Inhibition of hemoglobin-mediated lipid oxidation in washed fish muscle by cranberry components

Fractions enriched in phenolic acids (Fraction 1), anthocyanins (Fraction 2), flavonols (Fractions 3 and 4) and proanthocyanidins (Fractions 5 and 6) were prepared from cranberry powder using Sephadex LH-20 chromatography. Fractions 2, 3, 4, and 5 had nearly equivalent reactivity in the total phenolate assay employed per mg dry weight of each fraction while Fractions 1 and 6 were less reactive. The ability of cranberry fractions to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals as well as their inhibitory effects on hemoglobin (Hb)-mediated lipid oxidation in washed cod muscle were assessed. Addition of cranberry fractions at a level of 74 μmol quercetin equivalents per kg of washed cod muscle extended the induction time of thiobarbituric acid reactive substances (TBARS) formation in the order: Fraction 1, Fraction 3, Fraction 4 > Fraction 2 > Fraction 5 > Fraction 6. This suggests that oligomeric polyphenols (e.g., proanthocyanidins) were least effective at inhibiting Hb-mediated lipid oxidation in washed cod muscle compared to the other classes of polyphenolics in cranberry. The ability of the different cranberry fractions to scavenge DPPH radicals did not reflect their relative ability to inhibit lipid oxidation in the washed cod muscle system. Quercetin was tentatively identified as a component in cranberry that was especially effective at inhibiting Hb-mediated lipid oxidation. The ability of flavonol and proanthocyanidin-enriched fractions to inhibit Hb-mediated lipid oxidation in spite of efforts to wash away the added polyphenolics prior to Hb addition indicated these classes of polyphenolics had binding affinities for insoluble components of washed cod muscle.     

The effect of natural antioxidants on haemoglobin-mediated lipid oxidation during enzymatic hydrolysis of cod protein

Heating and changes in pH often practised during fish protein hydrolysis can cause lipid oxidation. The effect of natural antioxidants towards haemoglobin-mediated lipid oxidation during enzymatic hydrolysis of cod proteins was investigated. Different variants of a washed cod model system, containing different combinations of haemoglobin and natural antioxidants (l-ascorbic acid and Fuscus vesiculosus extract), were hydrolysed using Protease P “Amano” 6 at pH 8 and 36 °C to achieve 20% degree of hydrolysis. Lipid hydroperoxides and thiobarbituric acid reactive substances (TBARS) were analysed periodically during the hydrolysis process. The in vitro antioxidant activity of the final products was investigated. Results indicate that oxidation can develop rapidly during hydrolysis and antioxidant strategies are preferable to produce good quality products. Oxidation products did not have an impact on the in vitro antioxidant activity of the hydrolysates. The natural antioxidants inhibited oxidation during hydrolysis and contributed to the antioxidant activity of the final product.     

Interactions between hemoglobin and cod muscle constituents following treatment at extreme pH values

Acid and/or alkaline solubilization is a recent method developed to separate proteins from muscle foods with good functional properties. However, exposure of the muscle and its components to low pH values has been shown to promote lipid oxidation, limiting therefore the applications of this novel method. This research aimed primarily to study the physicochemical changes of the fish membranes brought about during acid or alkali solubilization processes. The effect on lipid oxidation and the possible role of the water soluble fraction of the muscle (press juice) as a potent antioxidant were also investigated. Model systems comprising minced cod muscle or cod microsomal suspensions were used. Results showed that acid or alkaline treatment (pH < 3.5 or pH > 10.5) of cod membranes significantly delayed lipid oxidation. Added triacylglycerols to washed cod system treated at low pH did not enhance hemoglobin-mediated lipid oxidation. Decreased precipitation of hemoglobin was observed with the alkali-treated membranes at all protein concentrations compared to the acid-treated and the untreated membranes. Finally, the addition of press juice to washed cod muscle tissue or to the membrane model system, significantly delayed hemoglobin lipid oxidation. PRACTICAL APPLICATION: The results of this study can be used to improve pH-shifting technologies to avoid or decrease lipid oxidation problems. Also, the use of press-juice from cod muscle as means of protecting the muscle against lipid oxidation is suggested.     

