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Fatty acid profiles of the total lipids and lipid oxidation in pork muscles as affected by canola oil in the animal diet and muscle location
Meat ";cience 23 (1988) 201-210
Fatty Acid Profiles of the Total Lipids and Lipid Oxiidation in Pork Muscles as Affected by Canola Oil in the Animal Diet and Muscle Location
K. S. Rhee, Y. A. Ziprin, G. Ordonez & C. E. Bohac* Meats and Muscle Biology Section, Department of Animal Science, Texas A&M University, College Station, Texas 77843, USA (Received 14 March 1988; revised version received 12 July 1988; accepted 13 July 1988)
ABSTRACT Twelve pigs at about 35 days of age were fed a control diet or test diets containing either 10% or 20% canola oil ( C O ) f o r 100 days. Four different muscles were excised from each carcass at 24 h post-mortem for analyses. Inclusion of 10% and 20% CO in the animal diet increased (P < 0"05) the relative amount (weight per cent) of unsaturatedfatty acids in the total lipids ( lipids extracted by 2:1 chloroform-methanol) by 6"7 and 15"8 percentage points, respectively,from 57"8%for the control and also increased (P < 0"05) that of polyunsaturated fatty acids by 5.5 and 9.7 percentage points, respectively, from 19"4% for the control The 20% CO treatment increased ( P < 0.05) the relative amount of monounsaturatedfatty acids (primarily CZ8:1) by 6"1 percentage points from 38"4% for the control, while the 10% CO treatment had no significant effect. The 10% or 20% CO treatment had n o significant effect on microsomal enzymic lipid peroxidation activity, heme pigment content, nonheme iron content and total lipid concentration. Overall lipid oxidation in ground muscle samples stored at 4°C tended to be higher for the 10% and20% CO treatment groups than for the control The tendency of increased lipid oxidation by the CO treatments apparently resulted from the increased percentages of polyunsaturated fatty acids, rather than from changes in catalytic constituents. * Present address: The Veterans Administration Medical Center, Denver, CO 80220, USA. 201
Meat Science 0309-1740/88/$03"50 © 1988 Elsevier Science Publishers Ltd, England. Printed in Great Britain
202
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INTRODUCTION Dietary monounsaturated fat is receiving an increasing attention from medical and scientific communities because of its promising health benefit. In certain areas of the Mediterranean region, the typical diet is high in olive oil, which is high in oleic acid, and the incidence of heart disease and levels of plasma cholesterol are fairly low (Grundy, 1986). Inclusion of oleic acid in the diet has been shown to lower the level of the undesirable ('harmful') plasma lipid, low density lipoprotein-cholesterol, without decreasing the desirable ('protective') plasma lipid, high density lipoprotein-cholesterol (Mattson & Grundy, 1985). In an effort to increase the monounsaturated fat content in pork, St. John et al. (1987) fed pigs a diet containing elevated levels ofcanola oil (with 64% oleic acid and 28% polyunsaturated fatty acids), and evaluated fatty acid profiles of the neutral lipids and sensory and carcass traits of tissues. The oxidation of meat lipids results in not only flavor deterioration in raw and cooked meat but also discoloration in raw meat. Any degree of lipid oxidation occurring in raw meat materials accelerates the oxidation reaction in cooked or processed meats because of the free radical chain reaction nature of lipid oxidation. Various catalytic factors have been found to influence lipid oxidation in red meat. Chief among them are meat pigments (hemoproteins), nonheme iron, and microsomal enzymic lipid peroxidation activity (Love, 1983; Rhee et al., 1986, 1987). However, most research studies dealing with catalytic factors of lipid oxidation in red meat have been conducted on beef and beef products, and little information is available concerning the catalysis of lipid oxidation in pork as affected by antemortem or post-mortem factors. The present study was a companion study to that of St. John et al. (1987), and was conducted to determine fatty acid profiles of the total lipids (rather than the neutral lipids), the levels of lipid oxidation catalysts, and the oxidative lipid stability of pork muscle tissue (uncooked and uncured) as affected by canola oil levels in the animal diet and muscle locations. Canola is the name reserved for rapeseed that is low in glucosinolates and erucic acid, both undesirable to humans.
MATERIALS AND METHODS Materials
Twelve pigs (four sets of littermates) at about 35 days of age were divided into three groups and each group was fed a control diet or test diets
Fatty acids and lipid oxidation in pork muscles
203
TABLE 1 Swine Diet Compositiona
Item
Ingredient Canola oil Sorghum Oats Soybean meal Ground limestone Defluorinated phosphate Salt Trace mineral premix Vitamin premix Aureo-SP-250b Total Calculated analysis Protein Lysine Calcium Phosphorus
Canola oil diets 0%
10%
20%
0 44.71 25.00 26.81 0-16 2"17 0"25 0-25 0'25 0'25 100"00
10"00 33"75 25-00 27'29 0.16 2'17 0.25 0-25 0-25 0.25 100"00
20.00 22'75 25-00 28.77 0'16 2-17 0'25 0-25 0-25 0"25 100.00
18.80 0-95 0-85 0"75
18.30 0.95 0-85 0"72
17.70 0-95 0"85 0"70
~From St. John et al. (1987). h Medication was removed from the diet 21 days before slaughter.
containing either 10% or 20% canola oil (CO). Pigs were on these diets for 100 days; they weighed approximately 100 kg prior to slaughter. Further details of the diet treatments which were previously reported by St. John et aL (1987) are shown in Table 1. The feeding trial period was from December to February. Longissimus dorsi (LD), psoas major (PM), semimembranosus (SM), and semitendinosus (ST) muscles were removed from each carcass at 24 h postmortem. All muscles were trimmed of outside fat and epimysium. Each muscle from each animal was divided into 150-200 g portions, which were then separately vacuum-packaged and stored at - 2 0 ° C until analyzed. Samples were analyzed first for microsomal enzymic lipid peroxidation activity and overall lipid oxidation potential and then for other variables.
