Effect of enzymatic treatment on phytate content and mineral bioacessability in soy drink

Accepted Manuscript Effect of enzymatic treatment on phytate content and mineral bioacessability in soy drink

Viviane Cristina Toreti Theodoropoulos, Mariana Amarante Turatti, Ralf Greiner, Gabriela Alves Macedo, Juliana Azevedo Lima Pallone PII: DOI: Reference:

S0963-9969(18)30185-6 doi:10.1016/j.foodres.2018.03.018 FRIN 7455

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Food Research International

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Please cite this article as: Viviane Cristina Toreti Theodoropoulos, Mariana Amarante Turatti, Ralf Greiner, Gabriela Alves Macedo, Juliana Azevedo Lima Pallone , Effect of enzymatic treatment on phytate content and mineral bioacessability in soy drink. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Frin(2017), doi:10.1016/j.foodres.2018.03.018

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ACCEPTED MANUSCRIPT EFFECT OF ENZYMATIC TREATMENT ON PHYTATE CONTENT AND MINERAL BIOACESSABILITY IN SOY DRINK

Viviane Cristina Toreti Theodoropoulos 1 ; Mariana Amarante Turatti 1 ; Ralf Greiner 1*

Department of Food Science, School of Food Engineering, University of Campinas

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1

Gabriela

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Alves Macedo 3 ; Juliana Azevedo Lima Pallone

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Max Rubner–Institut, Federal Research Institute of Nutrition and Food, Department of Food

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(UNICAMP), Monteiro Lobato Street, no. 80, Campinas, SP 13083-862, Brazil

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Technology and Bioprocess Engineering, Haid-und-Neu-Straße 9, 76131 Karlsruhe, Germany

Department of Food and Nutrition, School of Food Engineering, University of Campinas

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* Corresponding author

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(UNICAMP), Monteiro Lobato Street, no. 80, Campinas, SP 13083-862, Brazil

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E-mail address: [email protected]

ACCEPTED MANUSCRIPT ABSTRACT The objective of this study was to evaluate in vitro bioaccessibility of calcium (Ca), iron (Fe) and zinc (Zn) in soy drink after phytase treatment and correlate it with the content of myo-inositol phosphates. Samples of commercial soy drink products and one sample produced in the laboratory by maceration were evaluated. Phytase was applied using 300U per liter in 60 minutes

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considering the phosphate release. The content of myo-inositol tris -, tetrakis -, pentakis and

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hexakisphosphate was not observed after phytase treatment. The solubility assay showed an increase from 2.0% to 20.8% for Ca, 2.2% to 37.1% for Fe and 38.8% to 67.4% for Zn after

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phytase treatment with significant differences (p ≤ 0.05) for most samples. Dialysis assay

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demonstrated 1.0% to 9.5% for Ca after phytase treatment (p ≤ 0.05) except for one commercial sample. The phytase treatment is a valuable alternative process for improving mineral natural

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availability in soy drink and decreased the use of salts in the fortification.

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Keywords: Soy drink; Phytase; Myo-inositol phosphate; - Bioaccessibility; Calcium; Zinc; Iron.

ACCEPTED MANUSCRIPT 1 Introduction Myo-inositol hexakisphosphates (IP6 ) and myo-inositol pentakisphosphates (IP5 ) can act as antinutritional factors by forming insoluble complexes with cations at physiological pH values and inhibit mineral absorption (Greiner & Konietzny, 1999; Greiner & Konietzny, 2006; Kumar et al., 2010; Troesch et al., 2013).

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Decrease in the binding force between the mineral and phytate results in a higher

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solubility of dietary minerals (Griffiths & Thomas, 1981; Jaffe, 1981; Sandberg & Andlid, 2002,

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Kumar et al., 2010, Gupta et al., 2013) and one way to reduce phytates in food is using a biotechnological process by applying exogenous phytase.

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Soy drink is one valuable alternative in the beverage market for the consumption by people with of lactose intolerant , allergic to cow's milk protein, and who reduce or exclude food

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of animal origin from their diet (Slywitch, 2012; Rodrígues-Roque et al., 2013). Antinutritional factors present in soy drink can compromise mineral natural availability and its

salts. Besides

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being the elements calcium (Ca), iron (Fe) and zinc (Zn) that are most negatively affected by phytate (Reddy et al., 1989; Lihono et al., 1997; Greiner & Konietzny, 2006). The fortification of

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soy drinks by minerals, such as Ca and Zn intends to increase its nutritional value and make the beverage economically competitive (Casé et al., 2005).

