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Dietary manipulation of fatty acid composition in lamb meat and its effect on the volatile aroma compounds of grilled lamb
MEAT SCIENCE Meat Science 69 (2005) 233–242 www.elsevier.com/locate/meatsci
Dietary manipulation of fatty acid composition in lamb meat and its effect on the volatile aroma compounds of grilled lamb J. Stephen Elmore a,*, Sarah L. Cooper b, Michael Enser c, Donald S. Mottram a, Liam A. Sinclair b, Robert G. Wilkinson b, Jeffrey D. Wood c a School of Food Biosciences, The University of Reading, Whiteknights, Reading RG6 6AP, UK ASRC, School of Agriculture, Harper Adams University College, Edgmond, Newport, Shropshire TF10 8NB, UK Division of Food Animal Science, School of Veterinary Science, University of Bristol, Langford, Bristol BS40 5DU, UK b
c
Received 3 December 2003; received in revised form 8 July 2004; accepted 8 July 2004
Abstract The effect on lamb muscle of five dietary supplements high in polyunsaturated fatty acids (PUFA) was measured. The supplements were linseed oil, fish oil, protected lipid (high in linoleic acid (C18:2 n 6) and a-linolenic acid (C18:3 n 3)), fish oil/marine algae (1:1), and protected lipid/marine algae (1:1). Eicosapentaenoic acid (C20:5 n 3) and docosahexaenoic acid (C22:6 n 3) were found in the highest amounts in the meat from lambs fed diets containing algae. Meat from lambs fed protected lipid had the highest levels of C18:2 n 6 and C18:3 n 3, due to the effectiveness of the protection system. In grilled meat from these animals, volatile compounds derived from n 3 fatty acids were highest in the meat from the lambs fed the fish oil/algae diet, whereas compounds derived from n 6 fatty acids were highest in the meat from the lambs fed the protected lipid diet. 2004 Elsevier Ltd. All rights reserved. Keywords: Aroma volatiles; Polyunsaturated fatty acids; Algae; Fish oil; Linseed oil; Protected lipid; Lamb; n 3 fatty acids; n 6 fatty acids
1. Introduction The nutritional value of n 3 polyunsaturated fatty acids (PUFA) in the human diet is well recognised, and increased consumption of these fatty acids has been recommended (Department of Health, 1994). The precursor of the n 3 series of long-chain PUFA is the essential fatty acid a-linolenic acid (C18:3 n 3), from which man can synthesise the longer chain PUFA eicosapentaenoic acid (EPA, C20:5 n 3) and docosahexaenoic acid (DHA, C22:6 n 3). However, the conversion efficiency of C18:3 n 3 to EPA and DHA is poor (Enser, Hallett, Hewett, Fursey, & Wood, 1996;
*
Corresponding author. Fax: +44 118 931 0080. E-mail address: [email protected] (J.S. Elmore).
0309-1740/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2004.07.002
Enser et al., 1998; Esner, 2001). Hence, dietary sources of EPA and DHA are desirable. Linoleic acid (C18:2 n 6) and C18:3 n 3 are the PUFA predominantly found in the typical diets of ruminant animals, i.e., cereal or forage (Scott, Cook, & Mills, 1971). In the meat of these ruminants, concentrations of all PUFA are low, because of their biohydrogenation in the rumen (Wachira et al., 2002). However, a proportion of dietary PUFA will not be hydrogenated. Therefore, the inclusion in the animalÕs diet of oils high in n 3 PUFA will still result in elevated n 3 PUFA levels in ruminant tissue. Dietary sources of EPA and DHA include fish, marine microalgae and algae-like microorganisms (Zeller, Barclay, & Abril, 2001). In fact, algae are the major source of these fatty acids in fish. Unlike C18:2 n 6 and C18:3 n 3, EPA and DHA are not incorporated into the triacylglycerols of ruminants to any great extent
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but can be found in the structural phospholipids present in muscle (Wood et al., 1999). Elmore, Mottram, Enser, and Wood (2000) reported that when linseed and fish oils were fed as supplements to Suffolk and Soay lambs, increased levels of n 3 PUFAs were found in the phospholipids. Bruised whole linseed doubled the amount of C18:3 n 3 and the diet containing fish oil caused a two to fourfold increase of EPA and DHA. These changes in the fatty acid composition of the meat resulted in changes in the volatile composition of pressure-cooked muscle, particularly in the animals fed diets containing fish oil, where increases in a number of volatiles formed from the oxidation of n 3 PUFAs were observed. For example 2-(2-pentenyl)furan, 2-ethylfuran and l-penten-3-ol were present in the meat from animals fed the diet containing fish oil at levels 3 times higher than in the meat of the animals fed a control diet. In this paper the effects of five different dietary supplements high in n 3 PUFA on the volatile components of grilled lamb were examined. Two of these five diets include marine algae. The effects of diets containing algae on sheep meat composition have not previously been studied. However, Papadopoulos, Goulas, Apostolaki, and Abril (2002) fed an algae supplement high in DHA to ewes at three different levels and compared the levels of n 3 PUFA in the milk from these ewes with the milk from the ewes fed a control diet, not containing algae. Enrichment of the milk with PUFA occurred, resulting in elevated levels of DHA as well as EPA, whilst the concentrations of C22:5 n 3 and C22:5 n 6 also increased. The Rn 6:Rn–3 ratio decreased from 11.65 in the control milk to 2.40 in the milk of the ewes fed the highest levels of algae. Two of the diets we have examined in this work contain rumen-protected lipids. Cook, Scott, and Ferguson (1970) first showed that formaldehyde treated oil–protein particles could increase PUFA levels in ruminant meat, by reducing their hydrogenation in the rumen. In vitro and in vivo studies (Scott et al., 1971) subsequently demonstrated that virtually complete protection of both C18:2 n 6 and C18:3 n 3 in the rumen could be achieved.
PLS was encapsulated in formaldehyde-treated protein, using a refinement of the process developed by Scott et al. (1971). The five lipid supplements used in the dietary treatments were: (a) (b) (c) (d) (e)
linseed oil, fish oil, PLS, marine algae:fish oil (1:1), marine algae:PLS (1:1).
The PUFA composition of each supplement is shown in Table 1. All diets were offered ad libitum. The lambs were slaughtered at approximately 40 kg. A more comprehensive description of the feeding trials is published elsewhere (Cooper et al., 2004). 2.2. Fatty acid analysis The total fatty acid compositions of M. longissimus dorsi were measured using the method of Wachira et al. (2002). Values for total fatty acids were obtained from the sum of the fatty acids of the phospholipids and the neutral lipids. These lipids were extracted from the meat (10 g) using chloroform:methanol (2:1). Silicic acid solid phase extraction was used to prepare pure fractions of each type of lipid, which were then hydrolysed and cleaned up. Fatty acid methyl esters were prepared from both types of lipid, using diazomethane, and were analysed by gas chromatography. Duplicate analyses were performed on all 10 animals. 2.3. Grilling A portion of the loin muscle of five animals from each dietary treatment was studied. One loin chop from each animal was grilled, in order to analyse the volatile composition of the cooked meat. When required for analysis
Table 1 Polyunsaturated fatty acid compositions of the five diets used in this study (as percentage of total lipid composition) Fatty acid
2. Materials and methods 2.1. Diets Fifty Suffolk-cross wether lambs, weighing approximately 29 kg (SD ± 2.1 kg), were allocated one of five dietary treatments containing 60 g of oil per kg of dry matter, of which approximately 40 g was the test oil. Two of the diets contained a source of protected C18:2 n 6 and C18:3 n 3 (protected lipid supplement (PLS); CSIRO, Blacktown, NSW, Australia). The oil in the
18:2 18:3 18:4 20:4 20:5 22:5 22:6
n n n n n n n
6 3 3 6 3 3 3
Total PUFA Rn 6/Rn 3 P:S a
Fatty acid (%) Linseed
Fish
Fish/algae
PLSa
PLS/algae
22.12 45.63 – – – – –
14.61 3.12 2.98 0.29 6.12 0.59 5.93
14.61 2.48 1.98 0.16 4.10 0.46 15.87
44.39 15.97 – – – – –
35.88 12.05 – – – – 9.97
67.75 1.45 0.48
33.64 1.45 0.59
39.66 3.95 0.80
60.36 5.35 2.78
57.90 2.58 1.63
Protected lipid supplement.
