Testing food substances for the presence of lipids

Aim:

To establish which food substances contain lipids.

Hypothesis:

1. All diary products will contain lipids.

2. All animal tissue will contain lipids.

3. All plant storage organ products, i.e. seeds, will contain lipids.

Introduction:

Lipids are insoluble in water but soluble in organic solvents. Ethanol is an example of an organic polar solvent which will dissolve lipids to a limited extent. However ethanol is miscible with water so when a solution of lipid and ethanol is added to water the ethanol will combine preferentially to the water, leaving the ethanol to form tiny globules in the water. These globules then form a white emulsion.

Sudan III is a dye which binds preferentially to lipids and will stain them red. When lipids are in an aqueous suspension, the dye will colour them red whilst the water remains a light pink.

Method:

The presence of lipids was tested for in various food substances using the ethanol emulsion test, the grease spot test and the Sudan III test.

1. The liquid substances were all placed in 250cm3 beakers; vegetable oil, full fat milk, egg yolk and egg white. The watsits were ground using a mortar and pestle. All of the other substances were placed into petri dishes; the butter, margarine and lard were placed in directly; the bacon and cheese were thinly sliced using a scalpel and white tile and the chocolate was grated.

2. A boiling water bath should then be set up for use in the Sudan III test; A beaker containing 250cm3 of water should be set up on a tripod and gauze over a Bunsen burner. Once lit the air hole on the Bunsen burner should be opened so that it burns with a blue flame.

The Grease Spot Test

3. Two pieces of filter paper were folded into eight segments. Each segments was numbered to represent a particular food sample.

4. A small quantity of food material was then placed onto its segmented. If the material was a solid it was rubbed on to the filter paper. If the material was a liquid one drop was added using a 3cm3 plastic pipette. The filter paper was then left to dry before the results were recorded. A permanent, greasy translucent residue left on the paper indicted that lipids were present.

Sudan III Test

5. A spatula of the prepared food samples or a few drops of the liquid substances were added to labelled clean dry test tubes. The liquid substances were added using 3cm3 plastic pipettes.

6. 5cm3 of distilled water was then added to each test tube. This was then placed into the boiling water bath to allow for the food to break down. This caused the lipids to melt and enter the solution. The lipids then floated to the top surface of the water.

7. Tongs were then used to remove the test tubes from the water bath and to place them into a test tube rack.

8. 3 drops of Sudan III were then added to each test tube using a glass Pasteur pipette and they were carefully shaken.

9. The Sudan III stained any lipid present red. Final observations of the test tubes were then written into the results table.

Ethanol Emulsion Test

10. A small sample of the solid food materials and a few drops of the liquid material were placed into clean dry test tubes using either a spatula or 3cm3 plastic pipette.

11. 3cm3 of 100% ethanol was then added to each test tube using a pipette. The solution was then shaken vigorously.

12. The test tubes were then placed back into the test tube rack and left to allow for any solid material to settle.

13. A quantity of the supernatant liquid (ethanol containing dissolved lipids) was removed using a glass Pasteur pipette and added drop wise to half a test tube full of tap water.

14. The observations were then recorded in the results table. The presence of lipids was indicated by a white turbid suspension.

Safety Considerations:

Goggles were worn through out the experiment as Sudan III can be extremely harmful (it has known carcinogenic effects in humans). Inhalation of the solution was avoided as was contact with skin. After the experiment hands were washed to prevent any traces of Sudan III from being rubbed into eyes or contaminating food which would be eaten.

The ethanol was kept in a separate area to the Bunsen burners as it was extremely flammable. When it was not being used the bung was placed in the top of the container to prevent it from evaporating into the air which could potentially have been very dangerous.

After handling the raw bacon hands were washed to prevent any of the bacteria in it from being consumed and causing food poisoning.

