Structured Lipids Enriched with Omega-3 and Omega-6 Highly

Chapter 3

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Structured Lipids Enriched with Omega-3 and Omega-6 Highly Unsaturated Fatty Acids 1

S. P. J. NamalSenanayake1,2and Fereidoon Shahidi

2

1Departmentof Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3X9, Canada Current address: Martek Biosciences Corporation, 555 Rolling Hills Lane, Winchester, K Y 40391

Structured lipids are triacylglycerols (TAG) that have been modified to change the fatty acid compositions and stereospecific distribution of fatty acids from their native state. These tailor-made lipids may contain mixtures of short-, medium- and long chain fatty acids on the same glycerol molecule, and these can be saturated or unsaturated. They can be designed for nutritional and pharmaceutical purposes targeting specific diseases and pathological conditions. Structured lipids containing n-6 and n-3 polyunsaturated fatty acids (PUFA) were produced with both immobilized and non -immobilized lipases as biocatalysts. Among P U F A , eicosapentaenoic (EPA; 20:5n-3), docosahexaenoic(DHA; 22:6n-3) and γ-linolenic (GLA; 18:3n-6) acids have attracted much attention in recent years due to their beneficial health effects. Structured lipids containing these fatty acids may be desirable in certain health, nutritional and pharmaceutical applications.

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© 2003 American Chemical Society In Food Factors in Health Promotion and Disease Prevention; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


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Structured lipids are defined as triacylglycerols (TAG) containing mixtures of short- and/or medium- and long-chain fatty acyl residues attached to the glycerol backbone for specific functionality and produced via chemical or enzymatic reactions (1). The first structured lipids were produced, via chemical means, by mixing medium-chain triacylglycerols and long-chain triacylglycerols, allowing hydrolysis to free fatty acids, followed by random transesterification of the fatty acids into mixed triacylglycerol molecules. This results in the production of triacylglycerols containing combinations of short-, medium- and long-chain fatty acid moieties on the single glycerol backbone. These have unique physical, chemical and physiological properties, which differ from simple physical mixtures of the starting lipids. It is now possible to synthesize structured lipids via lipase- catalyzed reactions. As acyl donors for structured lipid production, both n-3 and n-6 polyunsaturated fatty acids (PUFA) may impart desirable benefits. For example, it is known that n-3 P U F A , mainly eicosapentaenoic ( E P A ; 20:5n-3) and docosahexaenoic (DHA; 22:6n-3) acids, exhibit various physiological functions. The potential health benefits of n-3 fatty acids include reduced risk of cardiovascular disease, hypertension, inflammatory and autoimmune disorders (2). E P A is an antagonist of the arachidonic acid cascade and competes with arachidonic acid to produce eicosanoids. It is believed that presence of D H A in both the brain and retina is important for proper nervous system and visual acuity in humans, respectively (3,4). The maintenance of adequate levels of D H A may be required for optimum neurological functions throughout the life span. It has been noted that there is a correlation between D H A deficiency and the incidence of Alzheimer's disease. A study of more than thousand elderly individuals indicated that a low D H A level is a significant risk factor in the onset of dementia (5). Studies have shown that an early dietary supplementation of D H A is a major determinant of improved performance on the mental development index (MDI) of term infants (6). Therefore, n-3 P U F A such as E P A and D H A should be included in the diet, perhaps in the form of structured lipids. Among n-6 P U F A , γ-linolenic acid (18:3n-6; G L A ) is an intermediate metabolite in the conversion of linoleic acid (LA; 18:2n-6) to arachidonic acid ( A A ; 20:4n6). G L A has the physiological functions of modulating immune and inflammatory response (7). G L A has also been used in the treatment of atopic eczema, rheumatoid arthritis, dermatitis, hypertension, diabetic neuropathy, cirrhosis of the liver, and premenstrual syndrome (8,9).

