Article pubs.acs.org/JAFC
Factors Affecting Lipid Oxidation Due to Pig and Turkey Hemolysate Haizhou Wu,†,‡ Jie Yin,‡ Jianhao Zhang,*,† and Mark P. Richards*,‡ †
National Center of Meat Quality, Safety Control, Jiangsu Innovation Center of Meat Production, Processing, College of Food Science, Technology, Nanjing Agricultural University, Nanjing 210095, P. R. China ‡ Department of Animal Sciences, Meat Science, Muscle Biology Laboratory, University of WisconsinMadison, 1805 Linden Drive, Madison, Wisconsin 53706, United States ABSTRACT: Turkey hemolysate promoted lipid oxidation in washed muscle more effectively than pig hemolysate, which was partly attributed to the greater ability of H2O2 that formed during auto-oxidation to oxidize the avian hemoglobin (Hb). Turkey and pig hemolysate (2.5 μM Hb) exposed to 10 μM H2O2 oxidized to 48% and 4% metHb, respectively. Catalase activity, which converts H2O2 to water, was elevated in the pig hemolysate. The larger difference in Hb oxidation when comparing turkey and pig hemolysate in washed muscle (relative to their auto-oxidation rates) suggested that lipid oxidation products facilitated formation of metHb. Turkey metHb released hemin more readily than pig metHb, which coincided with turkey metHb promoting lipid oxidation more effectively than pig metHb. Ferryl Hb was not detected during storage of turkey or pig hemolysate in washed muscle, which suggested a minor role for hypervalent forms of Hb in the oxidation of the lipids. KEYWORDS: oxidative rancidity, protein crystallography, poultry, swine, myoglobin
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INTRODUCTION The heme proteins hemoglobin (Hb) and myoglobin (Mb) can promote lipid oxidation in muscle foods. Lipid oxidation reactions need to be controlled during processing and storage of muscle foods because various parameters of quality are negatively affected, including odor, flavor, and nutritional value.1 Hb is a tetramer of two α chains and two β chains, with each chain containing one porphyrin (heme) moiety. Auto-oxidation of Hb describes when the reduced iron atom (Fe2+) of each heme moiety, liganded with oxygen (oxyHb) or free of a ligand (deoxyHb), is oxidized to the met state (Fe3+), forming metHb. For Mb, a monomer, the Fe2+ atom within the single porphyrin of oxyMb (or deoxyMb) is oxidized to form metMb. Auto-oxidation is a critical step in the onset of lipid oxidation because metHb and metMb formation weakens the porphyrin−globin linkage, which can facilitate hemin loss, particularly at acidic pH values.2 Subsequently, released hemin decomposes preformed lipid hydroperoxides into free radicals that readily stimulate the oxidation of lipids.3 MetMb and metHb formation also facilitates formation of ferryl forms of the heme proteins that can promote lipid oxidation.4 The addition of Mb and Hb to washed muscle has been used to assess aspects of Mb- and Hb-mediated lipid oxidation. For example, increasing iron release from Mb, using Mb mutants susceptible to porphyrin degradation, decreased lipid oxidation in washed muscle.5 This suggested that released iron did not contribute to Mb-mediated lipid oxidation. In a separate study, Hb promoted lipid oxidation more effectively compared to Mb in washed muscle, which was attributed to the lower hemin affinity of Hb.6 Little research has been done comparing the pro-oxidative characteristics of avian Hb to that of mammalian Hb. Hemoglobin comprised 25−54% of the total heme protein in various pork muscles, while Hb content in breast and thigh from chickens and turkeys was 22−86% of the total heme protein.7−10 Conversion of reduced Hb to metHb, dissociation © 2017 American Chemical Society
of the protoporphyrin moiety from metHb, and formation of ferryl Hb species are three pathways that can lead to oxidative rancidity. Our objective was to examine these pathways in relation to lipid oxidation mediated by pig and turkey Hb to better understand the key reactions that promote lipid oxidation when assessing mammalian and avian hemoglobins.