Microsomal lipid peroxidation activity Procedures for separation of microsomes and determination of microsomal protein and lipid peroxidation were those described previously (Rhee et al.,
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K.S. Rhee et al.
1984). The reaction mixtures (4 ml) contained 0"25 mg microsomal protein/ ml, 0.20mM NADPH, 0-20ram ADP, and 0.015mM FeC13 in 0.12M KC15ram histidine buffer (pH6"5) and were incubated at 36°C for 30min. Microsomal lipid peroxidation activity was expressed as nanomoles of malonaldehyde/mg microsomal protein.
Overall lipid oxidation potential A muscle sample was chopped finely (15s) using a Kitchen-Aid food processor (Model KFP 400; Hobart Corp.) with a plastic work-bowl and stainless steel blades, and mixed with 30ppm chlortetracycline to inhibit microbial growth. Thirty-gram portions of each comminuted muscle sample were placed in sterile Petri dishes (8"75-cm diameter) and pressed with the hand with a sterile glove to make the meat surface even. The Petri dishes were then covered with Saran wrap and stored in a cooler at 4°C in the dark for 0, 3 or 6 days (3 replications/storage period/sample). Lipid oxidation in stored, comminuted muscle samples was determined by the 2-thiobarbituric acid (TBA) procedure as described by Rhee (1978) using propyl gallate and EDTA during the blending step to protect the meat from further lipid oxidation which might occur during the assay.
Total lipids The total lipids were extracted using chloroform-methanol (2:1, v/v) according to the procedure of Folch et al. (1957). An aliquot of the total lipid extract (in triplicates) was freed of solvent and its lipid content was determined gravimetrically.
Fatty acid profile An aliquot of the total lipid extract was freed of solvent under nitrogen, and fatty acid methyl esters (FAMEs) were prepared as described by Morrison & Smith (1964). Methyl esters were analyzed using a flame ionization gas chromatograph (Varian 3400), with a fused silica capillary column, Supelcowax 10 (30 m length, 0"25 mm I.D., 0.25 #m film thickness; Supelco, Inc., Bellefonte, PA). Column temperature was 160-200°C (programmed at 4°C/min). Fatty acid methyl esters in hexane were delivered into the column using a Varian Autosampler 8034. The injection port and detector were maintained at 250°C and 300°C, respectively. Hydrogen was used as the carrier gas at 15 psi and nitrogen was the make-up gas. Chromatograms were recorded with a Varian 4270 computing integrator at a chart speed of 0"5 cm/min. The gas chromatograph system was calibrated with standard
Fatty acids and lipid oxidation in pork muscles
205
F A M E mixtures from Supelco, Inc., and palmitic acid methyl ester was arbitrarily chosen as the reference for detector response factors and relative retention times for other FAMEs. Identification of sample fatty acids was made by comparing the relative retention times of F A M E peaks from samples with those of standards. The following equations were used for calculation of response factors and for quantitation of sample FAMEs: RJ~ (from standard F A M E mixtures) = (Wi)(Ay (Wp)(Ai) Concentration (%) in sample = [(Ai)(RFi)/~(Ai)(RFi)] × 100 where, RF~ = I~ = A~ = Wj, = A j, =
response factor of the ith component, weight of the ith component, area of the Rh component, weight of palmitic acid methyl ester, and area of palmitic acid methyl ester.
Nonheme iron and total pigments Nonheme iron content was determined by the method of Schricker et al. (198211 as modified by Rhee & Ziprin (1987). Total pigment content was determined on LD and SM muscles only, by the procedure of Rickansrud & Henrickson (1967), because of insufficient amounts of samples for the PM and ST.
Statistical analysis Analysis of variance, mean separation by the Student-Newman-Keuls test, and c c)rrelation analysis, where appropriate, were performed by using the SAS program (SAS, 1982). The analysis of variance model included diet, animal (nested within diet), and muscle. 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 treatment means, while the mean square for the residual error was used as the error term to test muscle effect and to separate the means.
RESULTS A N D DISCUSSION Total lipid concentrations and relative weight percentages (based on total fatty acids) of total saturated, unsaturated, monounsaturated, and polyunsaturated fatty acids in total lipid extracts are shown in Table 2 and
K.S. Rhee et al.
206
TABLE 2 Total Lipid Concentrations and Percentages of Total Saturated, Unsaturated, unsaturated, and Polyunsaturated Fatty Acids
Mono-
Fatty' acid (%)d Total lipids (g/ lOO g muscle)
Total saturated
Total unsaturated
Mean
SD
Mean
SD
Mean
Diet ~ C ontrol 10% C O
4.9 ~ 4.3 ~
2-2 2.9
42.2° 35-4b
4"7 6.5
57"8¢ 64.5 b
20% CO
6-2"
2.4
26.4'
6-5
73"6"
Muscle y LD
5-3"b
3.2
34.1 "b
9.5
PM SM
5.1 °b 3.5 b
2-3 1.4
34"9°b 38.2°
lif0 7-2
ST
6-5"
2-5
31"6 h
7-9
Total rnonounsaturated
SD
Total polyunsaturated
Mean
SD
Mean
SD
4-7 6.4
38"4b 39"5 b
3-1 5"5
19-4~ 24-9b
4"6 4"3
6-5
44"5 °
3~4
29"1~
3'8
65.7"b
9.4
43-3°
4~2
22.5 b
6"2
65-1 °h 61.9 b
10.0 7-2
37.9 b 39-1 ~
5.6 3.6
27-2" 22"7 b
5-7 5"5
68"4"
8"0
42"8"
4-0
25"6°h
5"0
,,b,~ M e a n s in t h e s a m e c o l u m n w i t h i n t h e s a m e d a t a set (diet o r m u s c l e ) w h i c h a r e n o t f o l l o w e d by t h e s a m e s u p e r s c r i p t letter a r e significantly different ( P < 0"05). R e l a t i v e w e i g h t p e r c e n t b a s e d o n t o t a l f a t t y acids, C O : c a n o l a oil. s L D : Iongissimus dorsi; P M : psoas major; S M : semimembranosus; ST: semitendinosus.
