Suitability of saturated aldehydes as lipid oxidation markers in washed turkey meat

Meat Science 83 (2009) 412–416

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Suitability of saturated aldehydes as lipid oxidation markers in washed turkey meat G. Pignoli a, R. Bou b,c, M.T. Rodriguez-Estrada a, E.A. Decker c,* a

University of Bologna, Department of Food Science, Bologna, Italy University of Barcelona, Nutrition and Food Science Department, XaRTA-INSA, Barcelona, Spain c University of Massachusetts, Department of Food Science, Amherst, Chenoweth Laboratory, Amherst, MA 01003, USA b

a r t i c l e

i n f o

Article history: Received 31 October 2008 Received in revised form 5 June 2009 Accepted 8 June 2009

Keywords: Turkey meat HS-SPME-GC TBARs Volatile saturated aldehydes Lipid oxidation

a b s t r a c t The aim of this study was to evaluate the suitability of saturated aldehydes as lipid oxidation markers in washed turkey muscle, by means of headspace solid phase microextraction-gas chromatography (HSSPME-GC); the results were compared with the widely used thiobarbituric acid-reactive substances (TBARs) method. Changes in TBARs, propanal and hexanal concentrations were determined over time in a model system consisting of turkey muscle washed with a sodium phosphate buffer (pH 5.6). To stop oxidation from occurring during analysis, an antioxidant mixture (EDTA, trolox and propyl gallate) was added immediately before analyses. After antioxidant addition, propanal and TBARs concentrations did not increase during 8 h of further storage, while an unexpected decrease in hexanal was observed. To determine if aldehydes were interacting with washed turkey muscle, hexanal and propanal were added to either phosphate buffer or washed muscle and concentrations were monitored for 24 h. Neither propanal nor hexanal decreased in the phosphate buffer over time, but the headspace concentration of propanal and hexanal in washed turkey muscle were markedly lower (76% and 96%, respectively) at time zero and continued to decreased up to 24 h of storage. Because of this decrease in headspace aldehyde concentrations, TBARs were found to be a more sensitive and accurate marker of oxidative deterioration in washed turkey muscle. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Consumption of meat containing high amounts of polyunsaturated fatty acids (PUFA) has increased greatly in the last decade, due to the nutritionists’ recommendations to reduce intake of saturated fatty acids (SFA). However, a high degree of unsaturation accelerates oxidative processes, leading to deterioration of meat flavor, color, texture and nutritional value (Fenaille, Visani, Furmeaux, Milo, & Guy, 2003; Goodridge, Beaudry, Pestka, & Smith, 2003; Sanches-Silva, Rodríguez-Bernaldo de Quirós, López-Hernández, & Paseiro-Losada, 2004). Turkey meat has a moderately low fat content and is relatively rich in PUFA (Komprda et al., 2002; Taber, Chiu, & Whelan, 1998), but it is prone to oxidation due to its inefficient accumulation of dietary vitamin E (Mielnik, Olsen, Vogt, Adeline, & Skrede, 2006). The primary oxidation products for unsaturated fatty acids are the hydroperoxides, highly reactive compounds that decompose rapidly, yielding a complex mixture of non-volatile and volatile compounds, such as hydrocarbons, aldehydes and ketones, which affect the overall quality of the product (García-Llatas, Lagarda, * Corresponding author. Tel.: +1 413 545 1026; fax: +1 413 545 1262. E-mail address: [email protected] (E.A. Decker). 0309-1740/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2009.06.019

Romero, Abellán, & Farré, 2006). Aldehydes are particular important with respect to flavor alteration and from a toxicological standpoint (Frankel, 1980, 1982, 1993). Hexanal is a specific volatile oxidation product of n 6 PUFA, whereas propanal arises from n 3 PUFA oxidation (Romeu-Nadal, Castellote, & López-Sabater, 2004). n 6 and n 3 PUFA correspond to 26% and 3.0% of the total fatty acids in turkey meat, respectively (Taber et al., 1998). During oxidation, both the n 3 and n 6 fatty acids would be expected to oxidize and generate numerous volatile fatty acid decomposition products, including the saturated aldehydes, propanal and hexanal. Propanal and hexanal are often used as indicators of lipid oxidation in foods, because they can be measured in the sample headspace and their lack of double bonds makes them more oxidatively stable than unsaturated aldehydes. While most of the current research focuses on the prevention of oxidative deterioration of meats, rapid and inexpensive analytical methods to determine the extent of oxidative rancidity of raw meats are still lacking. Thiobarbituric acid-reactive substances (TBARs) method has been widely used to monitor secondary lipid oxidation in raw and cooked muscle foods, because it is easy to use and relatively fast; however, this method has been largely criticized due to its lack of specificity. Solid phase microextraction (SPME) potentially provides many advantages over conventional

