In Vitro Evaluation of Cu, Fe, and Zn Bioaccessibility in the Presence

J. Agric. Food Chem. , 2015, 63 (28), pp 6331–6337. DOI: 10.1021/acs.jafc.5b01947. Publication Date (Web): July 2, 2015. Copyright © 2015 American ...
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In vitro evaluation of Cu, Fe and Zn bioaccessibility in presence of babassu mesocarp Alexandre M Fioroto, Angerson N Nascimento, and Pedro Vitoriano Oliveira J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01947 • Publication Date (Web): 02 Jul 2015 Downloaded from http://pubs.acs.org on July 9, 2015

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Journal of Agricultural and Food Chemistry

In vitro evaluation of Cu, Fe and Zn bioaccessibility in presence of babassu mesocarp

Alexandre Minami Fiorotoa, Angerson Nogueria Nascimentob, Pedro Vitoriano Oliveiraa*

a

Instituto de Química, Universidade de São Paulo, CEP 05508-000, São Paulo, SP, Brazil.

b

Instituto de Ciências Ambientais, Químicas e Farmacêuticas,Universidade Federal de São Paulo, CEP 0641-5080, Diadema, SP, Brazil.

* Corresponding author address: (Tel.: +55 11 3091 8516; Fax: +55 11 3091 2161; E-mail: [email protected])

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ABSTRACT

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In vitro gastrointestinal digestion of babassu mesocarp in absence and

3

presence of milk and lignin was performed to evaluate the bioaccessibility

4

of Cu, Fe and Zn. Extractions using NaOH solutions (pH 7 and 12) were

5

carried out to evaluate the interactions of CuII, FeIII and ZnII with the

6

extracted compounds and with the washed mesocarp. The studies using

7

reference solutions showed a decrease in the free concentration of the

8

elements in the presence of mesocarp. Phytate, a component present in the

9

mesocarp, can be the main compound responsible for the elements

10

interactions with mesocarp. Lignin increases the elements soluble fractions;

11

however, the elements concentrations in the dialysed fractions, representing

12

the bioaccessible portion, were very low. On the other hand, Cu, Fe and Zn

13

bioaccessibility in milk was not influenced by mesocarp.

14 15

Keywords: Babassu, mesocarp; bioaccessibility; gastrointestinal digestion;

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copper; iron; zinc

17 18 19

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INTRODUCTION

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Babassu (Orbignya phalerata) is a Brazilian palm that grows in the

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wild of Northeast Brazil, which can mainly be found in Maranhão and

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Piauí states. The babassu palm has many applications and every part can be

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used, but nuts claim for the most attention, owing to their nutritional and

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commercial value. The babassu palm produces hard nuts that are an

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important source of oil, which can be used as a food, lubricant and biofuel,

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as well as in the cosmetic industry.1, 2

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Beside the kernels, the mesocarp is another part of babassu that has

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been added to animal feeding, as an alternative energy source 3 and has also

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been included in the human diet. It is used in the preparation of cakes,

31

breads and porridges, or it can be added to juices and milk. Babassu

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mesocarp has been used to replace 20% of wheat flour in the preparation of

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breads and added to milk for preparing a drink similar to chocolate milk.4

34

However, the babassu mesocarp contains a large amount of anti-nutritional

35

compounds (e.g. phytate and lignin), which may interact with mineral

36

nutrients, decreasing their bioavailability.5

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Phytic acid, for instance, can be found in abundance in cereals,

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legumes, oil seeds and nuts, comprising 1–5% by their weight.6 It has a

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function of reserving reactive phosphate groups and for energy storage in

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plants.7 At pH values close to intestinal region, phytic acid is strongly 3

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ionisable and has a high density of negative charge of phosphate groups,

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which enables it to interact with metal ions and form stable complexes,

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making them unavailable for absorption by human organism.8, 9

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The fibres are a class of compound derived from plants, which are

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not digested or absorbed by human organism. These fibres include

46

polysaccharides, oligosaccharides, lignin and other substances associated

47

with plants. All of them may negatively influence the bioavailability of

48

essential elements.8

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Lignin is a polyphenolic macromolecule present in cereal, fruits and

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vegetables. The rigid structure of lignin becomes resistant to digestion. In

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addition, it also has the ability to bind to metals ions, decreasing their

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bioavailability.10-12

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Several in vivo methods are used to estimate the bioavailability of

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nutrients, such as chemical balance, rate of depletion, plasma appearance

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and methods using radioisotopes or stable isotopes.13,14 On the other hand,

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in vitro methods are simpler, faster, lower cost and can provide information

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that is useful for planning in vivo experiments. Moreover, in vitro methods

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can provide information that cannot be achieved in animal studies due to a

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greater control of experimental conditions and a possibility of different

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digestion stages evaluation. In vitro methods provide information of

