PAGE 1 Telephone: +57 (2) 445 0000 Fax: +57 (2) 445...

Centro Internacional de Agricultura Tropical (CIAT) Kilómetro 17 Recta Cali – Palmira, Palmira, Valle del Cauca, Colombia Telephone: +57 (2) 445 0000 Fax: +57 (2) 445 0073

Laboratory Enhancements to Improve Human Nutrition

CIAT Progress Report to Monsanto Fund In December 2007, the Monsanto Fund approved a US$281,000 grant to the Centro Internacional de Agricultura Tropical (CIAT) based in Colombia to carry out laboratory enhancements to improve human nutrition. These funds have been used in the course of 2008 to purchase equipment for the newly formed Nutrition Quality Laboratory at CIAT. This document summarizes the advances the Laboratory has had from January 2009 to July 2009 and corresponds to the semi-annual progress report requested by the Monsanto Fund. As noted in the Laboratory’s 2008 Annual Report, 2009 has been dedicated to five major tasks:

1. Complete protocols and procedures 2. Analyze samples 3. Implement quality-control activities 4. Prepare articles 5. Develop proposals

Advances on these and other tasks are noted below. Complete protocols and procedures Three manuals have been drafted in the reporting period: Analytical Methods, General, and Quality. For beans, maize and rice, a homogeneous sample has been developed that will be used in all analyses as a quality-control measure. The Cost Analysis of Research and Breeding Operations (CARBO) methodology has been completed and prices set for Laboratory services. Analyze samples (Annex 1) A total of 3923 samples were received during the reporting period; 3895 (99%) of these have been analyzed (Annex 1). During the reporting period, several projects were begun, continued or completed (Annex 2). Implement quality-control activities As part of the Laboratory’s efforts to obtain certification by ISO, a quality-control system is being implemented under the leadership of Dayron Gutíerrez. New quality-control measures (such as use of the control card) have been implemented during the reporting period for the in vitro iron bioavailability method. With respect to inter-laboratory trials, contacts have been made with several international laboratories that will analyze the same food sample in the second semester of 2009 for tryptophan quantification, in vitro protein digestibility, in vitro iron dialyzability, in vitro carotenoid bioaccessibility, and carotenoid quantification. Prepare articles


The Laboratory’s first peer-reviewed scientific article was published (Annex 3). Several other manuscripts are in preparation (Annex 2). Develop proposals In response to Colciencia’s (Scientific Ministry of the Colombian government) call for concept notes in health, three notes were prepared, of which one was selected to compete in the next round, as a full proposal.

Evaluation of the carotenoid bioaccessibility of the main vitamin A dietary sources in Colombia’s Atlantic coast, for the purpose of improving pre-schooler’s vitamin A status. Colciencias code PRE00485021329. Selected for next round. Evaluation of the antioxidant contribution of fruits and vegetables in 5 regions of Colombia, and their promotion to address cardiovascular diseases. Colciencias code PRE00485021403. Evaluating NIRS technology to predict zinc concentration in beans. Colciencias code PRE00485021555.

Develop Laboratory brochures (Annexes 4 and 5) New Laboratory brochures were developed in English and in Spanish. Hire Laboratory personnel Chemist María Luisa Cortés joined the Laboratory in a part-time capacity in 2009. Effective 1 July 2009, Chemist Ingrid Aragón will join the Laboratory as a full-time staff person. During the January-July 2009 period, three students completed their internship or thesis activities in the Laboratory (Annex 6). Receive or impart trainings During the reporting period, Dayron Gutiérrez initiated his ISO/IEC 17025 52-hour training course in Certification Auditing. María Luisa Cortés trained Paulo Izquierdo, from the Andean Bean Program at CIAT, and Katherine Loaiza from CIAT’s Rice Breeding Program, on using NIRS. Offer presentations (Annex 7) An oral presentation and two poster presentations were given in Colombia during the reporting period. Further, several abstracts were submitted to upcoming conferences; confirmation of the acceptance of these abstracts has not been confirmed. The titles of these abstracts are listed below.


EVALUATION OF CAROTENOID BIOACCESIBILITY IN BEAN, CASSAVA, SWEET POTATO AND ALFALFA FOLIAR EXTRACTS. Ortiz D, Pico S, Pachón H, Chitchumroonchokchai C, Failla M. EVALUACIÓN DEL VALOR NUTRICIONAL DE DIFERENTES EXTRACTOS FOLIARES. Pico S, Gutiérrez D, Aragón I, Escobar A, Ortiz D, Sánchez T, Imbachí P, Pachón H.

EVALUACIÓN DE LA CALIDAD PROTEICA DE RECETAS TÍPICAS DEL DEPARTAMENTO DEL CAUCA - COLOMBIA ELABORADAS CON MAÍZ DE ALTA CALIDAD PROTEICA Y CON MAÍZ COMÚN. Imbachí P, Gutiérrez D, Ortiz D, Pachón H. VALIDACIÓN DE UN MÉTODO DE DIGESTIÓN DE HIERRO IN VITRO PARA LA EVALUACIÓN DE LA BIODISPONIBILIDAD DE HIERRO EN ALIMENTOS. Aragón I, Ortiz D, Pachón H. EFECTO DEL TRATAMIENTO DE FERMENTACION CON UNA MEZCLA DE MIELES EN LA CALIDAD PROTEÍCA DE ALIMENTOS CÁRNICOS Y LÁCTEOS. Gutiérrez D, Ortiz D, Pachón H.

Obtain complementary funding for Laboratory CIAT’s Tropical Fruits Program provided complementary funding to purchase inputs such as an HPLC column and reagents to establish in the Laboratory a method to quantify sugars (glucose, fructose, sacarose) and organic acids (malic acid, citric acid, oxalic acid). Host 86 visitors (Annex 8) Eighty six people visited the Nutrition Quality Laboratory between December 2008 and June 2009, as follows:

• December 15: CIAT’s Board of Directors (7 people) • December 19: Monsanto’s Head of Research and Development (1 person) • February 20: Centro de Investigaciones, Escuela de Nutrición y Dietética,

Universidad de Antioquia (2 people) • March 12: Caldono (Cauca Department) community members, Municipal Ministries of

Education and Agriculture, representatives of the Department’s PANES program (50 people)

• April 28: Instituto Colombiano de Bienestar Familiar from Cauca Department (7 people)

• May 8: Representatives from ADA Afro-Latino Development Alliance (5 people) • May 14: Delegation from the Nariño Governor’s office (8 people) • June 3: CGIAR Executive Committee (6 people)


Annex 1. Samples received in the Laboratory during the reporting period.

Institution Number of

Samples

Sample Type

Analyses Requested Analyses Completed

AgroSalud, Universidad del Cauca

360 Maize − Tryptophan quantification

− In vitro protein digestibility

− Soluble-protein quantification

− Tryptophan quantification



CIAT Cassava Improvement Program

1982 Cassava − Carotenoid quantification

− Determination of antioxidant activity

− Protein by NIRS

− Carotenoid quantification

− Determination of antioxidant activity

AgroSalud, Colombia 6 Maize − In vitro iron

dialyzability − In vitro iron

dialyzability AgroSalud, Universidad Industrial de Santander

12 Foliar extract

− In vitro iron dialyzability


AgroSalud, Empresa PROPOMIEL

57 Meat, dairy

products


− Soluble protein quantification


− Soluble protein quantification

AgroSalud, Clayuca 36 Maize − Tryptophan quantification






CIAT Biotechnology 24 Cassava − Carotenoid quantification

− Carotenoid quantification

AgroSalud, Cauca Project 75 Beans Maize



AgroSalud, Effect of cooking on iron bioavailability

36 Rice − In vitro iron dialyzability


CIAT Bean Program 1335 Beans − Iron quantification − Iron quantification Total 3923 3895


Annex 2. Research projects carried out in 2009. Project Responsible Collaborators Status Validation of an in vitro iron dialyzability method

Ingrid Aragón Dayron Gutiérrez, Darwin Ortiz, Universidad del Valle, AgroSalud

Completed

Comparison of an in vitro iron dialyzability method with in vivo iron bioavailability methods

Ingrid Aragón Dayron Gutiérrez, Darwin Ortiz, Universidad del Valle, AgroSalud

Thesis completed; manuscript in preparation

Evaluation of the nutritional quality of leaf extracts prepared from the foliage of different biofortified crops