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techniques for the extraction of volatile saturated aldehydes, including easy manipulation and experimental set up, short sampling times, easy automation and high sensitivity. The detection limit of headspace (HS)-SPME can be as low as parts per trillion (Zhang & Pawliszyn, 1993) and has been successfully used to estimate the hexanal content of cooked meats (Nilsen, Sørensen, Skibsted, & Bertlsen, 1997) and raw meat (Ahn, Jo, & Olson, 1999; Nam, Cordray, & Ahn, 2004). HS-SPME is able to extract organic compounds from virtually any matrix, as long as target compounds can be released from the matrix into the headspace (Olivier, Gauch, Mariaca, & Klein, 1995). The main disadvantage of HS-SPME is its inability to recover trace compounds and strongly bound semi volatile compounds. Chemical reactions via covalent and electrostatic reactions are known to be responsible for the irreversible linkage of volatiles to proteins (Fischer & Widder, 1997), which mainly depends on the polarity of proteins (Maier, 1975). This phenomenon can be partly overcome by salt addition (Flores, Gianello, PérezJuan, & Toldrá, 2007; Pérez-Juan, Flores, & Toldrá, 2007), which significantly reduces the ability of sarcoplasmic protein to bind branched aldehydes (3-methyl-butanal and 2-methylbutanal), hexanal and methional (Pérez-Juan et al., 2007). For all these reasons, it is necessary to monitor the formation of specific lipid oxidation markers, such as propanal and hexanal, to verify their actual reliability as oxidation indicators in complex food matrices. The aim of this study was to evaluate the suitability of saturated aldehydes as secondary lipid oxidation markers in washed turkey muscle, by using HS-SPME-gas chromatography (HS-SPME-GC); the results were compared with those obtained with the TBARs method. Changes in TBARs, propanal and hexanal concentrations were determined over time in refrigerated raw washed turkey muscle. 2. Materials and methods 2.1. Reagents and standards Hexanal, 2-thiobarbituric acid (TBA), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), n-propyl gallate, anhydrous monobasic sodium phosphate, pentanal, octanal, and 1,1,3,3-tetraethoxypropane, were purchased from Sigma–Aldrich (Sigma Chemical Co., St. Louis, MO, USA). Trichloroacetic acid (TCA), propanal, heptanal and nonanal were supplied by Acros (Acros Organics, Morris Plains, NJ, USA). Anhydrous dibasic sodium phosphate and potassium ferricyanide were purchased from Fisher (Fisher Scientific, Fair Lawn, NJ, USA). Ethylenediamine tetraacetic acid (EDTA) disodium salt was supplied by Curtin Matheson (Curtin Mahteson Scientific, Inc., Houston, TX, USA). Streptomycin sulfate was purchased from Spectrum Chemical (Spectrum Chemical MFG Corp., Gardena, CA, USA).

2.2. Sample preparation of washed muscle About 0.5 kg of organic turkey breast meat was purchased in a local supermarket located in Amherst (MA, USA); slaughtering conditions and the postmortem history of the turkey breast meat were not available. The two whole half breasts were trimmed to remove all remaining adipose tissue, cut into small pieces and minced using a commercial blender. The perfectly homogeneous, minced muscle was washed once with distilled deionized water at a 1:3 mince-to-water ratio (w/w) and stirred with a glass rod for 2 min. Subsequently, the mixture was allowed to stand for 15 min before dewatering with two layers of cotton cheesecloth. The mince was then washed twice with 50 mM sodium phosphate buffer (pH 5.6), as described above for the water washing. The washed mince (50 g) was then homogenized using a Tissue Tearor