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bioaccessibility (or availability) when physiological factors are not 4

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included.14 Nevertheless, an additional step using cell culture has been

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employed to the in vitro assay to simulate even more similar conditions to

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the organism, including physiological factors to the experiment. Caco-2

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cells, for instance, have been used for this proposal due to exhibit

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morphological and functional properties similar to intestinal cells

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responsible for nutrients absorption. Therefore, relative bioavailability

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information can be also achieved by in vitro methods.14-17

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In this context, studies have been carried out in order to evaluate the

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anti-nutritional compounds effects on mineral nutrients bioavailability/

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bioaccessibility.6-11,18,19 The low Ca bioaccessibility present in some Indian

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green leafy vegetables was correlated to anti-nutritional compounds

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contents.9 In other study, the factors responsible for inhibiting Fe and Zn

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bioaccessibility in pearl millet were accessed using phytate- and/or fibres-

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degrading enzymes incubation of insoluble residues remained from in vitro

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digestion. This study enabled to identify which compounds were chelated

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to the elements.18

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Some food with high nutrients content has been used as a supplement

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to meals in order to improve nutritional values. Nevertheless, anti-

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nutritional compounds (phytate, fibres, lignin and others) can affect the

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expected result of these supplementations. For example, bread prepared

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with amaranth flour (which has high content of iron) was evaluated using 5

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bioavailability studies in order to verify the contribution of this

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supplementation on its nutritional value. Furthermore, the bioavailable iron

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fraction was not increased due to the high phytate content in amaranth

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flour.19

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The babassu mesocarp has been used as food supplement, but its

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composition is not well known and also its effect over nutrients

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bioacessibility. This work aimed to study methods for assessing the

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interactions of Cu, Fe and Zn with mesocarp and its effect over the

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bioaccessibility of these elements in dry whole milk and lignin, with and

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without babassu mesocarp as supplement, using simulated gastrointestinal

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digestion.

94 95

MATERIAL AND METHODS

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Apparatus. An inductively coupled plasma optical emission spectrometer

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(ICP OES, iCAP 6300 Duo, Thermo Fisher Scientific, Cambridge,

98

England) equipped with axial and radial viewed plasma, a charge-injection

99

device (CID) detector, an Echelle polychromator, and a radiofrequency

100

source of 27.12 MHz was used for Cu, Fe, Zn, Ca and Mg determination.

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The instrumental conditions for the analysis were 1250 W of power, 12 L

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min-1 of plasma gas, 0.5 L min-1 of auxiliary gas and 0.2 L min-1 of

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nebuliser gas. A Babington nebuliser combined with a cyclonic spray 6

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chamber was used as sample introduction system. The selected

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wavelengths were 184.006 nm (Ca), 324.754 nm (Cu), 259.940 nm (Fe),

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285.213 nm (Mg) and 202.548 nm (Zn).

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A simultaneous atomic absorption spectrometer (SIMAA-6000®,

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PerkinElmer, Norwalk, CT, USA) equipped with a longitudinal Zeeman-

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effect background corrector, Echelle optical arrangement, solid-state

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detector and a standard THGA (transversely heated graphite atomiser) tube

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with a pyrolytically coated integrated platform was used when the analysis

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required a greater sensitivity, for instance in the determination of Cu and

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Fe. The spectrometer was operated using hollow cathode lamps and the

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solutions were delivered into the graphite tube by means of an AS-72

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autosampler. The instrumental conditions and heating program are shown

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in Table 1.

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Samples and standard reference materials (SRM) were digested using

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a high-pressure microwave oven (Speedwave® 4, Berghof, Eningen,

119

Germany) equipped with 12 TFMTM-PTFE vessels (Model DAC-17) and

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infrared sensors for individual temperature and pressure measurements.

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Mesocarp and milk drying procedures were carried out in a drying

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oven, Model Q317M-32 (Quimis®, São Paulo, SP, Brazil). A shaker table,

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Model Q225M (Quimis®), was used to solutions homogenisation and

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stirring during the extractions procedures. For simulated gastrointestinal 7

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digestion, a water bath at 36 ºC was used at 90 rpm for 120 min (Model

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Q226M2, Quimis®). A centrifuge, Model Q222TM (Quimis®), were used

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for phase separation. A pH meter, Model PH-710 (Unity, Guarulhos, SP,

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Brazil) was used for pH adjustment of the samples.

129 130

Reagents and samples. All solutions were prepared with high-purity

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deionised water (18.2 MΩ cm) obtained in a Milli-Q® water purification

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system (Millipore, Belford, MA, USA).

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Reference solutions were prepared from Titrisol® standard analytical

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solutions containing 1,000 mg L-1 iron (FeCl3), copper (CuCl2), zinc

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(ZnCl2), calcium (CaCl2) and magnesium (MgCl2) (Merck, Darmstadt,

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Germany) after dilution in HNO3 0.1% (v v-1) (Merck).

