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Influence of finishing diet on fatty acid profiles of intramuscular lipids, triglycerides and phospholipids in muscles of the Iberian pig
PII:
SO309-1740(96)OOlO2-7
Meat Science, Vol. 45, No. 2, 263-270, 1997 0 1997 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0309-1740/97 $17.00 +o.oo
ELSEVlER
Influence of Finishing Diet on Fatty Acid Profiles of Intramuscular Lipids, Triglycerides and Phospholipids in Muscles of the Iberian Pig Ramh
“Nutrition (Received
Cava,a* Jorge Ruiz,” Clemente Lbpez-Bote,h Lourdes Martin,a Carmen Garcia,a Jestis Ventana9 & Teresa Antequeraa “Tecnologia y Bioquimica de 10s Ahmentos, Facultad de Veterinaria, Universidad de Extremadura, 1007 1, Caceres, Spain Animal, Fact&ad de Veterinaria, Universidad Complutense, 28040, Madrid, 6 November
1995; revised version
received
24 July 1996; accepted
Spain
27 July 1996)
ABSTRACT Thirty Iberian x Duroc pigs allotted in groups of ten animals were.fed in three traditional d@erent management systems (‘Montanera’ (MO), fed on acorns; ‘Recebo’ (RE), fed on acorns and a commercial diet; and ‘Cebo’ (CE), fed on a commercial diet). Masseter muscle was obtained to evaluate the influence of management system on fatty acid (FA) composition of lean. The FA composition of the intramuscular total lipids, triglyceride (TG) andphospholipid (PL) fractions was evaluated. Muscle from MO pigs had greater quantities of monounsaturated fatty acids (MUFA) in the total lipids, triglyceride and phospholipid fractions than the other feedings. The percentage of saturatedfatty acids (SFA) of i.m. total lipids and TGs increased (P < .05) with duration of feeding on RE and CE, from 35.1335.10% in MO pigs to 37.47-37.84% in RE pigs and 39.98-41.11% in CE pigs. PLs from RE and CE pigs contained more Ct8:2 and Czo4 and less Cts:t than MO pigs. 0 1997 Elsevier Science Ltd. All rights reserved
INTRODUCTION The Iberian pig is an autochthonous porcine breed developed traditionally in the southwest of Spain. Principally, meat from Iberian pigs is produced to obtain dry cured hams characterized by a high sensorial quality and a high degree of consumer acceptance. According to the type of feeding in the final stage of fattening, different qualities of Iberian hams are obtained. Hams from pigs fattened with acorns (Quercus ilex, Q. rotundifoliu and Q. suber), denominated as ‘Montanera’, are more highly appreciated by consumers than those from acorn-grain and grain-fed pigs (the other two possible production systems). This preference is due to the intense and characteristic flavour that montanera hams possess because of the presence of numerous volatile compounds (Garcia et al., 1991; Lopez et al., 1992). *To whom correspondence
should be addressed. 263
264
R. Cava et al.
It is generally accepted that diet influences the fatty acid (FA) composition of the total fat and neutral fat fractions of muscle (Horstein et al., 1961; St John et al., 1987; Rhee et al., 1988a,b, 1990; Miller et al., 1990). The fatty acid composition of phospholipids (PLs) in pork, beef and chicken, principally polyunsaturated fatty acids (PUFA), depends on the composition of the dietary fat (Lin et al., 1989; Asghar et al., 1990; Monahan et al., 1992) and the duration of treatment (Miller et al., 1987; Larick & Turner, 1989, 1990). The fatty acid composition and the concentration of the fatty acids can influence the types and the concentration of volatiles produced in the heated fat (Selke et al., 1977, 1980) and dry cured products (Lopez et al., 1992). In this way, Larick et al. (1992) observed that increased levels of C1sZ2in the diet of pigs resulted in increases in the concentration of aldehydes in meat, especially pentanal and hexanal, which are indicators of increased oxidation. Furthermore, Larick and Turner (1990) demonstrated that the relationship between sensory response and fatty acid concentration was dependent on the phospholipid with which the fatty acid was associated. The objective of the present study was to investigate the effects of the finishing diet on the fatty acid profiles of the intramuscular total lipids, triglyceride and phospholipid fractions of porcine muscle.
MATERIALS
AND METHODS
Animals and diets
Thirty Iberian x Duroc (75%) pigs, at about 85-90 kg of initial weight, were divided into three groups of 10 animals each. According to the traditional types of feeding during the fattening period prior to slaughtering, groups were denominated ‘Montanera’ (MO), ‘Recebo’ (RE) and ‘Cebo’ (CE). Pigs of MO group were grown extensively on acorns (Quercus ilex, Q. rotundifoliu and Q. suber) during 60 days prior to slaughtering. CE pigs were kept throughout the fattening period in an intensive production system and fed a concentrate feed composed basically of a cereal and an oilseed (corn and soya). RE pigs were maintained in an intermediate fattening system: during the first 30 days animals were feeding on acorns, and finished on a concentrate feed, during the 30 days prior to slaughter. Pigs were slaughtered at 145-155 kg live weight. The chemical composition of diets (moisture, ash, crude fiber, crude protein, ether extract and nitrogen free extractives) was determined according to AOAC (1984) methods. Lipid analyses
Musseter muscles were removed from the carcass for lipid analyses. Samples were immediately placed in a freezer and stored at -80°C until analysis. Lipids of muscle and feed samples were extracted and purified according to the method described by Bligh and Dyer (1959). Triglyceride and phospholipid fractions of intramuscular fat were separated using NHz-aminopropyl minicolumns using the method described by Garcia-Regueiro et al. (1994). Fatty acid profiles
The fatty acid composition of chloroform extracts of acorn and commercial feed, intramuscular total fat, triglyceride and phospholipid fractions was determined by preparing fatty acid methyl esters (FAME) in acidic conditions (HaSOd:methanol).
