-trans-BC were the main proVA carotenoids, accounting for 22.33% (ranging from 17.39% to 26.88%) and 42.78% (ranging from 26.44% to 55.54%), respectively, of total carotenoids. BEH7 showed significantly higher all-trans-BC (7.12 μg g−1 DW) as compared to the other BEH (0.85 to 6.63 μg g−1 DW, BH6 and BH3, respectively) (Table 1). It is worth noting that all-trans-BC content was higher than BCX content and significant differences were observed for both BC isomers, 9-cis-BC and 13-cis-BC, in the kernels of the analyzed hybrids (Table 1). Up to 10 times differences between 9-cis-BC in CYH1 and BEH3 and BEH7 were observed, and more than 9 times difference for 13-cis-BC was observed between CYH1 and BEH7 (Table 1). Fate of Carotenoids during TN and NE Tortilla Production. Although there was genotypic variation in the carotenoid content of the analyzed hybrids (Table 1), the trend in the content through all stages of tortilla production (from dough or flour to tortilla) was similar between all BEH and CYH genotypes (see Tables 1S and 2S). Moreover, dry matter loss was similar (about 4%) for all genotypes during TN. CYH kernels had very low carotenoid content, and they decreased below the detection limit in dough, flour, and tortilla (see Tables 1S and 2S). To facilitate the presentation of results, we

0.11 0.22 1.23 0.80 1.36 1.00 0.95 0.46 1.52

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0.83 0.32 4.42 3.97 6.63 5.42 2.89 0.85 7.12

13-cis-BC

a a bc cd e b d a e

where xi is the original value, min(x ⃗) is the minimum carotenoid concentration for the given genotype, and max(x )⃗ is the maximum carotenoid concentration for the given genotype. All results were statistically analyzed using SigmaPlot ver. 11 (Systat Software, Inc.). Differences between variables were tested for significance using ANOVA. Differences between means were considered to be significantly different at p < 0.05. Nine replications were carried out for physical properties, and three replications were carried out for carotenoid analysis.

1.09 0.64 7.92 6.00 10.30 8.48 6.82 1.91 12.70

xi − min(x ⃗) max(x ⃗) − min(x ⃗)

genotype

Ind i =

a a b c d b e f g

between stages and processes of tortilla production, carotenoid data was normalized and data indices, between zero and one, were generated. Values close to one reflect higher metabolite content, while those close to zero reflect lower metabolite content. The following formula was used to create the index:

Different letter in the same column indicates significant difference at P < 0.05. Contents of chemical compounds are expressed on dry weight (DW) basis ± SD. FI, flotation index; HKW, hundred kernel weight; TW, test weight; LUT + ZEA, lutein + zeaxanthin; BCX, β-criptoxanthin; 9-cis-BC, 9-cis-β-carotene; all-trans-BC, all-trans-β-carotene; 13-cis-BC, 13-cis-β-carotene; CYH, Commercial Yellow Hybrid; BEH, Biofortified Experimental Hybrid.

Journal of Agricultural and Food Chemistry

DOI: 10.1021/acs.jafc.6b02951 J. Agric. Food Chem. 2016, 64, 8289−8295

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Journal of Agricultural and Food Chemistry Table 2. Carotenoid Content in the Different Stages of Tortilla Production by TN and NEa stage of tortilla production kernel nixtamal, TN dough, TN tortilla, TN flour, NE tortilla, NE

