Myoglobin and nonheme iron in fractions
As expected, the myoglobin concentration in beef loin was greater than that in chicken breast (Table 1). The myoglobin concentrations in both of LMW fractions from chicken breast and beef loin were not shown because those were undetectable. The concentrations of myoglobin and metmyoglobin percentage (Table 1) in all fractions from chicken breast did not change during storage. The myoglobin concentrations in all fractions from chicken breast meat in this study may be overestimated due to the turbidity of the extracts (Kranen and others 1999).
Table 1—. Myoglobin concentration (mg myoglobin/g meat), percent (%) metmyoglobin, and nonheme iron content (μg nonheme iron/g meat) in various fractions from chicken breast and beef loin during storage at 4 °C.
|Storage||Chicken breast||Beef loin||SEM|
|Myoglobin concentration||mg myoglobin/g meat|| |
| 0 d||1.16c||0.35de||0.56d||0.25dey||—||5.97ax||0.39dey||3.05bx||2.93bx||—||0.09|
| 5 d||1.11c||0.35e||0.58de||0.42dex||—||5.14ay||0.71dx||2.89bx||2.67bx||—||0.07|
| SEM||0.04||0.02||0.03||0.04|| ||0.18||0.08||0.08||0.08|| || |
|Percent (%) metmyoglobinB||% metmyoglobin|| |
| 0 d||62.15b||59.26bc||61.22b||46.70dy||—||57.71bcy||62.65b||51.50cdz||71.43ay||—||1.70|
| 5 d||64.60b||65.48bc||64.44b||60.79bx||—||68.39bxy||66.39b||67.86by||95.75ax||—||1.78|
| SEM||0.47||1.00||0.86||2.14|| ||3.63||0.54||3.70||3.34|| || |
|Nonheme iron content||μg nonheme iron/g meat|| |
| 0 d||1.02by||0.42cdz||0.32cd||0.27d||0.00e||2.06az||0.54cz||0.52cdy||0.50cdy||0.00ey||0.06|
| 5 d||1.30by||0.65cdy||0.34de||0.23ef||0.00f||3.03ay||1.24by||0.73cy||0.62cdy||0.00efy||0.08|
| 10 d||1.73bx||0.81cx||0.59cd||0.35cd||0.04d||4.34ax||1.87bx||1.60bx||0.86cx||0.41cdx||0.15|
| SEM||0.09||0.04||0.07||0.02||0.02||0.24||0.12||0.11||0.06||0.04|| |
The myoglobin concentrations in H, S, and HMW fractions from beef loin significantly decreased during storage, but increased in precipitate (P) fraction due probably to the deposition of denatured myoglobin. In addition, metmyoglobin percentage increased significantly in H, S, and HMW fractions from beef loin, but did not change in P fraction. The metmyoglobin percentage in the H fraction at day 10 was significantly lower than those in S and HMW fraction due to the presence of the P fraction. The P fraction including mitochodria and microsomes, which contain many enzymes including metmyoglobin-reducing enzyme system (Rhee 1988; Arihara and others 1995; Thomas 1995), retarded the autoxidation of myoglobin, which is a major source of catalysts/initiator such as ferrylmyoglobin, hematin, and free ionic iron for the development of lipid oxidation (Kim and Sevanian 1991; Baron and Andersen 2002; Min and Ahn 2005). In addition, the metmyoglobin percentage in the HMW fraction increased rapidly, but an increase in the metmyoglobin percentage in the S fraction was delayed at the early stage of storage due to the presence of the LMW fraction including low molecular weight reducing compounds. However, those compounds seemed to be unstable because metmyoglobin percentage in the S fraction increased rapidly at the late stage of storage. It was shown that LMW fractions of cytosol from fish and turkey have inhibitory effect on hemoglobin and myoglobin-catalyzed lipid oxidation due to reducing compounds and/or antioxidants in LMW fraction (Kanner and others 1991; Undeland and others 2003).