265
Infuence of diet on fatty acids in intramuscular lipids
FAMES were analyzed using a Hewlett Packard model HP-5890A gas chromatograph, equipped with a flame ionization detector (FID). FAMES were separated on a 30 m FFAP-TPA fused-silica column (Hewlett Packard) with an id. of 0.53 mm and a 1.0 ym film thickness. The injector and detector were maintained at 230°C. Column oven temperature was maintained at 225°C. The carrier gas was nitrogen at a flow rate of 15 ml/min. FAMES were quantified using tridecanoic acid as internal standard. Response factors were determined for all fatty acids by injecting samples containing a known amount of FAME standard and tridecanoate. Identification of fatty acid methyl esters was based on retention times of reference compounds (Sigma). Results are expressed as percentage of total fatty acid methylesters. Statistical
analysis
Statistical analyses were carried out using the STATGRAPHICS (Statistical Graphics System) statistical package run in a Hewlett Packard PC. Data relating to fatty acids were analyzed as one-way analysis of variance (ANOVA) that included in the model dietary treatment and animal (nested within diet). Tukey’s multiple range test was used to compare feeding regimen means when significant (P < 0.05) treatment effects were observed (Steel & Torrie, 1980). The mean square for the animal (nested within diet) term was used as the error term to test diet treatment effect and to separate the main effects due to diets. Results are presented as the mean of percentages of fatty acids on each type of feeding with the standard deviation (SD) for each parameter pooled across treatment.
RESULTS
AND DISCUSSION
Diet compositions
Data on chemical composition and fatty acid profiles of diets are given in Tables 1 and 2, respectively. The chemical composition of the acorns revealed high levels of nitrogen-free extractives (NFE) and a low protein percentage. However, crude protein levels in concentrate feed were three times higher than in acorns (12.95 versus 4.29%). In contrast to concentrate feed, acorns were higher in monounsaturated fatty acids, primarily oleic (Cis:,) acid (63.8 vs 24.2%). In comparison to other high-oleic vegetable oils, such canola oil (St John et al., 1987; Miller et al., 1990; Myer et al., 1992), acorn was
TABLE 1 Chemical Analysis of Swine Diets ‘Montanera’: acorn
Moisture Ash” Crude fiber” Crude protein* Ether extract0 NFE” “Expressed as % dry matter.
40.21 2.55 7.05 4.29 6.33 79.78
‘Recebo’/‘Cebo’: concentrate feed ____ 12.00 3.93 3.97 12.95 3.23 75.92
266
R. Cuva et al. TABLE 2
Fatty Acid Profiles of Swine Diets ‘Montanera’. acorn G2:o Cl40 c16:O CM0 L2o:o
Total saturated c16:l c18:l
Total monounsaturated CM:2 G&3
Total polyunsaturated
‘Recebo’l ‘Cebo’: concentrate feed
0.09 14.63 3.09 0.33 18.15
0.03 0.67 16.35 5.92 0.23 23.20
0.15 63.81 63.96
0.92 24.07 24.99
16.07 0.77 16.83
47.30 2.21 49.52
0.01
Results are expressed as percentage of total fatty acid methylesters.
similar in total monounsaturated fatty acids and was lower in total polyunsaturated fatty acids. Concentrate feed had higher proportions of unsaturated fatty acids, specifically C1sz2(53.7 versus 16.1%), compared to acorn. Levels of the other fatty acids were very similar between acorn and concentrate feed (Table 2). Muscle fatty acid profiles Relative percentages of individual fatty acids in intramuscular total lipids, triglyceride (TG) and phospholipid (PL) fractions of Masse&r muscle are shown in Tables 3-5, respectively. The results showed that the percentage of total monounsaturated fatty acids was highest in triglycerides, followed by total lipids and phospholipids, respectively; the percentage of total polyunsaturated fatty acids was highest in phospholipids, followed by total lipids and triglycerides, respectively. These data are in agreement with the findings of other authors (Sharma et al., 1987; Rhee et al., 1988~) who analyzed fatty acid compositions of total, neutral and polar lipids from pork muscles at different anatomical locations. Triglycerides, the major lipid class deposited during the fattening period, are responsible for higher total lipids. Total lipids and TGs showed similar percentages of most fatty acids; so they will be discussed concurrently. In both i.m. total lipids and TGs, monounsaturated fatty acids (MUFA) showed an increase (PC 0.05) in