LUT + ZEA 0.54 0.51 0.73 0.45 0.13 0.27

± ± ± ± ± ±

0.36 0.27 0.33 0.25 0.17 0.18

ab ab b abc c ac

BCX 0.60 0.78 0.93 0.84 0.02 0.09

± ± ± ± ± ±

0.28 0.10 0.10 0.07 0.04 0.07

all-trans-BC

9-cis-BC a b b b c c

0.58 0.81 0.97 0.77 0.09 0.20

± ± ± ± ± ±

0.29 0.11 0.05 0.10 0.10 0.18

a b b ab c c

0.71 0.54 0.56 0.26 0.40 0.47

± ± ± ± ± ±

0.38 0.37 0.20 0.22 0.31 0.15

a ab ab b ab ab

13-cis-BC 0.45 0.69 0.90 0.57 0.05 0.43

± ± ± ± ± ±

0.31 0.16 0.12 0.08 0.07 0.23

proVA a bc c ab d a

0.62 0.71 0.85 0.62 0.07 0.20

± ± ± ± ± ±

0.31 0.22 0.14 0.14 0.10 0.13

a a a a b b

Values are average of normalized data of all the 7 BEH ± SD. Different letter in the same column of each variable indicates significant difference at P < 0.05. Data closer to one means higher carotenoid content. BEH, Biofortified Experimental Hybrid; TN, traditional nixtamalization; NE, nixtamalization by extrusion; LUT + ZEA, lutein + zeaxanthin; BCX, β-criptoxanthin; 9-cis-BC, 9-cis-β-carotene; all-trans-BC, all-trans-β-carotene; 13-cis-BC, 13-cis-β-carotene; proVA, β-carotene equivalence of proVA carotenoids. a

have normalized the data to show the change in the chemical composition of the 7 BEH analyzed during tortilla production using both TN and NE (Table 2). We analyzed the dough (made using TN) and the flour (made using NE), as they can be used to make different food products, including tortillas, chips, porridge (atole), etc.8 There is ongoing controversy over the bioefficacy of cis-BC isomers; however, recent studies have shown that they yielded similar vitamin A stores in liver as the all-trans-BC isomer in Mongolian gerbil liver;23,24 although the bioefficacy was not one-to-one, it was higher than 0.5. Nevertheless, to facilitate comparison of proVA retention in maize products with that found in other studies, we included only 0.5 of cis-isomers in the computation, as described in Materials and Methods. LUT + ZEA, BCX, and 13-cis-BC content did not show significant variation during the different stages analyzed (Table 2). Corrales-Banuelos et al. (2016)17 recently reported up to 87.6% and 79.5% total carotenoid retention in tortillas prepared by TN and NE, respectively, made of pigmented landraces. Those results are in agreement with the findings in this study. Compared to the kernels, dough prepared using TN showed a significant slight increase in proVA content, but it was reduced in the tortillas (Table 2), indicating slight losses due to the cooking process. Contrary to our observations, De la Parra et al. (2007)13 reported that about 50% of proVA was lost during dough production. However, in their study the dough obtained using TN was dried at 60 °C before carotenoid analysis and the high temperature could have led to carotenoid losses;26 moreover, cis-BC isomers were not quantified and therefore not included in proVA calculations. We observed a significant decrease in proVA carotenoids in the NE flour produced from extruded kernels (Table 2). There was also a 30.14%, 7.32%, and 14.74% reduction in BCX, BC, and proVA, respectively (see Table 2 and Table 2S). Cooking the tortillas made from NE flour had a significant slight concentrating effect on their carotenoid content (Table 2; Figure 1). The mechanisms regulating carotenoid content in plants include localization of carotenoid biosynthetic enzymes in plastids (amyloplasts, in maize kernels), carotenoid catabolism, autoxidation, isomerization, thermal degradation, and photodegradation.25,26 Carotenoid degradation mechanisms may also play a role throughout kernel processing. One of the differences between TN and NE is that in the former the maize kernels remain intact during the strongest thermal and pH treatments (lime-cooking and steeping), while in NE the kernels are broken up into 1−2 mm grits before the alkali and extrusion process. Although breaking the kernel structures makes the carotenoids of the amyloplasts in the endosperm more available

Figure 1. Genotypic variation in proVA retention during different stages of traditional nixtamalization (TN) and nixtamalized extrusion (NE). D-TN: Dough made using TN. F-NE: Flour made using NE. TTN: Tortillas made using TN. T-NE: Tortillas made using NE.

