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Interference of sodium caseinate in the TBARS assay
Food Chemistry 124 (2011) 1284–1287
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Interference of sodium caseinate in the TBARS assay I. Caprioli, M. O’Sullivan, F.J. Monahan * UCD School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Dublin 4, Ireland
a r t i c l e
i n f o
Article history: Received 22 July 2009 Received in revised form 23 June 2010 Accepted 9 July 2010
Keywords: TBARS Sodium caseinate Meat Interference
a b s t r a c t One of the most common methods of measuring lipid oxidation in foods is the quantification of malonaldehyde by reaction with 2-thiobarbituric acid (TBA) in the so called TBA-reactive substances (TBARS) assay. Different variants of the TBARS assay include those in which the food sample reacts directly with the TBA reagent and those that involve collection of a distillate from the food before reaction with TBA. Interference of sodium caseinate (NaCas) in a TBARS assay involving direct reaction of TBA with a food sample was observed while exploring the possible retardation of lipid oxidation in sliced turkey meat coated with a NaCas-containing edible film. This short report shows the extent of interference encountered and illustrates the importance of validating the TBA method for particular applications. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction The most widely used methods for estimating lipid oxidation in meat involve determination of peroxide values, hexanal or malonaldehyde (MDA) (Fernandez, Perez-Halvarez, & Fernandez-Lopez, 1997). In the 2-thiobarbituric acid (TBA) assay MDA, a secondary decomposition product of polyunsaturated fatty acids with three or more double bonds, reacts with TBA to form a stable pink chromophore (Dahle, Hill, & Holman, 1962). The term TBA-reactive substances (TBARS) is more commonly used to describe the assay because the term takes into account the contribution of compounds other than MDA, such as 2,4-heptadienal, t-2-heptenal, t2-hexenal and hexanal, to chromophore formation following reaction with TBA (Sun, Faustman, Senecal, Wilkinson, & Furr, 2001). Since the discovery of the reaction between MDA and TBA and the relationship between MDA level and lipid oxidation (Bernheim, Bernheim, & Wilbur, 1948; Tarladgis & Watts, 1960; Yu & Sinnhuber, 1957) many different methods for measuring MDA have been reported in the literature. However, interference can occur in some of the assays due to constituents in food matrices (Fernandez et al., 1997). Nevertheless TBARS methods remain a useful measure of lipid oxidation in foods, especially when used in combination with other methods such as hexanal analysis (Caprioli, O’Sullivan, & Monahan, 2009). In the present study, preliminary tests were conducted prior to a larger scale shelf-life experiment (Caprioli, O’Sullivan, & Monahan, 2009) to examine the impact of sodium caseinate (NaCas) on the TBARS assay. Two different methods, a ‘‘direct method” of
* Corresponding author. Tel.: +353 1 7167090; fax: +353 1 7161147. E-mail address: [email protected] (F.J. Monahan). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.07.022
Buege and Aust (1970) and a ‘‘distillation method” of Tarladgis and Watts (1960), were used to observe possible interference of NaCas in the TBARS assay, using oxidised turkey meat as reference food matrix and tetramethoxypropane (TMP) as a MDA standard.
2. Material and methods 2.1. Chemical and reagents Sodium caseinate was obtained from DVM International (the Netherlands). The protein content of the NaCas was 88 g/100 g as certified by the supplier. Reagent grade glycerol, acetic acid, hydrochloric acid, TBA and TMP were supplied by Sigma–Aldrich Chemicals (Dublin, Ireland). Trichloroacetic acid (TCA) was supplied by Fluka through Lennox Chemicals Ltd. (Dublin, Ireland). Screw cap plastic tubes (50 mL) and screw cap Pyrex tubes (10 mL) were supplied by Sarstedt Ltd. (Wexford, Ireland).