J.S. Elmore et al. / Meat Science 69 (2005) 233–242
the chops, which had been stored at –18 C were defrosted overnight at 4 C. Lamb muscle (M. longissimus dorsi), attached to the bone, and surrounded by adipose tissue, was cooked to a core temperature of 70 C by grilling. Each sample was grilled separately under an electric grill (Belling, Glen Dimplex, Louth, Republic of Ireland). The grill was switched on for 15 min before the meat was cooked. Each chop was placed in the middle of the grilling tray in order to grill uniformly. Cooking to a fixed temperature compensated to an extent for any variations in the thickness between and within the samples, although thicker chops took longer to cook and hence received more surface heat than thinner chops. Samples ranged in average thickness from 25 to 34 mm, the maximum variation within a sample being 7 mm. Samples were turned over every 2 min during cooking. After cooking, the M. longissimus was separated and then chopped in an electric bowl chopper. The aroma volatiles were extracted from the cooked meat, using headspace concentration on Tenax and were analysed using gas chromatography-mass spectrometry. 2.4. Analysis of volatile material The minced lean muscle (10 g) was placed in a screw top conical flask (250 mL). A Dreschel head was attached to the flask, using an SVL fitting (Bibby, Stone, UK). The flask was held in a water bath at 60 C for 1 h while nitrogen, at 40 mL/min, swept the volatiles onto a glass-lined, stainless steel trap (105 mm · 3 mm i.d.) containing 85 mg Tenax TA (Scientific Glass Engineering Ltd., Ringwood, Australia). A standard (100 ng 1,2-dichlorobenzene in 1 ll methanol) was added to the trap at the end of the collection and excess solvent and any water retained on the trap were removed by purging the trap with nitrogen at 40 ml/min for 10 min. All analyses were performed on a Hewlett Packard 5972 mass spectrometer, fitted with a HP5890 Series II gas chromatograph and a G1034C Chemstation. A CHIS injection port (Scientific Glass Engineering Ltd.) was used to thermally desorb the volatiles from the Tenax trap onto a non-polar deactivated fused silica retention gap (5 m · 0.25 mm i.d.; Varian Chrompack International B.V., Middelburg, The Netherlands). The retention gap contained five small loops in a coil, which were cooled in solid carbon dioxide, contained within a 250 mL beaker. The retention gap was attached to a CP-Sil 8 CB low bleed/MS fused silica capillary column (60 m · 0.25 mm i.d., 0.25 lm film thickness; Varian Chrompack). During the desorption period of 5 min, the oven was held at 40 C. After desorption, the oven was held at 40 C for a further 2 min before heating at 4 C/min to 280 C, where it was held for 5 min. Helium at 16 psi was used as the carrier gas, resulting in a flow of 1.0 ml/min at 40 C. A series of n-alkanes (C5–C25) in
235
diethyl ether was analysed, under the same conditions, to obtain linear retention index (LRI) values for the lamb aroma components. The mass spectrometer operated in electron impact mode with an electron energy of 70 eV and an emission current of 35 lA. The mass spectrometer scanned from m/z 29 to m/z 400 at 1.9 scans/s. Compounds were identified by first comparing their mass spectra with those contained in the NIST/EPA/NIH Mass Spectral Database or in previously published literature. Wherever possible identities were confirmed by comparison of linear retention index (LRI) values, with either those of authentic standards, or published values. Quantities of the volatile compounds were approximated by comparison of their peak areas with that of the 1,2-dichlorobenzene internal standard, obtained from the total ion chromatograms, using a response factor of 1. 2.5. Statistical analysis One-way analysis of variance (ANOVA) was carried out on the quantitative data for each compound identified in the analyses of volatiles, and on the data for each fatty acid, in order to determine the effects exerted by the diets.
3. Results and discussion 3.1. Fatty acids The PUFA:saturated fatty acid (P:S) ratio and the Rn–6:Rn 3 ratio are indicators of the nutritional quality of the lipids in a food (Department of Health, 1984; Enser et al., 1996). Of the five lipid supplements studied, mainly due to its relatively high content of 18:3 n 3, the unprotected linseed possessed the highest P:S ratio and lowest Rn 6:Rn 3 ratio, both of which are nutritionally desirable (see Table 2). However, incorporation of 18:3 n 3 into lamb muscle was highest in the diets containing PLS and, in particular, the 100% PLS supplement. Although the linseed oil diet contained the highest levels of n 3 of the five diets, incorporation of n 3 fatty acids into the muscle was the lowest of the five diets. Protected PUFA in the two PLS diets were transferred to the muscle much more effectively than the PUFA in the linseed diet. Although the 18:3 n–3 level of the linseed diet was 3 times that of the 100% PLS diet, the lamb muscle from the animals fed the PLS diet contained 1.5 times as much 18:3 n 3 as the linseed diet. Unsurprisingly, the incorporation of 18:2 n 6 was also highest in the 100% PLS supplement, which had the highest 18:2 n 6 content of the five supplements. When comparing the total muscle lipid data for linseed with that obtained by Enser et al. (1998) for grass-fed lambs, there appeared to be higher amounts of 18:3 n 3, 18:2
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Table 2 Total fatty acid content of M. longissimus dorsi from lambs fed diets high in n 3 polyunsaturated fatty acids Fatty acid
Muscle (mg/100 g)a b
Standard error
Pc
Linseed
PLS
PLS/algae
Fish/algae
Fish oil
C14:0 C16:0 C16:1 n 7 C18:0 trans-C18:1 C18:1 n 9 C18:1 n 7 C18:2 n 6 C18:3 n 3 CLAd C20:1 n 9 C20:3 n 6 C20:4 n 6 C20:5 n 3 C22:4 n 6 C22:5 n 3 C22:6 n 3
77 716 57a 595 161b 1094 33a 157a 88b 37 2.4a 4.1a 28b 24a 1.1b 23a 7.7ab
78 770 59a 598 108a 1127 42b 545c 138c 26 3.1a 6.0b 43c 21a 1.8c 24ab 5.2a
92 891 65a 534 217c 1118 40b 396b 98b 33 3.7a 5.6b 26b 44b 0.3a 27bc 88c
104 896 85b 492 175bc 999 52c 150a 29a 28 35b 3.7a 19a 85c 0.2a 45d 94c
101 926 86b 502 176bc 1056 58c 120a 29a 27 56c 4.1a 19a 48b 0.4a 32c 23b
14 103 9.7 61 22 155 4.1 39 12 5.4 3.4 0.6 3.5 3.3 0.18 2.8 8.2
NS NS ** NS *** NS *** *** *** NS *** *** *** *** *** *** ***
Total P:Se Rn 6:Rn 3f
3401 0.17 1.33
3816 0.47 3.17
3931 0.33 1.66
3673 0.11 0.83
3706 0.10 1.10
438
NS
a
Means for the same fatty acid with different letters (a, b, c, d) are significantly different (P < 0.05); means are duplicate readings from 10 animals. Protected lipid supplement. c Probability that there is a difference between means; NS, no significant difference between means (P > 0.05); ** significant at the 1% level; *** significant at the 0.1% level. d Conjugated linoleic acid. e P:S ratio is (18:2 n 6 + 18:3 n 3)/(12:0 + 14:0 + 16:0 + 18:0). f Rn 6 consists of 18:2, 20:3, 20:4; Rn 3 consists of 18:3, 20:4, 20:5, 22:5, 22:6. b
n 6 and 18:1 n 9 in the linseed-fed sheep, compared with grass-fed sheep. Apart from these differences, the results for grass and unprotected linseed were similar. Levels of DHA and EPA were highest in the meat from the animals fed the combined fish oil/algae diet. Although this diet contained the highest levels of DHA, the fish oil diet was highest in EPA. The meat from the animals fed the two diets that did not contain EPA and DHA, i.e., the PLS diet and the linseed diet, contained significantly lower levels of EPA and DHA than the meat from the other three groups of animals. Diets containing algae led to the largest increases in the total n 3 content of the muscle, the results being similar whether fish oil or PLS was also present. A more comprehensive analysis of the lipid data is provided by Cooper et al. (2004). 3.2. Volatile compounds One hundred and eleven compounds were quantified in the lamb. Each of these compounds was present at a level of at least 20 ng per 100 g of sample in the headspace extract of at least one of the treatments (Table 3). Of these compounds, 78 were significantly affected by
dietary treatment, when examined by analysis of variance. Almost all of these compounds were formed from lipid oxidation. Although Maillard reaction products, such as pyrazines, were found in trace quantities, the only Maillard-derived compounds present above 20 ng in the headspace of 100 g of sample were simple sugar degradation products, such as 3-hydroxy-2-butanone and 2,3-butanedione, and the Strecker aldehydes, 2-methylpropanal, 2-methylbutanal and 3-methylbutanal. The compounds showing significant differences in ANOVA were visualised by principal component analysis (Fig. 1). The data was resolved into four principal components. Principal component (PC) 1 accounted for 87.4% of the variation in the data. The meat from the lambs fed algae was resolved from the other meat samples along PC1. In particular the fish/algae sample was well resolved from the other four samples and was strongly associated with most of the statistically significant volatile compounds. The high levels of DHA in the meat of the animals fed algae may account for the high levels of volatile compounds in these samples. Elmore, Mottram, Enser, and Wood (1999) suggested that highly unsaturated compounds readily form free radicals, which will then