Role of deoxyhemoglobin in lipid oxidation of washed cod muscle mediated by trout, poultry and beef hemoglobins

Deoxyhemoglobin content was measured in hemoglobins from trout, chicken and bovine sources between pH 5.5 and 7.5. With decreasing pH, deoxyhemoglobin content of trout was highest, low to intermediate in chicken, and lowest in beef hemoglobin. Each type of hemoglobin was added to washed cod muscle and lipid oxidation assessed during 2?°C storage. The lipid oxidation rate was trout > chicken > beef based on thiobarbituric reactive substances (TBARS) and lipid hydroperoxide formation. There was no significant difference in pro-oxidative activity of chicken compared to turkey hemoglobin. Hemoglobins from trout appeared to oxidize more rapidly compared to chicken hemoglobin in the washed cod muscle model system, as measured by a decrease in redness (a-value) during storage. Loss of red color was slowest in beef samples. These studies suggest that deoxyhemoglobin may be a major catalyst of lipid oxidation at post mortem pH values found in muscle foods, especially in fish and poultry compared to beef.     

Effect of caffeic acid on haemoglobin‐mediated lipid and protein oxidation in washed cod mince during ice and frozen storage

BACKGROUND: Little is known about the relation between haemoglobin (Hb)‐mediated lipid and protein oxidation in muscle foods and how these two reactions can be inhibited by naturally occurring antioxidants. This study was aimed at evaluating (1) lipid oxidation and protein oxidation induced by 20 µmol L^?1 Hb during chilled and frozen storage of washed cod mince and (2) the efficiency of 10–1000 ppm (0.063–6.3 mmol L^?1) caffeic acid in preventing these reactions. RESULTS: Addition of 20 µmol L^?1 Hb increased peroxide value (PV), rancid odour, protein carbonylation, protein insolubilisation, redness loss and α‐tocopherol loss in ice‐stored washed cod mince. Since both lipid and protein oxidation developed at the same time, it was not possible to conclude which reaction initiated the other. All studied reactions were efficiently inhibited by ≥ 50 ppm caffeic acid, which could be a result of α‐tocopherol regeneration, general radical scavenging, reduced formation of oxidised Hb forms and/or conformational changes in Hb structure. During frozen storage the only clear effect of Hb was increased PV, and here caffeic acid was less efficient as an antioxidant. CONCLUSION: Hb‐induced lipid and protein oxidation occurred quickly in ice‐stored washed cod mince, and the two reactions could not be separated in time. During frozen storage, Hb caused only limited lipid oxidation. Caffeic acid (≥50 ppm) was an efficient antioxidant during ice storage. Copyright © 2010 Society of Chemical Industry     

Activity of caffeic acid in different fish lipid matrices: A review

Caffeic acid, a hydroxycinnamic acid common in different vegetable sources, has been employed as a natural antioxidant for inhibiting oxidation of fish lipids present in different food matrices. The aim of this review is to discuss the mechanisms involved in the antioxidative and prooxidative effects of caffeic acid found in different model systems containing fish lipids. These model systems include bulk fish oils, liposomes from cod roe phospholipids, fish oil emulsions, washed cod mince, regular horse mackerel mince and a fish oil fortified fitness bar. The data reported show that the antioxidant activity depends on the physical state of the lipids and the composition of the intrinsic matrix in which they are situated. Caffeic acid significantly prevented rancidity in both unwashed and washed fish mince, the latter which was fortified with haemoglobin. In the unwashed mince, the activity was however clearly dependent on the lipid to antioxidant ratio. In these systems, an important redox cycle between caffeic acid and the endogenous reducing agents ascorbic acid and tocopherol were further thought to play an important role for the protective effects. The effect of caffeic acid was also highly dependent on the storage temperature, showing higher effectiveness above than below 0 °C. Caffeic acid was not able to inhibit oxidation of bulk fish oils, fish oil in water emulsions and the fish-oil enriched fitness bar. In the liposome system, caffeic acid inhibited haemoglobin (Hb)-promoted oxidation but strongly mediated Fe²⁺ mediated oxidation. In conclusion, caffeic acid can significantly prevent Hb-mediated oxidation in fish muscle foods but its activity in food emulsions and liposomes is highly dependent on the pH, the emulsifier used and the prooxidants present.     

