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Muscle membranal lipid peroxidation initiated by H2O2-activated metmyoglobin
Abstract
Metmyoglobin (MetMb), H2O2-activated by glucose-oxidase/glucose system, initiated membranal lipid peroxidation. No such peroxidation occurred in the presence of the glucose oxidase system, H2O2, or MetMb alone. Heated MetMb maintained its capacity to be activated by H2O2. The accumulation of thiobarbituric reactive substances (TBA-RS) and oxygen absorption showed a higher rate of lipid peroxidation by H2O2-activated MetMb in microsomes separated from turkey than from chicken muscle tissues. Membranal lipid peroxidation initiated by activated MetMb was inhibited by low concentrations of either ascorbyl palmitate, α-tocopherol, or butylated hydroxytoluene (BHT). Inhibition was also observed by very low concentrations of ascorbic acid in the presence of EDTA. Only very high concentration of EDTA (1-10 mM) inhibited significantly membranal lipid peroxidation by activated metmyoglobin.
... The reaction of metmyoglobin (MbFeIII) with hydrogen peroxide (H 2 O 2 ) has been a subject of study over many years as a model for the reaction of protein radicals, [1][2][3][4][5][6] heme alteration, 1,3,7 as a catalyst for the oxidation chemistry of organic molecules 8,9 and as a source of oxidative damage in tissues. [10][11][12][13] The mechanism underlying the reaction of metmyoglobin with hydrogen peroxide, although studied for more than 60 years, 14 remains obscure and has been reviewed recently using kinetic simulation in the presence of a nitroxyl as an antioxidant. 15 The overall mechanism developed in the latter study is used as a framework here to determine the kinetic effects of the natural antioxidants, ascorbate. ...
... There is no published rate constant for reaction (10). In addition to the ferryl IV center, • MbFeIVO has either a protein or porphyrin centered free radical. ...
Using software based on a stochastic approach to the kinetic simulation of complex reaction schemes having a wide dynamic range, existing experimental data on the reactions of ascorbate and Trolox C (a soluble form of vitamin E) on the metmyoglobin/hydrogen peroxide reactions have been used for simulation. To match both kinetic and static data, a scheme of 15 reactions was required of which five were either significant revisions of existing rate constants or the proposal of a new mechanism. In this way, new rate constants for the reactions of •MbFeIVO and MbFeIVO with both ascorbate and Trolox C have been estimated together with that for MbFeIVO + TrolO•. Simulations of the metmyoglobin/hydrogen peroxide reaction scheme in the presence of both ascorbate and Trolox C in the presence of ABTS (ABTS⁺•) were also used to establish an overall mechanism for the time‐resolved evolution of the ABTS cation radical, which can provide both a static or kinetic endpoint of the assay. Rate constants have thus been estimated for the reactions of the ABTS cation radical with both ascorbate and Trolox C, these fast reactions being a prerequirement for the appearance of distinct lag phases for formation of ABTS⁺• . In the case of the latter reaction with Trolox C, it is proposed that TrolO• reacts with the ABTS⁺• It is also clear from simulations that the reactions of the ABTS cation radical with antioxidants must have rate constants 10⁴ M⁻¹ s⁻¹ or greater to produce well‐defined lag phases. Lower values result in either no or indistinct lag phases, as sometimes observed with natural antioxidants. Consequences for factors affecting the sensitivity of the TEAC assay and the determination of inhibition factors are discussed.
... The latter metMb and metHb are Minh Van Nguyen 1, *, Le My Thi Phan 1 1 Nha Trang University, Faculty of Food Technology, 02 Nguyen Dinh Chieu,Nha Trang,Khanh Hoa,Vietnam. oxidised to yield perferryl-Mb and ferryl-Mb, perferryl-Hb and ferryl-Hb radicals, which catalyse lipid oxidation (Harel & Kanner, 1985;Kanner, German, Kinsella, & Hultin, 1987;Rao, Wilks, Hamberg, & Ortiz de Montellano, 1994). The iron ions released from heme proteins are thought to promote lipid oxidation (Faustman, Sun, Mancini, & Suman, 2010;Richards & Hultin, 2002). ...
... According to Richards et al. (1998), heme proteins, including myoglobin (Mb) and hemoglobin (Hb), play an important role in lipid oxidation as prooxidants. Various pathways by which Hb and Mb can promote lipid oxidation have been described, including pseudolipoxygenase activity (Everse & Hsia, 1997), perferryl-Mb and ferryl-Mb, perferryl-Hb and ferryl-Hb radicals catalysed lipid oxidation (Harel & Kanner, 1985;Kanner et al., 1987;Rao et al., 1994) and iron ions released from heme proteins to promote (Faustman et al., 2010;Richards & Hultin, 2002) as well as lipoxygenase products that are known to enhance lipid oxidation (Pettitt, Rowley, & Barrow, 1989). Moreover, the fish blood also contains large amount of white blood cells, which is believed to generate superoxide, hydrogen peroxide and hydroxyl radical (Gabig & Babior, 1981). ...
The effects of bleeding methods (iced water bleeding and air bleeding) on the quality and lipid degradation of cobia fillets during frozen storage for 24 weeks at -20 ± 2 °C were investigated. Bleeding significantly retarded lipid hydrolysis and oxidation progress, resulting in lower free fatty acid (FFA), lipid hydroperoxides (PV), thiobarbituric acid-reactive substances (TBARS) and higher phospholipids content (PL) obtained in bled samples. A higher polyunsaturated fatty acid (PUFA) content was observed in the bled samples compared to that of the unbled samples. Ice water bleeding showed the best bleeding method in terms of blood removal, leading to lower heme and non-heme iron contents in the fish muscle. Heme pigments in the fish muscle oxidised during frozen storage, resulting in decreased heme iron content and increased non-heme iron content. The development of lipid hydrolysis and oxidation was in high correlations with the heme and non-heme iron content remained in the fish muscle. Oxidation of lipid and heme pigments was the main cause of the flesh discolouration during frozen storage, resulting in decreased lightness (L* value) and increased yellowish (b* value). Cobia need to be bled before processing to maintain the quality of the product during frozen storage. © Published by Central Fisheries Research Institute (CFRI) Trabzon, Turkey in cooperation with Japan International Cooperation Agency (JICA), Japan.
... Of all the ROSs, OH are the most eminent oxidizing free radical. These radicals affect the in vitro biochemical features of myofibrillar protein and are formed by cellular reductants and iron, which are generally formed by the Fenton reaction in the existence of H 2 O 2 (Harel and Kanner 1985;Park et al., 2007;Uçar et al., 2022;Ö zgeriş et al., 2023). This retention is generally toxic by oxidation of proteins, DNA and PUFA. ...
The toxic effects of microplastic (MP) pollution, which is a growing threat to the aquatic ecosystem, are constantly recorded by scientific reports at the organism and cellular levels. Despite this, the action mechanism of MP toxicity remains ambiguous. This research was designed to investigate the interactions with multiple biomarkers in the tissues of Oncorhynchus mykiss exposed to polyethylene microplastics (MPs-PE) under controlled conditions. In this context, fish were fed with MP-PE added feeding at different levels [MP-PE-I (10%) and MP-PE-II (20%)]. It was aimed to elucidate the MP abundance in gills, gastrointestinal system, on growth and hematological indexes in fish, as well as possible oxidative, DNA damage in target tissues (brain, gill, liver and muscle) and a number of biochemical events underlying apoptosis. MPs-PE tested at different concentrations led to changes in growth parameters and hematologic indices in fish. In all tissues targeted for the follow-up of oxidative stress, inhibitions in GSH levels and antioxidant enzyme activities were determined, while MDA, ROS, DNA damage and apoptosis significantly changed the expression profile upwards. MPs-PE significantly inhibited neurotransmission in rainbow trout. In conclusion, the outcomes of this study revealed that MPs-PE induced dose-dependent ROS-mediated apoptotic responses/ DNA damage in rainbow trout. The data are also a first record for rainbow trout and will help unravel different mechanisms with the potential to model for other MPs-PE-based toxicity studies.
