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Use of mixed tocopherols during preparation of samples for analysis of thiobarbituric acid reactive substances
JOURNAL
OF FOOD COMPOSITION
AND ANALYSIS
4, 175-178 (1991)
NOTE Use of Mixed Tocopherols during Preparation of Samples for Analysis of Thiobarbituric Acid Reactive Substances ANNIE J. KING Avian
Sciences
Department,
University
of Cali$ornia,
Davis,
California
95616
Received July 17, 1990, and in revised form November 27, 1990 The efficacy of mixed tocopherols in retarding lipid oxidation during sample preparation for thiobarbituric acid reactive substances (TBRS) was investigated. In uncooked turkey muscle subjected to long-term storage and in cooked turkey muscle subjected to long-term storage prior to thawing and heating, mixed tocopherols (50 pg/mg of fat) were as effective as BHA (125 pg/mg of fat) in reducing TBRS values. Absorption spectra indicated reduction of color complexes at approximately 53 1 nm when tocopherols and BHA were added. The antioxidants were not effective in reducing TBRS values of uncooked turkey muscle subjected to short-term storage or in cooked catfish subjected to short-term storage prior to thawing and heating. o 1991 Academic press, I~C.
INTRODUCTION
The determination of thiobarbituric acid reactive substances (TBRS) by thiobarbituric acid has several limitations. However, the procedure is still widely used to determine the degree of rancidity in muscle foods. Several investigators improved the accuracy of the TBRS analysis by adding antioxidants to prevent lipid oxidation during preparation of samples (Moerck and Ball, 1974; Rhee, 1978; Pikul et al., 1983; Salih et al., 1987). Pikul et al. (1983) recommended the addition of 125 pg of butylated hydroxyanisole (BHA)/mg of fat to prevent autoxidation during heating of samples. Salih et al. ( 1987) added 125 pg of butylated hydroxytoluene (BHT)/mg of fat during development of their modified extraction 2-thiobarbituric acid method. Both groups of investigators reported a reduction in absorbance at 532 nm with samples containing an antioxidant compared to a control. Both BHA and BHT are major synthetic phenolic compounds used as antioxidants in foods. The compounds are highly soluble in oil and are known as lipophilic antioxidants which are effective in intact muscle tissue (Nawar, 1985). The various forms of vitamin E (phenolic compounds called tocopherols and tocotrienols) are also lipid-soluble antioxidants. They are natural compounds having high activity when added at a concentration approximating that found in vegetable oils (Nawar, 1985; Namiki and Osawa, 1986). As scavengers of peroxyl radicals, these compounds prevent the chain propagation step in the lipid oxidation reaction (N&i, 1987). As antioxidants in foods, the four common forms of tocopherols are ranked in the following order: 6 > y > ,0 > a! (Nawar, 1985; Namiki and Osawa, 1986). Manufacturers of mixed tocopherols claim that the tocopherols are particularly effective as antioxidants in processed foods. Reportedly, the improved solubility and heat stability of mixed tocopherols enhance the use of these natural antioxidants compared to the synthetic compounds. Mixed tocopherols are reported to be 100% (w/w) 175
0889-1575/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
176
NOTE
soluble in soybean oil at 25°C compared to 50% solubility of BHA under the same conditions (Anonymous, 1989). The increased solubility and heat stability of mixed tocopherols make them viable substitutes for BHA during preparation of stored muscle for determination of TBRS. It is possible that during sample preparation, the mixed tocopherols at low concentrations may retard lipid oxidation to a greater extent than the synthetic phenolic compounds now in use. An initial study was undertaken to test this hypothesis. BHA and varying amounts of mixed tocopherols were added during sample preparation of stored turkey and fish muscle for analysis of TBRS. MATERIALS
