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Study of the branched hydrocarbon fraction of intramuscular lipids from Iberian fresh ham
Meat Science 58 (2001) 175±179
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Study of the branched hydrocarbon fraction of intramuscular lipids from Iberian fresh ham J.F. Tejeda a,*, T. Antequera b, L. MartõÂn a, J. Ventanas b, C. GarcõÂa b Food Technology and Biochemistry, Escuela de IngenierõÂas Agrarias, Universidad de Extremadura, Ctra. de CaÂceres s/n, 06071 Badajoz, Spain b Food Technology and Biochemistry, Facultad de Veterinaria, Universidad de Extremadura, Av. Universidad s/n, 10071 CaÂceres, Spain
a
Received 10 July 2000; received in revised form 10 October 2000; accepted 28 October 2000
Abstract Branched hydrocarbons from the unsaponi®able lipid fraction of the Biceps femoris muscle of Iberian pigs were analyzed. Fifty®ve Iberian pigs were distributed to seven groups according to management system (short Montanera and long Montanera, i.e. fed on acorn and pasture for 55 and 75 days prior to slaughter, respectively, and Pienso, fed on a concentrate diet), and genetic type. A branched hydrocarbon, neophytadiene, was identi®ed only in samples from the pigs fed on the extensive system, with higher levels being found in pigs from the long Montanera group, than in those from the short Montanera group; this compound was not found in the Pienso group. Genotype did not aect the level of branched hydrocarbons found. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Iberian pig; Extensive system; Concentrate feed; Branched hydrocarbons
1. Introduction The lipids in the meat and dry-cured products from Iberian pigs have been extensively studied and re¯ect their importance in determining sensory quality (Cava, Ruiz, Ventanas, & Antequera, 1999; GarcõÂa, BerdagueÂ, Antequera, LoÂpez-Bote, CoÂrdoba, & Ventanas, 1991). Analysis, mainly of the fatty acids of the neutral and polar lipids, has been related to many qualities of Iberian pig products, which re¯ect the feeding regime in the ®nal stage of fattening (Cava et al., 1997; Ruiz, Cava, Antequera, MartõÂn, Ventanas, & LoÂpez-Bote, 1998). However, the unsaponi®able fraction of the lipids from Iberian pig products has not been studied although it contains a variety of compounds, of nutritional importance, such as sterols, tocopherols and/or carotenoids. Several authors have related the presence in animal tissues of speci®c compounds in this fraction, the hydrocarbons, to the feed, mostly of plant origin, consumed * Corresponding author. Tel.: +34-924-286200; fax: +34-924286201. E-mail address: [email protected] (J.F. Tejeda).
by the animals (Berdague & GarcõÂa, 1990; Tejeda, Antequera, Ruiz, Cava, Ventanas, & GarcõÂa, 1999; Tulliez & Bories, 1978). Hydrocarbons are inert in the digestive tract and are little modi®ed during digestion and metabolism (Mayes, Lamb, & Colgrove, 1988; Rembold, Wallner, Nite, Kollmannsberger, & Drawert, 1989; Van Straten, 1977). The absorption of hydrocarbons in the small intestine decreases with increasing length of the hydrocarbon chain (Mayes & Lamb, 1984). Normal biosynthesis is thought to be of limited importance in determining the presence of hydrocarbons in animal tissues (Bernardini, Boniforti, Citti, & Mosini, 1982). Analysis of the hydrocarbons in the unsaponi®able fraction is dicult as they are present at low concentrations (Tejeda et al., 1999). In a characterization of the volatiles from Iberian ham, GarcõÂa et al. (1991) and TimoÂn, Ventanas, MartõÂn, Tejeda, and GarcõÂa (1998) found a number of compounds belonging to the unsaponi®able fraction, such as linear and branched hydrocarbons, which were directly deposited in the fat from the feed (BerdagueÂ, Denoyer, Le Quere, & Semon, 1991). Tejeda et al. (1999) reported the presence of a
0309-1740/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(00)00148-0
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J.F. Tejeda et al. / Meat Science 58 (2001) 175±179
complete homologous series of n-alkanes from tetradecane (n-C14) to nonacosane (n-C29) in the subcutaneous fat of Iberian dry-cured hams. Results obtained on the intramuscular lipids of Iberian fresh meat, did not show dierences in n-alkane content between pigs fed on two dierent management systems (Montanera, fed on an extensive system, and Pienso, fed on a concentrate diet; Tejeda, 1999). However, branched hydrocarbons from Iberian fresh meat were not determined. The objectives of this work were: (1) to identify the branched hydrocarbons in Iberian fresh meat; and (2) to determine the dierences in this fraction between dierent lines and between two dierent management systems. 