Article pubs.acs.org/JAFC
Iron-Binding Capacity of Defatted Rice Bran Hydrolysate and Bioavailability of Iron in Caco‑2 Cells Lian-Chee Foong,† Mustapha Umar Imam,*,‡ and Maznah Ismail*,†,‡ †
Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences and ‡Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia ABSTRACT: The present study was aimed at utilizing defatted rice bran (DRB) protein as an iron-binding peptide to enhance iron uptake in humans. DRB samples were treated with Alcalase and Flavourzyme, and the total extractable peptides were determined. Furthermore, the iron-binding capacities of the DRB protein hydrolysates were determined, whereas iron bioavailability studies were conducted using an in vitro digestion and absorption model (Caco-2 cells). The results showed that the DRB protein hydrolysates produced by combined Alcalase and Flavourzyme hydrolysis had the best iron-binding capacity (83%) after 90 min of hydrolysis. The optimal hydrolysis time to produce the best iron-uptake in Caco-2 cells was found to be 180 min. The results suggested that DRB protein hydrolysates have potent iron-binding capacities and may enhance the bioavailability of iron, hence their suitability for use as iron-fortified supplements. KEYWORDS: defatted rice bran, hydrolysate, iron binding, iron bioavailability, iron uptake, Caco-2 cell culture
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INTRODUCTION Iron is an essential component of cytochromes, hemoglobin, myoglobin, and some enzymes, and its deficiency has serious implications for bodily functions. Iron deficiency is the topranking cause of anemia in the world,1 affecting billions of people, especially in the region of Southeast Asia.2−5 It can cause tiredness and difficulty in concentrating as well as impairment of children’s mental and motor development.1,6,7 In addition to the deleterious effects of iron deficiency on physiological systems in individuals, the resulting public health consequences can significantly affect economies in terms of public health costs, wasted educational resources, restraint of productivity, and loss of human capital formation.2 Insufficient dietary intake of iron is one of the main causes of iron deficiency.6 Oral iron supplementation is the first line of treatment and prevention for iron-deficiency anemia; however, low bioavailability and side effects such as nausea, abdominal cramps, and constipation have hindered its application.8 Food fortification such as adding peptide−iron complex to food has also been used as an alternative to reduce the prevalence of iron-deficiency diseases. Some food-based proteins and peptides have been reported to possess iron-binding properties including shrimp processing byproducts hydrolysates,9 soy protein isolate hydrolysate,10 porcine blood plasma protein hydrolysate,11 whey protein hydrolysate,12 chicken muscle protein,13 and casein phosphopeptide.14 To provide new opportunities for iron fortification of food, the application of defatted rice bran (DRB) protein hydrolysates was considered a suitable alternative. Rice bran (RB) is a byproduct of the rice milling process and constitutes about 10% of the rough rice. RB has been considered a waste in the past, but the discovery of healthpromoting bioactives has revolutionized its use. This “waste-tohealth” concept is the basis behind the heightened interest in RB as a potential source of functional ingredients. Moreover, RB has been recognized for some time now as a valuable © XXXX American Chemical Society
commodity for its oil. Its protein content after the extraction of oil is also of high quality, with a good balance of the essential amino acids. Additionally, treatment with food-based proteolytic enzymes has been demonstrated to yield bioactive peptides with different biological functions or physiological effects, including antioxidative,15 antidiabetic,16 cholesterollowering,17 and anticancer effects.18 Interestingly, DRB is rich in proteins and other nutritional components, making it a potentially good source of functional proteins and hydrolysates. The use of DRB protein and hydrolysates in the area of iron binding is of particular interest due to their availability and costeffectiveness, as well as being an environment-friendly alternative. Thus, the present study investigated the iron-binding property of DRB protein hydrolysates following enzymatic hydrolysis. In addition, iron bioavailability in the presence of DRB protein hydrolysates was determined using a model that combines both in vitro digestion and iron uptake by Caco-2 cells. Iron cell retention, transport, and uptake from DRB peptide hydrolysates after digestion were equally investigated.