AND
METHODS
Reagents. Mixed isomers of butylated hydroxyanisole (BHA, 2[3]-t-butyl 4-hydroxyanisole) and 1,1,3,3-tetraethoxypropane (TEP) were obtained from Sigma Chemical Co. (St. Louis, MO). 2-Thiobarbituric acid (TBA) and concentrated perchloric acid were obtained from Fisher Scientific Co. (Santa Clara, CA). A mixture of tocopherols ((Y,7.9%; p and y, 49.3%; 6, 15.6%; and vegetable oil constituents, 27.2%) was provided by Henkel Corp. (Kankakee, IL). Muscle. Short-term/frozen, uncooked muscle was from ground turkey thigh without skin stored at -20°C for 20 h. Long-term/frozen, uncooked muscle was from ground turkey thigh without skin stored at -20°C for 3 months. Long-term/frozen, cooked muscle was from ground turkey thigh without skin subjected to 3 months of frozen storage at -20°C prior to thawing and heating. Short-term/frozen, cooked muscle was from fillets of catfish stored for 20 h at -20°C prior to thawing and heating. Turkey
TABLE 1 EFFECTOF ANTIOXIDANTS ON TBRS VALUES OF TURKEY
AND FISH
MUSCLE
TBRS values’ Muscle subjected to long-term storage’ Antioxidants No antioxidant 125 pg BHA/mg of fat Mixed tocopherols, pg/mg of fat 50 75
100 125
Uncooked turkey
Cooked turkey
Muscle subjected to short-term storage’ Uncooked turkey
Cooked catfish
5.3
k 1.8"
17.7
f 3.8"
1.8
f 0.4"
I.0 * 0.2"
2.4
+ 0.4'
14.3
f 3.76
1.8
+ 0.3"
0.9
f 0.1 n.h
2.0 3.2 2.4 2.4
f + f k
14.3
f 4.2b
1.6
f 0.2"
0.9
f 0.1 a.b
o.3b 0.6' 0.3' o.2b
’ Means within columns with different superscripts differ significantly at P < 0.05. ’ Stored at -20°C for 3 months perior to thawing and cooking, followed by TBRS analysis; N = 3 for uncooked turkey, N = 10 for cooked turkey. ’ Stored at -20°C for 20 h prior to thawing and cooking, followed by TBRS analysis; N = 12 for cooked catfish, N = 7 for uncooked turkey.
177
NOTE
300
350
400
450
500
550
600
Wavelength (nm) FIG. I. Absorption spectra for solutions in the analysis of TBRS in cooked turkey muscle (long-term storage) with and without antioxidants.
muscle was heated in a microwave oven to an internal temperature of 77°C. Fillets of catfish were heated in a microwave oven to an internal temperature of 80°C. Fat in all muscle was determined (Method Number 24.005, AOAC, 1984). Analysisof TBRS. Procedures of Salih et al. (1987) were followed with some modifications. Antioxidants were mixed with ethanol so that 1 ml of solution contained 125 pg of BHA/mg of fat or 50, 75, 100, or 125 pg of mixed tocopherols/mg of fat. Muscle (10 g) was homogenized with perchloric acid (3.86%, 35 ml), an antioxidant solution (1 ml), and deionized, distilled water (1 ml) for 1 min. Absorptionspectra.A Shumadzu UV- 160 spectrophotometer (Shimidzu Corporation, Kyoto, Japan) was used to scan (400 to 700 nm) the same solutions prepared for analysis of TBRS in uncooked turkey muscle (short-term storage) and both types of cooked muscle (long-term storage). Statistical analysis.MSTAT subprograms were used to analyze all data (Power, 1983). The Anova subprogram was used to conduct a one-way analysis of variance for TBRS values of uncooked turkey thigh muscle subjected to long- and short-term storage. MSTAT Factor subprogram (randomized complete block design with replications as blocks) was employed to conduct an analysis of variance for TBRS values from duplicate analyses of cooked turkey muscle and triplicate analyses of cooked catfish. Duncan’s multiple range test was used to rank all means at P < 0.05. RESULTS AND