Table 3 Volatile compounds in the headspace of the grilled M. longissimus dorsi from lambs fed on diets high in polyunsaturated fatty acids Numbera
28 30 5
12 35 24 36 46 4 48 23 32 7 16 68 57 20 60
3 26 31 21 43 45 6
c
Pd
Method of identificatione
LRIm
551 553 600 602 649 654 659 659 682 700 700 700 702 703 707 724 731 730 744 753 766 765 769 789 800 804 808 811 818 819 820
Linseed
PLS
PLS/algae
Fish/algae
Fish
1251 82 650 22 112a 54a 33a 80 383a 123a 441a 26a 24 6a 3a 614 4a 35 18a 13a 311a 177 21a 16a 193a 1748a 3a 3a 3a 4a 2a
1425 39 1146 24 184ab 45a 37a 150 530ab 319ab 1463b 34a 21 10ab 4a 649 4a 40 11a 22ab 625bc 203 38a 20a 285a 5539cd 4a 3a 3a 7ab 2ab
971 151 1514 17 179ab 76ab 75ab 121 2829c 853bc 2908b 117b 29 23bc 25bc 576 12bc 27 43ab 84b 1328abc 171 157b 36bc 651ab 12917bcd 35b 9b 11b 19bc 10c
706 44 1786 28 278b 140b 73b 180 8108d 1716c 3106b 336c 38 28c 60c 165 23c 30 95b 255c 1318c 191 510c 60c 1499b 13638d 55b 48c 34c 33c 37b
813 87 747 14 148a 46a 30a 123 1153bc 345b 645a 70ab 16 8ab 13b 614 5ab 26 23 a 39ab 289a 129 66ab 21ab 315a 2008ab 7a 8b 5ab 8ab 5bc
NS NS NS NS * ** * NS *** ** ** *** NS ** ** NS *** NS ** *** * NS *** ** *** * ** * *** ** ***
MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI ms ms ms ms se
5a 9ab 9a 126 157a 23 4ab 5a 31a 21 54 34a
19b 6a 17a 83 124a 21 4a 6ab 41a 20 28 35a
28b 17bc 51ab 136 430ab 23 17b 23abc 82ab 23 54 55ab
26b 34c 128b 163 699b 30 50c 63c 84b 26 68 206c
7a 8ab 20a 70 200a 19 8b 12b 27a 17 25 75b
* *** *** NS * NS *** ** * NS NS ***
MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI ms ms MS + LRI MS + LRI MS + LRI MS + LRI
827 832 855 862 871 871 879 882 891 896 895 900 (continued on next page)
237
71
1-Propanol 2-Methylpropanal Butanaln + 2-butanone + hexane + diacetyl 2-Butanoln 3-Methylbutanal Benzene 1-Butanol 2-Methylbutanal 1-Penten-3-ol Heptane Pentanal 2-Ethylfuran 2-Pentanol 3-Pentanol 1,4-Cyclohexadienen Acetoin Ethylcyclopentane 3-Methyl-3-buten-1-oln 2,3-Diethyloxirane (E)-2-Pentenal 1-Pentanol Toluene 2-Penten-l-ol (E and/or Z) 1-Octenen Octane Hexanal 3-Hexenal (E and/or Z)n a 2,4-Octadienen a 2,4-Octadienen 2-Ethyl-(E)-2-butenaln 4-Propylcyclopentenen 67, 41 (30), 39 (24), 54 (24), 110 (22), 81 (20), 66 (17), 68 (14) Pentyl formaten Propylcyclopentane (E)-2-Hexenal Ethylbenzene 1-Hexanol 1,3-Dimethylbenzene 1,3,5-Octatriene 1,3,5-Octatriene 2-Heptanone 1,2-Dimethylbenzene Styrene Nonane
Mean concentration in headspace (ng/100 g)b
J.S. Elmore et al. / Meat Science 69 (2005) 233–242
39 22 8 56
Compound (m/z (relative intensity))
238
Table 3 (continued) Numbera
44 25 52
41 33
18 9 2 73 19 47 11 15
66 29 1 72 42 64 55 49
10 17 14 27
(Z)-4-Heptenal Heptanal 2-(1-Butenyl)furan (E and/or Z) 107, 122 (34), 77 (23), 55 (13), 108 (9), 39 (8), 121 (5), 65 (5) Isopropylbenzenen Propylcyclohexanen Butylcyclopentane a-Pinene Unknown 54, 55 (64), 71 (47), 39 (39), 83 (36), 41 (27), 43 (20), 53 (16), 29 (15), 126 (14) 6-Methyl-2-Heptanone Propylbenzene (E)-2-Heptenal 1-Ethyl-3-methylbenzenen 3-Ethylcyclopentanone Benzaldehyde 1-Heptanol (Z)-1,5-Octadien-3-oln 1-Octen-3-ol a-Methylstyrenen 2,3-Octanedione 6-Methyl-5-hepten-2-onen 2,2,4,6,6-Pentamethylheptanen 1-Decenen 2-Octanone 2-Pentylfuran 1,2,4-Trimethylbenzene Decane (E,Z)-2,4-Heptadienaln 2-(2-Pentenyl)furan (E and/or Z) Octanal (1-Methylpropyl)benzenen (E,E)-2,4-Heptadienal p-Cymenen 1-Propenylbenzenen Limonene 5-Ethyl-1-formylcyclopentene Butylbenzene (E)-2-Octenal 2-Octen-1-ol (E and/or Z) 1-Octanol (1,1-Dimethylethoxy)benzenen
Mean concentration in headspace (ng/100 g)b c
Pd
Method of identificatione
LRIm
Linseed
PLS
PLS/algae
Fish/algae
Fish
119a 852a 2ab
57a 829ab 1a
225ab 3876ab 8b
531b 7380b 32c
126ab 1778a 5b
* ** *
MS + LRI MS + LRI se
902 905 920
32 4a 6a 16 2ab
20 6ab 13ab 19 4a
33 16bc 32bc 12 29b
40 38c 73c 23 99c
18 9b 18b 25 15b
NS *** *** NS ***
MS + LRI MS + LRI ms MS + LRI
927 934 936 937 951
7a 42 16a 18 5a 331a 91a 13ab 151a 30 32a 37abc 19 14a 14ab 48a 20 41a 9a 4a 542a 18 10a 25 15 254 9a 35 19a 13a 142a 40
11a 29 46bc 16 17b 547b 90a 8a 315a 17 58b 30ab 18 13a 13a 126b 18 44ab 13a 3a 539a 13 22a 41 10 384 38bc 26 38a 15ab 101a 25
31ab 48 104abc 21 33ab 346a 319ab 68ab 662ab 31 118bc 44bc 14 21ab 23bc 239b 18 58b 44ab 13b 1226ab 19 90a 34 16 275 59bc 35 101ab 42ab 269ab 22
50b 58 115c 24 27b 540b 471b 215c 836b 40 177c 55c 32 35b 28c 205b 22 177c 127b 47c 2725b 25 285b 58 21 523 67c 47 164b 60b 461b 26
12a 27 21ab 16 7a 444ab 162a 28b 141a 15 39ab 33ab 9 20ab 20abc 37a 16 75b 20a 9b 870a 13 41a 42 7 484 17ab 22 28a 9a 164a 16
** NS * NS * * * *** * NS ** NS NS * ** * NS *** *** *** ** NS *** NS NS NS * NS ** * * NS
MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI msf MS + LRI MS + LRI ms + lrig MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI ms + lrih msi MS + LRI MS + LRI MS + LRI MS + LRI ms MS + LRI msj MS + LR1 MS + LRI MS + LRI MS + LRI ms
956 957 961 965 965 969 973 977 983 986 987 987 990 992 992 993 999 1000 1002 1002 1007 1014 1017 1030 1033 1035 1038 1061 1063 1071 1074 1074
J.S. Elmore et al. / Meat Science 69 (2005) 233–242
59
Compound (m/z (relative intensity))
74 54 63 53 67 37 38 62 58
34 65 61 40 76 51 13 50 70 77 69 75 78 a
11ab 28a 1100ab 8a 4a 16 42a 15a
10a 38ab 954a 7a 2a 13 37a 28a
13ab 49ab 1712b 28ab 9a 15 134ab 102ab
55c 90b 3959c 65b 37b 22 299b 252b
27bc 41ab 1391ab 19a 7a 11 76a 53a
*** * *** *** *** NS *** **
MS + LRI MS + LRI MS + LRI sek MS + LRI MS + LRI MS + LRI se
1093 1100 1109 1130 1159 1163 1165 1174
26a –
26a tr
37ab 8
65b 31
32a 4
** **
MS + LRI
1175 1190
14 23a tr
13 34a 2
16 52ab 12
22 98b 46
10 38a 8
NS ** ***
MS + LRI MS + LRI
1199 1200 1232
tr
2
19
69
9
***
24a 7a 39a 4a 17a 21a 24a 10a 10ab 7a 31
35a 9a 52ab 25b 30a 29a 38b 16ab 20abc 7a 34
103b 10a 101abc 54abc 86ab 39ab 43ab 12a 10a 14a 24
235b 28b 211c 81c 222b 87b 131c 29b 29c 547b 45
62a 20b 74b 12ab 53a 45b 135c 13a 20c 956c 38
** *** ** * ** ** *** *** ** *** NS
1236 MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI ms + lril
1268 1296 1300 1327 1370 1400 1501 1601 1701 1703 1785
Number refers to coordinate numbers in principal components analysis (Fig. 1). Means for the same volatile compound with different letters (a, b, c, d) are significantly different (P < 0.05); means are from five replicate samples; tr. less than 2 ng; –, less than 0.5 ng in the headspace of 100 g of cooked lamb. c Protected linseed supplement. d Probability that there is a difference between means; NS, no significant difference between means (P > 0.05); * significant at the 5% level; ** significant at the 1% level; *** significant at the 0.1% level. e MS + LRI, mass spectrum and LRI agree with those of authentic compound; ms + lri, mass spectrum identified using NIST/EPA/NIH Mass Spectral Database and LRI agrees with literature value; ms, mass spectrum agrees with spectrum in NIST/EPA/NIH Mass Spectral Database or with other literature spectrum as listed below; se, tentative identification from structure elucidation of mass spectrum. f Whitfield et al. (1982). g LRI value from Kondjoyan and Berdague´ (1996). h LRI value from Rychlik et al. (1998). i Smagula et al. (1979). j Werkhoff et al. (1993). k Elmore et al. (1999). l Urbach and Stark (1975). m Linear retention index on a CP-Sil 8 CB low bleed/MS column. n Reported for the first time in cooked lamb or mutton. b
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2-Nonanone Undecane Nonanal 1-Formyl-5-propylcyclopentene (E,Z)-2,6-nonadienaln Pentylbenzenen (E)-2-Nonenal 2-Ethylbenzaldehyde 134, 133 (71), 119 (57), 105 (56), 91 (38), 77 (25), 79 (17), 103(13) 1-Nonanol Unknown 137, 79 (71), 93 (35), 107 (23), 91 (21), 67 (20), 57(19), 77(17) Naphthalene Dodecane Unknown 135, 150 (66), 77 (19), 133 (15), 149 (10), 136(9), 121(9), 103(7) Unknown 79, 137 (77), 93 (41), 91 (24), 77 (22), 67 (21), 107(20), 109(17) (E)-2-Decenal 2-Undecanone Tridecane (E,E)-2,4-Decadienal (E)-2-Undecenal Tetradecane Pentadecane Hexadecane Heptadecane 2,6,10,14-Tetramethylpentadecane (pristane) 3,7,11,15-Tetramethyl-1-hexadecene (1-phytene)
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Fig. 1. Principal component analysis of the effect of dietary supplement on the volatile composition of grilled lamb chops (principal components 1 vs. 2) (PLS, protected lipid supplement).