Sayda Pico Darwin Ortiz, Ingrid Aragón, Dayron Gutiérrez, María Alejandra Lozano

Completed; manuscript in preparation

Protein-quality evaluation of different Colombian recipes prepared with biofortified maize

Paola Imbachí Dayron Gutiérrez, CIAT’s Cassava Program, CLAYUCA

Thesis completed; manuscript in preparation

The quantification of phytates via HPLC of a population of common beans (Phaseolus vulgaris) and identification of QTLs associated with them

María Alejandra Lozano

CIAT’s Bean Program, Universidad del Valle, AgroSalud

Thesis completed

Protein digestibility of meat and milk products processed with or without sugar-cane honey as a preservative

Dayron Gutíerrez AgroSalud, Empresa PROPOMIEL

Completed; manuscript in preparation

Evaluation of the anti-oxidant capacity of cassava

Hiroko Kunori CIAT’s Cassava Program In process

Developing NIRS equations to evaluate minerals in beans

María Luisa Cortés CIAT’s Bean Program, CIP In process; manuscript in preparation

Evaluation of the phytate concentration of rice lines

Dayron Gutíerrez, Darwin Ortiz

CIAT’s Rice Program Initiated, in process

Evaluation of the in vitro iron bioavailability of rice lines

Ingrid Aragón CIAT’s Rice Program Initiated, in process

NIRS analysis of 600 F1 cassava clones to develop calibration curves for crude protein

Darwin Ortiz CIAT’s Cassava Program Initiated, in process

NIRS evaluation of the carotenoid concentration of 80 high-carotenoid cassava clones


NIRS carotenoid-concentration evaluation of 350 F1 yellow-fleshed cassava clones


Annex 3. Laboratory article published in the first semester of 2009.

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JFS H: Health, Nutrition, and Food

Iron, Zinc, and Protein Bioavailability ProxyMeasures of Meals Prepared with NutritionallyEnhanced Beans and MaizeH. PACHON, D.A. ORTIZ, C. ARAUJO, M.W. BLAIR, AND J. RESTREPO

ABSTRACT: Nutritionally enhanced beans (NEB) with more Fe and Zn than conventional beans (CB) and nutri-tionally enhanced maize (NEM) with more tryptophan and lysine than conventional maize (CM) were developed aspart of a crop-biofortification strategy to improve human nutrition. Proxy measures were used to assess Fe and Znbioavailability and protein digestibility of a bean recipe (frıjol sancochado) and a maize–milk recipe (mazamorra)prepared with enhanced or conventional crops in Colombia. Fe concentration was similar in the cooked NEB andCB and in NEM and CM (P ≥ 0.05); in vitro Fe dialyzability was similar in cooked NEB (9.52%) and CB (9.72%) andgreater for NEM (37.01%) than CM (32.24%). Zn concentration was higher in the uncooked and cooked NEB than inthe CB (P < 0.05); phytate:Zn molar ratios were high in cooked NEB (36:1) and CB (47:1), suggesting low Zn bioavail-ability, and not different from each other (P = 0.07). There were no differences in Zn concentration or phytate:Znmolar ratio in the maize recipes. Nitrogen, tryptophan, and lysine concentrations were higher in the cooked NEMthan CM; nitrogen was higher in the cooked NEB than CB (P < 0.05). In vitro protein digestibility was comparable(82% to 83%) for NEM and CM and higher for NEB (84%) than for CB (82%). The higher nutrient concentrations+ similar bioavailability (protein in NEM, Zn in NEB), same nutrient concentrations + higher bioavailability (Fe inNEM) or higher nutrient concentrations + higher bioavailability (protein in NEB) can translate into more nutrientsabsorbed and utilized by the body.

Keywords: beans, bioavailability, biofortification, maize, nutrients

Introduction

Food-based approaches for addressing nutrient deficiencies in-clude food fortification, dietary diversity, and more recently,

crop biofortification. With biofortification, the nutrient levels ofstaple crops are naturally increased through conventional plantbreeding and modern biotechnology (Nestel and others 2006). Toachieve biofortified crops, high-nutrient plants are crossed withcommercially successful, locally important, and/or agronomicallysuperior plants. Through a succession of crosses that are closelymonitored by plant breeders, progeny are selected which maintainthe desirable characteristics of the parent plants, such as high nu-trient levels and agronomically favorable traits. The Intl. Center forMaize and Wheat Improvement (CIMMYT) in Mexico has followedthis path to develop maize with twice the levels of tryptophan andlysine found in conventional maize; this maize is known as qual-ity protein maize (QPM) or, its predecessor, opaque-2 (Krivanekand others 2007). The Intl. Center for Tropical Agriculture (CIAT)in Colombia has also used conventional plant breeding to developbeans with elevated iron and zinc levels in comparison with con-ventional beans (Blair and others 2009a).

Biofortified crops are those with higher nutrient levels andproven efficacy in improving human nutrition. The QPM used in

MS 20080957 Submitted 11/28/2008, Accepted 3/21/2009. Authors Pachon,Ortiz, and Blair are with Centro Internacional de Agricultura Tropical, AA6713, Cali, Colombia. Author Araujo is with Grupo de Nutricion, Univ. delValle, Calle 4B No 36-00, Cali, Colombia. Author Restrepo is with Fun-dacion para la Investigacion y Desarrollo Agrıcola, AA 6713, Cali, Colom-bia. Direct inquiries to author Pachon (E-mail: [email protected]).

this study meets this criteria; opaque-2 or QPM has been shownto improve the protein status of severely malnourished children orchildren recuperating from severe malnutrition, either comparedwith conventional maize (Graham and others 1989; Morales andGraham 1993), or compared with casein (Morales and Graham1993), or skim milk (Reddy and Gupta 1974). Further, a meta-analysis of 8 efficacy trials carried out with preschool children inLatin America or Africa estimated an 8% and 9% improvement inchildren’s height and weight, respectively, during the interventionperiod when they consumed QPM compared with conventionalmaize (Gunaratna 2007). The higher-mineral beans have not beenevaluated for their efficacy in improving human nutrition but haveshown 25 mg/kg and 10 mg/kg increments in iron and zinc concen-tration, respectively, over conventional beans (MW Blair, unpub-lished data). In this manuscript, the QPM and higher-mineral beanswill be referred to as nutritionally enhanced maize and beans,respectively.

The efficacy of the combination of these nutritionally enhancedcrops in improving the nutritional status of preschool childrenwas tested in Colombian daycare centers (Blair 2007). A substudywas carried out to evaluate nutrient bioavailability in meals pre-pared in the daycare centers with nutritionally enhanced or con-ventional beans and maize. The purpose was to explore if therewere differences in nutrient concentrations and bioavailability inthe meals served to the study children, which could explain theimpact of the crops on the children’s nutritional status. Prox-ies were used for bioavailability of zinc (phytate:zinc molar ra-tio), iron (in vitro iron dialyzability), and protein (in vitro proteindigestibility).

C© 2009 Institute of Food Technologists R© Vol. 74, Nr. 5, 2009—JOURNAL OF FOOD SCIENCE H147doi: 10.1111/j.1750-3841.2009.01181.xFurther reproduction without permission is prohibited

H:Health,Nutrition,&Food

Bioavailability of bean and maize meals . . .

Materials and Methods

Study contextThis study took place in the context of a larger efficacy study

whose objective was to evaluate the nutritional impact of nutrition-ally enhanced beans and maize on preschool children aged 2 to 5 y(Blair 2007). A total of 8 daycare centers from socioeconomic strata1 and 2 (where 1 is the lowest and 6 is the highest) in a large Colom-bian city were randomly assigned to receive for 6 mo high-mineralbeans and quality protein maize (n = 2), conventional beans andmaize (n = 3), or an iron supplement providing 10 mg of iron(n = 3) (Figure 1). The beans and maize were produced and pro-vided by the study team and distributed monthly to the centers;other ingredients for the meals prepared at the center were pur-chased with government-provided funds (through the InstitutoColombiano de Bienestar Familiar) or with private funds. The cen-ters receiving beans and maize prepared bean and maize meals orsnacks 2 times per week. The centers receiving the supplement pro-vided the iron to the children 1 time per week.