Hemoglobin-mediated lipid oxidation of protein isolates obtained from cod and haddock white muscle as affected by citric acid,calcium chloride and pH

Acid or alkali solubilization followed by isoelectric precipitation can be used to isolate proteins with good functional properties from muscle tissue. Both acid (pH 3.0) and alkali (pH 10.5) treatment of the muscle decreased lipid oxidation catalyzed by hemoglobin. Citric acid and calcium chloride improved the oxidative stability of both acid- and alkali-solubilized muscle protein isolates when added to the homogenized muscle before separating the membrane or directly to the isolated membranes in the assay. Citric acid may have functioned in part by lowering the pH and destroying preformed peroxides. Exposing the muscle and the hemoglobin together at pH 3 promoted lipid oxidation, while addition of citric acid/calcium chloride or press juice to washed cod prior to solubilization inhibited lipid oxidation even when the tissue and hemoglobin were acidified together.     

Novel interactions of caffeic acid with different hemoglobins: Effects on discoloration and lipid oxidation in different washed muscles

Caffeic acid (CA) accelerated methemoglobin (Hb) formation at pH 5.8 and 25 °C. This was attributed to electron donation from CA to liganded O₂ in Hb. CA inhibited hemin dissociation from metHb. Pig Hb remained mostly as oxyHb and did not promote lipid oxidation in washed cod muscle (WCM) nor washed turkey muscle (WTM) during iced storage at pH 5.8. Conversely, perch Hb rapidly was converted to metHb and readily promoted lipid oxidation based on lipid peroxide and hexanal formation. CA strongly inhibited perch Hb-mediated lipid oxidation during storage. Once metHb formation occurred in WCM, CA appeared to maintain the heme protein as metHb during the remainder of iced storage. CA may have become bound to perch Hb based on filtration analysis. CA facilitated the transfer of perch Hb (but not pig Hb) from the aqueous phase to the insoluble components of WCM. Collectively, these results suggest that CA inhibited Hb-mediated lipid oxidation by various mechanisms not related to inhibition of metHb formation.     

Factors affecting solubilisation and oxidation of proteins during equine metmyoglobin-mediated lipid oxidation in extensively washed cod muscle

Lipid and protein changes in extensively washed cod muscle were studied at pH 5.6 and 2 °C for 47 h in the absence and presence of horse metmyoglobin (metMb). MetMb significantly increased lipid peroxides and TBARS formation. MetMb addition reduced solubility of proteins, such as myosin heavy chain and its fragments, and slowed down the enzymatic release of soluble proteins and protein fragments, e.g. fragments from collagen.     

Antioxidant activity and phenolic composition of sumac (Rhus coriaria L.) extracts

Antioxidant activity and phenolic compounds of sumac extracts were investigated. Sumac was extracted in methanol and subjected to solvent–solvent partitioning to yield two fractions as ethyl acetate and aqueous. Methanol extract was further fractioned over Sephadex LH-20 column. Antioxidant activity of extracts and fractions were screened using ferric thiocyanate and DPPH radical scavenging methods. Phenolic composition of active fraction(s) was determined by HPLC–MS systems. Those fractions which exhibited strong antioxidant activity were rich in anthocyanins and hydrolysable tannins. While gallic acid was the main phenolic acid in the extracts, anthocyanin fraction contained cyanidin, peonidin, pelargonidin, petunidin, and delphinidin glucosides and coumarates. Pentagalloyl glucose was abundant in the hydrolysable tannin fraction. Effective scavenging concentration (EC₅₀) on DPPH radical was 0.70 μg/mL both in ethyl acetate and tannin fractions, and 5.33 μg/mL in anthocyanin rich fraction. Same extracts and fractions showed moderate lipid peroxidation inhibition effect compared with the synthetic antioxidants. The findings demonstrate that sumac can be used as a natural antioxidant.     

