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Effect of dietary vitamin E supplementation on cholesterol oxidation in vacuum packaged cooked beef steaks
Meat Science 55 (2000) 7±11
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Eect of dietary vitamin E supplementation on cholesterol oxidation in vacuum packaged cooked beef steaks K. Galvin, A.-M. Lynch, J.P. Kerry*, P.A. Morrissey, D.J. Buckley Department of Food Science and Technology, University College, Cork, Ireland Received 10 June 1999; accepted 3 November 1999
Abstract The eect of dietary vitamin E supplementation on cholesterol oxidation in vacuum packaged, cooked, refrigerated and frozen beef steaks, was investigated. Steers (FriesianCharolaisBlack Hereford) were fed diets providing 20 or 3000 mg a-tocopheryl acetate/head/day for 135 days prior to slaughter. a-Tocopherol concentrations in M. psoas major (PM) and M. longissimus dorsi (LD) were signi®cantly (p<0.05) increased by supplementation and were signi®cantly (p<0.05) higher in PM than LD. Cholesterol oxidation (monitored by measuring 7-ketocholesterol formation) increased during refrigerated and frozen storage in some, but not all, groups, and tended to be higher in PM than LD. Dietary vitamin E did not aect 7-ketocholesterol formation in LD, but signi®cantly (p<0.05) reduced concentrations in PM during refrigerated and frozen storage. Supplementation signi®cantly (p<0.05) reduced TBARS in PM and LD, indicating that vitamin E improved oxidative stability in both muscles. The results show that dietary vitamin E supplementation inhibits cholesterol oxidation in vacuum packaged, cooked beef during refrigerated and frozen storage, but may be in¯uenced by muscle type. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Vitamin E; Cholesterol oxidation; Beef
1. Introduction Lipid (fatty acid) oxidation is an important determinant of the quality of meats and meat products. However, in recent years, the formation of cholesterol oxidation products (COPs) in meats and other cholesterol-containing foods has attracted increasing attention. COPs exert a number of adverse eects in biological tissues and have been implicated in the initiation and progression of a number of diseases, particularly atherosclerosis (Kumar & Singhal, 1991). As COPs are known to be absorbed from the gastrointestinal tract (Emanuel, Hassel, Addis, Bergmann & Zavoral, 1991; Linseisen & Wolfram, 1998), factors determining their formation and distribution in foods are of considerable interest. Cholesterol oxidation is a free radical-mediated process and is promoted by factors which also accelerate fatty acid oxidation ( Kowale, Rao, Babu, Sharma & Bisht, 1996; Park & Addis, 1987; Pie, Saphis & Seillan, * Corresponding author. Tel.: +353-21-902256; fax: +353-21276318. E-mail address: [email protected] (J.P. Kerry).
1991). Hence, attempts to control cholesterol oxidation have focused on factors which are known to inhibit fatty acid oxidation. The antioxidant activity of vitamin E in meats has been widely investigated. Supplementation of animal diets with vitamin E (a-tocopheryl acetate) improves the vitamin E status of muscle, and improves the overall quality of meat products by inhibiting fatty acid oxidation and loss of desirable colour and ¯avour during both refrigerated and frozen storage (Morrissey, Sheehy, Galvin, Kerry & Buckley, 1998). In addition, studies have also shown that dietary vitamin E supplementation inhibits the formation of COPs in ®sh (Akhtar, Gray, Booren & Gomaa, 1998), minced pork (Monahan et al., 1992), veal (Engeseth, Gray, Booren & Asghar, 1993), and chicken (Galvin, Morrissey & Buckley, 1998; Lopez-Bote, Gray, Gomaa & Flegal, 1998; O'Neill, Galvin, Morrissey & Buckley, 1999), and beef round steak (Engeseth & Gray, 1994), during refrigerated storage. However, apart from these studies, little published information is available concerning the eects of dietary vitamin E on cholesterol oxidation in meat in general, and in intact cuts and frozen meat in particular. COPs are found in highest amounts in comminuted meats, while in pre-cooked, intact meats, oxides
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K. Galvin et al. / Meat Science 55 (2000) 7±11
are present at very low levels or are below detection limits. However, studies have shown that COPs concentrations in intact meat cuts can increase following storage (Engeseth & Gray, 1994). The purpose of this study was to examine the eect of dietary a-tocopherol supplementation on cholesterol oxidation in refrigerated and frozen vacuum packaged, cooked beef steaks.