2. Materials and methods 2.1. Material 2.1.1. Animals and diets Fifty-®ve Iberian and IberianDuroc (50%) pigs were divided into three groups according to feeding regime during the fattening period prior to slaughter. Two groups of pigs were grown extensively on acorns (Quercus ilex, Q. rotundifolia and Q. suber) and pasture for 55 and 75 days prior to slaughter, these groups were called: short Montanera and long Montanera, respectively. Pigs in the third group, Pienso, were kept throughout the fattening period on an intensive production system and fed a concentrate feed. Pienso and short Montanera groups were composed of pure Iberian pigs (Valdesequera genetic line) and Iberian (Valdesequera)Duroc crossbred (50%) pigs. The long Montanera group was formed by three genetic lines of pure Iberian pigs (Censyra, Torbiscal and Entrepelado). Table 1 shows the groups of Iberian pigs studied, based on genotype (genetic line and crossbreeding) and feeding regime. Diets (acorn, pasture and concentrate feed) were analysed for branched hydrocarbon content. After slaughter, the Biceps femoris muscles were dissected from the carcasses, placed under vacuum and held at 80 C until analysis. Table 1 Animal distribution according to feeding regime and genotype (Pienso: pigs fed on a concentrate diet; short Montanera and long Montanera: pigs fed on acorn and pasture for 55 and 75 days, respectively) Feeding system
Genotype
Pienso
Iberian Valdesequera line 10 Iberian Valdesequera lineDuroc (50%) 7 Iberian Valdesequera line 10 Iberian Valdesequera lineDuroc (50%) 7 Iberian Censyra line 7 Iberian Torbiscal line 7 Iberian Entrepelado line 7
Short Montanera Long Montanera
Samples (n)
2.1.2. Reagents and standards The solvents used were PRS (extra pure) grade supplied by Panreac (Barcelona, Spain). The absorbent used for column chromatography was silica gel 60 (Panreac, Barcelona, Spain). n-Eicosane (n-C20; Sigma Chemical Co., St. Louis, MO) was used as an internal standard. 2.2. Methods 2.2.1. Lipid extraction Intramuscular lipids were extracted with cloroform:methanol solution (1:2 v/v) following the procedure of Bligh and Dyer (1959). After evaporating the organic phase in a rotary evaporator and then under nitrogen, the residue (lipids) was saponi®ed. 2.2.2. Saponi®cation and hydrocarbon fraction extraction Hydrocarbons were extracted as described by Bories and Tulliez (1977). Samples (8 g) of intramuscular lipids were saponi®ed by re¯uxing for 2 h with 280 ml of 15% KOH in ethanol (w/v). The warm solution was transferred to a separating funnel, and 70 ml of distilled water added and the unsaponi®able fraction extracted with 70 ml of hexane. The organic layer was washed three times with 50 ml of distilled water, then dried over anhydrous sodium sulphate and concentrated to 2 ml. The extract was then transferred onto a chromatography column (1.5 cm i.d.) that had been prepared by adding successively 2 g of silica gel and 8 g of anhydrous sodium sulphate. Hydrocarbons were eluted with 50 ml of hexane. After evaporating to dryness under vacuum, the residue was dissolved in 1 ml of hexane, and 50 ml were taken for gas chromatographic analysis. 2.2.3. Identi®cation and quantitative determination of branched hydrocarbons Branched hydrocarbons were analysed by gas chromatography (GC) on a Hewlett Packard HP-5890A chromatograph, equipped with a ¯ame ionization detector and a Hewlett Packard fused silica capillary column (12 m0.2 mm i.d.) with a ®lm 0.33-mm thick stationary phase of methyl silicone. Helium was the carrier gas at a ¯ow rate of 14.9 ml/min. The oven program was from 100 to 270 C at 6 C/min and 25 min at 270 C. Injector and detector temperatures were 260 and 270 C, respectively. The split ratio was 1:25, inlet pressure 14 psi, and the volume injected 2 ml. Branched hydrocarbons were identi®ed by gas chromatography±mass spectrometry (GC±MS), using a Hewlett Packard HP-5890A chromatograph with a Hewlett Packard 5971A mass selective detector. The GC-MS transfer line temperature was held at 280 C. The mass spectrometer operated in the electron impact mode with an electron energy of 70 eV, a multiplier voltage of 1756 V
J.F. Tejeda et al. / Meat Science 58 (2001) 175±179
and collected data at 1 scan/s over a m/z range from 40 to 300. Spectra were compared with those of the standards and spectra from Wiley, Hewlett Packard and NIST (National Institute of Standards and Technology) libraries. Quantitative determination was performed by adding to the ®nal extract an appropriate amount of the internal standard (n-eicosane, n-C20). 2.2.4. Statistical analysis Results were subjected to analysis of variance according to the general linear model procedure, using SAS software (Statistical Analysis System). The model incorporated feeding system and genotype, and the statistical procedure was carried out using the SAS/SAT package (SAS Institute, 1989).