the MO group in comparison with CE group (particularly Ci&, accompanied by a decrease in the total saturated fatty acids (SFA) (Tables 3 and 4). The increase in monounsaturated fatty acids noted upon feeding acorn agrees with results of trials involving high-oleic acid vegetable oils (St John et al., 1987; Rhee et al., 1988~; Miller et al., 1990). The greater the number of days on concentrate feed (CE versus RE) the higher the levels (PC 0.05) of stearic (Cis:~) and total SFA in i.m. total lipids and the TG fraction. MO pigs showed lower levels of C1szoand total SFA than RE pigs; CE pigs showed the highest percentages. In all cases, i.m. total lipids and TG of RE pigs showed intermediate levels of this FA in comparison with MO and CE pigs. The variation that occurred within each treatment is the result of the type and duration of the feeding regimen. MUFA levels in the intramuscular total lipids and TGs from MO and RE pigs
Influence of diet on fatty acids in intramuscular lipids
267
are due to the direct deposition of monounsaturated
FA from acorn and those synthetized ‘de novo’ from stearate desaturation. Numerous authors have described an increase of stearoyl-CoA desaturase in tissues from rats (De Tomas et al., 1974; Jeffcoat et al., 1977), mice (Landschulz et al., 1994) and steers (Chang et al., 1992) when fed on high MUFA diets which resulted in a rise in tissue MUFA. In this sense, as C16:0 and C18:0 relative TABLE 3
Fat of Masseter Muscle from Pigs Fed ‘Montanera’, ‘Recebo’ and ‘Cebo’ Diets
Fatty Acid Profiles of Intramuscular
‘Montanera ’
_ Cl2.0
0.07”*0.01
Cl4:O
1.35”*0.09
‘Recebo
‘Cebo ’
c2o:o
23.60b f 0.67 9.92b f 0.43 0.16b+0.02
0.08” f 0.02 1.44”&0.15 24.62b i 1.6 11.14bf0.89 0 17a,b+0.02
0.08” It 0.01 1.46”10.19 26.09a f I .07 12.18”*0.98 0.18”zk0.02
Total saturated
35.13b* 1.01
3;.47b f 2.44
39.98” f 1.38
c16:I
C18:1
4.13”*0.27 48.38”* 1.24
3.57blt 0.4 45.77a f 2.1
3.57bi 0.49 44.86b i 1.30
Total monounsaturated
52.51”* 1.18
49.34b* 2.29
48.43bi 1.38
cm2
7.64”,b f 0 62 0.40a f 0:07 0.82”*0.19 8.86” f 0.72
cl6:O cl8:O
Cl8:3 c20:4
Total polyunsaturated
8.23” 0.36” 0.95a 9.54”
f f f f
1.70 0.05 0.76 2.44
Each value is the mean of 10 animals. Within rows, the means with superscripts differ significantly (P > 0.05). Results are expressed as percentage of total fatty acid methylesters.
Fatty
Acid Profiles
of the Triglyceride
6.44b i 0.95 0.22bi 0.03 0.95”*0.31 7.62” f 1.23 of different
letters
TABLE 4 Fraction of Masseter Muscle from Pigs Fed ‘Montanera’,
‘Recebo’ and ‘Cebo’ Diets ‘Montanera’
‘Recebo
‘Cebo ’
Cl2:O
0.08” i 0.01
0.08” i 0.00
0.08” f 0.01
Cl4:O
1.41ai0.07 23.74b •k 0.67 9.71bf0.51 0.1 7a i 0.02 35.10bi 1.09
1.40ai0.15 24.70b i 1.07 11.25bf 1.00 0.19”*0.02 37.84bi 1.88
1.47a f 0.11 26.85af 1.13 12.52” z!z0.94 0.19” f 0.02 41.11a*0.02
CM.1
4.25a f 0.27 50.50a f 1.43
3.67b i 0.49 47.95b* 1.53
3.63b f 1.87 47.68b f 0.36
Total monounsaturated
54.75” f 1.32
51 .62b f 1.67
51.32b* 1.22
6.69a f 0.68 0.41”*0.06 0.44a f 0.11 7.54af0.76
7.31a=k 1.08 0.35” f 0.05 0.48”iO.19 8.14ai 1.27
4.67b f 1.35 0.22b + 0.57 0.32a f 0.04 5.21b+0.11
cl6-0 Gs:o
c2o:o
Total saturated Cl6:I
CM:2 cl8:3 c20:4
Total polyunsaturated
Each value is the mean of IO animals. Within rows, the means with superscripts differ significantly (P > 0.05). Results are expressed as percentage of total fatty acid methylesters.
of different
letters
268
R. Cava et al. TABLE 5
Fatty Acid Profiles of the Phospholipid Fraction of Masseter Muscle from Pigs Fed ‘Montanera’, ‘Recebo’ and ‘Cebo’ Diets ‘Montanera’ c12:o c14:o cl6:O
Cl8:O c2o:o
Total saturated c16:I
Cl&l Total monounsaturated c18:2 cl8:3 c20:4
Total polyunsaturated
0.2Y f 0.60” f 28.48” f 18.24a f 0.44a f 48.01ak
0.23 0.30 6.02 4.48 0.11 10.85
‘Recebo’
‘Cebo ’
0.22”ztO.l6 0.23b*0.10 20.46b* 2.00 13.76bk3.00 0 36a-bk0 1 35.04%4:43
0.12a f 0.05 0.26b l 0.16 15.32bzt 1.98 9.32b*0.7 0.31bi0.05 25.32b + 0.56
l.23bzt0.14 20.43a f 3.44 21.66af3.48
l.12b*0.24 16.07bk3.44 17.19bi3.56
1.6O”ztO.l3 13.16bf0.94 14.76b f 0.98
15.30b*7.81 0.98” f 0.3 2.62b f 1.05 18.90b* 10.33
24.92a f 3.50 1.06a f 0.38 5.70af2.81 31 .74a f 5.66
27.12aIt 2.43 l.llaf0.17 8.16”ztO.98 36.34af 1.81
Each value is the mean of 10 animals. Within rows, the means with superscripts of different letters differ significantly (P > 0.05). Results are expressed as percentage of total fatty acid methylesters.
