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

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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])

1

ACS Paragon Plus Environment


1

ABSTRACT

2

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

22

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

23

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

24

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

25

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

26

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

27

as well as in the cosmetic industry.1, 2

28

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

29

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

30

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

32

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

33

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

37

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

38

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

39

function of reserving reactive phosphate groups and for energy storage in

40

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



41

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

42

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

43

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

44

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

45

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

49

Lignin is a polyphenolic macromolecule present in cereal, fruits and

50

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

51

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

52

bioavailability.10-12

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Several in vivo methods are used to estimate the bioavailability of

54

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

55

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

56

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

57

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

58

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

59

greater control of experimental conditions and a possibility of different

60

digestion stages evaluation. In vitro methods provide information of

61

bioaccessibility (or availability) when physiological factors are not 4


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

63

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

65

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

66

morphological and functional properties similar to intestinal cells

67

responsible for nutrients absorption. Therefore, relative bioavailability

68

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

69

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

70

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

79

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

88

composition is not well known and also its effect over nutrients

89

bioacessibility. This work aimed to study methods for assessing the

90

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

92

without babassu mesocarp as supplement, using simulated gastrointestinal

93

digestion.

94 95

MATERIAL AND METHODS

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Apparatus. An inductively coupled plasma optical emission spectrometer

97

(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.

101

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

102

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

103

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),

106

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

107

A simultaneous atomic absorption spectrometer (SIMAA-6000®,

108

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

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effect background corrector, Echelle optical arrangement, solid-state

110

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

114

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

115

autosampler. The instrumental conditions and heating program are shown

116

in Table 1.

117

Samples and standard reference materials (SRM) were digested using

118

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

119

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

120

infrared sensors for individual temperature and pressure measurements.

121

Mesocarp and milk drying procedures were carried out in a drying

122

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

123

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

124

stirring during the extractions procedures. For simulated gastrointestinal 7



125

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

126

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

127

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

128

Brazil) was used for pH adjustment of the samples.

129 130

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

131

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

132

system (Millipore, Belford, MA, USA).

133

Reference solutions were prepared from Titrisol® standard analytical

134

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

135

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

136

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

137

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

138

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

139

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

140

used for pH adjustments.

141

Gastrointestinal fluids were prepared using NaCl (Merck), pepsin

142

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

143

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

144

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

145

(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

149

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

150

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

151

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

152

Babassu mesocarp flour samples, purchased in Maranhão state

153

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

154

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

155

Aldrich) were used in simulated gastrointestinal digestion assays.

156 157

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

158

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

159

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).

163

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

164

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

165

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

166

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



167

were prepared using reference solutions in equivalents acid concentration

168

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

169

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

170

are considered macronutrients, and consequently if their presence during

171

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

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Zn.

173

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

174

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

175

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

176

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

177

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

178

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

179

mL) and later dried at 60 ºC.

180

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

181

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

182

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

183

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

184

scan.

185

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

186

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

187

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

189

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

190

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

191

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

192

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

193

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

194

recoveries of additions were calculated following: (obtained concentration

195

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

196

The in vitro gastrointestinal digestion procedure was employed to

197

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

198

fluids prepared according to US Pharmacopeia XXIV recommendations.20

199

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

201

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

202

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

203

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

204

water to 100 mL.

205

The general procedure followed in order to simulate gastrointestinal

206

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

207

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

208

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



209

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

210

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

211

and the extract solution was shaken again using the same conditions

212

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

213

Dialysis procedure was performed in order to simulate nutrient absorption

214

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

215

formed from cellulose membranes, tied with polyethylene tapes and

216

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

217

membrane was carefully opened and the solution was collected from inside

218

and analysed. The dialysed fraction was determined by subtracting the

219

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

220

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

221

in vitro gastrointestinal assays are shown in Table 2.

222

The calibrations for analyzes of in vitro gastrointestinal digestions

223

samples by ICP OES and SIMAAS were prepared using reference solutions

224

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

225

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

226

determination by SIMAAS, matrix interference effect was evaluated by

227

comparing calibrations curves using reference solutions in sample, blank

228

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

229

12


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RESULTS AND DISCUSSION

231

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

232

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

233

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

234

shown the method accuracy evaluation by SRMs analysis and

235

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

236

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

237

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

238

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

239

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.

246

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

248

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

249

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



251

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

259

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

260 261

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

262

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

270

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

273

extract.

274

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



293

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

315

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



335

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

355

18


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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



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


Page 20 of 35

Page 21 of 35


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

21



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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Dost, K.; Tokul, O. Determination of phytic acid in wheat and wheat

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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approach. Food Chemistry 1995, 54, 23-31.

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Physico-chemical characterization of lignins from different sources for use

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human digestion models for food applications. Food Chemistry 2011, 125,

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inductively coupled plasma mass spectrometry. Microchemical Journal

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

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Nascimento, A. N.; Naozuka, J.; Oliveira, P. V. In vitro evaluation of

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microwave digestion and inductively coupled plasma-atomic emission

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chelating treatments used on iron gall ink damaged manuscripts. Journal of

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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

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Page 27 of 35


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

519

nutrition 2013, 52, 1361-1368.

520

27.

521

ferric pyrophosphate to common iron salts and chelates as sources of

522

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

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and food chemistry 2009, 57, 5014-5019.

524

28.

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An in vitro method for estimation of iron availability from meals. The

526

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.

27



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.

28


Page 28 of 35

Page 29 of 35


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%

29



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


Soluble fraction


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.

30


Page 31 of 35


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)

31



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

32


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

Page 32 of 35

Page 33 of 35


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.

33



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


Page 34 of 35

Page 35 of 35


TOC Graphic

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

35


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

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