177
3. Results and discussion The principal group of branched hydrocarbons in the feeds corresponded to those having a linear chain and a branched methyl group (Table 2). In some cases, no positive identi®cation was possible, due to low concentration or the diculty of identi®cation by GC±MS. These peaks were designated ``branched hydrocarbon'' followed by an arabic number. In the literature, several authors have reported the same problems in identifying some of the branched hydrocarbons present in many animal tissues (Bastic, Bastic, Remberg, Skala, & Jovanovic, 1989; Berdague & GarcõÂa, 1990; Bernardini et al., 1982; GarcõÂa et al., 1991). Some dierences were found in the levels of branched hydrocarbons in the feeds, particularly in the case of
Fig. 1. Chromatograms of branched hydrocarbons identi®ed in the intramuscular lipids of Iberian fresh ham. Note the presence of the peak corresponding to neophytadiene (N) (noted with a circle) in the chromatogram from samples of pigs fed extensively (A) and in absence in pigs fed on a concentrate feed (B). The numbers correspond to unknown branched hydrocarbons.
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neophytadiene and squalene. Neophytadiene was identi®ed in all samples of pasture examined, being the most abundant hydrocarbon in this feed; Neophytadiene (3methylene-7,11,15-trimethylhexadecen-1-ene) was not present in acorn and the concentrate feeds. Neophytadiene, which belongs to the group of compounds known as phytanes, is abundant in plant cuticular waxes (Lintas, Balduzzi, Bernardini, & Di Muccio, 1979). Squalene was more abundant in concentrate than in acorn and pasture, and was the most abundant branched hydrocarbon in the concentrate feed. Table 2 Branched hydrocarbon content (expressed as peak area 100/internal standard peak area) of the feeds consumed during the fattening period Branched hydrocarbons
2,4-Dimethyl undecane 2-Methyl tridecane Branched hydrocarbon 1 Branched hydrocarbon 2 Branched hydrocarbon 3 3-Methyl pentadecane Branched hydrocarbon 4 Branched hydrocarbon 5 Neophytadiene Branched hydrocarbon 6 Branched hydrocarbon 7 Branched hydrocarbon 8 Squalene
Feeds Pasture
Acorn
Concentrate
0.50 0.34 tra ± tr 0.32 0.26 tr 136.23 ± ± tr 15.27
0.18 0.21 0.20 0.17 0.16 0.19 0.29 0.18 ± tr tr tr 23.90
0.31 0.27 0.23 0.26 0.26 0.33 0.67 0.44 ± ± 0.26 0.30 141.00
a tr, Trace levels (less than 0.05 peak area100/internal standard peak area).
Table 3 shows the content of branched hydrocarbons in the intramuscular lipids from the B. femoris muscles of Iberian pig. The existence of branched hydrocarbons in several tissues from pigs have been reported (Bastic et al., 1989; Bernardini et al., 1982). Shahidi, Rubin, and D'Souza (1986) found branched hydrocarbons in fresh pig meat, Berdague and GarcõÂa (1990) analysed the volatiles in dry-cured hams, and found a number of branched hydrocarbons while branched hydrocarbons of low molecular weight were isolated from Iberian dry-cured ham by GarcõÂa et al. (1991) and Ruiz, Ventanas, Cava, AndreÂs, and GarcõÂa (1999). TimoÂn et al. (1998) also found such compounds in Iberian dry-cured hams. In the adipose tissue of Iberian dry-cured hams some branched hydrocarbons were also found (Tejeda et al., 1999). A possible source of branched hydrocarbons is fatty acid oxidation occurring in the meat (Loury, 1972; Shahidi et al., 1986), and Gray and Pearson (1984) found an increase in branched hydrocarbon content in cured meat products during processing. However, Tejeda et al. (1999) suggested lipid oxidation is not the origin of most branched hydrocarbons in the adipose tissue of Iberian dry-cured hams, since the hydrocarbon chains are too long to be such oxidation products. The levels of neophytadiene in the B. femoris muscles showed signi®cant dierences (P<0.001) between groups. By contrast, although squalene was present at high levels in all the samples, no signi®cant dierences were found between groups. Feeding regime had a clear eect on the concentration of neophytadiene in the intramuscular lipids, however, genetic eects were not signi®cant. This compound was present in all pigs fed
