CERTIFICATE

This is to certify that the project work entitled Characterization of Edible Oils was carried out by Miss. Nagashri. H.V, during the academic year 2009 and submitted in partial fulfillment for the award of the Degree of Master of Science in Biotechnology, to the University of Mysore. This work is result of research work carried out by her under my guidance and supervision. This work has not been submitted for the award of any other Degree or Diploma.

Place: Mysore. Date:

Dr. B. R. Lokesh, Head of the Department Department of LSTF, CFTRI, Mysore.

CHARACTERIZATION OF EDIBLE OILS

Nagashri. H. V
M.Sc Biotechnology Haranahalli Ramaswami Institute of Higher Education, Hassan

Submitted for partial fulfillment for the award of the degree of Master of Science in Biotechnology University of Mysore Mysore, Karnataka.

Department of Lipid Science and Traditional Foods Central Food Technological Research Institute Mysore- 570 020, India April 2009

DECLARATION
I hereby declare that the report on the investigation titled characterization of the edible oils submitted to the University of Mysore, Mysore in partial fulfillment of the requirement for the award of degree in Master of Science in Biotechnology is the record of the original work carried out by me under the guidance of Dr. B. R. Lokesh, Head of the Department, Lipid Science and Traditional Foods Department of Central Food Technological Research Institute, Mysore, Karnataka. I further declare that the results of the work have not been previously submitted for the award of any degree or diploma.

Place: cftri, Mysore Date: 23/04/2009. NAGASHRI. H.V

ACKNOWLEDGEMENT
First of all I thank God Almighty for showering his blessings to me in completing my project work on Characterization of Edible Oils. I take this opportunity to thank my guide Dr. B. R. Lokesh., Head of the Department, Department of Lipid Science and Traditional Foods, for his valuable guidance, immeasurable help and constant encouragement throughout my project work. I am indebted to Dr. V. Prakash, Director, CFTRI, for providing me with the wonderful opportunity in such institution. This allowed me to gain good experience. At the same time, I am grateful to all the staffs of my department for their guidance. I gratefully acknowledge with thanks the help and encouragement by co-guide, Dr. Sakina Khatoon, Dr. A. G. Gopal Krishna, Dr. Nasirullah, Dr. L. R. Reddy and Dr. Sukumar Debnath, Scientists, LSTF, for helping me to complete this piece of work. Man bouquets, for the merit and gratitude to Reena, Meesha, Divya, Sugasini, Poorna Chandra Rao, Preethi and Rekha LSTF for their immense help and advice throughout my project work. I extend my thanks to Dr. M.C Vardharaj, Head, HRD and Dr. R.P Singh, Scientist, HRD Dept, CFTRI for their help and encouragement during the course of this project. My heartfelt gratitudes to my loving parents Venkatesha Murthy. H. K and Padmavathi J.S and my sisters Shree Lakshmi H.V Geetha H.V and Manasa H.V for their constant encouragement and moral support, without which this endeavor would not have been possible. CFTRI- Mysore Date NAGASHRI.H.V

INDEX
Sl.no Title page no.

1. 2. 3. 4. 5. 6.

Introduction Materials and Methods Prevention of Food Adulteration Rules for edible oils. Results and Discussion. Conclusion. Bibliography

4-18 19-26 27-32 33-46 47-47 48-49

INTRODUCTION
Fats and oils are predominantly triesters of fatty acids and glycerols, commonly called triglycerides. They are insoluble in water but soluble in most organic solvents, such as chloroform, carbon tetra chloride, petroleum ether and ethyl ether. There are four important Vitamins A, D, E & K, which are soluble in fats. Fats and oils constitute one of the three major categories of food components. The other two are proteins and carbohydrates. Fats and oils are essential nutrients of human diet and are concentrated sources of energy. They supply about 9 kilo calories/g as compared with about 4 kilo calories/g from proteins and carbohydrates. Fat is a part of structure of cells and membranes crucial to their proper functioning. Two fatty acids, which cannot be synthesized by body and must come from food are linoleic and linolenic acid, which are essential fatty acids. Sources of fats and oils: Fats and oils occur naturally in many of our foods, such as dairy products, meats, poultry, nuts, fish and oil seeds. Another source of vegetable oil is tree borne oil seeds. Some of the edible oils are Sunflower oil, fish oil, coconut oil, etc some are from plant origin and some are originated from animals. Oils and fats have lower densities than water and at normal temperatures range in consistency from liquid to solid appearing substances. Chemical composition of fats and oils: Triglycerides normally represent over 95%of the weight of most food fats and oils. The minor components include mono and diglycerides, free fatty acids, phosphatids, sterols, fatty alcohols, fat-soluble vitamins and other substances. The major components: Triglycerides: Triglycerides are composed of glycerol and three fatty acids. When all the fatty acids in a triglyceride are identical, it is termed as a simple triglyceride. The more common forms, however are the mixed triglycerides in which two or three kinds of fatty acid moieties are

present in the molecule. Illustrations of typical simple and mixed triglyceride molecular structures are: R1-COO, R2-COO and R3-COO represent the different fatty acid moieties as they are esterified with the glycerol moiety (-HOCH2 CHOH- CH2OH).

R1- COO - CH2 | R-COO- CH | R-COO- CH2 Simple triglyceride

R1 COO -CH2 | R2- COO -CH | R3 COO -CH2 Mixed triglyceride

The fatty acids in a triglyceride define the properties of the molecule. The minor components: 1. Mono and diglycerides: 2. Free fatty acids. 3. Phosphatids. 4. Sterols. 5. Phytosterols. 6. Fatty alcohols. 7. Tocopherols. 8. Carotenoid and chlorophyll. 9. Vitamins.. Mono and diglycerides: Mono and diglycerides are the mono and di esters of fatty acids with glycerol. R1-COO -CH2 | HO- CH | HO- CH2 1- monoglyceride R2-COO-CH2 | R1-COO-CH | HO- CH2 1,2-Diglyceride HO- CH2 | R1 - OOC-CH | HO- CH2 2- monoglyceride. R2-COO-CH2 | HO-CH | R1-COO-CH2 1,3- Diglyceride 7

1. Mono and diglycerides: are important as emulsifiers and are used frequently in foods for this purpose. They are prepared commercially by the hydrolysis of glycerol with triglycerides or by the esterification of glycerol with fatty acids. Mono and diglycerides are formed in the intestinal tract as the result of the normal digestion of triglycerides. They are also present in both animal and vegetable oils. 2. Free fatty acids: As the name suggests, free fatty acids (FFA) are the uncombined fatty acids present in a fat. Some unrefined oils may contain as much as several percentage of FFA. The levels of free fatty acids are reduced in the refining process. Refined fats and oils ready for use as foods usually have very low FFA content 3. Phosphatids: consists of polyhydric alcohols (usually glycerol), combined with fatty acids, phosphoric acid and a nitrogen containing compound. Lecithin and cephalin are the common phosphatids found in edible fats. In lecithin the nitrogen base is choline, and in cephalin, hydroxyl ethylamine. For all practical purposes, refining removes the phosphatids from fat or oil. 4. Sterols: sterols are a class of substances referred to as steroid alcohols, in that they contain the common steroid nucleus plus an 8 to 10 carbon atom side chain and an alcoholic group. Cholesterol is the primary animal fat sterol and is not found in vegetable oils. Vegetable oil sterols are collectively termed as Phytosterols. Sitosterols and stigma sterols are the best known members of vegetable oil sterols. The type and amount of vegetable oil sterols vary with the source of the oil. 5. Fatty alcohols : Long chain alcohols are of little importance in most edible fats. A small amount esterified with fatty acids is present in waxes found in some vegetable oil. Larger quantities are found in some marine oils. 6. Tocopherols: Tocopherols are important minor constituents of most vegetable fats. They serve as anti-oxidants to retard rancidity and as sources of an essential nutrient, Vitamin E. Among tocopherol, alpha tocopherol has the highest Vitamin E activity and lowest anti-oxidant activity. The anti-oxidant activities of other tocopherols in decreasing order are as follows: gamma, delta, beta and alpha. Tocopherols, naturally occurring in most vegetable fats, may be partially removed by processing and they are not present in

