Dietary fats are used to build every cell in the

body and cell membranes are made of a variety of

individual fatty acids which are carboxylic acids with

long hydrocarbon chains (usually C12-22). The

essential fatty acids from marine fish have protective

mechanisms against coronary heart disease, which

became apparent in the investigations of the health

status of Greenland Eskimos who consumed diets very

high in fat from seals,whales, fish etc, and yet had a

low rate of coronary heart disease

Chakraborty, Kajal

Iyapparajanarasimapallavan, G

Joseph, Dexy

Joy, Minju

Makkar, Fasina

Raola, Vamshi Krishna

Vijayagopal, P

CMFRI Digital Repository

Mar. Fish. Infor. Serv., T & E Ser., No. 223 & 224, 2015 3Long chain n-3 polyunsaturated fatty acid enriched oilemulsion from sardine oilKajal Chakraborty, Vijayagopal, P., Iyyapparajanarasimapallavan, G., Dexy Joseph, Fasina Makkar,Vamshi Krishna Raola and Minju JoyICAR-Central Marine Fisheries Research Institute, Kochi20:4n-6) and EPA are involved in the production andmodulation of eicosanoids respectively. DHA is a vitalcomponent of the phospholipids of cellularmembranes, especially in the brain and retina, andnecessary for their proper functioning. An imbalancein n-3/n-6 ratio can accentuate n-3 fatty aciddeficiency state, as shown by earlier studies. Theratio is found to have increased in industrializedsocieties because of increased consumption ofvegetable oils rich in n-6 fatty acids, ie, linoleic acid(18:2n-6) and reduced consumption of foods rich inn-3 fatty acids. Another important feature of n-3 fattyacids is their role in the prevention and modulationof certain diseases that are common. A partial list ofdiseases that may be prevented or ameliorated withn-3 fatty acids is given below.z Coronary heart disease and strokez Diabetesz Cancers of the breast, colon, and prostatez HypertensionThe LC - PUFAs are also recognized to havebeneficial therapeutic, physiological and nutritionaleffects on human health.Imported products (PUFA supplement) includeSeven Seas by a UK healthcare company producingPUFA rich Cod Liver Oil by Ocean Gold™ technology. Avalue-added PUFA concentrate named “fish oil-1000natural omega 3®” containing 100 capsulesmanufactured by Healtheries of New Zealand Ltd.priced  at about ` 1150 per pack, is currently beingmarketed in India by Perma Healthcare, Bangalore.“EPAX 1050 TG®”, a marine omega 3 formulaproduced from selected marine oils and marketed byEPAXAS, Norway is an imported product containingDietary fats are used to build every cell in thebody and cell membranes are made of a variety ofindividual fatty acids which are carboxylic acids withlong hydrocarbon chains (usually C12-22). Theessential fatty acids from marine fish have protectivemechanisms against coronary heart disease, whichbecame apparent in the investigations of the healthstatus of Greenland Eskimos who consumed diets veryhigh in fat from seals,whales, fish etc, and yet had alow rate of coronary heart disease. This paradox wasexplained by the fact that the Eskimos diet containedlarge quantities of the very-long-chain and highlypolyunsaturated fatty acids with C20-22 carbons and 5-6 olefinic bonds, which are abundant in marine fish,but scarce or absent in terrestrial animals and plants.Fatty acids with two or more double bonds are termedas polyunsaturated fatty acids (PUFAs) which arebroadly divided into two major families, n-3 and n-6PUFAs. The long chain C20-22 n-3 fatty acids are foundabundantly  in marine  fish and phytoplankton. Theseaffect many physiological processes includingcognitive function, visual acuity, immunosuppressiveand anti-thrombic activities along with a major rolein glucose and lipid metabolism. Research onexploring sources for long-chain C20-22 PUFAs (LC-PUFAs) such as Eicosapentaenoic acid (EPA, 20:5n-3) and Docosahexaenoic acid (DHA, 22:6n-3) fornutrition have received considerable attention (Fig.1). Since these PUFAs are usually low in abundancein humans, but  regarded as essential they have tobe supplied in the diet. The importance of PUFAs inhuman nutrition has been extensively investigatedduring the past 20 years. DHA one of the importantPUFAs maintains structural and functional integrityin larval cell membranes in addition to the neuraldevelopment and function, while Arachidonic acid (AA,Mar. Fish. Infor. Serv., T & E Ser., No. 223 & 224, 2015417% EPA for use as fish feed supplement. OMEGA XL®is marketed by DeColores in a bottle containing 