Synthesis of DHA Rich PUFA From Cod Liver Fish Oil

8/3/2019 Synthesis of DHA Rich PUFA From Cod Liver Fish Oil

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Synthesis of DHA Rich PUFA From Cod liver Fish oil

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Abstract

Docosahexaenoic acid (DHA) is one of the most useful polyunsaturated fatty acid (PUFA) with

pharmaceutical potential. The present paper investigates the synthesis of DHA (docosahexaenoic

acid) & EPA (eicosapentaenoic acid) enriched polyunsaturated fatty acid (PUFA) from cod liver

fish oil (CLO) by urea complexation method which is one of the most simplest and efficient

technique among all the available chemical methods. Synthesis of DHA & EPA rich PUFAs with

chemical method have been studied for cod liver fish oil. Total free fatty acids (FFA) of 2.79

mmoles per gram of oil have been recovered from total available FFAs (3.24 mmoles/g of oil) in

cod liver oil after hydrolysis, indicating 86.25% hydrolysis. After urea complexation method, a

recovery of 0.858 mmoles of PUFAs per gram of oil, indicating 26.48% recovery based on total

fatty acid content of oil initially. In esterification of PUFAs to FAMEs, 77.7% conversion has

been obtained, corresponding to 0.667 mmoles of FAMEs/g of oil.

Key words: Docosahexaenoic acid, Eicosapentaenoic acid, Urea complexation, Free fatty acids.

Aditi Sharma1 : Presently working as Lecturer in Department of Chemical Engineering,

Banasthali University, Banasthali, Rajasthan-304022

1. INTRODUCTION

DHA is one of the most useful polyunsaturated fatty acid (PUFA) with pharmaceutical potential

and important for the prevention & control of various human diseases and disorders such as

cardiovascular disease, inflammation, allergy, cancer, immune response, diabetes, hypertension

and renal disorders. DHA is also known as brain food as it is highly concentrated in the

membranes of brain cells and retinal cells of eye (Youdim et al., 2000; Uauy & Valenzuela, 2000

and Serhan et al., 2004). The most widely available source of DHA is cold water oily fishes such

as salmon, herring etc. because fish oils are rich in 3 polyunsaturated fatty acids (3 PUFA),

especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which is responsible

for most of the benefits offered by fish oils than EPA as recent studies shows that excessive

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consumption of EPA may even cause harmful health effects. Major components of Cod liver oil

are given in the Table-1 indicating fatty acid composition of CLO reported by DeWitt, Ackman,

Kingsbury and Klenk.

Table-1: Fatty Acid Composition of Cod liver Oil

Fatty

Acids

DeWitt et al., 1963 Ackman

et al.,

1964

Kingsbury

et al., 1962

Klenk

et al., 1962

Calculated

Average

Composition

Percentage

Composition

(Total 84.22)Min. Max.

14:0 2.46 3.06 3.5 2.4 3 2.92 3.47

16:0 10.77 12.50 10.4 11.5 12 11.38 13.51

16:1 6.56 10.31 12.2 7.8 5 8.36 9.93

18:1 21.96 28.30 19.6 25.6 24 23.58 27.99

20:1 9.69 17.01 14.6 11.7 9 13.37 15.87

20:5 7.19 12.03 5 8.2 8 5.70 6.77

22:1 4.76 7.09 13.3 4.9 5 7.28 8.64

22:5 0.79 1.43 1.9 1.3 1 1.33 1.58

22:6 6.94 10.68 10.5 7.4 19 10.30 12.24Total 71.12 102.71 91.0 80.8 86 84.22 100%

The National Institute of Health recently published recommended daily intakes of fatty acids

which include 650 mg of DHA. In the marine fishes, DHA are originated from the phytoplankton

and the seaweed that are the part of their food chain therefore DHA can also be synthesized from

some species of fungi, algae and bacterias (Calson, 1991; Connoe et al., 1992; Wanasundara &

Shahidi, 1998).

DHA can be purified & synthesized in the laboratory by both chemical and enzymatic methods.