Table 3 Branched hydrocarbon content in the intramuscular lipids of Biceps femoris muscles (expressed as peak area 100/internal standard peak area) from Iberian pigs (pure lines and crossbred Iberian pigs) fed on extensive (short and long Montanera) and intensive (Pienso) systems (meansstandard error) Concentrate feed Ib. Vald.a
short Montanera
Ib. Vald.a Ib. Vald.a Duroc (50%)
Ib. Vald. Duroc (50%)
long Montanera Ib. Censyra
Ib. Torbiscal
Eect Ib. Feeding Cross Line Entrepelado breeding
2,4-Dimethyl undecane 0.450.06 0.660.34 0.860.17 0.510.05 0.850.15 0.740.06 0.610.09 2-Methyl tridecane 0.430.06 0.730.32 0.920.17 0.540.07 0.980.19 0.870.12 0.880.07 Branched hydrocarbon 1 0.850.13 0.950.17 0.770.10 0.520.06 0.770.10 0.650.09 0.620.06 Branched hydrocarbon 2 0.500.08 0.640.22 0.790.13 0.480.06 0.990.14 1.090.08 0.880.06 Branched hydrocarbon 3 0.310.07 0.510.07 0.550.06 0.420.01 0.50.09 0.610.07 0.480.07 3-Methyl pentadecane 2.070.67 0.640.18 0.790.09 0.490.06 1.460.58 1.150.41 1.40.26 Branched hydrocarbon 4 0.720.14 0.870.11 0.630.07 0.670.12 0.30.05 0.260.08 0.310.12 3-Methyl heptadecane 0.580.08 0.670.11 0.560.07 0.560.07 0.510.07 0.680.08 0.460.07 Neophytadienec 0.000.00c 0.000.00c 1.300.15b 1.580.20b 1.740.15ba 2.180.29a 1.910.24a Branched hydrocarbon 5 0.250.05 0.300.09 0.320.03 0.290.04 0.350.03 0.260.05 0.250.04 Branched hydrocarbon 6 0.570.16 0.460.08 0.440.06 0.540.08 0.310.06 0.320.12 0.290.08 Branched hydrocarbon 7 0.860.22 0.980.11 0.730.12 1.170.35 0.380.12 0.40.22 0.450.15 Squalene 387.753.46 442.525.94 415.957.42 393.846.81 406.728.44 476.233.41 425.126.13 a
Ib. Vald., Iberian Valdesequera line pigs. ns, Not signi®cant. c Values with dierent letters dier signi®cantly. *P<0.001. b
nsb ns ns ns ns ns ns ns * ns ns ns ns
ns ns ns ns ns ns ns ns ns ns ns ns ns
ns ns ns ns ns ns ns ns ns ns ns ns ns
J.F. Tejeda et al. / Meat Science 58 (2001) 175±179
extensively (short and long Montanera), but not in Iberian pigs fed on an intensive system with a commercial diet (Pienso; Fig. 1). These results can be explained by the fact that neophytadiene is only found in pasture which is consumed by the pigs during fattening on Montanera but not in acorns or concentrate. Several peaks, smaller than those corresponding to branched hydrocarbons reported in Table 3, were also observed. Such peaks could not be identi®ed but their mass spectra suggests they could be branched or cyclic hydrocarbons. The presence of neophytadiene has been reported previously; Lintas et al. (1979) identi®ed neophytadiene in various bovine tissues, Bernardini et al. (1982), characterising the distribution of hydrocarbons in meats, found some branched hydrocarbons belonging to the phytanes in bovine meat. However, these compounds were not found in pork, probably because the pigs examined were fed intensively with concentrate diets, while the calves consumed pasture. In addition, Table 3 shows that the content of neophytadiene is higher in the B. femoris muscles from pigs raised extensively for a longer period of time (long Montanera) than in pigs raised for a shorter period with acorn and pasture (short Montanera), presumably because more pasture was consumed by the long Montanera group.
References Bastic, L. J., Bastic, M., Remberg, G., Skala, D., & Jovanovic, J. (1989). Hydrocarbon content of neutral lipid fractions of dierent hog and cattle tissues. In Proceedings 35th International Congress of Meat Science and Technology (pp. 587±590). 20±25 August 1989, Copenhagen, Denmark. BerdagueÂ, J. L., Denoyer, C., Le Quere, J. L., & Semon, E. (1991). Volatile components of dry-cured ham. Journal of Agricultural and Food Chemistry, 39, 1257±1261. BerdagueÂ, J. L., & GarcõÂa, C. (1990). Les components volatils du jambon sec. Viandes et Produits CarneÂs, 11, 319±320. Bernardini, M. P., Boniforti, L., Citti, G., & Mosini, V. (1982). Distribution of hydrocarbons and fatty acids in meats imported into Italy. Food Chemistry, 8, 51±60. Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and puri®cation. Canadian Journal of Biochemistry and Fisiology, 37, 911±917. Bories, G. F., & Tulliez, J. E. (1977). A versatile method for the determination of normal parans in foods. Journal Science of Food Agricultural, 28, 996±999.
