Effect of style of home cooking on retention and bioaccessibility of pro-vitamin A carotenoids in biofortified pumpkin (Cucurbita moschata Duch.)

    Effect of style of home cooking on retention and bioaccessibility of provitamin A carotenoids in biofortified pumpkin (Cucurbita moschata Duch.) Ediane Maria Gomes Ribeiro, Chureeporn Chitchumroonchokchai, Lucia Maria Jaeger de Carvalho, Fabiana F. de Moura, Jose Luiz Viana de Carvalho, Mark L. Failla PII: DOI: Reference:

S0963-9969(15)30172-1 doi: 10.1016/j.foodres.2015.08.038 FRIN 6001

To appear in:

Food Research International

Received date: Revised date: Accepted date:

30 June 2015 23 August 2015 26 August 2015

Please cite this article as: Ribeiro, E.M.G., Chitchumroonchokchai, C., de Carvalho, L.M.J., de Moura, F.F., de Carvalho, J.L.V. & Failla, M.L., Effect of style of home cooking on retention and bioaccessibility of pro-vitamin A carotenoids in biofortified pumpkin (Cucurbita moschata Duch.), Food Research International (2015), doi: 10.1016/j.foodres.2015.08.038

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT REVISED FOODRES-D-15-01851

IP

T

Effect of Style of Home Cooking on Retention and Bioaccessibility of Pro-Vitamin A Carotenoids in Biofortified Pumpkin (Cucurbita moschata Duch.)

SC R

Ediane Maria Gomes Ribeiroa†, Chureeporn Chitchumroonchokchaib† , Lucia Maria Jaeger de Carvalhoa, Fabiana F. de Mourac, Jose Luiz Viana de Carvalhod,

Rio de Janeiro Federal University, Pharmacy College, Av. Carlos Chagas Filho, 373,

MA

a

NU

and Mark L. Faillab*

Bloco L, subsolo, Lab. 17, 21949900, Rio de Janeiro, Brazil Human Nutrition Program, 325 Campbell Hall, The Ohio State University,1787 Neil

D

b

c

TE

Avenue, Columbus, OH USA 43210

HarvestPlus, International Food Policy Research Institute, 2033 K Street NW,

Embrapa Food Technology, Av. das Américas, 29.501, Guaratiba, Rio de Janeiro, RJ,

Brazil. †

AC

d

CE P

Washington DC 20006.

EMGR and CC contributed equally to this project.

*Corresponding author: [email protected] Running Title: Pro-vitamin A content and bioaccessibility in cooked pumpkin

1

ACCEPTED MANUSCRIPT ABSTRACT Pumpkin (Cucurbita moschata) is a food crop targeted for enrichment with pro-vitamin

T

A carotenoids. We investigated retention of pro-vitamin A carotenoids in pulp from

IP

orange fleshed pumpkin that was briefly steamed or boiled in either water or water

SC R

containing 60% sucrose in five genotypes grown in Brazil. Bioaccessibility of provitamin A carotenoids in cooked pulp was also determined by their transfer to mixed

NU

micelles during in vitro digestion and confirmed by their accumulation in Caco-2 human intestinal cells. Pulp from the biofortified genotypes contained 209-658 µg/g fresh

MA

weight pro-vitamin A and retention of the carotenes during cooking exceeded 78%. Bioaccessibility of β-carotene and α-carotene was poor (<4%), highly variable and

D

affected by food matrix and style of cooking. The estimated quantity of β-carotene

TE

equivalents transferred to mixed micelles during simulated digestion of cooked pulp from one genotype has the potential to provide more than 40% of the Estimated

CE P

Average Requirement of vitamin A for children 4-8 years of age per 100g serving.

AC

Possible causes for the low bioaccessiblity of pro-vitamin A in pumpkin are discussed.

Keywords: pumpkin, biofortification, pro-vitamin A carotenoids, bioaccessibility, Caco2 cells

2

ACCEPTED MANUSCRIPT 1. Introduction Vitamin A deficiency (VAD) continues to be a significant public health problem in

T

more than half of the countries in the world (WHO, 2009). Children and pregnant and

It has been estimated that 17.4% of

SC R

VAD (WHO, 2011; Sommer & Vyas, 2012).

IP

lactating women are at the greatest risk of developing the pathologies associated with

children under 5 years of age and 12.3% of women of reproductive age in Brazil are

NU

deficient in vitamin A (Ferraz et al., 2004; Ministry of Heath, Brazil, 2009). Several different strategies are being used to prevent VAD. Traditional interventions

MA

include supplementation with high doses of vitamin A and food fortification programs (Nestel et al., 2006). These approaches are costly, require appropriate infrastructure to

D

reach those at greatest risk, and non-sustainable. Because of such problems, there is

TE

increased interest in biofortification. This cost effective and sustainable strategy involves either conventional crossing of germplasm or the introduction of specific genes

CE P

to generate plants with edible plant tissues containing high amounts of targeted nutrients. This strategy is particularly important for relatively isolated rural regions that

AC

are difficult to reach for mainstream supplementation and fortification programs (Nestel et al., 2006; Saltzman et al., 2013). Pumpkin is one of the crops targeted by the Brazilian Research Agriculture Corporation (Embrapa) for enhancement of pro-vitamin A content by conventional breeding (Saltzman et al., 2013). Some varieties of pumpkin (Curcubita moschata) contain relatively high amounts of pro-vitamin A in comparison to other fruits and vegetables (Rodriguez-Amaya et al., 2008; Azevedo-Meleiro et al., 2007; Murkovic, Mulleder, & Neunteufl, 2002).

Boiling and steaming are the most common home

styles of cooking pumpkin pulp (Murkovic, Mulleder, & Neunteufl, 2002). Carvalho 3

ACCEPTED MANUSCRIPT and associates recently reported high retention of pro-vitamin A carotenoids in boiled pulp from a single landrace of C. moschata biofortified with pro-vitamin A carotenoids

T

(Carvalho et al., 2014).