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Nitric acid 65% (w v-1) (Merck) and hydrogen peroxide 30% (w v-1)

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(Merck) were used for sample digestion in a microwave oven, and

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hydrochloric acid (Merck) and sodium hydroxide (Merck) solutions were

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used for pH adjustments.

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Gastrointestinal fluids were prepared using NaCl (Merck), pepsin

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(CAS Number: 9001-75-6) (Sigma-Aldrich, Saint Louis, USA), HCl

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(Merck), NaHCO3 (Reagen, Rio de Janeiro, Brazil), K2HPO4 (Synth®,

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Diadema, Brazil), NaOH (Merck), pancreatin (CAS Number: 8049-47-6)

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(Sigma-Aldrich) and bile salts (Sigma-Aldrich). 8

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The dialysis procedure was performed using cellulose membranes (Sigma Aldrich) in 32 mm tubes with 12.4 kDa porosity.

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To check the accuracy of the analytical method used for the total

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determination of the elements, standards reference materials (SRM) of

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peach leaves (SRM® 1547) and bovine liver (SRM® 1577c) from the

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National Institute of Standards and Technology (NIST) were analysed.

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Babassu mesocarp flour samples, purchased in Maranhão state

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communities, dry milk (Ninho®, Nestlé, São Paulo, Brazil), alkaline lignin

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powder (Sigma Aldrich) and phytic acid solution 50% (w w-1) (Sigma-

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Aldrich) were used in simulated gastrointestinal digestion assays.

156 157

Procedure. For the total determination of the elements, a mass of 200 mg

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of mesocarp, milk or SRMs (SRM® 1547 and SRM® 1577c) were digested

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in a microwave oven with 2.0 mL of HNO3 + 1.0 mL of H2O2 + 3.0 mL of

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H2O, using a heating program in three steps (1st step: temperature = 140 ºC;

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ramp = 5 min; hold = 1 min, 2nd step: temperature = 180 ºC; ramp = 4 min;

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hold = 5 min, 3rd step: temperature = 200 ºC; ramp = 4 min; hold = 10 min).

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After digestion, the samples were diluted with deionised water until a final

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volume of 10 mL and analysed by ICP OES for Fe, Zn, Ca and Mg

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determination. The same solutions were used for Cu determination in a

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graphite furnace (SIMAAS-6000®). The calibrations for both techniques 9

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were prepared using reference solutions in equivalents acid concentration

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mediums of digestion procedure (HNO3 2% v v-1). The determination of Ca

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and Mg was performed in the mesocarp and milk because these elements

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are considered macronutrients, and consequently if their presence during

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the process can cause any influence on the bioaccessibility of Cu, Fe and

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Zn.

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A mass around 2 g of babassu mesocarp was used to extract phytate

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and other complexing substances with 200 mL of water at pH 7 and pH 12,

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adjusted with NaOH. The mixtures were agitated on a shaker table for 30

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min (400 rpm) and the mesocarp extract was separated using simple

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filtration. The residue from the extraction at pH 12 was washed again with

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NaOH (pH 12), and subsequently washed with three aliquots of water (200

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mL) and later dried at 60 ºC.

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To confirm the extraction of phytate, a standard solution of phytic

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acid was extracted from mesocarp (obtained at pH 7 and 12) and was

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scanned in a range between 260 and 800 nm by UV molecular absorption

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spectrophotometry. The pH of each solution was adjusted to 7 before the

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scan.

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Aliquots of Cu, Fe and Zn reference solutions were added to 2 mL of

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extracts, the pH was adjusted to 7 and diluted with water until to 3 mL for a

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final concentration in the 10 to 300 mg L-1 range. Then, the solutions were 10

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homogenised on a shaker table for 30 min (400 rpm), centrifuged at 1700

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rpm and the supernatants were analysed by ICP OES. A similar procedure

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was used for the washed mesocarp, adding Cu, Fe and Zn reference

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solutions to 50 mg of washed mesocarp and diluting until to 5 mL.

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The concentration of Cu, Fe and Zn were also determined in the

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extracts and in a blank prepared for washed mesocarp additions. Thus, the

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recoveries of additions were calculated following: (obtained concentration

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– blank concentration) / added concentration) x 100%.

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The in vitro gastrointestinal digestion procedure was employed to

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assess the bioaccessibility of the mineral nutrients (Cu, Fe and Zn) using

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fluids prepared according to US Pharmacopeia XXIV recommendations.20

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The gastric fluid was prepared by dissolving 0.2 g of NaCl and 0.32 g of

200

pepsin in water. Afterwards, 7.0 mL of HCl 10% (v v-1) was added, and this

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solution was diluted to 100 mL with deionised water. The intestinal fluid

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was prepared by dissolving 0.68 g of K2HPO4, 1.0 g of pancreatin, 1.25 g

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of bile salts and 7.7 mL of NaOH (0.2 mol L-1) and diluted in deionised

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water to 100 mL.