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In order to assess phytase effect on mineral availability, two analytical techniques were employed: the in vitro bioaccessibility technique, that provides an estimate on the amount of nutrients available from food, with quantification performed by Flame Atomic Spectrometry (FAAS) and myo-inositol phosphates evaluation by High-Performance Liquid Chromatography (HPLC) (Etcheverry et al., 2012). The use of the two analytical techniques are complementary and allow us to measure the solubility increase in dietary elements, evaluate the difference among ways of preparing food, verify the solubility of different salts employed in the fortification and investigate the effect of other food matrix components (Reykdal & Lee, 1991; Shen et al., 1995; Bossher et al., 1998; Roig et al., 1999; Jovaní et al., 2000; Chaiwanon et al.,

ACCEPTED MANUSCRIPT 2000; Jovaní et al., 2001; Bosscher et al., 2001; Perales et al., 2005; Ünal et al., 2005; Perales et al., 2006; Shiowatana et al., 2006). The soy drink contain antinutritional factors that compromise minerals availability. Phytate hydrolysis is an interesting process because it increases the availability of dietary minerals and improves the nutritional value of the beverage. Studies analyzing myo-inositol

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phosphates in soy drink and improvement mineral are still scarce. Therefore, the aim of this

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original study was to evaluate the effect of phytase treatment on mineral bioaccessibility and

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correlate it with the content of myo-inositol phosphates to reduce the use of salts in the

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fortification by biotechnological process.

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2 Material and methods

2.1 Materials

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Standards, reagents, acids, high-purity solvents and phytase used for sample and reagent preparation were purchased from Qhemis (Jundiai, Brazil), Synth (Diadema, Brazil), Sigma-

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Aldrich (St. Louis, MO, USA) and BASF (São Paulo, Brazil). All digestive enzymes were obtained from Sigma-Aldrich (St. Louis, MO, USA). Water, distilled and deionized to a

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resistance of ≥ 18.2 MΩ (Sartorius system, model Arium® comfort – USA), was used for sample and reagent preparation. A quantitative filter paper from Nalgon (9 cm diameter), was used to filter the digested solutions after mineralization by wet digestion. The equipment used for analysis included: pHmeter (Model TEC-11, Tecnal, Brazil); metabolic bath (Dusnoff, Model MA 093, Marcone, Brazil); centrifuge (Model J2-21, Beckamn Coulter, United States); lyophilizer (Model LS, Terroni, Brazil); block digester (Model M242 – Quimis, Brazil) and ultrasonic bath (Model 1510 – Brason, Brazil). The myo-inositol phosphate quantification was performed with high-performance liquid chromatography (HPLC – Shimadzu, Japan) by using a pump 2248 and gradient controller LCC

ACCEPTED MANUSCRIPT 2252 from Pharmacia LKB Biotechnology (Sweden), a high temperature oven from Knauer (Germany), a detector PN3150 Ri from Postnova Analytics GmbH (Germany) and a printer C R5A Chromatopac from Shimadzu (Japan). Mineral content analyses were performed with Flame Atomic Absorption Spectrometry (FAAS), Perkin Elmer (USA), Model AAnalyst-200, with a deuterium lamp for background

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radiation correction, and hollow cathode lamps for determination of calcium (422.67 nm), iron

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(248.3 nm) and zinc (213.86 nm).

2.2 Samples

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The commercial soy drink samples of three major local brands were purchased and mix five lots of each brand from local markets (Campinas, Brazil), and these were fortified with

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calcium and zinc. For comparison a soy drink sample without mineral fortification was produced in our laboratory by maceration according to the method of Mandarino & Carrão-Panizzi (1999).

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2.3 Experimental design

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All samples were lyophilized and stored at -18 °C for preservation.

Commercial and maceration samples were evaluated to verify the effectiveness of

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phytase in soy drink. Bioaccessibility of calcium, iron and zinc in soy drink samples with and without phytase treatment was determined as shown in Figure 1. “Figure 1”

2.4 Enzymatic treatments 2.4.1 Determination of activity of phytase Phytase activity measurements were performed according to Shimizu (1992) with adjustment. Briefly, 150 μL of an appropriately diluted phytase solution (0.02 mg mL-1 ; NATUPHOS® 10000 G, BASF) were incubated with 600 μL of phytate solution (0.002 mol L-1

ACCEPTED MANUSCRIPT of sodium phytate in 0.2 mol L-1 sodium acetate buffer, pH 5.5) at 37°C 30 minutes. Thereafter, the reaction was stopped by adding 750 µL 5.0% trichloroacetic acid (TCA), and the liberated inorganic phosphate (Pi) was measured using absorbance at 865 nm (He & Honeycutt, 2005). One unit of phytase activity was defined as the amount of enzyme required to release one μmoL Pi per minute at test conditions. The specific activity was expressed in units of enzyme activity

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per mg total protein.