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Cava, R., Ruiz, J., LoÂpez-Bote, C., MartõÂn, L., GarcõÂa, C., Antequera, T., & Ventanas, J. (1997). In¯uence of ®nishig diet on fatty acid pro®les of intramuscular lipids, triglycerides and phospholipids in muscles of 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 aected by feeding regime: extensive feeding and alpha-tocopheryl acetate supplementation. Meat Science, 52, 165±172. GarcõÂa, C., BerdagueÂ, J. J., Antequera, T., LoÂpez-Bote, C., CoÂrdoba, J. J., & Ventanas, J. (1991). Volatile components of dry cured Iberian ham. Food Chemistry, 41, 23±32. Gray, J. I., & Pearson, A. M. (1984). Cured meat ¯avor. Advances in Food Research, 29, 1±86. Lintas, C., Balduzzi, A. M., Bernardini, M. P., & Di Muccio, A. (1979). Distribution of hydrocarbons in bovine tissues. Lipids, 14, 298±303. Loury, M. (1972). Possible mechanisms of autoxidative rancidity. Lipids, 7, 671±675. Mayes, R. W., & Lamb, C. S. (1984). The possible use of n-alkanes in herbage as indigestible faecal markers. Proceedings of the Nutrition Society, 43, 39A. Mayes, R. W., Lamb, C. S., & Colgrove, P. M. (1988). Digestion and metabolism of dosed even-chain and herbage odd-chain n-alkanes in sheep. In Proceeding 12th General Meeting Eropean. Grasslands Fed. (pp. 159±163), July 1988, Dublin, Ireland. Rembold, H., Wallner, P., Nite, S., Kollmannsberger, H., & Drawert, F. (1989). Volatile components of chickpea (Cicer arietinun L.) seed. Journal of Agricultural and Food Chemistry, 37, 659±662. Ruiz, J., Cava, R., Antequera, T., MartõÂn, L., Ventanas, J., & LoÂpezBote, C. J. (1998). Prediction of the feeding backround of Iberian pigs using the fatty acid pro®le of subcutaneous, muscle and hepatic fat. Meat Science, 49, 155±163. Ruiz, J., Ventanas, J., Cava, R., AndreÂs, A. I., & GarcõÂa, C. (1999). Volatile compounds of dry-cured Iberian ham as aected by the length of the curing process. Meat Science, 52, 19±27. SAS Institute Inc. (1989). SAS/STAT user's guide. Version 6. (4th ed). NC: Cary. Shahidi, F., Rubin, L. J., & D'Souza, L. A. (1986). Meat ¯avor volatiles: a review of the composition, technique of analysis and sensory evaluation. CRC Critical Review in Food Science and Nutrition, 24, 219±227. Tejeda, J. F. (1999). A study of in¯uence of crossbreeding and feeding regime on intramuscular lipids from Iberian pigs. PhD thesis, University of Extremadura, CaÂceres, Spain. Tejeda, J. F., Antequera, T., Ruiz, J., Cava, R., Ventanas, J., & GarcõÂa, C. (1999). Unsaponi®able fraction content and n-alkane pro®les of subcutaneous fat from Iberian hams. Food Science and Technology International, 5, 229±233. TimoÂn, M. L., Ventanas, J., MartõÂn, L., Tejeda, J. F., & GarcõÂa, C. (1998). Volatile compounds in supercritical carbon dioxide extracts of Iberian ham. Journal of Agricultural and Food Chemistry, 12, 5143±5150. Tulliez, J. E., & Bories, G. F. (1978). Metabolism of a n-paran, heptadecane, in rats. Lipids, 13, 110±115. Van Straten, S. (1977). Volatile compounds in foods (4th ed.). Zeist, The Netherlands: CIVO-TNO Publisher.
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Study of the branched hydrocarbon fraction of intramuscular lipids from Iberian fresh ham J.F. Tejeda a,*, T. Antequera b, L. MartõÂn a, J. Ventanas b, C. GarcõÂa b Food Technology and Biochemistry, Escuela de IngenierõÂas Agrarias, Universidad de Extremadura, Ctra. de CaÂceres s/n, 06071 Badajoz, Spain b Food Technology and Biochemistry, Facultad de Veterinaria, Universidad de Extremadura, Av. Universidad s/n, 10071 CaÂceres, Spain
a
Received 10 July 2000; received in revised form 10 October 2000; accepted 28 October 2000
Abstract Branched hydrocarbons from the unsaponi®able lipid fraction of the Biceps femoris muscle of Iberian pigs were analyzed. Fifty®ve Iberian pigs were distributed to seven groups according to management system (short Montanera and long Montanera, i.e. fed on acorn and pasture for 55 and 75 days prior to slaughter, respectively, and Pienso, fed on a concentrate diet), and genetic type. A branched hydrocarbon, neophytadiene, was identi®ed only in samples from the pigs fed on the extensive system, with higher levels being found in pigs from the long Montanera group, than in those from the short Montanera group; this compound was not found in the Pienso group. Genotype did not aect the level of branched hydrocarbons found. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Iberian pig; Extensive system; Concentrate feed; Branched hydrocarbons
1. Introduction The lipids in the meat and dry-cured products from Iberian pigs have been extensively studied and re¯ect their importance in determining sensory quality (Cava, Ruiz, Ventanas, & Antequera, 1999; GarcõÂa, BerdagueÂ, Antequera, LoÂpez-Bote, CoÂrdoba, & Ventanas, 1991). Analysis, mainly of the fatty acids of the neutral and polar lipids, has been related to many qualities of Iberian pig products, which re¯ect the feeding regime in the ®nal stage of fattening (Cava et al., 1997; Ruiz, Cava, Antequera, MartõÂn, Ventanas, & LoÂpez-Bote, 1998). However, the unsaponi®able fraction of the lipids from Iberian pig products has not been studied although it contains a variety of compounds, of nutritional importance, such as sterols, tocopherols and/or carotenoids. Several authors have related the presence in animal tissues of speci®c compounds in this fraction, the hydrocarbons, to the feed, mostly of plant origin, consumed * Corresponding author. Tel.: +34-924-286200; fax: +34-924286201. E-mail address: [email protected] (J.F. Tejeda).