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(Biospec Products, Inc. Bartlesville, OK, USA). The homogenized mince was allowed to stand for 15 min and finally centrifuged (15,000g for 20 min at 4 °C), using an ultracentrifuge (Sorvall Ultra 80, DuPont, Wilmington, DE, USA). The resulting pellet was vacuum-packed in plastic bags and stored at 80 °C until analysis. 2.3. Experimental set-up Two sets of experiments were performed, in order to evaluate secondary lipid oxidation products over time (A), and the interaction between aldehydes and washed muscle after antioxidant addition (B). Each experimental set was repeated twice and three replicates were run per sampling point, using both analytical methods. 2.3.1. Experiment A Washed turkey muscle (350 mg and 700 mg for TBARs and HSSPME-GC analysis, respectively) was weighed into 10-mL screw capped tubes. Subsequently, a streptomycin solution (50 lL and 100 lL for TBARs and HS-SPME-GC analysis, respectively, with a final concentration of 300 mg/L) in 2 mM phosphate buffer (pH 5.6), was added to inhibit microbial growth. The tubes were stored at 4 °C for different time periods (0, 12, 36 and 54 h), followed by addition of an aqueous antioxidant mixture (75 lL and 150 lL for TBARs and HS-SPME-GC analysis, respectively) consisting of EDTA (1.0 mM), trolox (0.16 mM) and propyl gallate (0.5 mM). 2.3.2. Experiment B Samples were prepared as in experiment A, except that the aqueous antioxidants were added after 54 h of storage at 4 °C, followed by HS-SPME-GC and TBARs analysis at 0, 1, 2, 4 and 8 h after antioxidant addition. In addition, 700 mg of washed turkey muscle were weighed into 10-mL screw capped tubes along with 100 lL of streptomycin solution (final concentration = 300 mg/L). A control consisting of 700 lL of 2 mM phosphate buffer (pH 5.6) was also prepared. Both muscle sample and control were spiked with 2 concentration levels of propanal (0.18 and 0.52 mg/kg) and hexanal (0.02 and 0.06 mg/ kg), followed by 150 lL of the aqueous antioxidant mixture. Samples were then analyzed by HS-SPME-GC at different time intervals (0, 6, 12, 18 and 24 h). 2.4. Determination of thiobarbituric acid-reactive substances (TBARs) This determination was performed according to the modified method of Buege and Aust (1978). Three mL of a solution containing 1.3% TBA dissolved in 50% TCA were added to the samples contained in the tubes, capped, vortexed and incubated for 1 h at 65 °C. Samples were then stored at 4 °C for 1 h, followed by centrifugation at 2000g for 10 min. The absorbance of the supernatant was measured at 532 nm with a UV spectrophotometer (UV–Vis mod. UV-1601, Shimadzu, Kyoto, Japan) and TBARs concentration were calculated as mg of malonaldehyde (MDA) using the extinction coefficient of 532 = 1.56  105 M 1 cm 1 (Buege & Aust, 1978). TBARs results were expressed as mg of malonaldehyde/kg meat. 2.5. Determination of volatile aldehydes by HS-SPME-GC/FID Before analysis, 50 lL of a NaCl solution (3.42 mol/L) were added to the samples contained in the vials, which were capped and placed into the autosampler tray. An AOC-5000 Auto-injector (Shimadzu, Tokyo, Japan) suitable for HS-SPME analysis, was used. The sample vial was transported from the autosampler tray to the incubating chamber and held at 37 °C for 20 min. A 50/30 lm divynilbenzene/carboxen/polydimethylsiloxane (DVB/Carboxen/

