Omega-3 Fatty Acid Composition and Stability of Seal Lipids - ACS

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Omega-3 Fatty Acid Composition and Stability of Seal Lipids Fereidoon Shahidi, J. Synowiecki, R. Amarowicz, and Udaya Wanasundara Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3X9, Canada

Blubber and intramuscular lipids of seal meat contain a large proportion of highly unsaturated fatty acids (HUFA) of omega-3 type. The content of eicosapentaenoic acid (EPA, 20:5), docosapentaenoic acid (DPA, 22:5) and docosahexaneoic acid (DHA, 22:6) in mechanically separated seal meat (MSSM) was 6.9, 5.6 and 10.1%, respectively. Corresponding values for seal blubber prepared by a low-temperature rendering process were approximately 6.5, 4.3 and 8.8%, respectively. While the content of polar lipids in seal blubber was 1.1%, its content in MSSM was 21.2%. The ratio of omega-3 to omega-6 fatty acids was approximately 9.8 for blubber and 7.4 for meat lipids. Prolonged heat processing of seal blubber brought about degradation and/or isomerization of the sensitive HUFA. However, seal blubber oil was more stable to oxidative changes thanfishoil as evidenced by weight gain data, conjugated dien and peroxide values. Preservation of omega-3 fatty acids by either a low-temperature processing or controlled rendering may be required to prevent their quality deterioration and formation of undesirable flavors.

Seafood products play an important role in nutrition and health status of humans. Polyunsaturated fatty acids (PUFA) have long been recognized as desirable dietary components. The interest in long-chain omega-3 fatty acids namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are found abundantly in seafoods, began nearly 3 decades ago (i). These fatty acids are considered essential because they cannot be synthesized by humans and must be ingested in the diet (2). Greenland Eskimos consumed a considerable amount

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of seal meat and blubber and long-chain PUFAfromthese sources. The latter compounds were found to be responsible for health-pro biochemical and physiological changes in the body. The beneficial effects of PUFA have been ascribed to their ability to lower serum triacylglycerols and cholesterol. While DHA is essential for proper functioning of eye and may have a structural role in the brain, EPA serves as a precursor to eicosanoid compounds. The eicosanoids are hormone-like substances including prostaglandins, thromboxanes and leukotrienes, which act on cellular messengers and metabolic regulators and are produced by different cell types in response to physiological and non-physiological stimuli. Eicosanoids are involved in such diverse physiopathologic and physiologic processes as thrombosis, arteriosclerosis, inflammation, immune response, among others. EPA has also been recognized as having therapeutic benefits in human cardiovascular diseases (J). Docosapentaenoic acid (DPA) is an intermediary species between EPA and DHA (4) which is found in high proportions in seal blubber and intramuscular lipids and is also present in human milk (5). However, little information is available on its epidemiological properties. Nonetheless, long chain omega-3 fatty acids have been shown to possess beneficial effects in the prevention, or possible treatment, of coronary heart diseases, diabetes, high blood pressure, and autoimmune diseases (6). Beneficial physiological effects have also been observed in the inflammatory area, related to treatment of asthma, arthritis, migraine, psoriasis and nephritis. Potential applications have also been proposed in treatment of cancer of breast, prostate and colon (7). Long-chain omega-3 fatty acids of seal blubber are present mainly in 1and 3-positions of the triacylglycerol molecules. The omega-3 fatty acids of fish oils are known to be randomly distributed and are more abundant in the 2-position of the triacylglycerols. Therefore, seal blubber lipids may be assimilated more effectively thanfishoils. Based on the current trends in hunting of harp seal (Phoca groenlandica), approximately 1.2 million metric tons of blubber in annually available for processing (8). However, the potential production of blubber oil, based on the current 186,000 allowable catch is approximately 4 million metric tons. Hence, it is prudent to fully utilize this readily available source of omega-3 PUFA. Oxidative quality of marine oils is known to have significant effects on their storage stability and nutritional value. Because of the presence of HUFA, these oils easily undergo autoxidation during processing and storage, thus producing a wide array of undesirable compounds such as hydroperoxides and their breakdown products namely hydrocarbons, ketones, aldehydes, alcohols, epoxides, etc. The relative rate of autoxidation of oleate, linoleate and linolenate is reported to be in the order of 1:40-50:100 on the basis of oxygen uptake and in the order of 1:12:25 on the basis of peroxide formation (9). Polyunsaturated fatty acids, such as arachidonic acid, EPA and DHA, containing 4, 5, and 6 double bonds, respectively, are much less stable than linoleic and linolenic acids. Arachidonic acid was reported to be oxidized 2.9 times faster than linoleic acid (10). Ethyl esters of EPA and DHA were oxidized rapidly even at 5°C in the dark after an induction period of 3-4 days, whereas the induction period of linoleate and linolenate were 20 and 60 days, respectively. Similarly, oxygen