percentages differed slightly in feeds, variations in i.m. SFA and TGs between groups may be due to desaturation of Cis:e and Ci6:a in the tissues. MO and RE pigs showed higher levels of polyunsaturated fatty acids (PUFA) (P < 0.05), in i.m. lipids and TGs, than did pigs from the CE group. The percentage of Clsz2 showed a similar tendency to PUFA. The higher percentage of C18:2 in i.m. lipids and TGs from MO and RE pigs than in CE pigs is not well understood. Our findings are in accordance with results reported by Flores et al. (1988) who analyzed the fatty acid composition of subcutaneous tissue from Iberian pigs fed on acorns and commercial feed and showed higher C1sz2percentage in MO and RE pigs than in CE ones. Results do not agree with those reported by numerous authors who observed an increase in the C1sz2content in i.m. lipids and TG fraction which related to increases of this fatty acid in the feed (Miller et al., 1990; Larick et al., 1992; Monahan et al., 1992). These differing results among investigators could be due to the fact that the Iberian pig is a rustic and adipogenic breed that seems to possess a different metabolism from the pigs used in the other work. The fatty acid composition of the polar lipids is shown in Table 5. Oleic (Cis:r), palmitic (Cr6:J, stearic (Cis:J and linoleic (C& acids represented the major portion of total fatty acids in the phospholipids fraction. MO pigs showed higher values of MUFA (PC 0.05) than RE and CE pigs. The diet effect on the percentage of C18: 1 was similar to that on the percentage of monounsaturated fatty acids: feeding on acorn (MO) throughout the fattening period increased the C18:l percentage in PLs in contrast to RE and CE pigs. On the other hand, the percentages of palmitic (Cr&, stearic (Crs:e) and SFA differed significantly (P < 0.05) among the three groups: MO pigs had most saturated PLs while CE pigs had the lowest percentage of these fatty acids (Table 5). The PL fraction was higher in PUFA than the TG fraction. Muscles from pigs receiving commercial feed (RE and CE) increased the percentages of PUFA in their PLs. Results are in agreement with the work of Monahan et al. (1992) who observed variations in the composition of PLs when the PUFA content of the diet varied. Increased time on commercial
Influence of diet on fatty acids in intramuscular lipids
269
feeding (RE versus CE) increased percentages of PUFAs (P < 0.05) in the PL fraction, specifically linoleic (Cis2) and arachidonic (C2aZ4)acids. Commercial feed elevated particularly PUFA percentages in PLs from RE pigs in contrast to MO pigs due to a rapid incorporation of PUFAs into the membranes. This reflects the high metabolic activity and turnover of the PLs when FAs are rapidly substituted by the FAs of the feed (Pan & Storlien, 1993). The high concentrations of PUFAs in PLs and their association in the cell membrane (with enzymic and non-enzymic systems capable of generating pro-oxidative free-radical species) make PLs much more susceptible to oxidation than triglycerides (Love & Pearson. 1971; Igene & Pearson, 1979; Igene et al., 1980; Melton, 1983; Hare1 & Kanner, 1985). Mottram and Edwards (1983) reported that intramuscular triglycerides and phospholipids are the major source of volatile compounds. In this sense, the FA composition of both will be responsible for the volatile substances formed during the oxidation phenomena which occur during the processing of products from Iberian pig meat (Garcia et al., 1991; Antequera et al., 1992; Lopez et al., 1992). The lower PUFA percentage and higher Cis:, percentage in the phospholipids and triglycerides of MO pigs would produce dry products with high sensorial characteristics from the meat of Iberian pigs fed on acorn in comparison with those elaborated from the meat of pigs fed on the other two fattening systems (RE and CE).
ACKNOWLEDGEMENT This study was partially supported AAIR Action.
by the DIET-OX
project (CT94-1577, EU) of the
REFERENCES Antequera, T., Lopez-Bote, C. J., Cordoba, J. J., Garcia, C., Asensio, M. A., Ventanas, J., GarciaRegueiro, J. A. & Diaz, I. (1992). Food Chem., 45, 105. AOAC (1984). Ojicial Methods of Analysis, 14th edn. Association of Official Analytical Chemists, Washington, DC. Asghar, A., Lin, C. F., Gray, J. I., Buckley, D. J., Booren, A. M. & Flegal, C. J. (1990). J. Food Sci., 55, 46.
Bligh, E. G. & Dyer, W. J. (1959). Can. J. Biochem. Physiol., 37, 911. Chang, J. H. P., Lunt, D. K. & Smith, S. B. (1992). J. Nutrition, 122, 2074. De Tomas, M. E., Mercuri, 0. & Peluffo, R. (1974). Lipids, 10, 360. Flares, J., Biron, C., Izquierdo, L. & Nieto, P. (1988). Meat Sci., 23, 253. Garcia, C., Berdague, J. J., Antequera, T., Lopez-Bote, C., Cordoba, J. J. & Ventanas, J. (1991). Food Chem., 41,23.
Garcia-Regueiro, J. A., Gibert, J. & Diaz, I. (1994). J. Chromatography .A, 667, 225. Jeffcoat, R., Brawn, P. R., Safford, R. & James, A. T. (1977). Biochem. J., 161, 431. Harel, S. & Kanner, J. (1985). J. Agric. Food Chem., 33, 1188. Horstein, I., Crowe, P. F. & Heimberg, M. J. (1961). J. Food Sci., 26, 581. Igene, J. 0. & Pearson, A. M. (1979). J. Food Sci., 44, 1285. Igene, J. O., Pearson, A. M., Dugan, L. R. & Price, J. F. (1980). Food Chem., 5, 263. Landschulz, K. T., Jump, D. B., MacDougald, 0. A. & Lane, M. D. (1994). Biochem. Biophys. Res. Commun., 200, 763.
Larick, D. K. & Turner, B. E. (1989). J. Anim. Sci., 67, 2282. Larick, D. K. & Turner, B. E. (1990). .I. Food Sci., 55, 3 12. Larick, D. K., Turner, B. E., Schoenherr, W. D., Coffey, M. T. & Pilkington, D. H. (1992). J. Anim. Sci., 70, 1397.