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MATERIALS AND METHODS
Chemicals and Reagents. All of the chemicals used were of analytical reagent grade. Protease from Bacillus licheniformis (synonym: Alcalase), protease from Aspergillus oryzae (synonym: Flavourzyme), pepsin, pancreatin, bile acid, sodium bicarbonate, nonessential amino acids, penicillin−streptomycin, rat tail collagent type I, and amino acid standard mixture were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The peptidases used were Alcalase 2.4 L and Flavourzyme 1000 MG. Alcalase 2.4 L is an endopeptidase from B. licheniformis, with Subtilisin Carlsberg as the major enzymic component, having a specific Received: July 14, 2015 Revised: September 29, 2015 Accepted: October 4, 2015
A
DOI: 10.1021/acs.jafc.5b03420 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
with an inline column filter. The column temperature was maintained at 43 °C. The samples were separated and eluted by a gradient resulting from mixing mobile phases A and B. Mobile phase A consisted of 0.1 M ammonium acetate, pH 6.5, whereas mobile phase B consisted of 44:46:10 0.1 M ammonium acetate/acetonitrile/ methanol (v/v), pH 6.5. The flow rate was 1 mL/min throughout, and the gradient consisted of the following profiles: 100% A at start, 90% A and 10% B at 15 min, 60% A and 40% B at 30 min, 50% A and 50% B at 40 min, 100% B at 50 min, 100% A at 57−60 min. The samples eluted from the column were detected at 254 nm and recorded. The results were analyzed and compared with amino acid standards (AAS18, Sigma). The amino acid score (AAS) for the essential amino acid was calculated according to the formula
activity of 2.4 Anson units (AU) per gram. One AU is the amount of enzyme that under standard conditions digests hemoglobin at an initial rate that produces an amount of trichloroacetic acid-soluble product which gives the same color with the Folin reagent as 1 mequiv of tyrosine released per minute. Optimal endoprotease activity was obtained by application trials to pH 8.0. Flavourzyme 1000 MG is an exopeptidase and endoprotease complex with an activity of 1.0 leucine aminopeptidase unit (LAPU) per gram. One LAPU is the amount of enzyme that hydrolyzes 1 mmol of leucine−p-nitroanilide per minute. Optimal exopeptidase activity was obtained by application trials at pH 7.0. Preparation of DRB. RB mixed varieties were obtained from Padiberas National Berhad (BERNAS) rice milling factory, Tiram Jaya, Kuala Selangor, Malaysia. Freshly milled rice bran was stabilized and dried to constant weight at 100−105 °C in a custom-fabricated commercial stabilizer (Ansa Technology Sdn. Bhd, Johor, Malaysia). In brief, the rice bran powder was poured evenly into heat-resistant glass cookware and heated under high power (1000 W) in the stabilizer for approximately 180−220 s. After drying, RB was passed through a sieve (≤0.5 mm) and stored at 4 °C in a freezer prior to extractions. RB was then subjected to supercritical fluid carbon dioxide extraction (SFE; Thar 1000 F, Thar Technologies, Inc., Pitttsburgh, PA, USA) to obtain the DRB. Preparation of DRB Protein Concentrate. DRB was mixed with deionized water in the ratio of 1:10 (w/v) and then 3 M NaOH was added until the final pH of the mixture was 10.5. The mixture was stirred for 1 h at room temperature and then centrifuged at 4000g for 10 min. An additional extraction was carried out with the same volume of alkaline solution. Supernatants were pooled and the pH was adjusted to the isoelectric point (pH 4.5). The precipitate was then recovered by centrifugation at 4000g for 10 min, and washed with distilled water adjusted to the isoelectric point, after which it was freeze-dried until further use. Characterization of DRB Protein Concentrate. Determination of Yield and Protein Content of DRB Protein Concentrate. The Lowry assay was performed to obtain the protein content of DRB protein fractions.19 The absorbance of the sample or standard was measured at 630 nm using a microplate reader (Opsys MR, Thermo Labsystems, Franklin, MA, USA). The results were interpreted as milligrams of bovine serum albumin equivalents per gram of dried weight of sample (mg BSA/g). Amino Acid Analysis of DRB Protein Concentrate. The amino acid analysis of DRB protein fractions was carried out according to the method of Kwanyuen and Burton20 with some minor modifications. Briefly, sample with at least 16% protein content was placed in a sealable test tube. The test tube was acid-washed with 6 N hydrochloric acid (HCl) and dried prior to use. Then, the lyophilized protein was hydrolyzed with 6 N HCl at 110 °C for 24 h. After hydrolysis, L-2-aminobutyric acid (AABA; 0.2578 g of AABA was made up to 1 L of 0.1 N HCl solution) was added into the hydrolysate in a ratio of 10 ml per 15 ml of the hydrolysate. The hydrolysate was then filtered using Whatman filter paper, grade 1 (Sigma-Aldrich Co.), and the supernatant was topped up to 50 mL using deionized water. The hydrolyzed sample was stored at −20 °C. To prepare the derivatized solution, 10 μL of the hydrolyzed sample was dried under vacuum. The hydrolyzed sample was neutralized by adding 20 μL of a 2:2:1 mixture of methanol/water/TEA (v/v). The mixture was vortexed and fully dried under vacuum. Then, precolumn derivatization was performed by adding 20 μL of a mixture of 7:1:1:1 methanol/water/TEA/PITC (v/v) and mixing well with a vortex stirrer. The mixture was allowed to stand at room temperature for 20 min. Then, the sample was completely dried under vacuum. The sample was finally stored in a freezer prior to HPLC analysis. The dried samples were redissolved in 100 μL of buffer A containing 0.1 M ammonium acetate (pH 6.5) and filtered through a Millipore membrane (0.22 μm). Then, 20 μL of the sample was injected and analyzed with an HPLC system equipped with a column heater, autosampler, variable wavelength detector, and data acquisition software controller. The reverse-phase column used was a Pico-Tag (3.9 × 150 mm), dimethyloctadecylsilyl-bonded amorphous silica,
amino acid ratio mg of an essential amino acid in 1 g of sample = mg of the same essential amino acid in 1 g of a reference pattern
The AAS was determined based on the lowest amino acid ratio. The scoring patterns suggested by the FAO/WHO/UHU21 for adults were used in the present study. Preparation of DRB Protein Hydrolysates. DRB was treated batchwise with Alcalase and Flavourzyme by individual, combination, or sequential treatment. Table 1 shows the experimental conditions of
Table 1. Experimental Conditions of Hydrolysis for the Different Enzyme Systems enzyme systema DRB_A DRB_F DRB_A+F
DRB_A•F
detail partial hydrolysis using Alcalase partial hydrolysis using Flavourzyme partial hydrolysis with a combination of Alcalase and Flavourzyme sequential hydrolysis using Alcalase and Flavourzyme
pH
temperature (°C)
8.0
50
7.0
50
8.0
50
8.0 for Alcalase 7.0 for Flavourzyme
50
a
DRB_A+F corresponds to simultaneous hydrolysis with Alcalase and Flavourzyme. DRB_A•F represents sequential hydrolysis using Alcalase first for 1 h followed by Flavourzyme with intermediate pH adjustment.