2.2. Preparation and wrapping turkey breast meat slices Turkey breast meat slices were prepared and wrapped in edible films manufactured from NaCas as described in Caprioli et al. (2009). Briefly, entire turkey breasts were cooked in aluminium foil to an internal temperature of 74 °C in a domestic oven set at 190 °C (4 h). The cooked meat was vacuum packed and cooled to 4 °C by placing in a freezer for 2 h and then in a refrigerator for a further 2 h. The cooked meat was sliced into 5 mm thick slices and stored for 3 days at 4 °C with or without a NaCas film wrapping. The NaCas films were prepared by air drying an aqueous solution of NaCas (5% w/w containing glycerol at the glycerol/protein ratio of 0.32).
I. Caprioli et al. / Food Chemistry 124 (2011) 1284–1287
2.3. TBARS analysis 2.3.1. TBARS analysis by the direct method (Buege & Aust, 1970) The TBA reagent (0.026 M TBA, 0.92 M TCA) was prepared by dissolving the TBA and the TCA in 250 mL distilled water containing 62.5 mL 1 M HCl. Cooked turkey meat samples (3 g) were weighed into 50 mL screw cap plastic tube and blended with 15 mL distilled water using an Ultra Turrax T25 homogeniser (9000 rpm, 1 min). An aliquot of the homogenate (3 mL) was then added to 3 mL of the TBA reagent in a Pyrex screw cap test tube and vortexed thoroughly for 1 min. The tube contents were heated at 100 °C for 15 min in a water bath and then allowed to cool for 10 min. The flocculent precipitate was removed by centrifugation (3000 rpm, 15 min) and the absorbance of the supernatant was measured at 535 nm using a UV–Vis Spectrophotometer (Shimadzu UV-1201) against a blank containing TBA reagent with distilled water in place of sample supernatant. Absorbance values were used directly as a unit of comparison between different samples.
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weighed into 50 mL screw cap plastic tubes and blended in 25 g of distilled water using an Ultra Turrax T25 homogeniser (9000 rpm, 1 min). The homogenate was quantitatively transferred into a 500 mL Kjeldahl flask by washing with an additional 65 mL distilled water. HCl (10 mL, 1 M) was added to bring the pH to 1.5 and followed by a small amount (2 drops) of silicon antifoam. Each flask was connected to a distillation apparatus and heated with a Bunsen burner under such conditions as to collect 50 mL of distillate within 10–12 min of the initiation of boiling. A 3 mL aliquot of distillate was pipetted into a 10 mL screw capped, Teflon sealed glass tube with 3 mL TBA reagent. The tube was stoppered, vortexed and immersed in a boiling water bath for 35 min. After heating, tubes were allowed to cool and the absorbance was read using a UV–Vis Spectrophotometer (Shimadzu UV-1201) at 538 nm against a blank containing the TBA reagent with distilled water in place of the sample distillate. Absorbance values were used directly as a unit of comparison between different samples. 2.4. Statistical analysis
2.3.2. TBARS analysis by the distillation method (Tarladgis & Watts, 1960) The TBA reagent (0.02 M TBA in 90% acetic acid) was prepared by dissolving TBA in acetic acid, with gentle warming in a hot water bath and making up to volume with distilled water. On the day of analysis turkey meat was finely diced and 3 g samples were
For each of the four experiments represented by Figs. 1A, B, 2A and B a different turkey breast was used. Within each experiment each treatment was replicated three times using three different cooked turkey slices. For experiments with the MDA standard (represented by Figs. 3A and B), three different MDA solutions were
Fig. 1. Lipid oxidation (TBARS) measured by the direct method (A) and the distillation method (B) in cooked turkey meat slices after 3 days storage at 4 °C. Samples were either unwrapped or wrapped in a NaCas film (first and third bars, respectively) or unwrapped, with a portion of NaCas film added just prior to analysis of lipid oxidation (second bar). Each bar represents the mean (SD) of three replications. Bars with different letters are significantly different (P < 0.05).