propagate the breakdown of other fatty acid molecules. This means that highly unsaturated fatty acids, such as DHA, which contains six double bonds, will oxidise to give characteristic volatile compounds and will also catalyse the breakdown of more saturated fatty acids, resulting in increases of volatiles derived from, for example, 18:1 n 9, 18:2 n 6 and 18:3 n 3, that are present in greater amounts.
Principal component 2 accounted for 8.9% of the total variation in the data. The PLS-containing samples were resolved from the linseed sample and the two fish-containing samples along PC2, the PLS-containing samples having positive values on PC2, the fish and linseed samples having negative values. Resolution of the volatile compounds occurred mainly across PC2. Volatile compounds formed from n 6 fatty acids tended to
Table 4 Volatile compounds, derived from the decomposition of C18:2 n 6 and C18:3 n 3, which varied in the grilled muscle of lambs fed dietary supplements high in polyunsaturated fatty acids Polyunsaturated fatty acid C18:2 n 6
C18:3 n 3
Numbera
Compound
Number
Compound
1 3 4 5 6 7 8 9 10 11 13 14 15 17 21 25 37
2-Pentylfuran Pentyl formate 1-Pentanol 1-Butanol 2-Heptanone Hexanal Pentanal (E)-2-Heptenal 5-Ethyl-1-formylcyclopentene 1-Octen-3-ol (E,E)-2,4-Decadienal 2-Octen-1-ol (E and/or Z) 2,3-Octanedione (E)-2-Octenal 1-Hexanol Heptanal (E)-2-Nonenal
16 31 38 39 42 44 46 48 49 52 56 57 63 64 68 73
3-Hexenal (E and/or Z) (E)-2-Hexenal 2-Ethylbenzaldehyde 1-Penten-3-ol (E,Z)-2,4-Heptadienal (Z)-4-Heptenal (E)-2-Pentenal 2-Penten-1-ol (E and/or Z) (E,E)-2,4-Heptadienal 2-(1-Butenyl)furan (E and/or Z) 2-Ethylfuran a 2,4-Octadiene 2-(2-Pentenyl)furan (E and/or Z) (E,Z)-2,6-Nonadienal a 2,4-Octadiene Benzaldehyde
a
Number refers to coordinate numbers in principal components analysis (Fig. 1).
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be more associated with the PLS-containing samples, whereas those formed from n 3 fatty acids tended to be more associated with the fish-containing sample. Compounds reported in the literature as being formed from the decomposition of 18:2 n 6 and 18:3 n–3 fatty acids are listed in Table 4 (Elmore, Campo, Enser, & Mottram, 2002; Frankel, 1982; Grosch, 1987). One compound in Table 3, likely to result from the decomposition of n 3 fatty acids, is 1,5-octadien-3-ol (compound number 47 in Fig. 1), which contains one more double bond than its n 6 derived analogue 1-octen-3-ol. Other compounds, which appear likely to be formed from n 3 fatty acids, due to their high degree of unsaturation, include the 1,3,5-octatrienes (numbers 43 and 45), benzene (30) and 1,4-cyclohexadiene (35). Several long-chain hydrocarbons and ketones were also associated with the fish-containing samples, in particular 2,6,10,14-tetramethylpentadecane (pristane, number 78), which was at levels 50 times higher in the fish-containing samples than the others. This isoprenoid alkane is a major component of many fish oils; for example, it comprises 14% of shark liver oil (Budavari, 1989). It could be regarded as marker for meat from animals, which have been fed fish oil. A sample of the pure compound (Sigma–Aldrich) did not possess a potent aroma.
4. Conclusions This work has shown that large differences in the lipid composition of lamb muscle, and hence lipid-derived volatiles, can be caused by feeding dietary PUFA to sheep. The relatively low 18:2 n 6 content of the meat of the animals fed fish oil-containing diets meant that these diets gave the meat with the lowest Rn 6/Rn 3 ratio, although in all of the diets the Rn– 6/Rn 3 ratio was beneficially low (Enser et al., 1998). Lipid protection allowed large amounts of PUFA to enter the muscle, resulting in the meat from the PLS diets having the highest P:S ratio. The PLS diets were the only ones that gave meat with a P:S ratio above the minimum recommended value of 0.45 (Department of Health, 1984). When profiled by sensory assessors, the linseed-fed lamb was preferred to the other samples. It scored highest for lamb flavour and lowest for abnormal flavour, whereas algae-containing samples scored highest for abnormal flavour and rancid flavour. Meat from the animals fed fish oil-containing diets scored highest for fishy flavour and PLS-fed samples scored highest for grassy flavour, which was probably related to the high levels of hexanal in the PLS-fed samples. These results suggest that although elevation of PUFA levels in muscle may be nutritionally desirable, poor sensory quality may result, if incorporation of PUFA is excessive.
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Acknowledgements We are most grateful to K.G. Hallett (University of Bristol) for fatty acid determinations. We also gratefully acknowledge funding for this project from the Ministry of Agriculture, Fisheries and Food (Project Code: LS1804), as well as financial support from ABN Ltd., Roche Products Ltd., Tesco Stores Ltd., and Pedigree Petfoods.
References Budavari, S. (1989). The Merck Index (11th ed.). Rahway, NJ: Merck and Co Inc. Cook, L. J., Scott, T. W., & Ferguson, K. A. (1970). Production of poly-unsaturated ruminant body fats. Nature, 228, 178–179. Cooper, S. L., Sinclair, L. A., Wilkinson, R. G., Hallett, K. G., Enser, M., & Wood, J. D. (2004). Manipulation of the n–3 polyunsaturated fatty acid content of muscle and adipose tissue in lambs. Journal of Animal Science, 82, 1461–1470. Department of Health and Social Security. (1984). Report on health and social subjects No. 28. Diet and cardiovascular disease. London: HMSO. Department of Health. (1994). Report on health and social subjects No. 46. Nutritional aspects of cardiovascular disease. London: HMSO. Elmore, J. S., Campo, M. M., Enser, M., & Mottram, D. S. (2002). The effect of lipid composition on meat-like model systems containing cysteine, ribose and polyunsaturated fatty acids. Journal of Agricultural and Food Chemistry, 50, 1126–1132. Elmore, J. S., Mottram, D. S., Enser, M., & Wood, J. D. (1999). The effect of the polyunsaturated fatty acid composition of beef muscle on the profile of aroma volatiles. Journal of Agricultural and Food Chemistry, 47, 1619–1625. Elmore, J. S., Mottram, D. S., Enser, M., & Wood, J. D. (2000). The effects of diet and breed on the major volatiles present in lamb aroma. Meat Science, 55, 149–159. Enser, M., Hallett, K. G., Hewett, B., Fursey, G. A. J., & Wood, J. D. (1996). Fatty acid content and composition of English beef, lamb and pork at retail. Meat Science, 42, 443–456. Enser, M., Hallett, K. G., Hewett, B., Fursey, G. A. J., Wood, J. D., & Harrington, G. (1998). Fatty acid content and composition of UK beef and lamb muscle in relation to production system and implications for human nutrition. Meat Science, 49, 329–341. Esner, M. (2001). The role of fats in human nutrition. In J. B. Rossell (Ed.), Oils and fats, Vol. 2 – Animal carcass fats (pp. 77–122). Leatherhead, United Kingdom: Leatherhead Publishing. Frankel, E. N. (1982). Volatile lipid oxidation products. Progress in Lipid Research, 22, 1–33. Grosch, W. (1987). Reactions of hydroperoxides – products of low molecular weight. In H. W.-S. Chan (Ed.), Autoxidation of unsaturated lipids (pp. 95–139). London: Academic Press. Kondjoyan, N., & Berdague´, J.-L. (1996). A compilation of relative retention indices for the analysis of aromatic compounds. Saint Genes Champanelle,France: INRA de Theix. Papadopoulos, G., Goulas, C., Apostolaki, E., & Abril, R. (2002). Effects of dietary supplements of algae, containing polyunsaturated fatty acids on milk yield and the composition of milk products in dairy ewes. Journal of Dairy Research, 69, 357–365. Rychlik, M., Schieberle, P., & Grosch, W. (1998). Compilation of odor thresholds, odor qualities and retention indices of key food odorants. Garching, Germany: Deutsche Forschunganstalt fu¨r
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Lebensmittelchemie and Institut fu¨r Lebensmittelchemie der Technischen Universita¨t Mu¨nchen. Scott, T. W., Cook, L. J., & Mills, S. C. (1971). Protection of dietary polyunsaturated fatty acids against microbial hydrogenation in ruminants. Journal of the American Oil ChemistsÕ Society, 48, 358–364. Smagula, M. S., Chang, S. S., & Ho, C.-T. (1979). The synthesis of 2(2-pentenyl) furans and their effect on the reversion flavor of soy bean oil. Journal of the American Oil ChemistsÕ Society, 56, 516–519. Urbach, G., & Stark, W. (1975). The C-20 hydrocarbons of butter fat. Journal of Agricultural and Food Chemistry, 23, 20–24. Wachira, A. M., Sinclair, L. A., Wilkinson, R. G., Enser, M., Wood, J. D., & Fisher, A. V. (2002). Effects of dietary fat source and breed on the carcass composition, n–3 polyunsaturated fatty acid and conjugated linoleic acid content of sheep meat and adipose tissue. British Journal of Nutrition, 88, 697–709.