IP

Cooked pumpkin is being used as an ingredient in some processed foods. One such

SC R

product under development for the Brazilian School Feeding Program is a dessert that includes pumpkin boiled in 60% sucrose solution (Curado et al., 2009). In order for consumed pro-vitamin A carotenoids and their metabolites to be

NU

absorbed and delivered to peripheral tissues for storage or utilization, the carotenoids

MA

must be released from the food matrix and solubilized in oil droplets (Yonekura & Nagao, 2007; Fernandez-Garcia et al., 2012). During small intestinal digestion, a portion of the carotenoids and other dietary lipophiles, along with digestion products of The mixed micelles deliver

TE

D

co-consumed fat, partition into bile salt micelles.

carotenoids to absorptive epithelial cells that incorporate carotenoids and their esterified

CE P

retinoid metabolites into chylomicrons for secretion into lymph. The efficiency with which consumed carotenoids are transferred into mixed micelles is influenced by their

AC

physical state and sub-cellular localization within the food matrix, the extent to which cells walls are ruptured during processing of the plant tissue, and other components in the meal (Lemmens et al., 2014). The objective of this study was to investigate the effects of several styles of home cooking on the retention and bioaccessibility of pro-vitamin A carotenoids in five genotypes of biofortified C. moschata Duch. Bioaccessibility was determined by the partitioning of the pro-vitamin A carotenoids in mixed micelles during in vitro digestion and confirmed by measuring their uptake by enterocyte-like Caco-2 human intestinal cells. In vitro bioaccessibility of carotenoids has been shown to be a reliable predictor of carotenoid bioavailability in human subjects (Reboul et al., 2006). 4

ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Pumpkin

T

Five genotypes of pumpkin (Cucurbita moschata Duch.) were provided by the

IP

Biofortication Program of the Brazilian Research Agriculture Corporation (Embrapa).

SC R

Pumpkins were cultivated in the northeast region of Brazil with genotypes 58, 129 and 346 grown at Frei Paulo, Sergipe, and genotypes 12 and 13 grown at Petrolina, Pernambuco. The harvest cycle was 120 days with pumpkins harvested in March 2012

NU

(genotypes 58, 129 and 346) and March 2013 (genotypes 12 and 13). Pumpkins were

MA

transferred to Rio de Janeiro within two days of harvest, processed (see 2.2) and pulp was stored at -80°C under nitrogen gas. Frozen samples of raw and cooked pumpkins were shipped to Ohio State University (OSU) where they arrived frozen and stored at -

TE

D

80ºC. An orange fleshed pumpkin of unknown genotype was also purchased at a local market in Rio de Janeiro, prepared and shipped as above to determine the effect of

CE P

duration of cooking on the bioaccessibility of pro-vitamin A carotenoids. 2.2. Cooking conditions

AC

Pumpkins of each genotype were washed, peeled, and seeds were removed. Pulp was cubed (~5 cm2) and distributed into four sections.

One section (raw) was

homogenized and stored under nitrogen gas at -80°C. The other sections were cooked according to one of the three following procedures commonly used in homes. The first group of cubes from each genotype was immersed in 2 volumes of deionized water and boiled for 5 min with lid on the pan. The second group was steamed for 7 min and the third group was immersed in two volumes of water containing 60% sucrose (boiled + sucrose) with lid on the pan for 5 min.

The duration of boiling and steaming were

sufficient to soften the plant tissue as assessed by the penetration of tip of a knife without resistance (Carvalho et al., 2014). Pulp from each genotype was independently 5

ACCEPTED MANUSCRIPT cooked in triplicate, homogenized in a vertical mixer (IKA - Ultraturrax model T18 basic) to a puree and stored frozen under nitrogen gas. Moisture content of raw and

T

cooked pumpkins was determined as previously described (Carvalho et al., 2014).

IP

Retention of pro-vitamin A after cooking was calculated by dividing the quantities of

SC R

pro-vitamin A carotenoids per gram fresh weight (FW) in cooked pulp by the concentration per g in raw pulp (FW). Concentrations were not corrected for changes in moisture content after cooking as the differences between raw and cooked pulp were

Cooked pumpkin was digested in vitro to assess whether style

MA

2.3. In vitro digestion

NU

<10% and emphasis is given to amounts delivered to the plate (Table 1).

of home cooking affected the efficiency of their transfer into mixed micelles. Simulated digestion was performed for each set of cooked pumpkins as described by Garrett et al.

TE

D

(1999) and modified by Thakkar et al. (2007). The procedure consists of simulated oral, gastric and intestinal phases of digestion. Soybean oil (2.5% wt/wt) was added to

CE P

aliquots of thawed cooked pumpkin (1.0-1.3 g per digestion) prior to rehomogenization. After completion of the small intestinal phase of digestion, chyme was

AC

centrifuged (12,000g, 45 min, 4°C) to separate the aqueous and undigested fractions. Supernatant was filtered through cellulose acetate membranes (0.22 μm pores) to obtain the mixed micelle fraction. Aliquots of chyme and micelle fraction were stored under nitrogen gas at -80° C. Quantities of carotenoids in chyme divided by those in cooked pumpkin was used to determine stability during digestion.

Bioaccessibility was

calculated by dividing quantity of carotenoids in filtered aqueous fraction of chyme by that in pre-digested cooked pulp. All processing and manipulations during simulated digestion were performed under yellow light to minimize photo-oxidative reactions and a minimum of four aliquots of homogenate were independently digested for each cooked sample. 6

ACCEPTED MANUSCRIPT 2.4. Uptake of micellarized carotenoids by Caco-2 human intestinal cells Caco-2 human intestinal cells (HTB37) were purchased from American Type Culture

T

Collection at passage 19 and used for experiments at passages 26-28. Details for

IP

maintaining cultures are described elsewhere (Chitchumroonchokchai, Schwartz &

SC R

Failla, 2004). The micelle fraction generated during simulated digestion of cooked pumpkins was diluted 1:4 with HEPES buffered DMEM, pH 6.5, before adding 12 mL to T75 flasks containing washed monolayers of differentiated Caco-2 cells 11 days after

NU

the monolayer became 100% confluent. Possible cytotoxicity of the micelle fractions

MA

was assessed by observation of cell morphology (phase contrast microscopy) and protein content per flask (Bicinchoninic acid assay).

