Article pubs.acs.org/JAFC
Non-Heme Iron Loading Capacities of Anchovy (Engraulis japonicus) Meat Fractions under Simulated Gastrointestinal Digestion Liang Zhao,† Haohao Wu,*,† Mingyong Zeng,*,† and Hai Huang‡ †
College of Food Science and Engineering, Ocean University of China, 5 Yushan Road, Qingdao, Shandong Province 266003, China College of Food Engineering, Qinzhou University, 12 Binhai Road, Qinzhou, Guangxi Province 535011, China
‡
ABSTRACT: A ferric oxyhydroxide nanoparticle (FeONP)-mediated mechanism has been suggested recently for anchovy (Engraulis japonicus) meat (AM) enhancement of non-heme iron absorption. The current paper fractionates AM biomass into protein (70.67%), lipid (20.98%), and carbohydrate (i.e., glycogen and mucopolysaccharide, 1.07%) and evaluates their capacities in templating the formation of FeONPs under simulated gastrointestinal digestion. Results show that their iron-loading capacities (mg/g) follow the ascending order glycogen (2.43 ± 0.65), protein (20.16 ± 0.56), AM (28.19 ± 0.86), lipid (33.60 ± 1.12), and mucopolysaccharide (541.33 ± 32.33). Protein and lipid act in synergy to contribute the overwhelming majority (about 90%) of AM’s iron-loading capacity. L-α-Phosphatidylcholine and L-α-lysophosphatidylcholine are the predominant iron-loading fractions in the lipid digest. Dynamic light scattering and transmission electron microscopy exhibit coating of inorganic cores of the formed FeONPs with peptides or phospholipid-based mixed micelles. Overall, protein and phospholipid are key players in the nanoparticle-mediated “meat factor” mechanism. KEYWORDS: anchovy meat, gastrointestinal digestion, peptide, phospholipid, mixed micelle, non-heme iron, ferric oxyhydroxide nanoparticle
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INTRODUCTION Iron is a nutrient widely used in nature to transfer electrons for many functions (e.g., oxygen/CO2 transportation, cell respiration, and DNA synthesis). Iron deficiency, the most prevalent nutrient deficiency in both the developing and the developed world, is of great concern due to its adverse effects on immunity, thermoregulation, work performance, and cognition of sufferers, especially infants, young children, and women.1 Iron is in fact not rare in foods and can be classified as heme or non-heme iron. Heme iron (derived from hemoglobin and myoglobin) is highly bioavailable, but constitutes a minor part of dietary iron, especially in developing countries. Bioavailability of non-heme iron is greatly affected by various dietary components.2 Muscle foods (e.g., meat, poultry, and fish) have been well documented to promote non-heme iron absorption.3 However, despite numerous experimental efforts, the chemical nature and mechanism of action of the “meat factor”, which is responsible for the enhancing effect of muscle foods on nonheme iron absorption, are still not well understood at present. Free amino acids, especially cysteine, were at first supposed to account for the meat factor.4 Later studies revealed that rather than free amino acids, low-molecular-weight iron-binding peptides enriched in certain amino acid residues (e.g., cysteine, histidine, aspartic acid, and glutamic acid) could be responsible for the meat factor.5−7 Glycosaminoglycans and L-α-glycerophosphocholine released during digestion of carbohydrate and lipid in muscle foods, respectively, have also been proposed to contribute to the meat factor.8−10 As solubility is a prerequisite for iron bioavailability, the meat factor is usually regarded to chelate ferric iron or to reduce ferric iron to the more soluble ferrous form.11 However, no iron chelate of the meat factor has so far been characterized chemically or observed in the intestine. We previously found © 2016 American Chemical Society