Werkhoff, P., Bru¨ning, J., Emberger, R., Gu¨ntert, M., & Hopp, R. (1993). Flavor chemistry of meat volatiles: new results on flavor components from beef, pork and chicken. In R. Hopp & K. Mori (Eds.), Recent developments in flavour and fragrance chemistry (pp. 183–213). Weinheim, Germany: VCH. Whitfield, F. B., Freeman, D. J., Last, J. H., Bannister, P. A., & Kennett, B. H. (1982). Oct-1-en-3-ol and (5Z)-octa-1,5-dien-3-ol, compounds important in the flavour of prawns and sand-lobsters. Australian Journal of Chemistry, 35, 373–383. Wood, J. D., Enser, M., Fisher, A. V., Nute, G. R., Richardson, R. I., & Sheard, P. R. (1999). Manipulating meat quality and composition. Proceedings of the Nutrition Society, 58, 363–370. Zeller, S., Barclay, W., & Abril, R. (2001). Production of docosohexaenoic acid from microalgae. In F. Shahidi & J. W. Finley (Eds.), Omega-3 fatty acids: chemistry, nutrition and health effects (pp. 108–124). Washington, DC: American Chemical Society.
Dietary manipulation of fatty acid composition in lamb meat and its effect on the volatile aroma compounds of grilled lamb J. Stephen Elmore a,*, Sarah L. Cooper b, Michael Enser c, Donald S. Mottram a, Liam A. Sinclair b, Robert G. Wilkinson b, Jeffrey D. Wood c a School of Food Biosciences, The University of Reading, Whiteknights, Reading RG6 6AP, UK ASRC, School of Agriculture, Harper Adams University College, Edgmond, Newport, Shropshire TF10 8NB, UK Division of Food Animal Science, School of Veterinary Science, University of Bristol, Langford, Bristol BS40 5DU, UK b
c
Received 3 December 2003; received in revised form 8 July 2004; accepted 8 July 2004
Abstract The effect on lamb muscle of five dietary supplements high in polyunsaturated fatty acids (PUFA) was measured. The supplements were linseed oil, fish oil, protected lipid (high in linoleic acid (C18:2 n 6) and a-linolenic acid (C18:3 n 3)), fish oil/marine algae (1:1), and protected lipid/marine algae (1:1). Eicosapentaenoic acid (C20:5 n 3) and docosahexaenoic acid (C22:6 n 3) were found in the highest amounts in the meat from lambs fed diets containing algae. Meat from lambs fed protected lipid had the highest levels of C18:2 n 6 and C18:3 n 3, due to the effectiveness of the protection system. In grilled meat from these animals, volatile compounds derived from n 3 fatty acids were highest in the meat from the lambs fed the fish oil/algae diet, whereas compounds derived from n 6 fatty acids were highest in the meat from the lambs fed the protected lipid diet. 2004 Elsevier Ltd. All rights reserved. Keywords: Aroma volatiles; Polyunsaturated fatty acids; Algae; Fish oil; Linseed oil; Protected lipid; Lamb; n 3 fatty acids; n 6 fatty acids
1. Introduction The nutritional value of n 3 polyunsaturated fatty acids (PUFA) in the human diet is well recognised, and increased consumption of these fatty acids has been recommended (Department of Health, 1994). The precursor of the n 3 series of long-chain PUFA is the essential fatty acid a-linolenic acid (C18:3 n 3), from which man can synthesise the longer chain PUFA eicosapentaenoic acid (EPA, C20:5 n 3) and docosahexaenoic acid (DHA, C22:6 n 3). However, the conversion efficiency of C18:3 n 3 to EPA and DHA is poor (Enser, Hallett, Hewett, Fursey, & Wood, 1996;
*
Corresponding author. Fax: +44 118 931 0080. E-mail address: [email protected] (J.S. Elmore).
0309-1740/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2004.07.002
Enser et al., 1998; Esner, 2001). Hence, dietary sources of EPA and DHA are desirable. Linoleic acid (C18:2 n 6) and C18:3 n 3 are the PUFA predominantly found in the typical diets of ruminant animals, i.e., cereal or forage (Scott, Cook, & Mills, 1971). In the meat of these ruminants, concentrations of all PUFA are low, because of their biohydrogenation in the rumen (Wachira et al., 2002). However, a proportion of dietary PUFA will not be hydrogenated. Therefore, the inclusion in the animalÕs diet of oils high in n 3 PUFA will still result in elevated n 3 PUFA levels in ruminant tissue. Dietary sources of EPA and DHA include fish, marine microalgae and algae-like microorganisms (Zeller, Barclay, & Abril, 2001). In fact, algae are the major source of these fatty acids in fish. Unlike C18:2 n 6 and C18:3 n 3, EPA and DHA are not incorporated into the triacylglycerols of ruminants to any great extent
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but can be found in the structural phospholipids present in muscle (Wood et al., 1999). Elmore, Mottram, Enser, and Wood (2000) reported that when linseed and fish oils were fed as supplements to Suffolk and Soay lambs, increased levels of n 3 PUFAs were found in the phospholipids. Bruised whole linseed doubled the amount of C18:3 n 3 and the diet containing fish oil caused a two to fourfold increase of EPA and DHA. These changes in the fatty acid composition of the meat resulted in changes in the volatile composition of pressure-cooked muscle, particularly in the animals fed diets containing fish oil, where increases in a number of volatiles formed from the oxidation of n 3 PUFAs were observed. For example 2-(2-pentenyl)furan, 2-ethylfuran and l-penten-3-ol were present in the meat from animals fed the diet containing fish oil at levels 3 times higher than in the meat of the animals fed a control diet. In this paper the effects of five different dietary supplements high in n 3 PUFA on the volatile components of grilled lamb were examined. Two of these five diets include marine algae. The effects of diets containing algae on sheep meat composition have not previously been studied. However, Papadopoulos, Goulas, Apostolaki, and Abril (2002) fed an algae supplement high in DHA to ewes at three different levels and compared the levels of n 3 PUFA in the milk from these ewes with the milk from the ewes fed a control diet, not containing algae. Enrichment of the milk with PUFA occurred, resulting in elevated levels of DHA as well as EPA, whilst the concentrations of C22:5 n 3 and C22:5 n 6 also increased. The Rn 6:Rn–3 ratio decreased from 11.65 in the control milk to 2.40 in the milk of the ewes fed the highest levels of algae. Two of the diets we have examined in this work contain rumen-protected lipids. Cook, Scott, and Ferguson (1970) first showed that formaldehyde treated oil–protein particles could increase PUFA levels in ruminant meat, by reducing their hydrogenation in the rumen. In vitro and in vivo studies (Scott et al., 1971) subsequently demonstrated that virtually complete protection of both C18:2 n 6 and C18:3 n 3 in the rumen could be achieved.
PLS was encapsulated in formaldehyde-treated protein, using a refinement of the process developed by Scott et al. (1971). The five lipid supplements used in the dietary treatments were: (a) (b) (c) (d) (e)
linseed oil, fish oil, PLS, marine algae:fish oil (1:1), marine algae:PLS (1:1).
