Determination of FatB.K.K.K. Jinadasa

GS/M.Sc./FOOD /3608/08 1/16/2010

Determination of Fat 2.0 Introduction Most fat in the diet is in the form of triglyceride (three fatty acids esterified to a glycerol molecule backbone). There are also non-glyceride components such as sterols, e.g. cholesterol. Fat is used to describe those lipids that are solid at the specified temperature. Lipids have at least three important functions in foods; culinary, physiological and nutritional. Lipids have the ability to carry odours and flavours and contribute to the palatability of meats, to the tenderness of baked products and to the richness and texture of ice cream. Dietary lipids represent the most compact chemical energy to man. They contain twice the caloric value of an equivalent of sugar. Lipids are usually defined as food components that are insoluble in water and that are soluble in organic solvents. Solvents used to extract lipids include ether, petroleum ether, acetone, chloroform, benzene, alcohols and water saturated butanol. Successful extraction requires that bonds between lipids and other compounds be broken so that the lipids are freed and solubilized. Generally such solubility is attained when polarities of the lipid and the solvent are similar. There are several methods for use for determination fat. Continuous Solvent Extraction - GOLDFISCH Extraction Solvent from a continuously boiling solvent source flows over the sample held in a sample thimble. Fat content is measured by weight loss of the sample or by weight of fat removed. Ethyl ether, petroleum ether, hexane, or methylene chloride are common solvents Extraction times range from 4-16 hrs Sample is weighed, mixed with sand to increase surface area, and dried in a forced air oven. Lipid is extracted by the solvent Solvent is removed by evaporation or under reduced pressure, then dried at 100C for 30 min. Semi-Continuous Solvent Extraction - SOXHLET Extraction Similar sample prep to Goldfisch method Fat is extracted, semi-continuously, with an organic solvent Sample is in contact with the solvent in the extraction chamber for 5-min, then siphons back to the boiling flask (see diagram) Extraction time: 5-6 drops per second (4 hr). 2-3 drops per second (16 hrs) Fat content is measured by weigh loss of sample or weight of fat removed The Bligh, Dyer and Folch Method Mojonnier or Roese Gottlieb Fat Extraction (MoJo) Low resolution NMR (nuclear magnetic resonance) X-Ray Absorption Dielectric Infrared (used a lot) Ultrasonic Colorimetric Density (for oil seeds) Page 2 of 5

Determination of Fat 2.1 Determination of free fat 2.1.1. Materials Soxhlet extraction apparatus (flask extractor, thimble and condenser) Heating water bath Heating mantle Weighing scale Drying oven Petroleum ether Pumice chips Sample sausages Soxhlet method

2.1.2. Procedure 5 g of finely ground sample was taken in a motor and anhydrous sodium sulphate of twice the weight of the sample was added into it. Then the mixture was ground until a free flowing powder was obtained. Then the powder was transferred to a thimble and sealed the end. Extraction thimble with the sample was placed in the soxhelt apparatus and fixed a previously dried and weighed round bottom flask. 200 mL of extracting solvent (petroleum ether) was added to the flask containing pumice chips. Then the Flask and the condenser were connected to the soxhelt extractor. Sample was allowed to reflux for about five hours. After the extraction flask was removed from the apparatus and kept in the water bath and then in the oven. Then the flask was cooled and weight was taken. 2.1.3. Results and calculation Weight of ground sample Weight of flask + chips Weight of flask + chips + fat = = = 5.0357 g 120.9318 g 121.3958 g

X Weight of the flask with fat and chips F Weight of the flask and chips W- Weight of the sample

% crude fat content of the sample

= (121.3958 120.9318) 100 5.0357 = 9.21 %

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Determination of Fat 2.1.4. Discussion Soxhlet extraction is one of the most commonly used methods for determination of lipids in dried foods. This is mainly because it is fairly simple to use. The main disadvantages of the technique are that a relatively dry sample is needed (to allow the solvent to penetrate), it is destructive, and it is time consuming.

2.2 Determination of total fat 2.2.1. Materials Beakers (100 mL) Majonnier flasks Hydrochloric acid Ethanol 2.2.2. Procedure Three beakers were taken and 2 g of sample was placed in each beaker. Then 2 mL of 95% ethyl alcohol and 10 mL of prepared HCl (by mixing 25 mL of Conc. HCl and 11 ml of water) were added to each beaker and thoroughly mixed. Then the beakers were placed in a water bath (70C - 80 C) with stirring them frequently for about 30 - 40 minutes till the contents of beakers turn blackish brown in colour. Beakers were taken out from the water bath and cooled to room temperature. Then 10 mL of ethyl alcohol was added and contents were transferred to three majonnier flasks. Then each beaker was washed with 25 mL of diethyl ether in three portions and added to respective flasks. All the flasks were stoppered and shook vigorously for about 1 minute. 25 mL of PET ether was added to each flask and shook for 1 minute. Then the flasks were allowed to stand undisturbed until the upper ether layer is clear. Upper ether layers were taken into clean dried weighed flasks and dried them in a water bath at 80 C until a constant weight obtained. 2.2.3. Results and calculation

Weight of flask(g) Weight of sample(g) Weight of flask + extracted fat (after drying) (g) Total fat

A 50.9437 2.0035 51.2765 16.61

B 44.2183 2.0000 44.5494 16.56

C 56.7882 2.0015 57.1200 16.58 Page 4 of 5

Determination of Fat

Average total fat

= (16.61 + 16.56 + 16.58) 3 = 16.58 %

2.2.4. Discussion In total fat determination both free fat and bound fat are taken into account. Here the bound lipids can be made free by dissolving the food sample in polar solvents. Dissolution of food can be achieved by acid. In this method the food material is heated in a water bath with hydrochloric acid in the sample to break down the protein, and fat separates as a layer on the top of the acid liquid. The protein dissolves in the acid and the separated fat can be extracted by shaking at least three times with diethyl ether or diethyl ether and light petroleum mixture. Shape of the majonnier flask aids in settling down all the coagulated protein at the bottom

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