Initial (day 0) nonheme iron contents in H, P, S, and HMW fractions from chicken breast was significantly lower than their counterparts from beef loin during storage (Table 1). However, nonheme contents in LMW fractions from both meats were undetectable at days 0 and 5. Nonheme iron contents in the H and P fraction increased significantly during storage, but not in the S fraction. It is assumed that most of nonheme iron generated in chicken breast during storage may be stored in water-insoluble fraction, especially hemosiderin, and thus is not activated for lipid oxidation. Hemosiderin is a water-insoluble complex of iron, other metals and proteins, and considered as a ferritin decomposition or polymerization products (Decker and Hultin 1992).
Nonheme iron content in all fractions from beef loin significantly increased during storage, and the rates of increase in all fractions from beef loin were greatly higher than those from chicken breast (Table 1). Nonheme iron contents in the H and P fractions increased gradually throughout the storage time. However, increases in nonheme iron contents in the S, HMW, and LMW fractions were only observed from day 5 to day 10. The increase in nonheme iron content in LMW fraction was especially great in this period. Therefore, it is assumed that released nonheme irons in the beginning of storage may be captured in ferritin, which is subsequently converted to hemosiderin. However, the amount of nonheme iron not bound to proteins increased in S and LWM fraction from day 5 to day 10 because the amount of ferritin in beef is very limited (Hazell 1982). The major source of nonheme iron in beef is myoglobin and accounts for over 90% of heme proteins present in beef (Hazell 1982). The interaction of metmyoglobin with H2O2 or lipid hydroperoxide results in the release of free ionic iron (Harel and others 1988). Free ionic iron can serve as a catalyst in the production of •OH from H2O2 as well as in the degradation of lipid hydroperoxides to produce peroxyl and alkoxyl radicals, which can initiate lipid oxidation and/or be self-degraded to the secondary products of lipid oxidation (Min and Ahn 2005). However, reducing compounds are essential to convert ferric to ferrous ion, a catalyst for Fenton reaction. Ahn and Kim (1998) suggested that the status of ionic iron is more important than the amount of iron.
Total antioxidant capacity (TAC) and ferric ion reducing capacity (FRC)
Total antioxidant capacities (TAC) in H, S, HMW, and LMW fractions from chicken breast during storage were significantly higher than those in their counterparts from beef loin, but those in P fraction from both meats were not different (Table 2). TAC in all fractions from chicken breast did not change during storage, but TAC in those from beef loin decreased significantly. Chicken breast had higher initial TAC and stronger endogenous capability to stabilize TAC than beef loin. In addition, water-insoluble (P) fraction showed significantly higher TAC than water-soluble (S) fraction in both chicken breast and beef loin during storage. Reducing enzyme systems bound to mitochondria and microsome in P fraction may be contributed to higher TAC in P fraction and may have played an important role in maintaining oxidative stability of meat. HMW fraction had significantly higher TAC than LMW fraction in both meats during storage.
Table 2—. Total antioxidant capacity (TAC) and ferric ion reducing capacity (FRC) of various fractions from chicken breast and beef loin during storage at 4 °C. TAC was determined by the cupric reducing antioxidant capacity (CUPRAC) method. TAC and FRC were expressed as μg ascorbic acid equivalent/g meat.
