Carotenoid Retention of Biofortified Provitamin A Maize (Zea mays L

Article pubs.acs.org/JAFC

Carotenoid Retention of Biofortified Provitamin A Maize (Zea mays L.) after Zambian Traditional Methods of Milling, Cooking and Storage Luke Mugode,† Barbara Ha,‡ Augustine Kaunda,† Thelma Sikombe,† Sidney Phiri,§ Raphael Mutale,∥ Christopher Davis,⊥ Sherry Tanumihardjo,⊥ and Fabiana F. De Moura*,‡ †

National Institute of Scientific and Industrial Research (NISIR), P.O. Box 310158, 15302 Lusaka, Zambia HarvestPlus, c/o International Food Policy Research Institute (IFPRI) , 2033 K Street NW, Washington D.C. 20006, United States § Zambia Agriculture Research Institute (ZARI), Mount Makulu Research Station, Private Bag 7, Chilanga, Lusaka, Zambia ∥ HarvestPlus, WorldFish-Zambia Office, Katima Mulilo Road, Stand no. 37417, Olympia Park, Lusaka, Zambia ⊥ Department of Nutritional Sciences, University of WisconsinMadison, 1415 Linden Drive, Madison, Wisconsin 53706, United States ‡

S Supporting Information *

ABSTRACT: Provitamin A biofortified maize hybrids were developed to target vitamin A deficient populations in Africa. The purpose of this study was to evaluate the degradation of carotenoids after milling, cooking, and storage among biofortified varieties released in Zambia. The biofortified maize hybrids contained 7.5 to 10.3 μg/g dry weight (DW) of provitamin A as measured by β-carotene equivalents (BCE). There was virtually no degradation due to milling. The BCE retention was also high (>100%) for most genotypes when the maize was cooked into thick (nshima) and thin porridge, but showed a lower BCE retention (53−98%) when cooked into samp (dehulled kernels). Most of the degradation occurred in the first 15 days of storage of the maize as kernels and ears (BCE retention 52−56%) which then stabilized, remaining between 30% and 33% of BCE after six months of storage. In conclusion, most of the provitamin A degradation in biofortified maize hybrids occurred during storage compared with cooking and the magnitude of this effect varied among genotypes. KEYWORDS: β-carotene, β-cryptoxanthin, corn, carotenoid degradation, retention, nshima, samp

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INTRODUCTION Biofortified maize lines with higher provitamin A concentrations were developed to target vitamin A-deficient populations in areas of Africa where maize is a staple crop. HarvestPlus, which is part of the Consultative Group on International Agriculture Research (CGIAR) Centers on Agriculture for Nutrition and Health (A4NH) is breeding crops for better nutrition and has made remarkable progress in Zambia. Under the HarvestPlus program, the breeding target level of 17 μg of provitamin A per gram DW, or 15 μg/g fresh weight (FW), for maize was established to provide an incremental 50% of the estimated average requirement (EAR) of vitamin A for nonpregnant, nonlactating women of reproductive age and for preschool children 4−6 years old.1 The first wave of maize hybrids with enhanced provitamin A content were developed by the International Maize and Wheat Improvement Center (CIMMYT) and the International Institute of Tropical Agriculture (IITA) in collaboration with the Zambia Agriculture Research Institute (ZARI). Three biofortified maize varieties are currently released in Zambia that can provide 25% of the EAR of vitamin A (6−8 μg/g DW of provitamin A) for adult women and preschool children. The most recent developed lines have as much as 20 μg/g DW.2 The initial target level was set using assumptions based on the best available information on 50% losses of provitamin A with milling, processing, storage, and cooking, the amount of © 2014 American Chemical Society

maize consumed by children (200 g/d) and women (400 g/d), a 12:1 bioconversion factor of β-carotene to retinol, and 50% of the retinol EAR for children (275 μg/d) and women (500 μg/ d).3 Later on, results from a cross-sectional survey conducted in two districts in rural Zambia where maize is a staple crop reported maize consumption of 287 g/d among women and 182 g/d among children.4 A much higher conversion rate of 6.5:15 than originally assumed (12:1)6 overruled the lower than expected maize intake, confirming the original target of 15 μg/g FW of provitamin A. Few studies have reported the provitamin A retention in maize during processing. Provitamin A losses have been reported to be below 25% after the maize was processed and cooked according to African traditional cooking methods.7 Another study on different varieties of yellow maize reported decays of provitamin A of 15 to 45% after drying and 0 to 50% after four months of dark storage at 25 °C.8 The combined provitamin A losses from cooking and storage resulted in an overall 37% retention that was used in the calculation for the final target level. However, the studies were undertaken in the laboratory and with maize varieties not developed in Zambia. Received: Revised: Accepted: Published: 6317

March 17, 2014 June 13, 2014 June 15, 2014 June 16, 2014 dx.doi.org/10.1021/jf501233f | J. Agric. Food Chem. 2014, 62, 6317−6325