by the animals (Berdague & GarcõÂa, 1990; Tejeda, Antequera, Ruiz, Cava, Ventanas, & GarcõÂa, 1999; Tulliez & Bories, 1978). Hydrocarbons are inert in the digestive tract and are little modi®ed during digestion and metabolism (Mayes, Lamb, & Colgrove, 1988; Rembold, Wallner, Nite, Kollmannsberger, & Drawert, 1989; Van Straten, 1977). The absorption of hydrocarbons in the small intestine decreases with increasing length of the hydrocarbon chain (Mayes & Lamb, 1984). Normal biosynthesis is thought to be of limited importance in determining the presence of hydrocarbons in animal tissues (Bernardini, Boniforti, Citti, & Mosini, 1982). Analysis of the hydrocarbons in the unsaponi®able fraction is dicult as they are present at low concentrations (Tejeda et al., 1999). In a characterization of the volatiles from Iberian ham, GarcõÂa et al. (1991) and TimoÂn, Ventanas, MartõÂn, Tejeda, and GarcõÂa (1998) found a number of compounds belonging to the unsaponi®able fraction, such as linear and branched hydrocarbons, which were directly deposited in the fat from the feed (BerdagueÂ, Denoyer, Le Quere, & Semon, 1991). Tejeda et al. (1999) reported the presence of a
0309-1740/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(00)00148-0
176
J.F. Tejeda et al. / Meat Science 58 (2001) 175±179
complete homologous series of n-alkanes from tetradecane (n-C14) to nonacosane (n-C29) in the subcutaneous fat of Iberian dry-cured hams. Results obtained on the intramuscular lipids of Iberian fresh meat, did not show dierences in n-alkane content between pigs fed on two dierent management systems (Montanera, fed on an extensive system, and Pienso, fed on a concentrate diet; Tejeda, 1999). However, branched hydrocarbons from Iberian fresh meat were not determined. The objectives of this work were: (1) to identify the branched hydrocarbons in Iberian fresh meat; and (2) to determine the dierences in this fraction between dierent lines and between two dierent management systems. 2. Materials and methods 2.1. Material 2.1.1. Animals and diets Fifty-®ve Iberian and IberianDuroc (50%) pigs were divided into three groups according to feeding regime during the fattening period prior to slaughter. Two groups of pigs were grown extensively on acorns (Quercus ilex, Q. rotundifolia and Q. suber) and pasture for 55 and 75 days prior to slaughter, these groups were called: short Montanera and long Montanera, respectively. Pigs in the third group, Pienso, were kept throughout the fattening period on an intensive production system and fed a concentrate feed. Pienso and short Montanera groups were composed of pure Iberian pigs (Valdesequera genetic line) and Iberian (Valdesequera)Duroc crossbred (50%) pigs. The long Montanera group was formed by three genetic lines of pure Iberian pigs (Censyra, Torbiscal and Entrepelado). Table 1 shows the groups of Iberian pigs studied, based on genotype (genetic line and crossbreeding) and feeding regime. Diets (acorn, pasture and concentrate feed) were analysed for branched hydrocarbon content. After slaughter, the Biceps femoris muscles were dissected from the carcasses, placed under vacuum and held at 80 C until analysis. Table 1 Animal distribution according to feeding regime and genotype (Pienso: pigs fed on a concentrate diet; short Montanera and long Montanera: pigs fed on acorn and pasture for 55 and 75 days, respectively) Feeding system
Genotype
Pienso
Iberian Valdesequera line 10 Iberian Valdesequera lineDuroc (50%) 7 Iberian Valdesequera line 10 Iberian Valdesequera lineDuroc (50%) 7 Iberian Censyra line 7 Iberian Torbiscal line 7 Iberian Entrepelado line 7
Short Montanera Long Montanera
Samples (n)
2.1.2. Reagents and standards The solvents used were PRS (extra pure) grade supplied by Panreac (Barcelona, Spain). The absorbent used for column chromatography was silica gel 60 (Panreac, Barcelona, Spain). n-Eicosane (n-C20; Sigma Chemical Co., St. Louis, MO) was used as an internal standard. 2.2. Methods 2.2.1. Lipid extraction Intramuscular lipids were extracted with cloroform:methanol solution (1:2 v/v) following the procedure of Bligh and Dyer (1959). After evaporating the organic phase in a rotary evaporator and then under nitrogen, the residue (lipids) was saponi®ed. 2.2.2. Saponi®cation and hydrocarbon fraction extraction Hydrocarbons were extracted as described by Bories and Tulliez (1977). Samples (8 g) of intramuscular lipids were saponi®ed by re¯uxing for 2 h with 280 ml of 15% KOH in ethanol (w/v). The warm solution was transferred to a separating funnel, and 70 ml of distilled water added and the unsaponi®able fraction extracted with 70 ml of hexane. The organic layer was washed three times with 50 ml of distilled water, then dried over anhydrous sodium sulphate and concentrated to 2 ml. The extract was then transferred onto a chromatography column (1.5 cm i.d.) that had been prepared by adding successively 2 g of silica gel and 8 g of anhydrous sodium sulphate. Hydrocarbons were eluted with 50 ml of hexane. After evaporating to dryness under vacuum, the residue was dissolved in 1 ml of hexane, and 50 ml were taken for gas chromatographic analysis. 