Nutritional benefits of structured lipids Nutritional value and application of structured lipids containing mediumchain fatty acids at the sn-l and sn-3 positions and unsaturated fatty acids at the

In Food Factors in Health Promotion and Disease Prevention; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.


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sn-2 position have been of much interest. These structured T A G can be easily hydrolyzed by pancreatic lipase. Pancreatic lipase acts mainly on the sn-l and sn-3 positions of the T A G molecules to release 2-monoacylglycerols (2-MAG) and free medium-chain fatty acids. These free fatty acids are readily absorbed in the intestine and are transported into the liver to be consumed as a source of energy while the remaining 2 - M A G become the source of essential fatty acids. A study involving two types of structured lipids (randomized vs. specific product) revealed that the fatty acids located in the sn-2 position of T A G are preferentially absorbed (10). The randomized structured lipid used contained E P A , D H A and capric acid randomly distributed in the molecule. The specific structured lipid had E P A and D H A at the sn-2 position and capric acid at the sn1 and sn-3 positions of T A G . The concentrations of E P A and D H A in lymphatics were higher in the specific structured lipid than in the randomized structured lipid. Rapid hydrolysis and absorption of structured lipids containing medium-chain fatty acids at the sn-1 and sn-3 positions and long-chain fatty acids at the sn-2 position have also been reported (11).

Lipase-catalyzed reactions Lipase-catalyzed modification of T A G may be performed with several benefits over chemically assisted reactions to produced structured lipids. Through enzyme-catalyzed reactions, it is possible to incorporate a desired acyl group onto a specific position of the T A G (12,13), whereas chemically assisted reactions do not possess this regiospecificity due to the random nature of the reaction. In lipase-mediated reactions, lipases catalyze either the removal or exchange of fatty acyl groups on the glycerol backbone. Different lipases, however, show a preference for both the position of the fatty acyl group on the triacylglycerol and the nature of the fatty acid. This lipase specificity provides a means of classifying them. Based on the substrates involved in the lipase-catalyzed reactions, they can be classified into different categories: esterification, hydrolysis, acidolysis, alcoholysis and interesterification (1). Direct esterification reaction may be employed for the preparation of structured lipids by reacting free fatty acids with glycerol. However, this process is not commonly used in structured lipid production. The major problem is that the water molecules are formed as a result of the esterification reaction. The water molecules so produced need to be removed in order to prevent the hydrolysis of the product. Hydrolysis is the



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natural reaction for lipases, producing free fatty acids and glycerol. If a sn-l,3regiospecifc lipase is employed, free fatty acids and 2-monoacylglycerols will be produced. However, a non-specific lipase will catalyze complete hydrolysis of triacylglycerols to free fatty acids and glycerol. Acidolysis is the exchange reaction between an ester and free fatty acids, and the lipase will catalyze the transfer of the alcohol moiety of the ester to the new fatty acid until an equilibrium corresponding to the ratio of ester to fatty acid is obtained. Alcoholysis refers to the reaction of an alcohol with triacylglycerols and produces a new ester with another alcohol moiety. Glycerolysis is an important reaction that is used for the production of mono- and diacylglycerols. During this reaction, a triacylglycerol is reacted with excess glycerol and mono- and diacylglycerols are produced. Interesterification reaction is generally performed by mixing two types of esters and the lipase catalyzes redistribution of fatty acid moieties.

Synthesis of structured lipids via acidolysis For bench-scale synthesis of borage or evening primrose oil-based structured lipids, 297-300 mg oil were mixed with 115 mg E P A and/or 120 mg D H A . The mole ratio was 1:1 for oil to E P A or D H A . The reactions were conducted in 3 mL of hexane or in the absence of any organic solvent. Lipases from Candida antarctica (Novozym-435) or Pseudomonas sp. (PS-30) were added to the reaction mixture and incubated in an orbital shaker at 37°C for 24 h at 250 rpm.