No differences were noted

between the characteristics of monolayers exposed to control medium and those

TE

D

incubated for 4h in medium with micelle fraction. After 4h, washed monolayers were washed, scrapped from the surface of the flask into cold PBS and cells were collected

CE P

by centrifugation (400 x g, 4C, 10 min) and stored under nitrogen gas at -80C. 2.5. Extraction and analysis of carotenoids

AC

Raw and cooked pulp and chyme and micelle fraction of digested pulp were extracted using a modification of the methods of AOAC (AOAC, 1993) and Seo et al. (2005). Briefly, 5-10 mL of sample containing 0.8 µg apo-8’-carotenal as internal standard was transferred to polypropylene test tubes. Methanol (MeOH): tetrahydrofuran (10 mL, 50:50, v/v) was added and tubes vortexed (1 min) before addition of hexane (10 mL). The mixture was vortexed (1 min), centrifuged (1,200 x g, 4C, 10 min) and the upper layer transferred to screw capped glass vials. Extraction was repeated 2-3 times, pooled organic solvent was evaporated under nitrogen gas, and the residual film immediately re-solubilized in 1:1 (v:v) mixture of MeOH:methyl-tert-butyl ether and filtered for analysis by HPLC-DAD. Extraction of carotenes from cell pellets followed the protocol 7

ACCEPTED MANUSCRIPT of Chitchumroonchokchai et al. (2004). Carotenoids were separated using a YMC™ C30 column (4.6 x 150 mm, 5 µm; Waters, Milford, MA) coupled to diode array detector

T

monitoring absorbance at 350-500 nm and identified and quantified as previously

IP

reported (Chitchumroonchokchai, Schwartz & Failla, 2004; Thakkar, Huo, Maziya-

SC R

Dixon & Failla, 2007). Apo-8’-carotenal was used as internal standard to estimate the efficiency of carotenoid recovery during extraction (91-108%). 2.6. Statistical analysis

NU

Data are presented as mean  SEM. All data were analyzed using one way ANOVA

MA

follow by Tukey post-hoc test with significance set at p<0.05 by GraphPad Prism

D

Version 6.05 (GraphPad Software, Inc, CA, USA).

TE

3. Results

CE P

3.1. Carotenoid profile in pumpkin genotypes and retention after home cooking The most abundant carotenoids detected in all five genotypes of orange fleshed pumpkin were beta-carotene (βC) and alpha-carotene (C) (Table 1). Trace quantities

AC

(≤ 0.4% of total carotenoids) of lutein and zeaxanthin were detected in raw pumpkin and not further considered. Total concentrations of the pro-vitamin A carotenoids in the genotypes ranged from 209 µg/g FW (genotype 346) to 658 g/g FW (genotype 12) with βC accounting for 52% (genotype 13) to 90% (genotype 129) of the pro-vitamin A carotenoids. The amounts of αC in raw pulp ranged from 20 µg/g FW (genotype 129) to 206 µg/g FW (genotype 13). 9-, 13- and 15-Z βC were present in raw pulp and collectively accounted for ≤ 13% of total βC (Fig. 1). 13-Z-βC was the most abundant Z-isomer βC in raw pulp.

8

ACCEPTED MANUSCRIPT Boiled and steamed pulp from genotypes 12 and 13 contained 5-15% and 13-22% less total pro-vitamin A, respectively, than raw pulp (Table 1). These reductions were

T

due to significant losses of both all-E-βC and Z-βC. αC was also lower in boiled and

IP

steamed pulp from genotype 13, as well as steamed pulp from genotypes 12. Steaming

SC R

also significantly decreased total pro-vitamin A, all-E-βC and αC content in pulp from genotype 346. In contrast with the adverse impact of home cooking on pulp from genotypes 12, 13 and 346, there were minimal, if any, changes in total pro-vitamin A,

NU

all-E-βC and αC in cooked pulp from genotypes 58 and 129. The concentration of Z-βC (Table 1), and particularly 13-Z-βC (Fig. 1), increased (p <0.01) in cooked pulp from

MA

genotypes 58, 129 and 346, but accounted for ≤ 7% of total βC. In contrast, the concentration of Z-βC was lower in cooked pulp from genotypes 12 and 13 than in raw

TE

D

pulp (Table 1). Collectively, these data suggest that the impact of cooking on the profile and content of pro-vitamin A carotenoids in pumpkin varied according to

CE P

genotype, food matrix, and the style of home cooking. All-E-βC is cleaved into two molecules of retinal whereas cleavage of Z-βC and αC

AC

yields only a single molecule of retinal. Therefore, the amount of βC equivalents was calculated for raw and cooked pulp as follows: pro-vitamin A content in βC equivalents = all-E-βC + ½ (αC + Z-βC) (Table 1). Despite the presence of 43% more pro-vitamin A in raw pulp of genotype 12 than in genotype 58, βC equivalents in cooked pulp from these two genotypes only differed by 5-13%.

This was due to the presence of

considerably greater amounts of Z-isomers of βC and αC in the pulp from genotype 12.