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Lin, C. F., Gray, J. I., Asghar, A., Buckley, D. J., Booren, A. M. & Flegal, C. J. (1989). J. Food Sci., 54, 1457. Lopez, M. O., de la Hoz, L., Cambero, M. I., Gallardo, E., Reglero, G. & Ordoiiez, J. A. (1992). Meat Sci., 31, 267. Love, J. D. & Pearson, A. M. (1971). J. Am. Oil Chem. Sot., 48, 547. Melton, S. L. (1983). Food Technol., 37, 105. Miller, M. F., Shackelford, S. D., Hayden, K. D. & Reagan, J. 0. (1990). J. Animal Sci., 68, 1624. Miller, G. J., Field, R. A., Medieros, L. & Nelms, G. E. (1987). J. Food Sci., 52, 526. Monahan, F. J., Buckley, D. J., Morrissey, P. A., Lynch, P. B. & Gray, J. I. (1992). Meat Sci., 31, 229. Mottram, D. S. & Edwards, R. A. (1983). J. Sci. Food Agric., 34, 517. Myer, R. O., Johnson, D. D., Knauft, D. A., Gorbet, D. W., Brendemuhl, J. H. & Walker, W. R. (1992). J. Animal Sci., 70, 3734. Pan, D. A. & Storlien, L. H. (1993). J. Nutrition, 123, 512. Rhee, K. S., Davidson, T. L., Cross, H. R. & Ziprin, Y. A. (1990). Meat Sci., 27, 329. Rhee, K. S., Davidson, T. L., Knabe, D. A., Cross, H. R., Ziprin, Y. A. & Rhee, K. C. (1988a). Meat Sci., 24, 249. Rhee, K. S., Ziprin, Y. A., Ordonez, G. & Bohac, C. E. (1988b). Meat Sci., 23, 201. Steel, R. G. D. & Torrie, J. H. (1980). Principles and Procedures ofStatistics 2nd edn. McGraw-Hill Book Co., New York. Sharma, N., Gandemer, G. & Goutefongea, R. (1987). Meat Sci., 19, 121. St. John, L. C., Young, C. R., Knabe, D. A., Thompson, L. D., Schelling, G. T., Grundy, S. M. Jr Smith, S. B. (1987). J. Animal Sci., 64, 1441. Selke, E., Rohwedder, W. K. & Dutton, H. J. (1977). J. Am. Oil Chem. Sot., 54, 62. Selke, E., Rohwedder, W. K. & Dutton, H. J. (1980). J. Am. Oil Chem. SW., 57, 75.
SO309-1740(96)OOlO2-7
Meat Science, Vol. 45, No. 2, 263-270, 1997 0 1997 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0309-1740/97 $17.00 +o.oo
ELSEVlER
Influence of Finishing Diet on Fatty Acid Profiles of Intramuscular Lipids, Triglycerides and Phospholipids in Muscles of the Iberian Pig Ramh
“Nutrition (Received
Cava,a* Jorge Ruiz,” Clemente Lbpez-Bote,h Lourdes Martin,a Carmen Garcia,a Jestis Ventana9 & Teresa Antequeraa “Tecnologia y Bioquimica de 10s Ahmentos, Facultad de Veterinaria, Universidad de Extremadura, 1007 1, Caceres, Spain Animal, Fact&ad de Veterinaria, Universidad Complutense, 28040, Madrid, 6 November
1995; revised version
received
24 July 1996; accepted
Spain
27 July 1996)
ABSTRACT Thirty Iberian x Duroc pigs allotted in groups of ten animals were.fed in three traditional d@erent management systems (‘Montanera’ (MO), fed on acorns; ‘Recebo’ (RE), fed on acorns and a commercial diet; and ‘Cebo’ (CE), fed on a commercial diet). Masseter muscle was obtained to evaluate the influence of management system on fatty acid (FA) composition of lean. The FA composition of the intramuscular total lipids, triglyceride (TG) andphospholipid (PL) fractions was evaluated. Muscle from MO pigs had greater quantities of monounsaturated fatty acids (MUFA) in the total lipids, triglyceride and phospholipid fractions than the other feedings. The percentage of saturatedfatty acids (SFA) of i.m. total lipids and TGs increased (P < .05) with duration of feeding on RE and CE, from 35.1335.10% in MO pigs to 37.47-37.84% in RE pigs and 39.98-41.11% in CE pigs. PLs from RE and CE pigs contained more Ct8:2 and Czo4 and less Cts:t than MO pigs. 0 1997 Elsevier Science Ltd. All rights reserved
INTRODUCTION The Iberian pig is an autochthonous porcine breed developed traditionally in the southwest of Spain. Principally, meat from Iberian pigs is produced to obtain dry cured hams characterized by a high sensorial quality and a high degree of consumer acceptance. According to the type of feeding in the final stage of fattening, different qualities of Iberian hams are obtained. Hams from pigs fattened with acorns (Quercus ilex, Q. rotundifoliu and Q. suber), denominated as ‘Montanera’, are more highly appreciated by consumers than those from acorn-grain and grain-fed pigs (the other two possible production systems). This preference is due to the intense and characteristic flavour that montanera hams possess because of the presence of numerous volatile compounds (Garcia et al., 1991; Lopez et al., 1992). *To whom correspondence