Fig. 2. Effect of NaCas solution, added to the homogenised meat just prior to analysis, on lipid oxidation (TBARS) measured by the direct method (A) and the distillation method (B). Each bar represents the mean (SD) of three replications. Bars with different letters are significantly different (P < 0.05).
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I. Caprioli et al. / Food Chemistry 124 (2011) 1284–1287
Fig. 3. Effect of NaCas solution, added to a standard TMP solution just prior to analysis, on lipid oxidation (TBARS) measured by the direct method (A) and the distillation method (B). Each bar represents the mean (SD) of three replications. Bars with different letters are significantly different (P < 0.05).
prepared in order to have three replicates for each NaCas treatment. Data were evaluated using Minitab v14. Analysis of variance (ANOVA) was performed on the data and Tukey’s test (a = 0.05) was used to identify significant differences between samples.
3. Results and discussion The experiment involved the storage of 15 g slices of cooked turkey meat for 3 days at 4 °C. The objective was to determine whether wrapping the slices in a NaCas film had an antioxidant effect on them. After 3 days of storage a comparison of unwrapped with wrapped turkey slices showed a decrease in TBARS (Fig. 1A). The unwrapped sample stored for 3 days had a mean absorbance value of 0.90 whereas that of the sample wrapped in NaCas film had a lower absorbance value (0.45). However, the possibility that the antioxidant effect could partly or fully be caused by interference of NaCas in the TBARS assay was raised. Thus, an amount of NaCas film, equivalent to that which would be attached to a film-wrapped turkey slice during storage (approximately 26 mg/g turkey meat), was added directly to a sample of unwrapped turkey at the end of the 3 days storage and lipid oxidation was assessed as for the wrapped and unwrapped stored samples (Fig. 1A). An absorbance value of 0.67 was recorded for the turkey meat to which the NaCas was added directly after storage suggesting that about 50% of the antioxidant effect observed when turkey samples were wrapped with NaCas film could be attributed to the NaCas itself and not to prevention of lipid oxidation in the turkey slices during storage. These results suggest that NaCas interferes in the TBA–MDA reaction when the direct method is used, and possibly also in other methods that involve direct contact between the TBA and the food samples being assessed. Even with filtration methods, which involve filtering the food slurry before reaction with TBA, the possibility of interference exists because filtrates are likely to contain a complex mixture of components capable of interfering in the TBA–MDA reaction (Fernandez et al., 1997). In contrast to the direct method, no interference due to NaCas was observed when the distillation method was used to assess lipid oxidation (Fig. 1B) and a reduction in the absorbance value from 0.76 to 0.45 could be attributed to an antioxidant effect of the film on the formation of lipid oxidation products. The extent of potential interference from NaCas was demonstrated by using the direct method to assess lipid oxidation in turkey meat slices to which an increasing amount of NaCas was added following storage for 1 day at 4 °C (Fig. 2A). The experiment dem-
onstrated how NaCas, in the range 2.3–11 mg/g meat, can decrease the absorbance of a sample by up to half its value, using the direct method. In marked contrast, when assessed by the distillation method the TBARS values of the turkey meat were unaffected by the addition of NaCas (Fig. 2B) suggesting no interference in the reaction between TBA and MDA or similar TBA-reactive compounds. In fact increasing the concentration of NaCas up to 120 mg/g of meat resulted in no reduction in TBARS (data not shown). In the case of the direct method, when TMP, which functions as an MDA standard, was used in place of turkey meat it was also observed that the addition of NaCas reduced absorbance values (Fig. 3A); however, when the distillation method was used no such reduction was observed (Fig. 3B), an outcome similar to that depicted in Fig. 2B. The direct method has the advantage of being straightforward and easy to perform, requiring four steps: homogenisation, reaction, centrifugation and spectrophotometric reading. In contrast, the distillation method is more time consuming, requiring the use of