|Storage||Chicken breast||Beef loin||SEM|
|Total antioxidant capacity (TAC)||μg ascorbic acid equivalent/g meat|
| 0 d||2943a||1307c||651d||469e||109gh||2605bx||1336cx||369efx||278fgx||87hx||37|
| 5 d||2755a||1308c||663d||483e||107fh||2151by||1340cx||298fy||235fgy||52iy||34|
| 10 d||2763a||1301c||676d||477de||104f||1912by||1160cy||272efy||214efy||40fy||56|
| SEM|| 88|| 72||8|| 6|| 3|| 71|| 23|| 9|| 6|| 4|| |
|Ferric ion reducing capacity (FRC)|
| 0 d||24.72ax|| 6.93cd||12.33bx||2.02e||12.19bx||12.86b|| 7.76cx ||4.63dex||3.69ex||6.49cdx||0.54|
| 5 d||13.21ay||7.69b|| 5.38bcy||2.01d|| 4.08cdy||14.11a||5.44bcy|| 4.38cdxy|| 2.99cdy||3.91cdy||0.56|
| 10 d||13.90ay||6.92b|| 3.63cdz||2.05d|| 1.66dz||12.13a||4.89bcy||3.07cdy||2.00dz||3.03cdy||0.51|
| SEM||1.17 ||0.65 ||0.31||0.21 ||0.33||0.70||0.40 ||0.33 ||0.11 ||0.32 || |
As shown in Table 2, the initial (day 0) ferric ion reducing capacities (FRC) of H, S, and LMW fraction from chicken breast were twice or 3 times as high as those from beef loin, respectively, but rapidly decreased to the same level as those from beef loin at days 5 and 10. The rapid decrease of FRC in S and H fractions from chicken was due to the decrease of FRC in LMW fraction. These observations indicated that chicken breast has large amounts of unstable low molecular weight reducing compounds such as ascorbic acid, NAD(P)H, glutathione, and thiol compounds (Kanner and others 1991; Kanner 1994), which appears to be negligible in beef loin. The initial FRC in P and HMW fractions from chicken breast was not different from that from beef loin, and did not change during storage. After unstable reducing compounds in LMW fraction lose their FRC at the earlier stage of storage, FRC in H, S, and LMW fraction from chicken breast were not different from those of beef loin at days 5 and 10. This indicated that both chicken breast and beef loin had the same levels of stable systems to maintain the FRC during storage. It is assumed that FRC in H fraction from both meats at days 5 and 10 is due mostly to the water-insoluble fraction containing reducing enzyme systems in mitochondria and microsomes, and partly due to the stable compounds in water-soluble fraction. The FRCs in P, S, HMW, and LMW from beef loin decreased gradually during storage. The FRC in P and HMW fractions from beef loin decreased during storage due probably to oxidative damage in enzyme systems and sulfhydryl part of proteins. It is assumed that strong interactions among various reducing components, proteins, and stable low-molecular-weight reducing compounds in P, HMW, and LMW fractions might be contributed to the stability of FRC in H fraction from beef loin during storage.
The free radical scavenging activity of antioxidants such as α-tocopherol is achieved by 1 electron reduction of free radicals, resulting in the termination of lipid oxidation chain reaction. The TAC and FRC used in this study represented the reducing capacity of antioxidants to reduce cupric (Cu(II)) and ferric (Fe(III)) ions to cuprous (Cu(I)) and ferrous (Fe(II)) ions, respectively, and expressed as microgram ascorbic acid equivalent per gram meat. Huge differences between TAC and FRC for every fraction from chicken breast and beef loin were observed (Table 2), and those gaps should be primarily attributed to the differences in 1 electron reduction potential of copper ion and iron ion and their reactivity in redox reactions (Buettner 1993; Lynch and Frei 1995; Burkitt 2001; Apak and others 2004). The 1 electron reduction potential of Cu(II)/Cu(I) couple (0.615 volt at pH 7 in the presence of neocuproine; Burkitt 2001) is higher than that of Fe(III)/Fe(II) couple (0.11 V; Buettner 1993). Therefore, the substances with their reduction potentials (E°') between 0.11 and 0.65 V are not thermodynamically feasible to reduce Fe(III) to Fe(II), but Cu(II) to Cu(I). Because the reduction potentials of the major free radicals involved in free radical chain reactions of lipid oxidation such as hydroxyl radical (2.31 V), alkoxyl radical (RO •/ROH, 1.6 V), and peroxyl radical (ROO •/ROH, 1.0 V) (Koppenol 1990) are higher than that of copper ion (0.65 V), those are thermodynamically capable of scavenging free radicals and terminate lipid oxidation processes. Furthermore, copper ions are chemically more reactive than iron ions, resulting in faster kinetics than iron ions in redox reactions (Lynch and Frei 1995; Apak and others 2004). For example, the rate constant in Fenton reaction catalyzed by the copper ion would be 61.8 times faster than that by the iron ion (Halliwell and Gutteridge 1999). In addition, Mira and others (2002) reported that most of the flavonoids assessed in their study showed higher reducing capacity for copper ions than iron ions. Consequently, a large part of the TAC in each fraction may not participate in the reduction of Fe(III) to Fe(II) although the FRC is likely to be a part of TAC.
