Effects of hemopexin on hemin and hemoglobin-mediated lipid oxidation in washed fish muscle

Hemopexin (Hx) was isolated from pig blood using hemin-agarose chromatography. The effect of addition of Hx on hemin and hemoglobin (Hb) mediated lipid oxidation in washed cod muscle was investigated during iced storage at pH 5.5. At a 1:1 ratio of Hx to hemin, lipid oxidation as measured by development of thiobarbituric acid reactive substances (TBARS) was not significantly inhibited (p = 0.12). This may be attributed to a lack of hemin binding by Hx due to influence of pH and hemin precipitation. At a 1:2 ratio of Hx to Hb on a heme basis, TBARS development was significantly inhibited (p < 0.01) but not prevented. Equimolar addition of bovine serum albumin (BSA) in place of Hx did not inhibit TBARS development (p = 0.43). Hemin release from porphyrin-containing Hx, i.e. holoHx, and ferric sperm whale myoglobin (Mb) was measured at 37 °C, pH 5.7. Hemin release rates of 0.27 h^?1 and 0.05 h^?1, were calculated for holoHx and Mb, respectively. While the hemin affinity of Hx was greater than published values for trout Hb, the relatively low value measured for Hx compared to Mb may be caused by a combination of low pH and the absence of NaCl in the assays.     

Comparative studies of four different phenolic compounds on in vitro antioxidative activity and the preventive effect on lipid oxidation of fish oil emulsion and fish mince

Antioxidative activities of different phenolic compounds (catechin, caffeic acid, ferulic acid and tannic acid) at various levels were determined by different assays. Among all the phenolic compounds tested, tannic acid exhibited the highest 2,2-diphenyl-1-picryl hydrazyl (DPPH) and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities and ferric reducing antioxidant power (FRAP). Nevertheless, catechin showed the highest metal chelating activity (P < 0.05), whereas caffeic acid had the highest lipoxygenase (LOX) inhibitory activity (P < 0.05). The impact of different phenolic compounds at a level of 100 mg/l on lipid oxidation of menhaden oil-in-water emulsion and mackerel mince was investigated. Tannic acid showed the highest efficacy in retardation of lipid oxidation for both model systems as evidenced by the lower peroxide value (PV), conjugated diene (CD) and thiobarbituric acid-reactive substances (TBARS) values. This was also related with the lower non-heme iron content in tannic acid treated samples. Tannic acid was therefore considered as the most potential natural antioxidant for controlling oxidation of fish oil-in-water emulsion and fish mince, whereas ferulic acid seemed to possess the lowest preventive effect on lipid oxidation.     

Inhibition of haemoglobin-mediated lipid oxidation in washed cod muscle and cod protein isolates by Fucus vesiculosus extract and fractions

The effects of Fucus vesiculosus extract and fractions towards haemoglobin- (Hb-) catalysed lipid oxidation in washed cod muscle system and cod protein isolates during ice storage were examined. The extract and fractions were characterised in terms of total phlorotannin content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferrous ion-chelating ability and reducing power. Progression of oxidation was followed by determining rancid odour, thiobarbituric acid reactive substances (TBARS), redness and volatile oxidation compounds by gas chromatography (GC). In both washed cod muscle and protein isolates, phlorotannin-enriched ethyl acetate (EtOAc) fraction showed higher inhibitory effect than crude 80% ethanol (EtOH) extract. The addition of oligomeric phlorotannin-rich subfraction (LH-2) separated by Sephadex LH-20 chromatography, completely inhibited the initiation of lipid peroxidation in both systems throughout the entire study period (8 days). Its effectiveness at 300 mg/kg level was comparable to that of 100 mg/kg propyl gallate (PG), a highly effective synthetic antioxidant in muscle foods. Although polymeric phlorotannin-rich subfraction (LH-5) had similar level of TPC and chemical antioxidant activities as oligomeric subfraction LH-2, it was far less efficient in model systems. These results suggest that other factors rather than the intrinsic reactivity toward radicals could be responsible for the inhibitory effect of phlorotannins on lipid oxidation in fish muscle. This study highlights the great potential of oligomeric phlorotannins as novel natural antioxidants in fish and fish products.     