that the trypsin hydrolysate of anchovy muscle protein solubilizes ferric iron via mediating the formation of ferric oxyhydroxide nanoparticles (FeONPs) within a pH range resembling that of human intestinal lumen.12 In our later study, nanosized iron, which accounted for a significant portion of intraluminal iron in the rat small intestine, was proved to contribute to non-heme iron absorption.13 Intestinal absorption of nanosized iron via endocytosis or divalent transporter 1 has also been characterized both in vitro and in vivo recently.13−16 Based on these findings, it can be postulated that the meat factor−iron complex is possibly present in the form of colloidal FeONPs. Carboxyl groups are found to be the major nucleation sites for peptide-mediated biomineralization of FeONPs, and peptide backbones with certain length are also crucial for steric hindrance of crystal growth of FeONPs.12 Similarly, phospholipids and mucopolysaccharides in muscle foods, which possess acidic groups (e.g., phosphate and sulfate) and polymeric backbones, are also biopolymers with the potential of templating the formation of colloidal FeONPs. The current research characterized the in vitro gastrointestinal digestion of anchovy (Engraulis japonicus) muscle fractions (i.e., protein, lipid, and carbohydrate) and evaluated capacities of the digestive products in templating the formation of FeONPs, followed by an examination of colloidal and photophysical properties of the formed FeONPs. Received: Revised: Accepted: Published: 174
October 8, 2016 December 13, 2016 December 14, 2016 December 14, 2016 DOI: 10.1021/acs.jafc.6b04490 J. Agric. Food Chem. 2017, 65, 174−181
Article
Journal of Agricultural and Food Chemistry
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washed twice with 80% ethanol. After air-drying at 60 °C for 2 h, the precipitate was dissolved in water and desalted by dialysis in 1000 Da cutoff tubings for 24 h. The retentate was freeze-dried to make the anchovy meat mucopolysaccharide (AMM). In Vitro Gastrointestinal Digestion. To simulate human gastrointestinal digestion, the procedure described by Moreno et al.23 was followed, and some modifications were made according to the ionic constituents of human gastrointestinal fluids after eating.24 For the gastric digestion step, 5 g of lyophilized sample (e.g., AM, AMP, AMM, AMG, AML, etc.) was dispersed in 100 mL of simulated gastric fluid (SGF, 0.03 M NaCl, pH 2.5), and the suspension was preincubated at 37 °C for 10 min with magnetic stirring. Pepsin was then added at an enzyme-to-sample ratio of 1:100 (w/w), which was proved sufficient for full hydrolysis of AMP within 2 h by our preliminary tests. The gastric digestion proceeded for 2 h with the solution pH monitored every 30 min and adjusted to 2.5 using 1 M HCl if necessary. Aliquots (0.25 mL) withdrawn at 0 and 120 min were immediately mixed with 75 μL of 0.5 M NaHCO3 on ice to inactivate pepsin. For the intestinal digestion step, the pH of the gastric digest was raised to 6.5 with 0.5 M NaHCO3, followed by the addition of bile salts and CaCl2 at the final concentrations of 0.5 g/L and 0.25 mM, respectively. After addition of pancreatin at an enzymeto-sample ratio of 1:25 (w/w), the intestinal digestion was carried out at 37 °C for 30 min to simulate the extent of digestion in the duodenum, where most intestinal iron absorption occurs.1 The solution pH was monitored every 10 min and adjusted to 6.5 using 1 M HCl if necessary. To avoid overdigestion of the samples, enzymes in the digestion were heat inactivated by boiling water bath for 5 min. Digestion of reference standards including PC, LPC, triglyceride (TG), free fatty acid (FFA), and chondroitin sulfate was performed in the absence of pepsin and pancreatin. Glyceryl trioleate and oleic acid were used as standards for TG and FFA, respectively. The digests were centrifuged at 10000g for 20 min at 4 °C. The supernatants were stored at −20 °C or lyophilized for further analysis. Gel Chromatography. Molecular weight (MW) distributions of peptides and polysaccharides before and after in vitro gastrointestinal digestion were determined by gel chromatography on an Agilent 1260 high performance liquid chromatography (HPLC) system (Agilent Technologies, Palo Alto, CA, USA). The peptide MW distribution was analyzed using a TSK-GEL G2000 SWXL column (7.8 mm i.d. × 30 cm long, Tosoh, Tokyo, Japan) with a mobile phase of acetonitrile− water (45:55, v/v) containing 0.1% trifluoroacetic acid at a flow rate of 0.5 mL/min, and the eluent was monitored at 220 nm at 30 °C. A TSK-GEL Super AWM-H column (6.0 mm i.d. × 30 cm long, Tosoh, Tokyo, Japan) with a mobile phase of 0.2 M NaCl at a flow rate of 0.6 mL/min was used to determine the polysaccharide MW distribution, and the eluent was monitored by a refractive index detector (RID). Cytochrome c (12 588 Da), insulin (5733 Da), bacitracin (1423 Da), glutathione (307 Da), and glycine (75 Da) were used as standards to prepare the MW calibration curve for peptides. Dextran standards with MW of 5,000, 12,000, 50,000, 670,000, and 1,400,000 Da were used to make the MW calibration curve for polysaccharides. All digestive samples were diluted to a fixed volume before analysis for quantitative comparison. Analysis of Fatty Acid Composition. Fatty acid compositions of the digests were determined by the method of Lou et al.25 with slight modifications. To prepare fatty acid methyl esters, a 20−30 mg sample was dispersed in 1 mL of 10% concentrated sulfuric acid−methanol (v/v). After being sealed with nitrogen, the suspension was incubated at 60 °C for 15 min and then mixed with 1 mL of n-hexane. After phase separation, the n-hexane layer containing fatty acid methyl esters was removed, dried with anhydrous sodium sulfate, and filtered through a 0.22 μm membrane. The filtrate was immediately subjected to gas chromatography−mass spectrometry analysis using an INNOWax capillary column (30 m × 0.32 mm × 0.25 μm film thickness) on an Agilent 6890N GC system connected to an Agilent 5973 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). Electron impact ionization was performed at 70 eV. Carrier gas was helium at a flow rate of 1.0 mL/min. Temperatures of the injector and detector were 230 and 250 °C, respectively. The temperature of the
MATERIALS AND METHODS
Materials. Frozen Japanese anchovies (E. japonicus), 5−8 cm in length, were supplied by Allen Ship Service Co. Ltd. (Shandong, China) and stored frozen at −20 °C before use. Pepsin (3,820 hemoglobin units/mg protein) from porcine gastric mucosa, pancreatin (4× USP) from porcine pancreas, papain (3.1 Nαbenzoyl-L-arginine ethyl ester units/mg protein) from Carica papaya, bile salts (cholate:deoxycholate, 1:1), 3-(N-morpholino)propanesulfonic acid (MOPS), L-α-phosphatidylcholine (PC), L-αlysophosphatidylcholine (LPC), glyceryl trioleate, oleic acid, chondroitin sulfate, cytochrome C, insulin, bacitracin, glutathione, and dextran standards with known molecular weights were obtained from Sigma-Aldrich Co. (Shanghai, China). All other reagents used in this study were of analytical grade and commercially available. Chemical Analysis. After being thawed in ice water, the fish were beheaded, eviscerated, deboned, washed, and drained to obtain anchovy meat (AM). Moisture content in AM was analyzed by drying samples in an air oven at 105 °C to constant weight. Crude protein was assayed by Kjeldahl nitrogen determination with a multiplying factor of 6.25 according to AOAC method 981.10.17 Lipid was extracted as described later, and its content was calculated gravimetrically. Total carbohydrate content was determined by a phenol−sulfuric acid method.18 Extraction of Total Protein. Extraction of total protein was performed through a trichloroacetic acid (TCA)−acetone procedure.19 AM was minced and homogenized in 5 volumes (w/v) of ice-cold acetone containing 10% TCA and 0.07% (w/v) β-mercaptoethanol. Total protein was precipitated at −20 °C for 24 h, followed by centrifugation at 8000g for 15 min at 4 °C. Pellets were washed three times with ice-cold acetone (v/w = 5:1) for the purpose of removing TCA, followed by further washing with 7 volumes (v/w) of 95% ethanol to remove