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PDMS) Stable Flex SPME fiber (Supelco, Bellefonte, PA, USA) was inserted through the septum into the vial and exposed to the headspace. Vial penetration depth was set at 22 mm and, after 2 min of extraction, the SPME fiber was inserted into the injection port of the Shimadzu GC model GC-2014 (Shimadzu, Tokyo, Japan). The injection penetration depth was set at 54 mm. The SPME fiber was desorbed at 270 °C for 3 min in the split mode. The chromatographic separation of volatile aldehydes was performed on a fusedsilica capillary column (30 m  0.32 mm i.d.  1 lm) coated with 100% poly(dimethylsiloxane) (Equity-1, Supelco). The oven was programmed from 45 °C (kept for 2 min) to 255 °C at 15 °C/min and held for 1 min. The temperature of the injector and the flame ionization detector (FID) were set at 270 °C and 300 °C, respectively. Helium was used as carrier gas at a linear velocity of 40 cm/s; the split ratio was 1:8. For the quantitative determination of propanal and hexanal, standard calibration curves were used with a concentration range of 0–0.8 and 0–0.6 mg/kg sample, respectively. Regression equations were calculated for propanal (y = 259.4x + 10.33; R2 = 0.999) and hexanal (y = 4979x + 93.23; R2 = 0.995). The propanal and hexanal concentration in the samples was estimated by using the linear regression equation and the values were expressed as mg/kg sample. The limits of detection (LOD) of propanal and hexanal were 11.6 and 0.9 ng/g sample, respectively, whereas the limits of quantification (LOQ) were 44.8 and 3 ng/g sample. LOD and LOQ were calculated as 3 and 10 fold the standard deviation of the intercept of the calibration curve (Long & Winefordner, 1983). 2.6. Statistical analysis Data were analyzed using Statistica 6.0 software (2001, Statsoft, Tulsa, OK, USA) and underwent one-way-analysis of variance (ANOVA). Tukey’s test was performed at a 95% confidence level (p 6 0.05) to identify significant differences among samples within the evaluated parameters (TBARs and volatile aldehydes content) at various incubation times. Spearman correlations between TBARs and hexanal were determined at a 95% confidence level. 3. Results and discussion 3.1. Experiment A Fig. 1 shows the TBARs, hexanal and propanal (mg/kg meat) content in washed turkey muscle meat stored at 4 °C for 52 h. According to TBARs, washed turkey meat oxidized rapidly at 4 °C, reaching 0.37 mg MDA/kg meat after 52 h of storage. TBARs levels were slightly lower than those found in previous studies on raw

turkey meat (Ahn et al., 1999), which might be due to the removal of heme proteins during washing (Grunwald & Richards, 2006). The formation of volatile aldehydes in washed turkey muscle meat stored at 4 °C for 52 h, varied according to the type of saturated aldehyde (Fig. 1). Propanal did not significantly change over time (0.06 mg/kg meat), while hexanal significantly rose from 0.13 to 0.40 mg/kg meat. A relatively good, positive correlation between hexanal values and TBARs was confirmed (R = 0.62 at p = 0.0064), as stated by several authors (Brunton, Cronin, Monahan, & Durcan, 2000; Goodridge et al., 2003), despite the fact that TBARs is a nonspecific method and does not measure volatile compounds that contribute to rancidity in meats (Pearson, Love, & Shorland, 1977). The formation of lipid oxidation products in turkey meat is related to the high PUFA content in the membranes lipids (Genot et al., 1997), which are known to be similar to those of washed chicken muscle characterized by a higher level of n 6 PUFA (23.6% in mitochondria) than n 3 PUFA (6.4% in mitochondria) (Lauridsen, Buckley, & Morrissey, 1997). n 3 PUFA breakdown leads to the formation of propanal, whereas n 6 PUFA gives rise to hexanal. Hexanal formation was expected to be slower than propanal, since n 6 fatty acids in turkey (e.g. 18:2) are more oxidatively stable than n 3 fatty acids (20:5 and 22:6). Surprisingly, hexanal concentrations were observed to increase, while propanal concentrations did not change, suggesting that n 6 PUFA present in the sample are more prone to form aldehydes than n 3 PUFA. This is likely due to the high content of linoleic and arachidonic acids in the membranal fractions, as already mentioned (Komprda et al., 2002; Lauridsen et al., 1997). On the other hand, the different aldehyde vapor pressure and molecular weight, the interaction of the aldehydes with the muscle matrix and, finally, the breakdown of these aldehydes can also affect the headspace concentrations of the aldehydes in the stored washed muscle. 3.2. Experiment B To determine if headspace hexanal and propanal concentrations were affected by their interactions with other muscle components, antioxidants were added to the samples to stop the oxidative reactions after 54 h of storage and then headspace aldehyde and TBARs concentrations were monitored for an additional 1–8 h (Fig. 2). After antioxidant addition, TBARs concentrations did not increase during 8 h of further storage, indicating that the added antioxidants had stop further oxidation. Propanal concentrations also did not change, while a decrease of hexanal was observed.