a distillation apparatus initially to generate a distillate, which is subsequently reacted with TBA reagent. Moreover, the collection of a specific volume of distillate is somewhat empirical (Sinnhuber & Yu, 1977). The experimental conditions may also promote the formation of secondary oxidation products, thus increasing the final TBARS values (Pikul, Leszcynski, & Kummerow, 1983), although observed increases in TBARS on distillation have also been attributed to the liberation of MDA precursors bound to proteins (Rhee, 1978). Although direct methods have been used in the past to assess lipid oxidation in casein-containing systems (Cervato, Cazzola, & Cestaro, 1999) and in meat products containing non-meat ingredients including antioxidants (Kang et al., 2007) interference can occur during the analysis, as reported by Fernandez et al. (1997). Malonaldehyde and other TBA-reactive substances can react with amino acids, proteins and others food constituents, and even with water when specific conditions are present (Kwon, Menzel, & Olcott, 1965). As an example, t-2-hexenal and hexanal, which were found by Sun et al. (2001) to interact with TBA, were also found by Meynier, Rampon, Dalgalarrondo, and Genot (2004) to bind covalently with NaCas and whey proteins. In such situations where MDA is bound in the product matrix it may be unavailable for reaction with TBA, thus giving erroneously low TBARS absorbance values. Any one or a combination the above may account for the reduced absorbance values obtained when the direct method was used to assess lipid oxidation in the samples containing added NaCas.
I. Caprioli et al. / Food Chemistry 124 (2011) 1284–1287
4. Conclusions The results of this short study suggest that the presence of NaCas in food samples, and by extension perhaps other soluble proteins, can interfere with the determination of lipid oxidation by direct contact TBARS methods. In contrast distillation methods seem to be free from such interference. Thus it would seem wise for researchers to choose the method of TBARS determination carefully, having regard to the particular composition of their sample matrix. It would also be advisable to interpret the output of the TBARS method in conjunction with the results from other measurements of lipid oxidation, chosen, when possible, to evaluate both primary (such as hydroperoxides) and secondary (such as hexanal) products of lipid oxidation. Acknowledgements The technical assistance of Mr. Eamon Power and Mr. Michael Cooney is greatly appreciated. The financial support of Irish Department of Agriculture, Fisheries and Food under the Food Institutional Research Measure of the 2000–2006 National Development Plan is gratefully acknowledged. References Bernheim, F. M., Bernheim, L. C., & Wilbur, K. M. (1948). The reaction between thiobarbituric acid and the oxidation products of certain lipids. Journal of Biological Chemistry, 174, 257–264. Buege, J. A., & Aust, S. D. (1970). Microsomal lipid peroxidation. Methods in Enzymology, 52, 302–310.
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Caprioli, I., O’Sullivan, M., & Monahan, F. J. (2009). Use of sodium caseinate/glycerol edible films to reduce lipid oxidation in sliced turkey meat. European Food Research and Technology A, 228, 433–440. Cervato, G., Cazzola, R., & Cestaro, B. (1999). Studies on the antioxidant activity of milk caseins. International Journal of Food Sciences and Nutrition, 50, 291–296. Dahle, L. K., Hill, E. G., & Holman, R. T. (1962). The thiobarbituric acid reaction and the autoxidations of polyunsaturated fatty acid methyl esters. Archives of Biochemistry and Biophysics, 98, 253–261. Fernandez, J., Perez-Halvarez, J. A., & Fernandez-Lopez, J. A. (1997). Thiobarbituric acid test for monitoring lipid oxidation in meat. Food Chemistry, 59, 345–353. Kang, H. J., Jo, C., Kwon, J. H., Kim, J. H., Chung, H. J., & Byun, M. W. (2007). Effect of a pectin-based edible coating containing green tea powder on the quality of irradiated pork patty. Food Control, 18, 430–435. Kwon, T. W., Menzel, D. B., & Olcott, H. S. (1965). Reactivity of malonaldehyde with food constituents. Journal of Food Science, 30, 808–813. Meynier, A., Rampon, V., Dalgalarrondo, M., & Genot, C. (2004). Hexanal and t-2hexenal form covalent bonds with whey proteins and sodium caseinate in aqueous solution. International Dairy Journal, 14, 681–690. Pikul, J., Leszcynski, D. E., & Kummerow, F. A. (1983). Elimination