Antioxidant and antiradical properties of cranberry juice and extracts

The antioxidant capacities of cranberry juice and three extracts isolated from frozen cranberries containing anthocyanins, water-soluble and apolar phenolic compounds, were evaluated at pH 2.5 and 7, respectively. The free radical-scavenging (FRS) and the lipid peroxidation inhibition (LPI) activities of each samples, and extracts, were studied using the N,N-diethyl-p-phenylenediamine (DPD) decoloration test and the thiobarbituric acid reactive substances (TBARS) assay, respectively. The cranberry phenols displayed good free radical-scavenging properties, but were less efficient at inhibiting the peroxidation of lipids. Of all the samples tested, the water-soluble phenolic compounds showed the greatest free radical-scavenging (68.2 mmol TE/mg phenol) and antioxidant (13.4 mmol TE phenol) activities. The polarity of the phenols, the pH of the medium and the juice process had a great influence on the antioxidant activities. The phenols isolated from cranberries with an aqueous solvent have greater antioxidant properties than those extracted with an organic solvent mixture. The antioxidant activity of the cranberry samples adjusted at pH 2.5 was greater than those adjusted at pH 7. Compared to the cranberry extracts, the juice exhibited a much lower antioxidant activity, especially when compared with the extract containing water-soluble compounds which the extraction conditions were similar to those used to obtain the juice.     

Effects of juice processing on cranberry antioxidant properties

The effects of the industrial juice process on the antioxidant capacities of cranberry samples and of three phenolic extracts (polar phenolics (E1), apolar phenolics (E2) and anthocyanins (E3)) adjusted at pH 2.5 and 7.0 were investigated. The free radical-scavenging (FRS) and the lipid peroxidation inhibition (LPI) activities of each sample and extract, were studied using a N,N-diethyl-p-phenylenediamine discoloration test and the thiobarbituric acid reactive substances assay, respectively. The FRS and the LPI results, expressed in mM Trolox® equivalent (TE)/mg phenol and mg TE/mL/mg phenol, respectively, showed a negative effect of the juice process steps on E1. The E2 and E3 extracts were also affected by the process but not as much. The milling step increased the FRS capacity of E2 and E3 extracts, but decreased their LPI capacity. The evaporation of the juice did not have a significant effect on the FRS capacity, but lowered the LPI capacity. Before and after each step of the juice process, the FRS capacity of the cranberry extracts was greater than the LPI capacity. The results were consistent in classifying the FRS capacity of the extracts in the following order of polarity: E1 > E3 > E2; and their total phenolic (TP) content in the following order: E3 > E2 > E1. In general, the neutralization of the pH of the extracts did not significantly influence the changes observed during the juice process in the FRS and LPI capacity, except for E3 for which a decrease in the LPI capacity was observed.     

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.     

Investigation of plant extracts for the protection of processed foods against lipid oxidation. Comparison of antioxidant assays based on radical scavenging, lipid oxidation and analysis of the principal antioxidant compounds

 Antioxidant activities of plant extracts from spices, coffee, tea, grape skin, and tomato peel slurry were evaluated using a number of analytical methods including the quantification of principal compounds. Similar rankings in the activities of these extracts were obtained by evaluating their efficiencies as scavengers of stable free radicals: Fremy's salt, galvinoxyl or α,α-diphenyl-β-picrylhydrazyl (DPPH). Similar results were obtained with the lipid oxidation assays based on thermal acceleration (formation of conjugated dienes in methyl linoleate at 40  °C or the Rancimat test at 100  °C with lard). Rankings of the extract activity obtained by scavenging of hydroxyl radicals generated in the Fenton reaction were similar to those obtained by an oxygen consumption assay with linoleic acid as substrate and metmyoglobin as catalyst. However, the results of the latter two assays differed from those of the other assays. In the overall ranking, coffee and rosemary extracts were amongst the most potent extracts whereas the tomato peel slurry showed no activity. Received: 16 February 2000 / Revised version: 7 July 2000     