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2.4.2 Determination of Inorganic Phosphate

The method to quantify inorganic phosphate method was adapted from Thomas et al.

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(1989). 0.2 grams of lyophilized soy drink samples were transferred to clean Erlenmeyer flasks and mixed with deionized water to a final volume of 20 mL. Then, five mL of 50% TCA were

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added and the mixture was placed in a shaking water bath for 15 minutes at 37°C, and thereafter centrifuged (12,074 g) for 15 minutes at 5°C. The supernatant was used to determine inorganic

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phosphate content. The following solutions were added to a test tube in the order listed: one mL sample, one mL 1% ammonium molybdate, three mL 0.2M sodium acetate buffer pH 4.2, one

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mL 1% ascorbic acid. The absorbance at 865 nm (He & Honeycutt, 2005) was read after 20 minutes. Phosphate concentration was derived from a series of potassium phosphate standards

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ranging from 0.25 to 1.75 µmol mL-1 .

2.4.3 Incubation of soy drink with phytase Incubation of soy drink with phytase (Natuphos®) was performed according to Lihono et al. (1997) with some modifications. Briefly, the soy drink sample was resuspended to a final concentration of 10% (w/v in a citric acid solution (1 g L-1 ). This suspension was incubated for 0, 15, 30, 60, 90, 120 and 150 minutes with 300, 900, 1,200 and 1,800 U phytase per liter of soy drink).

ACCEPTED MANUSCRIPT The reaction was stopped with 1 M sodium hydroxide solution (NaOH) and samples were frozen, lyophilized to determine Pi according to the method of Thomas et al. (1989).

2.5 In vitro digestion 2.5.1 Solubility method

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The in vitro digestion model was adapted from Cámara et al. (2005). Five grams of

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lyophilized soy drink samples were transferred to clean Erlenmeyer flasks and mixed with

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deionized water to a final volume of 25 mL. The samples were acidified to pH 2.0 with 6 mol L-1 hydrochloric acid (HCl) and thereafter 1 mL of a porcine pepsin preparation (1.6 g of porcine

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pepsin in 10 mL 0.1 mol L-1 HCl) was added. The mixture was incubated at 37°C in a shaking waterbath for 2 hours. To stop the gastric digestion phase, the samples were maintained for 10

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minutes in an ice bath. To start the intestinal digestion, pH was increased to 5.0 with 1 M sodium bicarbonate (Na2 HCO 3 ) solution followed by the addition of 5.65 mL of pancreatin-bile salt

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mixture (4 mg porcine pancreatin and 25 mg bile bovine and ovine per 100 mL of 0.1 mol L-1 Na2 HCO 3 ). Samples were incubated in a shaking water bath at 37°C for 2 hours. To stop

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intestinal digestion, the samples were maintained for 10 minutes in an ice bath. The pH of each sample was increased to 7.2 with 0.5 mol L-1 NaOH. After the intestinal phase, samples were

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centrifuged (9,360 g) for 30 minutes at 4°C. The supernatant (soluble fraction) was collected, frozen (-40°C), lyophilized and the organic matter destroyed by wet digestion. Thereafter, the mineral content was measured by FAAS. Soluble mineral percentages were calculated as follows: Solubility (%) = 100 X S/C, where S = soluble mineral content (mg 100 mL-1 ) and C = total mineral content of the sample (mg 100 mL-1 ).

2.5.2 Dialysis method The in vitro digestion model was adapted from Cámara et al. (2005). The gastric digestion phase was maintained according to the procedure aforementioned. A gastric digest with

ACCEPTED MANUSCRIPT added pancreatin-bile solution was titrated with 0.5 mol L-1 Na2 HCO 3 solution to determine the volume of base needed to increase pH digests to about 7.2. Dialysis bags (Inlab, São Paulo, Brazil; cut-off from 12,000 to 16,000 and porosity of 25Å) containing 25 mL of freshly prepared 0.5 mol L-1

Na2 HCO 3 solution, were immersed in pepsin digests and incubated in a shaking

water bath at 37°C. After 30 minutes 5.65 mL of pancreatin-bile salt mixture (4 mg porcine

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pancreatin and 25 mg bile bovine and ovine per 100 mL of 0.1 mol L-1 Na2 HCO 3 ) were added

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and the incubation in a shaking water bath at 37°CC continued for 1 hour and 30 minutes. To

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stop intestinal digestion, the samples were maintained for 10 minutes in ice bath. Dialysates were frozen (-40 °C), lyophilized and used for determination of the minerals content and the results

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were expressed in percentages. Dialysis (%) = 100 X D/C, where D = dialyzed mineral content

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(mg 100 mL-1 ) and C = total mineral content of the sample (mg 100 mL-1 ).

