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Oxidative and lipolytic deterioration of different muscles from free-range reared Iberian pigs under refrigerated storage
Meat Science 65 (2003) 1157–1164 www.elsevier.com/locate/meatsci
Oxidative and lipolytic deterioration of different muscles from free-range reared Iberian pigs under refrigerated storage David Morcuende, Mario Este´vez, Jorge Ruiz, Ramo´n Cava* Tecnologı´a de los Alimentos. Facultad de Veterinaria, Universidad de Extremadura, Campus Universitario, Ca´ceres 10071, Spain Received 9 April 2002; received in revised form 10 February 2003; accepted 11 February 2003
Abstract Three different type of muscles, two glicolytic (Serratus ventralis and Longissimus dorsi) and one oxidative (Masseter) were displayed under refrigeration at +4 C during 10 days to evaluate differences in lipolytic and oxidative changes of different muscles with different metabolic pattern. Thiobarbituric acid reactive substances (TBARS), phospholipid content, hexanal content and fatty acid profiles of neutral and polar lipid fractions were analysed at day 0 and day 10. Phospholipid content (g phospholipids/g intramuscular fat) significantly (P< 0.035) decreased from day 0 to day 10 in m. Masseter (0.33 vs. 0.25, respectively), but not in m. L. dorsi (0.12 vs. 0.09, respectively) and m. S. ventralis (0.19 vs. 0.14, respectively). Changes in fatty acid profiles of neutral and polar lipids significantly differed among muscles after storage. Slight differences were found in neutral lipids from m. L. dorsi and m. S. ventralis, while neutral lipids from m. Masseter were highly altered. Great changes affected fatty acid profiles from polar lipids in the three muscles. m. Masseter muscle showed significantly higher (P< 0.000) TBARS values (1.13, 0.65 and 0.60 mg MDA/kg meat, respectively) and hexanal content (689.2, 241.2 and 355.8 mg/g meat, respectively) than m. L. dorsi and m. S. ventralis. In conclusion, oxidative meat is more prone to oxidative and lipolytic deterioration than glycolytic muscles during refrigerated storage and as a consequence of that a lower shelf-life. # 2003 Elsevier Ltd. All rights reserved. Keywords: Lipolysis; Lipid oxidation; Muscle type; Pork; Free-range rearing
1. Introduction Meat is refrigerated to increase its shelf-life and reduce microbial spoilage. However, even meat is stored under low temperatures its shelf-life is limited due to biochemical changes developed after slaughtering in which lipolysis and lipid oxidation of muscle lipids play an important role (Gray, Gomaa & Buckley 1996; Kanner, 1994; Morrissey, Sheehy, Galvin, Kerry & Buckley, 1998). Some specific enzymes of the adipose tissue and muscle fibres, named lipases, sterases, phospholipases and lysophospholipases, are involved in the hydrolytic processes of muscle lipids during refrigeration (Alasnier & Gandemer, 2000; Flores, Alasnier, Aristoy, Navarro,
* Corresponding author. Tel.: +34-927-257-169; fax: +34-927257-110. E-mail address: [email protected] (R. Cava). 0309-1740/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0309-1740(02)00344-3
Gandemer, & Toldra´, 1996; Herna´ndez, Navarro, & Toldra´, 1998; Monin, Horto´s, Diaz, Tibau, Diesdre, & Garcı´a-Regueiro, 1998) that release free fatty acids from both triacylglycerols and phospholipids (Alasnier, David-Briand, & Gandemer, 2000a; Alasnier, Meynier, Viau, & Gandemer, 2000b). Lipolysis has been considered as a promoter of lipid oxidation due to released free fatty acids that are very prone to lipid peroxidation, particularly long chain unsaturated free fatty acids (Nawar, 1996). Lipid oxidation in meat begins to develop soon after death and continues to increase on intensity until the meat becomes unacceptable to consumers due to adverse changes in meat flavour and meat colour produced by pigment oxidation. In addition to the implication of lipid oxidation for changes in meat flavours and colour, the oxidation of unsaturated lipids and cholesterol results in a significant generation of toxin and losses in nutritional value due to vitamins and essential fatty acid oxidation (Addis & Park, 1989; Gray et al., 1996; Kubow, 1992).
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The metabolic type of the fibres is a major factor involved in the heterogeneity of muscle quality within a carcass. In fact, several studies point out that oxidative muscles are more tasty and juicy and their lipids oxidise faster (Valin, Touraille, Vigneron, & Ashmore, 1982). This is partly related to their higher polyunsaturated fatty acid content (Alasnier & Gandemer, 1998). Moreover, oxidative muscles content more haem pigment and other prooxidants and their membrane lipids contain higher amounts of polyunsaturated fatty acids, factors that increase their susceptibility to lipid oxidation (Kanner, 1994). In reference to enzyme activity, some authors found differences in the lipolytic enzyme activities between oxidative and glycolytic muscles. Oxidative muscles exhibit a higher acid lipase, neutral lipase (Herna´ndez et al., 1998) phospholipase A and lysophospholipase activities than glycolytic muscles and consequently, oxidative muscles had a higher potential activity for post-mortem hydrolysis of phospholipids (Alasnier & Gandemer, 2000). According to the authors, this was consistent with the higher phospholipid content in oxidative muscles than in glycolytic muscles and with the involvement of phospholipases in the maintenance of membrane integrity by renewing damaged phospholipids. However, phospholipid hydrolysis occurrs at similar rate in both type of muscles during refrigeration, suggesting that there are differences in the regulation of phospholipase A activity in oxidative and glycolytic muscles. On the other hand, Flores et al. (1996) did not find a clear correlation between lipolytic activities and muscle metabolic pattern. The relationship between metabolic pattern and the content and location of muscle lipids has been a cause of controversy. Some authors suggest the existence of a relationship between allocation of triacylglycerols in function of the metabolic muscle type and the hydrolytic changes during the refrigeration of the meat (Alasnier et al., 2000a; Alasnier et al., 2000b; Herna´ndez et al., 1998). These authors put forward the hypothesis that intrafibre triacylglycerols could be more readily hydrolysed than triacylglycerols of adipose cells located between fibres because they are an in situ reserve of fatty acids quickly mobilised to supply energy. The influence of the metabolic muscle type on lipid composition and on the levels of enzyme activities could result in different concentration of flavour precursors and, consequently, different flavour development (Toldra´, Flores, & Aristoy, 1995) that could presumably affect the aptitude to conservation under refrigeration. The aim of the present work was to evaluate the lipolytic and oxidative changes in three muscles of different metabolic pattern from free-range reared Iberian pigs occurred during storage at +4 C for 10 days.
2. Material and methods 2.1. Animals Ten Iberian pigs ( 50 kg live weight) were free-range reared during autumn season, being fed on grass and a concentrate feed based on cereals without incorporation of any animal source of protein or fat. Feed analysis (AOAC, 1984) showed a protein content of 16.21 g/100 g dry matter (d.m.) and a fat content of 2.68 g/100 g d.m. The fatty acid composition of feed (expressed as percentage of total fatty acids identified) was as follows: palmitic acid (C16): 14.16%; stearic acid (C18): 3.13%; oleic acid (C18:1, n-9): 26.56%, linoleic acid (C18:2, n-6): 51.30% and linolenic acid (C18:3, n-3): 1.65%. Pigs were stunned and slaughtered at the end of the fattening period at a live weight of 85–90 kg. 2.2. Sampling In basis of myoglobin content of muscles (See Cava, Este´vez, Ruiz, & Morcuende, 2003), m. Masseter (oxidative metabolism), m. Longissimus dorsi and m. Serratus ventralis (glycolytic metabolism) were dissected from the carcass and freed of visible fat. Muscle slices (1.5 cm thick) were over-wrapped in PVC film and placed on Styrofoam meat trays and stored at +4 C under fluorescent light for 10 days. 2.3. Analytical methods Total intramuscular lipids from muscle were extracted according to the method described by Bligh and Dyer (1959), and the dry lipid extracts were weighed to estimate the lipid content. From the lipid extracted ( 30 mg lipids), the neutral lipid (NL) and polar lipid (PL) and free fatty acids (FFA) fractions were isolated using 100 mg amino-propyl minicolumns according to the method developed by Garcı´a-Regueiro, Gilbert, and Dı´az (1994). Fatty acid methyl esters (FAMEs) of lipid fractions were prepared by sterification in the presence of sulphuric acid (Cava et al., 1997). FAMEs were determined using a Hewlett-Packard (model HP-5890A) gas chromatograph, equipped with a flame ionisation detector (FID). FAMEs were separated on a HewlettPackard HP FFAP-TPA fused-silica column (30 m length0.53 mm i.d.1.0 mm film thickness). The injector and detector were maintained at 230 C. Column oven temperature was maintained at 220 C. The carrier gas was nitrogen at a flow rate of 1.8 ml/min. Identification of FAMEs was based on retention times of reference compounds (Sigma). Muscle phospholipid content were analysed according to Barlett (1959). Neutral lipid content was obtained by subtracting to intramuscular fat content the phospholipid
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content. Determination of TBARS numbers was done following the method described by Salih, Smith, Price, and Dawson (1987). 2.4. Hexanal content determination A Solid Phase MicroExtraction, SPME (Supelco Co., Bellefonte, PA) fibre (10 mm length) coated with Carboxen-poly(dimethylsiloxane) (75 mm thickness) was used for the determination of hexanal content of the samples. Prior to analysis the SPME fibre was preconditioned at 280 C for 45 min in the GC injection port. For HS-SPME extraction, muscle samples ham were ground with a commercial grinder. A 0.5-g portion was weighed into a 4-ml screw-capped vial. The fibre was inserted into the sample vial through the septum and then exposed to headspace. The extractions were carried out in an oven to ensure a homogeneous temperature for sample and headspace. Extraction was performed at 37 C for 30 min. Before extraction, samples were equilibrated for 15 min at the same temperature used for extraction. 2.5. Gas chromatography–mass spectrometry and hexanal quantification Analyses were performed using a Hewlett-Packard 5890 series II gas chromatograph coupled to a mass selective detector (Hewlett-Packard HP-5971 A). Volatiles were separated using a 5% phenyl–methyl silicone (HP-5) bonded-phase fused silica capillary column (Hewlett-Packard, 50 m0.32 mm i.d., film thickness 1.05 mm), operating at 6 psi of column head pressure, resulting in a flow of 1.3 ml min 1 at 40 C. The SPME fibre was desorbed and maintained in the injection port at 280 C during the whole chromatographic run. The injection port was in a splitless mode. The temperature program was isothermal for 10 min at 40 C, raised to 200 C at a rate of 5 C min 1, and then raised to 250 C at a rate of 15 C min 1, and held for 5 min. The transfer line to the mass spectrometer (MS) was maintained at 280 C. The mass spectra were obtained using a mass selective detector (Hewlett-Packard HP-5971 A) by electronic impact at 70 eV, a multiplier voltage of 1756 V, and collecting data at a rate of 1 scan s 1 over the m/z range of 30–300. Hexanal was tentatively identified by comparing its mass spectra with the contained in the NIST/EPA/NIH and Wiley libraries and by comparison of Kovats index with that reported in the literature. The quantification of the hexanal content was performed using an external standard. 2.6. Statistical analysis Main effects (muscle and storage) and interaction (musclestorage) were studied following a two-way
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ANOVA with interaction according General Linear Model (SPSS, 1999). When ANOVA was significant means were compared with the aid of Tukey’s test. SPSS 10.0 for Windows statistical package was used for statistical analysis.