2.2.3. Identi®cation and quantitative determination of branched hydrocarbons Branched hydrocarbons were analysed by gas chromatography (GC) on a Hewlett Packard HP-5890A chromatograph, equipped with a ¯ame ionization detector and a Hewlett Packard fused silica capillary column (12 m0.2 mm i.d.) with a ®lm 0.33-mm thick stationary phase of methyl silicone. Helium was the carrier gas at a ¯ow rate of 14.9 ml/min. The oven program was from 100 to 270 C at 6 C/min and 25 min at 270 C. Injector and detector temperatures were 260 and 270 C, respectively. The split ratio was 1:25, inlet pressure 14 psi, and the volume injected 2 ml. Branched hydrocarbons were identi®ed by gas chromatography±mass spectrometry (GC±MS), using a Hewlett Packard HP-5890A chromatograph with a Hewlett Packard 5971A mass selective detector. The GC-MS transfer line temperature was held at 280 C. The mass spectrometer operated in the electron impact mode with an electron energy of 70 eV, a multiplier voltage of 1756 V
J.F. Tejeda et al. / Meat Science 58 (2001) 175±179
and collected data at 1 scan/s over a m/z range from 40 to 300. Spectra were compared with those of the standards and spectra from Wiley, Hewlett Packard and NIST (National Institute of Standards and Technology) libraries. Quantitative determination was performed by adding to the ®nal extract an appropriate amount of the internal standard (n-eicosane, n-C20). 2.2.4. Statistical analysis Results were subjected to analysis of variance according to the general linear model procedure, using SAS software (Statistical Analysis System). The model incorporated feeding system and genotype, and the statistical procedure was carried out using the SAS/SAT package (SAS Institute, 1989).
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3. Results and discussion The principal group of branched hydrocarbons in the feeds corresponded to those having a linear chain and a branched methyl group (Table 2). In some cases, no positive identi®cation was possible, due to low concentration or the diculty of identi®cation by GC±MS. These peaks were designated ``branched hydrocarbon'' followed by an arabic number. In the literature, several authors have reported the same problems in identifying some of the branched hydrocarbons present in many animal tissues (Bastic, Bastic, Remberg, Skala, & Jovanovic, 1989; Berdague & GarcõÂa, 1990; Bernardini et al., 1982; GarcõÂa et al., 1991). Some dierences were found in the levels of branched hydrocarbons in the feeds, particularly in the case of
Fig. 1. Chromatograms of branched hydrocarbons identi®ed in the intramuscular lipids of Iberian fresh ham. Note the presence of the peak corresponding to neophytadiene (N) (noted with a circle) in the chromatogram from samples of pigs fed extensively (A) and in absence in pigs fed on a concentrate feed (B). The numbers correspond to unknown branched hydrocarbons.
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neophytadiene and squalene. Neophytadiene was identi®ed in all samples of pasture examined, being the most abundant hydrocarbon in this feed; Neophytadiene (3methylene-7,11,15-trimethylhexadecen-1-ene) was not present in acorn and the concentrate feeds. Neophytadiene, which belongs to the group of compounds known as phytanes, is abundant in plant cuticular waxes (Lintas, Balduzzi, Bernardini, & Di Muccio, 1979). Squalene was more abundant in concentrate than in acorn and pasture, and was the most abundant branched hydrocarbon in the concentrate feed. Table 2 Branched hydrocarbon content (expressed as peak area 100/internal standard peak area) of the feeds consumed during the fattening period Branched hydrocarbons
2,4-Dimethyl undecane 2-Methyl tridecane Branched hydrocarbon 1 Branched hydrocarbon 2 Branched hydrocarbon 3 3-Methyl pentadecane Branched hydrocarbon 4 Branched hydrocarbon 5 Neophytadiene Branched hydrocarbon 6 Branched hydrocarbon 7 Branched hydrocarbon 8 Squalene
Feeds Pasture
Acorn
Concentrate
0.50 0.34 tra ± tr 0.32 0.26 tr 136.23 ± ± tr 15.27
0.18 0.21 0.20 0.17 0.16 0.19 0.29 0.18 ± tr tr tr 23.90
0.31 0.27 0.23 0.26 0.26 0.33 0.67 0.44 ± ± 0.26 0.30 141.00
a tr, Trace levels (less than 0.05 peak area100/internal standard peak area).