TABLE 3 Percentages of Individual Fatty Acids
Fatty acid (%)d 14:0
16:0
16:1
18:0
18:1
18:2
18:3
20:1
20:4
Diet e Control 10% CO 20% CO
0'8 ~ 0'7 b 0"5 c
26.3 Q 22.3" 16"1 ~
1.2" 0.6 a 0.3 a
15"1" 12.5 b 9'8 c
33'9 b 36.2 b 42.1 a
15.4 c 19.0 b 23.1 ~
FO b 3.0 a 4.0"
3'3" 2-7 a 2.0 a
3.0 ~ 3.0 a 2.0 ~
Muscle I LD PM SM ST
0-7" 0"6 h 0"8 ~ 0-6 b
21.6 b 20.9 b 24"2 ~ 19"6 b
1.0" 0.6 b 0.6 b 0"8 "b
11"8 ~ 13.4" 13"2a 11"4 a
39.4 a 35.1 b 35-0 b 40.l a
17-9 b~ 21.4 a 17.2 ~ 20.0 °b
2.4" 2.7 ~ 2.8 ~ 2.7 a
2-9 a 2.2 ~ 3.6" 2&
2.2 b 3.0 ° 2-7 "b 2"8"
a,b.c M e a n s in t h e s a m e c o l u m n w i t h i n t h e s a m e d a t a set (diet or m u s c l e ) w h i c h are n o t f o l l o w e d by t h e s a m e s u p e r s c r i p t letter a r e significantly different ( P < 0'05). R e l a t i v e w e i g h t p e r c e n t b a s e d o n t o t a l f a t t y acids. ~CO: c a n o l a oil. f L D : longissimus dorsi; P M : psoas major; S M : semimembranosus; ST: semitendinosus,
Fatty acids and lipid oxidation in pork muscles
207
relative percentages of individual fatty acids in Table 3. The diet treatments had no significant (P > 0-05) influence on the total lipid content. However, the CO treatments affected the fatty acid profile of the total lipids from muscle tissue. Inclusion of 10%o and 20% CO in the animal diet increased (P < 0.05) the relative amount of total unsaturated fatty acids by 6-7 and 15.8 percentage points, respectively, from 57.8% for the control and also increased that of total polyunsaturated fatty acids by 5.5 and 9-7 percentage points, respectively, from 19-4% for the control. The 10% and 20% CO treatments increased the relative amount of C18:2 by 3-6 and 7.7 percentage points, respectively, from 15"4% for the control, slightly increased (P < 0-05) the C 18: 3 percentage, and had no significant effect on the C20:4 percentage. The 20% CO treatment increased the relative amount of total monounsaturated[ fatty acids by 6.1 percentage points from 38.4% for the control, but the 10%o CO treatment had no marked effect (P > 0.05). The diet treatment effect on the percentage of C18:1 (the major monounsaturate in pork muscle tissue) was similar to that on the percentage of total monounsaturated fatty acids, with the 20% CO treatment increasing C 18:1 by 8-2 percentage points from 33.9% for the control and the 10% CO treatment having no significant effect. Cencerning the differences among the muscles at different locations, the total lipids from the ST were more unsaturated than the total lipids from the SM, with the lipids from LD and PM muscles being intermediate. The PM had a higher percentage of total polyunsaturated fatty acids or C 18:2 than the LD and SM. In contrast, the LD and ST were higher in the percentage of total monounsaturated fatty acids or C18:1 than the PM and SM. When compared to fatty acid data on the neutral lipids of muscle tissue from the 0%, 10%oand 20% CO-fed pigs (St. John et al., 1987), the data shown in Tables 2 and 3 on the fatty acid composition of the total lipids of muscle tissue from the same animals that were used in the study by St. John et al. (1987) were higher in polyunsaturated fatty acids (specifically C18:2) I C 18:3 and C20:4 data on the neutral lipids were not reported by St. John et al. (1987)~and were lower in monounsaturated fatty acids (C16:1 and C18:1). Similarly, Sharma et al. (1987) who analyzed fatty acid compositions of total, neutral, and polar lipids from pork muscles at different anatomical locations, showed that the percentage of total monounsaturated fatty acids were highest in the neutral lipids, followed by the total lipids and the phospholipids, respectively; the percentage of total polyunsaturated fatty acids was highest in the phospholipids, followed by the total lipids and the neutral lipids, respectively. Da~La on lipid oxidation catalysts are presented in Table 4. Microsomal enzymic lipid peroxidation activity tended to decrease as the percentage of CO in the diet increased, but differences among the diet treatments were not
K.S. Rhee et al.
208
TABLE 4 Levels o f C a t a l y s t s in M u s c l e T i s s u e C o m p u t e d by D i e t T r e a t m e n t a n d M u s c l e L o c a t i o n
Microsomal lipid peroxidation activityc
Nonheme iron (#g/g)
Total heme pigments (mg/g)
Mean
SD
Mean
SD
Mean
SD
8.36 a 7.46" 4.90 a
5.68 4.00 3.78
3'20 a 2-96 a 3-75 a
1.37 1"37 2.43
0.84 a 0-92 a 0.65 a
0.26 0.30 0.22
6.26 ab 8.65" 4"86 b 7.87 ~
3.55 6.30 3"02 4.87
2'11 b 5.56 a 2.80 b 2"74 b
0'63 1.98 0-75 1-02
0-65 b -0-95" --
0"19 -0.28 --
Diet d Control 10% CO 20% CO Muscle e LD PM SM ST
~,b M e a n s in t h e s a m e c o l u m n w i t h i n t h e s a m e d a t a set (diet or m u s c l e ) w h i c h are n o t f o l l o w e d by t h e s a m e s u p e r s c r i p t letter a r e s i g n i f i c a n t l y different ( P < 0.05). c nmol malonaldehyde/mg microsomal protein. n C O : c a n o l a oil. e L D : Iongissimus'dorsi; P M : psoas major; S M : semimembranosus; ST: semitendinosus.