3. Results and discussion Fatty acid profiles of neutral, polar and free fatty acid fractions from fresh (day 0) and refrigerated at +4 C (day 10) muscles are shown in Tables 1, 2 and 3, respectively. 3.1. Chemical composition of raw muscles The intramuscular fat, neutral and phospholipid contents in the raw muscles depended on the metabolic type, as previously reported Cava et al. (2003). Phospholipid content was 0.33 g/g imf in the m. Masseter, 0.12 g/g imf in the m. L. dorsi and 0.19 g/g imf in the m. S. ventralis. Whereas the content of neutral lipids was 0.67, 0.88 and 0.81 g/g imf in the m. Masseter, m. L. dorsi and m. S. ventralis, respectively. In the same way, metabolic type of the muscle significantly affected the fatty acid profiles of neutral, phospholipid and free fatty acid fractions. Neutral lipids from the oxidative muscle contained less saturated fatty acids (SFA; 35.5, 40.3 and 38.7% for m. Masseter, m. L. dorsi and m. S. ventralis, respectively), monounsaturated fatty acids (MUFA; 42.8, 48.5 and 46.5% for m. Masseter, m. L. dorsi and m. S. ventralis, respectively) and more polyunsaturated fatty acids (PUFA; 22.1, 11.2 and 14.8% for m. Masseter, m. L. dorsi and m. S. ventralis, respectively) than the glycolytic muscles. Whereas polar lipids from m. Masseter contained the highest SFA proportions (36.4, 32.8 and 33.4% for m. Masseter, m. L. dorsi and m. S. ventralis, respectively) and the lowest MUFA proportions (13.3, 17.9 and 17.8%, for m. Masseter, m. L. dorsi and m. S. ventralis, respectively). Free fatty acid profiles did not show a clear relationship with metabolic type of fibre. The m. Masseter contained less SFA than m. L. dorsi and m. S. ventralis (38.7, 43.8 and 43.2%, respectively), and intermediate percentages of MUFA (40.79, 44.37 and 33.96%, for m. Masseter, m. L. dorsi and m. S. ventralis, respectively) and PUFA (20.98, 11.79 and 22.87%, for m. Masseter, m. L. dorsi and m. S. ventralis, respectively) than glycolytic muscles. Results reported for fatty acid profiles of the different lipid fractions agree with those reported by Cava et al. (1997) and Lesseigneur-Meynier and Gandemer (1991) in pig muscles and Alasnier, Re´mignon, and Gandemer (1996) in rabbit muscles with different oxidative pattern.
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Table 1 Changes in neutral lipid composition in m. Masseter, m. L. dorsi and m. S. ventralis from free-range reared Iberian pigs stored at +4 C for 10 days SEMa
Muscle
Masseter
Days of refrigeration
0
10
0
10
0
10
Muscle
Storage
Interaction
C12 C14 C16 C16:1 n-7 C17 C17:1 n-7 C18 C18:1 n-9 C18:2 n-6 C18:3 n-3 C20 C20:1 n-9 C20:2 n-6 C20:4 n-6
0.3a 1.0b 20.6c 2.6b 0.3a 0.2a 13.2a 38.6b 16.8a 0.4 0.2b 1.0b 0.7a 4.2a
0.1bc 1.1b 23.9ab 3.3ab 0.2ab 0.2ab 10.7b 44.6a 11.6bc 0.3 0.2b 1.4a 0.5ab 1.7b
0.1c 1.3a 24.6ab 3.5a 0.2b 0.2a 13.9a 43.8a 9.5cd 0.4 0.2b 1.0b 0.4b 1.0bc
0.1bc 1.3a 25.6a 3.5a 0.2b 0.2a 14.2a 43.8a 8.4d 0.3 0.3ab 1.0b 0.4b 0.7c
0.2ab 1.3a 23.5b 3.4ab 0.2ab 0.2a 13.3a 42.0ab 12.3b 0.4 0.2b 0.9b 0.4b 1.7b
0.1c 1.3a 25.1a 3.6a 0.2b 0.3a 13.2a 43.4a 9.5cd 0.4 0.3a 1.0b 0.4b 1.2bc
0.002 0.000 0.000 0.013 0.001 0.015 0.000 0.054 0.000 0.612 0.000 0.000 0.000 0.000
0.000 0.267 0.000 0.073 0.080 0.005 0.023 0.001 0.000 0.009 0.004 0.002 0.197 0.000
0.001 0.889 0.013 0.185 0.269 0.055 0.001 0.003 0.015 0.876 0.051 0.000 0.082 0.000
0.01 0.03 0.29 0.09 0.01 0.01 0.22 0.44 0.48 0.01 0.01 0.03 0.02 0.19
SFA MUFA PUFA MUFA/SFA PUFA/SFA MUFA/PUFA
35.6d 42.4b 22.1a 1.2b 0.6a 2.0c
36.3cd 49.5a 14.2bc 1.4a 0.4b 3.7b
40.3ab 48.5a 11.1cd 1.2ab 0.3d 4.4ab
41.7a 48.5a 9.8d 1.2b 0.2d 5.2a
38.7bc 46.5ab 14.8b 1.2ab 0.4bc 3.2bc
40.4ab 48.2a 11.4bcd 1.2ab 0.3cd 4.4ab
0.000 0.052 0.000 0.060 0.000 0.000
0.011 0.001 0.000 0.201 0.000 0.000
0.704 0.002 0.001 0.024 0.001 0.339
0.40 0.52 0.68 0.02 0.02 0.19
L. dorsi
P values
S. ventralis
Mean with different letters (a–c) differ significantly. Results expressed as percentage of total methyl esters identified. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. a Standard error of the mean.
Table 2 Changes in phospholipid composition in m. Masseter, m. L. dorsi and m. S. ventralis from free-range reared Iberian pigs stored at +4 C for 10 days SEMa
Muscle
Masseter
Days of refrigeration
0
10
0
10
0
10
Muscle
Storage
Interaction
C14 C16 C16:1 n-7 C17 C17:1 n-7 C18 C18:1 n-9 C18:2 n-6 C18:3 n-3 C20:4 n-6
0.4b 16.9a 0.7c 0.6b 0.6 18.4a 12.0b 33.1d 0.5ab 16.6c
0.4b 4.5c 2.2ab 1.3a 0.4 11.9b 9.2c 37.7bc 0.5ab 31.8a
0.4b 18.5a 1.0bc 0.7b 0.6 13.2b 16.3a 36.1cd 0.5b 12.8d
0.6ab 11.0b 2.7a 1.2a 0.7 8.2c 14.2ab 41.3ab 0.7ab 19.4b
0.5b 17.6a 1.0bc 0.8b 0.6 14.5b 16.2a 36.4cd 0.5ab 11.9d
0.9a 6.0c 2.9a 1.6a 0.7 11.4bc 13.0b 43.3a 0.8a 19.5b
0.005 0.000 0.243 0.036 0.453 0.000 0.000 0.000 0.339 0.000
0.010 0.000 0.000 0.000 0.876 0.000 0.000 0.000 0.004 0.000
0.082 0.000 0.794 0.259 0.493 0.168 0.646 0.434 0.104 0.000
0.04 0.84 0.17 0.06 0.07 0.57 0.42 0.62 0.03 0.98
SFA MUFA PUFA MUFA/SFA PUFA/SFA MUFA/PUFA
36.4a 13.3b 50.3c 0.4c 1.4c 0.3b
18.2b 11.8b 70.0a 0.7ab 4.1a 0.2a
32.8a 17.9a 49.4c 0.6bc 1.5c 0.4a
20.9b 17.6a 61.4b 0.9a 3.0b 0.3b
33.4a 17.8a 48.8c 0.5bc 1.5c 0.4a
19.8b 16.6a 63.6b 0.9a 3.3b 0.3b
0.746 0.000 0.000 0.003 0.035 0.000
0.000 0.117 0.000 0.000 0.000 0.000
0.003 0.672 0.001 0.993 0.006 0.658
1.14 0.46 1.25 0.03 0.17 0.01
C16 DMA C18 DMA C18:1 DMA DMA/FA (100)
56.8a 23.3d 19.9c 18.4b
46.6c 31.2a 22.2bc 37.3a
50.4b 25.6cd 24.1ab 20.6b
50.0b 28.7b 21.4c 35.2a
51.0b 24.4d 24.6ab 20.6b
46.8c 27.1bc 26.1a 37.8a
0.001 0.030 0.000 0.415
0.000 0.000 0.478 0.000
0.000 0.000 0.000 0.129
0.58 0.46 0.38 1.33
L. dorsi
P values
S. ventralis
C12, C20, C20:1 and C20:2: traces. Means with different letters (a–c) differ significantly. Results expressed as percentage of total methyl esters identified. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; DMA, dimethyl acetal; FA, fatty acids. a Standard error of the mean.
