Metmyoglobin and inorganic metals as pro-oxidants in raw and cooked muscle systems

Meat Science 15 (1985) 107-116

Metmyoglobin and Inorganic Metals as Pro-oxidants in Raw and Cooked Muscle Systems J. Z. Tichivangana* & P. A. Morrissey Department of Food Chemistry, University College, Cork, Ireland

(Received: 6 February, 1985)

SUMMARY The pro-oxidant activities of metmyoglobin ( Mb) and metal ions on the induction of lipid oxidation in raw and heated water-washed muscle systems from fish, turkey, chicken, pork, beef and lamb and during storage of these systems at 4°C, were investigated. Lipid oxidation was invariably faster in heated than in raw systems. In raw Mb-catalyzed systems, oxidation was slow over a 5-day period, except in fish, where significant (P < 0.05) increases in TBA values occurred; in contrast, significant (P < 0"05) increases in TBA values occurred in cooked fish, turkey, chicken and pork after 3 days of storage. Cooked beef and lamb, however, showed significant lipid oxidation only after 5 days of storage. Fe 2 ÷ was found to be highly catalytic in cooked muscle. Cu 2 ÷ and Co 2 + were less effective catalysts than Fe 2 ÷; the overall pro-oxidant activity was in the order Fe 2 ÷ > Cu 2 ÷ > Co 2 ÷ > Mb, and the susceptibility to lipid oxidation of the muscle systems was in the order: fish > turkey > chicken >pork > beef> lamb, probably reflecting the degree of unsaturation o f the constituent triglyceride fractions.

INTRODUCTION Traditionally, lipid oxidation in animal flesh has been attributed to haem catalysts such as haemoglobin, myoglobin and cytochromes (Tappel, 1962). However, it is still not certain that haem pigments are the main * Present address: Cold Storage Commission, PO Box 953, Bulawayo, Zimbabwe. 107 Meat Science 0309-1740/85/$03.30 © ElsevierApplied Science Publishers Ltd, England, 1985. Printed in Great Britain

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oxidative catalysts in muscle. Liu (1970a,b) observed that non-haem iron plays a dominant pro-oxidant role in shrimp flesh, whereas, in beef muscle, haem iron appeared to be the major catalyst. On the other hand, a number of workers (Sato & Hegarty, 1971; Love & Pearson, 1974) presented data which suggested that non-haem iron is the major prooxidant in cooked meat, and observed that meat pigments p e r se have no catalytic effect. Igene et al. (1979) also concluded that myoglobin is not the principal pro-oxidant in cooked meats. According to these authors, cooking destroys the haem pigments and releases a significant amount of non-haem iron from bound haem pigments and provides a source of free iron which accelerates lipid oxidation in cooked meats. Thus, they argue that the increased rate of lipid oxidation in cooked meat is due to the release of non-haem iron during cooking, which catalyzes lipid oxidation. In an earlier report (Tichivangana & Morrissey, 1984)we argue that if the hypothesis of Igene et al. (1979) is correct, then increased catalytic activity should also have been observed by the earlier workers (Sato & Hegarty, 1971 ; Love & Pearson, 1974) when metmyoglobin and haemoglobin were added to meat systems prior to cooking. Our preliminary studies (Tichivangana & Morrissey, 1984) showed that both metal ions and haem pigments catalyzed lipid oxidation in heated fish muscle systems. However, non-haem iron is the major pro-oxidant. The objective of the present study was to assess, in more detail, the relative contributions of haem and non-haem catalysts to lipid oxidation in raw and cooked muscle systems from lamb, beef, pork, chicken, turkey and fish. It is important to establish whether haem iron or non-haem, or both, promote oxidation in various meats. MATERIALS AND METHODS

Muscle Samples All samples were purchased fresh from local meat and fish markets and were processed immediately on arrival at the laboratory.

Preparation of Water-Washed Muscle Fibres (WF) Each muscle was trimmed of extramuscular fat, minced and extracted with deionised water as outlined previously (Tichivangana & Morrisey, 1984).

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Preparation and Treatment of Model Systems Various pro-oxidants were added to the WF preparations. A sample ( ~ 100 g) of extracted tissue was mixed thoroughly with the appropriate pro-oxidant; metmyoglobin was added at a level of 5 mg/g WF and the inorganic salts were added at the 5 mg/kg level.

Reagents Horse heart myoglobin (Mb) was purchased from Sigma Chemical Co. Ltd., Poole, Dorset, Great Britain. This preparation is metmyoglobin since the iron is primarily in the ferric form. All other chemicals were ~AnalaR' grade purchased from British Drug Houses, Poole, Dorset, Great Britain.