DISCUSSION
Neither BHA nor tocopherols prevented lipid oxidation during preparation of shortterm/frozen, uncooked turkey muscle or short-term/frozen, cooked catfish. During preparation of long-term/frozen, uncooked turkey muscle, 50 pg of tocopherols/mg of fat was as effective as greater amounts of tocopherols and 125 pg of BHA/mg of fat (Table 1). For long-term/frozen, cooked turkey muscle, the lowest level of tocopherols produced results similar to those at 125 pg of BHA (Table 1). The efficacy of 50 pg of tocopherols/mg of fat on extraction of TBRS from turkey muscle subjected to long-term storage seems to be related to the degree of oxidation in the muscle. Turkey muscle, having approximately 100 pg of a-tocopherols/g of extracted fat, is highly susceptible to lipid oxidation during storage (Mecchi et al., 1956). It is likely that more
178
NOTE
peroxyl radicals were produced during the sample preparation of muscle subjected to long-term storage alone or with heating than in muscle frozen for 20 h. Added mixed tocopherols scavenged radicals in the system undergoing the greatest degree of lipid oxidation. Thus, the most useful application of the mixed tocopherols may be in the preparation of samples from highly oxidized food systems. A more extensive study is underway to test this hypothesis and to determine if levels of mixed tocopherols less than 50 pg/mg of fat are effective in reducing lipid oxidation during preparation of turkey muscle for TBRS analysis. Absorption spectra of various solutions showed little yellow pigment (450 nm) formed by TBRS and alkanals, 2-alkenals, or 2,4 dienals in muscle (data not shown). A representative spectra of solutions prepared from cooked turkey muscle is shown in Fig. 1. The results revealed that antioxidants reduced maximum absorption at 530532 nm, usually accepted as the absorbance which is characteristic of the chromagen of TBA-malonaldehyde or TBRS (Gray, 1978; Kagan, 1988). REFERENCES ANONYMOUS (1989). Covi-ox Natural Antioxidants. Henkel Corp., La Grange, IL. AOAC (1984). Ojicial Methods of Analysis (S. Williams, Ed.), 14 ed. Association of Official Analytical Chemists, Washington, DC. GRAY, J. I. (1978). Measurement of lipid oxidation: A review. J. Am. Oil Chem. Sot. 55, 539-546. KAGAN, V. E. (1988). Lipid peroxidation products in viva. In Lipid Peroxidation in Membranes (V. E. Kagan, Ed.), CRC Press, Boca Raton, FL. MECCHI, E. P., POAL, G. L., BEHMAN, G. A., HAMACHI, M., AND KL.OSE,A. A. (1956). The role of tocopherol content in the comparative stability of chicken and turkey fat. Poult. Sci. 35, 1238-1246. MOERCK, K. E., AND BALL, H. R., JR. (1974). Lipid autoxidation in mechanically deboned chicken meat. .J. Food Sci. 39,876-879. NAMIKI, M., AND OSAWA, T. (1986). Antioxidants/antimutagens in foods. Basic Life Sci. 39, 131-142. NAWAR, W. W. (1985). Lipids. In Food Chemistry (0. R. Fennema, Ed.), Marcel Dekker, New York, NY. NIKI, E. (1987). Antioxidants in relation to lipid oxidation. Chem. Phys. Lipids. 44, 227-253. PIKUL, J., LESZCZYNSKI, D. E., AND KUMNEROW, F. A. (1983). Elimination of sample autoxidation by butylated hydroxytoluene additions before thiobarbituric acid assayfor malonaldehyde in fat from chicken meat. J. Agric. Food Chem. 31, 1338- 1342. POWER, P. (1983). User’s Guide to Mstat, Version 3.0. Michigan State University, East Lansing, MI. RHEE, K. S. (1978). Minimization of further lipid peroxidation in the distillation 2-thiobarbituric acid test of fish and meat. J. Food Sci. 43, 1776-1778. SALIH, A. M., SMITH. D. M., PRICE, J. F., AND DAWSON, L. E. (1987). Modified extraction 2-thiobarbituric acid method for measuring lipid oxidation in poultry. Poult. Sci. 66, 1483-1488.