Beans and maizeThe beans (Phaseolus vulgaris) and maize (Zea mays) used in the

study were developed by CIAT and CIMMYT, respectively, and weremultiplied for the efficacy study by the Fundacion para la Investi-gacion y Desarrollo Agrıcola (FIDAR). The nutritionally enhancedbeans provided during the time the meals were sampled wereprimarily NUA35 with some NUA45 (Table 1). Previous analyses

Daycare centers selected (n=8)

Nutritionally enhanced beans and maize (n=2)

Conventional beans and maize (n=3)

Fe-only supplement (n=3)

Cooked fríjoles sancochados bean recipe and mazamorra maize recipe

Obtained 2 75-g samples of each recipe at 2 times

Randomly assigned

Frozen at -80ºC, lyophilized for 4 d and stored at room temperature until analyzed

Nutrient bioavailability proxy measure: in vitro protein digestibility, in vitro iron dialyzability, phytate:zinc molar ratio

Nutrient determination: protein, tryptophan, lysine, iron, zinc, phytate

Figure 1 --- Study design.

Table 1 --- Uncooked bean and maize sample characteristics.

Mean (SEM)A

Fe (mg/kg), Zn (mg/kg), N (g/kg), Tryptophan LysineSampleB Name n = 3 n = 3 n = 3 (% total protein), n = 1 (% total protein), n = 1

BeansNutritionally enhanced NUA35 62.75 (0.127)a 28.74 (0.430)b 30.31 (0.726)a 0.202 NAC

NUA45 64.75 (1.947)a 23.99 (0.492)a 30.16 (1.365)a 0.203 NAConventional CAL96 57.12 (7.036)a 21.32 (1.086)a 31.3 (0.700)a 0.208 NA

MaizeNutritionally enhanced CML491 11.50 (0.388)a 14.89 (0.640)a 14.89 (0.868)a 0.084 0.366Conventional DK777 15.74 (0.788)b 22.82 (0.963)b 14.97 (0.149)a 0.054 0.254

AFor each crop and nutrient, values with no letters in common are statistically significantly different (P < 0.05). For beans, NUA35 and NUA45 were each comparedusing Student’s t-test to CAL96. No statistical tests were run for tryptophan and lysine as there was only 1 value per crop type.BThe beans were grown in FIDAR and CIAT fields in 5 sites in Colombia and the maize was grown in FIDAR fields in Palmira, Colombia.CNA = not analyzed.

suggested that these beans had mean iron concentrations of77.7 mg/kg (NUA35) and 73.7 mg/kg (NUA45) and mean zincconcentrations of 33.2 mg/kg (NUA35) and 28.7 mg/kg (NUA45)(Carolina Astudillo, CIAT, personal communication), while the con-ventional beans were CAL96 which had been characterized ashaving 60.4 and 30.9 mg/kg mean iron and zinc, respectively (Car-olina Astudillo, CIAT, personal communication). The nutrition-ally enhanced quality protein maize CML491 was selected for itshigher tryptophan (0.092%) and lysine (0.421%) content than con-ventional maize DK777 (tryptophan 0.054%, lysine 0.254%) (JoseRestrepo, unpublished observations). Beans and maize were har-vested by FIDAR, dried to 13% and 14% humidity for maize andbeans, respectively, sorted in 1 kg batches, packaged in polypropy-lene bags at the start of the efficacy trial and subsequently in paperbags for maize alone, labeled, and delivered to the Univ. del Vallewhich then distributed the foods to the corresponding daycarecenters.

Meals prepared with beans and maizeAll daycare centers receiving beans and maize were provided

with a recipe book and training to standardize preparation of mealswith these foods. For 2 of the centers, meals and snacks were pre-pared, on a rotating basis, by mothers of children attending thedaycare. For 3 of the centers, food preparation was done by cook-ing staff. Both of these groups will be referred to as cooking staff.Daycare centers often had to make adjustments to the standard-ized recipes given limitations in the kitchen facilities (for example,

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availability of blender). For this analysis, 2 relatively simple prepa-rations were selected, which were considered to require the leastamount of modifications by the cooking staff: frıjoles sancocha-dos (a bean stew) and mazamorra (a maize–milk combination)(Table 2). These, as well as the other bean and maize recipes pre-pared by the cooking staff, were prepared at most 4 times permonth, to avoid monotony and rejection of these foods by thechildren.

Meal sampling at daycare centersThe study was designed to collect, on 2 separate occasions, bean

and maize meal samples from the 5 centers providing these mealsto the children (Figure 1). At each sampling point, two 75 g sam-ples were obtained as follows. In the pots originally used to cookthe bean and maize recipes, the cooked meals were stirred thor-oughly by the cooking staff. The cooking staff served 2 portions ofthe meal in 2 separate acid-washed plastic containers (with 80 mLcapacity). Staff were asked what ingredients they used in the recipe;these were noted. Samples were refrigerated on ice, transported tothe Univ. del Valle, and frozen at −80 ◦C until transported on dry iceto CIAT for analyses.

Sample preparationAt CIAT, samples were maintained at −80 ◦C in their plastic con-

tainers. Samples were divided in 2 using a stainless steel knife. Halfof each sample was lyophilized (Labconco Corp., Kansas City, Mo.,U.S.A.) over 4 d and then ground to a homogenous flour with alocally produced zirconium-ball mill to avoid contamination withminerals. Two aliquots of each sample were used in subsequentanalyses. All chemicals and enzymes were purchased from SigmaChemical Co. (St. Louis, Mo., U.S.A.), unless otherwise stated and allwater used was 18M� (Synergy, Millipore SAS, Molshein, France).

In vitro iron dialyzability of cooked bean recipesDialyzable iron was measured using the method by Argyri and

others (2009), which is an adaptation of the method developed byKapsokefalou and Miller (1991). In vitro iron dialyzability meth-ods are highly correlated with in vivo iron bioavailability measures(r ≥ 0.92) and are considered appropriate for screening purposes(Sandberg 2005). In the adapted method, 1 g of the cooked recipeswas dissolved in 10 mL 18M� water and the pH adjusted to 2.8with 6 M HCl; 2 mL aliquots were transferred to 6-well plates(Corning Inc., Corning N.Y., U.S.A.). 1 mL of a pepsin solution (4g porcine pepsin suspended in 0.1 M HCl) was added to each well.Covered plates were placed in a 65 RPM reciprocal shaking waterbath (Thermo Fisher Scientific, Marietta, Ohio., U.S.A.) at 37 ◦C for2 h. Plates were removed from the water bath and dialysis mem-

Table 2 --- Ingredients in standard bean and maize recipes.

Bean recipe: Frıjoles Maize recipe:Sancochados Mazamorra

Ingredient Quantity Ingredient Quantity

Beans 888 g Maize 500 gCarrot 80 g Water 1200 mLWhite onion 120 g Whole milk 880 mLPumpkin 80 g Sugar or panelaa 60 gOil 10 gTomato 180 gScallion and 80 and 120 g

tomato pasteSalt, pepper, To taste

and cuminaSugar-cane juice that after repeated boiling solidifies; used as a sweetener.

brane (Spectrum Laboratories, Rancho Dominguez Calif., U.S.A.) of6000 to 8000 molecular weight cut-off was secured to an insert ringplaced over each well, allowing the membrane to have contact withthe well contents. A total of 2 mL of pH 6.3 PIPES solution (0.15 MPIPES adjusted to pH 6.3 using concentrated HCl) were added ontop of each insert, gradually diffusing through the membrane, andadjusting the pH of samples to 6.3. After 30 min in the 37 ◦C shak-ing water bath, the inserts were temporarily lifted to add 0.5 mLof a pancreatin-bile solution (0.2 g porcine pancreatin and 1.2 gbile extract suspended in 100 mL 0.1 M NaHCO3) to each well. In-serts were placed over the wells again and the plates were put in the37 ◦C shaking water bath for 2 h. Plates were removed from the wa-ter bath, inserts were removed, dialysates centrifuged (EppendorfAG, Hamburg, Germany) at 10000 × g for 20 min, and supernatantsplaced in 15 mL tubes.