Journal of Agricultural and Food Chemistry

Article

Milling Methods. From the remaining maize kernels, about 2/3 was milled into samp and 1/3 was milled into maize meal using a local hammer mill. Approximately 250 g from each of the milled products was collected and formed the raw samp and raw whole maize meal samples. The effect of milling on retention was evaluated at baseline and three and six months. Raw Maize Samp. First, 1700 g of whole dry kernels with 12% moisture (as-is-basis) was conditioned by steeping in cold water for 3− 5 min to loosen the bran for decortication by a PRL dehuller (380 V, 50 Hz, 250−500 g/h, Canada) using abrasive carborundum disks, revolving at high speed in a barrel, and then bran was removed by aspiration. The dehulled kernels were collected as maize samp and bran as byproduct. Whole Maize Meal (Mealie Meal). Whole maize kernels (400− 900 g, 12% moisture) was put on a hopper through the hammer mills (Model-Commercial, 50T/24 Hr 380 V, 50 Hz, China) fitted with sieve no. 2 (8 mm opening) to produce fine whole maize meal for use in cooking porridge and/or nshima. Cooking Methods. Three local women prepared the traditional Zambian maize dishes: porridge, nshima, and samp at NISIR in Lusaka. Their cooking methods were very similar; the recipes were standardized and each woman prepared only one dish in duplicate. Cooked products were prepared at baseline (time zero) and after the maize had been stored for three and six months. The cooking methods were standardized as follows: Porridge. First, 100 g of maize meal was added to 700 mL of water at 50 °C in an aluminum pot. Then the mixture was continuously stirred with a wooden spoon until it started boiling and formed a gel at 95 °C. The pot was then covered with a lid, and the boiling continued for about 15 min. Total cooking time was 20 min. Nshima. First, 150 g of maize meal was weighed and about 85 to 90 g was added to 600 mL of water at 50 °C in an aluminum pot. Then the mixture was continuously stirred with a wooden spoon until it started boiling and formed a gel at 95 °C and boiling was continued for 10 to 15 min. The remaining maize meal (60−65 g) was added to the pot with continuous stirring until a thick gel (nshima) was formed. The pot, covered with a lid, was allowed to simmer for about 5 min (total cooking time was 25 min). Cooked Samp. First, 250 g of samp was weighed, washed with tap water, and transferred to an aluminum pot with 2000 mL of tap water (enough to cover the samp). Then the samp was boiled at 97 °C with occasional stirring and replenishing with tap water as needed. The total cooking time was 180 min. Determination of Moisture. The moisture content of stored and processed samples was determined at baseline and three and six months by using a standard method (AOAC, 1984). Extraction and Analysis of Carotenoids. The carotenoid extraction method from maize flour and maize porridge was based on a previously published procedure11 with minor modifications. Whole maize samples were ground using a Knifetec 1095 sample mill (FOSS, Eden Prairie, MN, US). For all samples, 500 μL of 80% potassium hydroxide (w/v) was used for saponification. Saponification was performed to reduce the amount of lipid in the extract, which interferes with clean chromatography of carotenoids. Porridge samples were physically broken up after weighing but prior to the addition of ethanol and saponified for 45 min at room temperature. β-Apo-8′carotenal (Sigma-Aldrich, St. Louis, MO, US) was used as an internal standard. Samples were reconstituted in 500 μL of 50:50 methanol:dichloroethane (v/v) and 50 μL injected into the HPLC system for quantification. The HPLC system was composed of a 1525 binary pump, a 717 autosampler, and a 2996 PDA detector (Waters, Milford, MA, US). Two mobile phases were used in a 40 min gradient system run through a C30 carotenoid column (3 μm, 4.6 mm × 250 mm) (YMC America, Allentown, PA). Solvent A, methanol:water (92:8, v/v) with 10 mmol/L ammonium acetate, and solvent B, 100% methyl-tertiary-butyl ether, were used. The gradient was run at 1 mL/ min starting with 70% solvent A and transitioning to 40% within 30 min. The flow returned to 70% solvent A over 2 min, and then the column was equilibrated for 8 min prior to the next injection. Chromatograms were generated at 450 nm for quantification. β-

Furthermore, recent studies have demonstrated a genotype effect on retention9 and bioefficacy,10 underscoring the importance to conduct studies in the varieties ready for release. Therefore, the purpose of this study was to evaluate the retention of provitamin A carotenoids in biofortified maize hybrids from the National Performance Trials (NPTs) in Zambia taking into consideration the local conditions of processing, cooking and storage.

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MATERIALS AND METHODS

Biofortified Provitamin A Maize. Four medium maturing threeway hybrids with levels ranging from 6 to 8 μg/g DW of provitamin A were evaluated for their provitamin A retention after storage, milling, and cooking: two, GV662A (HP1002) and GV665A (HP1005), were hybrids released in Zambia in 2012, and two, HP1001 and HP1003, were advanced genotypes. Harvesting and Storage of Maize. The four biofortified maize genotypes were planted in November 2010 at the Golden Valley Agriculture Research Trust (GART) in Chisamba, Zambia. In April 2011, the maize was harvested at the “green” stage (60% moisture content), which were immediately boiled and roasted; two months later, the “dry maize” (12% moisture content) was harvested. On the day of harvest, ears of all four genotypes were labeled and transported to ZARI in Lusaka where half of them were shelled (kernels) and the other half kept unshelled (ears) for six months in a traditional storage bin at ambient temperature. The temperature and humidity inside the storage bin were recorded on a weekly basis during the duration of the study. Maize samples were collected at several intervals from the storage bin, and raw, processed, and cooked samples were kept in a liquid nitrogen tank until the samples were shipped for analysis. The baseline value (i.e., control) of provitamin A for all four genotypes was obtained from maize samples that were tested within 24 h of harvest. Subsequently, maize samples, stored as kernels and ears, were analyzed every two weeks (HP1002 and HP1005) and three and six months after storage (HP1001 and HP1003). Retention of Provitamin A. All processing and cooking of maize samples were performed at the National Institute of Scientific and Industrial Research (NISIR) in Lusaka. Samples were stored in liquid nitrogen until shipped to the laboratory at the University of WisconsinMadison for analysis. Processing of Green Maize. Green maize is commonly eaten either roasted or boiled. Before roasting or boiling, three rows of kernels of the raw green ear were removed and mixed to form the pool of maize kernels for the baseline values of the “raw roasted” or “raw boiled”. The maize kernels were mixed and divided into four quarters from which two opposite quarters were combined, discarding the other two quarters. This procedure was repeated, and 10 to 15 kernels were selected and packed in 5 mL vials with screw caps and stored in the freezer at −30 °C. After removing the kernels to obtain the “raw samples”, three ears from each genotype were placed on a brazier without the husk and roasted for 15−20 min until all sides had turned brown. After roasting, three rows of kernels were removed from each ear and mixed to obtain a sample of roasted maize. After removing three rows of kernels to obtain the “raw samples”, three ears for each variety containing a thin layer of husk were put in an aluminum pot with 1200 mL of tap water. The pots were covered with lids and the ears were boiled for 35 min at 95 °C. After boiling, three rows of kernels were removed from each ear and mixed to obtain a sample of boiled maize. Processing of Dry Maize. Most of the maize consumed in Zambia is in the form of “dry maize”, which is usually milled and cooked into mainly three products for consumption: porridge, nshima (thick porridge), and samp. Dry maize can be stored for long periods of time. In order to study the retention of provitamin A during milling and cooking, 25 maize ears of each genotype were shelled, producing about 2.6−3.1 kg of kernels. Approximately 250 g of kernel was sampled per genotype to form the baseline pool. 6318