OF FOOD COMPOSITION
AND ANALYSIS
4, 175-178 (1991)
NOTE Use of Mixed Tocopherols during Preparation of Samples for Analysis of Thiobarbituric Acid Reactive Substances ANNIE J. KING Avian
Sciences
Department,
University
of Cali$ornia,
Davis,
California
95616
Received July 17, 1990, and in revised form November 27, 1990 The efficacy of mixed tocopherols in retarding lipid oxidation during sample preparation for thiobarbituric acid reactive substances (TBRS) was investigated. In uncooked turkey muscle subjected to long-term storage and in cooked turkey muscle subjected to long-term storage prior to thawing and heating, mixed tocopherols (50 pg/mg of fat) were as effective as BHA (125 pg/mg of fat) in reducing TBRS values. Absorption spectra indicated reduction of color complexes at approximately 53 1 nm when tocopherols and BHA were added. The antioxidants were not effective in reducing TBRS values of uncooked turkey muscle subjected to short-term storage or in cooked catfish subjected to short-term storage prior to thawing and heating. o 1991 Academic press, I~C.
INTRODUCTION
The determination of thiobarbituric acid reactive substances (TBRS) by thiobarbituric acid has several limitations. However, the procedure is still widely used to determine the degree of rancidity in muscle foods. Several investigators improved the accuracy of the TBRS analysis by adding antioxidants to prevent lipid oxidation during preparation of samples (Moerck and Ball, 1974; Rhee, 1978; Pikul et al., 1983; Salih et al., 1987). Pikul et al. (1983) recommended the addition of 125 pg of butylated hydroxyanisole (BHA)/mg of fat to prevent autoxidation during heating of samples. Salih et al. ( 1987) added 125 pg of butylated hydroxytoluene (BHT)/mg of fat during development of their modified extraction 2-thiobarbituric acid method. Both groups of investigators reported a reduction in absorbance at 532 nm with samples containing an antioxidant compared to a control. Both BHA and BHT are major synthetic phenolic compounds used as antioxidants in foods. The compounds are highly soluble in oil and are known as lipophilic antioxidants which are effective in intact muscle tissue (Nawar, 1985). The various forms of vitamin E (phenolic compounds called tocopherols and tocotrienols) are also lipid-soluble antioxidants. They are natural compounds having high activity when added at a concentration approximating that found in vegetable oils (Nawar, 1985; Namiki and Osawa, 1986). As scavengers of peroxyl radicals, these compounds prevent the chain propagation step in the lipid oxidation reaction (N&i, 1987). As antioxidants in foods, the four common forms of tocopherols are ranked in the following order: 6 > y > ,0 > a! (Nawar, 1985; Namiki and Osawa, 1986). Manufacturers of mixed tocopherols claim that the tocopherols are particularly effective as antioxidants in processed foods. Reportedly, the improved solubility and heat stability of mixed tocopherols enhance the use of these natural antioxidants compared to the synthetic compounds. Mixed tocopherols are reported to be 100% (w/w) 175
0889-1575/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
176
NOTE
soluble in soybean oil at 25°C compared to 50% solubility of BHA under the same conditions (Anonymous, 1989). The increased solubility and heat stability of mixed tocopherols make them viable substitutes for BHA during preparation of stored muscle for determination of TBRS. It is possible that during sample preparation, the mixed tocopherols at low concentrations may retard lipid oxidation to a greater extent than the synthetic phenolic compounds now in use. An initial study was undertaken to test this hypothesis. BHA and varying amounts of mixed tocopherols were added during sample preparation of stored turkey and fish muscle for analysis of TBRS. MATERIALS