Chemical methods such as urea inclusion, molecular distillation, supercritical fluid extraction

and freezing crystallization have been employed and found to be limited in use due to high cost

and process complexicity (Guil-Guerrero et al., 2001). Among all the chemical methods urea

complexation is one of the most simplest and efficient technique (Klinkesorn et al., 2002). This

method of PUFA concentration encompasses four main steps including saponification of the oil;

use of urea inclusion adducts method to obtain PUFA, methylation of PUFA and argentation

silica gel column chromatography of the methylated PUFA.

Enzymatic processes such as selective hydrolysis, hydrolysis & selective esterification and

transesterification using lipases are another useful technique for the production of nutritionally

valuable fatty acids (DHA & EPA) because of their specificity and high activity at low

temperatures (Lee and Akoh, 1998; Soumanou et al., 1998). Liu et al., (2006) reported a total

content of DHA & EPA by urea complexation method was 89.38% at a urea to fatty acid ratio of

15.78 whereas Shimada et al., (2001) reported maximum 80% hydrolysis of DHA in tuna oil

with lipasesP. aeruginosa, C. rugosa andR. delemar. Experiments of selective hydrolysis with

selective esterification were conducted by Shimada et al., (1997) to achieve DHA of high purity

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i.e. >90% by R. delemarand Rhizomucor meihei lipases. The present study has been aimed for

the synthesis of DHA & EPA enriched PUFA concentrates from the cod liver fish oil with urea

complexation method.

2. MATERIALS & METHODS

2.1 Materials

Cod liver fish oil of refined grade (Seacod make) was purchased from local market. All the

chemicals including Butyl Hydroquinone (BTHQ), Potassium Hydroxide (KOH), Ethanol,

Hexane, Anhydrous Sodium Sulfate, Urea Crystals, Hydrogen Chloride (HCl), Methanol,

Sodium methoxide, Iso-Octane, were used of AR grade and CDH make.

2.2 Methods

2.2.1 Extraction of Total Free Fatty Acids

Preparation of free fatty acids from Cod Liver oil was carried out according to Senanayake and

Shahidi (1999) and Wanasundara and Shahidi (1998) with some modifications. According to this

method, first of all Cod Liver oil (25g) was treated with BTHQ (200ppm) and then saponified

with a mixture of KOH (5.75g), distilled water (11ml) and 95% aqueous ethanol (66ml) for 1 hr

at 62C in water bath. After saponification, distilled water (50ml) was added to the saponified

mixture and the unsaponifible matter was extracted with 2x100ml of hexane and discarded. The

saponifible matter in the aqueous layer was acidified to pH 1.0 with 6N HCl and the free fatty

acids were extracted into 50ml of hexane. Hexane layer containing free fatty acids were then

dried over anhydrous sodium sulfate. The remaining solvent in free fatty acids was removed by

heating the mixture at 80C in the water bath and then stored at 6C until use.

2.2.2 Separation of 3 Fatty Acids by Urea Complexation

The separation of 3 fatty acids from the hydrolyzed fatty acid mixture of Cod Liver oil was

carried out using urea-fatty acid adduct formation according to Senanayake and Shahidi (1999)

and Wanasundara and Shahidi (1998). Free fatty acids (5g) were added under constant stirring to

a hot (60C) solution of 15g urea in 75ml of 95% aqueous ethanol. The solution was heated and

stirred until clear. Then it was allowed to crystallize at room temperature for 3hr and kept at 6C

for 24hrs for further crystallization in incubator shaker. The formed crystals were separated from

the liquid by filtration. The filtrate was diluted with equal volume water and acidified to pH 4-5

with 6N HCl. Then equal volume of hexane was added and the mixture was stirred for 1hr. This

mixture was transferred to a separating funnel and hexane layer containing free fatty acids were

separated from the aqueous layer. This hexane layer was washed with distilled water to remove

any remaining urea and dried over anhydrous sodium sulfate. The solvent was subsequently

removed by heating at 80C under water bath.