3.2. Bioaccessibility of pro-vitamin A carotenoids in cooked pumpkin. The carotenes in cooked pumpkin were relatively stable during simulated oral, gastric and small intestinal digestion with retention ranging from 80-98%. 9

The

ACCEPTED MANUSCRIPT efficiency of micellarization (%) with which all-E-βC, Z-βC and C were transferred from cooked pulp to mixed micelles during simulated digestion was low, ranging from

T

0.4-3.3%, 0.6-7.6%, and 0.3-3.9%, respectively (Supplemental Table 1). However, the

IP

actual quantities of these carotenes in micelles were considerable in light of the

SC R

relatively high concentrations of concentrations of pro-vitamin A in cooked biofortified pumpkins (Table 2). All-E-βC, αC and βC equivalents that partitioned in the micelle fraction after digestion of cooked genotype 58 exceeded that of the other genotypes

NU

regardless of style of cooking (Table 2). All-E-βC, αC and βC equivalents in micelles

MA

were greater after digesting pulp from genotypes 12, 13, 58 and 346 boiled in water alone compared to pulp boiled in water + 60% sucrose. Similarly, all-E-βC, αC and βC

D

equivalents in micelles generated during digestion of pulp from genotypes 12 and 346

TE

boiled in water alone were greater than that after digesting steamed pulp from these genotypes. Correlation coefficients (R) for the quantity of βC equivalents in the

CE P

bioaccessible fraction of digested pulp versus βC equivalents in pulp from each genotype that had been boiled in water, steamed and boiled in water + 60% sucrose

AC

were 0.6142, 0.1651 and 0.3804, respectively. Monolayers of differentiated Caco-2 cells were used to confirm that pro-vitamin A carotenoids in the micelle fraction were available for uptake. Uptake (%) of all-E-βC and αC by Caco-2 cells from medium containing micelles generated during digestion of cooked pumpkin ranged from 12-19% and 11-16%, respectively (data not shown). ZβC uptake was below the limit of detection. Cellular content of both all-E-βC and αC (Fig. 2) was proportional to the quantities of these carotenoids present in the bioaccessible fraction (Table 2), and not necessarily proportional to the pro-vitamin A content in the different genotypes (Table 1). Caco-2 cells incubated in medium

10

ACCEPTED MANUSCRIPT containing mixed micelles generated during digestion of boiled and steamed pulp from

T

genotype 58 accumulated the highest concentrations of all-E-βC and αC (Fig. 2).

IP

3.3. Increased time of cooking pumpkin enhances the bioaccessibility of pro-vitamin A

SC R

in cooked pulp.

We assumed that the poor efficiency of micellarization of pro-vitamin A in pumpkin might be due to incomplete digestion and/or the brief duration of cooking that is

NU

common in Brazilian homes. Increasing the quantity of digestive enzymes and bile

MA

extract to three times that used in our standard protocol did not significantly (p >0.05) increase the efficiency of micellarization (data not shown). Pulp from an orange fleshed pumpkin purchased at a Brazilian local market was used to determine if extending time Total pro-vitamin A

TE

D

of cooking increased the micellarization of the carotenes.

carotenoid content of this pumpkin was 71 µg/g FW and the times for boiling and

CE P

steaming the cubes was varied from 5-20 min and 7-28 min, respectively. Retention of the carotenoids during prolonged boiling and steaming of pulp was 76 and 91%,

AC

respectively, and ≥ 93% of pro-vitamin A carotenoids in cooked pulp were recovered after simulated digestion. Bioaccessible βC equivalents in digested pulp also increased as time of cooking increased (Fig. 3). βC equivalents in the micelle fraction of digested pulp that was steamed for 28 min was 135% greater than that in digested pulp streamed for 7 min. βC equivalents in the micelle fraction generated during digestion of pulp boiled in water for 20 min was only 15% greater than after digestion of pulp boiled for 5 min.

4. Discussion

11

ACCEPTED MANUSCRIPT There are major qualitative and quantitative differences in the carotenoid content and profiles in Curcubita species and even varieties within the same species as a result

T

of genotype, pre-harvest environmental conditions and post-harvest processing

IP

(Rodriguez-Amaya et al., 2008). The present investigation of five different genotypes

SC R

of pumpkin biofortified with pro-vitamin A extends preliminary reports (Carvalho et al., 2012 & 2014) that pro-vitamin A carotenoids were well retained after common home styles of cooking orange fleshed C. moschata Duch. Three of the tested five genotypes

NU

contained higher amounts of pro-vitamin A (>450 µg/g FW) than previously reported

MA

for any variety of pumpkin (Table 1). Retention of pro-vitamin A carotenoids after boiling and steaming was relatively high (>78%), although the impact of cooking style varied among the genotypes. Consideration of the effect of cooking style on retention

TE

D

and isomeric profile of pro-vitamin A in foods is important as it affects the quantity of βC equivalents delivered to the plate. It is well established that heat, like light and

CE P

acidity, can oxidize, isomerize and degrade carotenoids (Mercadante 2007). Relatively high mean retention of pro-vitamin A carotenoids in cooked biofortified pumpkin

AC

(>78%) was similar to previous reports for boiled sweet potato (Failla, Thakkar & Kim, 2009; Berni et al., 2015), cassava (Thakkar, Huo, Maziya-Dixon & Failla, 2009; Failla et al, 2012; Gomes et al., 2013) and boiled (Carvalho et al., 2014), pressure cooked (Provesi, Dias & Amante, 2011) and candied pumpkin (Chavasit et al., 2002). Our observation that home cooking style differentially affected retention of pro-vitamin A in the different genotypes of pumpkin (Table 1) is similar to recent reports for biofortifed cassava and sorghum (Failla et al., 2012; Lipkie et al., 2013; Berni et al., 2014). It is important to note that the genotypes of pumpkin investigated in the present study were grown in two different areas and in different years. Possible interactions

12

ACCEPTED MANUSCRIPT between genotype and pre-harvest conditions on the physicochemical properties of the pulp that may affect retention during cooking warrant investigation.

T

We also found that the efficiency of transfer of pro-vitamin A from the cooked pulp

IP

from the food matrix to micelles during small intestinal digestion was relatively

SC R

inefficient (i.e., < 4%) and lower for pulp from several genotypes boiled in water + 60% sucrose compared to water alone (Table 2). The basis for this adverse influence of sucrose on micellarization of pro-vitamin A in sweetened pumpkin is unknown. A

NU

possibility that merits investigation is that sucrose penetrates the pulp during boiling to

MA

sufficiently increase the viscosity of chyme such that access of digestive enzymes is decreased resulting in reduced release of pro-vitamin A carotenoids.