(NB this table has lost its form)

Apparatus Used for Reason for Use

White tile Cutting cheese and bacon on Protects bench from scalpel

Scalpel Cutting cheese and bacon on Sharp

Glass Pasteur pipette Removing supernatant liquid from ethanol and food mixtureAdding Sudan III 1 drop º 0.1cm3 and glass wont get stained by Sudan III

3cm3 plastic graduated pipette Placing liquid material into test tubes Accuracy is not paramount. Can be disposed of if the solution gets stuck as they are relatively cheap.

Metal tongs Removing the test tubes from the boiling water Much safer than using hands

Bunsen burner Heating water Suitable piece of equipment

Glass Beaker- 250ml Containing boiling water Larger diameter so the water will heat up more quickly

Tripod and Gauze Holding Beaker Gives the beaker of boiling water a safe platform to rest on above the Bunsen flame

Test tubes (x33) Putting and heating the solution in Large surface area to volume ratio so contents heat more evenly and quickly during Sudan III test

Test tube rack Holding test tubes Secure place to put test tubes

Filter Paper Grease spot test Will rapidly absorb grease

Spatula Adding solid food materials to test tubes Smaller than diameter of test tube so it’s less likely that food will be dropped when transferring it

Petri dish Contains food samples Practical size making it easy to remove samples from

Mortar and Pestle Grinding up wotsits Offers safe and quick way to grind up food

Bottle of Distilled Water Adding to food material before Sudan III test Will not contaminate sample

Safety Goggles Protect eyes Sudan III may damage eyes

Results:

Food Appearance: Grease spot test Effects of adding ethanol to water Appearance after boiling and adding Sudan III dye

Vegetable oil Permanent translucent stain Turbid white emulsion formed on top of water Red globules floating on top of pale pink solution

Full fat milk Faint permanent translucent stain Turbid white emulsion formed on top of water Tiny red globules floating on top of white pink solution

Egg Yolk Opaque yellow stain Faint white emulsion Tiny red globules floating on top of white pink solution

Egg White Faint permanent translucent stain Clear solution produced Cloudy pink solution

Chocolate No translucent stain produced Turbid white emulsion formed on top of water Pink globules floating on surface

Bacon Permanent translucent stain Turbid white emulsion formed on top of water Pink globules floating on surface

Wotsits Permanent yellow translucent stain Turbid white emulsion formed on top of water Large layer of pink on surface

Cheese Permanent clear translucent stain Turbid white emulsion formed on top of water Distinct red globules floating on pink solution

Butter Permanent clear translucent stain Turbid white emulsion formed on top of water Distinct red globules floating over clear solution

Margarine Permanent clear translucent stain Turbid white emulsion formed on top of water Clear orangey red layer floating on surface

Lard Permanent clear translucent stain Turbid white emulsion formed on top of water Clear red layer floating on top of solution

Conclusions:

1. All diary products contained lipids, however within an egg the lipids were found to be concentrated in the yolk leaving relatively few in the white.

2. All products containing animal tissue contained lipids

3. All products containing plant storage organs contained lipids

Discussion:

All diary products contained lipids as they can be metabolised to yield twice as much energy, gram for gram compared to carbohydrates. For example the high saturated fat and cholesterol content in full fat milk is essential for young calves who need the energy to grow. It is also important to their survival as any fat which they gain can then be broken down in times of food shortage to make ATP in respiration. This happens because the fatty acids derived from triglycerides in the cytoplasm enter the mitochondria and become involved in the Krebs cycle through the beta oxidation pathway. The glycerol from the same triglycerides also becomes part of the Krebs cycle as they are made into pyruvate through glycolysis in the cytoplasm.

The Krebs cycle then yields ATP which can be broken down to supply energy for reactions through out the cell. The lipids will yield more energy than carbohydrates because there is a greater percentage of carbon and hydrogen in them where as carbohydrates have a greater percentage of oxygen, this affects the amount of energy for the metabolism of the molecule. A lipid can provided 38kJ/g. The fat content of milk is also important in the absorption of some of the vitamins as some need to be dissolved in lipids.

