Measurement of lipid oxidation in meat and meat products

Review

Measurement of lipid Lipid oxidation and the associated changes is a major cause of quality deterioration during the storage of meats and other fat-containing foods. Oxidative deterioration of lipids directly

oxidation in meat and

affects a number of quality characteristics in meat and meat products, including flavor, color, texture, nutritive value and safety. Consequently, the past 50 years have seen much

meat products

research activity in determining the factors that influence lipid oxidation in meat and other biological systems and in developing ways to minimize these oxidative changes. The extent

J. Ian Gray and Frank J. Monahan

of these changes can be measured by both chemical and physical means. The purpose of this short review is to discuss the principal methods used to assess lipid oxidation in meats and their limitations.

to the mechanism of lipid oxidation in foods and other biological materials are beyond the scope of this paper, but have been addressed recently in a number of excellent reviews 4 .8.9 • Hydroperoxides, the primary products of lipid oxidation, are colorless, tasteless and odorless. It is the breakdown of these peroxides that yields a complex mixture of low molecular weight compounds with distinctive odor and flavor characteristics, including alkanes, alkenes, aldehydes, ketones, alcohols, esters and acids 10.11. These compounds impart rancid, fatty, pungent and other off-flavor characteristics to meat l2 • The contribution that a particular compound makes to the flavor or aroma of a meat product depends on the concentration at which it is present and on its odor threshold. Aldehydes, unsaturated alcohols and vinyl ketones have relatively low odor thresholds; some have distinct aromas at concentrations below 1 ppb and are likely, therefore, to contribute flavor if present in food systems 10. Lipid oxidation is a very complex process, and there are many ways in which one could attempt to measure the extent of the oxidative changes that occur in food products. Ideally, it would be desirable to have a method of measuring lipid oxidation that permitted the prediction from a chemical or physical measurement of when a product would become unacceptable by sensory assessment. When the usefulness of a particular analytical procedure is evaluated, certain questions must be addressed.

Lipid oxidation leading to rancidity has long been recognized as a problem associated with the storage of fats and oils. Oxidative changes in food lipids primarily involve autoxidation reactions, which are accompanied by various oxidative and non-oxidative secondary reactions. Oxidation of unsaturated lipids has been well reported l - 3 and, unless mediated by other oxidants or enzyme systems, proceeds through a free radical chain mechanism involving initiation, propagation and termination stages". The initiation stage has been and still remains the subject of much research and general uncertainty. The direct reaction of unsaturated lipids with molecular oxygen is thermodynamically unfavorable 5 • However, the spin restriction that prohibits the interaction of groundstate oxygen with unsaturated fatty acids can be overcome by a number of initiating mechanisms, including singlet oxygen; partially reduced or activated oxygen species such as hydrogen peroxide, superoxide anion or hydroxyl radicals; active oxygen-iron complexes (ferryl iron); and iron-mediated homolytic cleavage of the hydroperoxides, which generate organic free radicals 4 . Examination of data relating to lipid oxidation in biological systems in vitro, particularly those published in the meat literature, suggests that the results generated over the past 30 years deal with hydroperoxide- • Does the property being measured adequately repdependent lipid oxidation - catalysis of the breakdown resent the extent of oxidation? of preformed lipid hydroperoxides, rather than initiation of lipid oxidation. For example, the mechanism pro- • Is the method specific for that particular property? posed by Tappel 6 for catalysis of lipid oxidation by • Would the property being measured arise under any hematin compounds depends on the presence of precircumstances other than oxidation? formed hydroperoxides. Likewise, catalysis of lipid oxidation by ferrous and ferric iron may be achieved by the Another factor that may dictate which method to select decomposition of lipid hydroperoxides, resulting in the is how well the method correlates with sensory analysis. production of free radicals capable of propagating the The purpose of this review is to present a brief outline oxidative reactions 7• These and additional issues related of some of the methods that have been used to monitor lipid oxidation in meat products. [To be consistent, J. Ian Gray is at the Department of Food Science and Human Nutrition, the term 'lipid oxidation' is used throughout the text, although it is recognized that 'lipid peroxidation' is Michigan State University, East Lansing, MI 48824, USA. Frank J. Monahan is more generally favored, particularly in the medical and at the Department of Food Science and Technology, University of California, Davis, CA 95616, USA. biochemical literature.]

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The methods used to measure lipid oxidation in animal tissues have traditionally been divided into those that measure primary changes and those that measure secondary changes (Box I). More recently, techniques have been developed that allow the detection of transient intermediates in the lipid oxidation reaction sequence. These latter techniques have been used to study more closely the mechanistic details of lipid oxidation and the effects of free radicals in lipid oxidation in vivo.