Method of Cooking Samples were sealed in 30 x 18 cm retortable bags (Seaward Laboratories, London, Great Britain) and then placed in a hot water bath to cook the samples to an internal temperature of 70°C x 30 min (Tichivangana & Morrissey, 1982). Following cooking, the samples were cooled and immediately assessed for lipid oxidation. Portions of the raw and cooked samples were stored at 4°C and the level of lipid oxidation assessed at regular intervals over a 5-day period.

Assessment of Lipid Oxidation Lipid oxidation was determined by the 2-thiobarbituric acid (TBA) method of Ke et al. (1977). The TBA values were expressed as milligrams of malonaldehyde per kilogram of tissue.

Statistical Analysis There were four replicates for each treatment in this study and all analyses were done in duplicate. Analysis of variance for TBA values was calculated using the University College, Cork, computer package program run on an IBM 4347 computer.

J. Z. Tichivangana, P. A. Morrissey

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RESULTS A N D DISCUSSION

Effect of Metmyogiobin The pro-oxidant activities of metmyoglobin (Mb) in raw and heated muscle systems were assessed over a period of 5 days and the results are shown in Fig. 1. The results for the heated systems catalyzed by metal ions are presented in Fig. 2. Linear equations were fitted to the data and regression analysis used to determine the rate of lipid oxidation (Table I) for each muscle system and treatment. No single equation could be fitted to all plots; hence, best estimates were obtained from linear equations. Fig. IA shows that Mb exerted very little catalytic activity in raw systems. Little or no increase in TBA values occurred over the 5 days of storage, thus indicating a long induction phase. The induction phase for the purpose of this study is defined as the time required to give a TBA value of 2. Analysis of variance of the data showed no significant effect due to sample treatment with Mb and storage time except in the fish system where there was a significant (P < 0.05) increase in TBA values after the fourth day of storage. Thus, the reactivity of muscle lipids appears to be influenced by the degree of unsaturation of the triglyceride fraction, as previously suggested (Tichivangana & Morrissey, 1982). 15

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Fig. 1. Effect of metmyoglobin (5 mg/g) on lipid oxidation in raw (A) and heated (B) water-extracted muscle fibres (WF) from various species, stored at 4°C. HI, fish; D, turkey; A, chicken; A, pork; O, beef and O, lamb.

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Fig. 2. Effect of Fe 2 + (A), Cu 2 + (B) and Co 2 + (C) on lipid oxidation in heated waterextracted muscle fibres (WF) from various species, stored at 4°C. II, fish: D , turkey: A, chicken; A , pork; O, beef and O , lamb.

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TABLE 1 Rates of Lipid Oxidation Catalyzed by Metmyoglobin (5 mg/g) and Metals (5 mg/kg) in Raw and Heated Water-Extracted (WF) Muscle Systems from Several Species Stored at 4°C for 5 Days. Increase in T B A values p e r d a y a Muscle

Fish Turkey Chicken Pork Beef Lamb

Control b

Mb

R

H

R

0.14 0.13 0.13 0.12 0.09 0.07

0.16 0.15 0.15 0.13 0.10 0.09

028 0.27 0.26 0.23 0.16 0.12

Fe 2 + H

2.70 2.18 1.98 1.82 1.19 0.86

R

2.71 1.89 1.77 1.42 0.62 0.44

Cu 2 ÷ H

4.37 3.84 3.73 3.27 2-13 1.09

R

2.30 1.61 1.58 1.04 0.49 0.33

H

388 3.45 3.32 2.84 1.77 1.06

Co 2 + R

1.48 1.04 0.98 0.61 0.33 0.24

H

3.29 2.79 2.38 2.10 1.29 0.89

a Mean TBA values per day of four replicates carried out in duplicate. b Muscle systems without additives. R, raw. H, heated.

In contrast to the stability of the raw muscle systems, the heated Mb systems were very susceptible to lipid oxidation (Fig. 1B). The increase in TBA values was low for both lamb and beef muscle even after 48 hours storage at 4 °C.These findings are similar to those previously reported for cooked beef systems stored for 48h (Sato & Hegarty, 1971; Love & Pearson, 1974). However, TBA values increased when the storage time was extended to 5 days. Table 1 shows that the oxidation rates in the raw and heated WF control samples were very low, and that only small, insignificant increases in the rate of oxidation occurred in the raw Mb-treated samples. The differences in rates of oxidation between the raw and heated Mbcatalyzed samples were highly significant (P < 0.001) for all muscle systems. The data in Fig. 1B and Table 1 also show that the rate and extent of lipid oxidation catalyzed by Mb in heated systems was in the order fish > turkey > chicken > pork > beef > lamb. Previous workers (Wilson et al., 1976) reported that turkey meat was most susceptible to oxidation, followed by chicken, pork, beef and mutton, in that order. The rates and extent of lipid oxidation are dependent on a number of factors, the most important being the level of polyunsaturated fatty acids