Ascorbic acid, an important biological reducing agent, can serve as an antioxidant or prooxidant in meat, depending upon its relative concentration to free ionic iron content: an antioxidant at higher concentrations and a prooxidant at lower concentrations (Decker and Hultin 1992; Gorelik and Kanner 2001). Therefore, it is assumed that the FRC can work as an antioxidant or prooxidant activity, depending upon the concentration of free ionic iron. The FRC in H fraction from chicken breast and beef loin were similar at days 5 and 10, but TBARS values at days 5 and 10 (Figure 2) were much higher in beef loin than in chicken breast. This indicated that greater increase in free ionic iron in beef loin during storage (Table 1) was partially responsible for the higher TBARS values in beef loin than those in chicken breast.
S and HMW fractions from beef loin showed significantly higher LOX-like activities than LMW fraction due to myoglobin in S and HMW fractions (Table 3). LOX-like activities in S and HMW fractions increased during storage. However, the changes of LOX-like activities in S and HMW fractions during storage were highly correlated with the metmyoglobin percentage (r= 0.90 and 0.94, respectively) and TAC (r=–0.83 and –0.88, respectively) in both fractions. LOX-like activities in S and HMW fractions from chicken breast were much lower than those from beef loin because of the low myoglobin content and high TAC in chicken breast. Reducing compounds such as ascorbic acid can reduce ferrylmyoglobin to metmyoglobin (Giulivi and Cadenas 1993; Kroger-Ohlsen and Skibsted 1997). Therefore, much higher LOX-like activity in beef loin than chicken breast was attributed to high myoglobin content and low TAC in beef loin.
Table 3—. Lipoxygenase-like activities of various fractions from chicken breast and beef loin during storage at 4 °C.
|Storage||Chicken breast||Beef loin|
Lipid oxidation potential of fractions
Lipid oxidation potential (LOP) is the ability to increase lipid oxidation in phospholipid liposome model system during incubation. LOP of each fraction can be determined by the interaction of prooxidant and antioxidant factors in each fraction. Differences in LOP among fractions could be derived from the differences in the balance of prooxidant and antioxidant factors. Metmyoglobin showed very high LOP in phospholipid liposome model systems (data not shown). A significantly higher LOP in H fraction from beef loin compared to that from chicken breast during storage (Figure 3) indicated that there could be a significant difference in the oxidative stability between the 2 meats.
Figure 3—. Lipid oxidation potential of various fractions from chicken breast (A1 to A3) and beef loin (B1 to B3) in the phospholipid liposome model system during storage 4 °C during the incubation at 37 °C for 90 min (expressed as TBARS value, mmol malondialdehyde [MDA]/kg phospholipid). The phospholipid liposome model system with 50 mM acetate buffer (pH 5.6) was used as a control (PL). Means with the standard deviation were indicated. n= 4. H = homogenate fraction; P = precipitate fraction; S = supernatant fraction; HMW = high molecular weight fraction from supernatant fraction; LMW = low molecular weight fraction from supernatant fraction.