animal fats. Anti-oxidants are commonly added after processing to retard rancidity in the finished product. 7. Carotenoids and Chlorophyll: Carotenoids are the colouring materials occurring naturally in fats and oils. Most range in colour from yellow to deep red. Chlorophyll is the green colouring matter of plants, which plays an essential part in the photosynthetic process. At times, the chlorophyll content of oils is increased and the oils may be tinged green. This has no effect on oil quality. 8. Vitamins: Generally speaking, most oils and fats are not good sources of vitamins other than vitamin E. The fat-soluble vitamins sometimes are added to foods which contain fat such as margarine and milk, because these foods serve s good carriers. Fatty acids: Fats are the esters of fatty acids and glycerol. Most fats occur in the form of triglycerides in which three fatty acids are attached to the glycerol. General formula RCOOH, where R is the aliphatic group. With few exceptions, fatty acids are linear, range in size from 4 - 24 carbons and contains even number of carbons. The fatty acids chain can be saturated (having no double bonds), mono unsaturated (one double bond) or poly unsaturated (two or more double bonds). The most common fatty acid in edible fats and oils are those containing 16 or 18 carbon atoms. These include the saturated palmitic (C 16:0) stearic acid (C 18:0), the monosaturated oleic acid (C 18:1) and polyunsaturated acids linoleic with two double bonds (C 18:2) and linolenic with three double bonds (C 18:3). The number of double bonds and the terminal methyl group is designated as Omega plus, a number. Eg: linoleic acid is termed as 6 fatty acid. CH3CH2CH2CH2CH2CH=CHCH2CH=CHCH2CH2CH2CH2CH2CH2CH2COOH 9, 12-octadecadienoic acid (Linoleic Acid) Similarly oleic acid is C18:1 9 and linolenic acid is C18: 33. The 3 fatty acid have some unique nutritional properties which are connected to their conversion to a particular group of prostaglandins.

The chemical reactivity of unsaturated fatty acids is determined by the position as well as the number of double bonds in the molecule. Reactivity increases markedly with an increase in the number of double bonds provided they are conjugated (separated by a single bond) or methylene interrupted (separated by a CH2- unit). H C C=CC= C H H H H H Conjugated H C=CCC=C H HH H H Methylene interrupted

If a fatty acid has two isolated double bonds (separated by 2 or more methylene units), its reactivity is slightly greater than that of fatty acids that has only one double bond. These differences are important when the fat is subjected to oxidation and also during hydrogenation process. In most naturally occurring unsaturated fatty acids, the double bonds are in cisconfiguration. This means that the carbon chains on the two sides of the double bonds are bent towards each other and the hydrogen atoms on the double bond are on the same side. The cis configuration structure Here is the diagra m for

Gamma-Linolenic acid (GLA): In trans, the hydrogen atoms on the double bonds are opposite to each other, as a result, the chain is nearly straight (with a slight kink on the double bond) as shown:

10

The trans configuration structure:

Comparison of the trans isomer (top) Elaidic acid and the cis-isomer oleic acid. Cis isomers prevail in all food fats and oils, though small amounts of trans isomers occur in fats from animals. Omega3 and Omega6 fatty acids: Omega3 and Omega6 fatty acids are essential fatty acids (EFAs) that need to be included in the diet because, the human metabolism cannot create them from other fatty acids. Since these fatty acids are polyunsaturated, the terms n-3 PUFAs and n-6 PUFAs are applied to Omega3 and Omega6 fatty acids respectively. These fatty acids use Greek alphabet (, , ..) to identify the location of the double bonds. The alpha () carbon is the carbon closest to the carboxyl group (carbon2) and the Omega is the last carbon of the chain because, Omega is the last letter of the Greek alphabet. Linoleic acid is Omega6 fatty acid because; it has a double bond at sixth carbon away from the Omega carbon. Linoleic acid plays an important role in lowering cholesterol levels. Alpha () linolenic acid is an Omega3 fatty acid because it has a double bond, three carbons away from the Omega carbon. By contrasting the highest double bond in the scientific name from the number of carbons in the fatty acid, we can obtain its classification. Eg: for Arachidonic acid, we subtract 14 from 20 to obtain 6, it is an Omega6 fatty acid. This type of terminology is sometimes applied to oleic acid which is an Omega9 fatty acid.

11

The structure of alpha linolenic( omega3)fatty acid Chemical Structure of Linolenic Acid

Structure of linoleic acid: CH3CH2CH2CH2CH2CH=CHCH2CH=CHCH2CH2CH2CH2CH2CH2CH2COOH 9,12-octadecadienoic acid (Linoleic Acid) In these simplified structural formulae of unsaturated fatty acids, each angle represents a carbon atom and here all the double bonds have cis- configuration.

Properties of fats and oils: The properties of fats and oils depend on factors such as
the source of fats/oils, degree of unsaturation, isomeric forms of the fatty acid, triglyceride molecular structure and processing technique. The most significant reason for the difference in between fats and oils is difference in their melting temperature and the degree of unsaturation (double bonds) of the fatty acids they contain. The introduction of double bonds into a hydrocarbon chain causes perturbations in the structure of the chain that decreases its ability to pack the chains closely into a solid structure. Eg: Sunflower oil contains far more unsaturated fatty acids than butter does and is thus liquid at room temperature and even in refrigerated temperatures. Fats and oils are an important part of the diet of most heterotrophs (including humans). Fats and oils or lipids are broken down in the body by enzymes called Lipases produced in the Pancreas. Oxidative rancidity: When a fat or an oil is allowed to stand in the open air, oxidation takes place slowly. During this process, aldehydes and other products are generated which impart off- flavor and rancid smell to oils. This type of rancidities called Oxidative rancidity. And this reaction is called Auto- oxidation.

12

This is observed more frequently in animal fat than in vegetable oil. This is because, in vegetable oil, natural anti- oxidants such as Vitamin E and Vitamin C, phenols, hydrophenols, tannins etc. are present which prevents rancidity. Hence, vegetable oils can be stored for a longer period than animal fats. Sometimes, substances like Vitamin E, C are added to foods to prevent rancidity. Such substances are called Anti-oxidants.

Characterization of fats and oils: The chemical and physical properties of fats and
oils are largely determined by the fatty acids that they contain and their position with the triacyl glycerol molecule. Chemically all fats and oils are the esters of glycerol and fatty acids; nevertheless, the physical properties of fats and oils vary widely because, 1. The proportions of fatty acids vary over wide range, 2. The triacyl glycerol structures vary for each individual oil and fat. The fatty acid components are distinguished in three ways: a. Chain length, b. Number and position of double bonds, c. Position of fatty acid within the glycerol molecule. Variations in these characteristics are responsible for the chemical and physical differences experienced with edible fats and oils. The structure of a fatty acid is commonly denoted by a name after the nomenclature of its parent hydrocarbon, by its common name, or by a short hand designation showing the number of carbon atoms and the number of double bonds. The fatty acid carbon chain lengths vary between 4 & 24 carbon atoms with up to 3 double bonds. The most prevalent saturated fatty acids are lauric (C-12:0), myristic (C-14:0), palmitic (C-16:0), stearic (C- 18:0), arachidic (C- 20:0), behenic (C 22:0) and lignoceric (C-24:0). The most important mono-unsaturated fatty acids are oleic (C-18:1) & erucic (C-22:1). The essential polyunsaturated fatty acids are linoleic (C-18:2) and linolenic (C-18:3). The triglyceride structure of an edible fat or oil is affected by fatty acids present and the point of attachment of each fatty acid to the glycerol. Triglycerides with three identical fatty acids are called monoacid triglycerides. Triglycerides containing more than one type of fatty acids are called mixed triglycerides.