2.6 Determination method for myo-inositol tris-, tetrakis-, pentakis-, hexakisphosphate(s)

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Freeze-dried samples (1 g) were extracted with 20 mL of 2.4% HCl for 3 h at room temperature. The slurries were centrifuged at 30,000 g for 30 minutes. 1 mL of the supernatants was

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diluted 1:25 with water and applied to a column (0.7 × 15 cm) containing AG1-X8, 100-200 mesh resin. The column was washed with 25 mL of water and 25 mL of 25 mM HCl. Then myo-inositol

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phosphates were eluted with 20 mL of 2 M HCl. The eluate obtained was concentrated in a vacuum evaporator to dryness and the residue was dissolved in 1 mL water. 20 µL of the samples were chromatographed on Ultrasep ES 100 RP18 (2 × 250 mm). The column was run at 45 °C and 0.2 mL min-1 of an eluent consisting of formic acid: methanol: water: tetrabutylammonium hydroxide (44:56:5:1.5 v/v), pH 4.25, as described by Sandberg & Ahderinne (1986). A mixture of the individual myo-inositol phosphate esters (IP3 -IP6 ) was used as standard. Calibration was performed with IP6 in the range of 0.2 - 2 µM.

ACCEPTED MANUSCRIPT 2.7 Determination of Mineral concentration For mineral determination, the glass and polyethylene material were washed with alkaline detergent (Det Lim 532, Chemco – Brazil) for 4 hours, soaked in nitric acid solution 10% (v/v) (sp. gr. 1.38) for 12 hours, rinsed with deionized water and dried at room temperature. All glassware used was calibrated.

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The mineralization was performed by wet digestion (Boen & Lima-Pallone, 2009). 0.3 g

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of the samples were used. After digestion, all samples were diluted to a final volume of 25 mL in

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volumetric flask with deionized water. For determination of minerals, samples were filtered using quantitative filter paper (9 cm diameter) and stored. 1.0% (w/v) lanthanum oxide solution

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was used to avoid interferences in the preparation of samples for Ca. Standards of calcium, iron and zinc solutions containing 0.5 to 4.5 mg L-1 , 0.3 to 1.8 mg L-1 and 0.4 to 2.1 mg L-1

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respectively, were prepared using 0.01 mol L-1 of HNO 3 solution. Both the standard solutions (Sigma-Aldrich, Calcium n° 69349, Iron n° 16596 and Zinc n° 18827) and the samples were

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analyzed using the Atomic Absorption Espectometer (FAAS). Each sample was placed into the

2.8 Statistical analysis

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nebulizer and mixed with air-acetylene flame (2.5/10 L h-1 ), at approximately 2,000 °C.

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All data is presented by means ( ± Standard Deviation - SD) of at least three independent experiments. For comparisons between samples, data was analyzed by ANOVA and Tukey’s test. A probability of 5% or less was accepted as statistical significance. Statistical analyses were performed using MiniTab 16 for Windows. 3 Results and Discussion 3.1 Activity of phytase and incubation of soy drink with phytase Enzymatic activity of the commercial phytase Natuphos® was determined to be 1.41U per mg total protein under assay condition. Release of inorganic phosphorus in soy drink produced by maceration was studied at various times with different concentration of phytase (Table 1).

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“Table 1” The condition for incubation of soy drink used was 300U of phytase activity per liter of soy drink for 60 minutes with statistical significance (p ≤ 0.05). At the condition of 900U of phytase activity per liter of soy drink for 150 minutes, it also presented statistical significance (p

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≤ 0.05). Comparing 300U at 60 minutes and 900U at 150 minutes, there was no statistical

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difference (p ≤ 0.05) and the difference in content of inorganic phosphorus released in the first

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condition was very small. Also, by using 3 times lower amount of enzyme in half the reaction time, the first condition described has been identified as the best in respect to phosphate release

All the samples contained

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3.2 Content of myo-inositol phosphate esters

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and economy on phytase use.

mainly myo-inositol hexakisphosphate besides partially

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phosphorylated myo-inositol phosphates. All four myo-inositol phosphates quantified were present in higher quantities in the commercial soy drink samples compared to the soy drink

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sample prepared by maceration. Table 2 summarizes the myo-inositol phosphate content of the soy drink samples under investigation. The values for the content of myo-inositol phosphates

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obtained for the majority of commercial soy drink samples are different from what was previously reported by Samsuddin, Yang, Vucenik 1996 apud Harland & Narula, 1999 (21.8 µmol g-1 IP6; 4.4 µmol g-1 IP5 and 0.9 µmol g-1 IP4), except for sample 1 before phytase treatment, in which close values were observed. The reason for observed differences can be the soybean composition, type of processing and ensuing processing conditions (high or low temperatures, short or prolonged temperature and cooking time, ultra-high temperature, spraydrying parameters, processing treatment combinations with alkali or other chemicals etc.) (Giri & Mangaraj, 2012). “Table 2”

ACCEPTED MANUSCRIPT Using 300U at 60 minutes, the concentration of all myo-inositol phosphate esters quantified were reduced below the threshold of the analytical method (p ≤ 0.05), indicating that the condition used was adequate for the purpose of this study.