However, the

quantity of βC equivalents transferred to mixed micelles during simulated digestion of

TE

D

genotype 58 suggested the potential to provide more than 40% of the EAR of vitamin A for children 4-8 y of age consuming 100 x g (Food & Nutrition Board, 2001). The

CE P

relatively inefficient micellarization of pro-vitamin A in cooked pumpkin differs markedly from an earlier report that the efficiency of micellarization of βC and αC were

AC

18% and 25%, respectively, during digestion of boiled pulp from a pro-vitamin A rich variety of C. moschata. (Priyadarshani & Chandrika, 2007). The basis for the marked difference between the present results and the previous report is unknown. Low (<10%) efficiencies of micellarization of pro-vitamin A carotenoids have been reported for sweet potato (Failla, Thakkar & Kim, 2009; Berni et al., 2015; Mills et al., 2009) and transgenic sorghum (Lipkie et al., 2013), whereas micellarization of βC during in vitro digestion of biofortified boiled cassava roots from conventionally bred and transgenic cassava ranged from 10-45% (Thakkar, Maziya-Dixon & Failla, 2007; Thakkar, Huo, Maziya-Dixon & Failla, 2009; Failla et al., 2012; Berni et al., 2014) and was 17% in digested porridge prepared from conventionally bred, biofortified maize (Thakkar & 13

ACCEPTED MANUSCRIPT Failla, 2008). Despite the relatively low efficiency of micellarization of pro-vitamin A carotenoids for biofortified pumpkins, βC equivalents in the micelle fraction were

T

considerable (Table 2). As uptake of pro-vitamin A from micelles by Caco-2 cells was

IP

proportional to the amount in micelles, our results suggest that inefficient release of pro-

SC R

vitamin A from pulp is the limiting factor for pro-vitamin A bioavailability of biofortified pumpkin and that provitamin A content of raw pumpkin is not the sole factor that determines bioaccessible βC equivalents.

NU

An interesting question that emerges from our results is whether a greater percentage of βC equivalents in biofortified pumpkin can be released from the matrix during

MA

digestion. Transfer of βC equivalents from pulp steamed longer than is conventional in homes into mixed micelles during simulated digestion was significantly increased (Fig.

TE

D

3). Release of carotenes from cooked carrot requires rupture of cell walls prior to digestion (Tydeman et al., 2010). The physical state and subcellular localization of pro-

CE P

vitamin A carotenoids, as well as the effect of styles of processing and cooking on the integrity of pumpkin cell walls, are unknown. Cells in pulp from butternut squash

AC

(Cucubita moschata Poir), like those in sweet potato, have fibrous walls with tubulous chromoplasts that may store protein bound carotenes (Jeffery, Holzenburg & King, 2012). Chromoplast substructure and the physical state of the accumulated carotenoids affect bioaccessibility (Schweiggert et al., 2012). Chromoplasts in carrot cells contain large crystalloid aggregates of βC, whereas βC and other carotenoids appear to be dissolved in lipid and lipid crystalline complexes in cells of mango and papaya. These differences are associated with the greater bioaccessibility of βC in mango and papaya compared to carrot. Analysis of cellular-ultrastructure and the physiochemical state of pro-vitamin A in biofortified pumpkin is expected to provide insights for processing methods that may increase the bioaccessibility of pro-vitamin A. 14

ACCEPTED MANUSCRIPT It is also important to note that we added a single concentration of soybean oil (2.5% wt/wt) to cooked pulp immediately before initiating in vitro digestion. Lipkie et al.

T

(2013) reported that increasing canola oil content from 5% to 10% in porridge prepared

IP

from sorghum biofortified with pro-vitamin A resulted in as much as a 5-fold

SC R

improvement in micellarization of all-E-βC during simulated digestion. The possibility that the presence of greater quantities of oil to pumpkin prior to cooking or during ingestion might increase pro-vitamin A bioaccessibility from biofortified pumpkin

NU

merits consideration.

In conclusion, the results from this study show that βC equivalents were well

MA

retained in pulp from pumpkins biofortified with high concentrations of all-E-βC and αC when briefly steamed, boiled in water or boiled in water + 60% sucrose. Although

TE

D

transfer of the provitamin A carotenoids from cooked pulp to mixed micelles during simulated digestion was relatively inefficient, total βC equivalents in the bioaccessible

CE P

fraction of digested pulp from one genotype appeared to have the potential to provide children 4-8 years of age consuming 100 x g cooked pumpkin with more than 40% of

AC

the daily EAR of vitamin A. Future research with this pro-vitamin A rich food should focus on methods to increase release of pro-vitamin A from pulp.

ACKNOWLEDGEMENTS The authors thank HarvestPlus and the Ohio Agricultural Research and Development Center for financial support of experiments conducted at The Ohio State University, the Embrapa - Monsanto Research Fund for support of the BioFORT project, the Chagas Filho Foundation for Research Support, and the Coordination for the Improvement of Higher Education Personnel, Brazil, for defraying the travel and living

15

ACCEPTED MANUSCRIPT expenses of EMGR. The authors were solely responsible for experimental design, data

T

collection and analysis and preparation and submission of the manuscript.

IP

References

SC R

Association of Official Analytical Chemistry (AOAC) (1993). Peer Verified Methods Program, Manual on politics and procedures. AOAC, Arlington: AOAC. pp. 33-83. Azevedo-Meleiro, C. H. & Rodriguez-Amaya, D. B. (2007). Qualitative and

NU

quantitative differences in carotenoid composition among Curcubita moschata,

Chemistry, 55, 4027-4033.

MA

Curcubita maxima and Curcubita pepo. Journal of Agricultural and Food

Berni, P., Chitchumroonchokchai, C., Canniatti-Brazaca, S. G., De Moura, F . F. &

TE

D

Failla, M. L. (2014). Impact of genotype and cooking style on the content, retention, and bioacessibility of β-carotene in biofortified cassava (Manihot

CE P

esculenta Crantz) conventionally bred in Brazil. Journal of Agricultural and Food Chemistry, 62, 6677-6686.