To prepare the samples for iron concentration analysis, 0.25 mLreducing protein precipitant solution (100 g trichloroacetic acid,50 g hydroxylamine monohydrochloride, and 100 mL concentratedHCl taken up to 1 L of solution with 18 M� water) were added to0.5 mL of the supernatant. After overnight storage at room temper-ature, the samples were centrifuged at 5000 × g for 10 min, and0.1 mL aliquots of the supernatant were transferred to a 96-wellplate (Corning Inc.). A total of 0.225 mL of a ferrozine solution (1part ferrozine solution 5 mg/mL and 8 parts HEPES buffer 0.3 M,pH 7.5) was added to each well. After 1 h, absorbances were readin a spectrophotometer (μQuant, Biotek Instrument, Winooski, Vt.,U.S.A.) at 562 nm. Iron concentration was calculated from a stan-dard curve generated with FeCl3 standards.

Results were expressed as percent dialyzable iron:

Total [Fe] in dialysate (mg/mL) × Total volume dialysate (mL)Total Fe in food sample (mg)

× 100

The iron concentration of the undigested food sample was de-termined as described subsequently; this value was multiplied bythe weight (expressed in kilograms) of the bean or maize sampleto generate the denominator in the equation. The numerator wascalculated from 10 replicates per sample, the denominator was cal-culated from 1 replicate per sample.

In vitro protein digestibility of cooked maize recipesThe method of Hsu and others (1977), modified by McDonough

and others (1990), was used. This method yields data that are highlycorrelated (r = 0.90) with in vivo results in rats (Hsu and others1977). Briefly, samples or a casein-sodium salt from bovine milkcontaining 10 mg of N were dissolved in 2.5 mL of water. To this,2.5 mL NaOH 0.2N were added. The solution was incubated for30 min in a 37 ◦C 65 RPM shaking water bath. Then 5 mL HCl 0.075Nwere added and the pH adjusted to 8. A total of 2 mL of a multien-zyme solution (4 mg trypsin, 4.48 mg chymotrypsin, 1.02 mg pep-tidase) were added. The pH was monitored for 10 min and the per-cent digestibility was calculated using the formula:

% digestibility = 210.46 − 18.10X, where X is the pH at 10 min .

4 replicates were run for each cooked recipe.

Nutrient determinationsThe iron and zinc concentrations (mg/kg) of uncooked maize

and beans and cooked maize and bean recipes were determined in2 replicates using atomic absorption spectrophotometry (Benton-Jones and others 1991). After acid digestion of the samples, nitro-gen (g/kg) was determined colorimetrically (Skalar Analytical BV1995). Colorimetric methods were also used to measure tryptophan

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(Villegas and others 1992 as modified by Nurit and others 2008)and lysine (Tsai and others 1972); these were determined in du-plicate and expressed as percent of total protein. Total phytateconcentration (mg/100 g) was determined colorimetrically by anadaptation (Blair and others 2009b) of standard methods (Xu andothers 1992; Burbano and others 1995). The intention was not todiscriminate among inositol phosphates (IPs), but rather to quan-tify total phytate concentration. For this purpose, the use of a col-orimetric method is appropriate.

Phytate:zinc molar ratioThe phytate:zinc molar ratio was calculated as follows (IZiNCG

2004):

Phytate concentration (mg/100g)/660Zinc concentration (mg/100g)/65.4

This molar ratio is considered a proxy zinc bioavailability mea-sure by several international organizations (WHO/FAO/IAEA 1996).Other researchers have used the molecular weight of IP6 (660) toestimate the molar ratio of total phytates to zinc for maize be-cause maize is composed of approximately 95% IP6 (Hambidge andothers 2004). Similarly, IP6 constitutes approximately 96% of the IPsin common bean (Alonso and others 2001). Therefore, it is appro-priate to use 660 as the molecular weight for total phytates for thesecrops because IP6 is the main IP.

Statistical analysesStatistical analyses were completed with Stata version 9 (Stat-

aCorp, Tex., U.S.A.). All values were log-transformed to bet-ter approximate a normal distribution and Student’s t-test wasperformed between the recipes prepared with nutritionally en-hanced and conventional crops. Means were considered to be sta-tistically significantly different if P < 0.05.

Results

Meal samplesFor both daycare centers in the nutritionally enhanced group,

bean and maize meal samples were obtained at 2 time points, asplanned (Table 3). For the 3 daycare centers in the conventionalcrops group, samples were obtained at 1 time point for all 3, and2nd samples were obtained for only 1 of the centers. Deviationsfrom the standard recipes, based on cooking staff’s report of whatingredients were used in the recipes, are summarized in Table 4.Staff added a variety of ingredients (n = 10) to the bean meal ascompared to the standardized recipe, omitting up to 3 ingredientsin the bean recipe (carrot, pumpkin, onion), adding up to 2 ingredi-ents to the maize recipe (sodium bicarbonate and cinnamon) andomitting no ingredients in the maize recipe.

Table 3 --- Daycares sampled.

Nutritionally Conventional TotalCrop Month enhanced (n) (n) (n)

Beans November 2006 2 3 5Beans December 2006 2 1 3Beans February 2007 0 0 0Maize November 2006 2 3 5Maize December 2006 1 1 2Maize February 2007 1 0 1

Nutrient profile of uncooked beans and maizeThe iron, zinc, nitrogen, tryptophan, and lysine profile of the

uncooked nutritionally enhanced beans and maize are listed inTable 1. The mean iron value for the conventional beans(57.1 mg/kg) was lower than the nutritionally enhanced beans(62.8 mg/kg for NUA35 and 64.8 mg/kg for NUA45); but thisdifference was not statistically significantly different (P ≥ 0.05).Mean zinc was higher for NUA35 (28.7 mg/kg) than for CAL96(21.3 mg/kg) (P < 0.05); there were no differences in zinc valuesbetween NUA45 (24 mg/kg) and CAL96 (P ≥ 0.05). Mean nitrogen(approximately 30 to 31 g/kg) values were comparable among the3 bean types (P ≥ 0.05); tryptophan values (approximately 0.20%)were similar among the bean types.

For maize, the nutritionally enhanced CML491 had lower meaniron (11.5 mg/kg) and zinc (14.9 mg/kg) values than the conven-tional maize (15.7 and 22.8 mg/kg, respectively) (P < 0.05). Nitro-gen levels were comparable in both maize types (approximately15 g/kg) (P ≥ 0.05) while tryptophan and lysine levels were higher inCML491 (0.084% and 0.366%, respectively) than in DK777 (0.054%and 0.254%, respectively) (P < 0.05).

Nutrient profile of cooked bean and maize recipesThe iron (approximately 45 mg/kg) and phytate concentration

(approximately 900 mg/100 g) in the cooked recipes prepared withnutritionally enhanced and conventional beans were statisticallycomparable (P ≥ 0.05); the nitrogen and zinc concentrations werehigher (P < 0.01) in the recipes prepared with nutritionally en-hanced beans as compared with conventional beans (Table 5).

In the cooked recipes, nitrogen, tryptophan, and lysine werestatistically higher (P < 0.05) in the maize recipe prepared withnutritionally enhanced maize than in the recipe prepared withconventional maize; there was no difference (P > 0.05) in the iron,zinc, and phytate concentration of the cooked recipes prepare withboth maize types.

Proxy bioavailability measures for iron,zinc, and protein

In vitro iron dialyzability was not different between the beanrecipes prepared with enhanced (9.52%) or conventional (9.72%)beans (P > 0.05) (Table 5). In vitro iron dialyzability was higherfor the recipes prepared with enhanced maize (37.01%) comparedwith conventional maize (32.24%) (P < 0.01). There was a trend forthe phytate:zinc molar ratio, a proxy for zinc bioavailability, of the

Table 4 --- Ingredients reported by cooking staff, com-pared with standardized recipe.

Ingredients added Ingredients omitted(n reported) (n reported)

Bean Maize Bean Maizerecipe recipe recipe recipe

Potato (6) Sodium Carrot (7) Nonebicarbonate (2) Pumpkin (3) reported

Cimarrona (1) Cinnamon (1) Onion (2)Cilantro (4)Red or green pepper (5)Garlic (6)Plantain (5)Artificial color (5)Bouillon cube (5)Spinach (1)Tomilloa (1)aAromatic spices.