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Table 3 Changes in free fatty acid composition in m. Masseter, m. L. dorsi and m. S. ventralis from free-range reared Iberian pigs stored at +4 C for 10 days SEMa
Muscle
Masseter
Days of refrigeration
0
10
0
10
0
10
Muscle
Storage
Interaction
C14 C16 C16:1 n-7 C17 C17:1 n-7 C18 C18:1 n-9 C18:2 n-6 C18:3 n-3 C20:4 n-6
1.4b 23.1b 3.2a 0.5b 0.5a 13.9bc 37.1abc 16.0b 0.9a 3.6ab
0.5c 26.5a 1.8b 0.7a 0.2b 24.4a 21.2d 20.8a 0.4c 3.4ab
1.8a 26.6a 2.9a 0.5ab 0.3b 15.1bc 41.3ab 9.7c 0.5bc 1.6c
0.8c 17.8c 3.2a 0.3b 0.2b 12.7c 44.4a 17.6bc 0.5bc 2.6bc
1.6ab 26.3a 2.8a 0.6ab 0.4a 14.8bc 30.7b 18.3bc 0.8ab 3.7a
0.8c 20.8bc 2.7a 0.5b 0.2b 15.7b 35.4bc 20.7a 0.6abc 2.7abc
0.000 0.003 0.004 0.014 0.047 0.000 0.000 0.000 0.049 0.000
0.000 0.000 0.003 0.944 0.000 0.000 0.072 0.000 0.002 0.649
0.349 0.000 0.000 0.002 0.026 0.000 0.000 0.024 0.087 0.002
0.07 0.54 0.09 0.03 0.02 0.61 1.28 0.63 0.05 0.14
SFA MUFA PUFA MUFA/ SFA PUFA/SFA MUFA/PUFA
38.7bc 40.8abc 20.5a 1.1b 0.5ab 2.2b
52.2a 23.2d 24.7a 0.5c 0.5b 0.9c
43.8b 44.4ab 11.8b 1.0b 0.3c 4.2a
31.6d 47.8a 20.6a 1.5a 0.7a 2.4b
43.2b 34.0c 22.9a 0.8b 0.5ab 1.5bc
37.7c 38.4bc 24.0a 1.1b 0.6a 1.7bc
0.000 0.000 0.000 0.000 0.001 0.000
0.164 0.037 0.000 0.364 0.000 0.000
0.000 0.000 0.008 0.000 0.000 0.006
1.04 1.35 0.73 0.05 0.02 0.18
L. dorsi
P values
S. ventralis
C12, C20, C20:1 and C20:2: traces. Means with different letters (a–c) differ significantly. Results expressed as percentage of total methyl esters identified. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. a Standard error of the mean.
3.1.1. Oxidative and lipolytic changes during refrigerated storage Lipolytic and oxidative changes after 10 days at +4 C affected initial phospholipid contents in a great extent ( 1.3-fold times lower in the three muscles), being statistically significant only in m. Masseter (0.33 vs. 0.25 g PLs/g imf, P=0.035) (Fig. 1). Numerous papers describe the importance of oxidative and hydrolytic processes of polar lipids during refrigeration (Alasnier & Gandemer, 1998; Alasnier et al., 2000a; Alasnier et al., 2000b; Currie & Wolfe, 1977; Sklan, Rubin, & Wood, D.F. 1983) or curing processes (Buscailhon, Gandemer, & Monin, 1994; Martı´n, Co´rdoba, Ventanas, & Antequera, 1999),
Fig. 1. Changes in phospholipid content in m. Masseter, m. L. dorsi and m. S. ventralis from free-range reared Iberian pigs stored at +4 C for 10 days. a,b: Different letters in the same days denote significant statistical differences among muscles (P <0.05).
which are responsible of the decrease of PL content due to fatty acid oxidation and release of free fatty acids, highly susceptible to oxidative processes as result of their high degree of unsaturation. The analysis of fatty acid profiles from neutral lipid fraction showed that these lipids were significantly affected by refrigerated storage; however changes did not affect in the same extent to the three muscles studied, as reflected a significant interaction in ANOVA P values for fatty acids C16, C18:2, C18:3 and C20:4. Refrigerated storage caused a decrease in C18:2 (P=0.023), C18:3 (P=0.009), C20:4 (P=0.000) percentages and total PUFA (P=0.000) with a concomitant increase in C16 percentages (P=0.000) (Table 1). Fatty acid profiles of polar lipids were affected in a different extent depending on the metabolic type of muscles. The changes affected percentages of C16, C18 and total SFA which surprisingly decreased and C18:2, C20:4 and total PUFA which increased at the end of the refrigerated storage (Table 2), contradicting the results of Alasnier et al. (2000b) who described an inverse behaviour, with a decrease of PUFAs and an increase of SFAs. In the polar lipids fraction, the ratio dimethyl acetals/ fatty acids (DMA/FA) significantly increased (P < 0.000) after 10 days of storage at +4 C being 1.7– 2.0 fold time higher than initial values (Table 2). Results indicate a low susceptibility of ether links (glycerol-fatty aldehyde) to hydrolytic processes derived of the activity of phospholipases A that produced an accumulation of fatty aldehydes and a reduction of fatty acids linked to sn-1 position in glycerol molecule.
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Free fatty acid profiles did not show a clear relationship with metabolic type of fibre. FFA fraction from the m. Masseter contained less SFA than the FFA fraction from m. L. dorsi and m. S. ventralis (38.7, 43.8 and 43.2%, respectively), and intermediate percentages of MUFA (40.79, 44.37 and 33.96%, for m. Masseter, m. L. dorsi and m. S. ventralis, respectively) and PUFA (20.98, 11.79 and 22.87%, for m. Masseter, m. L. dorsi and m. S. ventralis, respectively) than the other two muscles. Oxidative changes during refrigeration showed a significant increase (P < 0.05) in TBARS numbers in the three studied muscles (Fig. 2). TBARS numbers increased 2.9-, 1.6- and 1.8-fold times initial values in m. Masseter (0.39 vs. 1.13 mg MDA/kg muscle), m. L. dorsi (0.36 vs. 0.65 mg MDA/kg muscle) and m. S. ventralis (0.37 vs. 0.60 mg MDA/kg muscle) after 10 days at +4 C. The development of oxidation products reactive to TBA in pork during refrigerated storage is widely referred in the scientific bibliography and it is one of the most important factors responsible of reduction of shelf-life of meat and is associated to losses in nutritive and sensory quality of meat (Gray et al., 1996). After 10 days at 4 C, hexanal content increased, but only in a significant extent in m. Masseter, as a result of degradative processes took place on unsaturated lipids, mainly n-6 fatty acids family (C18:2 and C20:4). Results are in agreement with other works in which oxidation processes were monitorised using hexanal content in meat under refrigerated storage. The increase in hexanal content in the volatile fraction of meat is related to the development of rancid flavours in meat (Brewer, Inkins, & Harbers, 1992; Lai, Gray, Boored, Crackel, & Gill, 1995; Larick, Turner, Shoenherr, Coffey, & Pilkington, 1992).
3.1.2. Effect of metabolic type on oxidative and lipolytic processes during refrigerated storage Muscle type significantly affected changes in fatty acid profiles from neutral, polar lipids and free fatty acid fractions during refrigerated storage. In neutral lipids, C18:2 and C20:4 and total PUFA sharply decreased during refrigeration in m. Masseter, being less intense in m. S. ventralis; while neutral lipids from m. L. dorsi were not modified (Table 1). For m. Masseter, C18:2 and C20:4 percentages decreased from 16.79 to 11.73% (P < 0.05) ( 30.1%) and from 4.23 to 1.70% (P < 0.05) ( 59.8%), respectively. In m. S. ventralis C18:2 and C20:4 decrease accounted from 12.32 to 9.52% (P < 0.05) ( 22.7%) and from 1.65 to 1.15% (P < 0.05) ( 30.3%), respectively. Differences in the degree of triacylglyceride hydrolysis among type of muscles could be attributable both differences in lipolytic enzyme activities (acid and neutral lipases) and the different location of triacylglyceride depots in muscle. Recently, Herna´ndez et al. (1998) studying post-mortem activities of lipolytic enzymes in skeletal muscles reported a higher lipolytic activity of neutral and acid lipases and acid phospholipase in an oxidative muscle like m. Triceps brachii than in glycolytic (m. L. dorsi) or intermediate muscle (m. Biceps femoris). Furthermore, location of triacylglycerols in oxidative muscles is in droplets inside cell while fat depots in glycolytic muscles from fatty animals is between muscle fascicles. Intracellular triacylglycerols are more easily hydrolysable due to the presence of neutral lipase and lisosomal acid lipase and hormone sensible lipase than those from interfibrillar adipose tissue in which only hormone sensible hormone is present
Fig. 2. Changes in TBARS numbers and hexanal content in m. Masseter, m. L. dorsi and m. S. ventralis from free-range reared Iberian pigs stored at +4 C for 10 days. a,b: Different letters in the same days denote significant statistical differences among muscles (P <0.05).