RANCIDITY DEVELOPMENT IN A FISH MODEL SYSTEM AS AFFECTED BY PHOSPHOLIPIDS

Blood components demonstrated the ability to oxidize their own lipids. Blood plasma developed strong oxidation odor during storage apparently due to oxidation of its lipid components. Rancid odor and substantial thiobarbituric acid reactive substances (TBARS) development occurred in shaken solutions of trout blood containing approximately 0.01% lipid. When whole blood was added to washed cod muscle that was taken through a lipid reduction process, rancid odor still occurred during storage. Having at least six times more membrane phospho-lipids present did not enhance the rate or extent of rancidity development during storage of washed cod containing added blood. Contrary to sensory data, TBARS formed more rapidly in the reduced lipid washed cod compared to the washed cod containing approximately 0.6% lipid. The TBARS formation was greater in the reduced lipid washed cod than in the washed cod when expressed on a lipid basis but not when expressed on a wet weight basis. The lack of rancidity and TBARS development when hemolysate was added to a myosin preparation suggested that at least trace amounts of lipid were required for rancidity to occur. Results from these experiments indicate that rancid odor and extensive lipid oxidation can occur at remarkably low levels of fish lipid.     

Antioxidant activity of ethanolic extract of Cortex fraxini and use in peanut oil

Cortex fraxini was extracted with 95% ethanol to obtain a crude antioxidant extract. The antioxidant activity was evaluated using the linoleic acid peroxidation method and the free radical scavenging assays, namely 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals. Cortex fraxini extract (CFE) showed high inhibition of peroxidation of linoleic acid when compared with butylated hydroxytoluene (BHT). CFE also exhibited excellent scavenging activity on DPPH and hydroxyl radicals. Total antioxidant activity was measured by the reduction of Mo(VI) to Mo(V) by the extract, and subsequent formation of a green phosphate/Mo(V) complex at acid pH. CFE had significant total antioxidant activity and the effects were increased with increasing reaction time. The total phenolic content of the sample, analyzed by using Folin–Ciocalteu’s reagent, was 91.33 mg/g dry weight expressed as pyrocatechol equivalents. Then the suitability of CFE as an antioxidant was determined in peanut oil, and the decrease of lipid oxidation was monitored using thiobarbituric acid-reactive substances (TBARS) assay. CFE treatment significantly (P < 0.05) reduced lipid oxidation in peanut oil compared to the control. No significant differences (P = 0.05) in lipid oxidation were detected between CFE antioxidant and BHT antioxidant samples.     

Antioxidant activity of white and black sesame seeds and their hull fractions

The total phenolic content (TPC), total antioxidant status (TAS), free radical scavenging capacity, inhibition of low density lipoprotein (LDL) cholesterol and metal chelating capacity of extracts of whole black and whole white sesame seeds and their hull fractions in 80% aqueous ethanol were investigated. The TPC of whole black sesame seeds and hull extracts were 29.9 ± 0.6 and 146.6 ± 0.6 mg catechin equivalents/g crude ethanolic extract, respectively. The corresponding values for white sesame were 10.6 ± 1.6 and 29.7 ± 0.9 mg catechin equivalents/g crude ethanolic extract. The TAS as determined by Trolox equivalent antioxidant capacity assay and expressed as Trolox equivalents was highest for black sesame hulls (65.9 ± 1.7) while white seeds showed the lowest (4.4 ± 0.6). Free radical scavenging capacity of sesame extracts (5–40 μg/mL) was measured using 2,2-diphenyl-1-picrylhydrazyl radical. The highest scavenging capacity was obtained at 40 μg/mL and was 94.9 ± 0.8, 25.1 ± 0.4, 14.4 ± 0.9 and 2.5 ± 0.4 for black sesame hulls, black sesame seeds, white sesame hulls and white sesame seeds, respectively. Inhibition of LDL oxidation at 100 ppm level was highest for black sesame hulls (96.7%) followed by those for white sesame hulls (84.6%), black sesame (78.4%) and white sesame seeds (57.3%). Sesame products displayed good ferrous ion chelating capacities, which ranged from 12% to 46% and 17% to 62% at 50 and 100 ppm levels, respectively. Results demonstrated considerable antioxidant activity of sesame products tested especially black sesame hulls.     