Ability of whey protein isolate and/or fish gelatin to inhibit physical separation and lipid oxidation in fish oil-in-water beverage emulsion

The effect of pH on the capability of whey protein isolate (WPI) and fish gelatin (FG), alone and in conjugation, to form and stabilize fish oil-in-water emulsions was examined. Using layer-by-layer interfacial deposition technique for WPI–FG conjugate, a total of 1% protein was used to prepare 10% fish oil emulsions. The droplets size distributions and electrical charge, surface protein concentration, flow and dynamic rheological properties and physiochemical stability of emulsions were characterize at two different pH of 3.4 and 6.8 which were selected based on the ranges of citrus and milk beverages pHs, respectively. Emulsions prepared with WPI–FG conjugate had superior physiochemical stability compare to the emulsions prepared with individual proteins. Higher rate of coalescence was associated with reduction in net charge and consequent decrease of the repulsion between coated oil droplets due to the proximity of pH to the isoelectric point of proteins. The noteworthy shear thinning viscosity, as an indication of flocculation onset, was associated with whey protein stabilized fish oil emulsion prepared at pH of 3.4 and gelatin stabilized fish oil emulsion made at pH of 6.8. At pH 3.4, it appeared that lower surface charge and higher surface area of WPI stabilized emulsions promoted lipid oxidation and production of hexanal.     

Effect of grape antioxidant dietary fibre on the prevention of lipid oxidation in minced fish: Evaluation by different methodologies

The effect of grape antioxidant dietary fibre (GADF) addition to minced fish muscle (MFM) on lipid stability during frozen storage (6 months) was studied. Concentrations of 0%, 2%, and 4% GADF were added to MFM samples. Analyses were carried out immediately after preparation of samples and during and after storage at −20 °C. GADF was characterized in terms of dietary fibre, total polyphenols and antioxidant capacity, and multifunctional antioxidant assays were carried out on all the MFM samples. The addition of red grape fibre considerably delayed lipid oxidation in minced horse mackerel muscle during the first 3 months of frozen storage.     

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.     

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.     

Inhibition of oxidation of omega-3 polyunsaturated fatty acids and fish oil by quercetin glycosides

The antioxidant properties of naturally occurring flavonols, quercetin glycosides, were examined and compared with common food antioxidants butylated hydroxytoluene (BHT) and α-tocopherol. Antioxidants were incorporated into selected polyunsaturated fatty acids (PUFA) or fish oil in aqueous emulsions and bulk oil systems. The effectiveness of quercetin was similar to or greater than quercetin glycosides in inhibiting lipid oxidation in the oil-in-water emulsion systems when oxidation was induced by heat, light, peroxyl radical or ferrous ion. In bulk fish oil, C-3 glycosylation enhanced the antioxidant activity of quercetin. The effectiveness of quercetin and its glycosides was greater than that of α-tocopherol in the emulsions. Quercetin and quercetin-3-O-glucoside exhibited a better antioxidant activity than BHT in bulk fish oil; however, the reverse was observed in the emulsions of omega-3 PUFA and fish oil systems in agreement with the polar paradox theory. Quercetin and its glycosides were more effective than α-tocopherol in emulsion systems.     

Reduction in lipid oxidation by incorporation of encapsulated sodium tripolyphosphate in ground turkey

Ground turkey, with 1% NaCl, was incorporated with no sodium tripolyphosphate (control, nSTP), unencapsulated STP (uSTP; 0.3% or 0.5%), encapsulated STP (eSTP; 0.3% or 0.5% active, phosphate basis), or a blend (0.3% uSTP plus 0.2% eSTP). Encapsulate (hydrogenated vegetable oil) was designed to melt at 74 °C. Treatments were stored (4, 24 h at 3 °C) before being cooked to two different endpoints (EPT; 74, 79 °C) followed by post-cooked storage (0, 5, 10 days). An improvement of 77% (0.3% eSTP) and 80% (0.5% eSTP) in the reduction of TBARS was found in comparison to corresponding uSTP. The blend produced a 62% improvement compared to uSTP (0.5%) while maintaining cook yield. CIE a* values were highest at both EPT and post-cooked storage times beyond 0 day for eSTP. Meat manufacturing procedures that entail a delayed thermal processing step will benefit by an improvement in lipid oxidation control through the use of encapsulated phosphates.     