13

Non-glyceride components of fats and oils: the primary constituents in crude fats and oils are the triglycerides but they also contain varying amount of minor components, many of which significantly affect their physical and chemical properties. Crude vegetable oils commonly contain 2% or more non glyceride substances and animal fats contain much smaller quantities. These minor components, also referred to as the unsaponifiable fraction which contains phospholipids, tocopherol, sterols, and pigments such as carotene and chlorophyll. Some but not all of the non glyceride materials are undesirable; therefore, the objective in all edible oil processing is to remove objectionable impurities with the least damage to the desirable constituents. Blending of fats and oils and combining technologies: Blending of fat fractions or of fats and oils themselves is another approach to reformulating product with altered physical properties. Blending a hard fat into liquid oil increases the overall melting range and can extend the range of plasticity. Eg: When palm oil is blended with rice bran oil, the firmness is decreased, as both saturated and unsaturated fats are mixed together. Blending can also be used to manipulate polymorphism. The oils and fats have their own characteristics like free fatty acid, iodine value, acid value, etc. If an oil or fat has to be considered as a good one, it should bear all these characteristics within the specified limits.

14

Free fatty acids: (% of FFA):

Definition: Free fatty acids such as oleic, linoleic, stearic etc. that are not tied" into a glycerine molecule. Significance: % FFA indicates the care and control exercised during processing. It is an indication of fresh fat quality. In a well-defined shortening or oil (which does not contain emulsifier), the typical free fatty acid level will be less than 0.05%. With used fat, higher levels of FFA is an indication of the amount of hydrolysis that has taken place. Free fatty acids are the result of the reaction of water and fats at frying temperatures. The rate of development in FFA during frying is due primarily to the amount of moisture from the food that goes through the frying system and the temperature of frying fat.

Acid value:
Definition: Acid value is defined as the number of milligrams of KOH/NaOH required to neutralize the free fatty acids present in 1 g of fat. The value is also expressed as % of free fatty acids calculated as oleic acid. Principle: The acid value is determined directly by titrating the oil/fat in a medium against Std. KOH solution or NaOH solution. The value when expressed as % FFA is calculated as oleic, lauric, ricinoleic or palmitic acid. Importance: The value is a measure of the amount of fatty acids which have been liberated by the hydrolysis from their glyceride due to the action of moisture, temperature or lipolytic enzyme lipase.

Iodine value:
Definition: The iodine value of an oil /fat is the number of grams of iodine absorbed by 100 g of the fat, when determined by Wijs solution. Principle: The oil/ fat sample taken in carbon tetra chloride is treated with a known excess of iodine monochloride solution in glacial acetic acid (Wijs solution). The excess of iodine monochloride is treated with KI and the liberated iodine estimated by treating with Na2S2O3 solution.

15

Importance: The iodine value is mainly a measure of the amount of unsaturation (number of double bond) in a fat / oil, higher the iodine value, more the unsaturation of a specific fat or an oil.

Estimation of Oryzanol by spectrophotometer: The quantification was

accomplished by internal normalization. The Oryzanol presence is estimated in all the oils by Spectrophotometry, using a UV-Visible Dual Beam Spectrophotometer, to determine the presence and concentration of nutraceutical components. The concentration of -Oryzanol was determined that 314.5nm, as suggested by Seetha Ramaiah and Prabhakar in 1986, using heptane as a solvent. This Oryzanol percent is calculated using formula -Oryzanol%=O.D 100/ weight 358.9 At O.D at 314nm Wt= (oil (gms) 100 ml of hexane

Unsaponifiable matter determination:

Definition: Unsaponifiable matter includes those substances frequently found dissolved in fats and oils that can not be saponified by the usual caustic treatment, but are soluble in ordinary fat and oil solvents. Included in this group components are higher aliphatic alcohols, sterols, pigments and hydrocarbons. Objective and importance: The objective is to determine the amount of materials present in the oil that are not saponifiable. This is applicable to fats and oils containing higher levels of unsaponifiable matter than usually found in normal tallows and greases. This method is especially suited for marine oils, but it is also applicable to vegetable oil deodourizer distillates and sludges. This method is not applicable to feed grade fats.

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Colour determination by Tintometer (Lovibond):

Definition: This is the colour rationg of a fresh or used fat/oil, based on the comparisons with standard Lovibond colour tubes. Clear liquid samples are placed in standard glass colour tubes, the equipment used is specially designed for viewing and comparing samples and standards under a specific light source. The results are reported as values of yellow and red. Significance: The levels of Lovibond red colour is indicative of the quality and processing of the oil. Fats and oils are darkened in colour with use. Fry life kits have been developed to determine the status of frying fat.

Peroxide value:
Definition: Peroxide value determines all the substances in terms of milli equivalents of peroxide per 1.000 g of sample, which oxidize KI under the condition of the test. The substances are generally assumed to be peroxides or other similar products of fat oxidation. Significance: The peroxide value of an oil/fat is used as a measure of extent to which rancidity reactions have occurred during storage. The double bonds found in fats and oils play a major role in auto-oxidation. Oils with a high degree of unsaturation are more susceptible to auto-oxidation. The best test for auto-oxidation (oxidative rancidity) is determination of PV. Peroxides are intermediates in auto-oxidation reaction. Peroxide value, concentration of peroxide in an oil/ fat is useful for accessing the extent to which spoilage has advanced.

Determination

of

fatty

acid

composition

by

Gas

Liquid

Chromatography:
Determination of fatty acid composition is the fundamental step to characterize an oil/ fat: Fatty acid composition of the oils used in this study was analysed by GC (Fisons GC fitted with a flame ionization detector). The oils were saponified with 0.5 M KOH and methylated with 0.5ml BF3 methanol. The fatty acid methyl esters ware separated using a fused silica capillary column 25m 0.25mm (Parma bond FFAP-DF-0.25, Machery-Negal Gmbh co, Duren, Germany.

17

The operating conditions are as follows: Initial column temperature: 120C, raised by 15C/min to 220C, injection temperature 230C, and detector temperature 240C. Nitrogen was used as the carrier gas. Individual fatty acid was identified by comparing with the retention times of standards (Nuchek Prep, Elysin, MN, USA.) and was quantified by an online chromatopac CR-6A integrator. Significance: Determination of oil/ fat is a fundamental step to characterize an oil/fat. The components like fatty acids can give a fine picture about the quality and nutritional value of an oil/fat.