of sample autooxidation by butylated hydroxytoluene additions before thiobarbituric acid assay for malonaldehyde in fat from chicken meat. Journal of Agriculture and Food Chemistry, 31, 1338–1342. Rhee, K. S. (1978). Minimisation of further lipid peroxidation in the distillation 2thiobarbituric acid test of fish and meat. Journal of Food Science, 43, 1776–1778. Sinnhuber, R. O., & Yu, T. C. (1977). The 2-thiobarbituric acid reaction, an objective measure of the oxidative deterioration occurring in fats and oils. Journal Japan Oil Chemistry Society, 26, 259–267. Sun, Q., Faustman, C., Senecal, A., Wilkinson, A. L., & Furr, H. (2001). Aldehyde reactivity with 2-thiobarbituric acid and TBARS in freeze-dried beef during accelerated storage. Meat Science, 57, 55–60. Tarladgis, B. G., & Watts, B. M. (1960). Malonaldehyde production during controlled oxidation of pure unsaturated fatty acids. Journal of the American Oil Chemists’ Society, 37, 403–405. Yu, T. C., & Sinnhuber, R. O. (1957). 2-Thiobarbituric acid method for the measurement of rancidity in fishery products. Food Technology, 11, 104–108.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Short communication
Interference of sodium caseinate in the TBARS assay I. Caprioli, M. O’Sullivan, F.J. Monahan * UCD School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Dublin 4, Ireland
a r t i c l e
i n f o
Article history: Received 22 July 2009 Received in revised form 23 June 2010 Accepted 9 July 2010
Keywords: TBARS Sodium caseinate Meat Interference
a b s t r a c t One of the most common methods of measuring lipid oxidation in foods is the quantification of malonaldehyde by reaction with 2-thiobarbituric acid (TBA) in the so called TBA-reactive substances (TBARS) assay. Different variants of the TBARS assay include those in which the food sample reacts directly with the TBA reagent and those that involve collection of a distillate from the food before reaction with TBA. Interference of sodium caseinate (NaCas) in a TBARS assay involving direct reaction of TBA with a food sample was observed while exploring the possible retardation of lipid oxidation in sliced turkey meat coated with a NaCas-containing edible film. This short report shows the extent of interference encountered and illustrates the importance of validating the TBA method for particular applications. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction The most widely used methods for estimating lipid oxidation in meat involve determination of peroxide values, hexanal or malonaldehyde (MDA) (Fernandez, Perez-Halvarez, & Fernandez-Lopez, 1997). In the 2-thiobarbituric acid (TBA) assay MDA, a secondary decomposition product of polyunsaturated fatty acids with three or more double bonds, reacts with TBA to form a stable pink chromophore (Dahle, Hill, & Holman, 1962). The term TBA-reactive substances (TBARS) is more commonly used to describe the assay because the term takes into account the contribution of compounds other than MDA, such as 2,4-heptadienal, t-2-heptenal, t2-hexenal and hexanal, to chromophore formation following reaction with TBA (Sun, Faustman, Senecal, Wilkinson, & Furr, 2001). Since the discovery of the reaction between MDA and TBA and the relationship between MDA level and lipid oxidation (Bernheim, Bernheim, & Wilbur, 1948; Tarladgis & Watts, 1960; Yu & Sinnhuber, 1957) many different methods for measuring MDA have been reported in the literature. However, interference can occur in some of the assays due to constituents in food matrices (Fernandez et al., 1997). Nevertheless TBARS methods remain a useful measure of lipid oxidation in foods, especially when used in combination with other methods such as hexanal analysis (Caprioli, O’Sullivan, & Monahan, 2009). In the present study, preliminary tests were conducted prior to a larger scale shelf-life experiment (Caprioli, O’Sullivan, & Monahan, 2009) to examine the impact of sodium caseinate (NaCas) on the TBARS assay. Two different methods, a ‘‘direct method” of
* Corresponding author. Tel.: +353 1 7167090; fax: +353 1 7161147. E-mail address: [email protected] (F.J. Monahan). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.07.022
Buege and Aust (1970) and a ‘‘distillation method” of Tarladgis and Watts (1960), were used to observe possible interference of NaCas in the TBARS assay, using oxidised turkey meat as reference food matrix and tetramethoxypropane (TMP) as a MDA standard.