... The latter can recombine to produce H2O2 in aqueous or oil emulsion systems. According to Harel and Kanner (1985), when activated by H2O2, the heme pigments act as strong prooxidants. The reaction of MetMb with H2O2 generates ferric peroxide. ...
A meta-analysis of the effect of gamma irradiation on the microbial status and colour in raw chicken meat was performed. A total of 14 studies were selected and data about the total viable count (TVC), coliforms (TCC), instrumental colour measurements - lightness (L*), redness (a*) and yellowness (b*) was extracted for evaluation in gamma treated and untreated meat. Due to the high between study heterogeneity, the meta-analysis was conducted through random-effects model applied on the raw mean difference (effect size) for each outcome of interest. The results of the meta-analysis showed that gamma irradiation considerably decreased TVC and TCC (P<0.001). Gamma rays also significantly diminished L* (P=0.013), but increased a* and b* (P<0.001). The meta-regression models showed that the dose of the radiation contributed to the overall effect of irradiation TVC (P=0.006), TCC (P=0.074) and a*(P=0.007). The effect of gamma rays on TVC was further influenced by storage (P=0.007), while package affected the increase of a* (P=0.100).
... Research shows that during the storage of proteins, various reactive oxygen species are generated, including free radicals, lipid hydroperoxides, and reactive aldehydes, which are key factors for protein oxidation (Chen, Zhao, Sun, Ren et al., 2013;. During the production of soy protein, nearly 1% of lipids and highly reactive lipoxygenases (LOX) were left behind, resulting in a constant oxidative environment for soy protein, which made soybean protein susceptible to oxidation during storage (Harel & Kanner, 1985). After excessive oxidative denaturation, the solubility and interfacial activity of soybean protein would decrease significantly, resulting in the reduction of its functionality, which greatly limited the application of SPI in the field of food and produced severe economic losses for the food processing plants (Hinderink, Schrder, Sagis, Schron, & Berton-Carabin, 2021). ...
The emulsifying activity of soy protein would decrease after long-term storage, which caused huge economic losses to food processing plants. This study explored the temporal evolution mechanism of oxidation on the structure and function of soy protein aggregates, which would improve the application of soy protein in food industry. Decreased α-helix and increased random coil were observed at the initial oxidation stage (0–4 h), which induced increases in hydrophobicity and disulfide bond content. In addition, emulsibility increased significantly. However, when the oxidation time extended to 6–12 h, the soluble aggregates transformed into insoluble aggregates with large particle size, low solubility, and molecular flexibility. Surface hydrophobicity and emulsifying activity were reduced, resulting in bridging flocculation of emulsion droplets. Mutual transformation between components is affected by factors that include spatial conformation and intermolecular forces, which eventually lead to functional changes in the protein molecules.
... The antioxidant strength of phenolic compounds is generally caused by their main role in the adsorption and neutralization of free radicals and elimination of the singlet oxygen from reactions. Previously, it has been noted that oxidation in meat products is likely due to the presence of the components such as myoglobin and haemoglobin as pro-oxidants(Harel & Kanner, 1985). Pro-oxidant flavonoid compounds seem to catalyze the lipid oxidation(Dangles, Dufour, & Fargeix, 2000;Procházková, Boušová, & Wilhelmová, 2011). ...
... The antioxidant strength of phenolic compounds is generally caused by their main role in the adsorption and neutralization of free radicals and elimination of the singlet oxygen from reactions. Previously, it has been noted that oxidation in meat products is likely due to the presence of the components such as myoglobin and haemoglobin as pro-oxidants(Harel & Kanner, 1985). Pro-oxidant flavonoid compounds seem to catalyze the lipid oxidation(Dangles, Dufour, & Fargeix, 2000;Procházková, Boušová, & Wilhelmová, 2011). ...
Background and Objectives: Sodium nitrite and potassium nitrite have been traditionally used for inhibition of Clostridium botulinum and also as an agent to stabilize the color of meat products; however, usage of these additives at high levels could lead to toxicity and cancer originating from the formation of nitrosamines. Nowadays, application of natural preservatives in order to reduce the nitrite content in meat products is increasing. Thus, we used dry red grape pomace (DRGP) as a natural alternative to sodium nitrite.
Materials and Methods: The effect of two levels of DRGP (1 and 2%) on the proximate composition, microbial counts, pH values and residual nitrite level of the samples formulated with two levels of sodium nitrite (30 and 60 mg/kg), as well as the comparison of these sausages with the blank (nitrite-free) and control (full nitrite added) samples on the 1rst, 10th, 20th and 30th days of storage at 3-5 °C were evaluated.
Results: The results showed that all chemical compositions were in the ranges reported by other researchers, and nitrite was very effective in preventing the microbial growth. Also about 50 % of the ingoing nitrite could be analyzed in the samples after processing. Moreover, the residual nitrite level declined both during the storage of sausage and after the addition of DRGP.
Conclusions: The use of DRGP in combination with nitrite for sausages was more effective in keeping the quality and safety of the refrigerated consumer products as indicated by the lower nitrite levels, microbial count and similar composition as compared to the samples treated with nitrite and without nitrite.
Keywords: Dry red grape pomace (DRGP), Sausage, Nitrite, Microbial count
... The latter, on the other hand, spontaneously undergoes dismutation and forms hydrogen peroxide, which is a strong oxidant (Tajima and Shikama, 1987;Wazawa et al., 1992). Moreover, MetMb can be activated by hydrogen peroxide and initiate electron abstraction from polyunsaturated fatty acids (Harel and Kanner, 1985). The heme affinity of MetMb was reported to be approximately 60-fold lower than that of OxyMb . ...
... The antioxidant strength of phenolic compounds is generally caused by their main role in the adsorption and neutralization of free radicals and elimination of the singlet oxygen from reactions. Previously, it has been noted that oxidation in meat products is likely due to the presence of the components such as myoglobin and haemoglobin as pro-oxidants(Harel & Kanner, 1985). Pro-oxidant flavonoid compounds seem to catalyze the lipid oxidation(Dangles, Dufour, & Fargeix, 2000;Procházková, Boušová, & Wilhelmová, 2011). ...
... Next, the superoxide radical is rapidly dismutated to oxygen and hydrogen peroxide. Hydrogen peroxide can react with previously formed methemoglobin what causes the formation of a ferryl protein radicalknown as an initiator of lipid oxidation [35,37]. Furthermore, when a considerable amount of peroxides is present, iron can be released from hemin and participate in oxidation of lipids [36,38]. ...
The content and composition of lipids in different byproducts (skins, heads, and backbones) from mechanically processed farmed Atlantic salmon were determined and compared with that obtained from wild salmon. Three different procedures were used to establish the optimal conditions of oil extraction (at high temperature −95°C, “cold” extraction at temperature not exceeding 15°C and enzyme assisted with Alcalase). “Cold” extraction at temperature not exceeding 15°C was very efficient, yielding almost 95% of the oil from skins. In the case of heads the obtained yield of about 71% was not lower than that from extraction performed at 95 °C or extraction supported by enzyme treatment. The peroxide value of oil isolated from the heads using “cold” extraction was at the same level as in oil of the enzyme assisted process, but four times lower than in oil extracted at high temperature. The results showed that the content of lipids from in the farmed salmon byproducts the content of lipids was about 45–55% higher than in byproducts of wild salmon, however the EPA + DHA content was 10–33% lower.
Practical applications : With “cold” extraction heating which is commonly used for oil recovery in the fish industry could be eliminated and thus the cost of the process would be lower and oxidative changes in the oil reduced. Furthermore, this method based on rules of “green chemistry” can be more attractive and alternative procedure of oil isolation from fatty fish byproducts than those using organic solvents. The fatty fish byproducts such as heads, skins, and backbones may be used as a source of valuable oils rich in PUFA. The remaining material after oil isolation can be a source of collagen and gelatin used in the food, pharmaceutical, and cosmetic industries and finally of minerals preparation (in the case of heads and backbones) used for enriching animal feed.