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(Herna´ndez et al., 1998; Osca¨i, Essig, & Palmer 1992). That is a result of a different functionality of fatty depots, ‘in situ’ stocks easily mobilisable to provide substrates for b-oxidation in oxidative muscles and energy stocks in glycolytic muscles rarely used in their metabolism (Coppack, Jensen, & Miles, 1994). Fatty acid composition from phospholipids changed significantly after 10 days at +4 C. It is noticeable a higher reduction in palmitic acid (C16) percentages in m. Masseter than in m. L. dorsi (16.95% vs. 4.54%) (P < 0.05) ( 73.2%) and 18.47% vs. 10.95% (P < 0.05) ( 40.71%), respectively), and an inverse trend for stearic acid (C18) (18.43% vs. 11.94% (P < 0.05) ( 35.2%) and 13.22% vs 8.21% (P < 0.05) ( 37.9%, respectively), while for m. S. ventralis showed an intermediate values (17.63% vs. 5.96% (P < 0.05) ( 66.2%) for C16 and 14.53% vs. 11.37% (P < 0.05) ( 21.74%) for C18) (Table 2). Reduction in phospholipid contents was significantly affected by muscle type (P < 0.05). After 10 days of refrigeration, polar lipids from m. Masseter decreased significantly (P=0.035) (0.33 vs. 0.25 g PLs/g imf), while did not were statistically significant in m. L. dorsi (P=0.216) (0.12 vs. 0.09 g PLs/g imf) and m. Serratus ventralis (P=0.142) (0.19 vs 0.14 g PLs/g imf) (Fig. 1). These results suggest a higher activity of phospholipases A in oxidative muscles than in glycolytic muscles, according to previous findings of Curie and Wolfe (1977), Sklan, Tenne, and Budowski (1983) and Alasnier et al. (2000a); Alasnier et al. (2000b). In this sense, a higher phospholipase A activity in oxidative muscles than in glycolytic ones ( 10- to 25-fold times higher in rabbit muscles and 1.5-fold times in pig muscles) has been reported (Alasnier & Gandemer, 2000; Herna´ndez et al., 1998). These marked differences are a result of a higher activity of these enzyme in maintenance of cellular membrane integrity in the living muscle, repairing membrane polar lipids and regulating fatty acid composition (Ackerman & Dennis, 1995; Van den Bosch, 1980). Free fatty acid fraction at the end of refrigerated storage showed great differences as affected by muscle metabolic pattern. FFA in m. Masseter showed a significant higher proportion of total saturated fatty acids, palmitic and stearic acids than m. L. dorsi and m. S. ventralis. These results suggest a higher degradation of long chain polyunsaturated fatty acids by oxidative processes and the accumulation of saturated fatty acids in the m. Masseter, which are in accordance with the TBARS and hexanal content measured. Polyunsaturated fatty acids released from glycerol molecule by lipases are highly prone to oxidative degradation, increasing the oxidative measurements (Nawar, 1996). TBARS numbers after 10 days of refrigeration at 4 C depended on muscle type. TBARS numbers were sig-
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nificantly higher (P < 0.000) in the oxidative muscle than in the glycolytic muscles (1.13 vs. 0.65 and 0.60 mg MDA/kg muscle, for m. Masseter, m. L. dorsi and m. S. ventralis, respectively (Fig. 2). Hexanal content in refrigerated samples showed a significant (P < 0.05) higher content in the m. Masseter than in m. L. dorsi and m. S. ventralis (689.2, 241.2 and 355.8 mg/g, respectively). These results corroborate the higher measured values of malondialdehyde in the TBARS test. The relationship between TBARS numbers and hexanal content in the volatile fraction of the meat has been described by numerous authors (Lai et al., 1995; Shahidi et al., 1987). Our findings are in agreement with those described in previous papers confirming the higher tendency of oxidative muscles to oxidative deterioration than in glycolytic ones (Alasnier et al., 2000a; Alasnier et al., 2000b; Cava, Ruiz, Ventanas, & Antequera, 1999; Sklan et al., 1983). The higher oxidation in the m. Masseter could be attributable to a higher degree of hydrolysis of phospholipid—rich in polyunsaturated fatty acids, (see Table 2 and Fig. 1)—and triacylglyceride fractions—with a sharp decrease of C18:2 and mainly C20:4, (see Table 1)—that release fatty acids highly susceptible to oxidation together a higher content of heme pigments (see Cava et al., 2003) which are implied in the initiation and propagation of lipid oxidation phenomena. Linoleic acid is the major precursor of hexanal (Meynier, Genot, & Gandemer, 1999). Our results are in agreement with findings of Larick et al. (1992) who found an increase in the hexanal content in the volatile fraction of pork as a function of linoleic acid muscle content. In conclusion, meat shelf-life depends on its metabolic pattern among other factors. Muscles with an oxidative metabolism have a lower shelf-life than muscles with a glycolytic or intermediate metabolism, even under identical storage conditions. These differences are attributable to a different fat content, fatty acid profile of lipid fractions and prooxidant substances content. In muscles from pigs with a high tendency to fat accumulation and a high content of heme pigments, like Iberian pig muscles, these effects could be more intense than in lean pigs.
Acknowledgements Funded by a grant from Junta de Extremadura in the I Plan Regional de Investigacio´n (IPRD99001, ‘‘Establecimiento de para´metros predictores de la calidad de carne fresca de cerdo Ibe´rico y Alentejano destinada a la elaboracio´n de productos ca´rnicos curados y al consumo en fresco’’). The authors are grateful to AECERIBER and Miss Elena Die´guez and Mr Pedro Can˜uelo for providing the pigs. The authors also wish to thank Miss Ana Galaz and Miss Inmaculada Linares for their excellent technical assistance.
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References Ackerman, E. J., & Dennis, E. A. (1995). Mammalian calcium-independent phospholipase A2. Biochimica and Biophysica Acta, 1259, 125. Addis, P. B., & Park, S. W. (1989). A perspective on the relative risk. In S. L. Taylor, & R. A. Scanlan (Eds.), Food toxicology (pp. 297– 330). New York: Marcel Dekker. Alasnier, C., David-Briand, E., & Gandemer, G. (2000a). Lipolysis in muscles during refrigerated storage as related to the metabolic type of the fibres in the rabbit. Meat Science, 54, 127–134. Alasnier, C., & Gandemer, G. (1998). Fatty acid and aldehyde composition of individual phospholipid classes of rabbit skeletal muscles is related to the metabolic type of the fibre. Meat Science, 48, 225–235. Alasnier, C., & Gandemer, G. (2000). Activities of phospholipases A and lysophospholipases in glycolytic and oxidative skeletal muscles in the rabbit. Journal of the Science of Food and Agriculture, 80, 698–704. Alasnier, C., Meynier, A., Viau, M., & Gandemer, G. (2000b). Hydrolytic and oxidative changes in the lipids of chicken breast and thigh muscles during refrigerated storage. Journal of Food Science, 65, 9–14. Alasnier, C., Re´mignon, H., & Gandemer, G. (1996). Lipid characteristics associated with oxidative and glycolytic fibres in rabbit muscles. Meat Science, 43, 213–224. AOAC. (1984). S. Williams (Ed.). Association of Official Analytical Chemists. Official methods of analysis. Arlington, VA: Association of Official Analytical Chemists. Barlett, G. R. (1959). Phosphorus assay in column chromatography. Journal of Biological Chemistry, 234, 466–468. Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canandian Journal of Biochemistry and Physiology, 37, 911–917. Brewer, M. S., Inkins, W. G., & Harbers, C. A. Z. (1992). TBA values, sensory characteristics, and volatile in ground pork during longterm frozen storage: effects of packaging. Journal of Food Science, 57, 558–563. Buscailhon, S., Gandemer, G., & Monin, G. (1994). Time-related changes in intramuscular lipids of French dry-cured ham. Meat Science, 37, 245–255. Cava, R., Este´vez, M., Ruiz, J., & Morcuende, D. (2003). Physicochemical characteristics of three muscles from free-range reared Iberian pigs slaughtered at 90kg live weight. Meat Science, 63, 533– 541. Cava, R., Ruiz, J., Lo´pez-Bote, C., Martı´n, L., Garcı´a, C., Ventanas, J., & Antequera, T. (1997). Influence of finishing diet on fatty acid profiles of intramuscular lipids, triglycerides and polar lipids in muscles of the Iberian pig. Meat Science, 45, 263–270. Cava, R., Ruiz, J., Ventanas, J., & Antequera, T. (1999). Oxidative and lipolytic changes during ripening of Iberian hams as affected by feeding regime: extensive feeding and alpha-tocopheryl acetate supplementation. Meat Science, 52, 165–172. Coppack, S. W., Jensen, M. D., & Miles, J. M. (1994). In vivo regulation of lipolysis in humans. Journal of Lipid Research, 36, 85–101. Currie, R. W., & Wolfe, F. H. (1977). Evidence for differences in postmortem intramuscular phospholipase activity in several muscle type. Meat Science, 1, 185–193. Flores, M., Alasnier, C., Aristoy, M. C., Navarro, J. L., Gandemer, G., & Toldra´, F. (1996). Activity of aminopeptidase and lipolytic enzymes in five skeletal muscles with various oxidative patterns. Journal of the Science of Food and Agriculture, 70, 127–130. Garcı´a-Regueiro, J. A., Gilbert, J., & Dı´az, I. (1994). Determination of neutral lipids from subcutaneous fat of cured ham by capillary