statistically significant (P > 0.05) because of large variations in the activity between animals within each diet group. Neither nonheme iron content nor total heme pigment concentration in muscle tissue was significantly affected by the diet treatments. As for the differences among the muscles at different anatomical locations, the PM and ST had higher microsomal lipid peroxidation activity than the SM and the LD had intermediate activity. The PM was also higher in nonheme iron content compared to the other three muscles. The SM had higher total heme pigment concentrations than the LD. Lipid oxidation potential as measured by accumulation of TBA-reactive substances in refrigerated comminuted muscles, although not statistically different among the three diet treatments, tended to be higher for muscles from the 10% and 20% CO-fed animals (Table 5). Large variations between animals within each diet treatment group (Table 5) resulted in the statistical non-significance even when differences between the mean values of the 10% and 20% CO treatment groups and that of the control were marked. The PM had higher TBA values than the LD after 6 days of storage at 4°C; the SM and ST had similar TBA values at each storage time (Table 5). The tendency of increased lipid oxidation by the 10% and 20% CO treatments was apparently due to increases in the percentage of
Fatty acids and lipid oxidation in pork muscles
209
TABLE 5 T B A Values o f G r o u n d M u s c l e s stored at 4°C
TBA number (mg malonaldehyde/kg) 0 day
Diet c Control 10% C O 20% CO M uscl e a LD PM SM ST
3 days
6 days
Mean
SD
Mean
SD
Mean
SD
0-25 a
0.10
0"56a
0'42
1-51 a
1.54
0"24" 0"28"
0'08 0"12
0"78 a 1-75a
0-91 2.99
3-04" 4-91 a
5'20 7-58
0.25" 0'29" 0.24 ~ 0.25 a
0-08 0-12 0'11 0-08
0"41 a 1"84a |-05 a 0'81 a
0"12 3"29 1.15 1"18
0-88 b 6'52" 2"71 ~b 2.51 "b
0"57 8'79 3"03 4.66
a,b M e a n s in the s a m e c o l u m n within the s a m e d a t a set (diet or muscle) which are n o t followed by the, s a m e superscript letter are significantly different (P < 0-05). c CO: c-anola oil. a LD: longissimusdorsi; P M : psoas major; SM: semimembranosus; ST: semitendinosus.
polyunsaturated fatty acids by the treatments (cf, Tables 2 and 4). Even though the percentage of total monounsaturated fatty acids was also increased by the 20% CO treatment, monounsaturated fatty acids are far less ~,;usceptible to lipid oxidation than polyunsaturated fatty acids. The relative rates of autoxidation of methyl oleate, linoleate and linolenate at 20°C were shown to be 1:12:25 (Gunstone & Hilditch, 1945). The correlation coefficients between percentages of either C18:2 (the major polyunsaturated fatty acid in pork muscles) or total polyunsaturated fatty acids and TBA values at day 3 and day 6 were 0.36 (P < 0"01) and 0.39 (P < 0.01), respectively. Percentages of monounsaturated fatty acids were not correlated with TBA values of stored muscles (P > 0.01). It is apparent that when the level of monounsaturated fatty acids in pork is to be increased by inclusion, in the swine diet, of elevated levels of a fat or oil containing a large amount of monounsaturated fatty acids, the fat or oil should be low in polyunsaturated fatty acids so that the oxidative stability of the meat would not be adversely affected by the diet modification. A study is in progress in our laboratory to increase the monounsaturated fatty acid level in swine tissues by incorporating into the animal diet a sufficient amount of the oil from seeds of a new variety of sunflower oil (> 80% oleic acid, < 1 0 % polyunsaturated fatty acids) and to determine various
210
K.S. Rhee et al.
characteristics of resultant unprocessed and processed pork products. An alternative approach m a y be the inclusion of an antioxidant, along with the m o n o u n s a t u r a t e source such as CO, in the animal diet. The antioxidant for this purpose has to be one that would be safe for h u m a n consumption and deposited in the meat animal muscle tissue in significant amounts. Tocopherols have been tested in swine and poultry diets and proven to reduce the susceptibility of tissue lipids toward lipid oxidation (Webb et al., 1972; Tsai et al., 1978; Uebersax et al., 1978).
ACKNOWLEDGMENT Technical paper No. 23052, Texas Agricultural Experiment Station. Supported in part by the Natural Fibers and F o o d Protein Commission of Texas.
REFERENCES Folch, J., Lees, M. & Stanley, G. H. S. (1957). J. BioL Chem., 226, 497. Grundy, S. M. (1986). New England J. Med., 314(12), 745. Gunstone, F. D. & Hilditch, T. P. (1945). J. Chem. Soc. (London), 34, 836. Love, J. D. (1983). Food Technol., 37(7), 117. Mattson, F. H. & Grundy, S. M. (1985). J. Lipid Res., 26, 194. Morrison, W. R. & Smith, L. M. (1964). J. Lipid Res., 4, 600. Rhee, K. S. (1978). J. Food Sci., 43, 1776. Rhee, K. S. & Ziprin, Y. A. (1987). J. Food Sci., 52, 1174. Rhee, K. S., Dutson, T. R. & Smith, G. C. (1984). J. Food Sci., 49, 675. Rhee, K. S., Seideman, S. C. & Cross, H. R. (1986). J. Agric. Food Chem., 34, 308. Rhee, K. S., Ziprin, Y. A. & Ordonez, G. (1987). J. Agric. Food Chem., 35, 1013. Rickansrud, D. A. & Henrickson, R. L. (1967). J. Food Sci., 32, 57. SAS (1982). S A S User's Guide: Statistics. SAS Institute, Inc., Cary, NC. Schricker, B. R., Miller, D. D. & Stouffer, J. R. (1982). J. Food Sci., 47, 740. 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. & Smith, S. B. (1987). J. Anita. Sei., 64, 1441. Tsai, T. C., Wellington, G. H. & Pond, W. G. (1978). J. Food Sci., 43, 193. Uebersax, M. A., Dawson, L. E. & Uebersax, K. L. (1978). Poultry Sci., 57, 937. Webb, J. E., Brunson, C. C. & Yates, J. D. (1972). Poultry Sci., 51, 1601.