The oil was extracted from salmon byproducts: heads, backbones, and skins by using different methods. Conventional extraction of the oil at high temperatures ensures high yield but leads to low quality of the product. Enzymatic extraction is more preserving to oils rich in polyunsaturated fatty acids, but has special requirements. An attractive solution can be the “cold” extraction. This procedure allows achieving the oil from fatty fish byproducts with high yield and quality in a simple and cheap way.
... Oxymyoglobin, which generates H 2 O 2 during autooxida-tion, could activate its own molecule. The interaction of hydrogen peroxide with metmyoglobin very rapidly led to the generation of active species such as free radicals and reactive oxygen species, which promote membranal lipid peroxidation (Harel and Kanner 1985). Novelli and others (1998) studied lipid and cholesterol oxidation in frozen stored pork, and reported that pork shoulder and ham containing relatively higher myoglobin contents had higher POVs than belly and backfat containing relatively lower myoglobin content, and suggested that the differences in POVs among various muscles that might be linked with lean content are partially due to iron concentration. ...
Lipid oxidation and oxidative products as affected by pork meat cut, packaging method, and storage time were evaluated during refrigerated storage. Pork belly had higher pH and thiobarbituric acid reactive substance (TBARS) values than pork loin, and aerobic-packaged belly had higher TBARS than vacuum-packaged counterparts. Loin had higher free fatty acid (FFA) values than belly, and increased FFA values were observed with increased storage time. Peroxide values increased up to 7 d and decreased thereafter. Volatile compounds such as alkanes, aldehydes, ketones, and alcohols with high volatility in belly were higher than those in loin. Nonanoic acid, ethyl ester in belly, and hexadecanoic acid in loin might be considered as indices of lipid oxidation. Overall, vacuum packaging was better than aerobic packaging to retard lipid oxidation and production of oxidative products, and loin was more sensitive to lipid oxidation than belly.
... Heme-pigments, especially myoglobin, are responsible for meat color and also are considered as strong pro-oxidants when they are activated by hydrogen peroxide (Harel and Kanner, 1988). Changes in heme-pigments caused by irradiation could change color and generate off-flavors in raw meat (Ahn et al., 1998). ...
... nonheme iron content than the beef stored under HOMAP. Nonheme iron, i.e. free ion, is created by reaction of metmyoglobin with lipid hydroperoxides or H2O2 and has a direct relationship with lipid oxidation because of initiation and propagation by production of free radicals through Penton’s reaction (Harel and Kanner, 1985; Reeder and Wilson, 1998; Min and Ahn, 2005; Muhlisin et al., 2013). Thus, based on our results with this mechanism, the nonheme iron content in the stored-sliced beef may be highly correlated with activities of antioxidant enzymes, TRA, meat redness and degree of lipid oxidation as affected by packaging systems. ...
The effects of various packaging systems, vacuum packaging (VACP), medium oxygen-modified atmosphere packaging (50% O2/20% CO2/30% N2, MOMAP), MOMAP combined with vacuum skin packaging (VSP-MOMAP), high oxygen-MAP (80% O2/20% CO2/0% N2, HOMAP), and HOMAP combined with VSP (VSP-HOMAP), on the activity of antioxidant enzyme, and oxidation and color stabilities in sliced Hanwoo (Korean cattle) beef loin were investigated at 4°C for 14 d. Higher (p<0.05) superoxide dismutase activity and total reducing ability were maintained in VSP-MOMAP beef than in HOMAP beef. Lipid oxidation (2-thiobarbituric acid reactive substances, TBARS) was significantly (p<0.05) retarded in MOMAP, VSP-MOMAP, and VSP-HOMAP beef compared with HOMAP beef. Production of nonheme iron content was lower (p<0.05) in VSP-MOMAP beef than in HOMAP beef. Red color (a*) was kept higher (p<0.05) in VSP-MOMAP beef compared with MOMAP, HOMAP, and VSP-HOMAP beef. However, VACP beef was found to have the most positive effects on the antioxidant activity, oxidation and red color stabilities among the various packaged beef. These findings suggested that VSP-MOMAP was second to VACP in improving oxidation and color stabilities in sliced beef loin during chill storage.
... Soy protein, similar to other components such as lipids and pigments, is vulnerable to oxidative attack during processing and storage (Harel & Kanner, 1985). Protein oxidation is the structural modification induced directly by reactive oxygen species or indirectly by reaction with the by-products of lipid peroxidation (Shacter, 2000). ...
Soy protein isolate (SPI) was oxidized by peroxyl radicals derived from 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) and the structural and emulsifying properties of oxidized SPI were evaluated. Increasing extent of oxidation resulted in gradual carbonyl group generation, free sulfhydryl group degradation and dityrosine formation. Moderate oxidization could generate soluble protein aggregates with more flexible structure while over-oxidization would induce the formation of insoluble aggregates. Compared with the control, emulsions stabilized by moderately oxidized SPI had smaller droplet size and better thermal stability. Results from creaming index and microstructure measurement after 15 days indicated that emulsions stabilized by SPI of over-oxidation underwent severe droplet aggregation during storage while moderate oxidation had a positive effect on the emulsion stability.
... The oxidative stability study of isolated microsomes was carried out as described by Harel and Kanner (1985). Microsome fractions (1 mg ml 71 ) were mixed with metmyoglobin (30 mmol) and hydrogen peroxide (30 mmol) in a pH = 5.4 buffer (0.1 M KCl, 0.05 M NaOH and 0.13 M lactic acid). ...
The experiment was organized in a 3 x 2 factorial arrangement with three dietary fat blends and a basal (20 mg kg(-1) diet) or supplemented (220 mg kg(-1)) level of alpha-tocopheryl acetate. Dietary vitamin E and monounsaturated to polyunsaturated fatty acid ratio (dietary MUFA/PUFA) affected muscle alpha-tocopherol concentration (alpha-tocopherol [log mug g(-1)] = 0.18 (+/- 0.105) + 0.0034 (+/- 0.0003). dietary alpha-tocopherol [mg kg(-1) diet] (P < 0.0001) + 0.39 (+/- 0.122).dietary MUFA/PUFA (P < 0.0036)). An interaction between dietary alpha-tocopherol and dietary MUFA/PUFA exists for microsome alpha-tocopherol concentration alpha-tocopherol [log mug g(-1)] = 1.14 (+/- 0.169) (P < 0.0001) + 0.0056 (+/- 0.00099) . dietary alpha-tocopherol [mg kg(-1) diet] (P < 0.0001) + 0.54 (+/- 0.206) . dietary MUFA/PUFA (P < 0.0131) - 0.0033 (+/- 0.001])-dietary alpha-tocopherol [mg kg(-1))] x dietary MUFA/PUFA (P < 0.0067)), and hexanal concentration in meat (hexanal [ng . g(-1)] = 14807.9 (+/- 1489.8) - 28.8 (+/- 10.6) dietary alpha-tocopherol [mg . kg(-1)] (P < 0.01) - 8436.6 (+/- 1701.6) . dietary MUFA/PUFA (P < 0.001) + 24.0 (+/- 11.22) . dietary alpha-tocopherol . dietary MUFA/PUFA (P < 0.0416)). It is concluded that partial substitution of dietary PUFA with MUFA lead to an increase in the concentration of alpha-tocopherol in muscle and microsome extracts. An interaction between dietary alpha-tocopherol and fatty acids exists, in which at low level of dietary vitamin E inclusion, a low MUFA/PUFA ratio leads to a reduction in the concentration of alpha-tocopherol in microsome extracts and a concentration of hexanal in meat above the expected values.