AC

Berni, P., Chitchumroonchokchai, C., Canniatti-Brazaca, S. G., De Moura F. F. & Failla, M. L. (2015). Comparison of content and in vitro bioaccessibility of provitamin A carotenoids in home cooked and commercially processed orange fleshed sweet potato (Ipomea batatas Lam). Plant Foods and Human Nutrition, 70,1-8. Britton, G. (2011). UV/visible spectroscopy. In: G. Britton, S. L. Jensen & H. Pfander (Eds.), Carotenoids (pp. 13-62). Basal: Birkhauser. Carvalho, L. M. J., Gomes, P. B., Godoy, R. L. O., Pacheco, S., Monte, P. H. F., Carvalho, J.L. V., Nutti, M. R., Neves, A. C. L., Vieira, A. C. R. A. & Ramos, S. R. R. (2012). Total carotenoid content, α-carotene and β-carotene, of landrace 16

ACCEPTED MANUSCRIPT pumpkins (Cucurbita moschata Duch): A preliminary study. Food Research International, 47, 337-340.

T

Carvalho, L. M. J., Smiderle, L. A. S. M., Carvalho, J. L. V., Cardoso, F. S. N. &

IP

Koblitz, M. G. B. (2014). Assessment of carotenoids in pumpkins after different

SC R

home cooking conditions. Food Science and Technology, 34, 365-370. Chavasit, V., Pisaphab, R., Sungpuag, P., Jittinandana, S. & Wasantwisut, E. (2002). Changes in β-carotene and vitamin A contents of vitamin-A rich foods in Thailand

NU

during preservation and storage. Journal of Food Science, 6, 375-379.

MA

Chitchumroonchokchai, C., Schwartz, S. J. & Failla, M. L. (2004). Assessment of lutein bioavailability from meals and a supplement using simulated digestion and Caco-2 human intestinal cells. Journal of Nutrition, 134, 2280-2286.

TE

D

Curado, F. F., Queiroz, V. A. V., Dias, A. C. P., Silva, M. D. R. & Netto, R. S. M. (2009). Evaluation of the potential use of biofortified foods in school diets in

CE P

municipalities of Maranhao, Sergipe and Minas Gerais, Brazil. 3rd Annual Biofortification Meeting in Brazil. Aracaju, Brazil. htpp://www.biofort.com.br

AC

(accessed Nov. 20, 2013). Failla, M. L., Chitchumroonchokchai, C., Siritunga, D., De Moura, F. F., Fregene, M. & Sayre, R. (2012). Retention during processing and bioaccessibility of β-carotene in transgenic cassava root. Journal of Agricultural and Food Chemistry, 60, 3861-3866. Failla, M. L., Thakkar, S. K. & Kim, J. Y. (2009). In vitro bioaccessibility of betacarotene in orange fleshed sweet potato (Ipomoea batatas, Lam.). Journal of Agricultural and Food Chemistry, 57, 10922-10927. Fernández-Garcia, E., Carvajal-Lérida, I., Jarén-Galán, M., Garrido-Fernández, J., Pérez-Galvez, A. & Hornero-Méndez, D. (2012). Carotenoid bioavailability from 17

ACCEPTED MANUSCRIPT foods: From plant pigments to efficient biological activities. Food Research International, 46, 438-450.

T

Ferraz, I. S., Daneluzzi, J. C. Vannucchi, H., Jordao, A. A. Jr., Ricco, R. G., Del

IP

Ciampo, L. A., Martinelli, C. E. Jr., Engelberg, A. A, Bonilha, L. R. & Flores, H.

SC R

(2004). Detection of vitamin A deficiency in Brazilian preschool children using the serum 30-day dose-response test. European Journal of Clinical Nutrition, 58, 1372-1377.

NU

Food and Nutrition Board, Institute of Medicine. (2001). Dietary reference intakes for

MA

vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. Washington DC: National Academy Press (pp. 86-161).

TE

D

Garrett, D. A., Failla, M. L. & Sarama, R. J. (1999). Development of an in vitro digestion method to assess carotenoid bioavailability from meals. Journal of

CE P

Agricultural and Food Chemistry, 47, 4301-4309. Gomes, S., Torres A. G., Godoy, R., Pacheco S., Carvalho, J. & Nutti, M. (2013).

AC

Effects of boiling and frying on the bioaccessibility of β-carotene in yellowfleshed cassava roots (Manihot esculenta Crantz cv. BRS Jari). Food and Nutrition Bulletin, 34, 65-73. Jeffery, J. L., Holzenburg, A. & King, S. (2012). Physical barriers to carotenoid bioaccessibility: Ultra-structural survey of chromoplast and cell wall morphology in nine carotenoid-containing fruits and vegetables. Journal of the Science and Food Agriculture, 92, 2594-2602. Lemmens, L., Colle, I., Van Buggenhout, S., Palmero, P., Van Loey, A. & Hendrickx, M. (2014). Carotenoid bioaccessibility in fruit- and vegetable-based products as affected by product (micro) structural characteristics and the presence of lipids: A 18

ACCEPTED MANUSCRIPT review. Trends in Food Science and Technology, 38, 125-135. Lipkie, T. E., De Moura, F. F., Zhao, Z-Y., Albertsen, M. C., Che, P., Glassman, K. &

T

Ferruzzi, M. G.(2013). Bioaccessibility of carotenoids from transgenic pro-

IP

vitamin A biofortified sorghum. Journal of Agricultural and Food Chemistry,

SC R

61, 5764-5771.

Mercadante, A. Z. (2007). Carotenoids in foods: sources and stability during processing and storage. In S. E. Socaciu, Food Colorants: Chemical and Functional

NU

Properties (pp. 213-235). Boca Raton, FL: CRC Press.

Mills, J. P., Tumuhimbise, G. A., Jamil, K. M., Thakkar, S. K., Failla, M. L. &

MA

Tanumihardjo, S. A. (2009). Sweet potato β-carotene bioefficacy is enhanced by

TE

of Nutrtion, 139, 44-50.

D

dietary fat and not reduced by soluble fiber intake in Mongolian gerbils. Journal

Ministry of Health. Brazilian Center for Analysis and Planning. National Survey on

CE P

Demography and Health of Children and Women – PNDS/NRDHCW 2006: Dimensions of the reproduction and child health. Brazil, 2009.