gas chromatography and liquid chromatography. Journal of Chromatography A, 667, 225–233. Gray, J. I., Gomaa, E. A., & Buckley, D. J. (1996). Oxidative quality and Shelf life of meats. Meat Science, 43, S111–S123. Herna´ndez, P., Navarro, J. L., & Toldra´, F. (1998). Lipid composition and lipolytic enzyme activities in porcine skeletal muscles with different oxidative pattern. Meat Science, 49, 1–10. Kanner, J. (1994). Oxidative processes in meat and meat products: quality implications. Meat Science, 36, 169–189. Kubow, S. (1992). Routes of formation and toxic consequences of lipid oxidation products in foods. Free Radical Biology Medicine, 12, 63–81. Lai, S. M., Gray, J. I., Boored, A. M., Crackel, R. L., & Gill, J. L. (1995). Assessment of off-flavor development in restructured chicken nuggets using hexanal and, TBA-RS measurements and sensory evaluation. Journal of the Science of Food and Agriculture, 67, 447–452. Larick, D. K., Turner, B. E., Shoenherr, W. D., Coffey, M. T., & Pilkington, D. H. (1992). Volatile compound content and fatty acid composition of pork as influenced by linoleic acid content of the diet. Journal of Animal Science, 70, 1397–1403. Leseigneur-Meynier, A., & Gandemer, G. (1991). Lipid composition of pork muscle in relation to the metabolic type of the fibres. Meat Science, 29, 229–241. Martı´n, L., Co´rdoba, J. J., Ventanas, J., & Antequera, T. (1999). Changes in intramuscular lipids during ripening of Iberian drycured ham. Meat Science, 51, 129–134. Meynier, C., Genot, C, & Gandemer, G. (1999). Oxidation of muscle phospholipids in relation to their fatty acid composition with emphasis on volatile compounds. Journal of the Science of Food and Agriculture, 79, 797–804. Monin, G., Horto´s, M., Diaz, I., Tibau, J., Diesdre, A., & Garcı´aRegueiro, J. A. (1998). Lipolysis during chilled storage of pork. In Proceedings of the 44th ICOMST (B126, pp. 748–749). Barcelona, Spain, 30 August–4 September. Morrissey, P. A., Sheehy, P. J. A., Galvin, K., Kerry, J. P., & Buckley, D. J. (1998). Lipid stability in meat and meat products. Meat Science, 49, S73–S86. Nawar, W. W. (1996). Lipids. In O. R. Fennema (Ed.), Food chemistry (pp. 225–319). New York: Marcel Dekker. Osca¨i, L. B., Essig, D. A., & Palmer, W. K. (1990). Lipase regulation of muscle triglyceride hydrolysis. Journal of Applied Physiology, 69, 1571–1577. Salih, A. M., Smith, D. M., Price, J. F., & Dawson, L. E. (1987). Modified extraction 2-thiobarbituric acid method for measuring lipid oxidation in poultry. Poultry Science, 66, 1483–1489. Shahidi, F., Rubin, L. J., & Wood, D. F. (1987). Control of lipid oxidation in cooked ground pork with antioxidants and dinitrosyl ferrohemochrome. Journal of Food Science, 52, 564–567. Sklan, D., Tenne, Z., & Budowski, P. (1983). Simultaneous lipolytic and oxidative changes in turkey meat stored at different temperatures. Journal of the Science of Food Agriculture, 34, 93–99. SPSS. (1999). SPSS Base 10.0. user manual: application guide. Republic of Ireland: SPSS Inc. Toldra´, F., Flores, M., & Aristoy, M. C. (1995). Enzyme generation in free amino acids and its nutritional significance in processed pork meat. In G. Charalambous (Ed.), Food flavours: generation, analysis and processed influence (pp. 1303–1322). Amsterdam: Elsevier Science Publishers. Valin, B. R., Touraille, C., Vigneron, P., & Ashmore, C. R. (1982). Prediction of lamb meat quality traits based on muscle biopsy fibre typing. Meat Science, 6, 257–263. Van den Bosch, H. (1980). Intracellular phospholipases A. Biochimica and Biophysica Acta, 4, 191–246.
Oxidative and lipolytic deterioration of different muscles from free-range reared Iberian pigs under refrigerated storage David Morcuende, Mario Este´vez, Jorge Ruiz, Ramo´n Cava* Tecnologı´a de los Alimentos. Facultad de Veterinaria, Universidad de Extremadura, Campus Universitario, Ca´ceres 10071, Spain Received 9 April 2002; received in revised form 10 February 2003; accepted 11 February 2003
Abstract Three different type of muscles, two glicolytic (Serratus ventralis and Longissimus dorsi) and one oxidative (Masseter) were displayed under refrigeration at +4 C during 10 days to evaluate differences in lipolytic and oxidative changes of different muscles with different metabolic pattern. Thiobarbituric acid reactive substances (TBARS), phospholipid content, hexanal content and fatty acid profiles of neutral and polar lipid fractions were analysed at day 0 and day 10. Phospholipid content (g phospholipids/g intramuscular fat) significantly (P< 0.035) decreased from day 0 to day 10 in m. Masseter (0.33 vs. 0.25, respectively), but not in m. L. dorsi (0.12 vs. 0.09, respectively) and m. S. ventralis (0.19 vs. 0.14, respectively). Changes in fatty acid profiles of neutral and polar lipids significantly differed among muscles after storage. Slight differences were found in neutral lipids from m. L. dorsi and m. S. ventralis, while neutral lipids from m. Masseter were highly altered. Great changes affected fatty acid profiles from polar lipids in the three muscles. m. Masseter muscle showed significantly higher (P< 0.000) TBARS values (1.13, 0.65 and 0.60 mg MDA/kg meat, respectively) and hexanal content (689.2, 241.2 and 355.8 mg/g meat, respectively) than m. L. dorsi and m. S. ventralis. In conclusion, oxidative meat is more prone to oxidative and lipolytic deterioration than glycolytic muscles during refrigerated storage and as a consequence of that a lower shelf-life. # 2003 Elsevier Ltd. All rights reserved. Keywords: Lipolysis; Lipid oxidation; Muscle type; Pork; Free-range rearing
1. Introduction Meat is refrigerated to increase its shelf-life and reduce microbial spoilage. However, even meat is stored under low temperatures its shelf-life is limited due to biochemical changes developed after slaughtering in which lipolysis and lipid oxidation of muscle lipids play an important role (Gray, Gomaa & Buckley 1996; Kanner, 1994; Morrissey, Sheehy, Galvin, Kerry & Buckley, 1998). Some specific enzymes of the adipose tissue and muscle fibres, named lipases, sterases, phospholipases and lysophospholipases, are involved in the hydrolytic processes of muscle lipids during refrigeration (Alasnier & Gandemer, 2000; Flores, Alasnier, Aristoy, Navarro,
* Corresponding author. Tel.: +34-927-257-169; fax: +34-927257-110. E-mail address: [email protected] (R. Cava). 0309-1740/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0309-1740(02)00344-3
Gandemer, & Toldra´, 1996; Herna´ndez, Navarro, & Toldra´, 1998; Monin, Horto´s, Diaz, Tibau, Diesdre, & Garcı´a-Regueiro, 1998) that release free fatty acids from both triacylglycerols and phospholipids (Alasnier, David-Briand, & Gandemer, 2000a; Alasnier, Meynier, Viau, & Gandemer, 2000b). Lipolysis has been considered as a promoter of lipid oxidation due to released free fatty acids that are very prone to lipid peroxidation, particularly long chain unsaturated free fatty acids (Nawar, 1996). Lipid oxidation in meat begins to develop soon after death and continues to increase on intensity until the meat becomes unacceptable to consumers due to adverse changes in meat flavour and meat colour produced by pigment oxidation. In addition to the implication of lipid oxidation for changes in meat flavours and colour, the oxidation of unsaturated lipids and cholesterol results in a significant generation of toxin and losses in nutritional value due to vitamins and essential fatty acid oxidation (Addis & Park, 1989; Gray et al., 1996; Kubow, 1992).
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The metabolic type of the fibres is a major factor involved in the heterogeneity of muscle quality within a carcass. In fact, several studies point out that oxidative muscles are more tasty and juicy and their lipids oxidise faster (Valin, Touraille, Vigneron, & Ashmore, 1982). This is partly related to their higher polyunsaturated fatty acid content (Alasnier & Gandemer, 1998). Moreover, oxidative muscles content more haem pigment and other prooxidants and their membrane lipids contain higher amounts of polyunsaturated fatty acids, factors that increase their susceptibility to lipid oxidation (Kanner, 1994). In reference to enzyme activity, some authors found differences in the lipolytic enzyme activities between oxidative and glycolytic muscles. Oxidative muscles exhibit a higher acid lipase, neutral lipase (Herna´ndez et al., 1998) phospholipase A and lysophospholipase activities than glycolytic muscles and consequently, oxidative muscles had a higher potential activity for post-mortem hydrolysis of phospholipids (Alasnier & Gandemer, 2000). According to the authors, this was consistent with the higher phospholipid content in oxidative muscles than in glycolytic muscles and with the involvement of phospholipases in the maintenance of membrane integrity by renewing damaged phospholipids. However, phospholipid hydrolysis occurrs at similar rate in both type of muscles during refrigeration, suggesting that there are differences in the regulation of phospholipase A activity in oxidative and glycolytic muscles. On the other hand, Flores et al. (1996) did not find a clear correlation between lipolytic activities and muscle metabolic pattern. The relationship between metabolic pattern and the content and location of muscle lipids has been a cause of controversy. Some authors suggest the existence of a relationship between allocation of triacylglycerols in function of the metabolic muscle type and the hydrolytic changes during the refrigeration of the meat (Alasnier et al., 2000a; Alasnier et al., 2000b; Herna´ndez et al., 1998). These authors put forward the hypothesis that intrafibre triacylglycerols could be more readily hydrolysed than triacylglycerols of adipose cells located between fibres because they are an in situ reserve of fatty acids quickly mobilised to supply energy. The influence of the metabolic muscle type on lipid composition and on the levels of enzyme activities could result in different concentration of flavour precursors and, consequently, different flavour development (Toldra´, Flores, & Aristoy, 1995) that could presumably affect the aptitude to conservation under refrigeration. The aim of the present work was to evaluate the lipolytic and oxidative changes in three muscles of different metabolic pattern from free-range reared Iberian pigs occurred during storage at +4 C for 10 days.