Nutritional aspects: Role of fats and oils in storage and provision of metabolic
energy: The energy value of triacyl glycerols in the body is about 38kJ (9k Cal)/g compared with about 17kJ for proteins and 16kJ for carbohydrates. Although, the gross energies of fatty acids with different degrees of unsaturation differ slightly, these differences are insignificant nutritionally. The useful energy available to the body depends mainly on the digestibility of the fat and on the chain length of the fatty acid. Most common food fats are efficiently digested by most people with insignificant differences in their digestible energies. However, saturated (and to a lesser extent unsaturated) fatty acids with chain lengths of more than 18 carbons are less well digested and absorbed, the efficiency falling as chain lengths increases. The energy values of the medium and short chain fatty acids are considerably lower than those of longer chain acids. How much of a health problem is fat? The intense concern of fat is mainly due to much publicized associations between high fat intake and ill health. a. Obesity: Arguably, Obesity is one of the major health problems in industrialized countries and one that seems resistant to all public health attempts to control it. It is now recognized that central obesity is a significant risk factor for CHD where as lower body or overall obesity have virtually no relationship. Central obesity and the common disorders like glucose intolerance, (and its main clinical manifestation) insulin

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independent Diabetes mellitus, hypertriacylglycerolemia, and hypertension form what is sometimes dubbed the deadly quartet. The common link between the deadly quartet is thought to be due to hyperinsulinemia, i.e. habitually raised blood insulin concentrations. In the environmental factors, smoking and diet have been given most prominence, and of the various dietary components, most emphasis has been placed on fat. The consensus among nutritionists, epidemiologists, and medical practitioners is that SFA are the principal dietary components contributing to CVD through their influence on blood cholesterol, particularly LDL cholesterol. These particles are held to be a pre- requisite for the development of atherosclerotic plaque. Although the adherents to this hypothesis would be the first to accept the gross over simplicity of this view, the unbiased reader of the U.K Department of Healths most recent report, Nutritional Aspects of Cardio Vascular Disease, has been left in little doubt that those responsible for public health regard reduction in dietary SFA as the main plank in the strategy to reduce CVD. A considerable weakness in the concept of SFA- involvement in CVD is illustrated by the U.K department of Health report is the focus on total cholesterol and LDL in the fasting state. In practice, most human central obesity is characterized by extremely enlarged fat cells in the abdominal adipose tissue. The large fat cells are insulin resistant and need more insulin to sustain their metabolism. This contributes to the raised blood insulin. They also have a greater tendency to breakdown their triacyl glycerols liberating free fatty acids into the plasma. High concentrations of fatty acids in the blood are thought to reduce the insulin sensitivity of muscle as well as interfering with the removal of insulin by the liver. The excess of insulin and fatty acids causes the liver to turn the fatty acid into VLDL, which explains hyperlipidemia. Finally high insulin levels are thought to act upon the kidneys and the vascular systems in such a way as to produce high blood pressure, although the latter mechanisms are more obscure than those affecting blood lipids. b. Cardio Vascular diseases: It is recognized that many inherited and environmental factors interact to affect predisposition to the development of cardiovascular disease (CVD). In developed countries many spend most of their time in the postprandial state. Zilversmit proposed that the most atherogenic particles were not LDL but the remnants of

19

chylomicrons and VLDL after part of their triacyl glycerol has been removed. This idea has now been extended and refined, and current views of the role of lipoproteins in CVD are summarized in the concept of the atherogenic lipoprotein phenotype [ALP]. Briefly, ALP describes a collection of Abnormalities in lipoprotein metabolism that predispose apparently healthy people to increased CVD risk. Its characteristics are a moderately raised concentration of tri acyl glycerol, low concentration of HDL, and a predominance of unusually small, dense LDL and HDL particles in the post absorptive state. The ALP is associated with the condition of insulin resistance, in which tissues need more of the hormone than normal to accomplish a particular metabolic task. This causes the pancreas to secrete more insulin and results in habitually elevated blood insulin concentrations. There is a strong genetic basis for the ALP, which may mainly affect the enzyme lipoprotein lipase, which removes triacyl glycerol from circulating chylomicrons immediately. However, it is quite clear that development of condition involves diet, smoking and physical activity interacting with the predisposing genes. It is quite possible that certain fatty acids directly influence the expression of the genes involved in the ALP and this is currently the subject of vigorous research. c. Essential fatty acid deficiency: In 1929, Burr and Burr described how acute deficiency states could be produced in rats by feeding fat free diets and how these deficiencies could be eliminated by adding only certain specific fatty acids to the diet. It is now recognized that two primary essential fatty acids (EFA) for humans are Linoleic acid and linolenic acid; because the body is unable to synthesize them. Well documented EFA deficiency in humans is rare but was first seen in children given fat free diets when they developed skin conditions similar to those produced in rats. The skin abnormalities and other signs of EFA deficiency were disappeared. Therefore, oils and fats should be well balanced in terms of saturated, monounsaturated and essential fatty acids. This investigation therefore aims at evaluating the characteristics of edible oils used in our diet.

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MATERIALS AND METHODS

21

Materials: All the oil samples used in this study were collected from local market. The
laboratory grade solvents and chemicals used in the analysis were procured from Ranbaxy Fine Chemicals, New Delhi, India. The double distilled water was used for making solutions.

Methods: 1. Determination of Free fatty acids:

Weigh 5 g of oil sample Add 30 ml of neutralized alcohol to the sample Warm the solution, and then add drops of phenolphthalein Titrate the solution against NaOH solution (0.1 N) End point is appearance of pale pink colour which will persist at least for one min. FFA= CVN/M C= 1/10 of molecular weight appropriate fatty acid V= volume of the standard alkali used N= normality of standard alkali used M= mass of the test portion of oil Standardization of NaOH: Take two clean conical flasks. Weigh an accurate amount of PHP (Pot. Hydrogen phthalate). Add 10ml of distilled water to this (until the PHP solution dissolves, mix thoroughly) Then add one drop of phenolphthalein indicator. Titrate this against NaOH. Calculating the Normality of NaOH: Weight of PHP Burette reading 0.2044 (factor for PHP)

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2. Peroxide value:
Procedure: Weigh 5g 0.05g of sample into a 250 ml Erlenmeyer flask with glass stopper and add 30ml of the 3:2 acetic acid- chloroform mixture (solution) swirl to dissolve the sample. Add 0.5 ml of saturate KI solution using a suitable volumetric pipette. Allow the solution to stand with occasional shaking for exactly 1 minute and then immediately add 30ml of distilled water. Titrate with 0.1N solution of sodium thiosulphate, adding it gradually and with constant agitation. Continue the titration until the yellow iodine colour has almost disappeared. Add about 0.5ml of starch indicator solution; continue the titration with constant agitation, especially near the end point to liberate all of the iodine from the solvent layer. Add the thiosulphate solution drop wise until the blue colour just disappears. Calculations: Volume of alkali consumed N 1000 Weight of sample

3. Unsaponifiable matter determination:

Procedure: Accurately weigh about 2.0-2.5 g 0.1 mg of well mixed sample into a 250ml Erlenmeyer flask with ground glass joint. Add 25 ml of 95% ethyl alcohol and 1.5 ml of 50% KOH solution. Boil gently but steadily under reflux, using an air condenser for 1 hour or completely saponified. Complete saponification is essential. Transfer to the separatory funnel and wash with 15ml of ethyl alcohol. Complete the transfer with warm distilled water (20ml) till the total volume is 80ml. Wash out the flask with a small volume of (5ml) petroleum ether and add to the separatory funnel to room temperature (20-25C) and then add 50 ml of petroleum ether. Insert the stopper, shake vigorously for atleast 1 min and allow to settle until both layers are clear. Separate the upper petroleum ether layer. 23