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At day 0, the LOP of LMW fraction from chicken breast was the highest, and lipid oxidation by LMW fraction from chicken breast rapidly increased at beginning of incubation, but did not change after 30 min (Figure 3A1). The LOP of LMW fraction from chicken breast at both days 5 and 10 was very low compared to that at day 0. Table 2 showed that FRC of LMW fraction decreased rapidly during storage. Therefore, it is assumed that high FRC at day 0 was attributed to the low-molecular-weight reducing compounds such as ascorbic acid, which reduced the contaminated ferric ion in the reaction solution, leading to the rapid development of lipid oxidation. The LOPs of S and H fraction from chicken breast meat were significantly lower than that of the LMW fraction at day 0, indicating that the HMW and P fraction possessed antioxidant capacity. Kanner and others (1991) indicated that HMW fraction from turkey meat showed an antioxidant effect in model system containing ascorbic acid and ferric ion although LMW fraction acted as a prooxidant in the same system. The difference between LOPs of the H and LMW fractions was significantly greater than that of the S and LMW fractions, due to the higher TAC in the P fraction than that in the HMW fraction (Table 2). At day 10, the LOP of H fraction from chicken breast was significantly lower than those of S, P, and HMW factions, and was almost the same as that of control (Figure 3A3). Although prooxidant factors in S, P, and HMW fractions, respectively, were present, the interaction of antioxidant capacity between water-soluble (S), especially HMW fraction, and water-insoluble (P) fraction seemed to suppress the prooxidant activities in H fraction from chicken breast.
At day 0, HMW fraction from beef loin showed the highest LOP (Figure 3B1), and the LOP increased during storage (Figure 3B1 to 3B3). Metmyoglobin was most likely responsible for the high LOP of HMW fraction. An increase in metmyoglobin percentage in HMW fraction during storage (Table 1) are closely involved in the increase of the LOP. Myoglobin can be involved in catalyzing and accelerating lipid oxidation via the formation of ferrylmyoglobin or the release of hematin and free ionic iron in the presence of H2O2 or lipid hydroperoxides (Kanner and Harel 1985; Harel and others 1988; Kim and Sevanian 1991; Baron and others 2002; Min and Ahn 2005). Ferrylmyoglobin can initiate and propagate the free radical chain reaction of lipid oxidation (Harel and Kanner 1985; Reeder and Wilson 1998; Baron and Andersen 2002). The hematin released from myoglobin can react with lipid hydroperoxides or H2O2 to form high oxidation state (FeIV) of hematin, which can catalyze initiation and propagation of lipid oxidation (Dix and Marnett 1985; Kim and Sevanian 1991). The hematin may be more dangerous because it is more reactive than metmyoglobin and has a hydrophobic characteristic, which enables it to permeate into cell membrane to catalyze lipid oxidation (Kaschnitz and Hatefi 1975; Schmitt and others 1993; Baron and others 2002). Therefore, both ferrylmyoglobin and hematin can contribute to the LOX-like activity of HMW fraction. In addition, free ionic irons released from myoglobin can be a catalyst for lipid oxidation via the Fenton reaction in the presence of reducing agents (Harel and others 1988; Ahn and Kim 1998; Min and Ahn 2005). The free ionic irons can be responsible for the high LOP of HMW fraction from beef loin because of the FRC of the HMW fraction (Table 2). Therefore, it was suggested that myoglobin could be a major oxidative factor in beef loin. However, it is difficult to say which mechanism is dominant for the high LOP of HMW fraction from beef loin in this study.
LOPs of H and S fraction from beef loin were significantly lower than that of the HMW fraction during storage, due to the TAC of the LMW and P fraction (Table 2). Myoglobin can be stabilized by reducing agents, which can reduce ferrylmyoglobin to metmyoglobin and prevent the disruption of myoglobin to release hematin and/or free ionic iron (Giulivi and Cadenas 1993; Kroger-Ohlsen and Skibsted 1997). LMW fraction from beef loin at day 0 showed a similar pattern of increases in TBARS values to that from chicken breast, but its LOP was smaller due probably to its lower FRC at day 0 (Table 2). Consequently, the increase of lipid oxidation in beef loin during storage was caused by the prooxidant factors, probably myoglobin, in HMW fraction. Although the antioxidant activities in P and S (HMW + LMW) fractions lowered the LOP of HMW fraction, those antioxidant activities may be not enough to attenuate the prooxidative activities of myoglobin in HMW fraction from beef loin.