2. Material and methods 2.1. Animals Ten Iberian pigs ( 50 kg live weight) were free-range reared during autumn season, being fed on grass and a concentrate feed based on cereals without incorporation of any animal source of protein or fat. Feed analysis (AOAC, 1984) showed a protein content of 16.21 g/100 g dry matter (d.m.) and a fat content of 2.68 g/100 g d.m. The fatty acid composition of feed (expressed as percentage of total fatty acids identified) was as follows: palmitic acid (C16): 14.16%; stearic acid (C18): 3.13%; oleic acid (C18:1, n-9): 26.56%, linoleic acid (C18:2, n-6): 51.30% and linolenic acid (C18:3, n-3): 1.65%. Pigs were stunned and slaughtered at the end of the fattening period at a live weight of 85–90 kg. 2.2. Sampling In basis of myoglobin content of muscles (See Cava, Este´vez, Ruiz, & Morcuende, 2003), m. Masseter (oxidative metabolism), m. Longissimus dorsi and m. Serratus ventralis (glycolytic metabolism) were dissected from the carcass and freed of visible fat. Muscle slices (1.5 cm thick) were over-wrapped in PVC film and placed on Styrofoam meat trays and stored at +4 C under fluorescent light for 10 days. 2.3. Analytical methods Total intramuscular lipids from muscle were extracted according to the method described by Bligh and Dyer (1959), and the dry lipid extracts were weighed to estimate the lipid content. From the lipid extracted ( 30 mg lipids), the neutral lipid (NL) and polar lipid (PL) and free fatty acids (FFA) fractions were isolated using 100 mg amino-propyl minicolumns according to the method developed by Garcı´a-Regueiro, Gilbert, and Dı´az (1994). Fatty acid methyl esters (FAMEs) of lipid fractions were prepared by sterification in the presence of sulphuric acid (Cava et al., 1997). FAMEs were determined using a Hewlett-Packard (model HP-5890A) gas chromatograph, equipped with a flame ionisation detector (FID). FAMEs were separated on a HewlettPackard HP FFAP-TPA fused-silica column (30 m length0.53 mm i.d.1.0 mm film thickness). The injector and detector were maintained at 230 C. Column oven temperature was maintained at 220 C. The carrier gas was nitrogen at a flow rate of 1.8 ml/min. Identification of FAMEs was based on retention times of reference compounds (Sigma). Muscle phospholipid content were analysed according to Barlett (1959). Neutral lipid content was obtained by subtracting to intramuscular fat content the phospholipid
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content. Determination of TBARS numbers was done following the method described by Salih, Smith, Price, and Dawson (1987). 2.4. Hexanal content determination A Solid Phase MicroExtraction, SPME (Supelco Co., Bellefonte, PA) fibre (10 mm length) coated with Carboxen-poly(dimethylsiloxane) (75 mm thickness) was used for the determination of hexanal content of the samples. Prior to analysis the SPME fibre was preconditioned at 280 C for 45 min in the GC injection port. For HS-SPME extraction, muscle samples ham were ground with a commercial grinder. A 0.5-g portion was weighed into a 4-ml screw-capped vial. The fibre was inserted into the sample vial through the septum and then exposed to headspace. The extractions were carried out in an oven to ensure a homogeneous temperature for sample and headspace. Extraction was performed at 37 C for 30 min. Before extraction, samples were equilibrated for 15 min at the same temperature used for extraction. 2.5. Gas chromatography–mass spectrometry and hexanal quantification Analyses were performed using a Hewlett-Packard 5890 series II gas chromatograph coupled to a mass selective detector (Hewlett-Packard HP-5971 A). Volatiles were separated using a 5% phenyl–methyl silicone (HP-5) bonded-phase fused silica capillary column (Hewlett-Packard, 50 m0.32 mm i.d., film thickness 1.05 mm), operating at 6 psi of column head pressure, resulting in a flow of 1.3 ml min 1 at 40 C. The SPME fibre was desorbed and maintained in the injection port at 280 C during the whole chromatographic run. The injection port was in a splitless mode. The temperature program was isothermal for 10 min at 40 C, raised to 200 C at a rate of 5 C min 1, and then raised to 250 C at a rate of 15 C min 1, and held for 5 min. The transfer line to the mass spectrometer (MS) was maintained at 280 C. The mass spectra were obtained using a mass selective detector (Hewlett-Packard HP-5971 A) by electronic impact at 70 eV, a multiplier voltage of 1756 V, and collecting data at a rate of 1 scan s 1 over the m/z range of 30–300. Hexanal was tentatively identified by comparing its mass spectra with the contained in the NIST/EPA/NIH and Wiley libraries and by comparison of Kovats index with that reported in the literature. The quantification of the hexanal content was performed using an external standard. 2.6. Statistical analysis Main effects (muscle and storage) and interaction (musclestorage) were studied following a two-way
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ANOVA with interaction according General Linear Model (SPSS, 1999). When ANOVA was significant means were compared with the aid of Tukey’s test. SPSS 10.0 for Windows statistical package was used for statistical analysis.
3. Results and discussion Fatty acid profiles of neutral, polar and free fatty acid fractions from fresh (day 0) and refrigerated at +4 C (day 10) muscles are shown in Tables 1, 2 and 3, respectively. 3.1. Chemical composition of raw muscles The intramuscular fat, neutral and phospholipid contents in the raw muscles depended on the metabolic type, as previously reported Cava et al. (2003). Phospholipid content was 0.33 g/g imf in the m. Masseter, 0.12 g/g imf in the m. L. dorsi and 0.19 g/g imf in the m. S. ventralis. Whereas the content of neutral lipids was 0.67, 0.88 and 0.81 g/g imf in the m. Masseter, m. L. dorsi and m. S. ventralis, respectively. In the same way, metabolic type of the muscle significantly affected the fatty acid profiles of neutral, phospholipid and free fatty acid fractions. Neutral lipids from the oxidative muscle contained less saturated fatty acids (SFA; 35.5, 40.3 and 38.7% for m. Masseter, m. L. dorsi and m. S. ventralis, respectively), monounsaturated fatty acids (MUFA; 42.8, 48.5 and 46.5% for m. Masseter, m. L. dorsi and m. S. ventralis, respectively) and more polyunsaturated fatty acids (PUFA; 22.1, 11.2 and 14.8% for m. Masseter, m. L. dorsi and m. S. ventralis, respectively) than the glycolytic muscles. Whereas polar lipids from m. Masseter contained the highest SFA proportions (36.4, 32.8 and 33.4% for m. Masseter, m. L. dorsi and m. S. ventralis, respectively) and the lowest MUFA proportions (13.3, 17.9 and 17.8%, for m. Masseter, m. L. dorsi and m. S. ventralis, respectively). Free fatty acid profiles did not show a clear relationship with metabolic type of fibre. The m. Masseter contained less SFA than m. L. dorsi and m. S. ventralis (38.7, 43.8 and 43.2%, respectively), and intermediate percentages of MUFA (40.79, 44.37 and 33.96%, for m. Masseter, m. L. dorsi and m. S. ventralis, respectively) and PUFA (20.98, 11.79 and 22.87%, for m. Masseter, m. L. dorsi and m. S. ventralis, respectively) than glycolytic muscles. Results reported for fatty acid profiles of the different lipid fractions agree with those reported by Cava et al. (1997) and Lesseigneur-Meynier and Gandemer (1991) in pig muscles and Alasnier, Re´mignon, and Gandemer (1996) in rabbit muscles with different oxidative pattern.
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Table 1 Changes in neutral lipid composition in m. Masseter, m. L. dorsi and m. S. ventralis from free-range reared Iberian pigs stored at +4 C for 10 days SEMa
Muscle
Masseter
Days of refrigeration
0
10
0
10
0
10
Muscle
Storage
Interaction
C12 C14 C16 C16:1 n-7 C17 C17:1 n-7 C18 C18:1 n-9 C18:2 n-6 C18:3 n-3 C20 C20:1 n-9 C20:2 n-6 C20:4 n-6
0.3a 1.0b 20.6c 2.6b 0.3a 0.2a 13.2a 38.6b 16.8a 0.4 0.2b 1.0b 0.7a 4.2a
0.1bc 1.1b 23.9ab 3.3ab 0.2ab 0.2ab 10.7b 44.6a 11.6bc 0.3 0.2b 1.4a 0.5ab 1.7b
0.1c 1.3a 24.6ab 3.5a 0.2b 0.2a 13.9a 43.8a 9.5cd 0.4 0.2b 1.0b 0.4b 1.0bc
0.1bc 1.3a 25.6a 3.5a 0.2b 0.2a 14.2a 43.8a 8.4d 0.3 0.3ab 1.0b 0.4b 0.7c
0.2ab 1.3a 23.5b 3.4ab 0.2ab 0.2a 13.3a 42.0ab 12.3b 0.4 0.2b 0.9b 0.4b 1.7b
0.1c 1.3a 25.1a 3.6a 0.2b 0.3a 13.2a 43.4a 9.5cd 0.4 0.3a 1.0b 0.4b 1.2bc
0.002 0.000 0.000 0.013 0.001 0.015 0.000 0.054 0.000 0.612 0.000 0.000 0.000 0.000
0.000 0.267 0.000 0.073 0.080 0.005 0.023 0.001 0.000 0.009 0.004 0.002 0.197 0.000
0.001 0.889 0.013 0.185 0.269 0.055 0.001 0.003 0.015 0.876 0.051 0.000 0.082 0.000
0.01 0.03 0.29 0.09 0.01 0.01 0.22 0.44 0.48 0.01 0.01 0.03 0.02 0.19
SFA MUFA PUFA MUFA/SFA PUFA/SFA MUFA/PUFA
35.6d 42.4b 22.1a 1.2b 0.6a 2.0c
36.3cd 49.5a 14.2bc 1.4a 0.4b 3.7b
40.3ab 48.5a 11.1cd 1.2ab 0.3d 4.4ab
41.7a 48.5a 9.8d 1.2b 0.2d 5.2a
38.7bc 46.5ab 14.8b 1.2ab 0.4bc 3.2bc
40.4ab 48.2a 11.4bcd 1.2ab 0.3cd 4.4ab
0.000 0.052 0.000 0.060 0.000 0.000
0.011 0.001 0.000 0.201 0.000 0.000
0.704 0.002 0.001 0.024 0.001 0.339
0.40 0.52 0.68 0.02 0.02 0.19
L. dorsi
P values
S. ventralis
Mean with different letters (a–c) differ significantly. Results expressed as percentage of total methyl esters identified. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. a Standard error of the mean.
