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 14:0 16:0 16:1 18:1 20:1 20:5 22:1 22:5 22:6 Total DeWitt et al., 1963 Min. 2.46 10.77 6.56 21.96 9.69 7.19 4.76 0.79 6.94 71.12 Max. 3.06 12.50 10.31 28.30 17.01 12.03 7.09 1.43 10.68 102.71 Ackman et al., 1964 3.5 10.4 12.2 19.6 14.6 5 13.3 1.9 10.5 91.0 Kingsbury et al., 1962 2.4 11.5 7.8 25.6 11.7 8.2 4.9 1.3 7.4 80.8 Klenk et al., 1962 3 12 5 24 9 8 5 1 19 86 Calculated Average Composition 2.92 11.38 8.36 23.58 13.37 5.70 7.28 1.33 10.30 84.22 Percentage Composition (Total 84.22) 3.47 13.51 9.93 27.99 15.87 6.77 8.64 1.58 12.24 100%

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 lipases P. aeruginosa, C. rugosa and R. 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. delemar and 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.

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. 1 2 3 4 Property of Cod Liver Oil Density (kg/m3) Acid Value (mg KOH/g of oil) Saponification Value (SV) Iodine Value Literature Value (Klinkesorn et al., 2002; John 1999) 920 930 0.16 - 0.63 171 189 137 166 Experimental Value 928 0.561 181.9 142.82

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) 0.08-0.165 % 99.84 % 1.46% 90-95% 22-23% Acid Value (mgKOH/ g oil) 0.6 156.74 48.18 Estimated values based on Experimental Results mmoles/ g of oil 0.0106 2.79 0.858 Percentage (%) 0.3 % 99.7% 1.64% 86.17% 26.48 % Based on S. V. 53.47% Based on S. V. 20.61% Based on S. V. 30.74% Based on Total FFA obtained after hydrolysis 62.04% Based on Total FFA obtained after hydrolysis 77.7% Based on Total PUFA obtained after urea complexation

1 2 3 4 5

Free Fatty Acids (FFAs) content before hydrolysis Glycerides Content Unsaponifible Content (w/w %) FFAs after hydrolysis 3 Polyunsaturated Fatty Acids (PUFAs) Saturated and Monounsaturated Fatty Acids 3 Fatty Acid Methyl Esters (FAMEs)

77-78%

97.25

1.73

29%

37.45 Corresponding to acids converted to esters

0.667

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 1.73 mmoles/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; Klenk et 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
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