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bean and maize recipes cooked with nutritionally enhanced cropsto be lower than the recipes prepared with the conventional crops;however, these values were not statistically different (P > 0.05).In vitro protein digestibility was higher (P = 0.01) in the cookedrecipes prepared with nutritionally enhanced beans (84.15%) thanwith conventional beans (82.31%). In vitro protein digestibility wascomparable (P = 0.19) in the cooked recipes prepared with nutri-tionally enhanced (83.01%) and conventional maize (82.30%).

Discussion

Nutrient concentrations in uncookedcrops and in cooked recipes

Unexpectedly, the iron levels in the uncooked beans were notdifferent between the conventional and nutritionally enhancedsamples and the values (57.12 to 64.75 mg/kg) were within therange (54 to 74 mg/kg) observed by Ariza-Nieto and others (2007)for beans of the same Andean typology. These similarities carriedover to the cooked recipes where there were no differences in theiron levels of the cooked bean recipe prepared with the 2 differ-ent bean types. In other words, these data suggest that the iron-differentiated bean intervention was not delivered to the preschoolchildren.

In contrast, the higher zinc levels in the uncooked nutritionallyenhanced beans did result in higher zinc levels in the cooked beansprepared with the nutritionally enhanced beans. The zinc valuesobserved (21.32 to 28.74 mg/kg) for the uncooked beans were at thehigher end (17 to 25 mg/kg) observed for other Andean-type beans(Ariza-Nieto and others 2007).

Iron and zinc concentrations were higher in the uncooked con-ventional maize than in the nutritionally enhanced maize; however,the iron and zinc concentrations in the cooked recipes did not dif-fer between the maize types. The uncooked values were similar tothose found in CIMMYT germplasm pools and populations: 9.6 to18.3 mg/kg Fe and 14.5 to 30.3 mg/kg Zn (Banziger and Long 2000).

The higher nitrogen concentration in the recipes preparedwith nutritionally enhanced beans was unexpected as the un-cooked bean values were not different. This suggests that nitrogen-contributing ingredients in the frıjol sancochado recipe were dis-proportionately used when the nutritionally enhanced beans werecooked. The data collected on ingredients added or omitted to therecipe do not bear this out; however, amounts used in the recipeswere not quantified.

Tryptophan and lysine levels were higher in the raw nutrition-ally enhanced maize as compared to the conventional maize; ni-trogen levels were similar between the 2 maize types. As withthe zinc in beans, for the amino acids this translated into highertryptophan and lysine levels in the cooked maize recipes. Unex-pectedly, nitrogen levels were also higher in the cooked maizerecipes prepared with nutritionally enhanced maize. This differ-ence is unlikely due to systematically more milk being added tothe recipe prepared with the nutritionally enhanced maize, un-less the cooking staff noted a difference in cooking with thismaize and made adjustments to the recipe accordingly. Cook-ing amounts were not recorded; thus this hypothesis cannot betested.

Bioavailability proxy measures in cooked recipesThere was no difference in the percent dialyzable iron in

the cooked bean recipes prepared with enhanced or conven-tional beans, suggesting similar iron bioavailability. Using a similarin vitro methodology to the one we used, Lombardi and colleagues(1991, 1995) found the iron dialyzability of extruded mottled bean




flour and cooked mottled beans to be ≤ 1.2% and of cooked whitebeans to be 3.89%, lower than what we observed. This differencecould be due to the contamination iron from extrusion used inthe 1991 Lombardi study, which increased the denominator in thedialyzability equation thus decreasing the percent dialyzable ironand also that in contrast to the Lombardi studies, which used noingredients other than beans, the carotenoid- and ascorbic acid-contributing ingredients in the current study could have increasedthe dialyzability in the bean meals (Garcıa-Casal and others 1998;Cook and Reddy 2001).

In contrast, the in vitro iron dialyzability of maize was higherin the recipe prepared with nutritionally enhanced compared withconventional maize. This was not driven by differences in the phy-tate:iron molar ratio which was comparable (approximately 23 to24:1) in both recipes. There is data to suggest that lysine enhancesiron bioavailability in rats (Van Campen and Gross 1969); however,there are no in vivo comparisons of high-lysine maize comparedwith low-lysine maize on iron bioavailability. It is notable that thein vitro iron dialyzability of the maize recipes was 3 to 4 timeshigher than for the bean recipes; this could be due to the 3 to4 times lower phytate concentration in the maize recipes comparedwith the bean recipes.

Lower phytate:zinc molar ratios are suggestive of greater zincbioavailability. Lower phytate:zinc molar ratios were observed forthe recipes prepared with nutritionally enhanced crops comparedwith the recipes prepared with the conventional crops; however,these differences were not statistically significant. Several inter-national organizations offer a classification system for estimat-ing zinc bioavailability based on phytate:zinc molar ratio: < 5:1suggests high bioavailability, 5:1 to 15:1 medium, and > 15:1 low(WHO/FAO/IAEA 1996). Thus, the recipes analyzed with either typeof maize or beans would be classified as low bioavailability. Thephytate:zinc molar ratios observed for recipes prepared in thisstudy are in the 19:1 to 56:1 range noted by the Intl. Zinc NutritionConsultative Group (IZiNCG 2004) for beans and lentils and in the22:1 to 53:1 range noted by IZiNCG for whole-grain cereals such asmaize.

The in vitro protein digestibility of the maize–milk preparationwas in the order of 82% to 83%, regardless of the maize type used.These values are higher than other digestibility studies of QPMalone; this is not unexpected given the higher digestibility of milk(IOM 2005, 690 p), which was added to the maize recipes. For exam-ple, the in vitro protein digestibility was 77% to 80% for boiled con-ventional maize and 80% for boiled QPM (Fufa and others 2003).For nixtamilized QPM flour, in vitro protein digestibility rangedfrom 73% to 79% depending on the different processing condi-tions examined (Milan-Carrillo and others 2004). The in vitro pro-tein digestibility of cooked recipes was higher for the nutrition-ally enhanced beans than the conventional beans, but in the sameorder of magnitude as the maize. Rehman and Shah (2004) foundthe in vitro protein digestibility of cooked red and white kidneybeans to be approximately 64%, lower then what we found. How-ever, the methodology they used was different: they digested thesamples with pepsin alone, incubated for 23 h, filtered the residuethrough Celite, and used nitrogen content to determine digestibil-ity (Price and others 1979). Another study that used the samein vitro methodology for protein digestibility as in the current study,reported protein digestibility values in the 81% to 83% range forextruded whole pinto bean flour (Balandran-Quintana and others1998).

The protein digestibility-corrected amino acid score (PDCAAS)is one way to measure quality protein in a meal or diet (IOM 2005,

689 p). The formula for percent PDCAAS is as follows:

mg of limiting amino acid in 1 − g test proteinmg of same amino acid in 1 − g reference protein

× %true digestibility

Assuming that in the maize recipes the only amino acids with dif-ferent values between those prepared with nutritionally enhancedand conventional maize are tryptophan and lysine, and that pro-tein (N ∗ 6.25), tryptophan, lysine, and digestibility values are aslisted in Table 5, the PDCAAS is 64.1% for the enhanced maize and43.6% for the conventional maize recipes. These values are con-sistent with those obtained by researchers who calculated the PD-CAAS of 15 QPM and 5 commercial maize cultivars (Zarkadas andothers 2000); for those investigators, the digestibility portion of theequation was taken from published data, not data generated withthese varieties. In that study, PDCAAS ranged from 54% to 72% inthe lyophilized QPM varieties and 30% to 50% in the lyophilizedcommercial maize.

Potential of enhanced crops to improvehuman nutrition

Nutritionally enhanced crops can improve human nutrition ifthey translate into more nutrients absorbed and utilized by thebody. This can be achieved in 1 of 3 ways: higher nutrient con-centrations but same bioavailability as conventional crops, samenutrient concentrations but higher bioavailability as conventionalcrops, or higher nutrient concentrations combined with higherbioavailability.

The 1st option for improving human nutrition through en-hanced crops (higher nutrient concentration, same nutrientbioavailability) most closely describes the results observed in thisstudy with zinc in beans and quality protein in maize. Zinc con-centration was higher in the bean recipes prepared with enhancedcompared with conventional beans, and zinc bioavailability, asproxied by phytate:zinc molar ratio, was similar in the bean recipesprepared with both bean types. Given the high phytate:zinc mo-lar ratio, it is unlikely that statistically different ratios would lead togreater zinc bioavailability, unless the ratio could be reduced to be-low 15:1 for the enhanced bean recipe. Breeding strategies shouldfocus on increasing the zinc content in the enhanced beans and re-ducing the phytate:zinc molar ratio.