Antioxidant potential of ethanolic extract of Polygonum cuspidatum and application in peanut oil

Polygonum cuspidatum was extracted with 95% ethanol to obtain a crude antioxidant extract. P. cuspidatum extract (PCE) had a very high content of total phenol, which was 104.83 ± 8.58 mg/g dry weight, expressed as pyrocatechol equivalent. PCE exhibited excellent antioxidant activity, as measured using α,α-diphenyl-β-picryhydrazyl (DPPH) and total antioxidant assays. It also showed a high lipid antioxidant activity and hydroxyl radicals scavenging activity. A positive correlation was found between the reducing power and the antioxidant activity of PCE, which was found to be comparable to resveratrol and butylated hydroxytoluene (BHT). Then the suitability of PCE as an antioxidant was determined in peanut oil, and the decrease of oxidation was monitored using thiobarbituric acid-reactive substances (TBARS) assay. PCE treatment significantly (P <  0.05) reduced lipid oxidation in peanut oil compared to resveratrol and BHT.     

Extraction and application of antioxidants from black glutinous rice

This research aimed to determine optimum extraction condition of black glutinous rice crude extract and to determine its application as an antioxidant in fish oil enriched mayonnaise. Black glutinous rice flour was extracted twice with 70:30 acetone-water mixture (v/v) at pH 2 and 6.8 for 2, 4 and 8 h of total extraction times. Total phenolic content (TPC), total monomeric anthocyanin content (TMA) and antioxidant activities as determined by ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity assays of the crude extracts were measured. The extraction with pH 6.8 solvent for 4 h yielded the crude extract with significantly highest antioxidant activities analyzed by both FRAP and DPPH tests (p ≤ 0.05) although its TPC and TMA were not greatest. The freeze-dried extract from this condition was then added into fish oil enriched mayonnaise at 500 mg/kg and 1000 mg/kg (oil weight basis). Conjugated diene hydroperoxides (CDH), thiobarbituric acid reactive substance (TBARs) and color in CIELAB system of the mayonnaise samples stored at 30 °C were determined up to 30 days. The samples contained 1000 mg/kg crude extract had lowest rate of CDH and TBARs increase but had greatest extent of color deterioration, possibly due to anthocyanin degradation and Maillard reaction.     

Addition of partly reduced bovine serum albumin to a metmyoglobin-fortified washed cod system gives reduced formation of lipid oxidation products and increased degradation of proteins

The aim of the investigation was to study the pro-oxidative system of washed cod with added horse metmyoglobin (metMb) regarding lipid and enzyme oxidation plus protein degradation. The changes were studied at pH 5.6 and 2 °C for 47 h. Bovine serum albumin (BSA) with 0.2 or 3.5 thiol (reduced) groups per molecule was added as an antioxidant protein and as providers of free thiols. BSA with the higher amount of thiols decreased the formation of lipid peroxides and thiobarbituric acid reactive substances (TBARS) in the washed cod system. Metmyoglobin (metMb) addition slowed down the enzymatic release of proteins and protein fragments from insoluble washed cod proteins while the opposite was observed with added reduced BSA. Addition of metMb strongly inactivated the thiol proteases cathepsin B + L and effectively oxidized the low molecular weight thiols in the system while reduced BSA slowed down inactivation of the proteases. The endogenous proteases degraded BSA to a fragment of 41.7 kDa. Formation of this fragment was reduced when metMb was added. BSA (not reduced) did not influence TBARS formation in the washed cod system with metMb, but increased the formation of lipid peroxides after 47 h compared to the system without BSA.     

Retardation of myoglobin and haemoglobin-mediated lipid oxidation in washed bighead carp by phenolic compounds

Antioxidative activities of phenolic compounds (caffeic acid, gallic acid and tannic acid; 200 ppm) in washed mince (pH 6), with added myoglobin (Mb) and haemoglobin (Hb), from bighead carp (Hypophthalmichthys nobilis), during 9 days of iced storage, were studied. Tannic acid exhibited the preventive effect on discolouration of washed mince containing Mb or Hb during storage (P < 0.05). High peroxide value (PV) was found and large amount of, thiobarbituric acid-reactive substances (TBARS) and hexanal were formed in washed mince containing haem proteins, especially Hb. As determined by apo Streptococcal haem-associated protein, Hb had the lower haem affinity than Mb. Phenolic compounds, especially caffeic acid and gallic acid, could lower lipid oxidation induced by Mb or Hb throughout storage (P < 0.05). Prevention of haem release, as well as inhibition of lipid oxidation induced by haem proteins with selected phenolic compounds, should be an alternative means in lowering discolouration and lipid oxidation in fish muscle.     