should be addressed. 263
264
R. Cava et al.
It is generally accepted that diet influences the fatty acid (FA) composition of the total fat and neutral fat fractions of muscle (Horstein et al., 1961; St John et al., 1987; Rhee et al., 1988a,b, 1990; Miller et al., 1990). The fatty acid composition of phospholipids (PLs) in pork, beef and chicken, principally polyunsaturated fatty acids (PUFA), depends on the composition of the dietary fat (Lin et al., 1989; Asghar et al., 1990; Monahan et al., 1992) and the duration of treatment (Miller et al., 1987; Larick & Turner, 1989, 1990). The fatty acid composition and the concentration of the fatty acids can influence the types and the concentration of volatiles produced in the heated fat (Selke et al., 1977, 1980) and dry cured products (Lopez et al., 1992). In this way, Larick et al. (1992) observed that increased levels of C1sZ2in the diet of pigs resulted in increases in the concentration of aldehydes in meat, especially pentanal and hexanal, which are indicators of increased oxidation. Furthermore, Larick and Turner (1990) demonstrated that the relationship between sensory response and fatty acid concentration was dependent on the phospholipid with which the fatty acid was associated. The objective of the present study was to investigate the effects of the finishing diet on the fatty acid profiles of the intramuscular total lipids, triglyceride and phospholipid fractions of porcine muscle.
MATERIALS
AND METHODS
Animals and diets
Thirty Iberian x Duroc (75%) pigs, at about 85-90 kg of initial weight, were divided into three groups of 10 animals each. According to the traditional types of feeding during the fattening period prior to slaughtering, groups were denominated ‘Montanera’ (MO), ‘Recebo’ (RE) and ‘Cebo’ (CE). Pigs of MO group were grown extensively on acorns (Quercus ilex, Q. rotundifoliu and Q. suber) during 60 days prior to slaughtering. CE pigs were kept throughout the fattening period in an intensive production system and fed a concentrate feed composed basically of a cereal and an oilseed (corn and soya). RE pigs were maintained in an intermediate fattening system: during the first 30 days animals were feeding on acorns, and finished on a concentrate feed, during the 30 days prior to slaughter. Pigs were slaughtered at 145-155 kg live weight. The chemical composition of diets (moisture, ash, crude fiber, crude protein, ether extract and nitrogen free extractives) was determined according to AOAC (1984) methods. Lipid analyses
Musseter muscles were removed from the carcass for lipid analyses. Samples were immediately placed in a freezer and stored at -80°C until analysis. Lipids of muscle and feed samples were extracted and purified according to the method described by Bligh and Dyer (1959). Triglyceride and phospholipid fractions of intramuscular fat were separated using NHz-aminopropyl minicolumns using the method described by Garcia-Regueiro et al. (1994). Fatty acid profiles
The fatty acid composition of chloroform extracts of acorn and commercial feed, intramuscular total fat, triglyceride and phospholipid fractions was determined by preparing fatty acid methyl esters (FAME) in acidic conditions (HaSOd:methanol).
265
Infuence of diet on fatty acids in intramuscular lipids
FAMES were analyzed using a Hewlett Packard model HP-5890A gas chromatograph, equipped with a flame ionization detector (FID). FAMES were separated on a 30 m FFAP-TPA fused-silica column (Hewlett Packard) with an id. of 0.53 mm and a 1.0 ym film thickness. The injector and detector were maintained at 230°C. Column oven temperature was maintained at 225°C. The carrier gas was nitrogen at a flow rate of 15 ml/min. FAMES were quantified using tridecanoic acid as internal standard. Response factors were determined for all fatty acids by injecting samples containing a known amount of FAME standard and tridecanoate. Identification of fatty acid methyl esters was based on retention times of reference compounds (Sigma). Results are expressed as percentage of total fatty acid methylesters. Statistical
analysis
Statistical analyses were carried out using the STATGRAPHICS (Statistical Graphics System) statistical package run in a Hewlett Packard PC. Data relating to fatty acids were analyzed as one-way analysis of variance (ANOVA) that included in the model dietary treatment and animal (nested within diet). Tukey’s multiple range test was used to compare feeding regimen means when significant (P < 0.05) treatment effects were observed (Steel & Torrie, 1980). The mean square for the animal (nested within diet) term was used as the error term to test diet treatment effect and to separate the main effects due to diets. Results are presented as the mean of percentages of fatty acids on each type of feeding with the standard deviation (SD) for each parameter pooled across treatment.
RESULTS
AND DISCUSSION
Diet compositions
Data on chemical composition and fatty acid profiles of diets are given in Tables 1 and 2, respectively. The chemical composition of the acorns revealed high levels of nitrogen-free extractives (NFE) and a low protein percentage. However, crude protein levels in concentrate feed were three times higher than in acorns (12.95 versus 4.29%). In contrast to concentrate feed, acorns were higher in monounsaturated fatty acids, primarily oleic (Cis:,) acid (63.8 vs 24.2%). In comparison to other high-oleic vegetable oils, such canola oil (St John et al., 1987; Miller et al., 1990; Myer et al., 1992), acorn was
TABLE 1 Chemical Analysis of Swine Diets ‘Montanera’: acorn
Moisture Ash” Crude fiber” Crude protein* Ether extract0 NFE” “Expressed as % dry matter.