Cheese is made of milk which has been curdled causing it to have similar results. However the presence of lipid was more prominent in the grease spot test than with milk because the water in the milk has been removed in cheese so in a certain volume there will be much more fat present.

The tests on the eggs had different results as the two different parts tested had different functions. The yolk before an egg is fertilised is the nucleus of the single cell contains almost all of the lipid contained in the egg in the form of cholesterol, monounsaturated (44%), polyunsaturated (11%) and saturated (29%) fatty acids and the phospholipid lecithin. The yolk also contains half of the egg’s protein and vitamins D, A and E. The fat was detected in the Sudan III test as tiny red globules floated on top of a pink solution. This is because Sudan III combined preferentially to fat which had broken out of the yolk and floated to the surface of the water during heating The egg white formed a cloudy pink solution after the addition of Sudan III dye as it has an extremely low fat content and on heating in the boiling water chemical reactions would have occurred causing it to make the solution appear cloudy.

The main function of the egg white is to protect the egg yolk and provide the cell with some of the nutrients which it will need to grow as the egg white is the cytoplasm. Lipids are not needed in such abundance as in the yolk to fulfil this function. This is why it was shown to have no lipids present in them in both the ethanol emulsion test and the Sudan III test however the appearance of a faint permanent translucent stain in the grease spot test indicates that there must be a limited amount present. Only a faint yellow stain is produced during the grease spot test on egg yolk because as the water evaporates from the filter paper the plant pigment in the yolk, xanthophylls, colours the paper preventing the permanent translucent stain from being seen.

The basic ingredients of milk chocolate are sugar, cocoa butter, cocoa liquor, milk and vanilla. Both milk and cocoa butter have a high fat content however chocolate was not shown to contain lipids in the grease spot test. This is because it chocolate is solid at room temperature so grease cant escape and seep into the paper. It did however form a turbid white emulsion during the ethanol emulsion test. This is because some of the lipids dissolved into the ethanol as it is a polar solvent. The hydrocarbon tails on lipid molecules are strongly hydrophobic. When the ethanol and lipid solution is dropped into water the ethanol combines preferentially with water causing the lipid molecules to form a visible layer on the surface.

The cocoa bean contains fat so that it can be broken down to produce metabolic water for the embryo as it develops until it has an established root system by which it can gain water from. For similar reasons vegetable oils contain esters of glycerol and a varying blend of fatty acids. This is because they are often derived from oily seeds.

Butter, a diary product was found to contain lipids in all three food test. This is because it made by churning fresh cream. It consists of water and milk proteins in a matrix of fat. Most butters contain about 80%fat. The results of butter, margarine and lard, despite diverse origins were mostly the same as they all have similar physical properties and a high fat content. Margarine is often used as a substitute for butter as it contains less saturated fat. Its made from hydrogenated vegetable oil which has been passed over a nickel catalyst. It can also be made by passign . This is much healthier than lard which is traditionally made from animal fat produced from the fatty or otherwise unusable parts of pig carcasses.

Wotsits were shown to contain a large amount of fat in all three food tests. This is because they are made from heavily processed maize, vegetable oil, salt and cheese powder. Most of the 6.3g of fat in the 21g bag comes from the vegetable oil. The yellow tinge to the translucent stain in the grease spot test is from the cheese powder.

Bacon and lard are the two animal products tested. These products were both shown to contain fat. This is because fat is needed with in mammalian bodies to protect internal organs from sharp impacts. The fat is also stored to insulate animals for example the pig, which the bacon has come from would have a layer of subcutaneous fat beneath its skin to decrease the loss of metabolic heat. Fat would also be found through out a pig as a way of storing food which can later be broken down to give energy in times of famine.

Although the three tests carried out on the food are accurate in showing whether lipid is present they are limited in their usefulness as they do not differentiate between the different types of lipid present or give a quantitative analysis of the samples.