2.5. Statistical analysis
2. Materials and methods
3. Results and discussion
2.1. Reagents
The a-tocopherol concentrations of raw M. longissimus dorsi (LD) and M. psoas major (PM) are shown in Table 1. a-Tocopherol concentrations were signi®cantly (p<0.05) higher in supplemental groups than in corresponding basal groups. In addition, concentrations were signi®cantly (p<0.05) higher in basal and supplemental PM groups compared to corresponding LD groups. The eects of storage and dietary vitamin E supplementation on cholesterol oxidation in vacuum packaged cooked LD and PM during refrigerated and frozen storage are shown in Figs. 1 and 2. The COPs identi®ed in beef samples were 7-ketocholesterol, 7b-hydroxycholesterol, cholesterol-5a,6a-epoxide, cholesterol-5b,6bepoxide and 20a-hydroxycholesterol. 7-Ketocholesterol was present in highest amounts and was the only oxide consistently identi®ed in all samples. Therefore, it was used to monitor the extent of cholesterol oxidation during refrigerated and frozen storage. The initiation of cholesterol oxidation involves oxidation of the B-ring of the steroid nucleus, with the initial formation of a carbon-centred allylic C-7 radical, and the subsequent formation of epimeric C-7 hydroperoxides (Smith, 1996). The unstable hydroperoxides degrade to form 7a- and 7b-hydroxycholesterol and 7-ketocholesterol, with the latter predominating (Maerker, 1987). Therefore, 7ketocholesterol would be expected to be the dominant oxide formed during cholesterol oxidation in foods. Similar results were found in previous experiments.
All chemicals used were `AnalaR' grade, obtained from British Drug House, Poole, Dorset, England and Rathburn Chemicals, Walkerburn, Scotland. The atocopheryl acetate used in the diets was obtained from Roche Products, Welwyn Garden City, Herts., England. Cholesterol oxide standards were purchased from Sigma Chemical, Tallaght, Dublin, Ireland and Steraloids (UK), New Barnet, Herts., England. 2.2. Animals and diets Crossbreed (FreisianCharolaisBlack Hereford) steers (n=10) were divided into two groups (n=5) and fed silage/concentrate diets providing 20 (basal) or 3000 (supplemental) mg a-tocopheryl acetate/head/day for 135 days prior to slaughter. Feed was oered once a day and water was provided ad libitum. Slaughtering was carried out at a commercial butchers' slaughtering facility. Following slaughter, all carcasses were centrally split and held at 4 C for 10 days prior to muscle sampling. M. longissimus dorsi and M. psoas major muscles were removed and 2 steaks were cut from each muscle. All sub-primals were cut from the same primal positions. Steaks were cooked in a conventional oven (200 C) to an internal temperature of 75 C for 20 min. Following cooling, steaks were vacuum packaged, under full vacuum, in low oxygen permeable (45 cmÿ3 mÿ2 24 hÿ1 at STP) polyamide/polyethylene ®lm. Vacuum packaged steaks were stored in the dark at 4 or ÿ20 C. 2.3. Determination of -tocopherol The a-tocopherol content of raw muscle was determined as described by Sheehy, Morrissey and Flynn (1994). 2.4. Determination of lipid stability Cholesterol oxides were extracted from meat samples and determined by a GC method as described previously (O'Neill et al., 1999). The extent of lipid oxidation was also monitored using the TBA assay (Ke, Ackman, Linke & Nash, 1977).
All data were subjected to analysis of variance (ANOVA). The statistical signi®cance of the dierences between means was determined by the method of least signi®cant dierence (Snedecor & Cochran, 1967) for a probability of p<0.05.
Table 1 -Tocopherol content of M. longissimus dorsi (LD) and M. psoas major (PM) from steers fed a basal (20 mg/head/day) or supplemental (3000 mg/head/day) level of a-tocopheryl acetate for 135 days prior to slaughtera -Tocopherol (mg/g muscle)b,c Muscle
Basal
Supplemental
LD PM
0.840.15 2.450.12a
2.450.41* 5.750.25a*
a
Values are meanSEM of 5 determinations. Within rows, * indicates signi®cantly dierent from basal group (p<0.05). c Within columns, a indicates signi®cantly dierent from LD group (p<0.05). b
K. Galvin et al. / Meat Science 55 (2000) 7±11
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Fig. 1. Eect of dietary vitamin E supplementation on the formation of 7-ketocholesterol (7-Keto) in vacuum packaged cooked (a) M. longissimus dorsi and (b) M. psoas major during refrigerated storage. &, basal (20 mg/animal/day) a-tocopheryl acetate; &, supplemental (3000 mg/head/day) a-tocopheryl acetate. Values are meansSEM of 5 analyses. * Signi®cantly dierent from basal group (p<0.05).
Fig. 2. Eect of dietary vitamin E supplementation on the formation of 7-ketocholesterol (7-Keto) in vacuum packaged cooked (a) M. longissimus dorsi and (b) M. psoas major during frozen storage. See Fig. 1 for description of symbols. Values are meansSEM of 5 analyses. * Signi®cantly dierent from basal group (p<0.05).
