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A Possible Approach for Maintaining Effective Omega-6/ Omega-3 Fatty Acid Ratio from Mixed Vegetable Oils

M. M. Haque
Quality Control department, Igloo Ice-cream, Monem Group of Industries, Dhaka-1205, Bangladesh.

M. N. Rahman
Department of Food Technology and Nutritional Science, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902, Bangladesh.

M. J. Alam*
Department of Food Technology and Nutritional Science, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902, Bangladesh.

S. Akter
Department of Food Technology and Nutritional Science, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902, Bangladesh.

Abstract/ Executive Summary:

Vegetable oil rich in omega-3 and omega-6 fatty acids is an important element in the diet of most transitional countries. The ratio of omega-6 to omega-3 in modern diets is approximately 15:1, whereas ratios of 2:1 to 4:1 have been associated with reduced mortality from cardiovascular disease, suppressed inflammation in patients with rheumatoid arthritis, and decreased risk of breast cancer. The study was designed to investigate the fatty acid profile of six types of seed oils such as peanut, linseed, olive, soybean, sesame and sunflower oil. Afterwards the author prepared mixed vegetable oils with effective Omega-6 (n-6)/omega-3 (n-3) fatty acid ratio. It was found that the highest percentage (39.9%) of saturated fatty acid found in Linseed oil and the highest percentage (37.1%) of monounsaturated fatty acid found in Sesame oil. It was also observed that olive and soybean oil contain 100% polyunsaturated fatty acid and the lowest percentages (35.2%) of polyunsaturated fatty acid were found in Sesame oil. After preparing a mixed vegetable oil The ratio of n-6 to n-3 were 3.5:1 (soybean), 19:1 (olive), 0.43:1 (linseed), 0.13:1 (peanut) and sesame (16.5:1). It is also noted that n-3 was not detected in sunflower oil. Thus the investigation showed that Soybean oil contains the balanced omega-6/omega-3 fatty acid ratio than others.

Keywords: Cardiovascular disease, Edible oil, Essential fatty acids, Modern diet

Location: Department of Food Technology and Nutritional Science, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902, Bangladesh.

Start Date:

End Date:

Program Area: Quality and Nutrition

Commodity: Vegetable oil, Vegetable oil

Objectives:

To elucidate the fatty acid profile of six types of seed oils. Afterwards the author prepared mixed vegetable oils with effective n6/n-3 fatty acid ratio.

Methodology:

Reagents used in the analytical process All chemicals, biochemical standards, reagents and solvents were of analytical grade purchased from Dhaka, Bangladesh. Collection of fats and oils Six different sample such as soybean oil, sunflower oil, sesame oil, peanut oil, linseed oil and olive oil were used in this study which were collected from different parts of Bangladesh. All samples were preserved in dry, brown bottles. The plastic bottles were covered with carbon papers to prevent photo oxidation. The bottles were also stored at -28⁰ C until analysis to prevent auto oxidation. Determination of fatty acid composition of fats and oils Before determining the fatty acid composition of lipid by Gas Liquid Chromatography (GLC) the fatty acids were converted to their methyl esters. The procedure followed was essentially the same as described in details by Yusuf et al. (1993). Preparation of fatty acid methyl esters Preparation of ethanolic potassium hydroxide Potassium hydroxide (11.2g) was dissolved in 100 ml of 95% ethyl alcohol and the solution was filtered. Preparation of anhydrous methanolic HCl (5% w/w) Traces of water were removed from methanol by allowing it to stand overnight in contact with fused anhydrous coarse powdered calcium chloride and then either filtering or centrifuging the suspension. The dry methanol thus obtained was stored in a tightly Stopper brown bottle. Methanol HCl mixture was prepared in the following manner. Hydrochloric acid gas also dry was generated from NaCl contained in a quick fit round bottom flask, with concentrated H₂SO₄ added to the salt drop by drop from a separating funnel/burette. The gas produced was passed through a quick-fit conical flask filled with the fused CaCl₂ powder. The gas was introduced into a known amount of dry methanol contained in another bottle. The gas was passed until it was at least 5% (w/w) in solvent (methanol). Usually a 7% HCl in methanol mixture was prepared. Traces of CaCl₂ powder were found to sediment on standing and were removed by filtration. This HCl: methanol reagent was stored in a tightly stopper bottle. Methylation of fatty acids Total lipid (400-600 mg) was taken in a ground joint flask and saponified with 15-30 ml 2M KOH (ethanolic) in water bath at 70⁰ C for 1 hour by joining with a condenser. After cooling, the solution was diluted with equal volume of distilled water and acidified with concentrated HCl to PH <2 as ascertained with a PH meter. The liberated fatty acids (a mixture) were extracted with 30-60 ml of diethyl ether. Small amount of water was also extracted along with free fatty acids. This undesired water was removed by adding anhydrous sodium sulphate. The ether extract devoid of water was collected in another joint flask/filtration. The extract was then evaporated to dryness under N2. Dry methanolic HCl (25-50 ml) prepared as above, was added into the flask containing the fatty acid mixture and the content was heated at 85⁰ C under reflux for 2 hours. After cooling, the fatty acids methyl esters (FAME) were extracted three times with equal volume of petroleum spirit (bp40-600). All extracts were combined and evaporated to a small volume under N2. Purification of fatty acid methyl esters (FAME) FAME prepared as above was accompanied by free fatty acids, free cholesterol, cholesterol esters and other solvent impurities. Before analysis by GLC, they were purified by Thin-Layer Chromatography (TLC). A slurry of silica gel for thin layer chromatography is made with water (2 ml water per gm silica gel G) in a beaker (500ml capacity) and spread on 2 mm thick glass plates 20'20 cm by a TLC spreader. The silica gel coating is 250'm. The slurry thus spread is kept on the platform for about 10 minutes, transfer to the metal racks and dried in an oven at 110⁰ C for about an hour. The plates are now ready for use. Thin Layer Chromatographic (TLC) procedure Standard fatty acids preparation (~3-5 ml) is now spotted on the plates with a glass capillary taking precaution so that not more than 2-3 ml are spotted on the plates at a distance nearly ¾ for an inch from one edge on the plates. The gaps between two spots should be around half an inch and the spots should be as small as possible for better resolution of the fatty acids. The unknown should be spotted on the two locations. After air drying the plate is dipped in the solvents (n- hexane: Diethyl ether: glacial acetic acid 70:30:1) in the TLC jar which is pre-equilibrated with the solvent system for about an hour. The solvent rise up the silica gel ( ascending chromatography) and is allowed to rise approximately anywhere between 15-18 cm ( nearly one hours) at which point the plate is removed from the jar, air dried, placed in the iodine chamber for 5 minutes. The FAME band in the plate was visualized in the iodine chamber. The FAME in the sample can be identified by their Rf values when compared to standard. After the yellow color vanished the band was scraped into a centrifuge tube and eluted with methanol. The tube was then centrifuged and the supernatant was transferred into a dry flask. The FAME solution was evaporated to dryness under nitrogen. A small volume of dichloromethane solution was added to re-dissolve the FAME band and a 5-10 micro liter aliquot was analyzed in Gas-liquid chromatography. R_f = Distance traveled by particular fatty acid/Distance traveled by the solvent front Gas-Liquid Chromatographic (GLC) analysis of fatty acid methyl esters The fatty acid methyl esters, prepared and purified as above, were analyzed by gas-liquid chromatography (GLC). A 2x4 mm inside diameter column (Preferably glass) packed with 12-15 % (w/w) ethylene glycol succinate liquid phase coated on 100/200 mesh Gaschrom P was used. The injector temperature was 190⁰ C and the detector temperature was 260⁰ C. The temperature of the column was programmed initially at 170⁰ C for 8 minutes, then it was allowed to rise to 200⁰ C at a rate of 10 C/min and the isothermal final period was 55 minutes. Thermal conductivity detectors were excellent. Nitrogen was used as a carrier gas at a flow rate of 11.4 ml/min. Hydrogen flow was 10% above nitrogen flow. Standard fatty acid methyl esters were used for the identification of the sample fatty acid peaks. The following Standard fatty acids were used, the methyl esters of C 8:0 C 9:0 C 10:0 C 11:0 C 12:0 C 14:0 C 16:0 C 18:0 C 18:1 C 18:2 C 18:3 C 20:0 C 22:0. The peak area of each component was measured automatically by chromatograph machine. It was also measured by the actual physical measurement by the triangulation method (Yusuf et al, 1981). The total mm of all peak areas were taken as 100% and the percent population of a given fatty acid peak was calculated accordingly. The fatty acids were expressed as weight percentages of total fatty acids.

Source of Information: J. Environ. Sci. & Natural Resources, 9(2): 65-69, 2016 ISSN 1999-7361

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Achievement/Findings:

Sesame and olive oil have been supposed to possess the highest position in nutritional quality while compared to peanut or sunflower by the fatty acid composition. Soybean oil contains the balanced omega-6/omega-3 fatty acid ratio. Both olive and peanut oil could be consumed as vegetable oils like the soybean oil. Sunflower with peanut, linseed and soybean oil contained the effective ratio of n-6/n-3 fatty acids which may reduce the mortality from cardiovascular disease, suppressed inflammation in patients with rheumatoid arthritis and may decrease risk of breast cancer.

Knowledge Management: Journal