The PUFA composition of each supplement is shown in Table 1. All diets were offered ad libitum. The lambs were slaughtered at approximately 40 kg. A more comprehensive description of the feeding trials is published elsewhere (Cooper et al., 2004). 2.2. Fatty acid analysis The total fatty acid compositions of M. longissimus dorsi were measured using the method of Wachira et al. (2002). Values for total fatty acids were obtained from the sum of the fatty acids of the phospholipids and the neutral lipids. These lipids were extracted from the meat (10 g) using chloroform:methanol (2:1). Silicic acid solid phase extraction was used to prepare pure fractions of each type of lipid, which were then hydrolysed and cleaned up. Fatty acid methyl esters were prepared from both types of lipid, using diazomethane, and were analysed by gas chromatography. Duplicate analyses were performed on all 10 animals. 2.3. Grilling A portion of the loin muscle of five animals from each dietary treatment was studied. One loin chop from each animal was grilled, in order to analyse the volatile composition of the cooked meat. When required for analysis
Table 1 Polyunsaturated fatty acid compositions of the five diets used in this study (as percentage of total lipid composition) Fatty acid
2. Materials and methods 2.1. Diets Fifty Suffolk-cross wether lambs, weighing approximately 29 kg (SD ± 2.1 kg), were allocated one of five dietary treatments containing 60 g of oil per kg of dry matter, of which approximately 40 g was the test oil. Two of the diets contained a source of protected C18:2 n 6 and C18:3 n 3 (protected lipid supplement (PLS); CSIRO, Blacktown, NSW, Australia). The oil in the
18:2 18:3 18:4 20:4 20:5 22:5 22:6
n n n n n n n
6 3 3 6 3 3 3
Total PUFA Rn 6/Rn 3 P:S a
Fatty acid (%) Linseed
Fish
Fish/algae
PLSa
PLS/algae
22.12 45.63 – – – – –
14.61 3.12 2.98 0.29 6.12 0.59 5.93
14.61 2.48 1.98 0.16 4.10 0.46 15.87
44.39 15.97 – – – – –
35.88 12.05 – – – – 9.97
67.75 1.45 0.48
33.64 1.45 0.59
39.66 3.95 0.80
60.36 5.35 2.78
57.90 2.58 1.63
Protected lipid supplement.
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the chops, which had been stored at –18 C were defrosted overnight at 4 C. Lamb muscle (M. longissimus dorsi), attached to the bone, and surrounded by adipose tissue, was cooked to a core temperature of 70 C by grilling. Each sample was grilled separately under an electric grill (Belling, Glen Dimplex, Louth, Republic of Ireland). The grill was switched on for 15 min before the meat was cooked. Each chop was placed in the middle of the grilling tray in order to grill uniformly. Cooking to a fixed temperature compensated to an extent for any variations in the thickness between and within the samples, although thicker chops took longer to cook and hence received more surface heat than thinner chops. Samples ranged in average thickness from 25 to 34 mm, the maximum variation within a sample being 7 mm. Samples were turned over every 2 min during cooking. After cooking, the M. longissimus was separated and then chopped in an electric bowl chopper. The aroma volatiles were extracted from the cooked meat, using headspace concentration on Tenax and were analysed using gas chromatography-mass spectrometry. 2.4. Analysis of volatile material The minced lean muscle (10 g) was placed in a screw top conical flask (250 mL). A Dreschel head was attached to the flask, using an SVL fitting (Bibby, Stone, UK). The flask was held in a water bath at 60 C for 1 h while nitrogen, at 40 mL/min, swept the volatiles onto a glass-lined, stainless steel trap (105 mm · 3 mm i.d.) containing 85 mg Tenax TA (Scientific Glass Engineering Ltd., Ringwood, Australia). A standard (100 ng 1,2-dichlorobenzene in 1 ll methanol) was added to the trap at the end of the collection and excess solvent and any water retained on the trap were removed by purging the trap with nitrogen at 40 ml/min for 10 min. All analyses were performed on a Hewlett Packard 5972 mass spectrometer, fitted with a HP5890 Series II gas chromatograph and a G1034C Chemstation. A CHIS injection port (Scientific Glass Engineering Ltd.) was used to thermally desorb the volatiles from the Tenax trap onto a non-polar deactivated fused silica retention gap (5 m · 0.25 mm i.d.; Varian Chrompack International B.V., Middelburg, The Netherlands). The retention gap contained five small loops in a coil, which were cooled in solid carbon dioxide, contained within a 250 mL beaker. The retention gap was attached to a CP-Sil 8 CB low bleed/MS fused silica capillary column (60 m · 0.25 mm i.d., 0.25 lm film thickness; Varian Chrompack). During the desorption period of 5 min, the oven was held at 40 C. After desorption, the oven was held at 40 C for a further 2 min before heating at 4 C/min to 280 C, where it was held for 5 min. Helium at 16 psi was used as the carrier gas, resulting in a flow of 1.0 ml/min at 40 C. A series of n-alkanes (C5–C25) in
235
diethyl ether was analysed, under the same conditions, to obtain linear retention index (LRI) values for the lamb aroma components. The mass spectrometer operated in electron impact mode with an electron energy of 70 eV and an emission current of 35 lA. The mass spectrometer scanned from m/z 29 to m/z 400 at 1.9 scans/s. Compounds were identified by first comparing their mass spectra with those contained in the NIST/EPA/NIH Mass Spectral Database or in previously published literature. Wherever possible identities were confirmed by comparison of linear retention index (LRI) values, with either those of authentic standards, or published values. Quantities of the volatile compounds were approximated by comparison of their peak areas with that of the 1,2-dichlorobenzene internal standard, obtained from the total ion chromatograms, using a response factor of 1. 2.5. Statistical analysis One-way analysis of variance (ANOVA) was carried out on the quantitative data for each compound identified in the analyses of volatiles, and on the data for each fatty acid, in order to determine the effects exerted by the diets.
3. Results and discussion 3.1. Fatty acids The PUFA:saturated fatty acid (P:S) ratio and the Rn–6:Rn 3 ratio are indicators of the nutritional quality of the lipids in a food (Department of Health, 1984; Enser et al., 1996). Of the five lipid supplements studied, mainly due to its relatively high content of 18:3 n 3, the unprotected linseed possessed the highest P:S ratio and lowest Rn 6:Rn 3 ratio, both of which are nutritionally desirable (see Table 2). However, incorporation of 18:3 n 3 into lamb muscle was highest in the diets containing PLS and, in particular, the 100% PLS supplement. Although the linseed oil diet contained the highest levels of n 3 of the five diets, incorporation of n 3 fatty acids into the muscle was the lowest of the five diets. Protected PUFA in the two PLS diets were transferred to the muscle much more effectively than the PUFA in the linseed diet. Although the 18:3 n–3 level of the linseed diet was 3 times that of the 100% PLS diet, the lamb muscle from the animals fed the PLS diet contained 1.5 times as much 18:3 n 3 as the linseed diet. Unsurprisingly, the incorporation of 18:2 n 6 was also highest in the 100% PLS supplement, which had the highest 18:2 n 6 content of the five supplements. When comparing the total muscle lipid data for linseed with that obtained by Enser et al. (1998) for grass-fed lambs, there appeared to be higher amounts of 18:3 n 3, 18:2
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Table 2 Total fatty acid content of M. longissimus dorsi from lambs fed diets high in n 3 polyunsaturated fatty acids Fatty acid
Muscle (mg/100 g)a b
Standard error
Pc
Linseed
PLS
PLS/algae
Fish/algae
Fish oil
C14:0 C16:0 C16:1 n 7 C18:0 trans-C18:1 C18:1 n 9 C18:1 n 7 C18:2 n 6 C18:3 n 3 CLAd C20:1 n 9 C20:3 n 6 C20:4 n 6 C20:5 n 3 C22:4 n 6 C22:5 n 3 C22:6 n 3
77 716 57a 595 161b 1094 33a 157a 88b 37 2.4a 4.1a 28b 24a 1.1b 23a 7.7ab
78 770 59a 598 108a 1127 42b 545c 138c 26 3.1a 6.0b 43c 21a 1.8c 24ab 5.2a
92 891 65a 534 217c 1118 40b 396b 98b 33 3.7a 5.6b 26b 44b 0.3a 27bc 88c
104 896 85b 492 175bc 999 52c 150a 29a 28 35b 3.7a 19a 85c 0.2a 45d 94c
101 926 86b 502 176bc 1056 58c 120a 29a 27 56c 4.1a 19a 48b 0.4a 32c 23b