AC

Murkovic, M., Mulleder, U. & Neunteufl, H. (2002). Carotenoid content in different varieties of pumpkins. Journal of Food Composition and Analysis, 15, 633638.

Nestel, P., Bouis, H. E., Meenakshi, J. V. & Pfeiffer, W. (2006). Biofortification of staple food crops. Journal of Nutrition, 136, 1064-1067. Priyadarshani, A. M & Chandrika, U. G. (2007). Content and in-vitro accessibility of pro-vitamin A carotenoids from Sri Lankan cooked non-leafy vegetables and their estimated contribution to vitamin A requirement. International Journal of Food Sciences and Nutrition, 58, 659-667. Provesi, J. G., Dias, C. O. & Amante, E. R. (2011). Changes in carotenoids during 19

ACCEPTED MANUSCRIPT processing and storage of pumpkin puree. Food Chemistry, 128, 195-202. Reboul, E., Richelle, M., Perrot, E., Desmoulins-Malezet, C. Pirisi, V. & Borel, P.

T

(2006). Bioaccessibility of carotenoids and vitamin E from their main dietary

IP

sources. Journal of Agricultural and Food Chemistry, 54, 8749-8755.

SC R

Rodriguez-Amaya, D. B., Kimura, M., Godoy, H. T. & Amaya-Farfan, J. (2008). Updated Brazilian database on food carotenoids: Factors affecting carotenoid composition. Journal of Food Composition and Analysis, 21, 445-463.

NU

Saltzman, A., Birol, E., Bouis, H.E., Boy, E., De Moura, F. F., Islam, Y. & Pfeiffer, W.

MA

H. (2013). Biofortification : Progress toward a more nourishing future. Global Food Security, 2, 9-17.

Schweiggert, R.L.; Mezger, D.; Schimpf, F.; Steingass, C.B.; Carle, R. (2012).

TE

D

Influence of chromoplast morphology on carotenoid bioaccessibility of carrot, mango, papaya, and tomato. Food Chemistry, 135, 2736-2742.

CE P

Seo, J. S., Burri, B. J., Quan, Z. & Neidlinger, T.R. (2005). Extraction and chromatography of carotenoids from pumpkin. Journal of Chromatography. A,

AC

1073, 371- 375.

Sommer, A. & Vyas, K. S. (2012). A global clinical view on vitamin A and carotenoids. American Journal of Clinical Nutrition, 96, 1204S-1206S. Thakkar, S. & Failla, M.L. (2008). Bioaccessibility of pro-vitamin A carotenoids is minimally affected by non-pro-vitamin A xanthophylls in maize (Zea mays sp.). Journal of Agricultural and Food Chemistry, 56, 11441-11446. Thakkar, S. K., Huo, T., Maziya-Dixon, B. & Failla, M. L. (2009). Impact of style of processing on retention and bioaccessibility of β-carotene in cassava (Manihot esculanta, Crantz). Journal of Agricultural and Food Chemistry, 57, 13441348. 20

ACCEPTED MANUSCRIPT Tydeman, E. A., Parker, M. L., Wickham, M. S. J., Rich, G. T., Faulks, R. M., Gidley, M. J., Fillery-Travis, A. & Waldron, K. W. (2010). Effect of carrot (Daucus

T

carota) microstructure on carotene bioaccessibilty in the upper gastrointestinal

IP

tract. 1. In vitro simulations of carrot digestion. Journal of Agricultural and Food

SC R

Chemistry, 58, 9847-9854.

Thakkar, S. K., Maziya-Dixon, B., Dixon, A. G. O. & Failla, M. L. (2007). β-Carotene micellarization during in vitro digestion and uptake by Caco-2 cells is directly

NU

proportional to β-carotene content in different genotypes of cassava. Journal of

MA

Nutrition, 137, 2209-2233.

WHO. (2009). Global prevalence of vitamin A deficiency in populations at risk 1995-2005. WHO Global Database on Vitamin A Deficiency. Geneva: World

TE

D

Health Organization. 49pp.

WHO. (2011) Guideline: Vitamin A supplementation in infants and children 6–59

CE P

months of age. Geneva: World Health Organization. 29 pp. Yonekura, L. & Nagao, A. (2007) . Intestinal absorption of dietary carotenoids.

AC

Molecular Nutrition and Food Research, 51, 107-115.

21

ACCEPTED MANUSCRIPT Figure Legends Fig. 1. Profile of Z-βC in raw and cooked pumpkin. Data are mean ± SEM, n=4.

T

Different letters as superscripts above the bars for each genotype indicate that the

IP

estimated quantity of the Z-isomers in raw and cooked pumpkin differ significantly (p<

SC R

0.01)

Fig. 2. Uptake of all-E-βC and αC from medium containing micelles generated during

NU

simulated digestion of 1g (FW) cooked pumpkin by Caco-2 cells. Data are mean ± SEM, n = 4. Different lower case letters above bars indicate means for all-E-βC differ

MA

significantly across genotypes and cooking styles. Different upper case letters above bars indicate that means for αC differs significantly across genotypes and cooking styles

D

(p< 0.001).

TE

Fig. 3. Effect of increased cooking time on amount of bioaccessible βC equivalents in digested steamed (p< 0.001) and boiled (p<0.05) pulp from commercial pumpkin. Data

CE P

are mean ± SEM, n = 4. Significantly different means are indicated by presence of

AC

difference letters above bars for each cooking style (p  0.05).