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Fig. 1. TBARs (as MDA equivalents), hexanal and propanal (mg/kg meat) in washed turkey muscle meat stored at 4 °C. Data points are means (n = 3) and bars show ± standard deviation. For each determination, those points bearing the same letter are not significantly different (p 6 0.05).

Fig. 2. TBARs (as MDA equivalents), hexanal and propanal (mg/kg meat) content in washed turkey muscle meat added with an antioxidant mixture (EDTA, trolox and propyl gallate), after 54 h of storage at 4 °C. Data points are means (n = 3) and bars show ± standard deviation. For each determination, those points bearing the same letter are not significantly different (p 6 0.05).

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Fig. 3. Headspace propanal content in spiked phosphate buffer (control) and washed turkey muscle (meat) during 24-h storage. The spiking concentration levels were 0.18 and 0.52 mg of propanal/kg meat. Data points are means (n = 3) and bars show ± standard deviation. For each determination, those points bearing the same letter are not significantly different (p 6 0.05).

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anal nor hexanal decreased in the phosphate buffer over time, but the headspace concentration of propanal and hexanal in washed turkey muscle were markedly lower at time 0 (76% and 96%, respectively) and significantly decreased after 24 h of storage. Hexanal decreased more than propanal during storage. Other authors have reported that the reactivity of saturated aldehydes varies with increasing number of carbon atoms in the alkyl chain (Pokorny´, Svobodová, & Janícek, 1977). The decrease in headspace hexanal and propanal could be due to several different pathways. Relatively non-polar aroma compounds could bind to hydrophobic protein surface regions, resulting in reduced volatility. An aroma compound trapped within protein helix group also cannot exert an active role as an aroma constituent. In the case of aldehydes, they could also react with free amino- and SH-groups, thus resulting in reduced volatility. This hypothesis is, in fact, supported by various works; for instance, the e-amino groups of lysine residues of proteins were found to be modified to alkyl-substituted pyridinium rings upon exposure to vaporized hexanal (Kato, Kaneko, Okitani, Ito, & Hayase, 1986). Gianelli, Flores, and Toldrá (2003) proved that the extent of the aldehyde–protein interaction in a skeletal muscle model system, depended on the nature of peptides (carnosine and anserine), proteins (myoglobin as sarcoplasmic protein) and volatile compounds. Pérez-Juan et al. (2007) reported that the binding ability of sarcoplasmic proteins from pork muscle was higher than the ability shown by myofibrillar homogenates. In a recent study, it was confirmed that hexanal can form covalent bonds with whey proteins and sodium caseinate in aqueous solution (Meynier, Rampon, Dalgalarrondo, & Genot, 2004).

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Fig. 4. Headspace hexanal content in spiked phosphate buffer (control) and washed turkey muscle (meat) during 24-h storage. The spiking concentration levels were 0.02 and 0.06 mg of hexanal/kg meat. Data points are means (n = 3) and bars show ± standard deviation. For each determination, those points bearing the same letter are not significantly different (p 6 0.05).

The washed turkey muscle consists mainly of proteins and cell membranes. At equilibrium, the aldehydes would partition into the lipid, water and vapor phases. The reduction of hexanal recorded in the present work suggests that the hexanal chemically reacted with other muscle components, thus shifting its partitioning behavior and decreasing headspace concentrations. One possible cause of the decrease in headspace concentrations could be attributed to aldehyde interactions with amino acids in proteins via Schiff bases reactions (García-Llatas et al., 2006). In addition, the binding of volatiles to proteins could also include a wide range of interactions such as specific binding, adsorption, absorption, entrapment and covalent interaction, and depends on the physical–chemical properties of the volatile aldehydes and their relative concentration. Chemical reactions via covalent and electrostatic reactions are known to be responsible for the irreversible linkage of volatiles to proteins, and they are correlated to the polarity of proteins (Le Quach, Chen, & Stevenson, 1999). To determine if the aldehydes were becoming bond to components in the washed turkey muscle matrix leading to reduced volatility, two different levels of hexanal and propanal were added to either phosphate buffer or washed turkey muscle and their concentrations were monitored for 24 h (Figs. 3 and 4). Neither prop-