The issue of lipid changes in muscle foods under the action of atmospheric oxygen has captured the attention of researchers for over a century. Lipid oxidative processes initiate during the slaughtering of animals and persist throughout subsequent technological processing and storage of the finished product. The oxidation of lipids in muscle foods is a phenomenon extensively deliberated in the scientific community, acknowledged as one of the pivotal factors affecting their quality, safety, and human health. This review delves into the nature of lipid oxidation in muscle foods, highlighting mechanisms of free radical initiation and the propagation of oxidative processes. Special attention is given to the natural antioxidant protective system and dietary factors influencing the stability of muscle lipids. The review traces mechanisms inhibiting oxidative processes, exploring how changes in lipid oxidative substrates, prooxidant activity, and the antioxidant protective system play a role. A critical review of the oxidative stability and safety of meat products is provided. The impact of oxidative processes on the quality of muscle foods, including flavour, aroma, taste, colour, and texture, is scrutinised. Additionally, the review monitors the effect of oxidised muscle foods on human health, particularly in relation to the autooxidation of cholesterol. Associations with coronary cardiovascular disease, brain stroke, and carcinogenesis linked to oxidative stress, and various infections are discussed. Further studies are also needed to formulate appropriate technological solutions to reduce the risk of chemical hazards caused by the initiation and development of lipid peroxidation processes in muscle foods.
The objective of this study was to investigate the effects of cavitation jets on the structural, emulsifying, and rheological properties of soybean protein oxidation aggregates. The results showed that oxidation might induce the formation of larger particle sizes and molecular weight protein aggregates and the decrease of emulsifying properties. The cavitation jet at a short treatment time (<6 min) broke down the disulfide bonds and protein skeleton structures, which reduced the aggregate sizes and molecular weights and increased the emulsion activities, emulsion stabilities, apparent viscosity, and elastic modulus. The cavitation jet at a long treatment time (>6 min) supported disulfide bond formation among molecules by intermolecular interactions to form protein aggregates. In addition, the skeleton structure showed crosslinking aggregation. This increased the particle sizes and molecular weights and reduced the emulsion properties, consistency index K, and elastic modulus. The findings showed that a cavitation jet at 6 min on oxidized aggregates of soybean protein might enhance the structural, emulsifying, and rheological characteristics for the industry.
Nghiên cứu được thực hiện nhằm đánh giá ảnh hưởng của trạng thái nguyên liệu (làm choáng và không làm choáng), môi trường xả tiết (nước và không khí) và điều kiện xả tiết (nhiệt độ và thời gian) đến chất lượng của sản phẩm phi lê cá lóc. Các chỉ tiêu hóa lý được đánh giá gồm màu sắc (L*, a* và b*), hiệu suất thu hồi sau gia nhiệt, trạng thái cấu trúc, hàm lượng sắt heme, sắt non-heme và oxy hóa lipid (chỉ số peroxide-PV và TBARS). Kết quả cho thấy cá được làm choáng trước khi cắt tiết có giá trị L* cao hơn và giá trị a*, b* thấp hơn và hiệu quả loại máu tốt hơn (hàm lượng sắt heme và sắt non-heme thấp hơn) so với cá không được làm choáng. Nước là môi trường phù hợp để xả tiết cá lóc. Nhiệt độ và thời gian xả tiết là hai yếu tố quan trọng ảnh hưởng đến hiệu quả loại máu, điều kiện phù hợp để xả tiết cá lóc là nhiệt độ nước 23-25°C trong thời gian 20 phút.
The catalytic degradation of pentachlorophenol contaminated soil by heme and hydrogen peroxide has been reported. Here we studied evidence for the mechanism and mineralization by the heme catalyzed reaction for hazardous organopollutants. Ferryl heme radical and non-radical ferryl heme were generated rapidly by the interaction of heme and hydrogen peroxide (H2O2). The activated heme radical could initiate the oxidation of 5-aminosalicylic acid (5-ASA). The reactions by heme with H2O2 could support the redox cycling between the ferryl species of heme and 5-ASA as the mechanistic routes of the heme catalyzed reaction. Hazardous compounds such as pentachlorophenol, phenanthrene and benzo[a]pyrene were mineralized 20, 6, and 7%, respectively, with 30 mM heme and 1500 mM H2O2, after 24 hr reaction. This catalyzed degradation of organopollutants could be used as a novel technology for hazardous waste remediation.
The production of oxygen free radicals catalysed by non-haem iron was investigated in an in vitro mimetic model of the digestive tract using specific chemical traps. Superoxide radicals (O2∗-) and their protonated form (hydroperoxyl radicals, HO2∗) were detected by the reduction of nitroblue tetrazolium into formazan, and hydroxyl radicals (OH∗) were detected by the hydroxylation of terephthalate. Under gastric conditions, O2∗-/HO∗ were detected in higher quantity than OH∗. Increasing the pH from 3.5 to 6.5 poorly affected the kinetics of free radical production. The oxidations generated by these free radicals were estimated on myofibrils prepared from pork rectus femoris muscle. Myofibrillar lipid and protein oxidation increased with time and oxidant concentration, with a negative impact on the digestibility of myofibrillar proteins. Plant food antioxidants considerably decreased free radical production and lipid oxidation but not protein oxidation.
Background:
Metmyoglobin (MbFe(III)) reaction with H2O2 has been a subject of study over many years. H2O2 alone promotes heme destruction frequently denoted "suicide inactivation," yet the mechanism underlying H2O2 dismutation associated with MbFe(III) inactivation remains obscure.
Methods:
MbFe(III) reaction with excess H2O2 in the absence and presence of the nitroxide was studied at pH 5.3-8.1 and 25°C by direct determination of reaction rate constants using rapid-mixing stopped-flow technique, by following H2O2 depletion, O2 evolution, spectral changes of the heme protein, and the fate of the nitroxide by EPR spectroscopy.
Results:
The rates of both H2O2 dismutation and heme inactivation processes depend on [MbFe(III)], H2O2, and pH. Yet the inactivation stoichiometry is independent of these variables and each MbFe(III) molecule catalyzes the dismutation of 50±10 H2O2 molecules until it is inactivated. The nitroxide catalytically enhances the catalase-like activity of MbFe(III) while protecting the heme against inactivation. The rate-determining step in the absence and presence of the nitroxide is the reduction of MbFe(IV)O by H2O2 and by nitroxide, respectively.
Conclusions:
The nitroxide effects on H2O2 dismutation catalyzed by MbFe(III) demonstrate that MbFe(IV)O reduction by H2O2 is the rate-determining step of this process. The proposed mechanism, which adequately fits the pro-catalytic and protective effects of the nitroxide, implies the intermediacy of a compound I-H2O2 adduct, which decomposes to a MbFe(IV)O and an inactivated heme at a ratio of 25:1.
General significance:
The effects of nitroxides are instrumental in elucidating the mechanism underlying the catalysis and inactivation routes of heme proteins.
Numerous nonmeat additives have been evaluated for enhancement of the quality of meat products or for product development/modification. Some nonmeat additives function primarily as preservatives, as antimicrobials and/or antioxidants, while others mainly modify product attributes such as sensory, nutritional and processing properties. Such additives often have secondary effects (desirable or undesirable) on the quality of products involved. This paper examines the effects of various chloride salts, phosphates, salts of organic acids, and some fat substitutes on storage properties of meat products. Additionally, potential effects of microbial growth in test samples on lipid oxidation measurements and assessment of additive effects are discussed
When food products are subjected to decreasing temperatures, the ability of microorganisms on or in these foods to grow decreases. As a consequence, quality deterioration in many food products shifts from microbiological causes to chemical/enzymatic causes. Chief among these chemical causes is lipid oxidation. In this chapter this degradative pathway will be discussed by first addressing the basic mechanisms of lipid oxidation in frozen foods. The chapter will then focus on those factors, both physical and chemical, that influence the rate of lipid oxidation, followed by a discussion of the consequences of lipid oxidation in frozen foods.