Repeat the extraction at least 6 times, using 50ml portions of petroleum ether each time and shaking vigorously with each extraction. The petroleum ether fractions are combined in another 500ml separatory funnel. Wash the combined extracts in the separatory funnel three times, using 25ml portions of 10% ethyl alcohol in distilled water, shaking vigorously and drawing off the water, shaking vigorously and drawing off the aq. alcohol layer after each wash avoid removing any of the petroleum ether layer. Continue to wash with the 10% ethyl alcohol solution until the wash solution no longer gives a pink colour after the addition of one drop of phenolphthalein indicator. Transfer the petroleum ether extract to a tared soxhlet flask and evaporate the solvent in a flash evaporator and then to dry in water bath. After the entire sample has evaporated, complete the drying to constant weight in a vacuum oven at 7580C and an internal pressure of not more than 200mm of mercury. Cool in a desiccator and weigh. The result become A in the calculations. After weighing take up the residue in 50ml of warm ethyl alcohol, containing phenolphthalein indicator and previously neutralized to the end point. Titrate with 0.02N NaOH to the same final colour, correct the weight of the residue for free fatty acid content, using the following relationship. 1 ml of 0.02N NaOH is equivalent to 0.0056g of oleic acid. The gram of fatty acid determined by this titration becomes B in the calculation for determining percent unsaponifiable matter. A reagent blank correction should also be determined. Correct for any reagent blank by conducting the unsaponifiable matter procedure without any fat/oil present. The blank determined by this procedure becomes C in the calculation. Calculations: Unsaponifiable matter % = A-(B+C) 100 Wt. of sample Where, A = weight of residue, B = weight of fatty acid, C = weight of blank.

24

Expression of result: The result is expressed as % by weight. After condensation extracted with petroleum ether, then we take upper layer for washing. Lower layer is removed. While washing the upper layer with 15% ethyl alcohol removes the bottom layer, take upper layer for distillation.

4. Determination of Iodine Value:

Procedure: Weigh accurately an appropriate quantity of the dry oil/fat into a 250 ml collared neck conical flask with glass stopper, to which 15 ml of carbon tetra chloride (chloroform) have been added. Mix the contents well, the weight of the sample shall be such that there is an excess of 50-60% of Wijs solution over that actually needed. Pipette 25 ml of Wijs solution and replace the glass stopper after wetting with KI solution. Swirl for proper mixing and keep the flask in dark for half an hour for non drying and sunny drying oils, and 1 hour for drying oils. Carry out a Blank simultaneously, after standing add 20 ml of KI solution, followed by 150 ml of distilled water, rinsing it with the stopper also and titrate liberated iodine with standardized Na2S2O3 solution, using starch as an indicator at the end until the blue colour which is formed is disappeared after thorough shaking with the stopper on. Calculation: Iodine value = 12.69 (B-S) N W Where, B = volume in ml of Std. Sodium thiosulphate solution required for the Blank. S = volume in ml of Std. Sodium thiosulphate solution required for the sample. N = normality of the Std. Sodium thiosulphate solution. W = weight in grams of the sample.

25

5. Determination of Colour by Tintometer:

Procedure: Filter the oil through a filter paper to remove any impurities. Clean the glass cell with carbon tetra chloride and dry. Filter it with the oil and place the cell in position in the Tintometer. Match the colour with sliding Red, Yellow and Blue Colours. Report the colour of the oil in terms of Lovibond unit. Colour reading = Y+ 5R or Y+ 10R Where, Y+ 5R is the mode of expressing the colour of light coloured oils and Y= sum total of yellow slides used. R= sum total of Red slides used.

6. Determination of Acid value:

Procedure: Mix the oil or melted fat thoroughly before weighing. Weigh accurately about 2-2.5g of the sample in a 250 ml of ethyl alcohol and about 1 ml of phenolphthalein indicator solution. Boil the mixture for another 5 minutes and titrate while hot against Std. alkali solution shaking vigorously during the titration. The weight of the oil/fat taken for the estimation and the strength of the alkali required for the titration does not exceed 10 ml. Calculation: Acid value = 56.1 NV W 1.99FFA%. Where, V = volume in ml of Std. KOH or NaOH solution N = normality of the KOH or NaOH used. FFA as oleic acid percent by weight = 28.2N W or

26

FFA as recinoleic acid percent weight = 29.8N W FFA as Palmitic acid percent by weight = 25.6N W.

7. Estimation of Oryzanol by Spectrophotometer (%)

Procedure: Take 20 mg of sample into 30ml capacity stoppered tubes. Dissolve it in 10ml of hexane. Vortex the tubes. Using spectrophotometer, take the readings at 314 nm. Calculations: Oryzanol % = O.D 100 W 358.9 W= oil (gm)/ml 100 10 ml of hexane.

8.

Determination

of

Fatty

acid

composition

by

Gas

Liquid

Chromatography:
Procedure used for the preparation of Fatty acid Methyl Ester: Take a drop of oil. To that add 1ml of 0.5 M KOH in methanol. Vortex the tube carefully. Incubate it in water bath at 65C for 1 hour or 85C for 10 minutes. Add 2ml of hexane and vortex carefully. Discard the upper phase (unsaponifiable material). To the bottom layer, add 1ml of 0.7N HCl in methanol. Again add 2ml of hexane and vortex the tube. Keep it for centrifugation for 15 min, organic layer will separate. Keep upper layer in separate tube. Repeat the step again. 27

Pool the hexane layer. Evaporate the entire hexane layer. Add 0.2 ml of benzene + 0.5 ml BF3/ methanol. Incubate it at 86C for 10min or 65C for 45min. Then cool it and add 1ml of water. Extract thrice with 2ml of hexane. Pool and add 2-3 ml of water. Centrifuge, separate hexane layers. To the separated hexane layer add Na2SO4 in very little amount. Leave it for 5 min. Decant into the GC tube. Inject into gas chromatography.

28

PREVENTION OF FOOD ADULTERATION RULES FOR EDIBLE OILS

PFA REGULATIONS FOR EDIBLE OILS: Prevention of food adulteration cases 1995 and rules for edible oils: 1.A17.19- Palm oil:

29

Palm oil means the oil obtained from fleshy mesocarp of fruits of the oil palm (Elaeis guineensis) tree by the method of expression or solvent extraction. It shall be clear, free from rancidity, suspended or the other organic matter, separated water, added colouring and flavouring substances or mineral oil. It shall conform to the following standards namely: Iodine value: (Wijs method): Unsaponifiable matter: Acid value: Not more than 1.2%. Not more than 10.0. 45- 56.

Indigenously produced raw palm oil obtained by method of expression may be supplied for human consumption as such provided acid value more than 6.0. But palm oil imported into the country are produced by solvent extraction shall be refined before it is supplied for human consumption and it shall conform to the standards laid down under A.17.15. Additionally, it shall have flash point (Pensky Marten closed method) not less than 250C. A17.15: Refined vegetable oils This means any vegetable oil which is obtained by expression or solvent extraction of vegetable oil bearing materials, deacidified with alkali and /or by miscella refining using permitted grade solvents followed by bleaching adsorbent Earth/ carbon and deodourized with steam. No other chemical agent shall be used. The name of the vegetable oil from which the refined oil has been manufactured shall be clearly specified on the label of the container. In addition to the under mentioned standards to which refined vegetable oil shall conform to the standards prescribed in these rules for specified edible oils shall also apply except for acid value which shall not be more than 0.5.

A17.20. - Palmolein: Palmolein means the liquid fraction obtained by palm oil, obtained from the fleshy mesocarp of fruits of oil palm (Elaeis guineensis) tree by the method of expression of

30

solvent extraction. It shall be clear, free from rancidity, suspended or the other organic matter, separated water, added colouring and flavouring substances or mineral oils. It shall conform to the following standards, namely: Iodine value: (Wijs method): Unsaponifiable matter: Acid value: Not more than 1.2%. Not more than 6.0. 54-62.