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The general procedure followed in order to simulate gastrointestinal

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digestion using 3.0 mL of gastric fluid (pH ~ 1.5) added to 200 mg of

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mesocarp. The mixture was shaken continuously (100 rpm) in a

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thermostatic bath at 36 ºC for 2 h. To proceed with the intestinal digestion 11

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step, it was necessary to add 0.4 mL of NaHCO3 3% (w v-1) in order to

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adjust the pH to 6.8. Then, 3.0 mL of intestinal fluid (pH ~ 6.5) was added

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and the extract solution was shaken again using the same conditions

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reported in the gastric step and centrifuged to separate the supernatant.

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Dialysis procedure was performed in order to simulate nutrient absorption

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by human organisms. The supernatants (2 mL) were added into bags

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formed from cellulose membranes, tied with polyethylene tapes and

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immersed into tubes filled with 30 mL of water. After 12 hours, the

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membrane was carefully opened and the solution was collected from inside

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and analysed. The dialysed fraction was determined by subtracting the

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retained concentration in membrane from the soluble fraction.21 The varied

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conditions established to investigate the Cu, Fe and Zn bioaccessibility by

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in vitro gastrointestinal assays are shown in Table 2.

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The calibrations for analyzes of in vitro gastrointestinal digestions

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samples by ICP OES and SIMAAS were prepared using reference solutions

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in HNO3 0.1% (v v-1). For ICP OES analyzes, matrix interference effect

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was evaluated by addition and recovery test. Furthermore, for Fe and Cu

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determination by SIMAAS, matrix interference effect was evaluated by

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comparing calibrations curves using reference solutions in sample, blank

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(gastrointestinal fluid) and HNO3 0.1% (v v-1).

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RESULTS AND DISCUSSION

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Total determination of elements. The concentration of Cu, Fe, Zn, Ca,

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and Mg in mesocarp and milk samples was determined by ICP OES, after

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microwave oven acid digestion (Table 3). Furthermore, in Table 3 are also

234

shown the method accuracy evaluation by SRMs analysis and

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addition/recovery test results. The results of SRMs analyses present

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recoveries ranging from 83% to 99%. Good recoveries (84% to 116%)

237

were also obtained in addition/recovery test (Table 3).

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The concentrations of Fe and Ca in milk were similar to the

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information contained in the manufacturer's label (200 µg g-1 Fe and 9.4 mg

240

g-1 Ca). There was no information about other elements in milk label to be

241

compared. However, except Cu and Fe, all results were in agreement with

242

those described by the Brazilian Table of Food Composition (TACO) (1.1

243

µg g-1 Cu, 5.0 µg g-1 Fe, 27 µg g-1 Zn, 8.9 mg g-1 Ca and 0.77 mg g-1 Mg).22

244

The concentration of Fe was about 40 times greater than the TACO value,

245

owing to the milk sample being enriched with Fe pyrophosphate.

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Comparing the obtained concentrations for mesocarp with values

247

previously determined by our group (2.1 µg g-1 Cu, 16µg g-1 Fe, 2.4 µg g-1

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Zn, 432 µg g-1 Ca and 679 µg g-1 Mg),23 differences could be observed for

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the Cu and Mg concentrations, but for other elements, the results were

250

similar. When comparing the results with the TACO table values (2.2 µg g13

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1

Cu, 183 µg g-1 Fe, 3 µg g-1 Zn, 610 µg g-1 Ca and 390 µg g-1 Mg), only Zn

252

present similar values. These differences may be related to the factors that

253

can modify its mineral composition, such as origin, soil and climate.23

254

Reference daily intakes (RDIs) for essential human nutrition

255

elements have been established by Food and Drug Administration (FDA),

256

for instance Cu (2 mg), Fe (18 mg), Zn (15 mg), Ca (1000 mg) and Mg

257

(400 mg).24 Considering a portion of 26 g (two spoons) of mesocarp

258

consumed, the contribution percentage to RDIs are 5%, 2%, 1%, 1% and

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4% for Cu, Fe, Zn, Ca and Mg, respectively.

260 261

Evaluation of Cu, Fe and Zn interaction with the extract of babassu

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mesocarp. The mesocarp extract obtained at pH 12 had a darker brown

263

colour than that obtained at pH 7. This difference must be related to the

264

greater ability of the pH 12 solution to extract organic acids (mainly phytic

265

acid) that are bound to the matrix.

266

Phytic acid could be the main complexing substance extracted from

267

mesocarp, as this compound is found in high concentrations in vegetables,

268

because it has the function of storing phosphorus and energy. Phytic acid

269

also has the ability to bind to metal ions, forming stable complexes. 6-8

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Spectrophotometric analysis of the extracts showed that the spectra

271

of phytic acid and the extracts were similar, and both had a maximum 14

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absorbance at 272 nm, demonstrating the presence of this substance in the

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extract.