dx.doi.org/10.1021/jf501233f | J. Agric. Food Chem. 2014, 62, 6317−6325


6319

a Data are means ± SD of duplicated analyses. bDifferent lowercase letters indicate a statistically significant difference among the genotypes/varieties for the same harvesting stage. Different uppercase letters indicate a statistically significant difference between the two harvesting stages for the same genotype/variety. cGreen maize was harvested after 5 months of planting with a moisture content of 61% on average. dDry maize was harvested two months after the green maize with a final moisture content of 12% on average. eβ-carotene equivalent = [all-trans-β-carotene + 1/2 α-carotene + 1/2 βcryptoxanthin + 1/2 13-cis-β-carotene + 1/2 9-cis-β-carotene].

1.16A 0.21bA 0.09bcA 0.08cA 0.61aA ± ± ± ± ± 8.69 8.94 8.02 7.50 10.30 0.19A 0.06aA 0.03bA 0.06bA 0.09abB ± ± ± ± ± 1.12 1.37 0.95 0.99 1.17 0.25A 0.02aB 0.020bA 0.01bA 0.14aB ± ± ± ± ± 1.26 1.47 1.08 0.99 1.50 1.31A 0.04bA 0.06cA 0.08cA 0.27aB ± ± ± ± ± 4.18 5.03 3.13 2.83 5.72 0.30A 0.01bA 0.02aA 0.08bcA 0.10cB ± ± ± ± ± 0.40 0.34 0.88 0.22 0.16 1.06A 0.25bA 0.10aA 0.03aA 0.44aA ± ± ± ± ± 6.26 4.64 6.89 7.14 6.35 3.02B 0.47bB 0.09aB 0.22bA 0.29bB ± ± ± ± ± 0.94B 0.33bA 0.06bB 0.15aA 0.12cB ± ± ± ± ± 3.87 3.82 3.83 5.13 2.68

9.21 7.70 14.03 8.16 6.96

± ± ± ± ± 6.87 8.24 4.13 4.47 10.64 0.48A 0.22aA 0.10bB 0.12bA 0.13aA ± ± ± ± ± 1.25 1.58 0.70 0.93 1.78 0.53A 0.09bA 0.08cB 0.15cA 0.09aA ± ± ± ± ± 1.30 1.68 0.77 0.83 1.92 2.22A 0.69bA 0.20cB 0.39cA 0.40aA ± ± ± ± ± 4.58 5.84 2.50 2.57 7.41 0.11A 0.02aA 0.05aB 0.14aA 0.11aA ± ± ± ± ± 0.39 0.36 0.36 0.31 0.50 0.45B 0.06cB 0.16bcB 0.10bB 0.37aB ± ± ± ± ± 1.65 1.19 1.43 1.74 2.25 4.54A 0.41bA 0.61aA 2.94cA 0.82abA ± ± ± ± ± 14.83 14.30 19.78 8.42 16.83 1.18A 0.12cA 0.68aA 0.70bcA 1.23abA ± ± ± ± ± 5.00 4.03 6.11 4.16 5.70

Green Maizec all varieties HP1001 HP1002 HP 1003 HP1005 Dry Maized all varieties HP1001 HP1002 HP 1003 HP1005

all-trans-β-carotene α-carotene β-cryptoxanthin zeaxanthin lutein

Table 1. Baseline Carotenoid Content (μg/g DW) of Four Genotypes of Biofortified Maize at Two Harvesting Stagesa,b