Table 2 Changes in phospholipid composition in m. Masseter, m. L. dorsi and m. S. ventralis from free-range reared Iberian pigs stored at +4 C for 10 days SEMa
Muscle
Masseter
Days of refrigeration
0
10
0
10
0
10
Muscle
Storage
Interaction
C14 C16 C16:1 n-7 C17 C17:1 n-7 C18 C18:1 n-9 C18:2 n-6 C18:3 n-3 C20:4 n-6
0.4b 16.9a 0.7c 0.6b 0.6 18.4a 12.0b 33.1d 0.5ab 16.6c
0.4b 4.5c 2.2ab 1.3a 0.4 11.9b 9.2c 37.7bc 0.5ab 31.8a
0.4b 18.5a 1.0bc 0.7b 0.6 13.2b 16.3a 36.1cd 0.5b 12.8d
0.6ab 11.0b 2.7a 1.2a 0.7 8.2c 14.2ab 41.3ab 0.7ab 19.4b
0.5b 17.6a 1.0bc 0.8b 0.6 14.5b 16.2a 36.4cd 0.5ab 11.9d
0.9a 6.0c 2.9a 1.6a 0.7 11.4bc 13.0b 43.3a 0.8a 19.5b
0.005 0.000 0.243 0.036 0.453 0.000 0.000 0.000 0.339 0.000
0.010 0.000 0.000 0.000 0.876 0.000 0.000 0.000 0.004 0.000
0.082 0.000 0.794 0.259 0.493 0.168 0.646 0.434 0.104 0.000
0.04 0.84 0.17 0.06 0.07 0.57 0.42 0.62 0.03 0.98
SFA MUFA PUFA MUFA/SFA PUFA/SFA MUFA/PUFA
36.4a 13.3b 50.3c 0.4c 1.4c 0.3b
18.2b 11.8b 70.0a 0.7ab 4.1a 0.2a
32.8a 17.9a 49.4c 0.6bc 1.5c 0.4a
20.9b 17.6a 61.4b 0.9a 3.0b 0.3b
33.4a 17.8a 48.8c 0.5bc 1.5c 0.4a
19.8b 16.6a 63.6b 0.9a 3.3b 0.3b
0.746 0.000 0.000 0.003 0.035 0.000
0.000 0.117 0.000 0.000 0.000 0.000
0.003 0.672 0.001 0.993 0.006 0.658
1.14 0.46 1.25 0.03 0.17 0.01
C16 DMA C18 DMA C18:1 DMA DMA/FA (100)
56.8a 23.3d 19.9c 18.4b
46.6c 31.2a 22.2bc 37.3a
50.4b 25.6cd 24.1ab 20.6b
50.0b 28.7b 21.4c 35.2a
51.0b 24.4d 24.6ab 20.6b
46.8c 27.1bc 26.1a 37.8a
0.001 0.030 0.000 0.415
0.000 0.000 0.478 0.000
0.000 0.000 0.000 0.129
0.58 0.46 0.38 1.33
L. dorsi
P values
S. ventralis
C12, C20, C20:1 and C20:2: traces. Means with different letters (a–c) differ significantly. Results expressed as percentage of total methyl esters identified. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; DMA, dimethyl acetal; FA, fatty acids. a Standard error of the mean.
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Table 3 Changes in free fatty acid composition in m. Masseter, m. L. dorsi and m. S. ventralis from free-range reared Iberian pigs stored at +4 C for 10 days SEMa
Muscle
Masseter
Days of refrigeration
0
10
0
10
0
10
Muscle
Storage
Interaction
C14 C16 C16:1 n-7 C17 C17:1 n-7 C18 C18:1 n-9 C18:2 n-6 C18:3 n-3 C20:4 n-6
1.4b 23.1b 3.2a 0.5b 0.5a 13.9bc 37.1abc 16.0b 0.9a 3.6ab
0.5c 26.5a 1.8b 0.7a 0.2b 24.4a 21.2d 20.8a 0.4c 3.4ab
1.8a 26.6a 2.9a 0.5ab 0.3b 15.1bc 41.3ab 9.7c 0.5bc 1.6c
0.8c 17.8c 3.2a 0.3b 0.2b 12.7c 44.4a 17.6bc 0.5bc 2.6bc
1.6ab 26.3a 2.8a 0.6ab 0.4a 14.8bc 30.7b 18.3bc 0.8ab 3.7a
0.8c 20.8bc 2.7a 0.5b 0.2b 15.7b 35.4bc 20.7a 0.6abc 2.7abc
0.000 0.003 0.004 0.014 0.047 0.000 0.000 0.000 0.049 0.000
0.000 0.000 0.003 0.944 0.000 0.000 0.072 0.000 0.002 0.649
0.349 0.000 0.000 0.002 0.026 0.000 0.000 0.024 0.087 0.002
0.07 0.54 0.09 0.03 0.02 0.61 1.28 0.63 0.05 0.14
SFA MUFA PUFA MUFA/ SFA PUFA/SFA MUFA/PUFA
38.7bc 40.8abc 20.5a 1.1b 0.5ab 2.2b
52.2a 23.2d 24.7a 0.5c 0.5b 0.9c
43.8b 44.4ab 11.8b 1.0b 0.3c 4.2a
31.6d 47.8a 20.6a 1.5a 0.7a 2.4b
43.2b 34.0c 22.9a 0.8b 0.5ab 1.5bc
37.7c 38.4bc 24.0a 1.1b 0.6a 1.7bc
0.000 0.000 0.000 0.000 0.001 0.000
0.164 0.037 0.000 0.364 0.000 0.000
0.000 0.000 0.008 0.000 0.000 0.006
1.04 1.35 0.73 0.05 0.02 0.18
L. dorsi
P values
S. ventralis
C12, C20, C20:1 and C20:2: traces. Means with different letters (a–c) differ significantly. Results expressed as percentage of total methyl esters identified. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. a Standard error of the mean.
3.1.1. Oxidative and lipolytic changes during refrigerated storage Lipolytic and oxidative changes after 10 days at +4 C affected initial phospholipid contents in a great extent ( 1.3-fold times lower in the three muscles), being statistically significant only in m. Masseter (0.33 vs. 0.25 g PLs/g imf, P=0.035) (Fig. 1). Numerous papers describe the importance of oxidative and hydrolytic processes of polar lipids during refrigeration (Alasnier & Gandemer, 1998; Alasnier et al., 2000a; Alasnier et al., 2000b; Currie & Wolfe, 1977; Sklan, Rubin, & Wood, D.F. 1983) or curing processes (Buscailhon, Gandemer, & Monin, 1994; Martı´n, Co´rdoba, Ventanas, & Antequera, 1999),
Fig. 1. Changes in phospholipid content in m. Masseter, m. L. dorsi and m. S. ventralis from free-range reared Iberian pigs stored at +4 C for 10 days. a,b: Different letters in the same days denote significant statistical differences among muscles (P <0.05).
which are responsible of the decrease of PL content due to fatty acid oxidation and release of free fatty acids, highly susceptible to oxidative processes as result of their high degree of unsaturation. The analysis of fatty acid profiles from neutral lipid fraction showed that these lipids were significantly affected by refrigerated storage; however changes did not affect in the same extent to the three muscles studied, as reflected a significant interaction in ANOVA P values for fatty acids C16, C18:2, C18:3 and C20:4. Refrigerated storage caused a decrease in C18:2 (P=0.023), C18:3 (P=0.009), C20:4 (P=0.000) percentages and total PUFA (P=0.000) with a concomitant increase in C16 percentages (P=0.000) (Table 1). Fatty acid profiles of polar lipids were affected in a different extent depending on the metabolic type of muscles. The changes affected percentages of C16, C18 and total SFA which surprisingly decreased and C18:2, C20:4 and total PUFA which increased at the end of the refrigerated storage (Table 2), contradicting the results of Alasnier et al. (2000b) who described an inverse behaviour, with a decrease of PUFAs and an increase of SFAs. In the polar lipids fraction, the ratio dimethyl acetals/ fatty acids (DMA/FA) significantly increased (P < 0.000) after 10 days of storage at +4 C being 1.7– 2.0 fold time higher than initial values (Table 2). Results indicate a low susceptibility of ether links (glycerol-fatty aldehyde) to hydrolytic processes derived of the activity of phospholipases A that produced an accumulation of fatty aldehydes and a reduction of fatty acids linked to sn-1 position in glycerol molecule.