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The addition of elements in both extracts (pH 7 and 12 solutions)

275

was done with the final pH adjusted to 7, which is similar to intestinal

276

condition. After the addition of elements to the extracts, a brown precipitate

277

was formed and extract solutions discoloration was observed. In aqueous

278

solutions at pH 7, FeIII and CuII may hydrolyse and precipitate as

279

hydroxides. However, even with the possibility of FeIII and CuII hydrolysis,

280

the precipitate formed in the extract from mesocarp was not only provided

281

by hydrolysis of these ions, but also by complexation with phytate. The

282

discoloration of the extract solutions and the increasing volume of the

283

precipitate indicated that the compound present in the solution had

284

precipitated together with the elements.

285

The recovery of elements added (determined in supernatant) to the

286

babassu mesocarp extracts from pH 7 (Figure 1a) and pH 12 (Figure 1b)

287

solutions, after centrifugation, are shown in Figure 1. If only hydrolysis

288

was considered, high Cu and Fe recoveries were not expected, especially

289

for lower addition concentrations. However, the results show that

290

complexes formed between these elements and phytate until a certain

291

concentration, minimising the effect of hydrolysis, making them more

292

soluble and increasing recovery. The recovery is higher for the extract at 15

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pH 12 (Figure 1b), because it contains a higher concentration of phytate. It

294

is well known that the higher the concentration of cations bound to phytate,

295

the lower complex solubility. For example, mono-ferric phytate is highly

296

soluble, whereas tetra-ferric phytate is insoluble. 25

297

This experiment demonstrates that the phytate extract of the babassu

298

mesocarp can strongly interact with CuII and FeIII, and less so with ZnII, at

299

pH 7.

300 301

Evaluation of Cu, Fe and Zn interaction with washed mesocarp. The

302

recovery of CuII, FeIII and ZnII added to the washed mesocarp is shown in

303

Figure 1c. These additions were carried out in order to evaluate the

304

behaviour of these ions in the presence of solid mesocarp, after weakly

305

bound complexing compounds have been removed during the washing. It

306

can be seen that the recoveries of Cu and Fe in the supernatants were less

307

than 20%. These low recoveries cannot prove their interaction with washed

308

mesocarp because they can be attributed to the hydrolysis and precipitation

309

of CuII and FeIII at pH 7. On the other hand, at this pH the ZnII does not

310

hydrolyze. However, at lower added element concentrations, lower

311

recoveries for Zn were observed, demonstrating a possible interaction with

312

the complexing compounds that were not extracted from the mesocarp. The

313

increase in recovery for may be indicative of saturation of the binding sites 16

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in the mesocarp compounds. Therefore, low concentration of Zn was able

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to interact with mesocarp occupying all binding sites. Thus, when a higher

316

concentration was added, the excess of Zn remained in solution.

317 318

In vitro gastrointestinal digestion of babassu mesocarp. The addition

319

and recovery test shows that ICP OES analyses did not present significant

320

matrix interference effect. The obtained recoveries range from 87% to

321

107%. Matrix interference was also not observed for SIMAAS analyses.

322

The ratio between the slopes obtained from HNO3 0.1% (v v-1) solutions

323

and sample and blank mediums range from 1.0 to 1.2, thus no significant

324

difference was observed, allowing to calibrate with reference solutions in

325

HNO3 0.1% (v v-1).

326

The gastrointestinal digestion of babassu mesocarp showed that Fe

327

and Zn present in this material are not extracted, but Cu presented a soluble

328

fraction (96%). This indicates that, besides the presence of Fe and Zn in the

329

babassu mesocarp, as shown in Table 2, these elements are not

330

bioaccessible.

331

The supernatants of the gastrointestinal digestions with CuII, FeIII and

332

ZnII addition exhibited a dark colour, possibly caused by the extraction of

333

phytic acid and subsequent complexation with elements like Fe, as

334

observed in previous results. 17

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Table 4 presents the recoveries of Cu, Fe and Zn that were added to

336

the babassu mesocarp during gastrointestinal digestion, without and with

337

the addition of CaII and MgII. These elements were added in concentrations

338

similar to those present in milk. Without Ca and Mg, 65% of the Cu, 48%

339

of the Fe and 75% of the Zn remained in the supernatant solution. It is

340

likely that the other fraction of Cu, Fe and Zn were bound to the

341

compounds present in the mesocarp or precipitate. With the addition of 67

342

µg of MgII to the simulated gastrointestinal digestion, the recovery values

343

were not significantly changed (65% for Cu, 49% for Fe and 73% for Zn).