The lipids of animals have structural, metabolic or storage functions. The structural lipids are present in membranes where they exist as a lipid bilayer. Specialized structural lipids also occur as the myelin sheath surrounding nerves and in the brain. Except for these, structural lipids are present in tissues in relatively small amounts, approximately 0.5% of the weight of muscle or fatty tissue, and are therefore consumed in situ as part of the meat. Despite their low concentration they are important contributors to the species-specific cooked meat flavour and also to the rancid odour and flavour of meat, both raw and cooked, which has been stored too long. Many of the components of the structural lipids in meat will be incorporated into the structural lipids in the body of the consumer.
Lipid oxidation is a major deterioration reaction in foods which often results in a significant loss of quality. Oxidative deterioration of fats, oils and lipid-containing foods can result in the development of rancid off-flavours as well as the loss of the desirable characteristic flavour notes. It is also well known that oxidative reactions can cause discoloration of pigments, especially haem and carotenoids, destruction of certain nutrients and the possible formation of toxic by-products (Pearson et al., 1983; Addis, 1986). Generally, these changes in quality prevent consumer acceptance of oxidized food products.
The purpose of this study is to observe the change rule of lipid oxidation and meat color of three muscle parts from bluefin tuna at low frozen storage temperature, akami, chu-toro and o-toro, and then make correlation analysis for each index. The results showed that, with the extension of frozen storage time, the TBA values and metMb% of three parts both increased correspondingly. Besides, the red value (az.ast;), content of total pigment and sensory assessing values all appeared decreasing trend; O-toro changed the fastest, chu-toro the second, akama the slowest. metMb% and the red value (az.ast;) showed significant negative correlation (R=0.943, P<0.001), when with TBA values, they showed significant positive correlation (R=0.914, P<0.001). Through analyzing, we know three bluefin parts all had a trend of browning, and their speed was different from each other because of different fat contents; in order to make sure the perfect color of tuna meat, measures must be taken to prevent lipid and metmyoglobin from oxidation.
The influence of natural phytic acid extracted from wheat bran, and exogenous phytic acid on oxidative stability in chicken O/W emulsion was investigated. The antioxidant activity of phytic acid was evaluated by the thiobarbituric acid reactive substances (Tbars) values, oxygen absorption and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging methods. Phytic acid extracted from wheat bran (pae) was highly effective in reducing Tbars values (68% in comparison to control) and preventing lipid oxidation during storage of chicken O/W emulsion at 40°c for 2 weeks. Such antioxidant activity of exogenous phytic acid depends on its concentration in chicken O/W emulsion. Ascorbic acid at concentration 0.1% inhibited tbars formation by 56% compared to the control. Oxygen in the headspace of vials containing emulsion without phytic acid (control) was consumed faster than where phytic acid was added. Assuming a zero time reaction, the rate of oxidation without phytic acid (control) was 0.61% o 2/day, while that with phytic acid extract was 0.055% o 2/day or about 12 times slower (significantly different). Phytic acid extract and exogenous phytic acid (4 mm) were more effective in preventing lipid oxidation and oxygen absorption than ascorbic acid and BHA. Radical scavenging activity of phytic acid extract and pure phytic acid (4 mm) were 40.96±0.55 and 40.89±0.32%, respectively. Free radical scavenging ability of phyticacid was lower than other common antioxidants (ascorbic acid and bha). It can be concluded that phytic acid may substantially inhibit malondialdehyde formation, oxygen uptake, as well as warmed-over flavor development during storage of chicken meat or chicken meat products.
As with all dead organic matter, muscles from slaughtered animals, as a whole or in particulate form, may be modified by microorganisms during prolonged storage. Environmental conditions and storage time greatly influence the sort and extent of modification. For foods, there can be desirable and undesirable microorganisms, which bring about desirable and less-desirable changes. Desirable modifications are improvements in flavour, aroma, palatability, appearance and storage characteristics. Microbial activities that result in off-odours, strange taste, health- threatening metabolites, colour deterioriation, loss of consistency, and the growth of pathogenic and toxinogenic bacteria generally spoil the food and thus make it unsuitable for human consumption. Changes in microbial abundance, water activity (aw), pH, pO2 and concentration of chemical compounds may be due to real fermentation processes, i.e. incomplete anaerobic oxidations of organic substrates, or to aerobic microbial metabolism. However, it is common to call a food ‘fermented’ if microorganisms, be they truly fermenting or not, or enzymes had contributed significantly to its final characteristics (Campbell-Platt, 1987). Several meat enzymes are known to remain active in dead muscles, e.g. the glycolytic sequence, lipases and proteases. Dead or non-growing microorganisms may release or provide active enzymes, e.g. nitrate reductase and catalase.
Oxidative modification of soy protein isolate (SPI) by hydroxyl radicals derived from FeCl3/H2O2/ascorbic acid hydroxyl radical-generating systems (HRGS) at room temperature (20 °C) for 5 h was investigated. Increasing the H2O2 concentration resulted in the increase of protein carbonyl groups and dityrosine contents (P < 0.05), and the decrease of free sulfhydryl groups (P < 0.05). Circular dichroism spectra confirmed that oxidation resulted in a gradual loss of α-helical structure with a concomitant increase of β-sheet structure. Fluorescence spectroscopy revealed that oxidation treatment greatly increases the extent of exposed hydrophobic domains. The emulsifying activities of SPI were significantly improved at H2O2 concentrations up to 1.0 mM, and then declined at higher H2O2 concentration (P < 0.05). These results suggest that moderate oxidization could significantly enhance the emulsifying properties of SPI, which partly caused by the expose of surface hydrophobic groups and larger soluble aggregates, as well as mainly due to the enhancement of the electrostatic repulsive force between emulsion droplets.
The primary objective for packaging fresh and processed meats is to limit and delay both the growth of spoilage and pathogenic microorganisms and deteriorative chemical reactions through adequate containment, which is followed by the maintenance of the optimal sensory and other quality characteristics of the product within a specified shelf life through adequate protection and preservation. This book chapter will discuss the sensory quality changes that occur in fresh and processed meat products with respect to colour, flavour and texture, and also the typical packaging conditions that are used to maintain the sensory quality of these products throughout shelf life. Sensory quality changes that are not directly related to packaging conditions are also mentioned briefly for the sake of completeness.
Oxygen transport and storage proteins cause a continous flux of radicals in muscles. In meats these one-electron transfer reactions are mainly related to the heme pigment myoglobin, and they are accelerated by the post-mortem decrease in pH, One radical-generating pathway is the pseudoperoxidase reaction of myoglobin, whereby the pigment is activated by hydrogen peroxide to form a protein-based radical that is deactivated in two one-electron steps, leaving the oxidized substrates also as radicals. Possible substrates are proteins, lipids, and numerous smaller compounds such as ascorbate, reducing cofactors, carotenoids, and plant phenolics. The reactions involved are accelerated by acid, and a ferric protein radical in equilibrium with the Fe(IV)=O group may be the actual reacting species. In radical exchange reactions long-lived protein radicals seem to be formed in processes that could be accelerated by phase transitions in the meat caused by freezing, which could also lead to membranal damage.The nature of the substrate-derived radicals determines whether the reaction has an overall prooxidative or antioxidative effect in the meat system and will thus depend on the depletion of reductants in the meat.
L-ascorbyl palmitate (AsA-Pal-Enz) was synthesized by using an immobilized lipase from Aspergillus niger. A comparison of antioxidative effects between L-ascorbic acid (AsA) and AsA-Pal-Enz was determined in terms of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical–scavenging. The results indicate that the AsA-Pal-Enz was effective in preventing lipid oxidation, while the antioxidative activity in authentic AsA-Pal was lower. The activity of AsA-Pal-Enz was very stable than AsA-Pal standard during heating.
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This chapter discusses the basis for lipid and protein oxidation in fresh and processed red meat products. These processes lead to quality degradation, and a variety of antioxidant strategies have been developed to minimize and/or prevent this quality loss. The chapter provides an overview of the field’s status to date.