hydrolysis for different enzyme systems. Individual hydrolyses were developed over 4 h, whereas combination treatment was carried out by combining the two proteases for 4 h, and sequential treatment was carried out with an initial hydrolysis (1 h) using Alcalase and a second one (3 h) using Flavourzyme. The hydrolysis were carried out using the following parameters: (A) Alcalase hydrolysis, substrate concentration (S) = 10%; enzyme−substrate ratio (E/S) = 0.4 AU/g of protein; temperature T = 50 °C; pH 8.0; (F) Flavourzyme hydrolysis, S = 10%; E/S = 100 LAPU/g of protein, T = 50 °C, pH 7.0. Briefly, the suspension was adjusted initially to the appropriate temperature and pH before the proteases, Alcalase and Flavourzyme, were added individually or in combination. Once the enzymes were added, the pH of the reaction was monitored constantly and adjusted to the optimal level by adding 0.5 N NaOH every time it decreased 0.1 unit from the optimal pH. The amount of added NaOH was recorded and later used for the calculation of degree of hydrolysis (DH). The enzymes were subsequently deactivated by heating the mixture to 95 °C for 15 min. The resulting hydrolysates were centrifuged at 3000g for 30 min at 4 °C to separate the soluble hydrolyzed material from the insoluble residue. Determination of DH and Protein Content of DRB Protein Hydrolysates. The DH of DRB protein hydrolysates, defined as the percentage of peptide bonds cleaved, was calculated as B
DOI: 10.1021/acs.jafc.5b03420 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
some modifications. Briefly, DRB peptide hydrolysates (5 g) were incubated for 1 h at 37 °C in a 0.32% (w/v) pepsin solution containing 30 mM NaCl and 0.7% (v/v) of 0.2 N HCl, pH 1.2, in a 1/20 (w/w) enzyme/protein hydrolysate relation to mimic the gastric fluid digestion condition. Then, 5 mg of FeSO4 (37.5 μmol)13 was added and followed by sufficient water to top up the solution to 50 mL. To further simulate the intestinal fluid digestion, the pH of the pepsin digests was adjusted to 5.0 by dropwise addition of 0.05 M NaHCO3. Subsequently, the mixture was incubated for 3 h at 37 °C in a pancreatin−bile solution containing 0.005 g of pancreatin and 0.03 g of bile salts/g of sample. When the intestinal digestion was completed, the samples were kept for 10 min in an ice bath. The pH of the samples was then raised to 7.2 using 0.5 M NaOH. The digests were subjected to centrifugation (4000g, 25 min, and 4 °C). The supernatants were collected and filtered through a dialysis tubing cellulose membrane (D9777 Sigma, average flat width = 25 mm, typical molecular weight cutoff = 14000) using the method of Jovani et al.25 prior to iron uptake experiments. Iron Uptake by Caco-2 Cells. Cells were seeded at a density of (1−1.5) × 105 cells/cm2 in a collagen-coated 6-well transwell plates (Costar, High Wycombe, UK). The iron uptake experiment was conducted when the percent of phenol red diffusion was 500 Ω cm2. Briefly, growth medium was removed from the transwell by aspiration, and the upper and lower chambers were rinsed twice with phosphate-buffered saline (PBS) solution. This was followed by adding 1.5 mL of DMEM in the lower chamber and 0.5 mL of in vitro digested sample to the upper chamber. The apical samples and basal solutions were then collected after 4 h of incubation. The cells in the inset were lysed and harvested by adding 0.5 mL of 1 M NaOH. Total mineral content was measured in the apical solutions, cell monolayer, and basal solutions by using the formulas as provided in the following section. Iron contents in DRB hydrolysates and Caco-2 cell absorption were measured by inductively coupled plasma atomic emission spectrometry (ICP-AES; ELAN DRC-e, PerkinElmer). Iron standards were prepared by diluting the FeSO4 solution into series of concentrations (3.125−100.00 μg/L) with the same mixture used for the samples. The detection limit of iron analysis using ICP-MS was