Structured lipids containing n-6 and n-3 PUFA Borage, evening primrose, blackcurrant and fungal oils serve as major sources of γ-linolenic acid (18:3n-6; G L A ) . Among these, borage and evening primrose oils are used most frequently in nutritional and clinical studies. The contents of G L A present in borage and evening primrose oils are 23.5 and 9.1%, respectively (Table I). Borage and evening primrose oils containing G L A are used as ingredients of food materials, health foods, infant formula and cosmetics (14). There is evidence for therapeutic benefits of GLA-rich oils in the treatment of atopic eczema, dermatitis, hypertension and premenstrual syndrome (8). On the other hand, n-3 P U F A have potential for prevention of cardiovascular disease, arthritis, hypertension, immune and renal disorders, diabetes and cancer (4). Structured lipids containing both G L A and n-3 P U F A may be of interest because of their desired health benefits. Structured lipids containing G L A , E P A


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and/or D H A in the same glycerol backbone using borage and evening primrose oils were successfully produced as the main substrates (12,13).

Table I. Fatty acid compositions (mole %) of test oils


Major fatty acids

Borage oil

Evening primrose oil

9.6 3.5 15.5 37.8 23.5 4.2 2.3 1.5

6.2 1.8 8.7 72.6 9.1 0.3 0.1 ND

C16:0 C18:0 C18:l C18:2n-6 C18:3n-6 C20:l C22:l C24:l ND: not detected

Borage and evening primrose oils were transesterified with D H A at 1:1 mole ratio in hexane catalyzed by an immobilized Novozym-435 from Candida antarctica (Table II). The incorporation of D H A into borage and evening primrose oils was 27.4 and 25.2%, respectively. The amounts of G L A retained in the modified oils were 17.0 and 7.6%, respectively.

Table Π. Enzymatically transesterified borage (BO) and evening primrose oils (EPO) with DHA to produce structured lipids Reaction

Major fatty acids

Modified BO

Modified EPO

Novozym-435 lipase, 150 units Mole ratio of reactants, 1:1 Temperature, 37°C Incubation time, 24 h

C16:0 C18:0 C18:l C18:2n-6 C18:3n-6 C22:6n-3 n-3/n-6 ratio

6.9 2.6 11.3 27.0 17.0 27.4 0.6

4.7 0.7 4.2 54.3 7.6 25.2 0.4

The fatty acid compositions of borage and evening primrose oils were also modified by incorporation of E P A using lipase PS-30 from Pseudomonas sp. as


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the biocatalyst (Table ΙΠ). After acidolysis reaction, modified borage oil contained 26.8% E P A and 15.2% G L A . Meanwhile, modified evening primrose oil had E P A and G L A contents of 25.2 and 7.6%, respectively.


Table ΙΠ. Enzymatically transesterified borage (BO) and evening primrose oils (EPO) with EPA to produce structured lipids Reaction

Major fatty acids

Modified BO

Modified EPO

Lipase PS-30, 150 units Mole ratio of reactants, 1:1 Temperature, 37°C Incubation time, 24 h