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.     

Roles of lipid oxidation and pH on properties and yellow discolouration during storage of film from red tilapia (Oreochromis niloticus) muscle protein

Protein-based films prepared from red tilapia (Oreochromis niloticus) washed and unwashed mince solubilised at pH 3 and 11 were prepared and characterised. Tensile strength (TS) of films from washed mince was greater than that of films prepared from unwashed mince for both pH used (P < 0.05). TS of films prepared at pH 3 was higher than that of films prepared at pH 11 for both of washed and unwashed mince (P < 0.05). Film from washed mince with pH 3 showed the highest TS, while that from unwashed mince with pH 11 had the lowest TS with the highest elongation at break (EAB) (P < 0.05). Films from washed mince had the lower value of thiobarbituric acid reactive substances (TBARS) than did those from unwashed counterpart, regardless of pH used. Nevertheless, TBARS was much higher in films prepared at acidic pH, compared with those prepared at alkaline pH. During storage of 20 days at room temperature, films became yellowish as evidenced by the increases in b^∗ and ΔE^∗-values. Films prepared at pH 11 showed the higher b^∗ and ΔE^∗-values than did those prepared at pH 3, especially for those from unwashed mince. However, films prepared from washed mince at pH 3 showed higher b^∗ and ΔE^∗-values than did those prepared at pH 11 (P < 0.05). Films generally had the increase in TS but the decreases in water vapour permeability (WVP), film solubility and protein solubility after 20 days of storage (P < 0.05). Therefore, lipid oxidation more likely played a role in yellow discolouration of fish muscle protein film, mainly by providing the carbonyl groups involved in Maillard reaction, while pH regulated the rate of reaction.     

The effect of lutein, sesamol, ellagic acid and olive leaf extract on lipid oxidation and oxymyoglobin oxidation in bovine and porcine muscle model systems

The effect of lutein (100, 200, 300 μg/ml), sesamol (500, 1000, 2000 μg/ml), ellagic acid (300, 600, 900 μg/ml) and olive leaf extract (100, 200, 300 μg/ml) on oxymyoglobin oxidation and lipid oxidation in bovine and porcine muscle model systems (25% M. longissimus thoracis et lumborum homogenates) was examined. Radical scavenging activity, using the DPPH assay, and iron-chelating activities of lutein, sesamol, ellagic acid and olive leaf extract were assessed at concentrations ranging from 200 to 1000 ppm. The radical scavenging activity was of the order: ellagic acid > sesamol > olive leaf extract > lutein. None of the natural antioxidants examined exhibited iron chelating activity. Following induced lipid oxidation (FeCl₃/sodium ascorbate addition), lipid oxidation and oxymyoglobin oxidation were measured after 24 h at 4 °C. In bovine and porcine muscle model systems, lipid oxidation decreased (P < 0.001) following addition of each of the natural antioxidants relative to the control and antioxidant potency followed the order: sesamol > ellagic acid > olive leaf extract > lutein. Ellagic acid and olive leaf extract decreased oxymyoglobin oxidation (P < 0.001) while sesamol increased oxymyoglobin oxidation in both systems. The natural antioxidants examined may have applications in the development of nutritional enhanced meat products with enhanced shelf-life characteristics.     