Peroxynitrite is formed from the nearly diffusion limited reaction between nitric oxide and superoxide. This reactive nitrogen species has received intense interest in the biomedical field as an initiator of potentially harmful oxidation reactions. Peroxynitrite can also be viewed as an important pro-oxidant that affects food quality. Since most biological tissues used for foods contain systems that produce both nitric oxide and superoxide, conditions that enhance or suppress the formation of these precursors will influence the formation of peroxynitrite. The complex oxidative chemistry of peroxynitrite involves direct reactions with cellular constituents such as antioxidants, production of reactive species capable of initiating lipid oxidation, and catalyst-dependent nitration reactions which may redirect the reactivity of peroxynitrite to favor protein oxidations. Thus, peroxynitrite reactions in food may be deleterious, leading to a decline of quality parameters such as flavor, color, and functional properties.
The effect of extensive feeding, confinement and diet supplementation with a-tocopheryl acetate (100 mg/kg) on the fatty acid composition and tocopherol concentration of microsome extracts and their susceptibility to oxidation was studied in Iberian pigs. The diet of pigs raised extensively was mostly composed of acorn and grass. The a-tocopherol contents of acorn and grass were 20 and 171 mg/kg dry matter, respectively. Microsomal fatty acid composition showed no differences among groups. Pigs feeding extensively had a higher concentration of a-tocopherol in muscle and microsomes than pigs given mixed diet with the basal level of a-tocopheryl acetate (P < 0·05) but lower values than pigs given supplementary levels (100 mg/kg) (P < 0·05). Microsomal fractions from pigs given mixed diet with a basal level of a-tocopheryl acetate were significantly more susceptible to iron-induced lipid oxidation than extract from pigs given diets containing a supplementary level (P < 0·05). Microsomal extracts from pigs feeding extensively had the lowest oxidation rate (P < 0·05), suggesting that other dietary constituents may play a role in the stabilization of microsomal lipids.
Muscle microsomes were isolated from beef, pork and tuna and combined with oxymyoglobin to study oxymyoglobin and lipid oxidation interactions. Tuna muscle microsomes contained higher concentrations of long chain fatty acids and polyunsaturated fatty acids, and a lower α-tocopherol content than microsomes from the other two species (P < 0·05). Oxymyoglobin and lipid oxidation were greater in tuna followed by pork and beef (P < 0·05). The influence of high (HMW) and low molecular weight (LMW) cytosolic fractions from muscles of these species on oxymyoglobin and lipid oxidation was studied in an oxymyoglobin-phosphatidylcholine-liposome system. In each species, the LMW fraction resulted in greater OxyMb and lipid oxidation than the HMW fraction (P < 0·05). Within a given fraction type, there was no difference between species (P < 0·05). In lamb liver oxymyoglobin-microsomes, oxymyoglobin and lipid oxidation were delayed with increased microsomal α-tocopherol content (P < 0·05). These results suggest that differences in oxymyoglobin and lipid oxidation in vitro were more strongly influenced by oxidative stability of membrane components rather than cytosolic components.
Soy protein isolate (SPI) was modified by 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) oxidation pretreatment, and the in vitro digestibility of oxidised SPI was investigated. Results indicated that oxidation induced amino acid modification. The amount of most amino acids decreased, accompanied by decreasing digestive proteolysis susceptibility. Peptide size distribution implied that oxidation generated protein aggregates that could not be degraded by pepsin, but could be digested by pancreatin. Oxidation induced a maximum of 16.6% and 14.6% loss, respectively, for free essential and free total amino acid in the digests of oxidised SPI. Antioxidant activities evaluation of oxygen radical absorbance capacity (ORAC) value and DPPH scavenging activity showed that oxidation deteriorated the antioxidant activities of the digests from oxidised SPI.
A “blue” reaction intermediate (absorption maximum at 835 nm) is observed when ferrylmyoglobin is reacted with excess thiocyanate in neutral aqueous solution. The intermediate is formed in a bimolecular process (first-order in thiocyanate and ferrylmyoglobin) with a rate constant (0.119±0.006 M−1s−1at25°C) showing moderate temperature dependence (enthalpy of activation ΔH#=45±6 kJ mol−1, entropy of activation ΔS#=−113±19 J mol−1 K−1), but decays in a first-order, highly temperature-dependent reaction (1.53±0.05×10−4 s−1 with ΔH#=99±1 kJ mol−1 and ΔS#=14±3 J mol−1 K−1). Hypothiocyanite is not detected as an intermediate (as for peroxidase-catalyzed oxidation of thiocyanate), and the “blue” intermediate is proposed to be a protein radical with thiocyanate bound and which decays by intramolecular electron-transfer. ©
The demand for precooked, refrigerated ready-to-eat meat products is expected to increase both in the catering and retail sectors. In these products, the rate at which warmed-over flavour (WOF) develops often determines their shelf life. WOF is recognized by consumers as a ‘leftover’ or rancid taste that rapidly develops in cooked meats during refrigerated storage and is associated with oxidative deterioration of meat. Several approaches can be applied to retard the development of WOF. The purpose of this short review is to discuss how the development of WOF can be retarded, with special emphasis on the application of antioxidants and optimization of processing conditions.
Catalytic effects of different temperatures (55, 70, 85, and 100°C) on lipid oxidation were studied in aqueous- and chloroform/methanol-extracted beef model lipid systems containing iron forms inherent in beef (water-extractable, diffusate, nondiffusate, ferritin, myoglobin, hemoglobin), hematin, FeCl2, or FeCl3. Heating increased thiobarbituric acid and peroxide values in both systems. All forms of iron catalyzed lipid oxidation in aqueous systems, with greatest oxidation by heme and low molecular weight iron fractions. Oxidation in lipid extracts was not increased by ferritin, FeCl2, or FeCl3, but heme iron was the major oxidation catalyst. Lipid stability decreased with addition of any iron forms inherent in beef or with increased heating, which helps understanding of rapid oxidation of meat during refrigerated storage or after cooking.
Unwashed fillets from stage I rigor mackerel deteriorated extensively compared to unwashed fillets from stage III mackerel; this was likely related to the greater amount of blood contaminating the fillet surface of the fresher fish. Washing improved the quality of fillets from rigor fish but not stage III fish, which was attributed to the greater amount of blood removal from the fresher fish by washing. Having antioxidants in the washing solution doubled the shelf life of fillets compared to water-washed fillets. Filleting in an antioxidant solution slightly but significantly improved quality compared to cutting in air and rinsing with antioxidant solution 1 min after filleting with frozen but not refrigerated samples. Improvements with antioxidants were seen with fillets from stage I but not stage III mackerel. The pro-oxidative activity in extracts prepared from fillet surface tissue using linoleic acid as substrate was stimulated by lipid hydroperoxides. The heme proteins present in tissue extracts had the potential to account for much of the lipid oxidation observed. Keywords: Washing mackerel fillets; blood, role in quality; antioxidants, effect on mackerel quality; mackerel; quality of washed mackerel
A 23 factorial experimental design was used to investigate the effect of washing in combination with precooking on the oxidative stability of lipids in minced herring (Clupea harengus) at −18 °C. The following variables were studied: washing (no, yes), cooking time (38, 54 min), and cooking temperature (55, 100 °C).The responses monitored were peroxide value (PV), absorbance at 234 nm (A234), absorbance at 268 nm (A268), and lipid soluble fluorescent products (FP). A partial least-squares regression analysis (PLS) revealed that the best lipid stability was obtained at minimum cooking time and at lower temperature. On the basis of compositional analyses and in vitro experiments, this finding was proposed to be due to heat inactivation of catalytic enzymes, without simultaneous activation, for example, of hemoproteins. Washing reduced these benefits from precooking by removal of pro-oxidative enzymes and also through a reduction in the amount of antioxidants as well as a relative increase in phospholipids and free fatty acids in the fat. Keywords: Clupea harengus; herring; lipid oxidation; washing; precooking
This work reveals two biochemical effects of hydrogen peroxide treatment on hemoglobin, myoglobin, and cytochrome c. First, these heme proteins rapidly formed covalently crosslinked dimers and polymers detectable by detergent gel electrophoresis. Second, when treated in the presence of radioactive benzo[a]pyrene or 17β-estradiol, the heme proteins became covalently labeled. Nonheme proteins exhibited both cross-linking and radioactive labeling upon peroxide treatment in the presence but not the absence of heme protein or free hemin. Benzoyl peroxide or glucose and glucose oxidase effectively replaced direct addition of hydrogen peroxide. These results indicate that adventitious peroxidase activity expressed by oxygen carrying and electron transport proteins yields active oxygen species that can damage these heme proteins and nearby macromolecules, a possible biochemical mechanism for the lethal and other deleterious intracellular effects of perioxide.