AND
METHODS
Reagents. Mixed isomers of butylated hydroxyanisole (BHA, 2[3]-t-butyl 4-hydroxyanisole) and 1,1,3,3-tetraethoxypropane (TEP) were obtained from Sigma Chemical Co. (St. Louis, MO). 2-Thiobarbituric acid (TBA) and concentrated perchloric acid were obtained from Fisher Scientific Co. (Santa Clara, CA). A mixture of tocopherols ((Y,7.9%; p and y, 49.3%; 6, 15.6%; and vegetable oil constituents, 27.2%) was provided by Henkel Corp. (Kankakee, IL). Muscle. Short-term/frozen, uncooked muscle was from ground turkey thigh without skin stored at -20°C for 20 h. Long-term/frozen, uncooked muscle was from ground turkey thigh without skin stored at -20°C for 3 months. Long-term/frozen, cooked muscle was from ground turkey thigh without skin subjected to 3 months of frozen storage at -20°C prior to thawing and heating. Short-term/frozen, cooked muscle was from fillets of catfish stored for 20 h at -20°C prior to thawing and heating. Turkey
TABLE 1 EFFECTOF ANTIOXIDANTS ON TBRS VALUES OF TURKEY
AND FISH
MUSCLE
TBRS values’ Muscle subjected to long-term storage’ Antioxidants No antioxidant 125 pg BHA/mg of fat Mixed tocopherols, pg/mg of fat 50 75
100 125
Uncooked turkey
Cooked turkey
Muscle subjected to short-term storage’ Uncooked turkey
Cooked catfish
5.3
k 1.8"
17.7
f 3.8"
1.8
f 0.4"
I.0 * 0.2"
2.4
+ 0.4'
14.3
f 3.76
1.8
+ 0.3"
0.9
f 0.1 n.h
2.0 3.2 2.4 2.4
f + f k
14.3
f 4.2b
1.6
f 0.2"
0.9
f 0.1 a.b
o.3b 0.6' 0.3' o.2b
’ Means within columns with different superscripts differ significantly at P < 0.05. ’ Stored at -20°C for 3 months perior to thawing and cooking, followed by TBRS analysis; N = 3 for uncooked turkey, N = 10 for cooked turkey. ’ Stored at -20°C for 20 h prior to thawing and cooking, followed by TBRS analysis; N = 12 for cooked catfish, N = 7 for uncooked turkey.
177
NOTE
300
350
400
450
500
550
600
Wavelength (nm) FIG. I. Absorption spectra for solutions in the analysis of TBRS in cooked turkey muscle (long-term storage) with and without antioxidants.
muscle was heated in a microwave oven to an internal temperature of 77°C. Fillets of catfish were heated in a microwave oven to an internal temperature of 80°C. Fat in all muscle was determined (Method Number 24.005, AOAC, 1984). Analysisof TBRS. Procedures of Salih et al. (1987) were followed with some modifications. Antioxidants were mixed with ethanol so that 1 ml of solution contained 125 pg of BHA/mg of fat or 50, 75, 100, or 125 pg of mixed tocopherols/mg of fat. Muscle (10 g) was homogenized with perchloric acid (3.86%, 35 ml), an antioxidant solution (1 ml), and deionized, distilled water (1 ml) for 1 min. Absorptionspectra.A Shumadzu UV- 160 spectrophotometer (Shimidzu Corporation, Kyoto, Japan) was used to scan (400 to 700 nm) the same solutions prepared for analysis of TBRS in uncooked turkey muscle (short-term storage) and both types of cooked muscle (long-term storage). Statistical analysis.MSTAT subprograms were used to analyze all data (Power, 1983). The Anova subprogram was used to conduct a one-way analysis of variance for TBRS values of uncooked turkey thigh muscle subjected to long- and short-term storage. MSTAT Factor subprogram (randomized complete block design with replications as blocks) was employed to conduct an analysis of variance for TBRS values from duplicate analyses of cooked turkey muscle and triplicate analyses of cooked catfish. Duncan’s multiple range test was used to rank all means at P < 0.05. RESULTS AND
DISCUSSION