Further, if solvent is extracted from solvent extracted palm oil, it shall be refined before it is supplied for human consumption, and it shall conform to standards laid down under A.17.15. A.17.23:- Rice bran oil: means the oil obtained from the layer around the endosperm of rice extracted from paddy of Oryza sativa, Linn. Family: Gramineae, which is removed during the process of rice melting and is generally known as Rice bran. Refined rice bran oil shall be obtained from solvent extracted oil, neutralized with alkali, bleached with bleaching Earth or activated carbon or both and deodourised with steam. Alternatively, deacidification, bleaching and deodourisation may be done by physical means. The oil shall be clear from rancidity, adulterants, sediments, suspended and other foreign materials, separated water and colouring and flavouring substances. The clarity of the oil shall be judged by the absence of turbidity after keeping the filtered sample at 35C for 24 hours. Rice bran oil shall be sold for human consumption only after refining. It shall conform to the following standards. Namely: Iodine value: (Wijs method). Acid value: Not more than 0.5. 90- 105.

Prevention of Food Adulteration Act 1954: A.17.22- Sunflower seed oil: means the oil obtained from clear and round sunflower seeds or cake from the plant- Helianthus annuus, Linn, Family: (Compositae), by the

31

method of expression or solvent extraction. It shall be clear, free from rancidity, suspended or other solid matter, separated water, adding colouring or flavouring substances or mineral oil. It shall conform to the following standards: Iodine value: (Wijs method). Unsaponifiable matter: Acid value: Not more than 1.5%. Not more than 6.0. 100- 145.

Further, if the oil is obtained by the method of solvent extraction, it shall be supplied for human consumption only after refining and shall conform to the standards laid down under item A.17.15. A.17.24:- Blended vegetable oils: means an admixture of any two edible vegetable oils, where the proportion by weight of any edible vegetable oil used in the admixture is not less than 20%. The individual oils in the blend shall conform to the respective standards prescribed by these rules. The blend shall be clear, free from rancidity, suspended or insoluble matter or any other foreign matter, separated water, added colouring matter, flavouring substances, mineral oil or any other animal and non-edible fats, argemone oils, hydrocyanic acid, castor oil and tricresyl phosphate. It shall also conform to the following standards, namely: a) Moisture and volatile matter not more than 0.2% by weight. b) Acid value. Nature of oil 1. 2. 3. Both raw edible vegetable oils: One raw edible vegetable oil and one refined edible vegetable oil in the blend: Both refined edible vegetable oils in the blend. Not more than 0.5 Acid value: Not more than 6.0. Not more than 5.0

Unsaponifiable matter: Nature of oil 1. Blend with Rice bran oil: Unsaponifiable matter Not more than 3.0% by weight

32

2. Blend with other edible vegetable oils:

Not more than 1.5% by weight.

Labeling requirements:
Sub rule 4[5(Z Z)]: states that every package containing admixtures of edible oils shall carry following label, namely: i. .% by weight. ii. .% by weight. (Name and nature of edible vegetable oils i.e,.in raw or refined form), Date of packing.] According to Rule 43: Notice of addition, admixture or deficiency in food or oil: 1. Every advertisement and every price or trade list or label for an article of food or oil which contains an addition, admixture or deficiency shall describe the oil as containing such addition, admixture or deficiency. No such advertisements or price or trade list or label attached to the container of food or oil shall contain any words which might imply that the food/oil is pure. 2. Every package containing a food which is not pure by reason of any addition, admixture or deficiency shall be labeled with an adhesive label, which shall have the following declaration. [ DECLARATION] [THIS (a). CONTAINS AN ADMIXTURTE/ ADDITION OF NOT MORE THAN (b)..% OF 2[* * *] (c)[ * * * ] ] a) Here insert the name of oil/food sample. b) Here insert the quantity of admixture which may be present. c) Here insert the names of admixture or name of the ingredient which is deficient. 3. Unless the vendor of a food containing an addition, admixture or deficiency has reason to believe that the purchaser is able to read and understand the declaratory label, he shall give the purchaser, if asked the information contained in the declaratory label by the word of mouth at the time of sale. Prohibition and regulation of Sales:

33

According to Rule 44, sale of certain admixtures prohibited. Notwithstanding the provisions of Rule 43, no person shall either by any servant or agent sell: Eg: 1. A mixture of two or more edible oils as an edible oil. 2. Vanaspati to which ghee or any other substance has been added. 3. Milk that contains any added water. Rule 44-A: Provided also that in respect of clause (e), maximum tolerance of 10 red unit in 1 cm, cell on Lovibond scale is permitted when the oil is tested without dilution. Provided also that prohibition in clause (e) shall retain inoperative in respect of admixtures of any two edible vegetable oils as an edible vegetable oil. Where, a) The proportion by weight of any edible vegetable oil used in the mixture is not less than 20%. b) The admixtures of edible vegetable oils is processed are packed and sold by the Department of Civil Supplies, Government of India(Department of Vanaspati, Vegetable oils and fats) or by agencies in public private or joint sectors authorized by the Department, or by the National Dairy Development Board or by the State Co-operative Oil seed Growers Federation or Regional and Destrict Co-operative Oil seed Growers, Union Set-up under National Dairy Development Boards Oil seeds and Vegetable Oil project or by the Public Sector undertaking of Central and State Governments, in sealed packages weighing not more than 5 kgs under AGMARK Certification Mark compulsorily and bearing the label declaration as laid down in clause( Z Z ) of Rule 42 & c) The quality of each edible oil is used in the admixture conforms to the relevant standard prescribed by these rules.

34

RESULTS AND DISCUSSION:

35

The oils selected for this study were palm oil, Rice bran oil and oil blend A

Free fatty acid.

Samples FFA in % 1 0.0564% 0.5050% 0.223% 7.314% 0.489% 0.3376% 2 0.05623% 0.507% 0.2218% 7.55% 0.432% 0.3385% 0.436% 0.504% 0.224% 3 0.05631% 0.5053% 0.222% 7.432% 0.452% 0.3380% Mean

Sunflower oil

Linseed oil

palmolein

Palm oil

Oil blend A

Rice bran oil

The presence or absence of free fatty acids is an index of the quality of the fats and oils. Refined oils are largely devoid of free fatty acids, considerable amount of these constituents may be present in crude oils. Free fatty acids are the result of hydrolysis of glycerides, producing hydrolytic rancidity. Oils develop rancidity over a period of time due to enzymatic or non enzymatic hydrolysis of triglycerides. Measurement of free fatty acid content in oils indicates the degree of hydrolysis. The edibility of such oils is generally considered to be inversely proportional to the total amount of free fatty acids. According to the vegetable oil product controllers for India, no vegetable oil product should have more than 0.25% of the free fatty acids (calculated as oleic acid).

36

For palm oil, the free fatty acid content must be less than 5.0% as literature, but as for analysis the used oil is very crude oil, the result got is more than 5.0%. However our analysis shows that crude palm oil has FFA more than 5.0%. Hence such oils require further refining. Sunflower oil has minimum percentage of free fatty acid. As this is unsaturated and contain less amount of free fatty acid, it is good for consumption. Palmolein oil also showed free fatty acid percentage less than 0.25%. Blended oil is a mixture of two oils, which is used for these analytical studies. Blended oil had high percentage of free fatty acids than the refined oils.