Zubillaga and Maerker (1991) reported that 7-ketocholesterol accounted for over 50% of the total oxides found in raw beef, while Engeseth et al. (1993) showed that 7-ketocholesterol accounted for approximately 45% of total oxides in cooked veal steaks following refrigerated storage for 4 days. 7-Ketocholesterol concentrations were similar to those reported previously in beef. Concentrations ranged from 1.14 to 6.87 mg/g meat compared to 2.11 mg/g in freshly cooked minced beef (Pie et al., 1991), 4.84 mg/ g in refrigerated cooked beef patties (de Vore, 1988) and 4.80 mg/g in refrigerated cooked beef round steak (Engeseth & Gray, 1994). In general, 7-ketocholesterol concentrations tended to be lower in LD than PM. Overall, concentrations were 18±62% lower in basal LD than basal PM during refrigerated storage for up to 2 weeks, while in frozen meat, concentrations in basal LD were 10±26% lower than in basal PM during storage for 8 and 16 weeks. In terms of lipid stability, longissimus dorsi is a more oxidatively stable muscle than psoas, exhibiting comparatively greater colour stability and resistance to lipid oxidation during storage (Chan, Hakkarainen, Faustman, Schaefer, Scheller & Liu, 1996; Kerry, Lynch, Buckley, Morrissey, Monahan &
Allen, 1996). In the present experiment, 7-ketocholesterol concentrations were generally lower in LD than PM, indicating that cholesterol stability is also greater in LD. As it is thought that free radicals derived from fatty acids promote cholesterol oxidation, meat which is more susceptible to fatty acid oxidation would also be expected to be more susceptible to cholesterol oxidation. Similar eects have been observed in chicken, where COPs are generally higher in thigh meat (which is more susceptible to lipid oxidation) than breast meat (Galvin et al., 1998). 7-Ketocholesterol concentrations increased during storage in some, but not in all, groups. In frozen samples 7-ketocholesterol levels in basal LD and PM increased between 8 and 16 weeks, while storage had no eect on 7-ketocholesterol levels in supplemental samples (Fig. 2). During refrigerated storage, 7-ketocholesterol levels increased in supplemental groups, but tended to decrease in basal groups (Fig. 1). This trend was particularly pronounced in PM, where concentrations decreased from 6.62 to 2.5 mg/g meat during the 3 week storage period. Reductions in COPs levels during storage have been noted previously. For example, Wahle, Hoppe and McIntosh (1993) reported that
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K. Galvin et al. / Meat Science 55 (2000) 7±11
7-ketocholesterol levels decreased in powdered whole egg stored for up to 18 months. Decreases in COPs could be caused by their destruction or by their reaction with other molecules (Rodriguez-Estrada, Penazzi, Caboni, Bertacco & Lercker, 1997). The relatively high levels of 7-ketocholesterol detected at weeks 1 and 2 suggest that cholesterol oxidation in PM proceeded very rapidly prior to week 1, and an increase in concentration may have been observed if measurements had been carried out at an earlier point in the storage study. Despite the decreases observed in some groups, cholesterol oxidation did not appear to be inhibited by vacuum packaging or frozen storage. This agrees with previous studies conducted in vacuum packaged foods. Spanier, Vercellotti and James Jr. (1992) noted that while vacuum packaging reduces the formation of o¯avour compounds derived from fatty acid oxidation in beef, it does not completely inhibit lipid oxidation. Similarly, vacuum packaging has been observed to reduce, but not wholly inhibit, cholesterol oxidation in milk powder (Chan, Gray, Gomaa, Harte, Kelly & Buckey, 1993). In addition, the formation of COPs is not inhibited in raw and cooked minced beef, pork and
mutton during frozen storage (Pie et al., 1991; Kowale et al., 1996). Dietary vitamin E supplementation has been shown to inhibit COPs formation in meat during refrigerated storage (Engeseth et al., 1993; Galvin et al., 1998; Lopez-Bote et al., 1998; Monahan et al., 1992). In the present study, comparison of LD samples showed that there were no signi®cant dierences between basal and supplemental treatments at any point during 3 weeks of refrigerated storage [Fig. 1(a)] and 16 weeks of frozen storage [Fig. 2(a)]. 7-Ketocholesterol concentrations were signi®cantly (p<0.05) lower in supplemental PM than basal meat samples following refrigerated storage for 1 and 2 weeks, but not 3 weeks [Fig. 1(b)]. Supplementation also had no eect on 7-ketocholesterol concentrations in PM at 8 weeks of frozen storage, but signi®cantly (p<0.05) reduced concentrations at 16 weeks [Fig. 2(b)]. These results suggest that vitamin E had a greater protective eect in PM than LD. The extent of lipid oxidation was also monitored by measuring TBARS. In refrigerated (Fig. 3) and frozen (Fig. 4) LD and PM, dietary vitamin E supplementation signi®cantly (p<0.05) reduced TBARS values at all time
Fig. 3. Eect of dietary vitamin E supplementation on TBARS formation in vacuum packaged cooked (a) M. longissimus dorsi and (b) M. psoas major during refrigerated storage. See Fig. 1 for description of symbols. Values are meansSEM of 5 analyses. * Signi®cantly dierent from basal group (p<0.05).
Fig. 4. Eect of dietary vitamin E supplementation on TBARS formation in vacuum packaged cooked (a) M. longissimus dorsi and (b) M. psoas major during frozen storage. See Fig. 1 for description of symbols. Values are meansSEM of 5 analyses. * Signi®cantly different from basal group (p<0.05).