RESULTS AND DISCUSSION Carotenoid Profile of Biofortified Maize. Green versus Dry Maize. The total carotenoid content, on DW basis, ranged from 19.0 to 36.4 μg/g in the green maize and from 24.4 to 30.8 μg/g in the dry maize among all four genotypes (Table 1); the values in fresh weight are provided in Table 1A of the Supporting Information. As expected, the green maize had a higher (3−33%) total carotenoid content than the dry maize, except for genotype HP1003, which presented a 26% increase in the total carotenoid content in the dry maize. This was mainly due to the average decrease in the zeaxanthin and lutein levels from 14.8 to 9.2 μg/g DW and 5.0 to 3.87 μg/g DW, respectively. On the other hand, β-cryptoxanthin increased from 1.7 to 6.3 μg/g DW between the green and dry maturity stages among all four genotypes. Therefore, the BCE was higher in the dry maize compared to the green maize for most genotypes. The green maize is available during a short season and is consumed roasted or boiled, while the dry maize is consumed throughout the year on a daily basis. Unless specified, the results presented in the following sections refer to the dry maize. Dry Maize. Several carotenoids are present in maize (Table 1). The non-provitamin A carotenoids, lutein and zeaxanthin, are the major carotenoids in all four biofortified genotypes. Among the different genotypes, zeaxanthin and lutein accounted for 35% (28−46%) and 15% (11−20%) of total carotenoids, respectively. Although these carotenoids do not have vitamin A activity, they have an important role in maintaining good eye health and are associated with a reduced risk for age-related macular degeneration (AMD), the leading cause of blindness among elderly people.12−15 β-Cryptoxanthin and all-trans-β-carotene, were the main provitamin A carotenoids in the dry maize, accounting for 24% (19−28%) and 16% (10−23%) of total carotenoids, respectively. Collectively, the two provitamin A carotenoids accounted for 40% (33−49%) of total carotenoids. The amounts of α-carotene were negligible, accounting for only 1.5% (0.1−2.9%) of total carotenoids, while the cis-isomers of β-carotene, 9-cis and 13-cis, accounted for 4.3% and 4.9%, respectively. Overall, the β-carotene equivalents among all four genotypes ranged from 7.5 to 10.3 μg/g DW (7.0−9.5 μg/g FW). Genotype HP1005 (released in Zambia as variety GV665A) had the highest BCE (10.3 μg/g DW). Genotype HP1002 (released in Zambia as variety GV662A) presented BCE of 8.0 μg/g DW, not significantly different from either genotype HP1001 (8.9 μg/g DW) or HP1003 (7.5 μg/g DW). However, the genotypes showed different levels of all-trans-βcarotene and its cis-isomers as reported in the literature.26 It is important to note that high levels of β-cryptoxanthin in the biofortified maize were twice as high as all-trans-β-carotene

variety/genotype

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13-cis-β- carotene

9-cis-β-carotene

β-carotene equivalente

Carotene (Sigma-Aldrich, St. Louis, MO), α-carotene (isolated from freeze-dried carrots), β-cryptoxanthin (CaroteNature, GmbH, Lupsingen, Switzerland), lutein (GNC, Inc., Pittsburgh, PA), and zeaxanthin (GNC, Inc., Pittsburgh, PA) were identified using HPLC-purified standards. The β-carotene equivalents (BCE) were computed as follows: all-trans-β-carotene + 1/2 α-carotene + 1/2 β-cryptoxanthin + 1/2 13-cis-β-carotene + 1/2 9-cis-β-carotene. Statistical Analysis. All data were analyzed using STATA version 13.0 (StataCorp, 2013). Descriptive statistics including mean and SD from duplicate analyses were calculated for the retention of carotenoids from processed maize. Means were compared using Student t tests and one-way ANOVA. Differences were considered significant at P < 0.05.

2.83A 0.57bA 0.38cB 0.61cB 0.25aA

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Article

within the first 15 days of storage. A further 10% reduction within the next 15 days (total 30 days of storage) was observed, followed by a stabilization at 50% retention until 120 days of storage, thereafter declining to 40% retention between 120 and 180 days. The retention of BCE for genotypes HP1001 and HP1003 measured at baseline, 90 and 180 days of storage, followed a similar pattern achieving, respectively, 51.5% and 62.9% retention at 90 days and 40% and 63.1% retention at 180 days of storage. Weber19 reported an average total carotenoid retention of 58% among four inbred lines after 6 months of storage. Other provitamin A biofortified crops (e.g., sorghum, rice) also showed a comparable pattern of 40% degradation of provitamin A within the first few days of storage. Further postharvest research is needed to optimize storage conditions in order to minimize provitamin A degradation during storage, particularly immediately after harvest. A similar better performance of some genotypes was observed by Burt et al.,8 which reported an overall 25−60% decay of provitamin A among six genotypes after drying and four months of dark storage at 25 °C; total carotenoid loss ranged from 15 to 45% and 0 to 50% due to drying and storage, respectively. In this particular study, the researchers were able to categorize the genotypes into three groups, i.e., those with high losses due to drying and low loss due to storage, those with low losses due to drying, but high loss due to storage, and the intermediate genotypes with moderate losses due to both drying and storage. Another study showed a wide range of provitamin A degradation (7−45%) among six maize genotypes within the first four months of storage (unpublished data from Natalia Palacios). Effect of Milling. Milling into Mealie Meal. Maize kernels are usually milled into whole maize meal (named mealie meal), which is a fine flour used for making nshima and porridge. Most of the carotenoids were retained during this process except for β-cryptoxanthin, which showed a consistent 60% retention across all genotypes (Table 3). These findings were contrary to those reported by Pillay et al.,9 where a higher retention of βcryptoxanthin (95.3−113.5%) was observed in milled maize. Different maize genotypes and milling processes could be a source of variation with reported studies. Moreover, Pillay et al.20 reported that β-cryptoxanthin is the most abundant provitamin A carotenoid present in some biofortified varieties, and their range was higher than those reported by Li et al.7 Although lower β-cryptoxanthin retention was constant among genotypes in this study, Li et al.7 reported small significant losses (7−10%) of provitamin A carotenoids after soaking and milling to produce wet-milled flour. It was not surprising to observe degradation because the maize was soaked for 24 h prior to milling. Traditionally, Zambians mill the maize kernels directly, limiting exposure to oxygen and light. Although not significantly different, the β-carotene levels were higher in the mealie meal than in the intact raw maize kernels, which is a reason for figures above 100% BCE retention in some genotypes (HP1003 and HP1005). The higher carotenoid levels in the final milled products than those found in the raw samples could be due to extraction efficiency from processed samples compared with those of raw fresh foods as elaborated by Rodriguez-Amaya and Kimura Rodriguez.21 Overall, the BCE retention ranged from 80 to 110% among all four genotypes with HP1005 being the highest. Milling into Samp. Maize kernels can also be milled into samp after conditioning or tempering (soaking in water) for 5