22

ACCEPTED MANUSCRIPT Table 1. Pro-vitamin A content (µg/g FW) of raw and cooked pulp from biofortified

T

pumpkin. *βC equivalents = all-E-βC + ½ (Z-βC + αC). Data are mean ±

IP

SEM, n=4. Different letters as superscripts in column indicate significantly

Cooking method

346

387 ± 2.8ª

84 ± 1.6a

186 ± 2.9b

βC equivalents* 522 ± 5a

375 ± 3.2b

51 ± 1.6c

178 ± 6.6b

490 ± 7b

Steamed

72 ± 0.0

559 ± 9.0d

327 ± 7.0d

72 ± 3.9b

159 ± 2.3c

443 ±10cd

Boiled + sugar

71 ± 0.4m

625 ± 8.0b

365 ± 7.8c

73 ± 1.0b

187 ± 5.5b

495 ± 11b

Raw

80 ± 0.2h

459 ± 5.4h

230 ± 2.9e

23 ± 0.3e

206 ± 4.1a

345 ± 5g

Boiled

82 ± 0.7f

382 ± 1.3j

223 ± 0.6f

19 ± 0.4f

141 ± 1.0e

302 ± 1h

Steamed

79 ± 0.1i

357 ± 6.3k

187 ± 1.1i

19 ± 0.6f

150 ± 5.6d

272 ± 4i

Boiled + sugar

81 ± 0.1g

400 ± 4.2i

204 ± 5.3h

13 ± 1.0i

183 ± 3.9b

302 ± 8h

Raw

83 ± 0.2e

460 ± 3.3h

336 ± 3.1d

15 ± 0.3h

110 ± 4.4f

399 ± 6f

Boiled

84 ± 0.4d

498 ± 4.9f

365 ± 5.2c

19 ± 0.5f

114 ± 3.6f

432 ± 7de

Steamed

83 ± 0.1e

D

NU

604 ± 9.5c

487 ± 1.9g

359 ± 1.7c

18 ± 1.0fg

110 ± 4.8f

423 ± 5e

Boiled + sugar

78 ± 0.0

j

514 ± 2.7e

375 ± 2.0b

25 ± 0.3d

114 ± 2.0f

444 ± 3c

Raw

86 ± 0.3c

229 ± 2.3n

204 ± 3.1h

5 ± 0.3m

20 ± 2.4l

223 ± 4k

88 ± 0.0a

244 ± 3.9m

206 ± 5.9h

14 ± 0.9hi

27 ± 0.1k

225 ± 6k

b

244 ± 7.4m

202 ± 8.8h

13 ± 0.5j

29 ± 1.5j

223 ± 10k

Boiled + sugar

79 ± 0.1i

245 ± 3.2m

200 ± 3.8h

17 ± 0.1g

29 ± 0.1j

223 ± 4k

Raw

86 ± 0.2c

261 ± 1.6l

212 ± 2.3g

6 ± 0.7m

43 ± 0.3h

237 ± 3j

Boiled

87 ± 0.1

b

264 ± 9.3l

214 ± 10.5fg

14 ± 0.9hi

36 ± 4.5i

239 ± 13j

Steamed

86 ± 0.0c

209 ± 3.0o

159 ± 2.3j

10 ± 0.6l

40 ± 0.1i

184 ± 3l

Boiled + sugar

81 ± 0.1g

269 ± 4.7l

209 ± 10.5g

12 ± 0.3k

47 ± 2.2g

239 ± 12j

Boiled Steamed

AC

129

αC

l

CE P

58

all-Z-βC

658 ± 1.2a

Boiled

13

all-E-βC

72 ± 0.1l 74 ± 0.3k

Raw 12

Pro-vitamin A Carotenoids

MA

Moisture

TE

Genotype

SC R

different means (p< 0.01).

87 ± 0.0

23

ACCEPTED MANUSCRIPT Table 2. Estimated bioaccessibility of pro-vitamin A in a 100g serving of cooked pumpkin.* βC

T

equivalents = all-E-βC + ½ (Z-βC + αC). Data are mean ± SEM for n = 4 independent

IP

digestions. Different upper case letters as superscripts in the column indicate significantly

SC R

different means (p<0.05).

Cooking method

Bioaccessible pro-vitamin A (µg/100g) all-E-βC Z- βC C

12

Boiled Steamed Boiled + sugar

755 ± 28b 288 ± 8e 140 ± 10g

13

Boiled Steamed Boiled + sugar

376 ± 15d 349 ± 25d 81 ± 2h

58

Boiled Steamed Boiled + sugar

1180 ± 44a 1186 ± 24a 1119 ± 91a

129

Boiled Steamed Boiled + sugar

346

Boiled Steamed Boiled + sugar

βC equivalents*

531 ± 41b 139 ± 10g 68 ± 3j

1137 ± 23c 379 ± 11fg 268 ± 9i

165 ± 4f 253 ± 2e 56 ± 1k

500 ± 12e 485 ± 24e 119 ± 2k

81 ± 8d 47 ± 2e 100 ± 7c

600 ± 25a 329 ± 9d 392 ± 25c

1520 ± 37a 1374 ± 27b 1366 ± 89b

284 ± 17e 306 ± 10e 314 ± 22e

18 ± 2h 21 ± 1g 107 ± 4c

64 ± 3j 94 ± 5h 53 ± 4k

326 ± 18h 364 ± 11g 393 ± 21f

486 ± 14c 142 ± 12g 258 ± 24f

17 ± 1i 14 ± 1j 84 ± 7d

80 ± 4i 40 ± 2l 40 ± 2l

534 ± 14d 169 ± 12j 320 ± 22h

NU

232 ± 20a 43 ± 1f 187 ± 13b

TE

D

MA

84 ± 3d 19 ± 1h 19 ± 2gh

AC

CE P

Genotype

24

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 1

25

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 2

26

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 3

27

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Graphical abstract

28

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

29

ACCEPTED MANUSCRIPT

IP

T

Ribeiro et al. Effect of Style of Home Cooking on Retention and Bioaccessibility of Pro-Vitamin A Carotenoids in Biofortified Pumpkin (Cucurbita moschata Duch.)

SC R

Highlights

AC

CE P

TE

D

MA

NU

 Several tested genotypes of C. moshata contained more than 400 mg/kg provitamin A.  Pro-vitamin A retention in pulp cooked by common Brazilian home styles was 78%.  Bioaccessible βC equivalents/100g of one cooked pulp was > 40% EAR for 4-8y child.  Methods for more efficient release of pro-vitamin A in pumpkin merit investigation.

30