Measurement of primary changes Methods that measure primary changes may be classified as those that quantify the loss of reactants (unsaturated fatty acids or oxygen) or the formation of primary lipid oxidation products (hydroperoxides) and are generally more suited to measuring low levels of oxidation in uncooked products stored at low temperatures 13. For example, Moerck and Ball l4 and Igene et al. 15 found that changes in the phospholipid composition of mechanically de boned chicken muscle and beef muscle were indicative of the occurrence of oxidative changes during refrigerated storage. However, Melton l6 , in her review of methodology for following lipid oxidation in muscle foods, concluded that methods other than changes in the fatty acid composition are necessary to monitor lipid oxidation in foods, particularly lamb and beef. Measurement of oxygen absorption has also been used to follow lipid oxidation in muscle tissue homogenates I7- 19 • Hydroperoxides in muscle foods may be measured by a variety of methods 20 after the lipids have been extracted from the foods. The most common methods for measuring the content of hydroperoxides is the 'peroxide value' determination, which employs an iodometric technique similar to the AOAC (Association of Official Analytical Chemists) method 21 , in which the peroxide value is reported as milliequivalents (meq) iodine per kilogram of fat. Measurement of the peroxide value has been used to estimate lipid oxidation in a number of meat products, including pork fat n and muscle 23 , beef muscle 24 . ground chicken 2s and ground turkey26. However, the decomposition of peroxides to secondary

Box 1. Parameters used to measure lipid oxidation" Primary changes Oxygen uptake Loss of polyunsaturated fatty acids Formation of hydroperoxides (peroxide value)

Secondary changes Formation of carbonyls (as dinitrophenylhydrazones or by gas chromatography) Formation of malonaldehyde: 2-thiobarbituric acid (TBA) test Formation of hydrocarbons (e.g. pentane) Formation of fluorescent products (1-amino-3-iminopropene structures) a Adapted

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from Ref. 13

products can result in underestimation of the degree of oxidation. For example, A wad et al. 24 found that thc peroxide value of beef decreased after two weeks of frozen storage, but that other indices of oxidation increased. Melton l6 also concluded that the determination of peroxides may not be useful as a measure of lipid oxidation in muscle foods during prolonged storage, especially if the muscle has been ground. The relationship between the peroxide value and oxidized flavor in muscle foods apparently varies with the type of meat and the way it has been processed. The peroxide value procedure has not been used extensively in the study of oxidized flavors in meat productsn. Recently, the association of oxidation processes with aging, cancer and several clinically significant diseases has generated sizeable interest in the identification and determination of lipid hydroperoxides in biological fluids and tissues. This has led to the development of quantitative methods that indicate the presence of fatty acid hydroperoxides, rather than their decomposition products. Fatty acid hydroperoxides can be analysed by high-performance liquid chromatography (HPLC), but reduction to the corresponding hydroxy acids, readily achieved by treatment with triphenylphosphine or sodium borohydride, is required for gas chromatographic - mass spectrometric analysis 2x •29 • Other techniques include the chemiluminescence method, which is based on the detection of chemiluminescence generated during the oxidation of luminol by hydroperoxides 30 ; hydroperoxide oxidation of dichlorofluorescein to form the fluorescent dichlorofluorescein 31 ; activation of cyclooxygenase 32 ; formation of the iron thiocyanate complex"; and formation of oxidized glutathione 34 . Yang and coworkers 3s •36 have described a chromatographic procedure involving hydroperoxide separation by HPLC followed by post-column reaction with iodine or luminol. This method has been applied to both food systems and biological specimens 36 .

Measurement of secondary changes In situations where oxidation occurs at an accelerated rate, as in cooked meats, and primary products rapidly decompose to stable secondary products, it is more appropriate to measure these secondary products as an index of lipid oxidation. One of the oldest and the most frequently used test for assessing lipid oxidation in muscle foods (and other biological systems) is the 2-thiobarbituric acid (TBA) test I6.20 . The extent of lipid oxidation is reported as a 'TBA number' or 'TBA value' and is expressed as milligrams of malonaldehyde equivalents per kilogram of sample. Malonaldehyde is a relatively minor lipid oxidation product formed during the oxidation of polyunsaturated fatty acids, and reacts with TBA to produce a colored complex with an absorption maximum at 530-532 nm. The intensity of the color complex found on reacting the TBA reagent with lipid-containing foods, food extracts or steam distillates of foods was originally believed to be a measure of malonaldehyde concentration 37 .3x and has been reported to correlate well Trends in Food Science & Technology December 1992 [Vol. 31