K. Galvin et al. / Meat Science 55 (2000) 7±11
points examined, indicating that vitamin E supplementation did improve the oxidative stability of both LD and PM. Dierences in the inhibitory eect of vitamin E on COPs formation in LD and PM could be due to greater cholesterol stability in LD, as discussed earlier. Previous studies have indicated that dietary vitamin E has little or no eect on cholesterol oxidation when the rate of COPs formation is low (Engeseth et al., 1993). Similarly in the present study, the relatively low level of cholesterol oxidation in LD compared to PM may account for the lack of eect of dietary vitamin E in LD. In conclusion, the results showed that COPs were formed in vacuum packaged cooked beef steaks during refrigerated and frozen storage. Cholesterol oxidation was inhibited by dietary vitamin E supplementation. However, the results suggest that the eects of dietary vitamin E on cholesterol oxidation in beef may be dependent on muscle type. In view of these ®ndings, further examination of cholesterol stability in dierent muscle types and of the antioxidant eect of vitamin E on cholesterol in beef stored under dierent packaging conditions is required. Acknowledgements This research was part funded by grant aid under the Food Sub-Program of the Operational Programme for Industrial Development, which is administered by the Department of Agriculture, Food and Forestry, Dublin, and supported by national and European Union funds. References Akhtar, P., Gray, J. I., Booren, A. M., & Gomaa, E. A. (1998). The eects of dietary a-tocopherol and surface application of oleoresin rosemary on lipid oxidation and cholesterol oxide formation in cooked rainbow trout (Oncorhynchus mykiss) muscle. Journal of Food Lipids, 5, 59±71. Chan, S. H., Gray, J. I., Gomaa, E. A., Harte, B. R., Kelly, P. M., & Buckley, D. J. (1993). Cholesterol oxidation in whole milk powders as in¯uenced by processing and packaging. Food Chemistry, 47, 321±328. Chan, W. K. M., Hakkarainen, K., Faustman, C., Schaefer, D. M., Scheller, K. K., & Liu, Q. (1996). Dietary vitamin E eect on colour stability and sensory assessment of spoilage in three beef muscles. Meat Science, 42, 387±399. De Vore, V. R. (1988). TBA values and 7-ketocholesterol in refrigerated raw and cooked ground beef. Journal of Food Science, 53, 1058±1060. Emanuel, H. A., Hassel, C. A., Addis, P. B., Bergmann, S. D., & Zavoral, J. H. (1991). Plasma cholesterol oxidation products (oxysterols) in human subjects fed a meal rich in oxysterols. Journal of Food Science, 56, 843±847. Engeseth, N. J., Gray, J. I., Booren, A. M., & Asghar, A. (1993). Improved oxidative stability of veal lipids and cholesterol through dietary vitamin E supplementation. Meat Science, 35, 1±15.
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Engeseth, N. J., & Gray, J. I. (1994). Cholesterol oxidation in muscle tissue. Meat Science, 36, 309±320. Galvin, K., Morrissey, P. A., & Buckley, D. J. (1998). Cholesterol oxidation in processed chicken muscle as in¯uenced by dietary atocopherol supplementation. Meat Science, 48, 1±9. Ke, P. J., Ackman, R. G., Linke, B. H., & Nash, D. M. (1977). Differential lipid oxidation in various parts of frozen mackerel. Journal of Food Technology, 12, 37±47. Kerry, J. P., Lynch, M., Buckley, D. J., Morrissey, P. A., Monahan, F. J., & Allen, P. (1996). Eect of dietary vitamin E supplementation on the quality of fresh and vacuum packaged beef. In: Proceedings of the 42nd International Congress of Meat Science and Technology (pp. 96±97), Lillehammer, Norway, MATFORSK, Norwegian Food Research Institute. Kowale, B. N., Rao, V. K., Babu, N. P., Sharma, N., & Bisht, G. S. (1996). Lipid oxidation and cholesterol oxidation in mutton during cooking and storage. Meat Science, 43, 195±202. Kumar, N., & Singhal, O. P. (1991). Cholesterol oxides and atherosclerosis. A review. Journal of the Science of Food and Agriculture, 55, 497±510. Linseisen, J., & Wolfram, G. (1998). Absorption of cholesterol oxidation products from ordinary foodstus. Annals of Nutrition and Metabolism, 42, 221±230. Lopez-Bote, C. J., Gray, J. I., Gomaa, E. A., & Flegal, C. J. (1998). Eect of dietary administration of oil extracts from rosemary and sage on lipid oxidation in broiler meat. British Poultry Science, 39, 235±240. Maerker, G. (1987). Cholesterol autoxidation Ð current status. Journal of the American Oil Chemists' Society, 64, 388±392. Monahan, F. J., Gray, J. I., Booren, A. M., Miller, E. R., Buckley, D. J., Morrissey, P. A., & Gomaa, E. A. (1992). In¯uence of dietary treatment on lipid and cholesterol oxidation in pork. Journal of Agricultural and Food Chemistry, 40, 1310±1315. Morrissey, P. A., Sheehy, P. J. A., Galvin, K., Kerry, J. P., & Buckley, D. J. (1998). Lipid stability in meat and meat products. Meat Science, 49(Suppl. 