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Free fatty acid profiles did not show a clear relationship with metabolic type of fibre. FFA fraction from the m. Masseter contained less SFA than the FFA fraction from m. L. dorsi and m. S. ventralis (38.7, 43.8 and 43.2%, respectively), and intermediate percentages of MUFA (40.79, 44.37 and 33.96%, for m. Masseter, m. L. dorsi and m. S. ventralis, respectively) and PUFA (20.98, 11.79 and 22.87%, for m. Masseter, m. L. dorsi and m. S. ventralis, respectively) than the other two muscles. Oxidative changes during refrigeration showed a significant increase (P < 0.05) in TBARS numbers in the three studied muscles (Fig. 2). TBARS numbers increased 2.9-, 1.6- and 1.8-fold times initial values in m. Masseter (0.39 vs. 1.13 mg MDA/kg muscle), m. L. dorsi (0.36 vs. 0.65 mg MDA/kg muscle) and m. S. ventralis (0.37 vs. 0.60 mg MDA/kg muscle) after 10 days at +4 C. The development of oxidation products reactive to TBA in pork during refrigerated storage is widely referred in the scientific bibliography and it is one of the most important factors responsible of reduction of shelf-life of meat and is associated to losses in nutritive and sensory quality of meat (Gray et al., 1996). After 10 days at 4 C, hexanal content increased, but only in a significant extent in m. Masseter, as a result of degradative processes took place on unsaturated lipids, mainly n-6 fatty acids family (C18:2 and C20:4). Results are in agreement with other works in which oxidation processes were monitorised using hexanal content in meat under refrigerated storage. The increase in hexanal content in the volatile fraction of meat is related to the development of rancid flavours in meat (Brewer, Inkins, & Harbers, 1992; Lai, Gray, Boored, Crackel, & Gill, 1995; Larick, Turner, Shoenherr, Coffey, & Pilkington, 1992).
3.1.2. Effect of metabolic type on oxidative and lipolytic processes during refrigerated storage Muscle type significantly affected changes in fatty acid profiles from neutral, polar lipids and free fatty acid fractions during refrigerated storage. In neutral lipids, C18:2 and C20:4 and total PUFA sharply decreased during refrigeration in m. Masseter, being less intense in m. S. ventralis; while neutral lipids from m. L. dorsi were not modified (Table 1). For m. Masseter, C18:2 and C20:4 percentages decreased from 16.79 to 11.73% (P < 0.05) ( 30.1%) and from 4.23 to 1.70% (P < 0.05) ( 59.8%), respectively. In m. S. ventralis C18:2 and C20:4 decrease accounted from 12.32 to 9.52% (P < 0.05) ( 22.7%) and from 1.65 to 1.15% (P < 0.05) ( 30.3%), respectively. Differences in the degree of triacylglyceride hydrolysis among type of muscles could be attributable both differences in lipolytic enzyme activities (acid and neutral lipases) and the different location of triacylglyceride depots in muscle. Recently, Herna´ndez et al. (1998) studying post-mortem activities of lipolytic enzymes in skeletal muscles reported a higher lipolytic activity of neutral and acid lipases and acid phospholipase in an oxidative muscle like m. Triceps brachii than in glycolytic (m. L. dorsi) or intermediate muscle (m. Biceps femoris). Furthermore, location of triacylglycerols in oxidative muscles is in droplets inside cell while fat depots in glycolytic muscles from fatty animals is between muscle fascicles. Intracellular triacylglycerols are more easily hydrolysable due to the presence of neutral lipase and lisosomal acid lipase and hormone sensible lipase than those from interfibrillar adipose tissue in which only hormone sensible hormone is present
Fig. 2. Changes in TBARS numbers and hexanal content in m. Masseter, m. L. dorsi and m. S. ventralis from free-range reared Iberian pigs stored at +4 C for 10 days. a,b: Different letters in the same days denote significant statistical differences among muscles (P <0.05).
D. Morcuende et al. / Meat Science 65 (2003) 1157–1164
(Herna´ndez et al., 1998; Osca¨i, Essig, & Palmer 1992). That is a result of a different functionality of fatty depots, ‘in situ’ stocks easily mobilisable to provide substrates for b-oxidation in oxidative muscles and energy stocks in glycolytic muscles rarely used in their metabolism (Coppack, Jensen, & Miles, 1994). Fatty acid composition from phospholipids changed significantly after 10 days at +4 C. It is noticeable a higher reduction in palmitic acid (C16) percentages in m. Masseter than in m. L. dorsi (16.95% vs. 4.54%) (P < 0.05) ( 73.2%) and 18.47% vs. 10.95% (P < 0.05) ( 40.71%), respectively), and an inverse trend for stearic acid (C18) (18.43% vs. 11.94% (P < 0.05) ( 35.2%) and 13.22% vs 8.21% (P < 0.05) ( 37.9%, respectively), while for m. S. ventralis showed an intermediate values (17.63% vs. 5.96% (P < 0.05) ( 66.2%) for C16 and 14.53% vs. 11.37% (P < 0.05) ( 21.74%) for C18) (Table 2). Reduction in phospholipid contents was significantly affected by muscle type (P < 0.05). After 10 days of refrigeration, polar lipids from m. Masseter decreased significantly (P=0.035) (0.33 vs. 0.25 g PLs/g imf), while did not were statistically significant in m. L. dorsi (P=0.216) (0.12 vs. 0.09 g PLs/g imf) and m. Serratus ventralis (P=0.142) (0.19 vs 0.14 g PLs/g imf) (Fig. 1). These results suggest a higher activity of phospholipases A in oxidative muscles than in glycolytic muscles, according to previous findings of Curie and Wolfe (1977), Sklan, Tenne, and Budowski (1983) and Alasnier et al. (2000a); Alasnier et al. (2000b). In this sense, a higher phospholipase A activity in oxidative muscles than in glycolytic ones ( 10- to 25-fold times higher in rabbit muscles and 1.5-fold times in pig muscles) has been reported (Alasnier & Gandemer, 2000; Herna´ndez et al., 1998). These marked differences are a result of a higher activity of these enzyme in maintenance of cellular membrane integrity in the living muscle, repairing membrane polar lipids and regulating fatty acid composition (Ackerman & Dennis, 1995; Van den Bosch, 1980). Free fatty acid fraction at the end of refrigerated storage showed great differences as affected by muscle metabolic pattern. FFA in m. Masseter showed a significant higher proportion of total saturated fatty acids, palmitic and stearic acids than m. L. dorsi and m. S. ventralis. These results suggest a higher degradation of long chain polyunsaturated fatty acids by oxidative processes and the accumulation of saturated fatty acids in the m. Masseter, which are in accordance with the TBARS and hexanal content measured. Polyunsaturated fatty acids released from glycerol molecule by lipases are highly prone to oxidative degradation, increasing the oxidative measurements (Nawar, 1996). TBARS numbers after 10 days of refrigeration at 4 C depended on muscle type. TBARS numbers were sig-
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nificantly higher (P < 0.000) in the oxidative muscle than in the glycolytic muscles (1.13 vs. 0.65 and 0.60 mg MDA/kg muscle, for m. Masseter, m. L. dorsi and m. S. ventralis, respectively (Fig. 2). Hexanal content in refrigerated samples showed a significant (P < 0.05) higher content in the m. Masseter than in m. L. dorsi and m. S. ventralis (689.2, 241.2 and 355.8 mg/g, respectively). These results corroborate the higher measured values of malondialdehyde in the TBARS test. The relationship between TBARS numbers and hexanal content in the volatile fraction of the meat has been described by numerous authors (Lai et al., 1995; Shahidi et al., 1987). Our findings are in agreement with those described in previous papers confirming the higher tendency of oxidative muscles to oxidative deterioration than in glycolytic ones (Alasnier et al., 2000a; Alasnier et al., 2000b; Cava, Ruiz, Ventanas, & Antequera, 1999; Sklan et al., 1983). The higher oxidation in the m. Masseter could be attributable to a higher degree of hydrolysis of phospholipid—rich in polyunsaturated fatty acids, (see Table 2 and Fig. 1)—and triacylglyceride fractions—with a sharp decrease of C18:2 and mainly C20:4, (see Table 1)—that release fatty acids highly susceptible to oxidation together a higher content of heme pigments (see Cava et al., 2003) which are implied in the initiation and propagation of lipid oxidation phenomena. Linoleic acid is the major precursor of hexanal (Meynier, Genot, & Gandemer, 1999). Our results are in agreement with findings of Larick et al. (1992) who found an increase in the hexanal content in the volatile fraction of pork as a function of linoleic acid muscle content. In conclusion, meat shelf-life depends on its metabolic pattern among other factors. Muscles with an oxidative metabolism have a lower shelf-life than muscles with a glycolytic or intermediate metabolism, even under identical storage conditions. These differences are attributable to a different fat content, fatty acid profile of lipid fractions and prooxidant substances content. In muscles from pigs with a high tendency to fat accumulation and a high content of heme pigments, like Iberian pig muscles, these effects could be more intense than in lean pigs.
Acknowledgements Funded by a grant from Junta de Extremadura in the I Plan Regional de Investigacio´n (IPRD99001, ‘‘Establecimiento de para´metros predictores de la calidad de carne fresca de cerdo Ibe´rico y Alentejano destinada a la elaboracio´n de productos ca´rnicos curados y al consumo en fresco’’). The authors are grateful to AECERIBER and Miss Elena Die´guez and Mr Pedro Can˜uelo for providing the pigs. The authors also wish to thank Miss Ana Galaz and Miss Inmaculada Linares for their excellent technical assistance.
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