40.21 2.55 7.05 4.29 6.33 79.78
‘Recebo’/‘Cebo’: concentrate feed ____ 12.00 3.93 3.97 12.95 3.23 75.92
266
R. Cuva et al. TABLE 2
Fatty Acid Profiles of Swine Diets ‘Montanera’. acorn G2:o Cl40 c16:O CM0 L2o:o
Total saturated c16:l c18:l
Total monounsaturated CM:2 G&3
Total polyunsaturated
‘Recebo’l ‘Cebo’: concentrate feed
0.09 14.63 3.09 0.33 18.15
0.03 0.67 16.35 5.92 0.23 23.20
0.15 63.81 63.96
0.92 24.07 24.99
16.07 0.77 16.83
47.30 2.21 49.52
0.01
Results are expressed as percentage of total fatty acid methylesters.
similar in total monounsaturated fatty acids and was lower in total polyunsaturated fatty acids. Concentrate feed had higher proportions of unsaturated fatty acids, specifically C1sz2(53.7 versus 16.1%), compared to acorn. Levels of the other fatty acids were very similar between acorn and concentrate feed (Table 2). Muscle fatty acid profiles Relative percentages of individual fatty acids in intramuscular total lipids, triglyceride (TG) and phospholipid (PL) fractions of Masse&r muscle are shown in Tables 3-5, respectively. The results showed that the percentage of total monounsaturated fatty acids was highest in triglycerides, followed by total lipids and phospholipids, respectively; the percentage of total polyunsaturated fatty acids was highest in phospholipids, followed by total lipids and triglycerides, respectively. These data are in agreement with the findings of other authors (Sharma et al., 1987; Rhee et al., 1988~) who analyzed fatty acid compositions of total, neutral and polar lipids from pork muscles at different anatomical locations. Triglycerides, the major lipid class deposited during the fattening period, are responsible for higher total lipids. Total lipids and TGs showed similar percentages of most fatty acids; so they will be discussed concurrently. In both i.m. total lipids and TGs, monounsaturated fatty acids (MUFA) showed an increase (PC 0.05) in the MO group in comparison with CE group (particularly Ci&, accompanied by a decrease in the total saturated fatty acids (SFA) (Tables 3 and 4). The increase in monounsaturated fatty acids noted upon feeding acorn agrees with results of trials involving high-oleic acid vegetable oils (St John et al., 1987; Rhee et al., 1988~; Miller et al., 1990). The greater the number of days on concentrate feed (CE versus RE) the higher the levels (PC 0.05) of stearic (Cis:~) and total SFA in i.m. total lipids and the TG fraction. MO pigs showed lower levels of C1szoand total SFA than RE pigs; CE pigs showed the highest percentages. In all cases, i.m. total lipids and TG of RE pigs showed intermediate levels of this FA in comparison with MO and CE pigs. The variation that occurred within each treatment is the result of the type and duration of the feeding regimen. MUFA levels in the intramuscular total lipids and TGs from MO and RE pigs
Influence of diet on fatty acids in intramuscular lipids
267
are due to the direct deposition of monounsaturated
FA from acorn and those synthetized ‘de novo’ from stearate desaturation. Numerous authors have described an increase of stearoyl-CoA desaturase in tissues from rats (De Tomas et al., 1974; Jeffcoat et al., 1977), mice (Landschulz et al., 1994) and steers (Chang et al., 1992) when fed on high MUFA diets which resulted in a rise in tissue MUFA. In this sense, as C16:0 and C18:0 relative TABLE 3
Fat of Masseter Muscle from Pigs Fed ‘Montanera’, ‘Recebo’ and ‘Cebo’ Diets
Fatty Acid Profiles of Intramuscular
‘Montanera ’
_ Cl2.0
0.07”*0.01
Cl4:O
1.35”*0.09
‘Recebo
‘Cebo ’
c2o:o
23.60b f 0.67 9.92b f 0.43 0.16b+0.02
0.08” f 0.02 1.44”&0.15 24.62b i 1.6 11.14bf0.89 0 17a,b+0.02
0.08” It 0.01 1.46”10.19 26.09a f I .07 12.18”*0.98 0.18”zk0.02
Total saturated
35.13b* 1.01
3;.47b f 2.44
39.98” f 1.38
c16:I
C18:1
4.13”*0.27 48.38”* 1.24
3.57blt 0.4 45.77a f 2.1
3.57bi 0.49 44.86b i 1.30
Total monounsaturated
52.51”* 1.18
49.34b* 2.29
48.43bi 1.38
cm2
7.64”,b f 0 62 0.40a f 0:07 0.82”*0.19 8.86” f 0.72
cl6:O cl8:O
Cl8:3 c20:4
Total polyunsaturated
8.23” 0.36” 0.95a 9.54”
f f f f
1.70 0.05 0.76 2.44
Each value is the mean of 10 animals. Within rows, the means with superscripts differ significantly (P > 0.05). Results are expressed as percentage of total fatty acid methylesters.
Fatty
Acid Profiles
of the Triglyceride
6.44b i 0.95 0.22bi 0.03 0.95”*0.31 7.62” f 1.23 of different
letters
TABLE 4 Fraction of Masseter Muscle from Pigs Fed ‘Montanera’,
‘Recebo’ and ‘Cebo’ Diets ‘Montanera’
‘Recebo
‘Cebo ’
Cl2:O
0.08” i 0.01
0.08” i 0.00
0.08” f 0.01
Cl4:O
1.41ai0.07 23.74b •k 0.67 9.71bf0.51 0.1 7a i 0.02 35.10bi 1.09
1.40ai0.15 24.70b i 1.07 11.25bf 1.00 0.19”*0.02 37.84bi 1.88
1.47a f 0.11 26.85af 1.13 12.52” z!z0.94 0.19” f 0.02 41.11a*0.02
CM.1
4.25a f 0.27 50.50a f 1.43
3.67b i 0.49 47.95b* 1.53
3.63b f 1.87 47.68b f 0.36
Total monounsaturated
54.75” f 1.32
51 .62b f 1.67
51.32b* 1.22
6.69a f 0.68 0.41”*0.06 0.44a f 0.11 7.54af0.76
7.31a=k 1.08 0.35” f 0.05 0.48”iO.19 8.14ai 1.27
4.67b f 1.35 0.22b + 0.57 0.32a f 0.04 5.21b+0.11
cl6-0 Gs:o
c2o:o
Total saturated Cl6:I
CM:2 cl8:3 c20:4
Total polyunsaturated
Each value is the mean of IO animals. Within rows, the means with superscripts differ significantly (P > 0.05). Results are expressed as percentage of total fatty acid methylesters.