14 103 9.7 61 22 155 4.1 39 12 5.4 3.4 0.6 3.5 3.3 0.18 2.8 8.2
NS NS ** NS *** NS *** *** *** NS *** *** *** *** *** *** ***
Total P:Se Rn 6:Rn 3f
3401 0.17 1.33
3816 0.47 3.17
3931 0.33 1.66
3673 0.11 0.83
3706 0.10 1.10
438
NS
a
Means for the same fatty acid with different letters (a, b, c, d) are significantly different (P < 0.05); means are duplicate readings from 10 animals. Protected lipid supplement. c Probability that there is a difference between means; NS, no significant difference between means (P > 0.05); ** significant at the 1% level; *** significant at the 0.1% level. d Conjugated linoleic acid. e P:S ratio is (18:2 n 6 + 18:3 n 3)/(12:0 + 14:0 + 16:0 + 18:0). f Rn 6 consists of 18:2, 20:3, 20:4; Rn 3 consists of 18:3, 20:4, 20:5, 22:5, 22:6. b
n 6 and 18:1 n 9 in the linseed-fed sheep, compared with grass-fed sheep. Apart from these differences, the results for grass and unprotected linseed were similar. Levels of DHA and EPA were highest in the meat from the animals fed the combined fish oil/algae diet. Although this diet contained the highest levels of DHA, the fish oil diet was highest in EPA. The meat from the animals fed the two diets that did not contain EPA and DHA, i.e., the PLS diet and the linseed diet, contained significantly lower levels of EPA and DHA than the meat from the other three groups of animals. Diets containing algae led to the largest increases in the total n 3 content of the muscle, the results being similar whether fish oil or PLS was also present. A more comprehensive analysis of the lipid data is provided by Cooper et al. (2004). 3.2. Volatile compounds One hundred and eleven compounds were quantified in the lamb. Each of these compounds was present at a level of at least 20 ng per 100 g of sample in the headspace extract of at least one of the treatments (Table 3). Of these compounds, 78 were significantly affected by
dietary treatment, when examined by analysis of variance. Almost all of these compounds were formed from lipid oxidation. Although Maillard reaction products, such as pyrazines, were found in trace quantities, the only Maillard-derived compounds present above 20 ng in the headspace of 100 g of sample were simple sugar degradation products, such as 3-hydroxy-2-butanone and 2,3-butanedione, and the Strecker aldehydes, 2-methylpropanal, 2-methylbutanal and 3-methylbutanal. The compounds showing significant differences in ANOVA were visualised by principal component analysis (Fig. 1). The data was resolved into four principal components. Principal component (PC) 1 accounted for 87.4% of the variation in the data. The meat from the lambs fed algae was resolved from the other meat samples along PC1. In particular the fish/algae sample was well resolved from the other four samples and was strongly associated with most of the statistically significant volatile compounds. The high levels of DHA in the meat of the animals fed algae may account for the high levels of volatile compounds in these samples. Elmore, Mottram, Enser, and Wood (1999) suggested that highly unsaturated compounds readily form free radicals, which will then
Table 3 Volatile compounds in the headspace of the grilled M. longissimus dorsi from lambs fed on diets high in polyunsaturated fatty acids Numbera
28 30 5
12 35 24 36 46 4 48 23 32 7 16 68 57 20 60
3 26 31 21 43 45 6
c
Pd
Method of identificatione
LRIm
551 553 600 602 649 654 659 659 682 700 700 700 702 703 707 724 731 730 744 753 766 765 769 789 800 804 808 811 818 819 820
Linseed
PLS
PLS/algae
Fish/algae
Fish
1251 82 650 22 112a 54a 33a 80 383a 123a 441a 26a 24 6a 3a 614 4a 35 18a 13a 311a 177 21a 16a 193a 1748a 3a 3a 3a 4a 2a
1425 39 1146 24 184ab 45a 37a 150 530ab 319ab 1463b 34a 21 10ab 4a 649 4a 40 11a 22ab 625bc 203 38a 20a 285a 5539cd 4a 3a 3a 7ab 2ab
971 151 1514 17 179ab 76ab 75ab 121 2829c 853bc 2908b 117b 29 23bc 25bc 576 12bc 27 43ab 84b 1328abc 171 157b 36bc 651ab 12917bcd 35b 9b 11b 19bc 10c
706 44 1786 28 278b 140b 73b 180 8108d 1716c 3106b 336c 38 28c 60c 165 23c 30 95b 255c 1318c 191 510c 60c 1499b 13638d 55b 48c 34c 33c 37b
813 87 747 14 148a 46a 30a 123 1153bc 345b 645a 70ab 16 8ab 13b 614 5ab 26 23 a 39ab 289a 129 66ab 21ab 315a 2008ab 7a 8b 5ab 8ab 5bc
NS NS NS NS * ** * NS *** ** ** *** NS ** ** NS *** NS ** *** * NS *** ** *** * ** * *** ** ***
MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI ms ms ms ms se
5a 9ab 9a 126 157a 23 4ab 5a 31a 21 54 34a
19b 6a 17a 83 124a 21 4a 6ab 41a 20 28 35a
28b 17bc 51ab 136 430ab 23 17b 23abc 82ab 23 54 55ab
26b 34c 128b 163 699b 30 50c 63c 84b 26 68 206c
7a 8ab 20a 70 200a 19 8b 12b 27a 17 25 75b
* *** *** NS * NS *** ** * NS NS ***
MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI ms ms MS + LRI MS + LRI MS + LRI MS + LRI
827 832 855 862 871 871 879 882 891 896 895 900 (continued on next page)
237
71
1-Propanol 2-Methylpropanal Butanaln + 2-butanone + hexane + diacetyl 2-Butanoln 3-Methylbutanal Benzene 1-Butanol 2-Methylbutanal 1-Penten-3-ol Heptane Pentanal 2-Ethylfuran 2-Pentanol 3-Pentanol 1,4-Cyclohexadienen Acetoin Ethylcyclopentane 3-Methyl-3-buten-1-oln 2,3-Diethyloxirane (E)-2-Pentenal 1-Pentanol Toluene 2-Penten-l-ol (E and/or Z) 1-Octenen Octane Hexanal 3-Hexenal (E and/or Z)n a 2,4-Octadienen a 2,4-Octadienen 2-Ethyl-(E)-2-butenaln 4-Propylcyclopentenen 67, 41 (30), 39 (24), 54 (24), 110 (22), 81 (20), 66 (17), 68 (14) Pentyl formaten Propylcyclopentane (E)-2-Hexenal Ethylbenzene 1-Hexanol 1,3-Dimethylbenzene 1,3,5-Octatriene 1,3,5-Octatriene 2-Heptanone 1,2-Dimethylbenzene Styrene Nonane
Mean concentration in headspace (ng/100 g)b
J.S. Elmore et al. / Meat Science 69 (2005) 233–242
39 22 8 56
Compound (m/z (relative intensity))
238
Table 3 (continued) Numbera
44 25 52
41 33
18 9 2 73 19 47 11 15
66 29 1 72 42 64 55 49
10 17 14 27
(Z)-4-Heptenal Heptanal 2-(1-Butenyl)furan (E and/or Z) 107, 122 (34), 77 (23), 55 (13), 108 (9), 39 (8), 121 (5), 65 (5) Isopropylbenzenen Propylcyclohexanen Butylcyclopentane a-Pinene Unknown 54, 55 (64), 71 (47), 39 (39), 83 (36), 41 (27), 43 (20), 53 (16), 29 (15), 126 (14) 6-Methyl-2-Heptanone Propylbenzene (E)-2-Heptenal 1-Ethyl-3-methylbenzenen 3-Ethylcyclopentanone Benzaldehyde 1-Heptanol (Z)-1,5-Octadien-3-oln 1-Octen-3-ol a-Methylstyrenen 2,3-Octanedione 6-Methyl-5-hepten-2-onen 2,2,4,6,6-Pentamethylheptanen 1-Decenen 2-Octanone 2-Pentylfuran 1,2,4-Trimethylbenzene Decane (E,Z)-2,4-Heptadienaln 2-(2-Pentenyl)furan (E and/or Z) Octanal (1-Methylpropyl)benzenen (E,E)-2,4-Heptadienal p-Cymenen 1-Propenylbenzenen Limonene 5-Ethyl-1-formylcyclopentene Butylbenzene (E)-2-Octenal 2-Octen-1-ol (E and/or Z) 1-Octanol (1,1-Dimethylethoxy)benzenen
Mean concentration in headspace (ng/100 g)b c
Pd
Method of identificatione
LRIm
Linseed
PLS
PLS/algae
Fish/algae
Fish
119a 852a 2ab
57a 829ab 1a
225ab 3876ab 8b
531b 7380b 32c
126ab 1778a 5b
* ** *
MS + LRI MS + LRI se
902 905 920
32 4a 6a 16 2ab
20 6ab 13ab 19 4a
33 16bc 32bc 12 29b
40 38c 73c 23 99c
18 9b 18b 25 15b
NS *** *** NS ***
MS + LRI MS + LRI ms MS + LRI
927 934 936 937 951
7a 42 16a 18 5a 331a 91a 13ab 151a 30 32a 37abc 19 14a 14ab 48a 20 41a 9a 4a 542a 18 10a 25 15 254 9a 35 19a 13a 142a 40
11a 29 46bc 16 17b 547b 90a 8a 315a 17 58b 30ab 18 13a 13a 126b 18 44ab 13a 3a 539a 13 22a 41 10 384 38bc 26 38a 15ab 101a 25
31ab 48 104abc 21 33ab 346a 319ab 68ab 662ab 31 118bc 44bc 14 21ab 23bc 239b 18 58b 44ab 13b 1226ab 19 90a 34 16 275 59bc 35 101ab 42ab 269ab 22
50b 58 115c 24 27b 540b 471b 215c 836b 40 177c 55c 32 35b 28c 205b 22 177c 127b 47c 2725b 25 285b 58 21 523 67c 47 164b 60b 461b 26
12a 27 21ab 16 7a 444ab 162a 28b 141a 15 39ab 33ab 9 20ab 20abc 37a 16 75b 20a 9b 870a 13 41a 42 7 484 17ab 22 28a 9a 164a 16
** NS * NS * * * *** * NS ** NS NS * ** * NS *** *** *** ** NS *** NS NS NS * NS ** * * NS
MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI msf MS + LRI MS + LRI ms + lrig MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI ms + lrih msi MS + LRI MS + LRI MS + LRI MS + LRI ms MS + LRI msj MS + LR1 MS + LRI MS + LRI MS + LRI ms
956 957 961 965 965 969 973 977 983 986 987 987 990 992 992 993 999 1000 1002 1002 1007 1014 1017 1030 1033 1035 1038 1061 1063 1071 1074 1074
J.S. Elmore et al. / Meat Science 69 (2005) 233–242
59