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2.2.3 Preparation of 3 FAME (Fatty Acids Methyl Esters)

Preparation of 3 fatty acids methyl esters were carried out according to method given by Jham,

Teles, and Campos (1982) with slight modifications. The 3 fatty acids (5g) were esterified with

base i.e. 100 ml of KOH in methanol (0.5 N) at 100C for 5 minutes in tightly capped glass

bottles. After this, the mixture was esterified with acid i.e. 50ml of HCl in methanol (4:1 vol/vol)and then heated in a water bath for 15 minutes at 100C. The mixture was cooled and then 200ml

of distilled water was added. The fatty acid methyl ester was extracted with 2x100ml of hexane.

The hexane layer was dried over anhydrous sodium sulfate and the solvent was removed using

hot water bath at 80C. Then fatty acids methyl ester was stored at 6C in incubator shaker until

use. Standard ASTM test method has been used for estimating the acid value (ASTM D 1386

98,2004) of collected samples at different reaction time to determine moles of free fatty acids

(FFAs) formed.

3. RESULTS & DISCUSSION

Properties of cod liver oil such as density, acid value, saponification value and iodine value have

been evaluated using standard ASTM test methods and given in Table-2 along with the reported

literature values. As mentioned in the Table-2, the results obtained by various physicochemical

tests for Cod Liver oil indicate that the values obtained experimentally are very close to the

values reported in the literature and hence shows reliability of results obtained.

Table-2: Physicochemical Properties of Cod Liver Oil

S.

No.

Property of

Cod Liver Oil

Literature Value

(Klinkesorn et al., 2002; John1999)

Experimental

Value

1 Density (kg/m3) 920 930 928

2 Acid Value (mg KOH/g of oil) 0.16 - 0.63 0.561

3 Saponification Value (SV) 171 189 181.9

4 Iodine Value 137 166 142.82

The results obtained by the chemical transesterification method i.e. urea complexation of cod

liver fish oil, are given in Table-3. According to the experimental work conducted, the acid value

of cod liver fish oil initially found to be 0.6 mg KOH/g of oil which indicates the 0.0106 mmoles

of FFAs are present in 1 gram of oil before hydrolysis and also shows that initially cod liver oil

contains 99.7% of glycerides of fatty acids and other components.

After hydrolysis, acid value of oil was found to be 156.75 mg KOH/g of oil which corresponds

to 86.12% hydrolysis and 2.79 mmoles of FFAs per gram of oil. This hydrolysis was conducted

by the simple acid-base chemical method. Then, after hydrolysis total free fatty acids were

separated from the glycerides by solvent extraction method using hexane which separates

organic phase containing free fatty acids from the aqueous phase containing glycerides including

some other components.

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Table-3: Results obtained with Chemical Method to produce PUFA and

FAME from Cod liver oil

S.

No.

Parameter Literature

Value

(Klinkesorn

et al., 2002)

Acid Value

(mgKOH/

g oil)

Estimated values based on

Experimental Results

mmoles/

g of oil

Percentage (%)

1 Free Fatty Acids

(FFAs) content

before hydrolysis

0.08-0.165 % 0.6 0.0106 0.3 % -

2 Glycerides Content 99.84 % - - 99.7% -

3 Unsaponifible

Content

(w/w %)

1.46% - - 1.64% -

4 FFAs after

hydrolysis

90-95% 156.74 2.79 86.17% -

5 3 Polyunsaturated

Fatty Acids

(PUFAs)

22-23% 48.18 0.858 26.48 %

Based on

S. V.

30.74%

Based on Total

FFA obtained

after hydrolysis

6 Saturated and

MonounsaturatedFatty Acids

77-78% 97.25 1.73 53.47%

Based onS. V.

62.04%

Based on TotalFFA obtained

after hydrolysis

7 3 Fatty Acid

Methyl Esters(FAMEs)

29% 37.45

Correspondingto acids

converted toesters

0.667 20.61%

Based onS. V.

77.7%

Based on TotalPUFA obtained

after ureacomplexation

After separating PUFA from saturated and monounsaturated fatty acids by employing urea

complexation method, the acid value of PUFA fraction was found to be 48.18 mg KOH/g of oil

which corresponds to 0.858 mmoles of PUFA/g of oil. PUFA content was found to be 26.48%

based on the SV and 30.74% based on the total FFAs after hydrolysis. Similarly remaining

saturated & monounsaturated fatty acids in the form of urea complex have been obtained as 1.73mmoles/g of oil which corresponds to 53.47% based on SV and 62.04% based on the total FFAs

after hydrolysis.