for chemical extraction, it also makes them more prone to autoxidation, thermal degradation, and photodegradation. Carotenoid catabolism may also contribute to their degradation.26 In the TN process the catalytic enzymes that are present in the kernel germ or aleurone layer are inactivated by the boiling step, but in NE such enzymes may be present during milling. Nevertheless, proVA carotenoid losses during the NE process are lower compared with mealie meal (fine flour) preparation for African food products, where up to 20% of proVA is lost.22,27,28 Mugode et al.22 did not find reduction in proVA content when kernels were milled into samp. Milling into samp implies degermination and a subsequent decrease in enzymes that may also contribute to carotenoid loss. In addition, particle size in samp is higher than 2.3 mm.29 Taken together, all these results suggest that the extent of carotenoid degradation during milling depends on final particle size and on whether or not the aleurone layer and germ (live kernel structures) are present during processing. Lozano-Alejo et al. (2007)16 found a 36% reduction in proVA concentration in nixtamalized chips made from proVA-enhanced maize experimental varieties. Although the nixtamalized dough was not analyzed, the significant proVA loss reported in the chips most likely occurred during frying, a very strong thermal process. Additionally, no quantification of cis-BC isomers was presented, making it difficult to identify actual proVA retention in nixtamalized chips. Because dough made using TN has higher proVA carotenoid content (Table 2), it is recommended for making other food 8292

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Journal of Agricultural and Food Chemistry

Table 3. Percentage of Estimated Average Requirement (EAR) of RAEa Provided by Average Consumption of Tortilla in Mexico (2 Tortillas per Preschool Child per Day, 4 Tortillas per 4−8 Year Old Child per Day, and 8.4 Tortillas per 9−13 Year Old Child and Pregnant Woman per Day,8 Assuming Average Tortilla Weight of 11.7 g DW8) preschool children (1−3 years old)

children (4−8 years old)

children (9−13 years old)

pregnant women (19−50 years old)

genotype

T-TN

T-NE

T-TN

T-NE

T-TN

T-NE

T-TN

T-NE

CYH1 BEH2 BEH3 BEH4 BEH5 BEH7

0.45 10.30 16.28 15.54 13.48 20.33

0.52 9.92 15.19 13.49 10.84 18.76

0.69 15.73 24.86 23.73 20.59 31.05

0.80 15.14 23.20 20.61 16.56 28.66

0.89 20.42 32.26 30.76 26.73 40.30

1.03 19.65 30.11 26.74 21.49 37.19

0.72 16.52 26.10 24.92 21.62 32.61

0.84 15.90 24.36 21.64 17.39 30.09

a Retinol activity equivalents. One RAE = 6.48 μg of BC. bThe EAR recommended is 210, 275, 445, and 550 μg per day for preschool children, 4−8 year old children, 9−13 year old children, and pregnant women, respectively (Values from the DRI reports. Source: https://fnic.nal.usda.gov/sites/ fnic.nal.usda.gov/files/uploads/recommended_intakes_individuals.pdf).

Figure 2. Genotypic variation in proVA retention during different stages of traditional nixtamalization (TN) and nixtamalized extrusion (NE). DTN: Dough made using TN. F-NE: Flour made using NE. T-TN: Tortillas made using TN. T-NE: Tortillas made using NE. A different letter in each bar indicates a significant difference among genotypes at P < 0.05, lowercase letters for D-TN and uppercase letters for F-NE. There are no significant differences among genotypes for proVA retention in T-TN and F-NE.

per preschool child and 8.4 tortillas per pregnant woman per day) made from CYH only provides about 0.4% and 0.7% of the EAR to preschool children and pregnant women, respectively; whereas consuming tortillas made from BEH provides 10.3−20.3% of the EAR to preschool children and 16.5−32.6% to pregnant women, the two populations most prone to vitamin A deficiency (Table 3). Consumption of NE tortillas made from BEH provides slightly lower percentages of the EAR of vitamin A (up to 18.7% to preschool children and 30% to pregnant women; Table 3). Thus, tortillas not only provide 38.8% of proteins, 45.2% of calories, and 49.1% of calcium for an adult,9,10 but if they are prepared with proVAenriched maize, they can also provide between 10.3 and 40.3% of EAR.