Biological lipid autoxidation has been studied in a model system composed of sonicated phospholipids as substrate and electron transfer proteins found in membranes as possible catalysts. Heme compounds, flavoproteins, and iron-sulfur proteins were examined for their ability to initiate lipid autoxidation. Among many heme compounds tested, the most active were hematin ⩾microperoxidase ⪢ methemoglobin > cytochrome c. With fresh preparations of phospholipids, reaction rates (nanomoles of oxygen/minute nanomoles of heme) ranged from 5 (cytochrome c) to 350 (hematin). Only the oxidized heme compounds were active as catalysts. Reduced heme compounds, flavoproteins and riboflavin were inactive. In the presence of heme compounds, aged preparations of sonicated phospholipids were much more rapidly oxidized than fresh preparations. They also had a higher content of fatty acid hydroperoxides as judged from their characteristic diene absorption peak at 234 nm. This observation agrees with the postulated mechanism of lipid autoxidation by heme compounds, namely, homolytic scission of preformed fatty acid hydroperoxides. Iron-sulfur proteins were also active as initiators of lipid autoxidation when destabilized in the presence of an appropriate iron chelator (o-phenanthroline or 2,2′-bipyridine) or a chaotropic ion. Oxygen uptake rates (nanomoles of oxygen/minute × milligrams of protein) varied from about 200 for an iron-sulfur protein isolated from complex I to about 5500 for Clostridium pasteurianum ferredoxin. However, per nanomole of labile sulfide, the rates for all active iron-sulfur proteins were 4–7 nmol of oxygen/min × nmol of labile sulfide.
TBA (2-thiobarbituric acid) analysis demonstrated that turkey meat is most susceptible to WOF (warmed-over flavor) development, followed closely by chicken, then by pork, beef, and mutton in that order. Although freshly cooked muscle from all species except mutton had higher TBA numbers than fresh raw samples, the most dramatic change occurred during storage of cooked meat at refrigerated temperature (48 hr at 4°C). Red muscles had consistently higher TBA values than white muscles under these storage conditions, indicating that red muscles were more susceptible to oxidative deterioration. Correlation coefficients between TBA numbers and total lipid levels and between TBA values and phospholipids suggest that phospholipids play a major role in development of WOF in all cooked meats, except for pork, where total lipid levels seem to be the major contributor to WOF.
This chapter discusses “warmed–over” flavor (WOF) in meat, poultry, and fish. In the first section, the classification and significance of lipids is described. It explains the structure of lipids and the composition of animal fats. The role of lipids in meat flavor, both the desirable and undesirable, are presented. Next section discusses the mechanisms of lipid oxidation. It delves into the topics of autoxidation, catalysts of lipid oxidation, comparison of heme and nonheme iron as pro–oxdidants in muscle tissue, and phospholipid oxidations. In the subsequent section, development of WOF, the species differences in WOF, influence of deboned meat, influence of heating, influence of chopping and emulsifying and effects of curing are explained. A discussion on the prevention of WOF in meat, poultry, and fish is also presented in the last section.
The intermediate free radicals generated from phenols, naphthols and benzoate, in the peroxidase- and oxidase-reactions of horse radish peroxidase and in the peroxidase-reaction of methemoglobin, were studied by electron spin resonance spectroscopy.The difference between the peroxidase- and oxidase-reactions of HRP are demonstrated, i.e., the ferro horse radish peroxidase-O2 system attacks both phenols and benzoate yielding unidentified radicals, which may be hydroxy-cyclohexadienyl radicals, while the horse radish peroxidase-H2O2 system reacts only with phenols and naphthols producing the phenoxy-and naphthoxy-radicals.Phenoxy-radical formation from phenols, in the reactions horse radish peroxidase-H2O2 and methemoglobin-H2O2, occurs independently of the molecular sizes of phenols but dependently on their redox-potentials.On the basis of kinetic studies on methemoglobin-H2O2 system, the existence of a reactive intermediate complex between methemoglobin and H2O2 is proposed, which may be similar to compound-I or -II of horse radish peroxidase and which further degenerates to MetHb radical. The oxidation of phenols and naphthols takes place outside of the hemepocket of methemoglobin.
The original Lowry method of protein determination has been modified by the addition of sodium dodecyl sulfate in the alkali reagent and an increase in the amount of copper tartrate reagent. These alterations allowed the method to be used with membrane and lipoprotein preparations without prior solubilization or lipid extraction and with samples containing 200 mm sucrose or 2.5 mm EDTA.
The interaction of hydrogen peroxide (H2O2) with metmyoglobin (MetMb) led very rapidly to the generation of an active species which could initiate lipid peroxidation. The activity of this prooxidant decreased rapidly during the first minutes, but 50% of its activity remained stable for more than 30 min. In this model system, it was found that small amounts of H2O2 (1-10 microM) could activate MetMb for significant lipid peroxidation. The incubation of the sarcosomal lipids with activated MetMb caused oxygen absorption. No absorption of oxygen was determined in the presence of membrane with MetMb or H2O2 alone. Methemoglobin (MetHb) was also found to be activated by H2O2 and to initiate lipid peroxidation. Membranal lipid peroxidation initiated by activated MetMb was inhibited by several reducing compounds and antioxidants. However, several hydroxyl radical scavengers and catalase failed to inhibit this reaction.
Spectrophotometric changes in the extent of NADPH, but not NADH, reduction of microsomal cytochrome b5 are correlated with the utilization of oxygen and the accumulation of lipid peroxidation products. The results suggest that NADPH-cytochrome b5 reductase (NADPH-cytochrome c reductase) participates in the reduction of obligatory ferric chelates to their ferrous form prior to the initiation of lipid peroxidation. Further, an increased oxidation of cytochrome b5 observed only in the presence of peroxidation products implicates a peroxidase activity associated with b5 in the microsomal electron transport chain.
The acceleration and inhibition of unsaturated fatty acid oxidation by heme compounds was followed in model systems with an
oxygen analyzer. The linoleate to heme molar ratios for maximum catalysis were 100 for hemin and catalase, 250 for metmyoglobin,
400 for cytochrome c and 500 for methemoglobin. With heme concentrations of 2 to 4 times the optimum catalytic amount, no
oxidation occurred. Rapid heme destruction was observed with catalyzing ratios of lipid to heme, but with inhibitory ratios
a stable red compound formed, believed to be a lipid hydroperoxide derivative of the heme. The ratios of lipid to metmyoglobin
for maximum acceleration varied with the pH. Linolenate was much less sensitive to heme catalysis than linoleate. Colorless
products of heme degradation had a marked antioxidant effect. A possible mechanism for the antioxidant effect of hemes is
advanced, based on the formation of stable heme peroxide complexes or stable heme radicals, or both, during the early stages
of oxidation. Prooxidant activity is believed to occur only when the peroxide to heme ratio is so high that the oxidation
of the hemes goes beyond the initial stages.