(HP1002 and HP1003) or at equal levels with HP1001 and HP1005. During the germplasm screening for genetic variation, the sources of high provitamin A were found among maize from temperate regions, which were then bred into varieties suitable for tropical conditions. Moreover, these tropical varieties contain more β-cryptoxanthin and less β-carotene, and the emphasis was to enhance the β-carotene, which provides two vitamin A molecules per carotenoid molecule.2 However, animal studies have shown that β-cryptoxanthin is absorbed and stored in many tissues16 and it maintains the vitamin A status of gerbils as efficiently or better than βcarotene.17 Most of the yellow maize grown and consumed throughout the world has less than 2.0 μg/g DW of provitamin A carotenoids in kernels.2 The first wave of biofortified maize grown in Zambia contained 7.5 to 10.3 μg/g DW BCE and higher levels of β-cryptoxanthin, which is as efficient as βcarotene as a source of vitamin A. Most importantly was that the increase in β-cryptoxanthin and β-carotene did not compromise the levels of lutein and zeaxanthin, which have similar concentrations to those reported for yellow maize accounting for 30−50% of total carotenoids.18 Effect of Storage. At a household level, in Zambia, maize is harvested during the months of April−May, it is usually stored “as harvested” on the ears or shelled into kernels, and stored in bags under shelter for up to eight months at 16−27 °C and relative humidity 40 to 65%.27 These postharvest storage methods are practiced in specific regions of the country. For example, in Nyimba district in the Eastern Province, the majority of households (84%) stored maize on ears, covered with leaves and in a barn, while in Mkushi district in the Central Province, the majority of households (92%) store shelled maize in bags (F. F. De Moura, personal communication). Therefore, both postharvest storage practices were evaluated during storage (kernels and ears) on carotenoid retention over a period of 6 months. Weekly records of humidity and temperature inside the storage bin were taken and are provided in Table 2A of the Supporting Information. In general, the retention of the provitamin A carotenoids for all four genotypes did not differ significantly according to the postharvest storage method in which the maize was stored, either as ears or kernels (data not shown). Therefore, only kernel retention data over a 6-month period are presented herein. Figure 1 shows the retention of BCE in biofortified maize measured in 15-day intervals up to six months for stored ears of genotypes HP1002 and HP1005. All four genotypes followed a similar decay pattern for BCE with a 40% decrease occurring

Figure 1. Effect of storage on BCE retention of maize when stored as kernels for six months under ambient conditions in Zambia for genotypes HP1002 and HP1005 at 15-day intervals. Day 135 did not follow the projected decay line. 6320


raw-B boiled raw-R roasted


HP1002

HP1003

6321

6.74 5.79 4.67 4.09

4.73 6.27 3.58 4.24

5.72 4.68 6.50 5.53

4.11 6.16 3.95 4.60

5.33 5.72 4.68 4.61

0.66aAB 0.21aB 0.58aA 0.11aA 0.37bB 0.00aA 0.06cB 0.10bcB

± ± ± ±

± ± ± ± 0.51a 0.16abA 0.07bcB 0.25cB

0.05cB 0.19aA 0.11cB 0.02bB

± ± ± ±

± ± ± ±

1.12 0.68a 1.22a 0.61a

a

± ± ± ±

lutein

16.14 19.05 17.52 11.86

10.94 16.43 5.89 6.87

20.12 21.26 19.44 15.47

14.00 22.49 14.59 9.56

15.30 19.81 14.36 10.94 0.02bC 1.32aA 0.41bB 1.06cB 0.29aA 0.51aAB 0.75aA 0.11bA 0.54bD 0.13aC 0.18cC 0.36cC