3.3 Mineral availability by in vitro digestion

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Soy drink samples were subjected to an in vitro digestion procedure. The minerals

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analyzed were calcium (Ca), iron (Fe) and zinc (Zn), and these values can provide valuable

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information on their potential availability for absorption in vivo (O’Connell et al., 2007). The amount of each soy drink sample was expressed as a percentage of the respective mineral (Table

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3).

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“Table 3”

3.3.1 Calcium The result obtained by solubility method for Ca (Table 3) represents a variation of 9.1%

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to 56.8%, which can be attributed to the variation between the types of Ca salts used in the fortification, whereas in sample 4 the solubility was low because the element is naturally present

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in small concentration in the soy drink. Roig et al. (1999) reported 38.2% solubility for Ca in soy-based formula, values close to those found in our study.

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After phytase treatment, the solubility closed between 19.5% to 68.1% with significant difference (p ≤ 0.05) for the sample 2 and 4, demonstrating increase in Ca solubility of 12.6% and 20.8% (p < 0.05). However, the analysis of the myo-inositol phosphate fractions (Table 2) showed that in the samples 2 and 4 the content of IP6 and IP5 was low before the treatment with phytase and that after the enzymatic action partially phosphorylated myo-inositol phosphates were not detected, fact which contributed to the significant increase on the solubility of Ca (p ≤ 0.05). There are no studies showing the effect of phytase on phytate present in soy drink to promote the bioaccessibility increase of minerals. On the other hand, Frontela et al. (2008)

ACCEPTED MANUSCRIPT verified the effect of dephytinization on cereals for infant feeding in relation to the availability of Ca, Fe and Zn. They observed positive effect on the solubility of Ca and when they used water for reconstitution of the cereal, the value was between 22.9% to 85.6%. Reconstitution with infant formula ranged from 15.5% to 22.2%. The difference was attributed to the type of dietary calcium added to the fortification, as well as to matrix components such as lactose, casein, fat

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and the total Ca content.

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Through potentially available fraction to be absorbed, dialysis experiment was performed

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with Ca to predict the proportion of the element that is spread across a semipermeable membrane during the simulation of the intestinal phase (Kennefick & Cashman, 2000; Drago & Valencia,

below the threshold of the analytical method.

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2004). The content of iron and zinc could not be identified and quantified because they were

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The change of dialyzed fraction for Ca was between 3.3% and 5.4% (Table 3) before phytase treatment. After, phytase treatment values were from 4.3% and 13.6%. There was a

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significant increase (p ≤ 0.05) for all samples when compared before and after enzymatic treatment, except for sample 3, which was attributed to the type of salt used in the fortification,

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the presence of phytate (IP6) or the fact that the mineral is complexed with other components of the food matrix. When the dialysis data was related to those obtained in the solubility test, it was

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possible to conclude that the samples 1 and 2 that showed the highest dialyzed fraction were the same ones that presented greater solubility. Shen et al. (1995) obtained 6.0% dialysate for Ca in soybean-based formula, a value close to that obtained for sample 2 and 4 studied before treatment with phytase. Roig et al. (1999) and Jovaní et al. (2000) analyzed infant soybean-based formula and found values of 13.0% of dialyzed fraction for Ca, close results obtained for sample 1 after phytase treatment. Chaiwanon et al. (2000) achieve 11.0% of Ca dialyzed in soy drink, similar data found for sample 2 after phytase action.

ACCEPTED MANUSCRIPT 3.3.2 Iron The iron solubility varied 3.4% to 6.9% for commercial samples and 27.5% for soy drink prepared by maceration (Table 3). The low value detected can be attributed to the fact that commercial samples were not fortified, however the content naturally present in the soy drink prepared by maceration showed higher availability. After phytase treatment, the commercial

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samples had values ranging from 2.2% to 40.5%, while sample 4 showed 43.3% (p ≤ 0.05). Evaluating the fractions of myo-inositol phosphate (Table 2) the absence of partially

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phosphorylated myo-inositol phosphates was verified which contributed to the increase of the

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availability of the mineral except for sample 3 that did not present good solubility for the Fe. Sahuquillo et al. (2003) studied the bioaccessibility of Ca, Fe and Zn in legumes, showing

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the results of solubility for Fe of 52.0% for white beans; 58.8% for chickpeas and 11.5% for

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lentil and this result was different from the soy drink studied. Frontela et al. (2008) verified the effect of defitinization of cereals for infant feeding in relation to the availability of Ca, Fe and Zn

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and observed that there was an increase in Fe solubility after phytase treatment, results that

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follow the same trend as those obtained in this work.