1), S73±86. O'Neill, L. M., Galvin, K., Morrissey, P. A., & Buckley, D. J. (1999). Eect of carnosine, salt and dietary vitamin E on the oxidative stability of chicken meat. Meat Science, 49, 89±94. Park, S. W., & Addis, P. B. (1987). Cholesterol oxidation products in some muscle foods. Journal of Food Science, 52, 1500±1503. Pie, J. E., Saphis, K., & Seillan, C. (1991). Cholesterol oxidation products in meat products during cooking and frozen storage. Journal of Agricultural and Food Chemistry, 39, 250±254. Rodriguez-Estrada, M. T., Penazzi, G., Caboni, M. F., Bertacco, G., & Lercker, G. (1997). Eect of dierent cooking methods on some lipid and protein components of hamburgers. Meat Science, 45, 365±375. Sheehy, P. J. A., Morrissey, P. A., & Flynn, A. (1994). Consumption of thermally-oxidised sun¯ower oil by chicks reduces a-tocopherol status and increases susceptibility to lipid oxidation. British Journal of Nutrition, 71, 53±65. Smith, L. L. (1996). Review of progress in sterol oxidations: 1987± 1995. Lipids, 31, 453±487. Snedecor, G. W., & Cochran, W. G. (1967). Statistical methods. Ames, Iowa: Iowa State University Press. Spanier, A. M., Vercellotti, J. R., & James Jr., C. (1992). Correlation of sensory, instrumental and chemical attributes of beef as in¯uenced by meat structure and oxygen exclusion. Journal of Food Science, 57, 10±15. Wahle, K. W. J., Hoppe, P. P., & McIntosh, G. (1993). Eects of storage and various intrinsic vitamin E concentrations on lipid oxidation in dried egg powders. Journal of the Science of Food and Agriculture, 61, 463±469. Zubillaga, M. P., & Maerker, G. (1991). Quanti®cation of three cholesterol oxidation products in raw meat and chicken. Journal of Food Science, 56, 1194±1196.
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Eect of dietary vitamin E supplementation on cholesterol oxidation in vacuum packaged cooked beef steaks K. Galvin, A.-M. Lynch, J.P. Kerry*, P.A. Morrissey, D.J. Buckley Department of Food Science and Technology, University College, Cork, Ireland Received 10 June 1999; accepted 3 November 1999
Abstract The eect of dietary vitamin E supplementation on cholesterol oxidation in vacuum packaged, cooked, refrigerated and frozen beef steaks, was investigated. Steers (FriesianCharolaisBlack Hereford) were fed diets providing 20 or 3000 mg a-tocopheryl acetate/head/day for 135 days prior to slaughter. a-Tocopherol concentrations in M. psoas major (PM) and M. longissimus dorsi (LD) were signi®cantly (p<0.05) increased by supplementation and were signi®cantly (p<0.05) higher in PM than LD. Cholesterol oxidation (monitored by measuring 7-ketocholesterol formation) increased during refrigerated and frozen storage in some, but not all, groups, and tended to be higher in PM than LD. Dietary vitamin E did not aect 7-ketocholesterol formation in LD, but signi®cantly (p<0.05) reduced concentrations in PM during refrigerated and frozen storage. Supplementation signi®cantly (p<0.05) reduced TBARS in PM and LD, indicating that vitamin E improved oxidative stability in both muscles. The results show that dietary vitamin E supplementation inhibits cholesterol oxidation in vacuum packaged, cooked beef during refrigerated and frozen storage, but may be in¯uenced by muscle type. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Vitamin E; Cholesterol oxidation; Beef
1. Introduction Lipid (fatty acid) oxidation is an important determinant of the quality of meats and meat products. However, in recent years, the formation of cholesterol oxidation products (COPs) in meats and other cholesterol-containing foods has attracted increasing attention. COPs exert a number of adverse eects in biological tissues and have been implicated in the initiation and progression of a number of diseases, particularly atherosclerosis (Kumar & Singhal, 1991). As COPs are known to be absorbed from the gastrointestinal tract (Emanuel, Hassel, Addis, Bergmann & Zavoral, 1991; Linseisen & Wolfram, 1998), factors determining their formation and distribution in foods are of considerable interest. Cholesterol oxidation is a free radical-mediated process and is promoted by factors which also accelerate fatty acid oxidation ( Kowale, Rao, Babu, Sharma & Bisht, 1996; Park & Addis, 1987; Pie, Saphis & Seillan, * Corresponding author. Tel.: +353-21-902256; fax: +353-21276318. E-mail address: [email protected] (J.P. Kerry).