Fatty Acid Profiles of the Total Lipids and Lipid Oxiidation in Pork Muscles as Affected by Canola Oil in the Animal Diet and Muscle Location
K. S. Rhee, Y. A. Ziprin, G. Ordonez & C. E. Bohac* Meats and Muscle Biology Section, Department of Animal Science, Texas A&M University, College Station, Texas 77843, USA (Received 14 March 1988; revised version received 12 July 1988; accepted 13 July 1988)
ABSTRACT Twelve pigs at about 35 days of age were fed a control diet or test diets containing either 10% or 20% canola oil ( C O ) f o r 100 days. Four different muscles were excised from each carcass at 24 h post-mortem for analyses. Inclusion of 10% and 20% CO in the animal diet increased (P < 0"05) the relative amount (weight per cent) of unsaturatedfatty acids in the total lipids ( lipids extracted by 2:1 chloroform-methanol) by 6"7 and 15"8 percentage points, respectively,from 57"8%for the control and also increased (P < 0"05) that of polyunsaturated fatty acids by 5.5 and 9.7 percentage points, respectively, from 19"4% for the control The 20% CO treatment increased ( P < 0.05) the relative amount of monounsaturatedfatty acids (primarily CZ8:1) by 6"1 percentage points from 38"4% for the control, while the 10% CO treatment had no significant effect. The 10% or 20% CO treatment had n o significant effect on microsomal enzymic lipid peroxidation activity, heme pigment content, nonheme iron content and total lipid concentration. Overall lipid oxidation in ground muscle samples stored at 4°C tended to be higher for the 10% and20% CO treatment groups than for the control The tendency of increased lipid oxidation by the CO treatments apparently resulted from the increased percentages of polyunsaturated fatty acids, rather than from changes in catalytic constituents. * Present address: The Veterans Administration Medical Center, Denver, CO 80220, USA. 201
Meat Science 0309-1740/88/$03"50 © 1988 Elsevier Science Publishers Ltd, England. Printed in Great Britain
202
K.S. Rhee et al.
INTRODUCTION Dietary monounsaturated fat is receiving an increasing attention from medical and scientific communities because of its promising health benefit. In certain areas of the Mediterranean region, the typical diet is high in olive oil, which is high in oleic acid, and the incidence of heart disease and levels of plasma cholesterol are fairly low (Grundy, 1986). Inclusion of oleic acid in the diet has been shown to lower the level of the undesirable ('harmful') plasma lipid, low density lipoprotein-cholesterol, without decreasing the desirable ('protective') plasma lipid, high density lipoprotein-cholesterol (Mattson & Grundy, 1985). In an effort to increase the monounsaturated fat content in pork, St. John et al. (1987) fed pigs a diet containing elevated levels ofcanola oil (with 64% oleic acid and 28% polyunsaturated fatty acids), and evaluated fatty acid profiles of the neutral lipids and sensory and carcass traits of tissues. The oxidation of meat lipids results in not only flavor deterioration in raw and cooked meat but also discoloration in raw meat. Any degree of lipid oxidation occurring in raw meat materials accelerates the oxidation reaction in cooked or processed meats because of the free radical chain reaction nature of lipid oxidation. Various catalytic factors have been found to influence lipid oxidation in red meat. Chief among them are meat pigments (hemoproteins), nonheme iron, and microsomal enzymic lipid peroxidation activity (Love, 1983; Rhee et al., 1986, 1987). However, most research studies dealing with catalytic factors of lipid oxidation in red meat have been conducted on beef and beef products, and little information is available concerning the catalysis of lipid oxidation in pork as affected by antemortem or post-mortem factors. The present study was a companion study to that of St. John et al. (1987), and was conducted to determine fatty acid profiles of the total lipids (rather than the neutral lipids), the levels of lipid oxidation catalysts, and the oxidative lipid stability of pork muscle tissue (uncooked and uncured) as affected by canola oil levels in the animal diet and muscle locations. Canola is the name reserved for rapeseed that is low in glucosinolates and erucic acid, both undesirable to humans.
MATERIALS AND METHODS Materials
Twelve pigs (four sets of littermates) at about 35 days of age were divided into three groups and each group was fed a control diet or test diets
Fatty acids and lipid oxidation in pork muscles
203
TABLE 1 Swine Diet Compositiona
Item
Ingredient Canola oil Sorghum Oats Soybean meal Ground limestone Defluorinated phosphate Salt Trace mineral premix Vitamin premix Aureo-SP-250b Total Calculated analysis Protein Lysine Calcium Phosphorus
Canola oil diets 0%
10%
20%
0 44.71 25.00 26.81 0-16 2"17 0"25 0-25 0'25 0'25 100"00
10"00 33"75 25-00 27'29 0.16 2'17 0.25 0-25 0-25 0.25 100"00
20.00 22'75 25-00 28.77 0'16 2-17 0'25 0-25 0-25 0"25 100.00
18.80 0-95 0-85 0"75
18.30 0.95 0-85 0"72
17.70 0-95 0"85 0"70
~From St. John et al. (1987). h Medication was removed from the diet 21 days before slaughter.
containing either 10% or 20% canola oil (CO). Pigs were on these diets for 100 days; they weighed approximately 100 kg prior to slaughter. Further details of the diet treatments which were previously reported by St. John et aL (1987) are shown in Table 1. The feeding trial period was from December to February. Longissimus dorsi (LD), psoas major (PM), semimembranosus (SM), and semitendinosus (ST) muscles were removed from each carcass at 24 h postmortem. All muscles were trimmed of outside fat and epimysium. Each muscle from each animal was divided into 150-200 g portions, which were then separately vacuum-packaged and stored at - 2 0 ° C until analyzed. Samples were analyzed first for microsomal enzymic lipid peroxidation activity and overall lipid oxidation potential and then for other variables.
Microsomal lipid peroxidation activity Procedures for separation of microsomes and determination of microsomal protein and lipid peroxidation were those described previously (Rhee et al.,
204
K.S. Rhee et al.