3.3.3 Zinc The range of zinc solubility was 7.6% to 22.2% (Table 3). The greater solubility was

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observed in sample number 4 without fortification. Trademarks are fortified but do not declare on the label which salt they use, thus explaining the low solubility of the element, besides the presence of phytate that may be complexed with Zn. After phytase treatment, the variation of the soluble Zn content was between 49.3% to 89.6%. All the samples investigated presented increase on bioaccessibility of Zn (p ≤ 0.05) indicating the effectiveness of phytase. The partially phosphorylated inositol phosphates with different numbers of phosphate residues were not verified (Table 2), which contributed to the increase of bioaccessibility of this mineral.

ACCEPTED MANUSCRIPT Frontela et al. (2008), verified the defitinization of cereals for infant feeding in relation to the availability of Zn and observed a positive effect on solubility, a result that is in agreement with the one obtained in this work, even using a different food matrix. In the work presented by Akhter et al. (2012), the defitinization of some varieties of wheat was evaluated and the

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application, being in accordance with the findings of this work.

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bioaccessibility of Ca, Fe and Zn observed the increase of Zn availability after phytase

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4 Conclusion

In conclusion phytase treatment resulted in a decrease of the content of myo-inositol

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phosphates and improved the nutritional value of soy drink by improving the solubility of Ca, Fe and Zn. Thus, phytase can become a viable alternative in fortification for the food industry due

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to its action that increases the availability of minerals and also allows the salt reduction in the fortification. Nonetheless, it should be remembered that in vitro bioaccessibility is the estimation

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of the fractions of minerals available and it is necessary to investigate the soy drink minerals

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bioavailability in humans in order to correlate the studies.

ACCEPTED MANUSCRIPT Acknowledgements The authors also would like to thanks for BASF for providing the phytase enzyme. This work was supported by the Fundação de Amparo a Pesquisa do Estado de São Paulo

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(FAPESP) [grant number: 2013/16643-8] and PROEX/Capes [grant number: 3300301702P1].

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Organic

Phosphates.

ACCEPTED MANUSCRIPT Jaffe, G. (1981). Phytic acid in soybeans. Journal of the American Oil Chemists’ Society, 58 (3), 493–495. Jovaní, M., Alegría, A., Barberá, R., Farré, R., Lagarda, M.J., & Clemente, G. (2000). Effect of proteins, phytates, ascorbic acid and citric acid on dialysability of calcium, iron, zinc and copper

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in soy-based infant formulas. Nahrung, 44, 114-117. Jovaní, M.; Barberá, R.; & Farré, R. (2001) Review: Effect of some components of milk- and infant

formulas

on

mineral bioavailability.

Science

and

Technology

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International, 7 (3), 191–198.

Food

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soy-based

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Kennefick, S., & Cashman, K D. (2000). Investigation of an in vitro model for predicting the effect of food components on calcium availability from meals. International Journal of Food

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Sciences and Nutrition, 51 (1), 45–54.

Kumar, V., Sinha, A.K., Makkar, H.P.S., & Becker, K. (2010). Dietary roles of phytate and

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phytase in human nutrition: A review. Food Chemistry, 120 (4), 945–959.

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Lihono, M.A., Serfass, R.E., Sell, J.L., & Palo, P.R. (1997). Bioavailability of calcium citrate malate added to microbial phytase-treated, hydrothermally cooked soymilk. Journal of Food Science, 62(6), 1226–1230.

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Lihono, M.A.; Serfass, R.E.; Sell, J.L.; Palo, P.R. (1997) Bioavailability of calcium citrate malate added to microbial phytase-treated, hydrothermally cooked soymilk. Journal of Food Science, 62 (6), 1226-1230.

Mandarino, J. M. G., & Carrão-Panizzi, M.C. (1999). A soja na cozinha. Embrapa Soja, Doc. 136, 13. O’Connell, O.F., Ryan, L., & O’Brien, N.M. (2007). Xanthophyll Carotenoids are more bioaccessible from fruits than dark green vegetables. Nutrition Research, 27, 258 – 264.