60capsules of refined combination of omega-3’s costingabout $50 per pack, as a nutraceutical. Great HealthWorks manufactures a concentrate of PUFA from BlueGrenadier Fish that is also a costly PUFA supplement.DSM Nutritional Products, Switzerland manufacturesand sells PUFA concentrate under the trade nameROPUFA® produced from refined vegetable andmarine oils. These products are currently importedto meet the domestic demand of PUFA concentrateformulation for fish feed supplements andnutraceutical purposes. This underlied the need todevelop an indigenous n-3 (PUFA) supplement forwhich the Marine Bioprospecting section of MarineBiotechnology Division screened locally available low-value fish for n-3 PUFAs, concentrating theseessential fatty acids therefrom by chemical and/orenzymatic process.The aim was to develop anindigenous n-3 PUFA enriched formulation(s)comprising fatty acid concentrate and individual orcombination of additives with potential antioxidantproperties to form a stabilized and concentrated formof long chained n-3 PUFAs which may be a cheaperalternative to the imported PUFA supplements, andwill be useful as nutraceuticals and in maricultureoperations for fish larval nutrition. It is believed that the optimal formulations forthe first-feeding fish larvae should simulate the yolkcomposition and to some extent reflect the nutrientrequirements and metabolic capacities of the pre-feeding fish. Dietary long chain PUFAs play animportant role as vital sources of essential fattyacids, needed for normal growth and suvival. Larvaeof many marine fishes require highly unsaturatedfatty acids of the n-3 series, such as, DHA due to theabsence of essential enzymes required forbiosynthesis of C20-22 LC-PUFAs from their short chainanalogues (Fig. 2). Some investigations have shownthat DHA is superior to EPA for larval fish suggestinga different physiological function.The important natural sources of n-3 LC-PUFAsare fishes such as mackerel, sardines, sharks;microalgae, polychaetes, etc. Among these, sardinesare inexpensively available and contain considerableamount of PUFAs, particularly 20:5n-3. Hence sardineoil was preferred as the raw material to formulateFig. 1.  C20-22 polyunsaturated fatty acids (Eicosapentaenoic and Docosahexaenoic acids)Mar. Fish. Infor. Serv., T & E Ser., No. 223 & 224, 2015 5the n-3 C20-22 LC- PUFA concentrates based on differentphysicochemical properties associated with theolefinic bonds in fatty acids and/or acyl chain length.The objective was to purify EPA and DHA from sardineoil by saponification of fish oil to derive free fattyacids, enrichment of PUFA content from the mixedfatty acid concentrate by amide fractionation andchromatography by utilizing the silica gel complexedwith a d-block element.The unique substratespecificity of microbial triacylglycerol acyl hydrolaseswas utilized for the enhancement of PUFA content intriglycerides to further enrich the C20-22 LC PUFAs.Triacylglycerol acyl hydrolases specifically hydrolysecarboxyl esters of triglycerides into free fatty acidsand partial acylglycerols. The unique characteristicsof this group of enzymes such as positional andstereospecificity were utilized to selectivelyconcentrate targeted fatty acids in triglycerides thatcan be readily absorbed into plasma triglycerides.PUFA enrichment by different physicochemicalproceduresThe crude sardine oil was clarified by a sequentialprocess of degumming, decolorization, andFigure 2. Biosynthetic pathways of the long chain polyunsaturated fatty acids (LC-PUFA)Mar. Fish. Infor. Serv., T & E Ser., No. 223 & 224, 20156deodorization, and was found to contain LC-PUFAs,particularly 20:5 n-3 or EPA (17.80 ± 1.57%) of totalfatty acids, TFA) and 22:6 n-3 or DHA (7.67 ± 1.50%of TFA) along with other n-3 and n-6 PUFAs likeLinolenic acid (LA or 18:3 n-3; 4.47 ± 0.84% TFA),Linoleic acid (18:2 n-6; 0.71 ± 0.23% TFA), andDocosapentaenoic acid (DPA or 22:5 n-3; 1.14 ± 0.08%TFA) (Fig. 3). The n-6 fatty acids have a minor shareof the total fatty acid content of sardine oil (0.81%TFA). The PUFAs containing C18– C22 acyl chain lengthcontributed a major share of the total fatty acids ofthe sardine oil (>30% TFA). Among the saturated fattyacids (SFAs), 14:0 was found to be predominant (7.04± 0.22% TFA), while 16:1 n-7 contributed the majorshare (31.56 ± 2.59% TFA) among themonounsaturated fatty acids (MUFAs). When fattyacids are required in free form for further analyses,lipids were hydrolyzed in alkaline medium