of different
letters
268
R. Cava et al. TABLE 5
Fatty Acid Profiles of the Phospholipid Fraction of Masseter Muscle from Pigs Fed ‘Montanera’, ‘Recebo’ and ‘Cebo’ Diets ‘Montanera’ c12:o c14:o cl6:O
Cl8:O c2o:o
Total saturated c16:I
Cl&l Total monounsaturated c18:2 cl8:3 c20:4
Total polyunsaturated
0.2Y f 0.60” f 28.48” f 18.24a f 0.44a f 48.01ak
0.23 0.30 6.02 4.48 0.11 10.85
‘Recebo’
‘Cebo ’
0.22”ztO.l6 0.23b*0.10 20.46b* 2.00 13.76bk3.00 0 36a-bk0 1 35.04%4:43
0.12a f 0.05 0.26b l 0.16 15.32bzt 1.98 9.32b*0.7 0.31bi0.05 25.32b + 0.56
l.23bzt0.14 20.43a f 3.44 21.66af3.48
l.12b*0.24 16.07bk3.44 17.19bi3.56
1.6O”ztO.l3 13.16bf0.94 14.76b f 0.98
15.30b*7.81 0.98” f 0.3 2.62b f 1.05 18.90b* 10.33
24.92a f 3.50 1.06a f 0.38 5.70af2.81 31 .74a f 5.66
27.12aIt 2.43 l.llaf0.17 8.16”ztO.98 36.34af 1.81
Each value is the mean of 10 animals. Within rows, the means with superscripts of different letters differ significantly (P > 0.05). Results are expressed as percentage of total fatty acid methylesters.
percentages differed slightly in feeds, variations in i.m. SFA and TGs between groups may be due to desaturation of Cis:e and Ci6:a in the tissues. MO and RE pigs showed higher levels of polyunsaturated fatty acids (PUFA) (P < 0.05), in i.m. lipids and TGs, than did pigs from the CE group. The percentage of Clsz2 showed a similar tendency to PUFA. The higher percentage of C18:2 in i.m. lipids and TGs from MO and RE pigs than in CE pigs is not well understood. Our findings are in accordance with results reported by Flores et al. (1988) who analyzed the fatty acid composition of subcutaneous tissue from Iberian pigs fed on acorns and commercial feed and showed higher C1sz2percentage in MO and RE pigs than in CE ones. Results do not agree with those reported by numerous authors who observed an increase in the C1sz2content in i.m. lipids and TG fraction which related to increases of this fatty acid in the feed (Miller et al., 1990; Larick et al., 1992; Monahan et al., 1992). These differing results among investigators could be due to the fact that the Iberian pig is a rustic and adipogenic breed that seems to possess a different metabolism from the pigs used in the other work. The fatty acid composition of the polar lipids is shown in Table 5. Oleic (Cis:r), palmitic (Cr6:J, stearic (Cis:J and linoleic (C& acids represented the major portion of total fatty acids in the phospholipids fraction. MO pigs showed higher values of MUFA (PC 0.05) than RE and CE pigs. The diet effect on the percentage of C18: 1 was similar to that on the percentage of monounsaturated fatty acids: feeding on acorn (MO) throughout the fattening period increased the C18:l percentage in PLs in contrast to RE and CE pigs. On the other hand, the percentages of palmitic (Cr&, stearic (Crs:e) and SFA differed significantly (P < 0.05) among the three groups: MO pigs had most saturated PLs while CE pigs had the lowest percentage of these fatty acids (Table 5). The PL fraction was higher in PUFA than the TG fraction. Muscles from pigs receiving commercial feed (RE and CE) increased the percentages of PUFA in their PLs. Results are in agreement with the work of Monahan et al. (1992) who observed variations in the composition of PLs when the PUFA content of the diet varied. Increased time on commercial
Influence of diet on fatty acids in intramuscular lipids
269
feeding (RE versus CE) increased percentages of PUFAs (P < 0.05) in the PL fraction, specifically linoleic (Cis2) and arachidonic (C2aZ4)acids. Commercial feed elevated particularly PUFA percentages in PLs from RE pigs in contrast to MO pigs due to a rapid incorporation of PUFAs into the membranes. This reflects the high metabolic activity and turnover of the PLs when FAs are rapidly substituted by the FAs of the feed (Pan & Storlien, 1993). The high concentrations of PUFAs in PLs and their association in the cell membrane (with enzymic and non-enzymic systems capable of generating pro-oxidative free-radical species) make PLs much more susceptible to oxidation than triglycerides (Love & Pearson. 1971; Igene & Pearson, 1979; Igene et al., 1980; Melton, 1983; Hare1 & Kanner, 1985). Mottram and Edwards (1983) reported that intramuscular triglycerides and phospholipids are the major source of volatile compounds. In this sense, the FA composition of both will be responsible for the volatile substances formed during the oxidation phenomena which occur during the processing of products from Iberian pig meat (Garcia et al., 1991; Antequera et al., 1992; Lopez et al., 1992). The lower PUFA percentage and higher Cis:, percentage in the phospholipids and triglycerides of MO pigs would produce dry products with high sensorial characteristics from the meat of Iberian pigs fed on acorn in comparison with those elaborated from the meat of pigs fed on the other two fattening systems (RE and CE).
ACKNOWLEDGEMENT This study was partially supported AAIR Action.
by the DIET-OX
project (CT94-1577, EU) of the
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