Peroxide value
Table of PV in milli equivalence peroxide/kg sample.

Sl. no

Samples

1 1.980 23.24 4.038 8.025 3.005 18.131

2 2.020 24.35 3.037 8.063 2.004 19.182

3 2.003 23.32 3.042 8.064 2.016 17.101

mean 2.001 23.63 3.372 8.050 2.431 18.138

Sunflower oil

Linseed oil

Rice bran oil.

Palm oil

palmolein

Oil blend A

The peroxide value is a measure of the extent of oxidation undergone by oil or a fat. In case of oils the peroxide number was found to be high in crude oils. This peroxide accumulation depends on the storage because, if storage is not proper, then the oil will go bad (rancid).

37

In these analysis the linseed oil showed more peroxide value than other oils. If the peroxide value is high, it represents the state of deterioration or the extent of rancidity in them. Peroxides are intermediary unstable compounds in the chain of oxidations of glycerides. The peroxide value of sunflower oil is low composed to other oils and linseed oil has high peroxide value. The analytical procedure for the determination of the peroxides in oxidized fats, utilizes the oxidation of ferrous salts, stannous chloride etc. Normally used technique is volumetric method.

Acid value:
Sl. No sample 1 14.455 2 14.710 mean 14.562

Palm oil

Sunflower oil

0.112 0.781 0.444 0.975

0.118 0.725 0.443 0.920

0.115 0.753 0.4435 0.9475

Rice bran oil

palmolein

Blended oil

The acid value is a measure of the amount of fatty acids which have been liberated by hydrolysis from glyceride due to the presence of moisture, temperature or lipolytic enzyme lipase. According to PFA, the acid value of palm oil should not be more than 10.0, and the results got in this study is 14.5. The palm oil used for the analysis was a crude oil.

38

For sunflower oil, the results got are about 0.115, which has come within the range as stated in PFA. The given acid value range in PFA is that, it should not be more than 6.0. The results for Rice bran oil is 0.75 and PFA value prescribes not more than 0.5. For Palm olein as per the PFA statement acid value should not be more than 6.0, the results got is 0.44. And for the Blended oil, showed acid value 0.94.

Unsaponifiable matter:

Sl.no

Samples

Unsaponifiable matter 1.34% 3.217% 0.471% 1.10% 2.91%

For free fatty acid 1.30% 3.20% 0.37% 1.09% 2.89%

1 2 3 4 5

Sunflower oil Rice bran oil Palm oil Palm olein Blended oil

In an oil/fat, 90-98% is made up of triglycerides and the remaining portion of the oil is considered as unsap which cannot be saponified. Unsap matter present in each oil is specific to that oil. Unsap matter includes components like oryzanol, tocopherol, tocotrienol etc Each oil is particular in its effect; some of them are having nutraceutical effect beneficial to health present in unsap matter. Eg: Oryzanol has hypo cholesterolemic effect, suppressing the HMG Co-A reductase which is present uniquely in RBO. In this study unsaponifiable matter found in RBO is comparatively more than other oils tested. According to PFA statement unsap matter of Sunflower oil should be not more than 1.5%, for Rice bran oil it should be 3-5. and for palm oil not more than 1.4%, for palm 39

olein not more than 1.4%, for blended oil, if the oil is blended with the rice bran oil it can be more than 3 but less than 5.

Colour determination by Tintometer:

Sl. no 1. 2. 3. 4. 5. Samples Sunflower oil Palm oil Palm olein Rice bran oil Oil blend Red 0.2 21 0.5 0.6 2 Yellow 1.2 20 8 9 10 Blue 0.0 0.0 0.0 0.0 0.0 White 0.2 0.2 0.1 0.2 0.1 Y+5R 2.2 125 9.5 12.0 20.0

Lovibond colour determination is the colour rating of a fresh or used fat or oil, based on comparisons with the standard lovibond colour tubes. The equipment used is especially designed for viewing and comparing samples and standards under a specific light source. The results are reported as given values of yellow and red. In this study, the oils like Palm oil, Rice bran and Blended oil all have values more than 10. As sunflower oil is having very low value, it has better clarity than other oils like Palm oil, Rice bran, Blended oils etc. The level of lovibond Red colour is indicative of the quality and processing of the oil. But this is not an indicator of fry life.

40

Iodine value:

Sl.no

Iodine Value Samples 1 2 3

Mean

Rice bran oil

105.02

104.19

104.17

104.17

palmolein Palm oil Sunflower

60.54 53.79 128.16 91.43

61.10 54.00 128.03 90.48

61.44 -

61.02 53.895 128.09 90.95

oil Oil bland A

Iodine value is a measure of the amount of unsaturation (number of double bonds) present in an oil/fat. If the unsaturation is high, the amount of iodine consumed is also high. So iodine value is directly proportional to the amount of unsaturation. Iodine value of an oil or a fat is the number of grams of iodine absorbed by 100gms of fat, when determined by Wijs solution.

41

Oryzanol estimation by Spectrophotometric method:

Sl.no 1 2 Samples 1 Rice Bran oil Blended Oil 1.3817 0.950 Oryzanol% 2 1.415 0.9684 1.39 % 0.9592 % Mean

Rice bran oil contains Oryzanol, which is an antioxidant and it reduces the cholesterol level. Blended oil is the blend/mixture of two edible oil which is also containing Oryzanol This Oryzanol present in the oil has the capacity to reduce LDL in the body which is a bad lipo- protein and HDL in the body is good lipoprotein. -Oryzanol in rice Bran oil has been reported to lower serum cholesterol. Oryzanol identified in the rice bran consist of ferulic acid and triterpene derived compounds, which are combined by an ester bond. Cyclo artenyl Ferulate, 24-methalyne cycloartanyl ferulate and campesteryl ferulate are the three major components and account for 80% of -Oryzanol in rice bran. The nutritional function of -Oryzanol components may be related to their antioxidant property because of the ferulic acids structure . Ferulic acids is a phenolic acid antioxidant The Oryzanol in RBO was 1.39% and the Oryzanol percent of blended oil is 0.959%. The quantification was accomplished by internal normalization. The Oryzanol presence is estimated in all the oils by Spectrophotometry, using a UV-Visible Dual Beam Spectro photo meter, to determine the presence and concentration of nutraceutical components. The concentration of -Oryzanol was determined that 314.5nm, as suggested by Seetha Ramaiah and Prabhakar in 1986, using heptane as a solvent The Oryzanol percent is calculated using formula -Oryzanol%=O.D 100/ weight 358.9 At O.D at 314nm Wt= (oil (gms) 100 ml of hexane

42

Determination

of

fatty

acid

composition

by

Gas

Liquid

Chromatography:

Table of fatty acid composition of oils by Gas Liquid Chromatography:

Fatty acids 8:0 10:0 12:0 14:0 16:0 18:0 18:1 18:2 18:3 SFA MUFA PUFA

Palm oil nd nd nd 0.5 40.4 2.3 42.0 14.7 nd 43.2 42.0 14.7

Palm olein nd nd nd 0.8 44.0 1.1 40.9 13.1 nd 45.9 40.9 13.1

SNO nd nd nd nd 6.5 1.6 26.5 65.4 nd 8.1 26.5 65.4

CNO 0.1 2.9 51.3 23.6 10.7 2.4 7.1 1.9 Nd 91 7.1 1.9

RBO nd nd nd 0.2 20.5 0.1 40.6 38.5 nd 20.8 40.6 38.5

Oil blend A Nd nd nd 0.5 27.0 1.5 42.1 28.9 nd 29.0 42.1 28.9

43

Determination of fatty acid composition of Edible oils:

[mV]

2.940

10 8 Voltage 6 4 2 0

4.453

Nag 24-Feb-2009N CNO (R) 24.02.0915_46 - Detector 1

2.253

7.807

16.047

15.127

1.953

10 Time

15

18.810 20 [min.]
7 [min.]