1984). The reaction mixtures (4 ml) contained 0"25 mg microsomal protein/ ml, 0.20mM NADPH, 0-20ram ADP, and 0.015mM FeC13 in 0.12M KC15ram histidine buffer (pH6"5) and were incubated at 36°C for 30min. Microsomal lipid peroxidation activity was expressed as nanomoles of malonaldehyde/mg microsomal protein.
Overall lipid oxidation potential A muscle sample was chopped finely (15s) using a Kitchen-Aid food processor (Model KFP 400; Hobart Corp.) with a plastic work-bowl and stainless steel blades, and mixed with 30ppm chlortetracycline to inhibit microbial growth. Thirty-gram portions of each comminuted muscle sample were placed in sterile Petri dishes (8"75-cm diameter) and pressed with the hand with a sterile glove to make the meat surface even. The Petri dishes were then covered with Saran wrap and stored in a cooler at 4°C in the dark for 0, 3 or 6 days (3 replications/storage period/sample). Lipid oxidation in stored, comminuted muscle samples was determined by the 2-thiobarbituric acid (TBA) procedure as described by Rhee (1978) using propyl gallate and EDTA during the blending step to protect the meat from further lipid oxidation which might occur during the assay.
Total lipids The total lipids were extracted using chloroform-methanol (2:1, v/v) according to the procedure of Folch et al. (1957). An aliquot of the total lipid extract (in triplicates) was freed of solvent and its lipid content was determined gravimetrically.
Fatty acid profile An aliquot of the total lipid extract was freed of solvent under nitrogen, and fatty acid methyl esters (FAMEs) were prepared as described by Morrison & Smith (1964). Methyl esters were analyzed using a flame ionization gas chromatograph (Varian 3400), with a fused silica capillary column, Supelcowax 10 (30 m length, 0"25 mm I.D., 0.25 #m film thickness; Supelco, Inc., Bellefonte, PA). Column temperature was 160-200°C (programmed at 4°C/min). Fatty acid methyl esters in hexane were delivered into the column using a Varian Autosampler 8034. The injection port and detector were maintained at 250°C and 300°C, respectively. Hydrogen was used as the carrier gas at 15 psi and nitrogen was the make-up gas. Chromatograms were recorded with a Varian 4270 computing integrator at a chart speed of 0"5 cm/min. The gas chromatograph system was calibrated with standard
Fatty acids and lipid oxidation in pork muscles
205
F A M E mixtures from Supelco, Inc., and palmitic acid methyl ester was arbitrarily chosen as the reference for detector response factors and relative retention times for other FAMEs. Identification of sample fatty acids was made by comparing the relative retention times of F A M E peaks from samples with those of standards. The following equations were used for calculation of response factors and for quantitation of sample FAMEs: RJ~ (from standard F A M E mixtures) = (Wi)(Ay (Wp)(Ai) Concentration (%) in sample = [(Ai)(RFi)/~(Ai)(RFi)] × 100 where, RF~ = I~ = A~ = Wj, = A j, =
response factor of the ith component, weight of the ith component, area of the Rh component, weight of palmitic acid methyl ester, and area of palmitic acid methyl ester.
Nonheme iron and total pigments Nonheme iron content was determined by the method of Schricker et al. (198211 as modified by Rhee & Ziprin (1987). Total pigment content was determined on LD and SM muscles only, by the procedure of Rickansrud & Henrickson (1967), because of insufficient amounts of samples for the PM and ST.
Statistical analysis Analysis of variance, mean separation by the Student-Newman-Keuls test, and c c)rrelation analysis, where appropriate, were performed by using the SAS program (SAS, 1982). The analysis of variance model included diet, animal (nested within diet), and muscle. 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 treatment means, while the mean square for the residual error was used as the error term to test muscle effect and to separate the means.
RESULTS A N D DISCUSSION Total lipid concentrations and relative weight percentages (based on total fatty acids) of total saturated, unsaturated, monounsaturated, and polyunsaturated fatty acids in total lipid extracts are shown in Table 2 and
K.S. Rhee et al.
206
TABLE 2 Total Lipid Concentrations and Percentages of Total Saturated, Unsaturated, unsaturated, and Polyunsaturated Fatty Acids
Mono-
Fatty' acid (%)d Total lipids (g/ lOO g muscle)
Total saturated
Total unsaturated
Mean
SD
Mean
SD
Mean
Diet ~ C ontrol 10% C O
4.9 ~ 4.3 ~
2-2 2.9
42.2° 35-4b
4"7 6.5
57"8¢ 64.5 b
20% CO
6-2"
2.4
26.4'
6-5
73"6"
Muscle y LD
5-3"b
3.2
34.1 "b
9.5
PM SM
5.1 °b 3.5 b
2-3 1.4
34"9°b 38.2°
lif0 7-2
ST
6-5"
2-5
31"6 h
7-9
Total rnonounsaturated
SD
Total polyunsaturated
Mean
SD
Mean
SD
4-7 6.4
38"4b 39"5 b
3-1 5"5
19-4~ 24-9b
4"6 4"3
6-5
44"5 °
3~4
29"1~
3'8
65.7"b
9.4
43-3°
4~2
22.5 b
6"2
65-1 °h 61.9 b
10.0 7-2
37.9 b 39-1 ~
5.6 3.6
27-2" 22"7 b
5-7 5"5
68"4"
8"0
42"8"
4-0
25"6°h
5"0
,,b,~ M e a n s in t h e s a m e c o l u m n w i t h i n t h e s a m e d a t a set (diet o r m u s c l e ) w h i c h a r e n o t f o l l o w e d by t h e s a m e s u p e r s c r i p t letter a r e significantly different ( P < 0"05). R e l a t i v e w e i g h t p e r c e n t b a s e d o n t o t a l f a t t y acids, C O : c a n o l a oil. s L D : Iongissimus dorsi; P M : psoas major; S M : semimembranosus; ST: semitendinosus.