± ± ± ± ± ± ± ± ± ± ± ± 0.12cB 0.47aBC 0.35bA 0.29B

3.58 2.53a 5.55b 3.40b

± ± ± ±

± ± ± ±

ab

zeaxanthin

2.56 4.03 1.95 1.58

1.76 3.92 1.71 2.14

1.29 2.60 1.56 2.17

1.14 1.44 1.24 1.50

1.69 3.00 1.61 1.85

± ± ± ±

± ± ± ±

± ± ± ±

± ± ± ±

± ± ± ±

0.15bA 0.02A 0.09cA 0.08cB

0.08bcB 0.12aA 0.14cAB 0.04bA

0.04dC 0.07aB 0.05cBC 0.06bA

0.03bC 0.05aC 0.04abC 0.11aB

0.59 1.13a 0.28b 0.33b

b

β-cryptoxanthin

0.58 1.07 0.42 0.30

0.43 1.22 0.19 0.21

0.33 0.90 0.40 0.30

0.38 1.69 0.35 0.40

0.43 1.22 0.34 0.30

± ± ± ±

± ± ± ±

± ± ± ±

± ± ± ±

± ± ± ±

0.06aA 0.97aA 0.05aA 0.02aB

0.03bAB 0.02aA 0.01cB 0.02cC

0.04bB 0.04aA 0.03bA 0.00bB

0.10bB 0.03aA 0.02bA 0.02bA

0.11 0.48a 0.10b 0.08b

b

α-carotene

b

7.06 4.91 7.75 3.78

2.91 3.11 2.24 2.41

2.35 2.15 2.66 2.98

5.26 4.12 6.41 6.15

4.39 3.57 4.77 3.83

± ± ± ±

± ± ± ±

± ± ± ±

± ± ± ±

± ± ± ±

0.04bA 0.02cA 0.01a 0.27dB

0.10aC 0.06aC 0.04bC 0.06bC

0.15bD 0.07bD 0.03abC 0.19aC

0.07bB 0.03cB 0.30aB 0.11aA

2.02 1.11a 2.54a 1.53a

a

all-trans-β-carotene

1.91 2.10 1.93 1.66

0.96 1.34 0.70 0.97

0.70 0.88 0.84 1.24

1.75 1.59 1.61 2.60

1.33 1.48 1.27 1.62

± ± ± ±

± ± ± ±

± ± ± ±

± ± ± ±

± ± ± ±

0.12abA 0.04aA 0.09abA 0.11bB

0.10bB 0.00aC 0.04cC 0.08bC

0.03cB 0.01bD 0.01bcC 0.08aC

0.04bA 0.04cB 0.02cB 0.01aA

0.55 0.47a 0.55a 0.66a

a

13-cis-β-carotene

1.67 1.57 1.89 1.61

1.02 1.04 0.84 0.94

0.62 0.63 0.77 1.03

1.77 1.64 1.40 2.29

1.27 1.22 1.22 1.47

± ± ± ±

± ± ± ±

± ± ± ±

± ± ± ±

± ± ± ±

0.02aA 0.13aA 0.01aA 0.29aB

0.08aB 0.03aB 0.08aC 0.02aC

0.02bC 0.02bB 0.06bC 0.10aBC

0.06bA 0.20bA 0.10bB 0.05aA

0.50 0.45a 0.49a 0.59a

a

9-cis-β-carotene

10.42 9.29 10.85 6.35

4.99 6.87 3.95 4.54

3.82 4.65 4.45 5.34

7.78 7.30 8.71 9.54

6.75 7.03 6.99 6.44

± ± ± ±

± ± ± ±

± ± ± ±

± ± ± ±

± ± ± ± a

0.07aA 0.60aA 0.03aA 0.51bB

0.17bC 0.11aB 0.10cC 0.00bC

0.15cD 0.09abC 0.06bcC 0.31aBC

0.04cB 0.13cB 0.32bB 0.15aA

2.74 1.78a 3.10a 2.05a

BCE

58.6

89.2

115

138

120

122

110

93.9

92.1

104

BCE retention rate (%)

Data are means ± SD for three independent experiments of green raw maize and green roasted maize. Different lowercase letters indicate a statistically significant difference among final products (raw-B vs boiled uses “a” and “b”; raw-R vs roasted uses “c” and “d”) within each genotype/variety. Different uppercase letters indicate a statistically significant difference among the genotypes/varieties for the same final product.

a


HP1001

raw-B boiled raw roasted


all varieties

HP 1005

final product

variety/genotype

Table 2. Carotenoid Content (μg/g DW) of Four Genotypes of Biofortified Green Maize in Raw Kernels and Boiled and Roasted Productsa,b

Journal of Agricultural and Food Chemistry Article


raw kernel raw meal nshima porridge raw samp cooked samp





all varieties

HP1001

HP1002

HP1003

HP1005

6322

2.68 3.89 7.59 4.96 3.99 3.00

5.13 6.42 15.09 12.14 5.82 6.26

3.83 4.59 9.27 5.42 3.97 3.78

3.82 4.89 6.62 6.78 3.43 2.87

3.87 4.95 9.64 7.33 4.30 3.98

c

0.94 1.10bc 3.82a 3.21ab 0.98bc 1.60bc 0.33abcB 1.22abcA 0.34abB 1.43aB 0.24bcB 0.12cA 0.06bB 0.11abA 2.90aAB 0.18abB 0.22bB 0.09bA 0.15bA 0.24bA 2.04aA 2.07aA 0.16bA 1.71bA 0.20bC 0.012bA 1.68aAB 0.54abB 0.31bB 0.40bA

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

lutein

0.09aA 0.27aA 9.85aA 0.12aA 0.01aA 1.41aA 0.22bB 0.10bB 2.56aA 3.05aA 0.17bB 0.53bAB 0.29bB 0.02bB 4.50aA 1.74abA 0.53bB 0.40bBC

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6.96 8.42 18.14 12.83 9.91 7.48