Chromatogram of the fatty acid composition of Coconut oil

[mV] 4 Nag 24-Feb-2009N SNO 24.02.0914_35 - Detector 1 15 3.783

Voltage

10

2.770

0 0 1 2 3 4 Time 5 6

Chromatogram of the fatty acid composition of Sunflower Oil.

3.413

3.533

44

[mV] 2 2 .7 7 3 12 10 V o lta g e 8 6 3 .4 1 3 4 2 0 2 4 Time 6 8 [min.] 3 .7 7 7 1 3 Nag 24-Feb-2009N Palm oil 24.02.09 11_55 - Detector 1 4 3 .5 3 3
4

Chromatogram of the fatty acid composition of Palm oil.

[mV] Nag 24-Feb-2009N palm olein 24.02.0912_26 - Detector 1 3.410

15 Voltage

2.660

20

2 .4 1 3

0 0 2 4 Time 6 8 [min.]

Chromatogram of the fatty acid composition of Palm olein.

2.303

3.290

3.643

10

45

[mV] 2 4 3.420 25 2.677 24-Feb-2009N nutri R 24.02.0916_08 - Detector 1

20

Voltage

15

10 1 3 3 Time 3.297

0 0 1 2 4 5 6 7 [min.]

Chromatogram of the fatty acid composition of Oil Blend A

[mV] 10 8 V o lta g e 6 1 4 2 2 3 .4 9 0 4 3 .7 3 7 5 Nag 24-Feb-2009N RBO 24.02.0912_36 - Detector 1

2 .7 4 0

2.327

2 .3 5 7

3 .3 6 3

4 Time

3.663

8 [min.]

Chromatogram of the fatty acid composition of Rice Bran Oil

46

Palm oil: Fatty acid analysis of palm oil showed that it contain 43.2% saturated fatty acids and it contain almost equal amount of oleic (42%), linoleic acid content about 14.7%. According to literature, the fatty acid composition of palm oil is 14:0 is in the range 0.52.0, palmitic acid is 39.3-47.5, stearic acid is 3.5-6.0, oleic acid is 36.0-44.0, linoleic acid is 9.0-12.0. The oil used in this study is a crude oil. Palm olein oil: Fatty acid composition of palm olein showed that it contains saturated fatty acids about 45.9%. MUFA about 40.9%, and PUFA content about 13.1%. As per the literature, the fatty acid composition of palm olein is 14:0 should be in between 0.5-1.5, palmitic acid is 38.0-43.5, stearic acid is 3.5-5.0, oleic acid is 39.8-46.0, linoleic acid is 10.0-13.5. Sunflower oil: Sunflower oil fatty acid analysis showed that it contain saturated fatty acid about 8.1%and 91.9% of unsaturated, which includes MUFA about 26.5%, and PUFA about 65.4%. According to literature, sunflower oil is containing fatty acid composition like; palmitic acid 5.0-7.6, stearic acid 2.7-6.5, oleic acid 14.0-39.4, linoleic acid. 48.3-74.0, linolenic is ND-0.3. Coconut oil: The fatty acid composition of Coconut oil is 8:0 is 0.1, 10:0 2.9, 12:051.3, 14:0.. 23.6, 16:0.. 10.7, 18:0 2.4, 18:17.1, 18:2..1.9. i.e., SFA about 91%, MUFA about 7.1, and PUFA about 1.9. The standard range as the literature says for coconut oil is 8:0 4.6-10, 10:05.0-8.0, 12:0.. 45.1-53.2, 14:0 16.8-21.0, 16:0. 7.5-10.2, 18:02.0-4.0, 18:15.0-10.0, 18:2 1.0-2.5. Rice bran oil: The fatty acid analysis of Rice bran oil is found to be 14:0 is 0.2, 16:0 is 20.5, 18:0 is 0.1, 18:1 is 40.6, 18:2 is 38.5. i.e., SFA 20.8, MUFA about 40.6, and PUFA about 38.5%.

47

Blended oil: The fatty acid analysis of blended oil has shown that it contains about 29.0% of saturated fatty acid and unsaturated fatty acid about 71.0%. Which includes MUFA about 42.1% and PUFA about 28.9%. Fatty acid determination is the main criteria for the preparation of Blended oil.

48

CONCLUSIONS
In this study, Characteristics of edible oils like Sunflower oil, palm oil, Rice bran oil, palm olein and oil blend A are studied by measuring physical or analytical parameters like Free fatty acid, fatty acid composition, unsaponifiable matter, peroxide value, colour, oryzanol, acid value, Iodine value etc. By studying these physical parameters, we can conclude that the sunflower oil used has superior quality and the crude oils had more Free fatty acid percentage and peroxide value. The iodine value shows the presence of unsaturation in the oils. In this study, it was shown that Sunflower oil has more double bonds and Rice bran oil also has more unsaturation. Characterization of Edible oils gives us all the physical characteristics of oil, which tells and guides us about the quality of oil, which helps for marketing and for indicating safety of oil for human consumption.

49

BIBLIOGRAPHY:
1. Dr. S.K. Handoo and K.P. Sharma, Modern Technology in the oils and fat industry, Oil Technologies Association of India, New Delhi, 1-4 2. F.D. Gunstone, J L Hardwood and A J Dijkstra, The Lipid Handbook, 3rd edition, CRC Press (Taylor and Francis group), 2007; 3-6, 49-53, 66-67, 3. Harry .W. Lawson, Standards for Fats and Oils, AVI Publishing Company, INC, 1985; 4-9, 44-47. 4. J. Devine and P.N Williams, Chemistry and Technology of Edible Oils and Fats, Symposium Publication Division Press, 1961; 2-4. 5. Frank D Gunstone, Fred .B. Pandley, Lipid Technologies and Applications. 6. Ching Kuang Chow, Fatty acids in Food and their Health Implications, 3rd edition, CRC Press (Taylor and Francis group), 2008; 3-9. 7. Kirschenbauer, Fats and Oils, Reinhold Publishing Corporation, USA, 1948; 2629. 8. Frank. D. Gunstone, Modifying Lipids for use in Food, CRC Press, Wood Head Publishing Limited, England, 2006; 14-21, 130-131. 9. P.R. Yadav, Rajiv Tyagi, Lipid Biotechnology, Discovery Publishing House, New Delhi, 2006; 11-12. 10. Casimir .C. Akoh, Hand book of Functional Lipids. 11. K.T. Achaya, Oils and Fats Today, Nutrition and Health aspects of Fats, 2003; 5: 1-2.

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12. Reena MB, Lokesh BR. Hypolipidemic effect of oils with balanced amounts of fatty acids obtained by blending and interesterification of coconut oil with rice bran oil or sesame oil. J Agric Food Chem 2007; 55: 1046110469. 13. Bauro Yang, Natural Vitamin E: Activities and Sources Lipid Technology 2003; 15: 125-130. 14. Prevention of Food Adulteration Cases 1995 (Regd R.N. 23756/72 of Registrar of News Papers of India), Edited by Ms Swarn Bhatia Nijhawan, ILB Co.s Publication, 1, 51-59, 170-176. 15. Prevention of Food Adulteration Act 1954, 23rd Edition, Eastern Book Company, Lucknow, 2002; 201-213.

51