1991). Hence, attempts to control cholesterol oxidation have focused on factors which are known to inhibit fatty acid oxidation. The antioxidant activity of vitamin E in meats has been widely investigated. Supplementation of animal diets with vitamin E (a-tocopheryl acetate) improves the vitamin E status of muscle, and improves the overall quality of meat products by inhibiting fatty acid oxidation and loss of desirable colour and ¯avour during both refrigerated and frozen storage (Morrissey, Sheehy, Galvin, Kerry & Buckley, 1998). In addition, studies have also shown that dietary vitamin E supplementation inhibits the formation of COPs in ®sh (Akhtar, Gray, Booren & Gomaa, 1998), minced pork (Monahan et al., 1992), veal (Engeseth, Gray, Booren & Asghar, 1993), and chicken (Galvin, Morrissey & Buckley, 1998; Lopez-Bote, Gray, Gomaa & Flegal, 1998; O'Neill, Galvin, Morrissey & Buckley, 1999), and beef round steak (Engeseth & Gray, 1994), during refrigerated storage. However, apart from these studies, little published information is available concerning the eects of dietary vitamin E on cholesterol oxidation in meat in general, and in intact cuts and frozen meat in particular. COPs are found in highest amounts in comminuted meats, while in pre-cooked, intact meats, oxides
0309-1740/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(99)00119-9
8
K. Galvin et al. / Meat Science 55 (2000) 7±11
are present at very low levels or are below detection limits. However, studies have shown that COPs concentrations in intact meat cuts can increase following storage (Engeseth & Gray, 1994). The purpose of this study was to examine the eect of dietary a-tocopherol supplementation on cholesterol oxidation in refrigerated and frozen vacuum packaged, cooked beef steaks.
2.5. Statistical analysis
2. Materials and methods
3. Results and discussion
2.1. Reagents
The a-tocopherol concentrations of raw M. longissimus dorsi (LD) and M. psoas major (PM) are shown in Table 1. a-Tocopherol concentrations were signi®cantly (p<0.05) higher in supplemental groups than in corresponding basal groups. In addition, concentrations were signi®cantly (p<0.05) higher in basal and supplemental PM groups compared to corresponding LD groups. The eects of storage and dietary vitamin E supplementation on cholesterol oxidation in vacuum packaged cooked LD and PM during refrigerated and frozen storage are shown in Figs. 1 and 2. The COPs identi®ed in beef samples were 7-ketocholesterol, 7b-hydroxycholesterol, cholesterol-5a,6a-epoxide, cholesterol-5b,6bepoxide and 20a-hydroxycholesterol. 7-Ketocholesterol was present in highest amounts and was the only oxide consistently identi®ed in all samples. Therefore, it was used to monitor the extent of cholesterol oxidation during refrigerated and frozen storage. The initiation of cholesterol oxidation involves oxidation of the B-ring of the steroid nucleus, with the initial formation of a carbon-centred allylic C-7 radical, and the subsequent formation of epimeric C-7 hydroperoxides (Smith, 1996). The unstable hydroperoxides degrade to form 7a- and 7b-hydroxycholesterol and 7-ketocholesterol, with the latter predominating (Maerker, 1987). Therefore, 7ketocholesterol would be expected to be the dominant oxide formed during cholesterol oxidation in foods. Similar results were found in previous experiments.
All chemicals used were `AnalaR' grade, obtained from British Drug House, Poole, Dorset, England and Rathburn Chemicals, Walkerburn, Scotland. The atocopheryl acetate used in the diets was obtained from Roche Products, Welwyn Garden City, Herts., England. Cholesterol oxide standards were purchased from Sigma Chemical, Tallaght, Dublin, Ireland and Steraloids (UK), New Barnet, Herts., England. 2.2. Animals and diets Crossbreed (FreisianCharolaisBlack Hereford) steers (n=10) were divided into two groups (n=5) and fed silage/concentrate diets providing 20 (basal) or 3000 (supplemental) mg a-tocopheryl acetate/head/day for 135 days prior to slaughter. Feed was oered once a day and water was provided ad libitum. Slaughtering was carried out at a commercial butchers' slaughtering facility. Following slaughter, all carcasses were centrally split and held at 4 C for 10 days prior to muscle sampling. M. longissimus dorsi and M. psoas major muscles were removed and 2 steaks were cut from each muscle. All sub-primals were cut from the same primal positions. Steaks were cooked in a conventional oven (200 C) to an internal temperature of 75 C for 20 min. Following cooling, steaks were vacuum packaged, under full vacuum, in low oxygen permeable (45 cmÿ3 mÿ2 24 hÿ1 at STP) polyamide/polyethylene ®lm. Vacuum packaged steaks were stored in the dark at 4 or ÿ20 C. 2.3. Determination of -tocopherol The a-tocopherol content of raw muscle was determined as described by Sheehy, Morrissey and Flynn (1994). 2.4. Determination of lipid stability Cholesterol oxides were extracted from meat samples and determined by a GC method as described previously (O'Neill et al., 1999). The extent of lipid oxidation was also monitored using the TBA assay (Ke, Ackman, Linke & Nash, 1977).