8.16 8.50 20.76 18.17 9.21 10.56

14.03 13.10 27.07 18.23 12.22 14.20

6.35 3.87 1.83 0.72 7.08 1.23

7.14 4.01 2.07 1.04 7.46 1.21

6.89 4.23 2.02 0.77 7.77 1.30

4.64 2.66 0.97 0.66 5.29 0.69

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

0.47bcB 1.36abcB 0.38abA 4.14aA 0.00bcC 0.99cC

± ± ± ± ± ±

7.70 9.93 13.56 15.97 6.40 5.12

0.44aA 0.05bA 0.46cA 0.06dA 0.07aA 0.03cdA

0.02aA 0.08bA 0.32cA 0.19dA 0.02aA 0.10dA

0.01aA 0.04bA 0.75cA 0.00cA 0.26aA 0.10cA

0.25bB 0.16cB 0.04dA 0.14dA 0.22aB 0.01dB

1.06 0.70b 0.59c 0.18c 1.03a 0.27c

± ± ± ± ± ±

6.26 3.69 1.72 0.80 6.90 1.11

3.02 2.09b 6.71a 3.12a 2.23b 3.71b

± ± ± ± ± ±

9.21 9.99 19.88 16.30 9.44 9.34

a

β-cryptoxanthin

b

zeaxanthin

0.16 0.43 0.48 0.91 0.30 0.56

0.22 0.20 0.90 1.13 0.17 0.29

0.88 0.16 0.67 0.50 0.21 0.28

0.34 0.23 0.61 0.83 0.31 0.33

0.40 0.25 0.67 0.84 0.25 0.37

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

0.01bC 0.01bA 0.19abA 0.02aA 0.18bA 0.03abA

0.08cBC 0.01cB 0.17abA 0.34aA 0.01cA 0.03bcB

0.02aA 0.01cB 0.24abA 0.03abcA 0.10cA 0.00bcB

0.01aB 0.08aB 0.01aA 0.47aA 0.03aA 0.11aAB

0.30 0.11c 0.21ab 0.33a 0.10c 0.13bc

bc

α-carotene

5.72 7.47 13.47 9.25 6.03 7.42

2.83 4.25 7.32 6.73 3.99 3.89

3.13 3.29 5.43 3.44 2.89 2.53

5.03 5.48 7.36 8.90 5.24 4.12

4.18 5.12 8.40 7.08 4.54 4.49

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

0.27bA 0.02abA 3.48aA 1.37abA 0.611bA 0.699abA

0.08cC 0.01bcC 1.22aA 1.38abA 0.11bcBC 0.12bcB

0.06aC 0.02aD 2.02aA 0.12aA 0.06aC 0.08aB

0.04abB 0.44abB 0.16abA 2.51aA 0.21abAB 0.55bB

1.31 1.68ab 3.61a 2.75ab 1.30b 1.95b

b

all-trans-β-carotene

1.50 1.86 2.23 1.30 1.40 1.70

0.99 1.53 2.11 1.78 1.31 1.09

1.08 1.13 1.42 0.66 0.86 0.74

1.47 1.58 1.55 1.74 1.42 1.07

1.26 1.53 1.83 1.37 1.25 1.15

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

0.14aA 0.02aA 0.62aA 0.11aA 0.156aA 0.085aA

0.01bB 0.02abB 0.39aA 0.45abA 0.05abA 0.02bB

0.02aB 0.02aC 0.56aA 0.02aA 0.01aB 0.09aC

0.02aA 0.14aAB 0.05aA 0.61aA 0.07aA 0.07aB

0.25 0.28ab 0.51a 0.56ab 0.25ab 0.38b

ab

13-cis-β-carotene

1.17 1.55 2.28 1.28 1.41 1.83

0.99 1.31 1.66 1.60 1.49 1.49

0.95 0.83 1.48 0.66 0.75 1.07

1.37 1.44 1.75 1.67 1.39 1.71

1.12 1.28 1.79 1.30 1.26 1.53

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

0.09bAB 0.02abA 0.53aA 0.22bA 0.00983abB 0.177abA

0.06aB 0.01aB 0.42aA 0.01aA 0.01aA 0.14aA

0.02aB 0.01aC 0.56aA 0.02aA 0.01aC 0.08aA

0.06aA 0.08aAB 0.08aA 0.81aA 0.02aB 0.34aA

0.19 0.30ab 0.46a 0.53ab 0.32ab 0.35ab

b

9-cis-β-carotene

10.30 11.32 16.88 11.36 11.12 10.08

7.50 7.77 10.69 9.51 9.20 5.93

8.02 6.47 8.23 4.73 7.68 4.22

8.94 8.44 9.80 11.35 9.44 6.02

8.69 8.50 11.40 9.23 9.36 6.56

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ±

± ± ± ± ± ± ab

0.61aA 0.04aA 4.38aA 1.57aA 0.567aA 0.654aA

0.08abC 0.02abB 1.86aA 1.87abA 0.09abB 0.15bB

0.09aBC 0.01aC 3.08aA 0.10aA 0.24aC 0.21aB

0.21aB 0.58aB 0.25aA 3.52aA 0.36aB 0.80aB

1.16 1.91ab 4.11a 3.32ab 1.33ab 2.34b

BCE

NA 110 164 110 108 97.8

NA 104 143 127 123 79.1

NA 80.6 103 58.9 95.7 52.6

NA 94.3 110 127 106 67.3

NA 97.8 131 106 108 75.5

BCE retention (%)

Data are means ± SD, n = 2 independent analyses. bDifferent lowercase letters indicate a statistically significant difference among final products within each genotype/variety. Different uppercase letters indicate a statistically significant difference among the genotypes/varieties for the same final product. cMoisture content (as percentage of wet weight) of raw maize kernels (12%), raw whole maize meal (12%), raw samp (13%), cooked samp (70%), nshima (75%), and porridge (86%).

a

final productc

variety/genotype

Table 3. Baseline Carotenoid Content (μg/g DW) of Four Genotypes of Biofortified Dry Maize in Raw Kernels and Processed Productsa,b

Journal of Agricultural and Food Chemistry Article



Article

Figure 2. Retention of BCE of traditional Zambian cooked products from four genotypes of biofortified provitamin A maize (HP1001, HP 1002, HP1003, and HP1005) at baseline and after the maize was stored for 90 days at room temperature before cooking. For each cooking method, the different letters among genotypes indicates significant difference (P < 0.05) at baseline (lower case letters) and after 90 days (upper case letters). The error bars indicate standard deviations.