TABLE 3 Percentages of Individual Fatty Acids
Fatty acid (%)d 14:0
16:0
16:1
18:0
18:1
18:2
18:3
20:1
20:4
Diet e Control 10% CO 20% CO
0'8 ~ 0'7 b 0"5 c
26.3 Q 22.3" 16"1 ~
1.2" 0.6 a 0.3 a
15"1" 12.5 b 9'8 c
33'9 b 36.2 b 42.1 a
15.4 c 19.0 b 23.1 ~
FO b 3.0 a 4.0"
3'3" 2-7 a 2.0 a
3.0 ~ 3.0 a 2.0 ~
Muscle I LD PM SM ST
0-7" 0"6 h 0"8 ~ 0-6 b
21.6 b 20.9 b 24"2 ~ 19"6 b
1.0" 0.6 b 0.6 b 0"8 "b
11"8 ~ 13.4" 13"2a 11"4 a
39.4 a 35.1 b 35-0 b 40.l a
17-9 b~ 21.4 a 17.2 ~ 20.0 °b
2.4" 2.7 ~ 2.8 ~ 2.7 a
2-9 a 2.2 ~ 3.6" 2&
2.2 b 3.0 ° 2-7 "b 2"8"
a,b.c M e a n s in t h e s a m e c o l u m n w i t h i n t h e s a m e d a t a set (diet or m u s c l e ) w h i c h are n o t f o l l o w e d by t h e s a m e s u p e r s c r i p t letter a r e significantly different ( P < 0'05). R e l a t i v e w e i g h t p e r c e n t b a s e d o n t o t a l f a t t y acids. ~CO: c a n o l a oil. f L D : longissimus dorsi; P M : psoas major; S M : semimembranosus; ST: semitendinosus,
Fatty acids and lipid oxidation in pork muscles
207
relative percentages of individual fatty acids in Table 3. The diet treatments had no significant (P > 0-05) influence on the total lipid content. However, the CO treatments affected the fatty acid profile of the total lipids from muscle tissue. Inclusion of 10%o and 20% CO in the animal diet increased (P < 0.05) the relative amount of total unsaturated fatty acids by 6-7 and 15.8 percentage points, respectively, from 57.8% for the control and also increased that of total polyunsaturated fatty acids by 5.5 and 9-7 percentage points, respectively, from 19-4% for the control. The 10% and 20% CO treatments increased the relative amount of C18:2 by 3-6 and 7.7 percentage points, respectively, from 15"4% for the control, slightly increased (P < 0-05) the C 18: 3 percentage, and had no significant effect on the C20:4 percentage. The 20% CO treatment increased the relative amount of total monounsaturated[ fatty acids by 6.1 percentage points from 38.4% for the control, but the 10%o CO treatment had no marked effect (P > 0.05). The diet treatment effect on the percentage of C18:1 (the major monounsaturate in pork muscle tissue) was similar to that on the percentage of total monounsaturated fatty acids, with the 20% CO treatment increasing C 18:1 by 8-2 percentage points from 33.9% for the control and the 10% CO treatment having no significant effect. Cencerning the differences among the muscles at different locations, the total lipids from the ST were more unsaturated than the total lipids from the SM, with the lipids from LD and PM muscles being intermediate. The PM had a higher percentage of total polyunsaturated fatty acids or C 18:2 than the LD and SM. In contrast, the LD and ST were higher in the percentage of total monounsaturated fatty acids or C18:1 than the PM and SM. When compared to fatty acid data on the neutral lipids of muscle tissue from the 0%, 10%oand 20% CO-fed pigs (St. John et al., 1987), the data shown in Tables 2 and 3 on the fatty acid composition of the total lipids of muscle tissue from the same animals that were used in the study by St. John et al. (1987) were higher in polyunsaturated fatty acids (specifically C18:2) I C 18:3 and C20:4 data on the neutral lipids were not reported by St. John et al. (1987)~and were lower in monounsaturated fatty acids (C16:1 and C18:1). Similarly, Sharma et al. (1987) who analyzed fatty acid compositions of total, neutral, and polar lipids from pork muscles at different anatomical locations, showed that the percentage of total monounsaturated fatty acids were highest in the neutral lipids, followed by the total lipids and the phospholipids, respectively; the percentage of total polyunsaturated fatty acids was highest in the phospholipids, followed by the total lipids and the neutral lipids, respectively. Da~La on lipid oxidation catalysts are presented in Table 4. Microsomal enzymic lipid peroxidation activity tended to decrease as the percentage of CO in the diet increased, but differences among the diet treatments were not
K.S. Rhee et al.
208
TABLE 4 Levels o f C a t a l y s t s in M u s c l e T i s s u e C o m p u t e d by D i e t T r e a t m e n t a n d M u s c l e L o c a t i o n
Microsomal lipid peroxidation activityc
Nonheme iron (#g/g)
Total heme pigments (mg/g)
Mean
SD
Mean
SD
Mean
SD
8.36 a 7.46" 4.90 a
5.68 4.00 3.78
3'20 a 2-96 a 3-75 a
1.37 1"37 2.43
0.84 a 0-92 a 0.65 a
0.26 0.30 0.22
6.26 ab 8.65" 4"86 b 7.87 ~
3.55 6.30 3"02 4.87
2'11 b 5.56 a 2.80 b 2"74 b
0'63 1.98 0-75 1-02
0-65 b -0-95" --
0"19 -0.28 --
Diet d Control 10% CO 20% CO Muscle e LD PM SM ST
~,b M e a n s in t h e s a m e c o l u m n w i t h i n t h e s a m e d a t a set (diet or m u s c l e ) w h i c h are n o t f o l l o w e d by t h e s a m e s u p e r s c r i p t letter a r e s i g n i f i c a n t l y different ( P < 0.05). c nmol malonaldehyde/mg microsomal protein. n C O : c a n o l a oil. e L D : Iongissimus'dorsi; P M : psoas major; S M : semimembranosus; ST: semitendinosus.