Suitability of saturated aldehydes as lipid oxidation markers in washed turkey meat

The aim of this study was to evaluate the suitability of saturated aldehydes as lipid oxidation markers in washed turkey muscle, by means of headspace solid phase microextraction-gas chromatography (HS-SPME-GC); the results were compared with the widely used thiobarbituric acid-reactive substances (TBARs) method. Changes in TBARs, propanal and hexanal concentrations were determined over time in a model system consisting of turkey muscle washed with a sodium phosphate buffer (pH 5.6). To stop oxidation from occurring during analysis, an antioxidant mixture (EDTA, trolox and propyl gallate) was added immediately before analyses. After antioxidant addition, propanal and TBARs concentrations did not increase during 8 h of further storage, while an unexpected decrease in hexanal was observed. To determine if aldehydes were interacting with washed turkey muscle, hexanal and propanal were added to either phosphate buffer or washed muscle and concentrations were monitored for 24 h. Neither propanal nor hexanal decreased in the phosphate buffer over time, but the headspace concentration of propanal and hexanal in washed turkey muscle were markedly lower (76% and 96%, respectively) at time zero and continued to decreased up to 24 h of storage. Because of this decrease in headspace aldehyde concentrations, TBARs were found to be a more sensitive and accurate marker of oxidative deterioration in washed turkey muscle.     

Oxidative properties of lactoferrins of different iron-saturation in an emulsion consisting of metmyoglobin and cod liver oil

Lactoferrins (LFs) at iron-saturation 8 (native) and 100%, respectively, were present in an oil-in-water (O/W) emulsion composed of 5% (w/v) cod liver oil (CLO) and metmyoglobin (metMb) in 50 mM phosphate buffer at pH 6.0. Initially both LFs acted as antioxidants and reduced initial peroxide formation, but after 48 h holo LF revealed the most peroxides but the least trienes. Native LF (0.8 mg/ml) gave the highest (p < 0.05) amounts of lipid derived volatiles after 48 h incubation at 4 °C. Both LFs gave similar increases in adducts to metMb with time. The most extensive aggregation induced by radicals or peroxides was found for native LF. The results pointed at reactions at the O/W interphase as highly influential for lipid and protein oxidation kinetics. Added ascorbic acid (1 mM), however, behaved as an antioxidant in the pro-oxidative oil-in-water emulsion system and prevented lipid degradation and protein adductation as well as protein aggregation.     

Casein hydrolysis by Bifidobacterium longum KACC91563 and antioxidant activities of peptides derived therefrom

Milk protein is a well-known precursor protein for the generation of bioactive peptides using lactic acid bacteria. This study investigated the antioxidant activity of bovine casein hydrolysate after fermentation with Bifidobacterium longum KACC91563 using the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay and total phenolic content (TPC). The antioxidant activities of the 24-h and 48-h hydrolysates were higher than that of the 4-h hydrolysate (2,045.5 and 1,629.3 μM gallic acid equivalents, respectively, vs. 40.3 μM) in the ABTS assay. In contrast, TPC values showed activities of 43.2 and 52.4 μM gallic acid equivalents for the 4-h and 24-h hydrolysates, respectively. Three fractions (≥10 kDa, ≥3 but <10 kDa, and <3 kDa) were separated from the 24-h hydrolysate by ultrafiltration. Among these fractions, the <3 kDa fraction exhibited the highest antioxidant activity (936.7 μM) compared with the other fractions (42.1 and 34.2 μM for >10 kDa and 3–10 kDa fractions, respectively). Through liquid chromatography-electrospray ionization-tandem mass spectrometry analysis, 2 peptides, VLSLSQSKVLPVPQK and VLSLSQSKVLPVPQKAVPYPQRDMPIQA, containing the fragment VLPVPQ that has antioxidant properties, were identified in the <3 kDa fraction after 24 h of hydrolysis. The present study demonstrates the possibility of antioxidant peptide production from bovine casein using Bifidobacterium longum.