For protein quality, the same trend was observed: higher aminoacid and protein levels in the cooked maize recipes prepared withenhanced maize yet similar in vitro protein digestibility values asmaize recipes cooked with conventional maize. The PDCAAS cal-culation of the cooked recipes supports the assertion that higheramino acid levels from enhanced maize coupled with similar di-gestibility values as conventional maize yield more quality pro-tein in the diet. This enhanced maize is likely to benefit childrenwho consume a low proportion of dietary protein from animal-source foods. Using FAO food-balance data, the Latin American andCaribbean countries with the lowest proportion of dietary proteinfrom animal sources from 2001 to 2003 were as follows (FAO 2007a),where the total proportion of animal and plant sources of proteinwas approximately 90% (not 100%): El Salvador (28%), Guatemala(22%), Haiti (14%), Honduras (33%), and Nicaragua (22%). With theexception of Haiti, these countries are also high maize-consuming(FAO 2007b), as defined by the proportion of per capita energy in-take consumed from maize: El Salvador (31%), Guatemala (39%),Honduras (31%), and Nicaragua (21%). QPM cultivars have beencommercially released in Nicaragua in 2007, in El Salvador, Haiti,Honduras, and Panama in 2008, and are planned for Guatemala


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in 2009 (Gary Atlin, CIMMYT, personal communication). The nu-tritional impact of these QPM cultivar releases on young children’smaize intake and nutritional status should be monitored.

The 2nd option for improving human nutrition through en-hanced crops (same nutrient concentration, higher nutrientbioavailability) describes the results observed in this study withiron in the cooked maize recipe. The greater in vitro iron dialyzabil-ity may have more to do with the other ingredients in the recipe,or the cooking preparation, than with the maize per se, however, ithighlights the importance of examining the bioavailability of bio-fortified crops that are cooked using local recipes and methods.Further, it is worthwhile mentioning that during the years-long pro-cess of developing biofortified crops through conventional plantbreeding, there may be unintended consequences, positive or neg-ative, of selecting for crops that are agronomically and nutrition-ally superior. For example, a high correlation between iron and zincconcentration is found in beans (Beebe and others 2000), suggest-ing that selection for crops with high levels of 1 of these nutrientswill yield crops with high levels of the other nutrient. Therefore, it ispossible that selection for maize with higher tryptophan and lysinecan unintentionally influence other maize constituents that lead togreater iron dialyzability; this requires further study.

The 3rd option for improving human nutrition through en-hanced crops (higher nutrient concentration, higher nutrientbioavailability) describes the results obtained with nutritionally en-hanced beans for protein. As with nutritionally enhanced maize,these beans can be promoted in those countries where they con-tribute importantly to protein intakes.

The small sample size limited the statistical power to detect dif-ferences in nutrient values between nutritionally enhanced andconventional crops. While attempts were made to standardizepreparation methods and ingredients, these varied among the day-care centers. However, these varied methods better reflect the cook-ing conditions that these crops will be exposed to in real-life, non-experimental settings.

AcknowledgmentsThe daycare directors and cooks for allowing us to take samples ofthe meals they prepared. Researchers from the parent efficacy studywho permitted the addition of this meal analysis substudy. LydiaChild from Cornell Univ. for design of the sampling protocol and forcollecting samples. Piedad Murillo from Univ. del Valle for collec-tion of meal samples. Natalia Palacios at CIMMYT for conductingthe protein, tryptophan, and lysine analyses. Monica Gutierrez andIngrid Aragon from Univ. del Valle for carrying out the in vitro pro-tein digestibility and iron dialyzability analyses, respectively. CIAT’sServicios Analıticos staff and Carolina Astudillo for analysis of ironand zinc levels. Support from the Monsanto Fund for CIAT’s Nutri-tion Quality Lab. where the bioavailability studies were conducted.Consejo Directivo del Fondo Regional de Tecnologıa Agropecuaria(FONTAGRO FTG-05/2003), Inst. Colombiano para el Desarrollo dela Ciencia y la Tecnologıa “Francisco Jose de Caldas” (COLCIEN-CIAS 6295-12-16793), and Proyecto Fondo para la Accion Ambi-ental (000068) for funding of the agronomic studies and efficacytrial, and the Canadian Intl. Development Agency (CIDA 7034161),through the AgroSalud Project, for funding the staff at CIAT’s Nutri-tion Quality Lab.

ReferencesAlonso R, Rubio LA, Muzquiz M, Marzo F. 2001. The effect of extrusion cooking on

mineral bioavailability in pea and kidney bean seed meals. Anim Feed Sci Technol94:1–13.

Argyri, K, Birba A, Miller DD, Komaitis M, Kapsokefalou M. 2009. Predicting relativeconcentrations of bioavailable iron in foods using in vitro digestion: new develop-ments. Food Chem 113:602–7.

Ariza-Nieto M, Blair MW, Welch RM, Glahn RP. 2007. Screening of iron bioavailabilitypatterns in eight bean (Phaseolus vulgaris L.) genotypes using the Caco-2 cell invitro model. J Agric Food Chem 55:7950–6.

Balandran-Quintana RR, Barbosa-Canovas GV, Zazueta-Morales JJ, Anzaldua-Morales A, Quintero-Ramos A. 1998. Functional and nutritional properties of ex-truded whole pinto bean meal (Phaseolus Vulgaris L.). J Food Sci 63:113–6.

Banziger M, Long J. 2000. The potential for increasing the iron and zinc density ofmaize through plant-breeding. Food Nutr Bull 21:397–400.

Beebe S, Gonzalez AV, Rengifo J. 2000. Research on trace minerals in the commonbean. Food Nutr Bull 21:387–91.

Benton-Jones J, Wolf B, Mills HA. 1991. Plant analysis handbook: a practical sampling,preparation, analysis, and interpretation guide. Athens, Ga.: Micro-Macro Publish-ing, Inc. 213 p.

Blair MW. 2007. Mejoramiento de la nutricion humana en comunidades pobres deAmerica Latina utilizando maız (QPM) y frıjol comun biofortificado con micronu-trientes: informe de avance al Consejo Directivo del Fondo Regional de TecnologıaAgropecuaria (FONTAGRO), ano 1. Cali, Colombia: Centro Internacional de Agri-cultura Tropical (CIAT). 17 p.

Blair MW, Astudillo C, Grusak M, Graham R, Beebe S. 2009a. Inheritance of seediron and zinc content in common bean (Phaseolus vulgaris L.). Mol Breed 23:197–207.

Blair MW, Sandoval TA, Caldas GV, Beebe SE, Paez MI. 2009b. Quantitative trait locusanalysis of seed phosphorus and seed phytate in a recombinant inbred line popu-lation of common bean. Crop Sci 49:237–46.

Burbano, C, Muzquiz M, Osagie A, Ayet G, Cuadrado C. 1995. Determination of phy-tate and lower inositol phosphates in Spanish legumes by HPLC methodology. FoodChem 52:321–5.

Cook JD, Reddy MB. 2001. Effect of ascorbic acid intake on nonheme-iron absorptionfrom a complete diet. Am J Clin Nutr 73:93–8.

[FAO] Food and Agriculture Org. 2007a. FAO food security statistics on food con-sumption pattern (dietary protein) of main food items. Rome, Italy: FAO. Availablefrom: http://www.fao.org/faostat/foodsecurity/Files/DietFoodItemsProtein en.xlsAccessed Jun 20, 2007.

[FAO] Food and Agriculture Org. 2007b. FAO food security statistics on food con-sumption pattern (dietary energy) of main food items. Rome, Italy: FAO. Availablefrom: http://www.fao.org/faostat/foodsecurity/Files/DietFoodItemsEnergy˙en.xlsAccessed Jun 20, 2007.

Fufa H, Akalu G, Wondimu A, Taffesse S, Gebre T, Schlosser K, Noetzold H, Henle T.2003. Assessment of protein nutritional quality and effects of traditional processes:a comparison between Ethiopian quality protein maize and five Ethiopian adaptednormal maize cultivars. Nahrung 47(4):269–73.