phospholipid. The residue ethanol and acetone was allowed to evaporate in a fume hood overnight, and the pellet was ground thoroughly to a very fine powder to produce the anchovy meat protein (AMP). Extraction of Total Lipid. Total lipid in AM was extracted following the Bligh and Dyer method.20 Briefly, 100 g of aqueous suspension of AM containing about 20% dry matter was homogenized with a mixture of 100 mL of chloroform and 200 mL of methanol for 2 min (monophasic system). Then another 100 mL of chloroform was added to the solution, and after blending for 30 s, 100 mL of water was added with blending once again for 30 s. After suction filtration, the final biphasic system separated into two layers and the chloroform layer was collected. For quantitative lipid extraction, the lipid withheld in the tissue residue was recovered by blending the residue and filter paper with 100 mL of chloroform, followed by suction filtration, and the filtrate was combined with the original filtrate. The collected chloroform phase was evaporated to dryness under nitrogen to produce the anchovy meat lipid (AML). Carbohydrate Extraction. AM was first degreased with 1.5 volumes of isopropyl alcohol (v/w). Free glycogen was then extracted from AM by hot water.21 Briefly, 50 g of defatted sample was boiled in 1500 mL of water for 3 h, followed by centrifugation at 8000g at room temperature for 15 min, and the insoluble residue was treated again as mentioned above. The supernatants were combined, concentrated, and precipitated with 3 volumes of absolute ethanol at 4 °C for 12 h. The precipitate was then dissolved in water, deproteinated with Sevag reagent (chloroform:n-butanol = 5:1, v/v) at least five times, alcoholprecipitated, and air-dried to produce the anchovy meat glycogen (AMG). Extraction of mucopolysaccharides in AM was performed according to a previously reported method.22 Briefly, 1 g of defatted AM was suspended in 30 mL of 0.1 M acetate buffer (pH 6.0) containing 100 mg of papain, 5 mM cysteine, and 5 mM EDTA and incubated at 60 °C for 24 h. The mixture was then centrifuged at 8000g for 15 min, and 1.6 mL of 10% cetylpyridinium chloride solution was added into the supernatant to precipitate mucopolysaccharides. The precipitate was dissolved in 15 mL of 2.1 M NaCl−ethanol (v/v, 100:15) solution, reprecipitated by the addition of 30 mL of ethanol, and 175
DOI: 10.1021/acs.jafc.6b04490 J. Agric. Food Chem. 2017, 65, 174−181
Article
Journal of Agricultural and Food Chemistry oven was raised from 120 to 210 °C at a rate of 3 °C/min and held at 210 °C for 10 min. The interface temperature was maintained at 280 °C, and the source temperature was kept at 230 °C. The mass scan range was 50−500 u (m/z). The duration of analysis was 40 min. Peaks were automatically integrated, and the components were identified by standard database and characteristic ions. Lipid Separation by Thin Layer Chromatography (TLC). TLC analysis was performed on glass plates (10 cm × 5 cm) prefabricated with silica gel F 254 (Yantai Chemical Industrial Institute, Yantai, China) according to Parker and Peterson’s report26 with some modifications. Samples and reference lipids were solubilized in appropriate amounts of chloroform and were applied to the TLC plate with a microliter syringe. Subsequently, the air-dried plate was placed in a special solvent system (chloroform:methanol:water = 65:25:4, by volume) closed in a chromatographic tank, and stood for approximately 30 min. Followed by air-drying, the plate was exposed to iodine vapor, and the spots were immediately outlined. Glyceryl trioleate and oleic acid were used as standards for TG and FFA, respectively. Iron-Loading Capacity (ILC) Assay. ILCs of the digests were determined based on our previous study.12 Briefly, freshly prepared 1 mM FeCl3 was added dropwise into a vortex-stirred sample solution buffered with 30 mM MOPS (pH 7.0) at room temperature, and after the solution was filtered through 0.22 μm cellulose acetate filters, FeONPs in