All data were subjected to analysis of variance (ANOVA). The statistical signi®cance of the dierences between means was determined by the method of least signi®cant dierence (Snedecor & Cochran, 1967) for a probability of p<0.05.
Table 1 -Tocopherol content of M. longissimus dorsi (LD) and M. psoas major (PM) from steers fed a basal (20 mg/head/day) or supplemental (3000 mg/head/day) level of a-tocopheryl acetate for 135 days prior to slaughtera -Tocopherol (mg/g muscle)b,c Muscle
Basal
Supplemental
LD PM
0.840.15 2.450.12a
2.450.41* 5.750.25a*
a
Values are meanSEM of 5 determinations. Within rows, * indicates signi®cantly dierent from basal group (p<0.05). c Within columns, a indicates signi®cantly dierent from LD group (p<0.05). b
K. Galvin et al. / Meat Science 55 (2000) 7±11
9
Fig. 1. Eect of dietary vitamin E supplementation on the formation of 7-ketocholesterol (7-Keto) in vacuum packaged cooked (a) M. longissimus dorsi and (b) M. psoas major during refrigerated storage. &, basal (20 mg/animal/day) a-tocopheryl acetate; &, supplemental (3000 mg/head/day) a-tocopheryl acetate. Values are meansSEM of 5 analyses. * Signi®cantly dierent from basal group (p<0.05).
Fig. 2. Eect of dietary vitamin E supplementation on the formation of 7-ketocholesterol (7-Keto) in vacuum packaged cooked (a) M. longissimus dorsi and (b) M. psoas major during frozen storage. See Fig. 1 for description of symbols. Values are meansSEM of 5 analyses. * Signi®cantly dierent from basal group (p<0.05).
Zubillaga and Maerker (1991) reported that 7-ketocholesterol accounted for over 50% of the total oxides found in raw beef, while Engeseth et al. (1993) showed that 7-ketocholesterol accounted for approximately 45% of total oxides in cooked veal steaks following refrigerated storage for 4 days. 7-Ketocholesterol concentrations were similar to those reported previously in beef. Concentrations ranged from 1.14 to 6.87 mg/g meat compared to 2.11 mg/g in freshly cooked minced beef (Pie et al., 1991), 4.84 mg/ g in refrigerated cooked beef patties (de Vore, 1988) and 4.80 mg/g in refrigerated cooked beef round steak (Engeseth & Gray, 1994). In general, 7-ketocholesterol concentrations tended to be lower in LD than PM. Overall, concentrations were 18±62% lower in basal LD than basal PM during refrigerated storage for up to 2 weeks, while in frozen meat, concentrations in basal LD were 10±26% lower than in basal PM during storage for 8 and 16 weeks. In terms of lipid stability, longissimus dorsi is a more oxidatively stable muscle than psoas, exhibiting comparatively greater colour stability and resistance to lipid oxidation during storage (Chan, Hakkarainen, Faustman, Schaefer, Scheller & Liu, 1996; Kerry, Lynch, Buckley, Morrissey, Monahan &
Allen, 1996). In the present experiment, 7-ketocholesterol concentrations were generally lower in LD than PM, indicating that cholesterol stability is also greater in LD. As it is thought that free radicals derived from fatty acids promote cholesterol oxidation, meat which is more susceptible to fatty acid oxidation would also be expected to be more susceptible to cholesterol oxidation. Similar eects have been observed in chicken, where COPs are generally higher in thigh meat (which is more susceptible to lipid oxidation) than breast meat (Galvin et al., 1998). 7-Ketocholesterol concentrations increased during storage in some, but not in all, groups. In frozen samples 7-ketocholesterol levels in basal LD and PM increased between 8 and 16 weeks, while storage had no eect on 7-ketocholesterol levels in supplemental samples (Fig. 2). During refrigerated storage, 7-ketocholesterol levels increased in supplemental groups, but tended to decrease in basal groups (Fig. 1). This trend was particularly pronounced in PM, where concentrations decreased from 6.62 to 2.5 mg/g meat during the 3 week storage period. Reductions in COPs levels during storage have been noted previously. For example, Wahle, Hoppe and McIntosh (1993) reported that
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7-ketocholesterol levels decreased in powdered whole egg stored for up to 18 months. Decreases in COPs could be caused by their destruction or by their reaction with other molecules (Rodriguez-Estrada, Penazzi, Caboni, Bertacco & Lercker, 1997). The relatively high levels of 7-ketocholesterol detected at weeks 1 and 2 suggest that cholesterol oxidation in PM proceeded very rapidly prior to week 1, and an increase in concentration may have been observed if measurements had been carried out at an earlier point in the storage study. Despite the decreases observed in some groups, cholesterol oxidation did not appear to be inhibited by vacuum packaging or frozen storage. This agrees with previous studies conducted in vacuum packaged foods. Spanier, Vercellotti and James Jr. (1992) noted that while vacuum packaging reduces the formation of o¯avour compounds derived from fatty acid oxidation in beef, it does not completely inhibit lipid oxidation. Similarly, vacuum packaging has been observed to reduce, but not wholly inhibit, cholesterol oxidation in milk powder (Chan, Gray, Gomaa, Harte, Kelly & Buckey, 1993). In addition, the formation of COPs is not inhibited in raw and cooked minced beef, pork and