Garcıa-Casal MN, Layrisee M, Solano L, Baron MA, Arguello F, Llovera D, RamırezJ, Leets I, Tropper E. 1998. Vitamin A and β-carotene can improve nonheme ironabsorption from rice, wheat and corn by humans. J Nutr 128:646–50.

Graham GG, Lembcke J, Lancho E, Morales E. 1989. Quality protein maize: digestibil-ity and utilization by recovering malnourished infants. Pediatrics 83:416–21.

Gunaratna NS. 2007. Evaluating the nutritional impact of maize varieties geneticallyimproved for protein quality [PhD thesis]. West Lafayette, Ind.: Purdue Univ. 118 p.Available from: Univ. Microfilms, Ann Arbor, Mich.

Hambidge KM, Huffer JW, Raboy V, Grunwald GK, Westcott JL, Sian L, Miller LV,Dorsch JA, Krebs NF. 2004. Zinc absorption from low-phytate hybrids of maize andtheir wild-type isohybrids. Am J Clin Nutr 79:1053–9.

Hsu HW, Vavak DL, Satterlee LD, Miller GA. 1977. A multienzyme technique for esti-mating protein digestibility. J Food Sci 42(3):1269–73.

[IOM] Inst. of Medicine. 2005. Dietary reference intakes for energy, carbohydrate,fiber, fat, fatty acids, cholesterol, protein, and amino acids. Washington, D.C.: Natl.Academy Press. 1357 p.

[IZiNCG] International Zinc Nutrition Consultative Group. 2004. Assessment of therisk of zinc deficiency in populations and options for its control. Food Nutr Bull25:S94–203.

Kapsokefalou M, Miller DD. 1991. Effects of meat and selected food componentson valence of nonheme iron during in vitro digestion. J Food Sci 56(2):352–5,58.

Krivanek AF, De Groote H, Gunaratna NS, Diallo AO, Friesen D. 2007. Breeding anddisseminating quality protein maize (QPM) for Africa. Afr J Biotechnol 6(4):312–24.

Lombardi-Boccia G, Di Lullo G, Carnovale E. 1991. In vitro iron dialysability fromlegumes: influence of phytate and extrusion cooking. J Sci Food Agric 55:599–605.

Lombardi-Boccia G, De Santis N, Di Lullo G, Carnovale E. 1995. Impact of processingon Fe dialysability from bean (Phaseolus vulgaris L.). Food Chem 53:191–5.

McDonough FE, Sarwar G, Steinke FH, Slump P, Garcia S, Boisen S. 1990. In vitro assayfor protein digestibility: interlaboratory study. J Assoc Off Anal Chem 73(4):622–5.

Milan-Carrillo J, Gutierrez-Dorado R, Cuevas-Rodrıguez EO, Garzon-Tiznado JA,Reyes-Moreno C. 2004. Nixtamilized flour from quality protein maize (Zeamays L.): optimization of alkaline processing. Plant Foods Hum Nutr 59:35–44.

Morales E, Graham GG. 1993. Maız peruano de alta calidad proteıca: digestibilidad yutilizacion en ninos malnutridos. Arch Latinoamer Nutr 43(2):176–83.

Nestel P, Bouis HE, Meenakshi JV, Pfeiffer W. 2006. Biofortification of staple foodcrops. J Nutr 136:1064–7.

Nurit E, Tiessen A, Pixley K, Palacios-Rojas N. 2008. Unpublished methodology: al-ternative colorimetric method for tryptophan determination in maize kernels. Tex-coco, Mexico: Centro Internacional de Mejoramiento de Maız y Trigo.

Price M L, Butter LG, Rogler JC, Featherson WR. 1979. Overcoming the nutritionallyharmful effects of tannin in sorghum grains by treatment with inexpensive chemi-cals. J Agric Food Chem 27:441–5.

Reddy V, Gupta CP. 1974. Treatment of kwashiorkor with opaque-2 maize. Am J ClinNutr 27:122–4.

Rehman Z, Shah WH. 2005. Thermal heat processing effects on antinutrients, proteinand starch digestibility of food legumes. Food Chem 91:327–31.

Sandberg A-S. 2005. Methods and options for in vitro dialyzability; benefits and limi-tations. Int J Vitam Nutr Res 75:395–404.




Skalar Analytical BV. 1995. The SANplus segmented flow analyzer: soil and plant anal-ysis. Breda, The Netherlands: Skalar Analytical BV. 160 p. Publication number0102003B.

Tsai CY, Hansel LW, Nelson OE. 1972. A colorimetric method of screening maize seedsfor lysine content. Cereal Chem 49:572–9.

Van Campen D, Gross E. 1969. Effect of histidine and certain other amino acids on theabsorption of iron-59 by rats. J Nutr 99:68–74.

Villegas E, Vasal SK, Bjarnason M. 1992. Quality protein maize: what is it and how wasit developed. In: Mertz ET, editor. Quality protein maize. St. Paul, Minn.: AmericanAssoc. of Cereal Chemists. p 27–48.

[WHO, FAO, IAEA] World Health Org., Food and Agriculture Org., Intl. Atomic EnergyAssoc. 1996. Trace elements in human health and nutrition. Geneva, Switzerland:World Health Org.

Xu P, Price J, Aggett PJ. 1992. Recent advances in methodology for analysis of phytateand inositol phosphates in foods. Prog Food Nutr Sci 16(3):245–62.

Zarkadas CG, Hamilton RI, Yu ZR, Choi VK, Khanizadeh S, Rose NGW, Pattison PL.2000. Assessment of the protein quality of 15 new northern adapted cultivars ofquality protein maize using amino acid analysis. J Agric Food Chem 48(11):5351–61.


Annex 4. New Laboratory brochure developed in English.

International Center for Tropical Agriculture Centro Internacional de Agricultura Tropical (CIAT)

Kilómetro 17 Recta Cali–PalmiraPalmira, Valle del Cauca, Colombia

Telephone: + 57 (2) 4450000 ext. [email protected] / www.AgroSalud.org

Evaluating the nutritionalquality of food.

laboratory * laboratory * laboratory * laboratory * laboratory * laboratory * laboratory * laboratory * laboratory * laboratory * laboratory * laboratory * laboratory * laboratory * laboratory

Principal activitiesPrincipal activities

analytical servicesanalytical services

Bioavailability is that fraction of an ingested nutrient that is available for use by the human body. In other words, the term refers to the percentage of the nutrients the body consumes that can be digested, assimilated, and used for normal biological functions.

To measure bioavailability, the Laboratory simulates the phases of human digestion-oral, gastric and intestinal-using in vitro methods.

¿what isbioavailability?¿what is

bioavailability?

Darwin Ortiz, Chemist, e-mail: [email protected] Pachón, Nutritionist, e-mail: [email protected]

quipmentquipment

Quantification of soluble protein.Quantification of tryptophan.Determination of in vitro protein digestibility.Determination of in vitro iron dialyzability.Determination of in vitro carotene bioaccessibility.Identification of carotenes.Quantification of total carotenes.Quantification of antioxidant activity.Quantification of phytic acid.

xamples of researchprojects conducted

xamples of researchprojects conducted

Evaluating the in vitro bioavailability of iron and zinc in a typical Colombian bean dish (assessing both biofortified and conventional beans).

Evaluating the in vitro digestibility of protein in mazamorra, a typical Colombian porridge made with maize and milk (assessing both high-quality protein and conventional maize).

Validating an in vitro digestion method for evaluating iron bioavailability in crops.

Evaluating the nutritional value of extracts prepared from foliage of diverse crops.

Evaluating the protein quality of typical dishes from the Department of Cauca (Colombia) prepared with biofortified or conventional maize.

Quantifying phytates, using HPLC, in a population of common beans (Phaseolus vulgaris) and identifying QTLs associated with phytate contents.

Evaluating the quality of proteins in meat and dairy products treated with honey as a possible preservation method and comparing these with untreated products.

Implementing a methodology to evaluate antioxidant activity in food.

The Laboratory has at its disposal the most recent technology. It can obtain results that are precise, reliable, and quick. The technology includes:

High Performance Liquid Chromatography (HPLC)

Near Infrared Reflectance Spectroscopy (NIRS)

UV/Vis spectrophotometer

¿who we are?¿Who we are?