2. Material and methods 2.1. Chemical and reagents Sodium caseinate was obtained from DVM International (the Netherlands). The protein content of the NaCas was 88 g/100 g as certified by the supplier. Reagent grade glycerol, acetic acid, hydrochloric acid, TBA and TMP were supplied by Sigma–Aldrich Chemicals (Dublin, Ireland). Trichloroacetic acid (TCA) was supplied by Fluka through Lennox Chemicals Ltd. (Dublin, Ireland). Screw cap plastic tubes (50 mL) and screw cap Pyrex tubes (10 mL) were supplied by Sarstedt Ltd. (Wexford, Ireland).
2.2. Preparation and wrapping turkey breast meat slices Turkey breast meat slices were prepared and wrapped in edible films manufactured from NaCas as described in Caprioli et al. (2009). Briefly, entire turkey breasts were cooked in aluminium foil to an internal temperature of 74 °C in a domestic oven set at 190 °C (4 h). The cooked meat was vacuum packed and cooled to 4 °C by placing in a freezer for 2 h and then in a refrigerator for a further 2 h. The cooked meat was sliced into 5 mm thick slices and stored for 3 days at 4 °C with or without a NaCas film wrapping. The NaCas films were prepared by air drying an aqueous solution of NaCas (5% w/w containing glycerol at the glycerol/protein ratio of 0.32).
I. Caprioli et al. / Food Chemistry 124 (2011) 1284–1287
2.3. TBARS analysis 2.3.1. TBARS analysis by the direct method (Buege & Aust, 1970) The TBA reagent (0.026 M TBA, 0.92 M TCA) was prepared by dissolving the TBA and the TCA in 250 mL distilled water containing 62.5 mL 1 M HCl. Cooked turkey meat samples (3 g) were weighed into 50 mL screw cap plastic tube and blended with 15 mL distilled water using an Ultra Turrax T25 homogeniser (9000 rpm, 1 min). An aliquot of the homogenate (3 mL) was then added to 3 mL of the TBA reagent in a Pyrex screw cap test tube and vortexed thoroughly for 1 min. The tube contents were heated at 100 °C for 15 min in a water bath and then allowed to cool for 10 min. The flocculent precipitate was removed by centrifugation (3000 rpm, 15 min) and the absorbance of the supernatant was measured at 535 nm using a UV–Vis Spectrophotometer (Shimadzu UV-1201) against a blank containing TBA reagent with distilled water in place of sample supernatant. Absorbance values were used directly as a unit of comparison between different samples.