ACCEPTED MANUSCRIPT Perales, S.; Barberá, R.; Lagarda, M.J.; & Farré, R. (2005). Availability of iron from milk-based formulas and fruit juices containing milk and cereals estimated by in vitro methods (solubility, dialysability) and uptake and transport by Caco-2 cells. Food Chemistry, 102 (4),1296–1303. Perales, S.; Barberá, R.; Lagarda, M.J.; & Farré, R. (2006). Fortification of milk with calcium: effect on calcium bioavailability and interactions with iron and zinc. Journal of Agricultural and

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Food Chemistry, 4, 4901-4906.

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Reddy, N.R.; Pierson, M.D.; Sathe, S.K.; Salunkhe, D.K. (1989). Occurrence, distribution,

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content, and dietary intake of phytate. In.: Reddy, N.R.; Pierson, M.D.; Sathe, S.K.; Salinkhe,

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D.K. Phytates in Cereals and Legumes, Boca Raton, CRC Press, 39-56. Reykdal, O.; & Lee, K. (1991). Soluble, dialyzable and ionic calcium in raw and processed skim

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milk, whole milk and spinach. Journal of Food Science, 56 (3), 864–866. Rodríguez-Roque, M. J., Rojas-Graü, M.A., Elez-Martínez, P., & Martín-Beloso, O. (2013).

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Soymilk phenolic compounds, isoflavones and antioxidant activity as affected by in vitro

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gastrointestinal digestion. Food Chemistry, 136(1), 206–212. Roig, M.J., Alegría, A., Barberá, R., Farré, R., & Lagarda, M.J. (1999). Calcium bioavailability in human milk, cow milk and infant formulas – comparison between dialysis and solubility

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methods. Food Chemistry, 65, 353-357. Sahuquillo, A., Barberá, R., & Farré, R. (2003). Bioaccessibility of calcium, iron and zinc from three legume samples. Nahrung–Food, 47 (6), 438–441. Sandberg, A.S., & Ahderinne, R. (1986). HPLC method for determination of inositol triphosphates, tetraphosphates, pentaphosphates and hexaphosphates in foods and intestinal contents. Journal of Food Science, 51 (3), 547–550. Sandberg, A.S., & Andlid, T. (2002). Phytogenic and microbial phytases in human nutrition. International Journal of Food Science and Technology, 37 (7), 823–833.

ACCEPTED MANUSCRIPT Shen, L., Robberecht, H., Vandael, P., & Deelstra, H. (1995). Estimation of selenium bioavailability from human, cow’s, goat and sheep milk by an in vitro method. International Journal of Food Sciences and Nutrition, 47 (1), 75–81. Shimizu, M. (1992). Purification and characterization of phytase from Bacillus subtilis (natto) N-

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77. Bioscience, Biotechnology and Agrochemistry, 56 (8), 1266-1269. Shiowatana, J.; Kitthikhum, W.; Sottimai, U.; Promchan, J.; & Kunajiraporn, K.(2006).

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of mineral bioavailability of food. Talanta, 68 (3), 549–557.

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Dynamic continuous-flow dialysis method to simulate intestinal digestion for in vitro estimation

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Slywitch, E. (2012). Guia alimentar de dietas vegetarianas para adultos. São Paulo, Sociedade Vegetariana Brasileira. http://www.svb.org.br/livros/guia-alimentar.pdf

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Thomas, R., deMan, J.M., & deMan, L. (1989). Soymilk and tofu properties as influenced by soybean storage conditions. Journal of the American Oil Chemists’ Society, 66 (6), 777-782.

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Troesch, B., Jing, H., Laillaou, A., & Fowler, A. (2013). Absorption studies show that phytase

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from Aspergillus niger significantly increases iron and zinc bioavailability from phytate-rich foods. Food and Nutrition Bulletin, 34 (2), 90–101. Ünal, G.; El, S.N.; & Kiliç, S. (2005). In vitro determination of calcium bioavailability of milk,

13-22.

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dairy products and infant formulas. International Journal of Food Sciences and Nutrition, 56 (1),

Captions of Figures Figure 1. Experimental design of soy drink and analyses.

ACCEPTED MANUSCRIPT Table 1. Study of some conditions for phosphorus release with Natuphos ®. Inorganic phosphorus (mg 100 mL-1 ) 300U

900U

50.2 ± 2.7 d, A 50.2 ± 2.7 c, A c, A 167.8 ± 12.3 182. 6 ± 16.3 b, A 184.7 ± 7.3 bc, A 185. 2 ± 6.4 b, A ab, A 211.3 ± 6.2 185.1 ± 11.1 b, AB 211.5 ± 10.4 ab, A 200.8 ± 13.5 ab, A 219.1 ± 2.6 a, A 206.3 ± 12.3 ab, A a, AB 222.1 ± 10,0 240.2 ± 19.5 a, A