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present in the particular lipid system (Allen & Foegeding, 1981). Since lamb and beef are relatively low in polyunsaturated fatty acids (Pearson et al., 1977), it is not surprising that the induction periods of these systems are relatively long; the more rapid rates of oxidation for the other muscle systems examined correspond to their higher contents of polyunsaturated fatty acids. Earlier studies conclude that triglycerides and phospholipids are important in the development of rancidity in chicken (Igene et al., 1980) and fish (Tichivangana & Morrissey, 1982). The influence of triglycerides on the development of rancidity was shown to depend upon the degree of unsaturation (Igene et al., 1980). Thus, it appears that triglycerides, which contain significant amounts of polyunsaturated fatty acids, may be much more important than previously considered. The present results may help to explain the earlier findings (Sato & Hegarty, 1971; Love & Pearson, 1974) where haem iron, even at concentrations of 10 mg/g, had essentially no effect on the development of oxidation in beef systems. It is also suggested that the storage time used by Sato & Hegarty (1971) and Love & Pearson (1974) was not sufficiently long for a significant increase in oxidation to occur. Effect of Metals on Lipid Oxidation

The influence of F e 2 +, C u 2 + or Co 2 ÷ on lipid oxidation in raw and heated muscle systems of several species was also examined and the data for the heated systems are presented in Fig. 2. In the raw muscle systems (result not presented) induction of oxidation was notably slow in the lamb and beef systems where little oxidation occurred throughout the storage period. However, significant (P < 0.01) increases in TBA values occurred in pork, and highly significant increases (P < 0-001) occurred in chicken, turkey and fish after 5 days of storage. The catalytic effectiveness of Fe 2 + was very pronounced in all heated systems (Fig. 2A). With the exception of lamb and beef, TBA values immediately after heating (0 days) were above the arbitrary threshold value of 2. Analysis of variance showed highly significant (P < 0-001) effects due to F e 2 + and storage time in all muscle systems except lamb in which lesser, but still significant (P < 0.01), increases occurred. There are only three reports (Ke & Ackman, 1976; Ke et al., 1977; Tichivangana & Morrissey, 1984) on the r61e of other transition metals in lipid oxidation, probably due to the fact that iron is the dominant metal in muscle. However, copper and cobalt also occur in muscle, and, by their

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nature, could play a significant part in lipid oxidation. The effects o f C u 2 + and Co 2+ in heated muscle systems are shown in Figs. 2B and 2C, respectively. The data indicates that Cu 2 +-catalyzed oxidation followed a pattern similar to that for Fe z+ catalysis, but Cu 2+ was slightly less effective. Co 2 +-catalyzed lipid oxidation (Fig. 2C) was lower (P < 0-05) than that for Cu 2+ but followed essentially the same trend. The rates of oxidation (Table 1) show that pro-oxidant activity was in the order: F e 2 + > C u 2 + > C o 2 + > Mb, and that differences in activity between Fe 2 + and Cu 2 +, Fe 2 + and Co 2 + and Fe 2 + and Mb were significant at the P < 0.05, P < 0 . 0 1 and P<0.001 levels, respectively, in all muscle systems. While it would be difficult to completely separate effects due to haem and metal ions in intact muscle, the above experiments may aid in assessing the importance of some catalysts in muscle systems. It appears from the present study that haem iron is not a major pro-oxidant in muscle. The haem levels employed in the present study are higher than those reported for a range of animal species (Livingston & Brown, 1981). While the pro-oxidant activity of haem compounds is well recognised, it has been reported that high concentrations of haem compounds (low ratio of lipid to haem) may actually be anti-oxidant via some unknown mechanisms (Kendrick & Watts, 1969; Hirano & Olcott, 1971). The former reported optimum linoleate-to-haem ratios of 100 for haemin and catalase, 250 for metmyoglobin, 400 for cytochrome c and 500 for methaemoglobin. At haem concentrations of two to four times the optimum catalytic amount, complete inhibition occurred. According to Liu & Watts (1970), the ratio of polyunsaturated fatty acids and Mb in meats would normally be expected to fall in the catalytic range. It may be argued that, in the present study, the use of Mb at a concentration of 5 mg per gram of muscle could have an inhibitory effect in some meat systems. However, in an earlier study we observed that lipid oxidation catalysed by Mb increased with increasing concentration of Mb over the range 1-10 mg/g in heated fish muscle (Tichivangana & Morrissey, 1984) and in heated beef muscle (unpublished results). Thus, it appears that the prooxidant activity of Mb is independent of the ratio of Mb to unsaturated fatty acid in heated muscle systems. The non-haem iron, copper and cobalt used in the present study were added as soluble salts. However, it is unlikely that much free iron is present in muscle. According to Hazell (1982) and Bogunjoko et al. (1983) iron in meat is distributed between five main components in muscle--an