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MATERIALS AND METHODS
Chemicals. Heparin, streptomycin sulfate, ammonium thiocyanate, ferrous sulfate, sodium sulfide, and sucrose were obtained from Sigma Chemical A/S (St. Louis, MO). Chloroform, methanol, potassium ferricyanide, and tris(hydroxymethyl) aminomethane (Tris) were obtained from Fisher Scientific (Pittsburgh, PA, U.S.A). All other chemicals were reagent grade. Preparation of Washed Cod Muscle. Washed muscle from turkey or pork was not used because the residual heme content has been found to be substantial in these washed muscles, which can interfere with reactivity of added heme proteins. Washed cod muscle (WCM) contains negligible amounts of residual heme, providing a useful matrix to assess effects of added heme proteins. The lipid fraction of the washed cod muscle contains mostly phospholipids with smaller amounts of free fatty acids and neutral lipids.11 Washed cod muscle from fresh cod (Gadus morhua) fillets was prepared as described previously.12 Preparation of Hemolysates and Purified Hb’s. Four volumes of blood from turkey and swine, both of which were obtained from campus sources (University of WisconsinMadison), were mixed with 1 vol of anticoagulant containing 150 mM NaCl and sodium heparin (120 units/mL). Hemolysates were prepared as described previously.13 The hemolysate was then passed through a 10-DG column (Bio-Rad, Hercules, CA) to remove components less than 6 kDa and buffer-exchange the Hb into 1 mM Tris (pH 8.0). A Received: Revised: Accepted: Published: 8011
June 14, 2017 August 18, 2017 August 22, 2017 August 22, 2017 DOI: 10.1021/acs.jafc.7b02764 J. Agric. Food Chem. 2017, 65, 8011−8017
Article
Journal of Agricultural and Food Chemistry millimolar extinction coefficient of 500 mM−1 cm−1 at 415 nm was used to quantify the concentration of oxyHb.14 Purified Hb’s were prepared using an fast protein liquid chromatography (FPLC) system equipped with a HiLoad gel-filtration column containing Superdex 200 (GE Healthcare, Inc., Uppsala, Sweden) to remove contaminants (column dimensions 2.6 cm × 60 cm). Elution buffer was 10 mM Tris (pH 8.0) containing 50 mM NaCl. The Hb-containing fraction was then buffer-exchanged into 1 mM Tris (pH 8.0) prior to use. The purified turkey Hb fraction consisted of HbA (αA2β2) and HbD (αD2β2) at a 3:1 ratio. Preparation of metHb. metHb was prepared as described previously.15 Briefly, 4 mol of potassium ferricyanide were added per mol of reduced Hb in the hemolysate (on a heme basis) and mixed. After incubation on ice for 1 h, ferricyanide was removed using a 10DG desalting column and 1 mM Tris, pH 8.0 elution buffer (Bio-Rad, Hercules, CA). A millimolar extinction coefficient of 716 mM−1 cm−1 at 405 nm was used to quantify the concentration of metHb.14 Addition of Reduced Hb in Hemolysates and metHb to Washed Cod Muscle. Hb’s were mixed with washed cod muscle as described previously.6 The final Hb concentration was 10 μmol/kg of washed cod muscle. Determination of Lipid Hydroperoxides. Lipid hydroperoxides were determined as previously described.11,16 Optical density was measured at 500 nm after 20 min incubation of the reaction mixture at room temperature in the dark. A standard curve was constructed using cumene hydroperoxide. There was ∼0.7 g total lipid per 100 g of washed muscle, wet weight.11 Determination of Thiobarbituric Acid Reactive Substances. Thiobarbituric acid reactive substances (TBARS) were determined according to the modified method of Lemon.17 One modification was that 0.5 g samples were homogenized with 6 mL of the extracting solution. A standard curve was constructed using tetraethoxypropane. Determining Hb Auto-oxidation Rates (MetHb Formation). Auto-oxidation (kox) rates for Hb were determined as described previously.18 Briefly, Hb was buffered with 200 mM sodium phosphate (pH 6.3) and stored at 4 °C. Samples were stored in polystyrene cuvettes. Spectra were obtained at regular time intervals between 700 and 500 nm using the UV-2401 spectrophotometer (Shimadzu Instruments, Inc., Columbia, MD). The percentage of methemoglobin was calculated according to the equations of Benesch et al.19 The existence of ferrous hemoglobin spectra was verified for each heme protein prior to initiating the experiment. Hb solutions were incubated as treatment I, presence of added 3 mmol of superoxide dismutase (SOD) and catalase (CAT) per mol of heme, and treatment II, absence of added SOD and CAT. Added SOD and CAT removed any superoxide and hydrogen peroxide that was produced during incubation. The slopes obtained during storage were used to determine the relative rates of Hb auto-oxidation (kox). Measuring Hemin Loss from metHb. Hemin loss was determined as described by Grunwald and Richards.5 Hemin loss rates at 25 °C were measured by mixing the apoglobin form of H64Y sperm whale Mb with the oxidized forms of the turkey and pig holoHb’s (10 μmol/L) at pH 6.3, and time courses for the uptake of hemin by the apoMb reagent were measured spectrophotometrically following previous procedures.20,18 Color Measurement. The Hunter L (lightness), a (redness), and b (yellowness) values of WCM containing turkey or pig Hb were measured daily using a Minolta CR-300 chroma meter using the D65 illuminant and 8 mm aperture (Minolta Camera Co., Osaka, Japan). The chroma meter was calibrated against a white board prior to use. At the beginning of each experiment, 1 g of the reaction from each treatment was added to the Petri dish (35 mm × 10 mm). The samples were then wrapped in aluminum foil and stored in the dark at 2 °C. Quantifications of Ferryl Hb in WCM. Washed cod muscle containing added hemolysate (1.0 g) was centrifuged at 16100 × g for 10 min at 4 °C using a tabletop microcentrifuge. The supernatant (0.1 mL) was diluted in 0.85 mL of 50 mM phosphate buffer, pH 7.4, and scanned from 370 to 700 nm. After scanning, 50 μL of 40 mM Na2S in 50 mM phosphate buffer, pH 7.4, was added into the same cuvette to
achieve the final concentration of 2 mM Na2S. Following mixing, the solution was immediately scanned from 370 to 700 nm. Ferryl Hb reacts with Na2S to yield sulfhemoglobin with a characteristic peak at 618 nm (ε618 nm = 21.1 mM−1 cm−1).21 Statistical Analysis. Igor Pro software (WaveMetrics, Portland, OR) was used to calculate rates of auto-oxidation and hemin loss by fitting observed data into respective functions. For auto-oxidation and hemin loss, three reactions were examined per treatment (n = 3 replicates). The MIXED procedure of SAS was used to analyze data, and means were separated using p-diff test. For lipid oxidation measurements during storage, three separate reactions per treatment were examined at each time point. The level of significance for statistical evaluations was set at P < 0.05.
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RESULTS MetHb Formation in Pig and Turkey Hemolysate during Auto-oxidation and Reaction with Hydrogen Peroxide. Auto-oxidation rates of reduced Hb in pig and turkey hemolysate (2.5 μM) were measured during storage at 4 °C (pH 6.3) in the presence and absence of SOD and CAT (Table 1). Turkey Hb auto-oxidized more rapidly in the Table 1. Auto-oxidation Rate (kox) for Turkey and Pig Hemoglobin (2.5 μM Hb) in Hemolysates during 4 °C Storage at pH 6.3a hemoglobin
kox (day−1)
SOD/CATb
turkey pig turkey pig
0.0148 ± 0.0005 c 0.0222 ± 0.0012 b 0.0343 ± 0.0014 a 0.0243 ± 0.0006 b
+ + − −
a
Means bearing different designations (a, b, and c) in a column differ significantly (P < 0.05). The Hb concentration was 2.5 μM in 200 mM sodium phosphate (pH 6.3). Replicates per treatment were n = 3. Means and standard deviations are shown. b+, addition of superoxide dismutase and catalase (3 mmol per mol of heme from Hb); −, no addition of superoxide dismutase or catalase to the hemolysates.
absence of SOD and CAT versus in the presence of SOD and CAT (p < 0.05). However, there was no significant difference in auto-oxidation rates for pig Hb when comparing the presence and absence of added SOD and CAT. Hb in pig hemolysate auto-oxidized more rapidly than Hb in turkey hemolysate when in the presence of SOD and CAT (p < 0.05), whereas in the absence of SOD and CAT turkey Hb had more rapid auto-oxidation rate than pig Hb (p < 0.05). SOD and CAT removed superoxide radical and hydrogen peroxide, respectively, that were produced during incubation. These results suggest that Hb in turkey hemolysate was more sensitive to oxidation in the presence of superoxide radical and/or H2O2 as compared to Hb in pig hemolysate. To further substantiate the role of hydrogen peroxide (H2O2), pig and turkey hemolysate (2.5 μM Hb) were incubated with 2.5−40 μM H2O2 for 1 h at 4 °C. Pig hemolysate was weakly reactive with H2O2, indicating