In Lipids in Food Flavors; Ho, Chi-Tang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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uptake of EPA and DHA esters after induction period was 5.2 and 8.5 times faster than that of ethyl linolenate (11). Therefore, inhibition of oxidation is a major criterion when marine oils are incorporated into food products. The toxicity studies of oxidized oil has been of considerable interest for the last few decades. Oxidation of oil results in the development of off-flavors which can be evaluated by sensory tests. However, the routine evaluation of flavor by taste panels is tedious, sometimes subjective, expensive and difficult to compare results from different laboratories (12). Therefore, different chemical methods have been suggested for assessing oxidative quality of oils either by measuring their primary or secondary oxidation products (12,13). However, there is no information available on the oxidative stability of seal oil with respect to other marine oils. The present study was undertaken to examine the fatty acid composition of seal blubber and intramuscular lipids. Since lipids are the major precursors of aroma in muscle foods, the composition of lipids in meat and subcutaneous fat play an important role in their oxidative deterioration and off-flavor development. Hence, oxidative stability of blubber lipids was also studied.

Materials and Methods Extraction of Seal Blubber and Intramuscular Lipid. Seal oil was prepared from raw blubber of harp seal (Phoca groenlandica). In the laboratory, protein residues were separated and after repeated washing, the resultant oil was treated with a bicarbonate solution and washed again with water. Thefinalproduct was dried over anhydrous sodium sulfate. In the industrial scale, the blubber was rendered using a steam injection process with subsequent removal of residues from the bottom of the tanks and removal of oil by phase separation. The rendering process took approximately 2.5 h at a temperature of 100°C. Intramuscular lipids of seal meat were extracted into a chloroform-methanol-water mixture as described by Bligh and Dyer (14). Isolated lipids were quantified (15) and used for experimentation. Seal blubber and intramuscular lipids of meat, extracted according to Bligh and Dyer (14), were subjected to transmethylation in acidified methanol and methyl esters so prepared were subsequently quantified as described elsewhere (16). Separation of lipid classes was done according to Christe (16). Sample Preparation. For weight gain studies, two grams of oil samples (in triplicates) were placed in a petri dish, traces of water were removed in a vacuum oven at 35°C, reweighed and stored in a forced air oven at 65°C. The weight gain of sample was recorded at 24 h intervals. Twentyfivemillilitres of oil were stored separately under the same conditions in small open glass containers for other chemical analyses. Official methods (17) were used for determination of peroxide value (PV), 2-thiobarbituric acid reactive substances (TBARS) and iodine value (IV). The conjugated dienes (CD) and conjugated trienes (CT) were determined using

In Lipids in Food Flavors; Ho, Chi-Tang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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IUPAC (18) methods of analyses. The cholesterol content of samples was determined as described previously (15).

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Results and Discussion The contents of neutral and polar lipids as well as cholesterol in blubber and intramuscular fat of harp seal (Phoca groenlandica) are summarized in Table I. Approximately 98.9% of blubber lipids were composed of triacylglycerols. However, presence of 21.2% polar components in seal intramuscular lipids indicated the very lean nature of the meat; polar lipids are derived mainly from structural components of cells. This corresponds well with low lipid content of seal meat which averages between 1.8 and 2.7%. The total lipid content of raw blubber was 95.5 ±0.7%, of which 84.2 ± 3 . 1 % were recovered after aqueous washing. Upon caustic refining and bleaching 71.1 ± 3.7% refined, bleached seal oil (RBSO) was obtained.