mutton during frozen storage (Pie et al., 1991; Kowale et al., 1996). Dietary vitamin E supplementation has been shown to inhibit COPs formation in meat during refrigerated storage (Engeseth et al., 1993; Galvin et al., 1998; Lopez-Bote et al., 1998; Monahan et al., 1992). In the present study, comparison of LD samples showed that there were no signi®cant dierences between basal and supplemental treatments at any point during 3 weeks of refrigerated storage [Fig. 1(a)] and 16 weeks of frozen storage [Fig. 2(a)]. 7-Ketocholesterol concentrations were signi®cantly (p<0.05) lower in supplemental PM than basal meat samples following refrigerated storage for 1 and 2 weeks, but not 3 weeks [Fig. 1(b)]. Supplementation also had no eect on 7-ketocholesterol concentrations in PM at 8 weeks of frozen storage, but signi®cantly (p<0.05) reduced concentrations at 16 weeks [Fig. 2(b)]. These results suggest that vitamin E had a greater protective eect in PM than LD. The extent of lipid oxidation was also monitored by measuring TBARS. In refrigerated (Fig. 3) and frozen (Fig. 4) LD and PM, dietary vitamin E supplementation signi®cantly (p<0.05) reduced TBARS values at all time
Fig. 3. Eect of dietary vitamin E supplementation on TBARS formation in vacuum packaged cooked (a) M. longissimus dorsi and (b) M. psoas major during refrigerated storage. See Fig. 1 for description of symbols. Values are meansSEM of 5 analyses. * Signi®cantly dierent from basal group (p<0.05).
Fig. 4. Eect of dietary vitamin E supplementation on TBARS formation in vacuum packaged cooked (a) M. longissimus dorsi and (b) M. psoas major during frozen storage. See Fig. 1 for description of symbols. Values are meansSEM of 5 analyses. * Signi®cantly different from basal group (p<0.05).
K. Galvin et al. / Meat Science 55 (2000) 7±11
points examined, indicating that vitamin E supplementation did improve the oxidative stability of both LD and PM. Dierences in the inhibitory eect of vitamin E on COPs formation in LD and PM could be due to greater cholesterol stability in LD, as discussed earlier. Previous studies have indicated that dietary vitamin E has little or no eect on cholesterol oxidation when the rate of COPs formation is low (Engeseth et al., 1993). Similarly in the present study, the relatively low level of cholesterol oxidation in LD compared to PM may account for the lack of eect of dietary vitamin E in LD. In conclusion, the results showed that COPs were formed in vacuum packaged cooked beef steaks during refrigerated and frozen storage. Cholesterol oxidation was inhibited by dietary vitamin E supplementation. However, the results suggest that the eects of dietary vitamin E on cholesterol oxidation in beef may be dependent on muscle type. In view of these ®ndings, further examination of cholesterol stability in dierent muscle types and of the antioxidant eect of vitamin E on cholesterol in beef stored under dierent packaging conditions is required. Acknowledgements This research was part funded by grant aid under the Food Sub-Program of the Operational Programme for Industrial Development, which is administered by the Department of Agriculture, Food and Forestry, Dublin, and supported by national and European Union funds. References Akhtar, P., Gray, J. I., Booren, A. M., & Gomaa, E. A. (1998). The eects of dietary a-tocopherol and surface application of oleoresin rosemary on lipid oxidation and cholesterol oxide formation in cooked rainbow trout (Oncorhynchus mykiss) muscle. Journal of Food Lipids, 5, 59±71. Chan, S. H., Gray, J. I., Gomaa, E. A., Harte, B. R., Kelly, P. M., & Buckley, D. J. (1993). Cholesterol oxidation in whole milk powders as in¯uenced by processing and packaging. Food Chemistry, 47, 321±328. Chan, W. K. M., Hakkarainen, K., Faustman, C., Schaefer, D. M., Scheller, K. K., & Liu, Q. (1996). Dietary vitamin E eect on colour stability and sensory assessment of spoilage in three beef muscles. Meat Science, 42, 387±399. De Vore, V. R. (1988). TBA values and 7-ketocholesterol in refrigerated raw and cooked ground beef. Journal of Food Science, 53, 1058±1060. Emanuel, H. A., Hassel, C. A., Addis, P. B., Bergmann, S. D., & Zavoral, J. H. (1991). Plasma cholesterol oxidation products (oxysterols) in human subjects fed a meal rich in oxysterols. Journal of Food Science, 56, 843±847. Engeseth, N. J., Gray, J. I., Booren, A. M., & Asghar, A. (1993). Improved oxidative stability of veal lipids and cholesterol through dietary vitamin E supplementation. Meat Science, 35, 1±15.
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