the filtrate were relatively quantified by the optical absorption at 310 nm where oxo-bridged diferric centers (Fe−O−Fe) in FeONPs have a characteristic absorption band. The amount of FeONPs formed in the presence of a fixed concentration of a digest increases linearly with increasing ferric concentration until no more iron can be loaded by the digest, and excess ferric ions added into the solution can coprecipitate the already-formed FeONPs. ILC can thus be inferred from the dose-dependent formation of FeONPs in the presence of a fixed concentration of a digest as follows: ILC (mg/g) = CFe‑max/Csample, where CFe‑max was the ferric concentration (mg/L) corresponding to the peak optical density at 310 nm (OD310 nm) and Csample was the concentration (g/L) of a digest. Characterization of FeONPs. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to characterize FeONPs. The DLS analysis was performed on a Zetasizer Nano ZS 90 (Malvern Instruments, Herrenberg, Germany) equipped with a 633 nm He−Ne laser using a constant scattering angle of 90° at 25 ± 0.1 °C. For TEM measurements, sample solution was dropped on the carbon-coated copper grids, air-dried, and then examined in a JEM 1200 EX (JEOL, Tokyo, Japan) electron microscope at 100 kV. Statistical Analysis. Data were presented as means ± standard deviations of triplicate determinations. The least significant difference (LSD) mean comparison was performed using the SPSS software program (SPSS Inc., Chicago, IL, USA) to evaluate the mean differences between the measurements at P = 5%.
contents of AM were 15.83%, 4.70%, and 0.24% in the present study, respectively, within their reported ranges of 12−23%, 0.9−19%, and 0.1−2.4%.27−34 Protein accounted for the largest part (about 71%) of the dry matter of AM in this study, and lipid was the second largest component (about 21%). Carbohydrate constituted only a minor fraction (about 1%) of AM dry matter. Fatty acids in AM were found to be longchain ones (13 to 22 carbons) (Table 2), which is in Table 2. Fatty Acid Composition of Anchovy Meat fatty acids
contents (%)
Saturated Fatty Acids C14:0 C15:0 C16:0 C17:0 C18:0 C20:0 total saturated fatty acids Monounsaturated Fatty Acids C16:1 C18:1 C20:1 total monounsaturated fatty acids Polyunsaturated Fatty Acids C13:3 C18:2 C18:3 C20:4 C20:5 C22:6 total polyunsaturated fatty acids
2.45 0.48 23.79 0.82 3.86 0.88 32.28 3.58 7.33 1.05 11.96 0.53 0.98 0.70 1.36 10.11 39.46 53.14
accordance with previous reports.31−35 The predominant fatty acids in AM were docosahexaenoic acid (C22:6, 38.69%) and palmitic acid (C16:0, 23.29%), similar to the results of Zlatanos and Laskaridis32 and Kaya and Turan.35 In Vitro Gastrointestinal Digestion. Figure 1 shows gel filtration elution profiles of the soluble fractions from AMP and
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RESULTS AND DISCUSSION Proximate Composition of AM. AM composition is usually affected by species, sexual maturity, feed, and sea temperature.27−35 Table 1 shows the moisture, protein, lipid, and carbohydrate contents of AM used in this study. Water content in AM varies from 62% to 78% according to previous studies,27−30 and in this study, it was 77.60%, at the higher end of the reported range. The protein, lipid, and carbohydrate
Figure 1. Gel chromatography traces of the digests from (a) the anchovy meat protein and (b) the anchovy meat glycogen.
AMG during a two-stage simulated gastrointestinal digestion process. The MW calibration curve for peptides was set up as log(MW) = 6.904 − 0.239t (R2 = 0.958; t, retention time, min), and the MW distributions of peptides released during AMP digestion (Table 3) were calculated accordingly from Figure 1a. Before pepsin was added, a minor portion of AMP was solubilized in SGF, and nearly half of this portion was larger than 10 kDa. Myofibrillar proteins, which constitute 50−60% of total muscle proteins, are usually regarded to be insoluble in low ionic strength solutions (10 kDa 5−10 kDa 1−5 kDa 0.5−1 kDa