Secondary lipid oxidation products were monitored by means of TBARs and headspace volatile aldehyde determinations in washed turkey muscle with added antioxidants. The addition of antioxidants was able to stop the oxidative reactions, allowing a steady point to be reached in the TBARs method. However, hexanal present in the washed turkey meat significantly decreased after 8 h of incubation. This reduced volatility of hexanal seems to be due to interactions between the aldehyde and the amine and sulfhydryl groups of the washed turkey muscle proteins. This hypothesis is supported by the fact that when washed turkey muscle was spiked with a known amount of propanal and hexanal, significant decreases were observed in both propanal and hexanal (76% and 96%, respectively). Despite the lack of specificity, TBARs were found to be a more appropriate marker of lipid oxidation in washed turkey muscle than headspace propanal or hexanal, as the latter was influenced by the matrix over a short period time. Further studies are required to evaluate the extent of the interaction between different aldehydes deriving from lipid oxidation and meat proteins. Acknowledgments This research project was supported by the Marco Polo Program (University of Bologna). The authors want to thank Jean Alamed (University of Massachusetts) for her technical support. References Ahn, D. U., Jo, C., & Olson, D. G. (1999). Headspace oxygen in sample vials affects volatiles production of meat during the automated purge-and-trap/GC analyses. Journal of Agricultural and Food Chemistry, 47, 2776–2781. Brunton, N. P., Cronin, D. A., Monahan, F. J., & Durcan, R. (2000). A comparison of solid-phase microextraction (SPME) fibers for measurement of hexanal and pentanal in cooked turkey. Food Chemistry, 68, 339–345. Buege, J. A., & Aust, S. D. (1978). Microsomal lipid peroxidation. Methods in Enzymology, 52, 302–310.

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Fenaille, F., Visani, P., Furmeaux, R., Milo, C., & Guy, P. A. (2003). Comparison of mass spectrometry-based electronic nose and solid phase microextraction gas chromatography–mass spectrometry technique to assess infant formula oxidation. Journal of Agricultural and Food Chemistry, 51, 2790–2796. Fischer, N., & Widder, S. (1997). How proteins influence food flavours. Food Technology, 51, 68–70. Flores, M., Gianello, M., Pérez-Juan, M., & Toldrá, F. (2007). Headspace concentration of selected dry-cured aroma compounds in model system as affected by curing agents. Food Chemistry, 102, 488–493. Frankel, E. N. (1980). Lipid oxidation. Progress in Lipid Research, 19, 1–22. Frankel, E. N. (1982). Volatile lipid oxidation products. Progress in Lipid Research, 22, 1–33. Frankel, E. N. (1993). Formation of headspace volatiles by thermal decomposition of oxidized fish oils vs. oxidized vegetable oils. Journal of the American Oil Chemists’ Society, 70, 767–772. García-Llatas, G., Lagarda, M. J., Romero, F., Abellán, P., & Farré, R. (2006). A headspace solid-phase microextraction method of use in monitoring hexanal and pentane during storage: Application to liquid infant foods and powdered infant formulas. Food Chemistry, 1001, 1078–1086. Genot, C., Meynier, A., Viau, M., David, E., Métro, B., Rémignon, et al. (1997). Acides gras alimentaires, composition des lipids intramusculaires et sensibilité à l’oxydation de la viande de dinde. In Deuxièmes journées de la recherché avicole (pp. 231–234), 8–10 April, Tours, France. Gianelli, M. P., Flores, M., & Toldrá, F. (2003). Interactions of soluble peptides and proteins from skeletal muscle on the release of volatile compounds. Journal of Agricultural and Food Chemistry, 51, 6828–6834. Goodridge, C. F., Beaudry, R. M., Pestka, J. J., & Smith, D. M. (2003). Solid phase microextraction-gas chromatography for quantifying headspace hexanal above freeze-dried chicken myofibrils. Journal of Agricultural and Food Chemistry, 51, 4185–4190. Grunwald, E. W., & Richards, M. P. (2006). Studies with myoglobin variants indicate that released hemin is the primary promotor of lipid oxidation in washed fish muscle. Food Chemistry, 54, 4452–4460. Kato, H., Kaneko, S., Okitani, A., Ito, M., & Hayase, F. (1986). Modification of lysine residues to alkyl-substituted pyridiniums on exposure of proteins to vaporized hexanal. Agricultural and Biological Chemistry, 50, 1223–1228. Komprda, T., Šarmanová, I., Kelenka, J., Bakaj, P., Fialová, M., & Fajmonová, E. (2002). Effect of sex and age on cholesterol and fatty acid content in turkey meat. Archiv für Geflügelkunde, 66, 263–273. Lauridsen, C., Buckley, D. J., & Morrissey, P. A. (1997). Influence of dietary fat and vitamin E supplementation on alpha-tocopherol levels and fatty acid profiles in chicken muscle membranal fractions and on susceptibility to lipid peroxidation. Meat Science, 46, 9–22. Le Quach, M., Chen, X. D., & Stevenson, R. J. (1999). Headspace sampling of whey protein concentrate solutions using solid-phase microextraction. Food Research International, 31, 371–379.