Effect of Cooking. Green Maize. After roasting the green maize for 20 min, the retention of BCE was above 100%, except for genotype HP1005, yielding a 59% BCE retention. This lower retention can be attributed to the reduction of almost 50% in all-trans-β-carotene content of raw green maize (Table 2). The cis-isomers of β-carotene increased after roasting, the 13-cis increased by 65% among genotypes HP1001, HP1002, and HP1003, and the 9-cis increased by 60% in genotype HP1001. Similar retention was obtained after boiling the green maize for 35 min at 95 °C. Genotypes HP1002 and HP1003 obtained

min for ease of bran removal to obtain the dehulled kernels. No significant carotenoid losses were observed after milling into samp, resulting in retention of BCE, in most cases, higher than 100%. Both β-carotene and β-cryptoxanthin were maintained in samp; therefore, yielding a final product with a higher retention range (95.7−154.7%) compared to maize milled into mealie meal (91.6−130.7%), although these values showed no significant difference at (P < 0.05) (Table 3). On the other hand, Pillay et al.,9 reported that maize milled into mealie meal resulted in higher retention of zeaxanthin, β-carotene, and βcryptoxanthin compared with milling into samp. 6323



Article

initial drop, the retention of provitamin A was maintained consistent throughout the six months of storage. Therefore, the 50% of provitamin A degradation assumed in the original calculation of the target levels for provitamin A for maize was somewhat accurate, concluding that the values for retention were satisfactory in ultimately providing adequate amounts of vitamin A through biofortification. In addition, we recognize the need for upstream research identifying genotypes with higher provitamin A retention during storage in order to achieve the maximum benefit of biofortified maize. Further research could also identify the mechanisms that make one genotype more resistant to provitamin A degradation than another. An effort to optimize postharvest storage methods is also recognized in order to minimize provitamin A degradation, particularly soon after harvest where the decay pattern for BCE is in excess of 40-50% in the first 15 days. More improvements and optimization of cooking methods could also increase retention of carotenoids in provitamin A biofortified maize for the benefit of populations who consume maize in the rural settings in Africa. Overall, these results are very encouraging for the biofortification program in Zambia.

100% of the BCE retention and 90% of the BCE retention for genotypes HP1001 and HP1005 (Table 2). HP1005 lost almost 50% of the all-trans-β-carotene after boiling. No consistent trend was observed for the cis-isomers after boiling. Muzhingi et al.22 also reported similar results on retention levels above 100% for all individual carotenoids, except for βcryptoxanthin (92%), for yellow maize boiled for 1 h at 100 °C. Dry Maize. The retention of individual carotenoids and BCE of all three cooked products, nshima, porridge, and cooked samp, are shown in Table 3. Nshima, by far the most popular maize dish consumed in Zambia, showed high BCE retention levels (103−164%) despite the lower retention (21−29%) of βcryptoxanthin. Lutein and zeaxanthin were also significantly higher in nshima than raw maize. Porridge, which is consumed mostly by children at breakfast, likewise had high BCE retention (110−127%) except for genotype HP1002 (59%). The lower BCE retention observed in genotype HP1002 can be attributed to the drastic drop in the levels of β-cryptoxanthin from 6.9 to 0.8 μg/g DW. Similar results of high retention (>100%) of individual carotenoids were reported by Muzhingi et al.22 for cooked products of yellow maize for “sadza”, which is Zimbabwe’s version of nshima, as well as porridge.23−25 In particular in porridge, but not in sadza, the β-cryptoxanthin was significantly lower than in the raw kernels while the all-trans-βcarotene was constant in both products. Additionally, the muffins, which were baked for 25 min at 232 °C, showed significantly lower retention for all individual carotenoids, including 25% and 51% retention for all-trans-β-carotene and βcryptoxanthin, respectively. Lastly, the cooked samp in the current study showed higher BCE retention when the maize was milled into samp (96−155%), although the combination of high temperature with prolonged cooking (3 h) resulted in a lower BCE retention range (53−98%) among the four genotypes. Retention of β-Carotene Equivalents (BCE) among All Cooked Products. In this study, the BCE retention of cooked products from just harvested maize was compared with those of cooked products from maize stored for 90 and 180 days. The BCE retention did not differ significantly between the cooked products made from maize stored either at 90 or 180 days. Therefore, Figure 2 shows the BCE retention of different cooked products of maize that have just been harvested, including green and dry maize, and cooked products made from dry maize that had been stored for 90 days. On average, the genotypes, HP1001 and HP1003, showed high BCE retention among the different maize cooked products, except for a lower BCE retention in cooked samp as discussed in the previous section. Furthermore, genotype HP1002, besides lower BCE retention for samp, had 60% retention in cooked porridge. Interestingly, genotype HP1005 was the only type that showed a 60% BCE retention for roasted product, while the other three genotypes had BCE retention higher than 100%; however, it had the highest BCE retention, almost 100% when cooked as samp. The BCE retention of the cooked product was not affected by the maize being stored for 90 days, although the carotenoid levels decreased substantially (see the previous section on the Effects of Storage). In summary, the carotenoids in the provitamin A biofortified maize showed virtually no degradation when the maize was cooked into nshima and porridge, for most of the maize genotypes. When the maize was cooked into samp, the degradation was more significant but still showed a wide range among genotypes from 50% to 100% retention. After an

■

ASSOCIATED CONTENT

S Supporting Information *

Baseline carotenoid content (μg/g FW) of four genotypes of biofortified maize at two harvesting stages. Weekly humidity and temperature readings. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Phone: 1-202-862-5693. Fax: 1-202-467-4439. E-mail: f. [email protected]. Funding

Funding was provided by HarvestPlus (www.HarvestPlus.org), a global alliance of agriculture and nutrition research institutions working to increase the micronutrient density of staple food crops through biofortification. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We are greatly thankful to Eliab Simpungwe for his assistance and support. ABBREVIATIONS USED BCE, β-carotene equivalents = [all-trans-β-carotene + 1/2 αcarotene +1/2 β-cryptoxanthin +1/2 13-cis-β-carotene +1/2 9cis-β-carotene]; DW, dry weight basis; FW, fresh weight basis

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REFERENCES

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