Effect of antioxidant on the fatty acid composition and lipid oxidation of intramuscular lipid in pressurized pork

To investigate the effect of antioxidants on lipid oxidation and fatty acid composition in pressurized pork, minced pork with or without 1% Na(2)EDTA was pressurized at 500MPa before 7days storage at 4°C. TBARS value, lipid content and fatty acid composition in untreated and high-pressure (HP) treated samples were analyzed. HP treatment induced marked increases in TBARS values and lipolysis of partial phospholipids causing an increase of free fatty acid content. Preferential hydrolysis for polyunsaturated fatty acids (PUFA) in phospholipids resulted in the percentage of PUFA in phospholipids decreasing markedly and thereby that in free fatty acids increasing significantly. Addition of 1% Na(2)EDTA to minced pork before HP significantly decreased the TBARS values in pressurized samples, but did not inhibit the lipolysis of phospholipids, causing the fatty acid composition of phospholipids and free fatty acids to change similarly to those samples without Na(2)EDTA.     

Inhibition of protein and lipid oxidation by rapeseed, camelina and soy meal in cooked pork meat patties

In this study protein-containing by-products of deoiling processes rich in phenolics were applied to meat to be used as potential food ingredients in developing meat products with antioxidant effect. The effect of rapeseed meal (Brassica rapa L.), camelina meal (Camelina sativa), soy meal and soy flour (from soybean, Glycene max L.), in inhibiting oxidation of lipids and proteins was tested in cooked pork meat patties. A commercial CO₂ extract from rosemary (Rosmarinus officinalis) was used as a reference material alone and in combination with the other plant materials. The cooked pork meat with added plant materials was oxidized for 10 days at 5 °C under light. The oxidation was followed by measuring the formation of hexanal, pentanal and propanal by headspace gas chromatography and the formation of protein carbonyls by converting them to 2,4-dinitrophenylhydrazones (DNPH). Rapeseed meal (0.5 and 0.7 g/100 g meat) and camelina meal (0.7 g/100 g meat) as such and their combination (addition of 0.5 g/100 g) with rosemary extract (0.04 g/100 g) were effective antioxidants toward both protein and lipid oxidation while soy meal and flour were effective only in combination with rosemary extract.     

The capability of rosemary extract in preventing oxidation of fish lipid

The effects of rosemary extract at different levels (%1, R1, and %2, R2) on the quality of vacuum‐packed sardine in terms of sensory, biochemical (thiobarbituric acid, total volatile basic nitrogen, peroxide value and free fatty acids) and microbiological analyses (total viable counts) were investigated. Fish were filleted and divided into three groups. First group was used as the control (C) without rosemary extract, second group was treated with 1% rosemary extracts (10 g L^?1) for 2 min (R1), and the third was treated with 2% rosemary extracts (20 g L^?1) for 2 min (R2). Thirty fillets per litre were used. After that, all groups were vacuum‐packed in polyethylene bags. The samples were stored in the refrigerator condition (4 ± 1 °C) over the storage period of 20 days. The results showed that the use of rosemary extract improved the sensory quality of both raw and cooked sardine, most preferably sardine treated with 1% of rosemary. Biochemical analysis showed that the use of 2% of rosemary extract were found to be most effective (P < 0.05) in controlling the rate of lipid oxidation.     

Effect of hydroxycinnamic acids on lipid oxidation and protein changes as well as water holding capacity in frozen minced horse mackerel white muscle

The antioxidant effectiveness of different hydroxycinnamic acids for inhibiting the formation of off-flavours associated to rancidity in minced frozen horse mackerel white muscle stored at −10 and −18 °C was studied. The influence of lipid oxidation on protein aggregation, protein denaturation and water holding capacity was also determined. Caffeic acid, o-coumaric acid and ferulic acid were the antioxidants tested. The order of antioxidant effectiveness was caffeic acid > ferulic acid > o-coumaric acid accordingly with previous results obtained in chilled horse mackerel. A strong dependence of antioxidant effectiveness to the ratio lipid/antioxidant concentration was observed. There was no evidence to suggest that antioxidants reduce protein aggregation or water holding capacity changes. Protein aggregation was not accompanied by gross protein denaturation, as monitored by differential scanning calorimetry. No evidence was found for a link between extent of lipid oxidation and degree of protein aggregation or water holding capacity changes.