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weighed into 50 mL screw cap plastic tubes and blended in 25 g of distilled water using an Ultra Turrax T25 homogeniser (9000 rpm, 1 min). The homogenate was quantitatively transferred into a 500 mL Kjeldahl flask by washing with an additional 65 mL distilled water. HCl (10 mL, 1 M) was added to bring the pH to 1.5 and followed by a small amount (2 drops) of silicon antifoam. Each flask was connected to a distillation apparatus and heated with a Bunsen burner under such conditions as to collect 50 mL of distillate within 10–12 min of the initiation of boiling. A 3 mL aliquot of distillate was pipetted into a 10 mL screw capped, Teflon sealed glass tube with 3 mL TBA reagent. The tube was stoppered, vortexed and immersed in a boiling water bath for 35 min. After heating, tubes were allowed to cool and the absorbance was read using a UV–Vis Spectrophotometer (Shimadzu UV-1201) at 538 nm against a blank containing the TBA reagent with distilled water in place of the sample distillate. Absorbance values were used directly as a unit of comparison between different samples. 2.4. Statistical analysis
2.3.2. TBARS analysis by the distillation method (Tarladgis & Watts, 1960) The TBA reagent (0.02 M TBA in 90% acetic acid) was prepared by dissolving TBA in acetic acid, with gentle warming in a hot water bath and making up to volume with distilled water. On the day of analysis turkey meat was finely diced and 3 g samples were
For each of the four experiments represented by Figs. 1A, B, 2A and B a different turkey breast was used. Within each experiment each treatment was replicated three times using three different cooked turkey slices. For experiments with the MDA standard (represented by Figs. 3A and B), three different MDA solutions were
Fig. 1. Lipid oxidation (TBARS) measured by the direct method (A) and the distillation method (B) in cooked turkey meat slices after 3 days storage at 4 °C. Samples were either unwrapped or wrapped in a NaCas film (first and third bars, respectively) or unwrapped, with a portion of NaCas film added just prior to analysis of lipid oxidation (second bar). Each bar represents the mean (SD) of three replications. Bars with different letters are significantly different (P < 0.05).
Fig. 2. Effect of NaCas solution, added to the homogenised meat just prior to analysis, on lipid oxidation (TBARS) measured by the direct method (A) and the distillation method (B). Each bar represents the mean (SD) of three replications. Bars with different letters are significantly different (P < 0.05).
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Fig. 3. Effect of NaCas solution, added to a standard TMP solution just prior to analysis, on lipid oxidation (TBARS) measured by the direct method (A) and the distillation method (B). Each bar represents the mean (SD) of three replications. Bars with different letters are significantly different (P < 0.05).
prepared in order to have three replicates for each NaCas treatment. Data were evaluated using Minitab v14. Analysis of variance (ANOVA) was performed on the data and Tukey’s test (a = 0.05) was used to identify significant differences between samples.
3. Results and discussion The experiment involved the storage of 15 g slices of cooked turkey meat for 3 days at 4 °C. The objective was to determine whether wrapping the slices in a NaCas film had an antioxidant effect on them. After 3 days of storage a comparison of unwrapped with wrapped turkey slices showed a decrease in TBARS (Fig. 1A). The unwrapped sample stored for 3 days had a mean absorbance value of 0.90 whereas that of the sample wrapped in NaCas film had a lower absorbance value (0.45). However, the possibility that the antioxidant effect could partly or fully be caused by interference of NaCas in the TBARS assay was raised. Thus, an amount of NaCas film, equivalent to that which would be attached to a film-wrapped turkey slice during storage (approximately 26 mg/g turkey meat), was added directly to a sample of unwrapped turkey at the end of the 3 days storage and lipid oxidation was assessed as for the wrapped and unwrapped stored samples (Fig. 1A). An absorbance value of 0.67 was recorded for the turkey meat to which the NaCas was added directly after storage suggesting that about 50% of the antioxidant effect observed when turkey samples were wrapped with NaCas film could be attributed to the NaCas itself and not to prevention of lipid oxidation in the turkey slices during storage. These results suggest that NaCas interferes in the TBA–MDA reaction when the direct method is used, and possibly also in other methods that involve direct contact between the TBA and the food samples being assessed. Even with filtration methods, which involve filtering the food slurry before reaction with TBA, the possibility of interference exists because filtrates are likely to contain a complex mixture of components capable of interfering in the TBA–MDA reaction (Fernandez et al., 1997). In contrast to the direct method, no interference due to NaCas was observed when the distillation method was used to assess lipid oxidation (Fig. 1B) and a reduction in the absorbance value from 0.76 to 0.45 could be attributed to an antioxidant effect of the film on the formation of lipid oxidation products. The extent of potential interference from NaCas was demonstrated by using the direct method to assess lipid oxidation in turkey meat slices to which an increasing amount of NaCas was added following storage for 1 day at 4 °C (Fig. 2A). The experiment dem-