forextracting the unsaponifiable material. Sardine oilwas saponified with NaOH/Na2EDTA to yield free fattyacids. Na2EDTA appeared to form complex with tracesof metal ions (Cu, Fe), which catalyze oxidation ofunsaturated fatty acids during saponification, andsubsequently removed by extraction with water thushindering the interferences of metal ions during thecourse of further purification process. Relatively largevolumes of n-hexane were added to the aliquot ofthe salt of fatty acid mixtures for better phaseseparation, thus removing the unsaponificablematerials. Among saturated fatty acids (SFAs), 14:0was found to be predominant (7.04% TFA), while16:1n-7 contributed the major share among allindividual fatty acids in the crude sardine oil (>31%TFA). EPA and DHA were found to be the major n-3PUFAs contributing to 17.8% and 7.67% of TFA,respectively. The n-6 fatty acids have minor share inthe total fatty acid content of sardine oil. Solventextraction resulted in marginal increase ofunsaturation (0.85%) in the fatty acid profile.  ThePUFA exhibited an increase of 6.49%, while MUFA andSFA reduced by 2.96% and 6.91% respectively. Then-3 fatty acids exhibited an increase of 5.73% in thesolvent extract of fatty acids.The free fatty acids derived from sardine oil weresubjected to amide fractionation using methanol assolvent at three different temperatures and urea-fatty acid ratios to obtain PUFAs of high purity. Theinterfering SFAs and most of the MUFAs were removedin the form of amide inclusion compound. Further,as oxidized products do not form amide adducts, theperoxidation of n-3 PUFAs could be avoided duringthe extraction of free acids from fish oil triglycerides.Amide fractionation resulted in total reduction ofSFAs (>95%) (14:0, 16:0, and 17:0), moderatereduction of MUFAs (>65%). The U/FA ratio of 4:1(w/w) was found to be optimal for getting high-purityEPA (47.8%) while that at 3:1 (at 20 C) yielded highercontent of DHA (>20 %) when crystallized at 20C.Based on these results, DHA obtained from sardineoil at 2°C temperature of amide-crystallization byusing a U/FA ratio of 3:1 was selected for subsequentpurification of C22 fatty acid DHA. It is likely that atthe lower temperature (20C), the reaction kineticsto form amide-inclusion complex with SFAs andMUFAs was relatively lower resulting in higher DHAin the extract.Fig. 3. Fatty acid composition of crude and solventextracted sardine oil, and PUFA concentrateobtained by the amide fractionation (U/FA:amide/fatty acid ratio)Mar. Fish. Infor. Serv., T & E Ser., No. 223 & 224, 2015 7Change in fatty acid composition as a function ofmicrobial triacylglycerol acyl hydrolase-catalyzedhydrolysis of fatty acids from sardine oilThe free fatty acids were esterified by using amixture of ethyl alcohol and 0.1 (N) H2SO4  resultingin the ethyl ester of the fatty acids. An extracellulartriacylglycerol acyl hydrolase derived from Bacillussubtilis isolated from marine macroalga, Turbinariaconoides, was used to prepare C22 n-3 polyunsaturatedfatty acid concentrates from the ester fraction. Theenzyme was purified 132-fold with specific activityof 386 triacylglycerol acyl hydrolase units/mg. Theurea fractionated fatty acyl esters were hydrolyzedwith enzyme purified from the bacterium Bacillussubtilis and the TFA content of fatty acyl esters afterlipase hydrolysis were analyzed. SFA levels showed areduction to 0.05% after 3 hours (h) of hydrolysis.The decrease in the content of SFAs and MUFAs inthe fatty acyl ester mixture with the progress ofhydrolysis suggested that SFAs and MUFAs were moreeasily hydrolyzed by the lipase than those in esterifiedfatty acids that contain DHA, resulting in theenrichment of the latter in the ester fraction.The variations of PUFA content of sardine oiltriglycerides as a function of time during the microbialtriacylglycerol acyl hydrolase-catalyzed hydrolysis areillustrated (Fig. 4). The total DHA of fatty acyl esterfraction increased with time up to 3 h of enzyme-catalyzed hydrolysis (35.27% TFA), beyond which itslowly decreased (9.81% TFA after 9 h). The purifiedtriacylglycerol acyl hydrolase was able to enrich DHAwith 35.27% 22:6 n-3 after 3 h of hydrolysis. Theresults also suggested that the esteritic bonds of C22acyl chain lengthened n-3 PUFAs are resistant tohydrolysis by the lipase. However, after prolongedhydrolysis (>9 h), when only a few target fatty acidester bonds (n-6 fatty acyl ester bonds and estersother than C22 n-3 fatty acids) were available