statistically significant (P > 0.05) because of large variations in the activity between animals within each diet group. Neither nonheme iron content nor total heme pigment concentration in muscle tissue was significantly affected by the diet treatments. As for the differences among the muscles at different anatomical locations, the PM and ST had higher microsomal lipid peroxidation activity than the SM and the LD had intermediate activity. The PM was also higher in nonheme iron content compared to the other three muscles. The SM had higher total heme pigment concentrations than the LD. Lipid oxidation potential as measured by accumulation of TBA-reactive substances in refrigerated comminuted muscles, although not statistically different among the three diet treatments, tended to be higher for muscles from the 10% and 20% CO-fed animals (Table 5). Large variations between animals within each diet treatment group (Table 5) resulted in the statistical non-significance even when differences between the mean values of the 10% and 20% CO treatment groups and that of the control were marked. The PM had higher TBA values than the LD after 6 days of storage at 4°C; the SM and ST had similar TBA values at each storage time (Table 5). The tendency of increased lipid oxidation by the 10% and 20% CO treatments was apparently due to increases in the percentage of
Fatty acids and lipid oxidation in pork muscles
209
TABLE 5 T B A Values o f G r o u n d M u s c l e s stored at 4°C
TBA number (mg malonaldehyde/kg) 0 day
Diet c Control 10% C O 20% CO M uscl e a LD PM SM ST
3 days
6 days
Mean
SD
Mean
SD
Mean
SD
0-25 a
0.10
0"56a
0'42
1-51 a
1.54
0"24" 0"28"
0'08 0"12
0"78 a 1-75a
0-91 2.99
3-04" 4-91 a
5'20 7-58
0.25" 0'29" 0.24 ~ 0.25 a
0-08 0-12 0'11 0-08
0"41 a 1"84a |-05 a 0'81 a
0"12 3"29 1.15 1"18
0-88 b 6'52" 2"71 ~b 2.51 "b
0"57 8'79 3"03 4.66
a,b M e a n s in the s a m e c o l u m n within the s a m e d a t a set (diet or muscle) which are n o t followed by the, s a m e superscript letter are significantly different (P < 0-05). c CO: c-anola oil. a LD: longissimusdorsi; P M : psoas major; SM: semimembranosus; ST: semitendinosus.
polyunsaturated fatty acids by the treatments (cf, Tables 2 and 4). Even though the percentage of total monounsaturated fatty acids was also increased by the 20% CO treatment, monounsaturated fatty acids are far less ~,;usceptible to lipid oxidation than polyunsaturated fatty acids. The relative rates of autoxidation of methyl oleate, linoleate and linolenate at 20°C were shown to be 1:12:25 (Gunstone & Hilditch, 1945). The correlation coefficients between percentages of either C18:2 (the major polyunsaturated fatty acid in pork muscles) or total polyunsaturated fatty acids and TBA values at day 3 and day 6 were 0.36 (P < 0"01) and 0.39 (P < 0.01), respectively. Percentages of monounsaturated fatty acids were not correlated with TBA values of stored muscles (P > 0.01). It is apparent that when the level of monounsaturated fatty acids in pork is to be increased by inclusion, in the swine diet, of elevated levels of a fat or oil containing a large amount of monounsaturated fatty acids, the fat or oil should be low in polyunsaturated fatty acids so that the oxidative stability of the meat would not be adversely affected by the diet modification. A study is in progress in our laboratory to increase the monounsaturated fatty acid level in swine tissues by incorporating into the animal diet a sufficient amount of the oil from seeds of a new variety of sunflower oil (> 80% oleic acid, < 1 0 % polyunsaturated fatty acids) and to determine various
210
K.S. Rhee et al.
characteristics of resultant unprocessed and processed pork products. An alternative approach m a y be the inclusion of an antioxidant, along with the m o n o u n s a t u r a t e source such as CO, in the animal diet. The antioxidant for this purpose has to be one that would be safe for h u m a n consumption and deposited in the meat animal muscle tissue in significant amounts. Tocopherols have been tested in swine and poultry diets and proven to reduce the susceptibility of tissue lipids toward lipid oxidation (Webb et al., 1972; Tsai et al., 1978; Uebersax et al., 1978).
ACKNOWLEDGMENT Technical paper No. 23052, Texas Agricultural Experiment Station. Supported in part by the Natural Fibers and F o o d Protein Commission of Texas.
REFERENCES Folch, J., Lees, M. & Stanley, G. H. S. (1957). J. BioL Chem., 226, 497. Grundy, S. M. (1986). New England J. Med., 314(12), 745. Gunstone, F. D. & Hilditch, T. P. (1945). J. Chem. Soc. (London), 34, 836. Love, J. D. (1983). Food Technol., 37(7), 117. Mattson, F. H. & Grundy, S. M. (1985). J. Lipid Res., 26, 194. Morrison, W. R. & Smith, L. M. (1964). J. Lipid Res., 4, 600. Rhee, K. S. (1978). J. Food Sci., 43, 1776. Rhee, K. S. & Ziprin, Y. A. (1987). J. Food Sci., 52, 1174. Rhee, K. S., Dutson, T. R. & Smith, G. C. (1984). J. Food Sci., 49, 675. Rhee, K. S., Seideman, S. C. & Cross, H. R. (1986). J. Agric. Food Chem., 34, 308. Rhee, K. S., Ziprin, Y. A. & Ordonez, G. (1987). J. Agric. Food Chem., 35, 1013. Rickansrud, D. A. & Henrickson, R. L. (1967). J. Food Sci., 32, 57. SAS (1982). S A S User's Guide: Statistics. SAS Institute, Inc., Cary, NC. Schricker, B. R., Miller, D. D. & Stouffer, J. R. (1982). J. Food Sci., 47, 740. 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. & Smith, S. B. (1987). J. Anita. Sei., 64, 1441. Tsai, T. C., Wellington, G. H. & Pond, W. G. (1978). J. Food Sci., 43, 193. Uebersax, M. A., Dawson, L. E. & Uebersax, K. L. (1978). Poultry Sci., 57, 937. Webb, J. E., Brunson, C. C. & Yates, J. D. (1972). Poultry Sci., 51, 1601.