onstrated how NaCas, in the range 2.3–11 mg/g meat, can decrease the absorbance of a sample by up to half its value, using the direct method. In marked contrast, when assessed by the distillation method the TBARS values of the turkey meat were unaffected by the addition of NaCas (Fig. 2B) suggesting no interference in the reaction between TBA and MDA or similar TBA-reactive compounds. In fact increasing the concentration of NaCas up to 120 mg/g of meat resulted in no reduction in TBARS (data not shown). In the case of the direct method, when TMP, which functions as an MDA standard, was used in place of turkey meat it was also observed that the addition of NaCas reduced absorbance values (Fig. 3A); however, when the distillation method was used no such reduction was observed (Fig. 3B), an outcome similar to that depicted in Fig. 2B. The direct method has the advantage of being straightforward and easy to perform, requiring four steps: homogenisation, reaction, centrifugation and spectrophotometric reading. In contrast, the distillation method is more time consuming, requiring the use of a distillation apparatus initially to generate a distillate, which is subsequently reacted with TBA reagent. Moreover, the collection of a specific volume of distillate is somewhat empirical (Sinnhuber & Yu, 1977). The experimental conditions may also promote the formation of secondary oxidation products, thus increasing the final TBARS values (Pikul, Leszcynski, & Kummerow, 1983), although observed increases in TBARS on distillation have also been attributed to the liberation of MDA precursors bound to proteins (Rhee, 1978). Although direct methods have been used in the past to assess lipid oxidation in casein-containing systems (Cervato, Cazzola, & Cestaro, 1999) and in meat products containing non-meat ingredients including antioxidants (Kang et al., 2007) interference can occur during the analysis, as reported by Fernandez et al. (1997). Malonaldehyde and other TBA-reactive substances can react with amino acids, proteins and others food constituents, and even with water when specific conditions are present (Kwon, Menzel, & Olcott, 1965). As an example, t-2-hexenal and hexanal, which were found by Sun et al. (2001) to interact with TBA, were also found by Meynier, Rampon, Dalgalarrondo, and Genot (2004) to bind covalently with NaCas and whey proteins. In such situations where MDA is bound in the product matrix it may be unavailable for reaction with TBA, thus giving erroneously low TBARS absorbance values. Any one or a combination the above may account for the reduced absorbance values obtained when the direct method was used to assess lipid oxidation in the samples containing added NaCas.
I. Caprioli et al. / Food Chemistry 124 (2011) 1284–1287
4. Conclusions The results of this short study suggest that the presence of NaCas in food samples, and by extension perhaps other soluble proteins, can interfere with the determination of lipid oxidation by direct contact TBARS methods. In contrast distillation methods seem to be free from such interference. Thus it would seem wise for researchers to choose the method of TBARS determination carefully, having regard to the particular composition of their sample matrix. It would also be advisable to interpret the output of the TBARS method in conjunction with the results from other measurements of lipid oxidation, chosen, when possible, to evaluate both primary (such as hydroperoxides) and secondary (such as hexanal) products of lipid oxidation. Acknowledgements The technical assistance of Mr. Eamon Power and Mr. Michael Cooney is greatly appreciated. The financial support of Irish Department of Agriculture, Fisheries and Food under the Food Institutional Research Measure of the 2000–2006 National Development Plan is gratefully acknowledged. References Bernheim, F. M., Bernheim, L. C., & Wilbur, K. M. (1948). The reaction between thiobarbituric acid and the oxidation products of certain lipids. Journal of Biological Chemistry, 174, 257–264. Buege, J. A., & Aust, S. D. (1970). Microsomal lipid peroxidation. Methods in Enzymology, 52, 302–310.
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