Long, G. L., & Winefordner, J. D. (1983). Limit of detection. Analytical Chemistry, 55, 712A–724A. Maier, H. G. (1975). Binding of volatile aroma substances to nutrients and foodstuffs. In Proceedings of the international symposium on aroma research (pp. 143–157), 26–29 May, Zeist, The Netherlands. 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. Mielnik, M. B., Olsen, E., Vogt, G., Adeline, D., & Skrede, G. (2006). Grape seed extract as antioxidant in cooked, cold stored turkey meat. Lebensmittel-Wissenschaft und Technologie, 39, 191–198. Nam, K.-C., Cordray, J., & Ahn, D. U. (2004). Automated dynamic headspace/GC–MS analyses affect the repeatability of volatiles in irradiated turkey. Journal of Agricultural and Food Chemistry, 52, 1735–1741. Nilsen, J. H., Sørensen, B., Skibsted, L. H., & Bertlsen, G. (1997). Oxidation in precooked minced pork as influenced by chill storage of raw muscle. Meat Science, 46, 191–197. Olivier, L., Gauch, R., Mariaca, R., & Klein, B. (1995). Comparison of various sample treatments for the analysis of volatile compounds by GC–MS: Application to the swiss emmental cheese. Mitteilungen aus Gebiete der Lebensmitteluntersuchung und Hygiene, 86, 627–698. Pearson, A. M., Love, J. D., & Shorland, F. B. (1977). Warmed-over flavor in meat poultry and fish. Advances in Food Research, 23, 1–74. Pérez-Juan, M., Flores, M., & Toldrá, F. (2007). Effect of ionic strength of different salts on the binding of volatile compounds to porcine soluble protein extracts in model systems. Food Research International, 40, 687–693. Pokorny´, J., Svobodová, H., & Janícek, G. (1977). Reaction of lower alkanals with proteins. Correlation of flavour changes and nonenzymic browning during storage of model systems. Sbornik Vysoke Skoly Chemicko-Technologicke v Praze, E49, 5–21. Romeu-Nadal, M., Castellote, A. I., & López-Sabater, M. C. (2004). Headspace gas chromatographic method for determining volatile compounds in infant formulas. Journal of Chromatography A, 1046, 235–239. Sanches-Silva, A., Rodríguez-Bernaldo de Quirós, A., López-Hernández, J., & PaseiroLosada, P. (2004). Determination of hexanal as indicator of the lipidic oxidation state in potato crisps using gas chromatography and high-performance liquid chromatography. Journal of Chromatography A, 1046, 75–81. Taber, L., Chiu, C.-H., & Whelan, J. (1998). Assessment of the arachidonic acid content in foods commonly consumed in the American diet. Lipids, 33, 1151–1157. Zhang, Z., & Pawliszyn, J. (1993). Headspace solid-phase microextraction. Annals of Chemistry, 65, 1843–1852.