344

However, with the addition of 880 µg CaII, FeIII and ZnII recoveries

345

undergo alteration, decreasing to 41% for Fe and 56% for Zn. In this case,

346

the concentration of soluble Cu was also almost the same under all

347

conditions.

348

Upon the addition of CaII, the colour of the solution was clearer than

349

that without CaII. The dark colour of the solution can be related to soluble

350

phytate complexes. Therefore, discoloration caused by the addition of CaII

351

indicates a decrease in phytate complex solubility. Calcium forms

352

complexes with phytate, and these complexes (Ca–phytate) can bind ZnII

353

and FeIII, forming complexes that are even more insoluble than just Fe–

354

phytate and Zn–phytate complexes.25

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In vitro gastrointestinal digestion of milk with mesocarp and lignin. In

357

principle, the studies performed to evaluate the bioaccessibility of the

358

elements with babassu mesocarp were all utilised standard solutions, which

359

were very important to a better understanding of the system.

360

To simulate a situation closer to reality, tests were conducted to

361

assess the bioaccessibility of Cu, Fe and Zn in the gastrointestinal digestion

362

of whole dried milk and milk in the presence of mesocarp and lignin. The

363

Cu, Fe and Zn bioaccessibility values are shown in Figure 2. In each case,

364

the first column in the graphs (white bar) refers to the total concentration of

365

elements present in the weighed mass of milk and mesocarp. The second

366

column (gray bar) represents the soluble fractions obtained during

367

gastrointestinal digestions, after centrifugation and analysis of supernatant,

368

and the last column (black bar) represents the concentration of these

369

elements dialysed in the cellulose membrane (the fraction that migrated

370

throughout membrane).

371

The concentration of Cu in milk is low, but it is fully bioaccessible,

372

as shown in Figure 2a. A comparison of the total and soluble Fe and Zn

373

contents (Figures 2b and 2c) shows that they are not fully solubilised

374

during the milk gastrointestinal digestion. Furthermore, after dialysis these

375

elements were completely retained inside the membrane, indicating they

376

were associated to high molecular weight compounds, which probably 19

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377

could decrease their bioaccessibility. The results observed for Fe are very

378

interesting, as the milk used in this research was enriched with ferric

379

pyrophosphate by manufacture. This iron compound is widely used for

380

fortify foods. Although it is known that it presents low bioavailabity when

381

compared to others iron species, such as ferrous sulphate, studies

382

demonstrate that it suffers less negative effect of anti-nutritional

383

compounds.26,27

384

When the milk was mixed with mesocarp, the total concentration of

385

Cu increases meaning fully, owing to the presence of mesocarp that has a

386

concentration 20 times higher than in milk. Consequently, the soluble and

387

dialysed fractions observed in Figure 2a are from mesocarp. Thus, it was

388

not possible to evaluate the effect on Cu bioaccessibility in milk. The

389

addition of mesocarp to milk increased the concentration of soluble Fe

390

(Figure 2b) and its dialysed fraction compared to the digestion of milk

391

alone. Fe hydrolyses and precipitates at intestinal pH, however nutrients

392

present in food, such as amino acids and peptides, can bind to Fe, turning it

393

soluble and, consequently bioavailable.28 Thus, substances present in milk,

394

such as casein or other binding compounds, can form a soluble complex

395

with Fe, thereby enhancing the Fe bioaccessibility in mesocarp. Another

396

suggested explanation is that compounds present in the mesocarp (e.g.,

397

phytate) may also form soluble compounds of the elements, depending on 20

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398

their concentrations. The soluble and dialysed fractions of Zn in the

399

mixture of milk and mesocarp remained unchanged compared to the milk

400

alone (Figure 2c); thus, the mesocarp does not influence Zn bioaccessibility

401

in milk.

402

Although lignin increases the elements soluble fractions, the dialysed

403

fractions were still very low. However, these elements, even when soluble,

404

may not be bioaccessible for absorption by the body, because they are

405

strongly associated to lignin, which is a compound of high molecular

406

weight.

407

In conclusion, total elemental determination showed that mesocarp

408

babassu contained Fe, Cu and Zn, however, Fe and Zn are not available

409

after in vitro gastrointestinal digestion. Therefore, the mesocarp cannot be

410

considered as a source of these nutrients. Only Cu was extracted during the

411

simulated digestion, having a soluble fraction of 96%. Furthermore, it was

412

observed that the mesocarp could interact with these elements, and phytate

413

was the main compound responsible for these interactions. It was also

414

noted that FeIII and CuII ions may hydrolyse and precipitate at intestinal pH,

415

inhibiting them from being absorbed by human organisms. The negative

416

effects on Cu, Fe and Zn bioaccessibility observed in the studies using

417

reference solutions did not occur when digestion of the milk mixtures were

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418

performed. The presence of mesocarp did not reduce the available fractions

419

of elements.