with sensory scores of oxidized flavors in muscle foods 39-42. However, several confounding factors can also affect the intensity of the color complex. For example, other products of lipid oxidation, such as alka2,4-dienals, also react with TBA to form a red complex with the same absorption maximum as the malonaldehyde-TBA complex 43 • For this reason, Gray and Pearson"" suggested that the TBA procedure should be used to assess the extent of lipid oxidation in general, rather than to quantify malonaldehyde, and the teon 'thiobarbituric acid-reactive substances' (TBARS) is now commonly used in place of the TBA number or value. Although the TBA test has been used almost slavishly by meat technologists and by medical researchers to assess lipid oxidation, many reviews have pointed out the limitations of the procedure. Hoyland and Taylor 45 comprehensively reviewed the various TBA test methodologies that have been used and indicated that, despite copious literature references and its widespread usage, there are still uncertainties over the exact chemistry of the reaction and its applicability. In an excellent, but seldom-quoted critique of the test, Ward46 concluded that 'without the knowledge of (a) the exact nature of the TBA active substrate(s); (b) what TBA-adduct(s) are formed; (c) the compositional profile of the lipid system in question; (d) the oxidative pathways taken by components of the lipid system leading to the formation of TBA active substrate(s); (e) the relationship of the TBA active substrate(s) to flavor producing molecules, if the assay is to be used to indicate flavor degradation; the TBA assay can only be of limited value, either in assessing the degree of chemical oxidation or in relation to organoleptic response'. Ward 46 further concluded from literature observations that the assay is operator dependent, method dependent and suffers from interference. The reaction of TBA with other food components such as aldehydes, sugars and wood smoke components has been well established, as has the decomposition of many amino acids and carbohydrates in the presence of iron during heating to provide TBA-reactive residues 20 • Problems with the TBA test also arise with cured meats as a result of nitrosation of malonaldehyde by the residual nitrite during the analysis itself47. The addition of sulfanilamide to cured meats prior to distillation was suggested as a way of circumventing the possible underestimation of the malonaldehyde content in cured meats 47 . Shahidi et al. 4K further evaluated the effects of nitrite and sulfanilamide on TBA values in aqueous model systems and cured meat systems and concluded that the TBA test for the evaluation of the oxidative state of cured meats may only be used when the content of nitrite in the cure or final product is known. Addition of sulfanilamide was necessary for products prepared with the addition of ~IOO mg nitrite per kilogram of meat to prevent underestimation of the TBA value. However, in the absence of nitrite, sulfanilamide may interact with malonaldehyde to produce a l-amino-3iminopropene derivative 48 • The TBA test has also been extensively scrutinized by researchers involved in studying oxidation in biological

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systems, and again caution has been advocated in interpreting TBA data 8 .49. As pointed out in these publications, some of the malonaldehyde detected in the TBA test is formed during the oxidation process itself, but most is generated by the decomposition of lipid peroxides and by further oxidation during the acid-heating stage of the test. Peroxide decomposition requires the presence of iron ions, which may be found as contaminants in the TBA reagents5(). While some researchers add antioxidants (to eliminate oxidation during the test) or iron (to facilitate complete decomposition of the peroxides) to the TBA test reagents, there is no version of the test that is suitable for all applications. Gutteridge and Halliwe1l 49 have discussed the limitations of the TBA assay, particularly when it is applied to measure oxidation in human materials. For example, plasma contains many substances that react in the TBA test, including bile pigments, amino acids and carbohydrates. The assay will also measure endoperoxides in human bodily fluids that arise enzymatically through the prostaglandin biosynthetic pathway49. Should we continue to use the TBA assay to quantify the extent of lipid oxidation in meats? In our opinion, yes, as the TBA test can provide useful data on the state of lipid oxidation in foods - provided that it is used wisely and that one recognizes the limitations of the method. If all TBARS are deteonined by a single method, the change in TBARS for that particular situation and type of meat can show the relative amount of lipid oxidation occurring during storage and/or processing - for example, to evaluate the efficacy of antioxidants from different sources, or of different packaging methods on product stability. However, it is preferable to quantify the extent of lipid oxidation by a complementary analytical procedure, such as hexanal measurement, and to relate both sets of analytical data to sensory (organoleptic) scores. Similar views on alternative procedures have been expressed by Gutteridge and Halliwe1l 49 with regard to studying lipid oxidation in human materials. In order to define as precisely as possible what reactions are occurring, it is important to use techniques that give specific information about the compounds present. Thus, an increasing number of research groups are using HPLC and gas chromatographic procedures to obtain more precise chemical information in studies of complex mixtures. For example, Tamura and Shibamot0 51 recently developed a new gas chromatographic method to monitor the formation of reactive carbonyl compounds such as malonaldehyde and 4-hydroxy-2-nonenal in oxidizing arachidonic acid and linoleic acid model systems. The two aldehydes were reacted with N-methylhydrazine to yield l-methylpyrazole and 5-( )'-hydroxyhexyl)-l-methylpyrazoline, respectively, which were subsequently analysed by capillary gas chromatography. Such information on known lipid oxidation products is needed to clarify more fully the specific roles played by lipid oxidation in cell injury and in human disease. Other degradation products of lipid hydroperoxides