Table 3 shows the content of branched hydrocarbons in the intramuscular lipids from the B. femoris muscles of Iberian pig. The existence of branched hydrocarbons in several tissues from pigs have been reported (Bastic et al., 1989; Bernardini et al., 1982). Shahidi, Rubin, and D'Souza (1986) found branched hydrocarbons in fresh pig meat, Berdague and GarcõÂa (1990) analysed the volatiles in dry-cured hams, and found a number of branched hydrocarbons while branched hydrocarbons of low molecular weight were isolated from Iberian dry-cured ham by GarcõÂa et al. (1991) and Ruiz, Ventanas, Cava, AndreÂs, and GarcõÂa (1999). TimoÂn et al. (1998) also found such compounds in Iberian dry-cured hams. In the adipose tissue of Iberian dry-cured hams some branched hydrocarbons were also found (Tejeda et al., 1999). A possible source of branched hydrocarbons is fatty acid oxidation occurring in the meat (Loury, 1972; Shahidi et al., 1986), and Gray and Pearson (1984) found an increase in branched hydrocarbon content in cured meat products during processing. However, Tejeda et al. (1999) suggested lipid oxidation is not the origin of most branched hydrocarbons in the adipose tissue of Iberian dry-cured hams, since the hydrocarbon chains are too long to be such oxidation products. The levels of neophytadiene in the B. femoris muscles showed signi®cant dierences (P<0.001) between groups. By contrast, although squalene was present at high levels in all the samples, no signi®cant dierences were found between groups. Feeding regime had a clear eect on the concentration of neophytadiene in the intramuscular lipids, however, genetic eects were not signi®cant. This compound was present in all pigs fed
Table 3 Branched hydrocarbon content in the intramuscular lipids of Biceps femoris muscles (expressed as peak area 100/internal standard peak area) from Iberian pigs (pure lines and crossbred Iberian pigs) fed on extensive (short and long Montanera) and intensive (Pienso) systems (meansstandard error) Concentrate feed Ib. Vald.a
short Montanera
Ib. Vald.a Ib. Vald.a Duroc (50%)
Ib. Vald. Duroc (50%)
long Montanera Ib. Censyra
Ib. Torbiscal
Eect Ib. Feeding Cross Line Entrepelado breeding
2,4-Dimethyl undecane 0.450.06 0.660.34 0.860.17 0.510.05 0.850.15 0.740.06 0.610.09 2-Methyl tridecane 0.430.06 0.730.32 0.920.17 0.540.07 0.980.19 0.870.12 0.880.07 Branched hydrocarbon 1 0.850.13 0.950.17 0.770.10 0.520.06 0.770.10 0.650.09 0.620.06 Branched hydrocarbon 2 0.500.08 0.640.22 0.790.13 0.480.06 0.990.14 1.090.08 0.880.06 Branched hydrocarbon 3 0.310.07 0.510.07 0.550.06 0.420.01 0.50.09 0.610.07 0.480.07 3-Methyl pentadecane 2.070.67 0.640.18 0.790.09 0.490.06 1.460.58 1.150.41 1.40.26 Branched hydrocarbon 4 0.720.14 0.870.11 0.630.07 0.670.12 0.30.05 0.260.08 0.310.12 3-Methyl heptadecane 0.580.08 0.670.11 0.560.07 0.560.07 0.510.07 0.680.08 0.460.07 Neophytadienec 0.000.00c 0.000.00c 1.300.15b 1.580.20b 1.740.15ba 2.180.29a 1.910.24a Branched hydrocarbon 5 0.250.05 0.300.09 0.320.03 0.290.04 0.350.03 0.260.05 0.250.04 Branched hydrocarbon 6 0.570.16 0.460.08 0.440.06 0.540.08 0.310.06 0.320.12 0.290.08 Branched hydrocarbon 7 0.860.22 0.980.11 0.730.12 1.170.35 0.380.12 0.40.22 0.450.15 Squalene 387.753.46 442.525.94 415.957.42 393.846.81 406.728.44 476.233.41 425.126.13 a
Ib. Vald., Iberian Valdesequera line pigs. ns, Not signi®cant. c Values with dierent letters dier signi®cantly. *P<0.001. b
nsb ns ns ns ns ns ns ns * ns ns ns ns
ns ns ns ns ns ns ns ns ns ns ns ns ns
ns ns ns ns ns ns ns ns ns ns ns ns ns
J.F. Tejeda et al. / Meat Science 58 (2001) 175±179
extensively (short and long Montanera), but not in Iberian pigs fed on an intensive system with a commercial diet (Pienso; Fig. 1). These results can be explained by the fact that neophytadiene is only found in pasture which is consumed by the pigs during fattening on Montanera but not in acorns or concentrate. Several peaks, smaller than those corresponding to branched hydrocarbons reported in Table 3, were also observed. Such peaks could not be identi®ed but their mass spectra suggests they could be branched or cyclic hydrocarbons. The presence of neophytadiene has been reported previously; Lintas et al. (1979) identi®ed neophytadiene in various bovine tissues, Bernardini et al. (1982), characterising the distribution of hydrocarbons in meats, found some branched hydrocarbons belonging to the phytanes in bovine meat. However, these compounds were not found in pork, probably because the pigs examined were fed intensively with concentrate diets, while the calves consumed pasture. In addition, Table 3 shows that the content of neophytadiene is higher in the B. femoris muscles from pigs raised extensively for a longer period of time (long Montanera) than in pigs raised for a shorter period with acorn and pasture (short Montanera), presumably because more pasture was consumed by the long Montanera group.
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