420 421

ACKNOWLEDGMENTS We thank Nova Analítica Imp. Exp. LTDA for providing the

422 423

microwave oven and ICP OES.

424 425

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426

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Gupta, R. K.; Gangoliya, S. S.; Singh, N. K. Reduction of phytic acid

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Kumar, V.; Sinha, A. K.; Makkar, H. P.; Becker, K. Dietary roles of

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Torre, M.; Rodriguez, A. R.; Saura-Calixto, F. Interactions of Fe (II),

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Ca (II) and Fe (III) with high dietary fibre materials: A physicochemical

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Thompson, B.; Sharp, P.; Elliott, R.; Al-Mutairi, S.; Fairweather-

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to evaluate the bioaccessibility of Al, Cd, Fe and Zn in lettuce and cole by

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Relative contribution of phytates, fibers, and tannins to low iron and zinc in

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vitro solubility in pearl millet (Pennisetum glaucum) flour and grain

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supplemented with amaranth (Amaranthus cruentus): effect of phytates on

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in vitro iron absorption. Plant foods for human nutrition 2012, 67, 50-56.

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da Silva, E. D.; Heerdt, G.; Cidade, M.; Pereira, C. D.; Morgon, N.

Lestienne, I.; Caporiccio, B.; Besançon, P.; Rochette, I.; Trèche, S.

Sanz-Penella, J. M.; Laparra, J. M.; Sanz, Y.; Haros, M. Bread

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microwave digestion and inductively coupled plasma-atomic emission

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Analytical Atomic Spectrometry 2011, 26, 2434-2441.

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Grathwohl, D.; Hurrell, R. F. Ferrous ammonium phosphate (FeNH4PO4)

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as a new food fortificant: iron bioavailability compared to ferrous sulfate

NEPA/UNICAMP

(Núcleo

de

Estudos

e

Pesquisas

em

Naozuka, J.; Carvalho Vieira, E.; Nascimento, A. N.; Oliveira, P. V.

Dolan, S. P.; Capar, S. G. Multi-element analysis of food by

Rouchon, V.; Pellizzi, E.; Duranton, M.; Vanmeert, F.; Janssens, K.

Walczyk, T.; Kastenmayer, P.; Bonsmann, S. S.; Zeder, C.;

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Journal of Agricultural and Food Chemistry

518

and ferric pyrophosphate from an instant milk drink. European journal of

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nutrition 2013, 52, 1361-1368.

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27.

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ferric pyrophosphate to common iron salts and chelates as sources of

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bioavailable iron in a Caco-2 cell culture model. Journal of agricultural

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and food chemistry 2009, 57, 5014-5019.

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American Journal of Clinical Nutrition 1981, 34, 2248-2256.

Zhu, L.; Glahn, R. P.; Nelson, D.; Miller, D. D. Comparing soluble

Miller, D. D.; Schricker, B. R.; Rasmussen, R. R.; Van Campen, D.

527 528

The authors are grateful to Conselho Nacional de Desenvolvimento

529

Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do

530

Estado de São Paulo (FAPESP) for financial support. The authors also

531

thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico

532

(CNPq) for the research opportunities and fellowships provided.

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Figure Captions

Figure 1. Recovery of Cu (

), Fe (

) and Zn (

) that was added to

the extract prepared at (a) pH 7, (b) pH 12 and (c) in the washed mesocarp.

Figure 2. (a) Cu, (b) Fe and (c) Zn bioaccessibility in milk, a mixture of milk and mesocarp as well as a mixture of milk and lignin.

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Journal of Agricultural and Food Chemistry

Table 1: Instrumental Conditions and Heating Program for Simultaneous Determination of Cu and Fe by SIMAASa Element Cu Fe

Wavelength (nm) Current (mA) Slit width (nm) 324.8 12 0.7 248.3 25 0.7 Heating program Temperature Ramp Hold Ar flow Step (ºC) (s) (s) (mL min-1) Drying I 110 1 30 250 Drying II 130 15 30 250 Pyrolysis 1200 10 20 250 Atomisation 2000 0 5 0 Cleaning 2500 1 3 250 a Pyrolysis and atomization temperatures were selected based on the most and least volatile analyte to be simultaneously determined. No chemical modifier was used. The injection temperature of the graphite tube was 30 oC and the pipette speed of the injection was 100%

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Table

2:

Experimental

Conditions

Used

to

Page 30 of 35

Investigate

the

Bioaccessibility of Cu, Fe and Zna Assay Cu, Fe, and Zn bioaccessibility in mesocarp Cu, Fe and Zn recovery after addition to the washed mesocarp (pH=12)