The Nutrition Quality Laboratory (Laboratorio de Calidad Nutricional) is the first in Latin America to offer a set of analytical methods that determine the in vitro concentration and bioavailability of iron, zinc, vitamin A, and protein. The Laboratory:

Evaluates the nutritional quality of biofortified (i.e., nutritionally improved) crops. Evaluates the nutritional quality of natural or industrially processed foods.

Through this Laboratory, foods whose nutrients are more readily assimilated by the human body can be identified.

Quantify the concentrations of nutrients such as carotenes, soluble protein, and tryptophan, using chromatographic and spectroscopic techniques.

Determine the bioavailability of nutrients, using previously validated in vitro methodologies such as in vitro bioaccessibility of carotenes, in vitro dialyzability of iron, in vitro digestibility of protein, and the molar ratio phytate:zinc as an indicator of zinc bioavailability.

Develop methods for determining bioactive compounds, including their bioavailability, in foods of interest to different audiences.

Provide technical assistance in the evaluation of the nutritional quality of foods according to the needs of individuals, companies or food programs.

Train researchers and students through inter-laboratory trials, internships, thesis proposals, and other research projects.

Ensure the quality of results by implementing a Quality Management System based on the ISO 17025 standard.

Annex 5. New Laboratory brochure developed in Spanish.

Centro Internacional de Agricultura Tropical (CIAT)Kilómetro 17 Recta Cali-Palmira

Palmira, Valle del Cauca, ColombiaTeléfono: + 57 (2) 4450000 ext. 3675

[email protected] / www.AgroSalud.org

Evaluación de la calidadnutricional de alimentos

laboratorio * laboratorio * laboratorio * laboratorio * laboratorio * laboratorio * laboratorio * laboratorio * laboratorio * laboratorio * laboratorio * laboratorio * laboratorio * laboratorio

Principales actividadesPrincipales actividades

Servicios de análisisServicios de análisis

Biodisponibilidad es la fracción ingestada de un nutriente que es disponible para el cuerpo. Es decir que se refiere al porcentaje de los nutrientes que el cuerpo consume que pueda digerir, asimilar y utilizar en sus funciones biológicas normales.

Para medir la biodisponibilidad en el laboratorio, se simula condiciones de la digestión humana, tanto en su fase oral, como estomacal e intestinal, por métodos in vitro.

¿qué esbiodisponibilidad ?

¿qué esbiodisponibilidad ?

Darwin Ortiz, Químico: [email protected] Pachón, Nutricionista: [email protected]

quiposquipos

Cuantificación de proteína soluble.Cuantificación de triptófano.Determinación de digestibilidad in vitro de proteína.Determinación de dializabilidad in vitro de hierro.Determinación de bioaccesibilidad in vitro de carotenos.Identificacion de carotenos.Cuantificación de carotenos totales.Cuantificación de actividad anti-oxidante.Cuantificación de ácido fítico.

Algunas investigacionesrealizadas

Algunas investigacionesrealizadas

Evaluación de la biodisponibilidad in vitro de hierro y zinc en una receta típica colombiana de fríjol (biofortificado y convencional) y de digestibilidad in vitro de proteína de una receta típica colombiana de maíz con leche -mazamorra- (de alta calidad de proteína y convencional).

Validación de un método de digestión in vitro para la evaluación de biodisponibilidad de hierro de cultivos.

Evaluación del valor nutricional de extractos foliares preparados a partir del follaje de diversos cultivos.

Evaluación de la calidad proteica de recetas típicas del departamento del Cauca (Colombia), elaboradas con maíz biofortificado.

Cuantificación de fitatos por HPLC en una población de fríjol común (Phaseolus vulgaris) e identificación de QTLs asociados a su contenido.

Evaluación de la calidad proteica en alimentos cárnicos y lácteos tratados con y sin miel, como un método de preservación.

Implementación de una metodología para evaluar la actividad anti-oxidante en alimentos.

El laboratorio cuenta con la más reciente tecnología que brinda la capacidad de obtener resultados más precisos, confiables y rápidos. Algunos de ellos:

HPLC (High Performance Liquid Chromatography)

NIRS (Near Infrared Reflectance Spectroscopy)

Espectrofotómetro UV/VIS

¿quiénes somos?¿quiénes somos?

El Laboratorio de Calidad Nutricional (Nutrition Quality Laboratory) es el primero en Latinoamérica en ofrecer un conjunto de métodos de análisis para determinar la concentración y biodisponibilidad in vitro de hierro, zinc, vitamina A y proteína para:

Evaluar la calidad nutricional de cultivos nutricionalmente mejorados (biofortificados).

Evaluar la calidad nutricional de alimentos naturales o procesados industrialmente.

Con este laboratorio es posible identificar los alimentos cuyos nutrientes serán más asimilados por el cuerpo humano.

Cuantificar la concentración de nutrientes como carotenos, proteína soluble y triptófano, mediante técnicas cromatográficas y espectroscópicas.

Determinar la biodisponibilidad de nutrientes a través de análisis in vitro de metodologías validadas previamente como la bioaccesibilidad in vitro de carotenos, la dializabilidad in vitro de hierro, la digestibilidad in vitro de proteína y la relación molar fitato:zinc como indicador de la biodisponibilidad de zinc. Desarrollar métodos para determinar compuestos bio-activos, incluyendo su biodisponibilidad, que tengan los alimentos de interés para diferentes públicos.

Brindar acompañamiento técnico en evaluaciones de calidad nutricionalde alimentos de acuerdo a las necesidades de grupos de personas,empresas o programas alimentarios.

Capacitar a investigadores y estudiantes a través de intercambios,pruebas inter-laboratorio, pasantías, proyectos de tesis y otros proyectosde investigación.

Asegurar la calidad de los resultados emitidos mediante la implementación de un Sistema de Gestión de Calidad,

basado en la norma ISO 17025.

Annex 6. Students who contributed to the Laboratory’s activities in the first semester of 2009. Name University Academic Program Role Dates in

Laboratory Project in Laboratory Presentations Given

Ingrid Aragón

Universidad del Valle

Chemistry Intern, Thesis student

10 March 2008-31 May 2009

Validation of an in vitro method to assess the iron bioavailability of biofortified crops

Validation of an in vitro method to assess the iron bioavailability of biofortified crops, CIAT, 4 March 2009

Paola Imbachí

Universidad del Cauca

Agroindustrial Engineer Intern, Thesis student

12 May 2008-31 May 2009

Protein-quality evaluation of different Colombian recipes prepared with biofortified maize

Protein-quality evaluation of different Colombian recipes prepared with biofortified maize, CIAT, 13 May 2009

Hiroko Kunori

Tokyo University of Agriculture

Tropical Horticulture Japan CGIAR Fellow

18 November 2008-15 January 2009

Implementation of an analytic method to evaluate the anti-oxidant capacity of biofortified crops

None

Annex 7. Presentations of Laboratory findings in the first semester of 2009. Event Place Date Authors Presentation Title I Congreso Internacional, XI Nacional & Olimpiadas Académicas y Deportivas Universidad de Santander Sede Valledupar

Valledupar, Colombia

24 April 2009 Darwin Ortiz

Investigaciones de alimentos en pro de la nutrición humana: Ejemplo cultivos biofortificados dentro del marco del Proyecto AgroSalud

CIAT Knowledge-sharing Week and Board of Trustees Meeting

Palmira, Colombia

18-29 May 2009 Dayron Gutierrez, Darwin Ortiz, Helena Pachón

Nutrition Quality Laboratory: Projects and developments

CIAT Knowledge-sharing Week and Board of Trustees Meeting

Palmira, Colombia

18-29 May 2009 Ingrid Aragón Darwin Ortiz, Helena Pachón

Validación de un Método de Digestión in Vitro para la Evaluación de Biodisponibilidad de Hierro en alimentos

Annex 8. Select photos of Laboratory visitors in December 2008 and the first semester of 2009. December 2008 visit of Andrés Laignelet,

Monsanto April 2009 visit of nutritionists from the

Cauca Department’s Instituto Colombiano de Bienestar Familiar

May 2009 visit of representatives from ADA Afro-Latino Development Alliance

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