C16:0 C18:0 C18:l C18:2n-6 C18:3n-6 C20:5n-3 n-3/n-6 ratio

6.4 2.6 12.0 25.3 15.2 26.8 0.7

4.0 1.1 5.6 55.6 7.6 25.2 0.4

In another study, borage and evening primrose oils were modified using combinations of E P A and D H A to produce structured lipids in an acidolysis reaction catalyzed by lipase PS-30 from Pseudomonas sp. (Table IV). Borageoil based structured lipid contained 23.1 and 8.7% E P A and D H A , respectively, and 18.4% G L A . On the other hand, evening primrose oil-based structured lipid had 23.5% E P A , 9.2% D H A and 7.4% G L A . The modified borage and evening primrose oils thus obtained may have potential health benefits. Lipase-catalyzed acidolysis has been employed for the incorporation of E P A and capric acid (10:0) into borage oil using two immobilized lipases, SP435 from Candida antarctica and IM60 from Rhizomucor miehei as biocatalysts (75). Higher incorporation of E P A (10.2%) and 10:0 (26.3%) was achieved with IM60 lipase, compared to 8.8 and 15.5%, respectively, with SP435 lipase (75). By a two-step process, which involved Candida rugosa lipase-catalyzed selective hydrolysis of borage oil and subsequent acidolysis of the resulting products with n-3 fatty acids, 72.8% of n-3 and n-6 fatty acids in borage oil acylglycerols was obtained (76). The contents of G L A , E P A and D H A in the structured lipid so prepared were 26.5, 19.8 and 18.1%, respectively. The n-3/n6 ratio increased from 0 to 1.09, following acidolysis (16).


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Table IV. Enzyme-catalyzed transesterification of borage (BO) and evening primrose oils (EPO) with n-3 fatty acids to produce structured lipids


Reaction Lipase PS-30, 150 units Mole ratio of oil:EPA:DHA, 1:0.5:0.5 Temperature, 37°C Incubation time, 24 h

Major fatty acids C16:0 C18:0 C18:l C18:2n-6 C18:3n-6 C20:5n-3 C22:6n-3 n-3/n-6 ratio

Modified BO 4.0 2.0 10.7 26.5 18.4 23.1 8.7 0.7

Modified EPO 3.5 1.0 5.9 49.4 7.4 23.5 9.2 0.6

Stereospecific analyses The stereospecific distribution of fatty acids in T A G molecules of D H A , E P A and EPA+DHA-enriched structured lipids synthesized in our laboratory was determined. Tables V and V I report the positional distribution of fatty acids in structured lipids examined. The results of this study showed that D H A was randomly distributed over all three positions (34.6% at sn-l, 33.5% at sn-2 and 35.9% at sn-3) of the T A G molecules of DHA-enriched borage oil (Table V ) . In DHA-enriched evening primrose oil, however, this fatty acid was mainly occupied by the sn-2 position (38.2%), followed by sn-3 (33.1%) and sn-l (24.5%) positions (Table VI). It should be noted that these DHA-enriched structured lipids were prepared using Novozym-435 from Candida antarctica as the biocatalyst. The positional specificity of Novozym-435 depends on the type of substrates used in various reactions. In some reaction systems, this enzyme behaves as a nonspecific lipase whereas in other systems it exhibits sn-l,3 regiospecificity (17). Based on the reaction conditions employed in this study, Novozym-435 functions as a nonspecific lipase. The stereospecific distribution of fatty acids in the native borage and evening primrose oils have previously been reported (18). In native borage oil, G L A was distributed asymmetrically and preferentially located at the sn-2 and sn-3 positions (18). In native evening primrose oil, G L A was concentrated in the sn-3 position (18). Linoleic acid (LA; 18:2n-6) was fairly evenly distributed in all positions of native evening primrose oil, but was preferentially located in the sn-l position of native borage oil (18). The results of our study showed that in DHA-enriched borage oil, G L A was mainly located in the sn-2 (18.4%) and sn-3