in theenzyme hydrolysate that were susceptible to hydrolysisby the bacterial hydrolase, the microbialtriacylglycerol acyl hydrolase could cleave bonds highlyresistant to hydrolysis, i.e., DHA. It can thereforebe concluded that it is possible to separate andconcentrate C22 PUFAs with n-3 double bonds like DHAusing lipase from seaweed associated bacteria likeBacillus subtilis.Separation of phospholipid fraction from the seerfish roe and preparation of DHA rich oil emulsion wasalso done.The total lipids of seer fish roe wereseparated into different lipid classes by silicic acidcolumn chromatography. The lipid fractions werequalitatively analyzed by Thin Layer Chromatography(TLC) for identifying triglycerides, glycolipids andphospholipid components. Fatty acid methyl esters(FAMEs) of the total lipid and the individual lipidclasses were prepared by transesterification process.The DHA enriched fatty acid concentrate was enrichedthrough biochemical and microbiological proceduresto formulate enrichment emulsions which containedroughly, 90% DHA enriched fatty acid concentrate and10% phospholipids fraction extracted from seer  fishroe. The aggregate content of DHA in fish roephospholipidic fraction was recorded as 24.3% DHAalong with 5.2% EPA. The polyunsaturated fatty acidconcentrate of the fish body oil after adding fish roewas found to contain greater than 35% DHA withsignificantly lesser content of saturated fatty acids(0.22%) and monounsaturated fatty acids (~7% oftotal fatty acids).Fig. 4. Fatty acid composition of urea concentratedfatty acids, triacylglycerol acyl hydrolase-catalyzed fatty acid concentrate, seer fish roe,and DHA rich concentrateMar. Fish. Infor. Serv., T & E Ser., No. 223 & 224, 20158Fig. 5. Polyoxyethylene derivative of sorbitanmonolaurate (or Polysorbate 20)Use of emulsifiers to increase the stability ofenriched PUFA emulsion An emulsion is a mixture of two or more liquidsthat are normally immiscible and  an emulsifier is asubstance that stabilizes an emulsion by increasingits kinetic stability. Emulsion should be used whenboth the dispersed and the continuous phase areliquids. An experiment was conducted to understandthe effect of emulsifier to stabilize the DHAconcentrate. The emulsifier used was Tween 20 (orPolysorbate 20), which was able to contain thestability of the preparation for an extended timeperiod (Figs. 5 & 6). Longer chain TAGs such aspolysorbate 20 are more hydrophobic and thereforehave higher oil/water interfacial tension than shorterchain ones. The stability of the PUFA concentrate (assuch without Polysorbate 20) decreased after 15minutes whereas the same appended with Polysorbate20 was able to maintain the stability for an extendedperiod of time due to increased kinetics (increasedBrownian movement)ConclusionsA fatty oil from the livers of various fishes (ascod, halibut or sharks) used chiefly as a source offatty acids, vitamin A and also of vitamin D is calledfish liver oil. Fish oil is short for “fish body oil” andnot the same thing as “cod liver oil” available in themarket. Cod liver oil contains greater concentrationsof vitamin A. Taking cod liver oil in the same amountsthat are recommended for fish oil can be toxic, andeven more so in people who have chronic renal failure(because vitamin A can build up to toxic levels). TheC20-22 n-3 polyunsaturated fatty acid concentrateprepared from the inexpensively available marinesources, such as sardine oil can be a potentialsubstitute of the imported PUFA supplements asfunctional food product.The PUFA enrichedformulation (Fig. 7) from low value “fish body oils”will also overcome the risks associated withhypervitaminosis (A, D) and exposure toenvironmental toxins (mercury, PCBs, dioxins etc.)associated with “liver oils” available in market. Theindigenous n-3 polyunsaturatated fatty acidsemulsion developed from the locally available low-value fish may also serve as a cheaper alternative tothe imported fatty acid emulsions used in fish larvalnutrition during mariculture operations.Fig. 6. Photomicrograph of a water-in-oil emulsion byusing Tween 20Fig. 7. Polyunsaturated fatty acid concentrateprepared from sardine oil. (A) Crude sardineoil (B) PUFA concentrate(A) (B)

Long chain n-3 polyunsaturated fatty acid enriched oil

emulsion from sardine oil

http://eprints.cmfri.org.in/10619/1/223-224-1.pdf

Long chain n-3 polyunsaturated fatty acid enriched oil emulsion from sardine oil

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