products that have to be analyzed for their proVA content, which can be affected by the cooking process. Processing and cooking methods that result in good proVA retention need to be identified and recommended. Estimated Average Requirement of Vitamin A from Nixtamalized Tortillas. Determining proVA retention in food products made from biofortified crops is important for estimating their bioconversion especially if the corresponding non-biofortified crop has very low levels of the micronutrient or none at all. Based on estimated average requirement (EAR)30 of vitamin A, the retinol activity equivalents (RAE) determined for maize (6.48 μg BC to 1 RAE),31 and the proVA retention values determined here, we estimated the percentage of EAR provided by tortillas.31,32 Consuming TN tortillas (2 tortillas 8293

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Journal of Agricultural and Food Chemistry Genotypic Variation in ProVA Carotenoid Retention in Tortillas Made by TN and NE. Although the trend in proVA carotenoid content through all stages of tortilla production was similar among all BEH and CYH genotypes (Tables 1S and 2S), slightly significant (p < 0.05) proVA carotenoid retention was observed in TN dough and NE flour among genotypes (Figure 1). ProVA retention values in tortillas made from BEH genotypes using both methods ranged from 81% to 114% (Figure 2), and no significant differences among genotypes were observed in proVA retention in tortillas made by either TN or NE. BEH3 had the lowest proVA retention value in TN dough, while BEH4, BEH5, and BEH6 had the highest retention in TN dough (>120%) compared to other genotypes (Figure 2). Such differences in proVA retention among genotypes were also observed in flour prepared using NE, where hybrids BEH3, BEH4, and BEH6 had less than 90% proVA retention in NE flour. Genotypic differences in carotenoid retention during food preparation have been previously suggested not only in maizederived food products but also in other food products derived from biofortified staple crops such as cassava and sweet potato.6,22,33,34 This suggests that carotenoid catabolism (enzymatic degradation) may play an important role, given that nonenzymatic degradation depends on the external conditions to which the genotypes were exposed like the tortilla cooking process. Of course, genotypic variations in other metabolites (antioxidants, fatty acids, starch pathways) can also contribute to variations in carotenoid retentions.26 ProVA retention in CYH was about 40%. This may be explained by the rather low proVA carotenoid content in CYH kernels, which may have been affected during processing, but reached the lowest quantification limit of the analytical method of carotenoids. The above-mentioned genotypic differences in proVA retention are important, from the plant breeding perspective, for selecting varieties that are less prone to degradation. Food technology is also challenged to maximize carotenoid retention and bioavailability while at the same time minimizing carotenoid isomerization.

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ACKNOWLEDGMENTS

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REFERENCES

The authors wish to thank the staff at CIMMYT’s Maize Nutritional Quality Laboratory for their technical assistance. We also thank Julieta Espinoza from the Universidad Autónoma de Sonora for her support in the extrusion process and Reina Flores Corona for her help in the nixtamalization process. We thank Angela Pacheco for her support on statistical analysis. The authors also acknowledge CIMMYT’s HarvestPlus breeding program for the seed used in the experiments. We are grateful to Gricelda Vázquez and Fabiana DeMoura for reading the manuscript and to Alma Mcnab for the English editing.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b02951. Carotenoid content during the different stages of tortilla production by traditional nixtamalization and by extrusion nixtamalization (PDF)

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Article

AUTHOR INFORMATION

Corresponding Author

*Tel: +52 (55) 58042004. Fax: +52 (55) 58047558. E-mail: n. [email protected]. Funding

Partial funding for this study was provided by HarvestPlus through a cooperative agreement with the International Maize and Wheat Improvement Center (CIMMYT). CRP MAIZE also supported this research. A.R. would like to thank the ́ Consejo Nacional de Ciencia y Tecnologia-Mé xico (CONACyT) for its financial support (417567) of his graduate studies (M.Sc. degree). Notes

The authors declare no competing financial interest. 8294

DOI: 10.1021/acs.jafc.6b02951 J. Agric. Food Chem. 2016, 64, 8289−8295

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DOI: 10.1021/acs.jafc.6b02951 J. Agric. Food Chem. 2016, 64, 8289−8295