Neither BHA nor tocopherols prevented lipid oxidation during preparation of shortterm/frozen, uncooked turkey muscle or short-term/frozen, cooked catfish. During preparation of long-term/frozen, uncooked turkey muscle, 50 pg of tocopherols/mg of fat was as effective as greater amounts of tocopherols and 125 pg of BHA/mg of fat (Table 1). For long-term/frozen, cooked turkey muscle, the lowest level of tocopherols produced results similar to those at 125 pg of BHA (Table 1). The efficacy of 50 pg of tocopherols/mg of fat on extraction of TBRS from turkey muscle subjected to long-term storage seems to be related to the degree of oxidation in the muscle. Turkey muscle, having approximately 100 pg of a-tocopherols/g of extracted fat, is highly susceptible to lipid oxidation during storage (Mecchi et al., 1956). It is likely that more
178
NOTE
peroxyl radicals were produced during the sample preparation of muscle subjected to long-term storage alone or with heating than in muscle frozen for 20 h. Added mixed tocopherols scavenged radicals in the system undergoing the greatest degree of lipid oxidation. Thus, the most useful application of the mixed tocopherols may be in the preparation of samples from highly oxidized food systems. A more extensive study is underway to test this hypothesis and to determine if levels of mixed tocopherols less than 50 pg/mg of fat are effective in reducing lipid oxidation during preparation of turkey muscle for TBRS analysis. Absorption spectra of various solutions showed little yellow pigment (450 nm) formed by TBRS and alkanals, 2-alkenals, or 2,4 dienals in muscle (data not shown). A representative spectra of solutions prepared from cooked turkey muscle is shown in Fig. 1. The results revealed that antioxidants reduced maximum absorption at 530532 nm, usually accepted as the absorbance which is characteristic of the chromagen of TBA-malonaldehyde or TBRS (Gray, 1978; Kagan, 1988). REFERENCES ANONYMOUS (1989). Covi-ox Natural Antioxidants. Henkel Corp., La Grange, IL. AOAC (1984). Ojicial Methods of Analysis (S. Williams, Ed.), 14 ed. Association of Official Analytical Chemists, Washington, DC. GRAY, J. I. (1978). Measurement of lipid oxidation: A review. J. Am. Oil Chem. Sot. 55, 539-546. KAGAN, V. E. (1988). Lipid peroxidation products in viva. In Lipid Peroxidation in Membranes (V. E. Kagan, Ed.), CRC Press, Boca Raton, FL. MECCHI, E. P., POAL, G. L., BEHMAN, G. A., HAMACHI, M., AND KL.OSE,A. A. (1956). The role of tocopherol content in the comparative stability of chicken and turkey fat. Poult. Sci. 35, 1238-1246. MOERCK, K. E., AND BALL, H. R., JR. (1974). Lipid autoxidation in mechanically deboned chicken meat. .J. Food Sci. 39,876-879. NAMIKI, M., AND OSAWA, T. (1986). Antioxidants/antimutagens in foods. Basic Life Sci. 39, 131-142. NAWAR, W. W. (1985). Lipids. In Food Chemistry (0. R. Fennema, Ed.), Marcel Dekker, New York, NY. NIKI, E. (1987). Antioxidants in relation to lipid oxidation. Chem. Phys. Lipids. 44, 227-253. PIKUL, J., LESZCZYNSKI, D. E., AND KUMNEROW, F. A. (1983). Elimination of sample autoxidation by butylated hydroxytoluene additions before thiobarbituric acid assayfor malonaldehyde in fat from chicken meat. J. Agric. Food Chem. 31, 1338- 1342. POWER, P. (1983). User’s Guide to Mstat, Version 3.0. Michigan State University, East Lansing, MI. RHEE, K. S. (1978). Minimization of further lipid peroxidation in the distillation 2-thiobarbituric acid test of fish and meat. J. Food Sci. 43, 1776-1778. SALIH, A. M., SMITH. D. M., PRICE, J. F., AND DAWSON, L. E. (1987). Modified extraction 2-thiobarbituric acid method for measuring lipid oxidation in poultry. Poult. Sci. 66, 1483-1488.