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have been quantified and used as indices of lipid oxidation in foods 16.20. Hexanal, one of the major secondary products formed during the oxidation of linoleic acid 52 , and other aldehydes have been used successfully to follow lipid oxidation in meat products; increased levels of the aldehydes are related to off-flavor development. Shahidi et al. 53 reported a linear relationship between hexanal content, sensory scores and TBA numbers of cooked ground pork, and St Angelo et al. 54 established a similar correlation for cooked beef. The latter also demonstrated that levels of 2,3-octanedione, total volatiles and hexanal were significantly correlated to sensory scores and TBARS of roast beef. They proposed that many compounds usually associated with lipid oxidation reactions could be used as marker compounds to follow the development of rancid flavors in meats. Melton l6 had earlier concluded that direct quantification of peroxide decomposition products by gas chromatography may be a more accurate method than either the total carbonyl assay or the TBA test for determining oxidative changes in foods. Several other methods exist to measure lipid oxidation, including the measurement of hydrocarbons (ethane and pentane) and fluorescent products 16.20. While they have been applied to meat products 1\ they have not been used extensively and thus are not considered a routine part of the analytical arsenal of the meat scientist. Similarly, the conjugated diene method is not routinely used by meat scientists, although it is a popular method for measuring lipid oxidation end products in human body fluids 8 •

it to a more stable, detectable radical adduct. Most of the commonly used spin traps are nitroso or nitrone compounds58 . The application of ESR and the spin trap procedure to various biological and model systems has been reviewed by Davies 58 • The free radicals generated during the lipoxygenase-catalysed breakdown of unsaturated fatty acids 59 , NADPH-stimulated lipid oxidation in rat hepatic microsomes 6(I, iron-stimulated lipid oxidation in rat hepatocytes 61 , aerobic incubation of hepatic microsomes from normal and malignant hyperthermiasusceptible pigs62, and the oxidation of microsomal fractions from pigs fed control and vitamin E-supplemented diets 6 ] have been identified. The identity of the trapped radical is not always easy to ascertain 62 , and confirmative studies are often required. Schaich and Borg56 outlined a number of critical considerations regarding the interpretation of spintrapping data. For example, the detection of a particular trapped radical does not necessarily indicate that it is a major intermediate or part of a major reaction pathway, merely that the trapped radical reacts most rapidly and/or stably with the trap used. The application of ESR to the study of food systems is a relatively new development, but is becoming increasingly relevant. For example, the detection of free radicals by ESR is currently being investigated as a method for detecting food that has been irradiated 64 • ESR spectroscopy may offer a means of elucidating the role of free radicals in the formation of specific flavor compounds and mutagenic compounds in meats.

Measurement of free radical intermediates Lipid oxidation processes in foods and biological systems have conventionally been studied by analysis of primary and secondary lipid oxidation products, but in the past 20 years, advances in pulse radiolysi s55 and electron spin resonance (ESR)56 techniques have facilitated the detection and study of short-lived free radical intermediates. ESR spectroscopy only detects species with an unpaired electron, such as free radicals. The technique relies on the absorption of microwave energy (which arises from the promotion of an electron to a higher energy level) when the sample is placed in a variable magnetic field 57 . A major limitation in the detection of free radicals by ESR is the requirement that radical concentrations remain higher than 10-8 M. Radical lifetimes in solution are very short (
Summary A variety of methods are available for assessing lipid oxidation in meats. The most commonly used procedure, the TBA test, is an enigma in that it is routinely criticized but still remains the method of choice in most applications. It is simple to use, and offers considerable sensitivity and versatility for detecting the occurrence of lipid oxidation and other radical reactions. However, it must be used and the data interpreted with caution. We recommend that when the TBA test (regardless of the particular method) is used on a comparative basis, it should be combined with another measurement (e.g. quantification of hex anal levels) when assessing lipid oxidation in meats. Similar caution has been advocated for its use in studies of other biological tissues. While measurement of TBA-reactive species is perhaps the most convenient way of assessing lipid oxidation in vitro, reliance on methods of analysis based on gas chromatography - mass spectrometry and HPLC will increase because of the superior chemical specificity provided by these techniques.

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