1200U

1800U

50.2 ± 2.7 b, A 159.0 ± 1.3 a, A 170.7 ± 8.5 a, A 179.2 ± 6.4 a, B 183.3 ± 24.1 a, A 193.0 ± 17.2 a, A 198.2 ± 10.3 a, B

50.2 ± 2.7 b, A 183.8 ± 3.0 a, A 186.1 ± 16.5 a, A 190.4 ± 9.3 a, AB 195.4 ± 10.2 a, A 197.1 ± 1.0 a, A 199.1 ± 9.4 a, AB

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Reaction Time (minutes) 0 15 30 60 90 120 150

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Different small letters denote significant differences (p < 0.05) between the different reaction time and the same enzyme concentrations. Different capital letters denote significant differences (p < 0.05) between the different enzyme concentrations and the same reaction time.

ACCEPTED MANUSCRIPT Table 2. Content of myo–inositol tris-, tetrakis, pentakis, hexakisphosphate(s) in soy drink samples. IP6 (µmol g-1) a

IP4 (µmol g-1)

IP3 (µmol g-1)

a

0.9 ± 0.02

0.2 ± 0.01

ND

ND

5.2 ± 0.2

0.1 ± 0.02

a

0.3 ± 0.02

b

14.58 ± 0.1

ND 1.2 ± 0.05

a

a

ND

ND

ND

ND

0.3 ± 0.02

0.1 ± 0.01

a

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5.0 ± 0.1

ND

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ND

a

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19.7 ± 0.3

IP5 (µmol g-1)

a

a

a

ND

ND

ND

ND

0.49 ± 0.02

0.2 ± 0.02

0.2 ± 0.01

0.1 ± 0.01a

a

a

ND

ND

a

ND

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Samples 1 Before phytase treatment After phytase treatment 2 Before phytase treatment After phytase treatment 3 Before phytase treatment After phytase treatment 4 Before phytase treatment After phytase treatment

ND

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Samples 1, 2 and 3: Commercial soy drink. Sample 4: Soy drink produced by maceration. ND: Not Detected. Data represent mean ± SD from three determinations (N = 3). Different small letters denote significant differences (p < 0.05)

ACCEPTED MANUSCRIPT 23 Table 3. Content of Ca, Fe and Zn on in vitro bioaccessibility (Solubility and Dialysis methods) in soy drink samples. Bioaccessibility Fe

Ca

Zn

Samples Solubility (%) 1 Before phytase treatment After phytase treatment 2 Before phytase treatment After phytase treatment 3 Before phytase treatment After phytase treatment 4 Before phytase treatment After phytase treatment

Increase Ca (%)

56.8 ± 13.9

Dialysis (%)

a

4.1 ± 1.3

68.1 ± 6.9

a

41.1 ± 8.1

b

61.9 ± 5.2

a

17.5 ± 4.5

a

19.5 ± 4.4

a

9.1 ± 1.4

Solubility (%)

b

11.3

6.9 ± 2.3

b

U N

a

20.8

a

3.4 ± 0.4

5.6 11.0 ± 2.5 a

3.3 ± 0.3 2.0

12.6

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a

a

5.3 ± 0.4

b

8.3 ± 1.8

a

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3.0

b

12.9 ± 0.1

41.5

54.4 ± 11.8 a

7.6 ± 0.7

b

37.1 61.3 ± 8.5

a

---

10.5 ± 1.4

b

a

2.2 ± 1.7

a

2.2

43.3 ± 1.6 a

53.7

38.8 49.3 ± 11.2 a

27.5 ± 8.1 b

Increase Zn (%)

b

40.5 ± 4.1

1.0

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4.3 ± 2.0

a

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24.1 ± 4.1

Solubility (%)

T P

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17.2

13.6 ± 1.1

5.4 ± 0.5

Increase Fe (%)

b

9.5

b

21.7 ± 1.6

Increase Ca (%)

22.2 ± 2.6

b

89.6 ± 4.3

a

15.8

67.4

Sample 1,2 and 3: Commercial soy drink. Sample 4: Soy drink preparation by maceration. Calculation increase was performed by difference of the sample after phytase treatment of the sample before phytase treatment. Data represent mean ± SD from three determinations (N=3). Different letters denote signficant differences (p ≤ 0.05).

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ACCEPTED MANUSCRIPT 24

HIGHLIGHTS



Decrease of the content of myo-inositol phosphates improving the solubility of Ca, Fe and

Zn. The phytase treatment is a valuable alternative process for improving mineral natural

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

Phytase actions increase the bioacessiblity of minerals and also allow the salt reduction in

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availability in soy drink.

the fortification of soy drink.

“In vitro bioaccessibility is makes it possible to investigate the formulation of soy drink

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that are fortified”

Graphics Abstract

Figure 1