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insoluble (haemosiderin) fraction, ferritin, haemoglobin, myoglobin and a low molecular weight fraction. Wills (1966) observed that pure ferritin is ineffective as a catalyst. Thus, in raw muscle it is unlikely that ferritin and other non-haem complexes function as pro-oxidants. However, the iron can be released from ferritin by ascorbic acid to become an effective catalyst (Wills, 1966). It is well recognised that denaturation of myoglobin by heat leads to a release of free iron (Igene et al., 1979; Morrissey & Tichivangana, 1985) and increased catalytic activity. One can only speculate about the changes occurring in the non-haem complexes during heating. However, since iron is released from ferritin by ascorbic acid (Wills, 1966), it is probable that iron may also be released from ferritin and haemosiderin on heating, thereby rendering the system more susceptible to oxidation. Low levels of free iron (,-~ 1 mg/kg) are highly effective as pro-oxidants (Tichivangana & Morrissey, 1984). The present findings show that the pro-oxidant effects of metal ions and haem iron were invariably more pronounced in heated than in raw muscle. This probably reflects a redistribution of pro-oxidants resulting from denaturation of lipoproteins and destabilization of the muscle structure. Susceptibility of the raw and heated muscle systems to lipid oxidation catalysed by the various pro-oxidants was in the order: fish > turkey > chicken > pork > beef > lamb, which is generally consistent with polyunsaturated fatty acid content of these tissues. ACKNOWLEDGEMENT This work was supported by a grant from the Department of Foreign Affairs, Dublin, to J. Z. Tichivangana. REFERENCES Allen, C. E. & Foegeding, E. A. (1981). Food Tech., 35(5), 253. Bogunjoko, F. E., Neale, R. J. & Ledward, D. A. (1983). Br. J. Nutr., 50, 511. Hazell, T. (1982). J. Sci. Food Agric., 33, 1049. Hirano, Y. & Olcott, H. S. (1971). J. Am. Oil Chem. Soc., 48, 523. Igene, J. O., King, J. A., Pearson, A. M. & Gray, J. I. (1979). J. Agric. Food Chem., 27, 838. Igene, J. O., Pearson, A. M., Dugan, L. R. & Price, J. F. (1980). Food Chem., 5, 263.

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Ke, P. J. & Ackman, R. G. (1976). J. Am. Oil Chem. Soc., 53, 636, Ke, P. J., Ackman, R. R., Linke, B. A. & Nash, D. M. (1977). J. Food Tech., 12, 37. Kendrick, J. & Watts, B. M. (1969). Lipids, 4, 454. Liu, H. (1970a). J. Food Sci., 35, 590. Liu, H. (1970b). J. Food Sci., 35, 593. Liu, H. & Watts, B. M. (1970). J. Food Sci., 35, 596. Livingston, D. J. & Brown, W. D. (1981). Food Tech., 35(5), 244. Love, J., D. & Pearson, A. M. (1974). J. Agric. Food Chem., 22, 1032. Morrissey, P. A. & Tichivangana, J. Z. (1985). Meat Sci.(ln press.) Pearson, A. M., Love, J. D. & Shorland, F. B. (1977). Adv. Food Res., 23, 1. Sato, K. & Hegarty, G. R. (1971). J. Food Sci., 36, 1098. Tappel, A. L. (1962). In 'Symposium on foods." Lipids and their oxidation' (Schuitz, H. W., Day, E. A. & Sinnhuber, R. O. (Eds.)), Avi Publ. Co., Westport, Connecticut, 122. Tichivangana, J. Z. & Morrissey, P. A. (1982). It. J. Food Sci. Tech., 6, 157. Tichivangana, J. Z. & Morrissey, P. A. (1984). Ir. J. Food Sci. Tech., 8, 47. Wills, E. D. (1966). Biochem. J., 99, 667. Wilson, B. R., Pearson, A. M. & Shorland, F. B. (1976). J. Agric. Food Chem., 24, 7.