Table I. Content of neutral and polar lipids, cholestérol and iodine value seal blubber and intramuscular lipids Component in 100 g sample Neutral Lipids (g) Polar Lipids (g) Cholesterol (mg) Iodine Value (g)

Blubber 98.9 1.1 105.7 146.3

± ± ± ±

0.2 0.2 22.0 1.0

Intramuscular Lipids 78.8 ± 1.1 21.2 ± 0.5

-

Table Π summarizes different classes of lipid fatty acidsfrommechanically separated seal meat (MSSM). Corresponding results for seal blubber lipids are shown in Table m . A close scrutiny of the data presented in Tables Π and ΙΠ indicates that a) the content of HUFA in both subcutaneous and intramuscular lipids of seal is in the order of DHA >EPA>DPA; b) more than 50% of lipids in seal aie monounsaturates; c) the ratio of the ω3 to ω6 polyunsaturates in the neutral fraction is higher than that in the polar lipid; d) the ratio of saturates to unsaturates is much higher in the polarfractionas compared with the neutral fraction. Furthermore, the polar lipids of seal blubber and intramuscular lipids, contrary to the general expectation, possessed less HUFA than the neutral triacylglycerol components. Figure 1 shows the effect of steam rendering over a period of 2.5 h, in an industrial-scale operation (Carino Company, Dildo, NF), on the fatty acid composition of seal blubber oil. The content of long chain omega-3 fatty acids in the oil decrease progressively as the duration of heat processing increased. Isomerization and/or oxidation of these highly sensitive fatty acids is contemplated. Incorporation of a food-grade antioxidant/chelator system and/or low-temperature processing of blubber is expected to minimize undesirable structural changes in

In Lipids in Food Flavors; Ho, Chi-Tang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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the oil. In contrast to heat processing, caustic or bicarbonate refining of the oil enhanced its content of long-chain omega-3 fatty acids. Possible removal of saturated fatty acids by precipitation/crystallization might be involved (results not shown).

Table Π. Classes of lipids of mechanically separated seal meat

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Fatty Acids, Area % Total Saturates (TS) Total Unsaturates (TU) Total Monounsaturates Total Polyunsaturates EPA DPA DHA Total ω3 Total b)6 ω3/ω6 TS/TU

Total

Neutral

Polar

20.1 79.9 52.3 27.6 6.9 5.6 10.1 24.4 3.3 7.39 0.25

18.2 81.8 54.1 27.7 6.5 6.3 10.6 25.4 2.4 10.58 0.22

32.2 67.8 43.1 24.7 8.8 1.6 5.6 16.8 7.9 2.13 0.48

Figure 2 shows the weight gain data of RBSO under Schaal oven test over a 16-day period. The weight gain of the oil was minimal over a 24 h storage. Possible presence of natural antioxidants in the oil might be responsible for better stability of seal oil compared to cod liver oil. Cod liver oil showed weight gain of 2.0% (w/w) over the same period. The weight gain of the oil continued rapidly from day 1 to day 3, after which much smaller oxygen uptake was noticed. The increase in the weight gain may be due to the addition of oxygen to lipid molecules to form hydroperoxides during primary stages of oxidation. Further storage of the oil for up to 16 days resulted in relatively small loss of weight, perhaps due to the breakdown of heat-labile hydroperoxides and volatilization of some secondary oxidation products under experimental conditions. Privett and Nickell (19) have reported that addition of oxygen to lipid to form hydroperoxides is reasonably quantitative during initial stages of oxidation. Olcott and Einset (20) have reported that the weight gain serves as a useful technique to evaluate the oxidative stability of edible oils. Ke and Ackman (22) reported that the methods is simple, has a satisfactory reproducibility and can be used to compare oxidation of lipids obtainedfromdifferent parts of fish. However, surface exposure of the sample is very important in carrying out the experiments (22). As expected, the weight gain data correlated well (r = 0.912) with corresponding peroxide values for RBSO (Figure 3). The weight gain of the oil arisesfromthe uptake of oxygen and formation of lipid hydroperoxides. Similar to the weight gain data, PV of seal blubber oil were generally smaller in magnitude than those for cod liver oil.

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0

0.5

1.0

2.5

Heating time (h)

Figure 1. Effect of steam rendering (at 100°C) on the long-chain omega-3 fatty acids of seal blubber oil (sample used in this study was from a different batch than that used in other studies).

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Storage Time (Days)

Figure 2. Weight gain of seal blubber and cod liver oils stored at 65°C.