Sample 200 mg of mesocarp 200 mg of mesocarp

Addition

Fraction analysed

Determination

-

Soluble fraction

Cu and Fe by SIMAAS Zn by ICP OES

Soluble fraction

Cu, Fe and Zn by ICP OES

Soluble fraction

Cu, Fe and Zn by ICP OES

Soluble fraction

Cu, Fe and Zn by ICP OES

Soluble and dialysable fraction

Cu by SIMAAS Fe and Zn by ICP OES

0.018 mg of Cu 0.28 mg of Fe

III

0.14 mg of Zn

II

0.018 mg of Cu Effect of Ca on Cu, Fe and Zn bioaccessibility

200 mg of mesocarp

0.28 mg of Fe

200 mg of mesocarp

II

III

0.14 mg of Zn

II

0.88 mg of Ca

II

0.018 mg of Cu Effect of Mg on Cu, Fe and Zn bioaccessibility

II

0.28 mg of Fe

II

III

0.14 mg of Zn

II II

0.067 mg of Mg Cu, Fe and Zn bioaccessibility in milk

100 mg of milk

-

Cu, Fe and Zn bioaccessibility in presence of milk + mesocarp

100 mg of Soluble and Cu by SIMAAS milk dialysable Fe and Zn by 100 mg of fraction ICP OES mesocarp 100 mg of Cu, Fe and Zn Soluble and Cu by SIMAAS milk bioaccessibility in dialysable Fe and Zn by 3,2 mg of presence of milk + lignin fraction ICP OES lignin a The gastrointestinal digestion used 3.0 mL of gastric fluid (pH ~ 1.5) added to sample. The mixture was shaken (100 rpm) in a thermostatic bath at 36 ºC for 2 h added 0.4 mL of NaHCO3 3% (w v-1) in order to adjust the pH to 6.8. An aliquot of 3.0 mL of intestinal fluid (pH ~ 6.5) was added and the extract solution was shaken and centrifuged to separate the supernatant.

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Journal of Agricultural and Food Chemistry

Table 3: Concentration of Elements in Mesocarp, Milk, SRMs, as Well as Addition and Recovery Test and Limits of Detectiona a

b b b b Cu Fe Zn Ca Mg -1 -1 -1 -1 (µg g ) (µg g ) (µg g ) (mg g ) (mg g-1) Certified 275.2 ± 4.6 197.94 ± 0.65 181.1 ± 1.0 0.131 ± 0.010 0.620 ± 0.042 Bovine liver Found 177 ± 4 173 ± 4 0.130 ± 0.007 0.55 ± 0.01 229 ± 4 (SRM® 1577c) Recovery 83% 90% 95% 99% 90% Certified 3.7 ± 0.4 218 ± 14 17.9 ± 0.4 15.6 ± 0.2 4.32 ± 0.08 Peach leaves Found 3.5 ± 0.4 195 ± 23 16.73 ± 0.07 14.1 ± 0.09 3.89 ± 0.04 (SRM® 1547) Recovery 95% 89% 93% 90% 90% Conc. 0.23 ± 0.03 236 ± 6 33 ± 0.7 9.1 ± 0.2 0.77 ± 0.01 Milk Addition 2.5 250 25 5 0.5 Recovery 84% 110% 84% 95% 109% Conc. 4.07 ± 0.07 16 ± 1 2.9 ± 0.3 0.40 ± 0.02 0.57 ± 0.01 Mesocarp Addition 5 10 1.0 0.25 0.5 Recovery 99% 116% 85% 95% 104% Limit of Detection (LOD) 0.1 0.2 0.02 0.0006 0.0006 a The total determination of the elements was used a mass of 200 mg of mesocarp, milk or SRMs for digestion in a microwave oven. After digestion the solution was diluted with deionised water up to 10 mL and analysed. a Analysis by SIMAAS; b Analysis by ICP OES (n = 3 samples)

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Table 4: Addition and Recovery Tests for Fe, Cu and Zn in Gastrointestinal Digestion of Babassu Mesocarp in Presence of Ca and Mga Element

Recoveries mass (µg) Recoveries mass (µg) (without Ca and Mg) (with Ca addition) Fe 134 ± 3 115 ± 2 Cu 11.7 ± 0.2 11.6 ± 0.3 Zn 78 ± 6 105 ± 5 a Addition of 18 µg of Cu, 280 µg of Fe and 140 µg of Zn

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Recovery mass (µg) (with Mg addition) 138 ± 7 11.5 ± 0.4 103 ± 3

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Journal of Agricultural and Food Chemistry

Figure 1. Recovery of Cu (

), Fe (

) and Zn (

) that was added to

the extract prepared at (a) pH 7, (b) pH 12 and (c) in the washed mesocarp.

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Figure 2. (a) Cu, (b) Fe and (c) Zn bioaccessibility in milk, a mixture of milk and mesocarp as well as a mixture of milk and lignin. 34

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TOC Graphic

In vitro evaluation of Cu, Fe and Zn bioaccessibility in presence of babassu mesocarp

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