By taking the FFAs composition of CLO from literature where DHA & EPA is reported to be

present in 19-20% and PUFA in 22-23% approximately (Kingsbury et al., 1962; Klenket al.,

1962; DeWitt et al., 1963 and Ackman et al., 1964) and assuming maximum DHA & EPA to be

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20% and PUFA to be 23% in CLO, there would be 0.558 mmoles of DHA & EPA/g of oil and

0.642 mmoles of PUFA/g of oil. The PUFA content obtained by the experimental work i.e.

26.48% based on SV and 30.7% based on the total FFAs after hydrolysis, indicating a

comparatively higher value than the data given in the literature i.e. 23%. The deviation observed

may be due to the incomplete complexation of saturated and monounsaturated fatty acids with

urea and possibility of uncomplexed saturated and monounsaturated fatty acids remaining with

PUFA fraction separated from hydrolyzed fatty acids by the solvent extraction and the urea

complexation method.

It has been possible to convert 0.667 mmoles PUFA rich DHA & EPA per g of oil into fatty

acids methyl esters (FAMEs) by chemical transesterification method with sodium methoxide

which corresponds to 20.61% 3-FAMEs based on S.V. of cod liver oil and 77.7% based on the

PUFA obtained after urea complexation.

4. CONCLUSIONS

Synthesis of DHA rich PUFAs from cod liver fish oil, by urea complexation method has been

studied. Recovery of 2.79 mmoles of total FFAs per gram of oil, indicating 86.25% hydrolysis

has been obtained with respect to total FFAs present in cod liver oil i.e. 3.24 mmoles/g of oil.

Similarly 0.858 mmoles PUFAs/g of oil and 0.667 mmoles of FAMEs/g of oil after urea

complexation and chemical transesterification method have been obtained respectively.

5. REFERENCES

1. Ackman, R. G., R. D. Burgher and Can., J. Biochem. Physiol.41:2501-2505(1963).

2. ASTM D 1386 98, ASTM International, 100 Barr Harbor Drive, PO Box C700, West

Conshohocken, PA 19428-2959, United States (2004).

3. Calson S.E. and G. J. Nelson, Health Effects of Dietary Fatty Acids, American Oil

Chemists Society, Champaign, 4249 (1991).

4. Connoe W., E. Neuringer and S. Reisbick, Nutr. Rev., 50:2129(1992).

5. DeWitt K. W., J. Sci. Fd. Agric., 14:92-98(1963).

6. Guil-Guerrero J.L., and E.H. Belarbi, J. Am. Oil Chem. Soc., 78:477484(2001).

7. Jham G. N., F. F.Teles and L. G. Campos, Journal of American Oil Chemists Society,

59(3):32-133(1982).

8. John A. D., Langes Handbook of chemistry, fifteenth edition, McGRAW-HILL, INC.New York, (1999).

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9. Kingsbury K. J., T. D. Heyes, D. M. Morgan, C. Aylott, P. A. Burton, R. Emmersen and

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11.Klinkesorn U., A. H. Kittikum, P. Chinachoti and P. Sophanodora, ChemicalTransestrification of tuna oil to enrich omega-3 polyunsaturated fatty acids,Department of Food Science, University of Massachusetts, Amherst, MA 01003,

USA(2002).

12.Lee K., and C.C. Akoh, J. Am. Oil Chem. Soc., 75:495499(1998).

13.Liu S., C. Zhang, P. Hong and H. Ji, Journal of Food Engineering, 73:203-209(2006).

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73(3-4):15572(2004).

16.Shimada Y., A. Sugihar, H. Nakano, T. Kuramoto, T. Nagao, M. Gemba and Y.Tominaga, J. Am. Oil Chem. Soc., 74:97-101(1997).

17.Soumanou M.M., U.T. Bornscheuer and R.D. Schmid, Ibid., 75:703710(1998).

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health implication, (2000).

Document By: Bharadwaj

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Synthesis of DHA Rich PUFA From Cod Liver Fish Oil