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(19.2%) positions of T A G (Table V ) . In DHA-enriched evening primrose oil, however, G L A was mainly located in the sn-2 (7.5%) position (Table VI). L A was randomly distributed over all three positions of T A G in both oils. The positional distribution of fatty acids in T A G of EPA-enriched oils (Tables V and VI) was also determined. In this work, EPA-enriched oils were synthesized using lipase PS-30 from Pseudomonas sp. as the biocatalyst. The E P A of EPA-enriched borage oil was randomly distributed in the T A G (33.4% at sn-l; 32.5% at sn-2; 30.9% at sn-3) (Table V ) . In EPA-enriched evening primrose oil, however, this fatty acid was mainly esterified at the primary positions (39.5% at sn-l and 42.1% at sn-3) of T A G (Table VI) and was also present in appreciable amounts (23.2%) at the sn-2 position. Therefore, it is assumed that Pseudomonas sp. lipase shows no specificity and may incorporate E P A in all three positions of T A G of the oils. In both oils, G L A was esterified preferentially at the sn-2 position (18.6 and 7.2% in EPA-enriched borage and evening primrose oils, respectively). In EPA-enriched borage oil, L A in T A G was distributed randomly while in EPA-enriched evening primrose oil it was mainly placed at the sn-2 position (Tables V and VI). In another study, the positional distribution of fatty acids in n-3 fatty acids (EPA and DHA)-enriched oils were determined (Tables V and VI). In this study, preparation of EPA+DHA-enriched structured lipids was catalyzed by lipase PS30 from Psuedomonas sp. In EPA+DHA enriched borage oil, G L A was mainly located at the sn-2 position (32.9%) (Table V ) . However, E P A and D H A were preferentially esterified at the primary positions (sn-l and sn-3) of T A G molecules (Table V ) and their quantities were E P A , 26.1 and 30.8%; and D H A , 8.3 and 9.8%, respectively. In EPA+DHA enriched evening primrose oil, G L A was located mainly at the sn-2 (10.8%) and sn-3 (9.0%) positions of T A G (Table VI). E P A was preferentially esterified at the sn-l (31.5%) and sn-3 (24.1%) positions while approximately half of the D H A was located in the sn-3 position (10.5%) (Table VI). Therefore, lipase PS-30 from Pseudomonas sp., under the reaction conditions employed in this study, has the ability to incorporate n-3 fatty acids (EPA and D H A ) preferentially at the £«-1 and sn-3 positions of the T A G molecules.

Oxidative Stability Structured lipids containing n-3 fatty acids were highly susceptible to oxidation probably because they lost some of the endogenous antioxidants, such as tocopherols, present in borage and evening primrose oils used for the synthesis (19) and also partly due to the increase in their degree of unsaturation. Therefore, it is suggested that appropriate antioxidants of choice be added back to structured lipids as this may improve their oxidative stability and preserve the integrity of nutritionally important n-3 and n-6 fatty acid components present.



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Table V . Stereospecific distribution of fatty acids (mole %) in modified and unmodified borage oils Positional distribution

C18:2n-6

C18:3n-6

C20:5n-3

C22:6n-3

Unmodified borage o i f sn-l sn-2 sn-3

38.5 53.4 34.2

3.5 32.2 17.4

ND ND ND

ND ND ND

DHA-enriched sn-l sn-2 sn-3

23.9 22.8 25.3

13.2 18.4 19.2

ND ND ND

34.6 33.5 35.9

EPA-enriched sn-l sn-2 sn-3

23.6 21.2 20.2

11.5 18.6 13.4

33.4 32.5 30.9

ND ND ND

EPA+DHA-enriched sn-l sn-2 sn-3

22.9 26.2 15.3

4.1 32.9 15.1

26.1 15.2 30.8

8.3 3.8 9.8

Reference (18) ND: not detected


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Table VL Stereospecific distribution of fatty acids (mole %) in modified and unmodified evening primrose oils Positional distribution

C18:2n-6

C18:3n-6

C20:5n-3

C22:6n-3

Unmodified evening primrose oil" sn-l sn-2 sn-3

70.0 81.5 71.6

4.9 10.3 10.2

ND ND ND

ND ND ND

DHA-enriched sn-l sn-2 sn-3

45.1 44.9 41.5

5.5 7.5 4.8

ND ND ND

24.5 38.2 33.1

EPA-enriched sn-l sn-2 sn-3

39.1 48.4 37.5

4.4 7.2 4.8

39.5 23.2 42.1

ND ND ND

EPA+DHA-enriched sn-l sn-2 sn-3

40.1 61.0 32.6

4.2 10.8 9.0

31.5 17.2 24.1

5.8 4.8 10.5

Reference (18) ND: not detected

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Structured Lipids Enriched with Omega-3 and Omega-6 Highly

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