In Lipids in Food Flavors; Ho, Chi-Tang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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Table Π Ι . Classes of Upids and seal blubber Fatty Acid, Area %

Total

Neutral

Total Saturates (TS) Total Unsaturates (TU) Total Monounsaturates Total Polyunsaturates EPA DPA DHA Total ω3 Total ω6 ω3/ω6 TS/TU

13.0 87.0 63.3 23.6 6.5 4.3 8.9 21.4 2.2 11.09 0.15

13.2 86.8 63.1 23.7 7.0 4.1 8.7 21.9 2.0 10.95 0.15

Polar

24.1 75.9 47.7 28.2 6.1 3.2 5.0 17.1 3.4 5.03 0.32

Lipid radicals formed during the initiation step may undergo rearrangement. Thus, the methylene-interrupted feature of HUFA of seal blubber is lost in favor of formation of conjugated dienes. Figure 4 represents the variation of CD in RBSO over a 16-day storage and Figure 5 represents the correlation (r = 0.966) of conjugated dienes, expressed as the absorbance at 234 nm, with corresponding peroxide values. Meanwhile, formation of CT (absorbance at 268 nm) followed a trend similar to that observed for CD (Figure 4). As hydroperoxides are the primary products of lipid oxidation (25), PV provides a clear indication concerning die initial oxidation potential of different lipids. Conjugated diene value may also be used to determine the initial rate of oxidation (9). Jackson (24) indicated that formation of hydroperoxides normally coincides with CD formation in oils upon oxidation. The CD assay is faster that PV determination and does not depend on chemical reactions such as color development for its determination. Therefore, CD content may be used as a measure of primary oxidation products for both seal blubber and cod liver oils. According to both of these methods, seal blubber oil is more stable than cod liver oil. Structural differences between the two oils as well as possible presence of stabilizers in seal blubber may be responsible for this observation. Figure 6 summerizes changes in the content of TBARS of seal blubber oil over a 16-day storage period at 65°C. It is interesting to note that the content of TBARS remained relatively unchanged between days 3 and 12 during the storage period. Therefore, it is concluded that the rate of formation of TBARS is equivalent to that of their disproportionation and/or further reaction. In contrast, TBARS of cod liver oil increased progressively over the entire storage period. These results are in agreement with sensory characteristics of seal blubber and fish oils. While cod liver oil attained an intense off-flavor after one day of storage, the blubber oil showed a delayed response (2 days) to off-flavor development.

In Lipids in Food Flavors; Ho, Chi-Tang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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10

20

30

40

50

Peroxide Value (meq/kg oil)

Figure 3. Relationship between peroxide value and weight gain data (corr. coeff. r = 0.912) of seal blubber oil during accelerated oxidation at 65°C.

J

I

I

I

I

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I

L_

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Storage Time (Days)

Figure 4. Conjugated diene and triene values of seal blubber oil during accelerated oxidation at 65°C.

In Lipids in Food Flavors; Ho, Chi-Tang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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ν

C o d liver oil



S e a l blubber oil

— J

10

ι

ι

ι

l

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_

Peroxide Value (meq/kg oil)

Figure 5. Relationship between peroxide value and conjugated diene value of seal blubber oil (corr. coeff. r = 0.966) and cod liver oil (corr. coeff. r = 0.953) during accelerated oxidation at 65°C. •

C o d liver oil



S e a l blubber oil

2

4

6

8

10 12 14 16

Storage Time (Days) Figure 6. The 2-thiobarbituric acid reactive substances (TBARS) values of seal blubber and cod liver oils stored at 65 °C.

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Elucidation of the chemical identity of major contributors to off-flavor development in seal blubber oil is currently under investigation. Polyunsaturated fatty acids are among die most easily oxidizable components of foods and cells and many of the oxidized products including peroxides, free radicals and aldehydes are highly toxic and mutagenic (25). The ease of nonenzymatic free radical oxidation of fatty acids is proportional to the number of methylene groups between double bonds, thus DHA, DPA and EPA are highly prone to oxidation. Therefore, protection of HUFA of the omega-3 type is essential in order to counterbalance any harmful effects and to take full advantage of their nutritional and health-related benefits.

Conclusions and Further Research Needs Seal blubber lipids consist of approximately 22% long-chain omega-3 fatty acids, namely EPA, DPA and DHA. These fatty acids are dominant in positions of 1 and 3 of the triacylglycerol and thus are susceptible to hydrolysis by pancreatic lipase. Infishoils DHA is mostly in the 2-position but the EPA may be less specifically located. Seal blubber lipids also contains considerable amounts of monoenes and DPA. The oxidative stability of seal blubber lipids is better than other marine oils. Further research is needed to explore possibilities for valueadded utilization of this readily available source of omega-3 fatty acids for food and pharmaceutical applications.

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