Compound (m/z (relative intensity))
74 54 63 53 67 37 38 62 58
34 65 61 40 76 51 13 50 70 77 69 75 78 a
11ab 28a 1100ab 8a 4a 16 42a 15a
10a 38ab 954a 7a 2a 13 37a 28a
13ab 49ab 1712b 28ab 9a 15 134ab 102ab
55c 90b 3959c 65b 37b 22 299b 252b
27bc 41ab 1391ab 19a 7a 11 76a 53a
*** * *** *** *** NS *** **
MS + LRI MS + LRI MS + LRI sek MS + LRI MS + LRI MS + LRI se
1093 1100 1109 1130 1159 1163 1165 1174
26a –
26a tr
37ab 8
65b 31
32a 4
** **
MS + LRI
1175 1190
14 23a tr
13 34a 2
16 52ab 12
22 98b 46
10 38a 8
NS ** ***
MS + LRI MS + LRI
1199 1200 1232
tr
2
19
69
9
***
24a 7a 39a 4a 17a 21a 24a 10a 10ab 7a 31
35a 9a 52ab 25b 30a 29a 38b 16ab 20abc 7a 34
103b 10a 101abc 54abc 86ab 39ab 43ab 12a 10a 14a 24
235b 28b 211c 81c 222b 87b 131c 29b 29c 547b 45
62a 20b 74b 12ab 53a 45b 135c 13a 20c 956c 38
** *** ** * ** ** *** *** ** *** NS
1236 MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI MS + LRI ms + lril
1268 1296 1300 1327 1370 1400 1501 1601 1701 1703 1785
Number refers to coordinate numbers in principal components analysis (Fig. 1). Means for the same volatile compound with different letters (a, b, c, d) are significantly different (P < 0.05); means are from five replicate samples; tr. less than 2 ng; –, less than 0.5 ng in the headspace of 100 g of cooked lamb. c Protected linseed supplement. d Probability that there is a difference between means; NS, no significant difference between means (P > 0.05); * significant at the 5% level; ** significant at the 1% level; *** significant at the 0.1% level. e MS + LRI, mass spectrum and LRI agree with those of authentic compound; ms + lri, mass spectrum identified using NIST/EPA/NIH Mass Spectral Database and LRI agrees with literature value; ms, mass spectrum agrees with spectrum in NIST/EPA/NIH Mass Spectral Database or with other literature spectrum as listed below; se, tentative identification from structure elucidation of mass spectrum. f Whitfield et al. (1982). g LRI value from Kondjoyan and Berdague´ (1996). h LRI value from Rychlik et al. (1998). i Smagula et al. (1979). j Werkhoff et al. (1993). k Elmore et al. (1999). l Urbach and Stark (1975). m Linear retention index on a CP-Sil 8 CB low bleed/MS column. n Reported for the first time in cooked lamb or mutton. b
J.S. Elmore et al. / Meat Science 69 (2005) 233–242
2-Nonanone Undecane Nonanal 1-Formyl-5-propylcyclopentene (E,Z)-2,6-nonadienaln Pentylbenzenen (E)-2-Nonenal 2-Ethylbenzaldehyde 134, 133 (71), 119 (57), 105 (56), 91 (38), 77 (25), 79 (17), 103(13) 1-Nonanol Unknown 137, 79 (71), 93 (35), 107 (23), 91 (21), 67 (20), 57(19), 77(17) Naphthalene Dodecane Unknown 135, 150 (66), 77 (19), 133 (15), 149 (10), 136(9), 121(9), 103(7) Unknown 79, 137 (77), 93 (41), 91 (24), 77 (22), 67 (21), 107(20), 109(17) (E)-2-Decenal 2-Undecanone Tridecane (E,E)-2,4-Decadienal (E)-2-Undecenal Tetradecane Pentadecane Hexadecane Heptadecane 2,6,10,14-Tetramethylpentadecane (pristane) 3,7,11,15-Tetramethyl-1-hexadecene (1-phytene)
239
240
J.S. Elmore et al. / Meat Science 69 (2005) 233–242
Fig. 1. Principal component analysis of the effect of dietary supplement on the volatile composition of grilled lamb chops (principal components 1 vs. 2) (PLS, protected lipid supplement).
propagate the breakdown of other fatty acid molecules. This means that highly unsaturated fatty acids, such as DHA, which contains six double bonds, will oxidise to give characteristic volatile compounds and will also catalyse the breakdown of more saturated fatty acids, resulting in increases of volatiles derived from, for example, 18:1 n 9, 18:2 n 6 and 18:3 n 3, that are present in greater amounts.
Principal component 2 accounted for 8.9% of the total variation in the data. The PLS-containing samples were resolved from the linseed sample and the two fish-containing samples along PC2, the PLS-containing samples having positive values on PC2, the fish and linseed samples having negative values. Resolution of the volatile compounds occurred mainly across PC2. Volatile compounds formed from n 6 fatty acids tended to
Table 4 Volatile compounds, derived from the decomposition of C18:2 n 6 and C18:3 n 3, which varied in the grilled muscle of lambs fed dietary supplements high in polyunsaturated fatty acids Polyunsaturated fatty acid C18:2 n 6
C18:3 n 3
Numbera
Compound
Number
Compound
1 3 4 5 6 7 8 9 10 11 13 14 15 17 21 25 37
2-Pentylfuran Pentyl formate 1-Pentanol 1-Butanol 2-Heptanone Hexanal Pentanal (E)-2-Heptenal 5-Ethyl-1-formylcyclopentene 1-Octen-3-ol (E,E)-2,4-Decadienal 2-Octen-1-ol (E and/or Z) 2,3-Octanedione (E)-2-Octenal 1-Hexanol Heptanal (E)-2-Nonenal
16 31 38 39 42 44 46 48 49 52 56 57 63 64 68 73
3-Hexenal (E and/or Z) (E)-2-Hexenal 2-Ethylbenzaldehyde 1-Penten-3-ol (E,Z)-2,4-Heptadienal (Z)-4-Heptenal (E)-2-Pentenal 2-Penten-1-ol (E and/or Z) (E,E)-2,4-Heptadienal 2-(1-Butenyl)furan (E and/or Z) 2-Ethylfuran a 2,4-Octadiene 2-(2-Pentenyl)furan (E and/or Z) (E,Z)-2,6-Nonadienal a 2,4-Octadiene Benzaldehyde
a
Number refers to coordinate numbers in principal components analysis (Fig. 1).
J.S. Elmore et al. / Meat Science 69 (2005) 233–242
be more associated with the PLS-containing samples, whereas those formed from n 3 fatty acids tended to be more associated with the fish-containing sample. Compounds reported in the literature as being formed from the decomposition of 18:2 n 6 and 18:3 n–3 fatty acids are listed in Table 4 (Elmore, Campo, Enser, & Mottram, 2002; Frankel, 1982; Grosch, 1987). One compound in Table 3, likely to result from the decomposition of n 3 fatty acids, is 1,5-octadien-3-ol (compound number 47 in Fig. 1), which contains one more double bond than its n 6 derived analogue 1-octen-3-ol. Other compounds, which appear likely to be formed from n 3 fatty acids, due to their high degree of unsaturation, include the 1,3,5-octatrienes (numbers 43 and 45), benzene (30) and 1,4-cyclohexadiene (35). Several long-chain hydrocarbons and ketones were also associated with the fish-containing samples, in particular 2,6,10,14-tetramethylpentadecane (pristane, number 78), which was at levels 50 times higher in the fish-containing samples than the others. This isoprenoid alkane is a major component of many fish oils; for example, it comprises 14% of shark liver oil (Budavari, 1989). It could be regarded as marker for meat from animals, which have been fed fish oil. A sample of the pure compound (Sigma–Aldrich) did not possess a potent aroma.
4. Conclusions This work has shown that large differences in the lipid composition of lamb muscle, and hence lipid-derived volatiles, can be caused by feeding dietary PUFA to sheep. The relatively low 18:2 n 6 content of the meat of the animals fed fish oil-containing diets meant that these diets gave the meat with the lowest Rn 6/Rn 3 ratio, although in all of the diets the Rn– 6/Rn 3 ratio was beneficially low (Enser et al., 1998). Lipid protection allowed large amounts of PUFA to enter the muscle, resulting in the meat from the PLS diets having the highest P:S ratio. The PLS diets were the only ones that gave meat with a P:S ratio above the minimum recommended value of 0.45 (Department of Health, 1984). When profiled by sensory assessors, the linseed-fed lamb was preferred to the other samples. It scored highest for lamb flavour and lowest for abnormal flavour, whereas algae-containing samples scored highest for abnormal flavour and rancid flavour. Meat from the animals fed fish oil-containing diets scored highest for fishy flavour and PLS-fed samples scored highest for grassy flavour, which was probably related to the high levels of hexanal in the PLS-fed samples. These results suggest that although elevation of PUFA levels in muscle may be nutritionally desirable, poor sensory quality may result, if incorporation of PUFA is excessive.
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Acknowledgements We are most grateful to K.G. Hallett (University of Bristol) for fatty acid determinations. We also gratefully acknowledge funding for this project from the Ministry of Agriculture, Fisheries and Food (Project Code: LS1804), as well as financial support from ABN Ltd., Roche Products Ltd., Tesco Stores Ltd., and Pedigree Petfoods.
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