In fresh muscle foods, phagocytic cells may conceivably initiate and accelerate lipid oxidation. Fish leukocytes were obtained by density gradient centrifugation. The isolated neutrophils were rich in myeloperoxidase which was extracted from the leukocytes at pH 4.0 in the presence of 0.3 M sucrose. The crude enzyme showed a peroxidase activity of about 160 nmol of purpurogallin (formed from pyrogallol) (mg of protein)-1 min-1. An oxidation system using discoloration of β-carotene as an index of lipid peroxidation was developed. Myeloperoxidase from fish leukocytes caused rapid discoloration of β-carotene in the presence of H2O2 and iodide or bromide ions. Purified myeloperixidase cooxidized β-carotene in the presence of chloride ions. No destruction of β-carotene occurred when halogen ions, H2O2, or the enzyme was omitted from the system. The data indicate that leukocytes may be a focus for initiation of lipid peroxidation in biological tissues.
Haemoglobin catalyses at low concentrations (0.01–1 μM) a quasi‐lipoxygenase reaction with remarkably high substrate specificity. Formation of lipohydroperoxides was demonstrated only with free 9‐ cis ,12‐ cis ‐octadecadienoic acid (Linoleic acid), its glycerol monoester, or 9‐ cis ,12‐ cis ‐octadecadienol (Linoleoyl alcohol). All‐ cis ,‐9,12, 15‐octadecatrienoic acid (α‐linolenic acid), all‐ cis ,‐5,8,11,14‐eicosatetraenoic acid (arachidonic acid) and all‐ cis ,‐9,12, 15‐ eicosatetraenoic acid (dihomo‐ν‐linolenic acid), phospholipids and biological membranes were not attacked. Saturated and unsaturated free fatty acids other than linoleic acid were strong inhibitors of the haemoglobin‐catalyzed oxygenation of linoleate. The reaction was also inhibited by cyanide, carbon monoxide, phenolic antioxidants and the lipoxygenase inhibitor salicylhydroxamic acid, as well as by some anti‐inflammatory drugs (prednisolone, dexamethasone, acetylsalicylic acid); indomethacin did not inhibit. Protein inhibitors of liproxygenase reactions were found in lung homogenates of various species. The molecular activity of the haemoglobin‐catalyzed oxygenation of linoleate was comparable with those of true lipoxygenases and was strongly suppressed by denaturation (heating, urea, pronase treatment, acid splitting). The apparent S 0.5 , the activation energy and the pH‐optimum did not differ from those of most of the true lipoxygenases.
The reaction product hydroperoxylinoleic acid but not hydrogen peroxide was an activator of the haemoglobin‐catalyzed oxygenation of linoleate. Hydroxylinoleic acid was a competitive inhibitor of the activation. At haemoglobin concentrations higher than 1 μM the quasi‐lipoxygenase activity was completely suppressed presumably owing to the formation of inhibitory concentrations of hydroxylinoleate via the lipohydroperoxidase [21] activity of haemoglobin.
The quasi‐lipoxygenase reaction of haemoglobin exhibits a suicidal behaviour, caused by destruction of haem groups. The properties of the free haemin‐catalyzed reaction were similar to those of haemoglobin. Among other haemoproteins tested myoglobin and cytochrome P‐450 LM show comparable activities. The mechanism of the haemoglobin‐catalyzed oxygenation of linoleate seems to involve a change of valency of the haem iron.
Sarcoplasmic reticulum, isolated from the muscle of winter flounder (Pseudopleuronectes americanus), was preincubated with phospholipase A2 to determine the effects of enzymic and non-enzymic lipid-peroxidation systems. Eicosapentaenoic acid (20:5) was preferentially released by phospholipase A2, but the percentage of docosahexaenoic acid (22:6) in the free fatty acids was similar to the percentage in membrane total fatty acids. A decrease in the production of both thiobarbituric acid-reactive substances and lipid hydroperoxides was observed in both the enzymic and non-enzymic peroxidation systems upon preincubation with phospholipase A2. Addition of palmitic acid or lysophosphatidylcholine did not inhibit either peroxidation system. Non-enzymic peroxidation was inhibited by the addition of beta-glycerophosphate, but enzymic peroxidation was apparently unaffected. A model is proposed to explain the inhibitory effect of phospholipid hydrolysis on lipid peroxidation in both systems, suggesting that fatty acids are structurally realigned upon hydrolysis, leading to decreased free-radical chain propagation. Additional factors appear to contribute to inhibition of enzymic peroxidation.
A lactoperoxidase/H2O2/halide system caused the initiation of linoleate peroxidation as indicated by diene conjugation. Coupled lipid peroxidation was accelerated by iodide, chloride and bromide ions at pH 4.0 and 6.2. No peroxidation occurred in the presence of H2O2 or lactoperoxidase alone. The rate of linoleate peroxidation by lactoperoxidase in the presence of chloride depended on the concentration of H2O2. Linoleate peroxidation by the enzymatic system was inhibited by high concentration of H2O2 by methionine, tryptophan and BHT. Oxygen was absorbed during peroxidation and the major products were the 13-hydroperoxides. The mechanisms of the initiation of lipid peroxidation by a peroxidase/H2O2/halide system are discussed.
During primate evolution, a major factor in lengthening life-span and decreasing age-specific cancer rates may have been improved protective mechanisms against oxygen radicals. We propose that one of these protective systems is plasma uric acid, the level of which increased markedly during primate evolution as a consequence of a series of mutations. Uric acid is a powerful antioxidant and is a scavenger of singlet oxygen and radicals. We show that, at physiological concentrations, urate reduces the oxo-heme oxidant formed by peroxide reaction with hemoglobin, protects erythrocyte ghosts against lipid peroxidation, and protects erythrocytes from peroxidative damage leading to lysis. Urate is about as effective an antioxidant as ascorbate in these experiments. Urate is much more easily oxidized than deoxynucleosides by singlet oxygen and is destroyed by hydroxyl radicals at a comparable rate. The plasma urate levels in humans (about 300 microM) is considerably higher than the ascorbate level, making it one of the major antioxidants in humans. Previous work on urate reported in the literature supports our experiments and interpretations, although the findings were not discussed in a physiological context.
The microsomal fraction from fish muscle has previously been shown to catalyze the oxidation of its lipid. In this study we have studied the rate of the reaction in the frozen state. The rate was dependent on temperature, decreasing with decreasing temperature. When the microsomes were frozen in the presence of NaCl there was greater activity than when they were frozen in the presence of KCl. The specific activity of the oxidation decreased with increasing protein concentration. This is possibly due to the limitation of oxygen in the frozen system. Lipid oxidation is a complex reaction and both initial products (lipid hydroperoxides) and breakdown products (those reacting with malondialdehyde) were measured. This ratio was relatively constant over a variety of conditions indicating that the rate-limiting step of the reaction occurred prior to the formation of lipid hydroperoxide. A study of the reaction at above-freezing temperatures and below-freezing temperatures in the presence of miscible solvents to prevent freezing at temperatures below 0 °C gave results which were consistent with the hypothesis that ice crystal formation had an accelerating effect on the reaction. Presumably this is due to concentration of reactants since freezing and thawing of the microsomes did not affect their rates of lipid oxidation. Potent inhibitors of the lipid oxidation reaction were found in the soluble fraction of the muscle tissue. These were both high-molecular and low-molecular-weight compounds. The low-molecular-weight inhibitors were more effective in the frozen state while the high-molecular-weight compounds were relatively more effective in the reaction catalyzed at temperatures above freezing.
The mechanism of linoleate oxidation in the presence of the catalysts hemin, cytochrome c, hemoglobin, and catalase was the subject of this investigation. Catalytic activity decreased in the order: cytochrome c > hemin > hemoglobin > catalase. Kinetically, the oxidation of colloidal linoleate followed the relationship: The linoleate peroxides resulting from hemin-catalyzed linoleate oxidation were characterized by production of carbonyl compounds. The hemin-catalyzed oxidation of methyl oleate, methyl linoleate, and methyl linolenate was zero order. Induction periods decreased and reaction rates increased as the reactivity of the fatty acid esters increased.The postulated mechanism of linoleate-oxidation initiation involves a direct reaction of linoleate peroxide with hematin catalysts. Propagation may take place in a manner similar to autocatalytic linoleate oxidation. The termination reaction probably involves interaction of linoleate peroxide radicals and hematin compounds.