Experiment to Investigate the Action of Bile Salts on Fat in Milk

Planning

Variables

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Temperature – room temperature

Full cream milk concentration – 2.5 cm3 per test tube

Bile salts concentration – We will investigate this variable at different values

Lipase concentration – 0.5 cm3 per test tube

Sodium carbonate – 3.5 cm3 per test tube

Distilled water – This will vary according to the amount of bile salts in each tube

Phenolphthalein – 3 drops

We can easily change the concentrations of bile salts by changing the amount of bile salts in each test tube.

We can easily keep the temperature constant by doing all the experiments at the same time in the same part of the laboratory. Because we are going to work at room temperature this will keep the temperatures roughly similar. We have chosen room temperature because it is the easiest temperature to keep constant. We are using test tubes to carry out the experiment in so the values of the milk, lipase and sodium carbonate work best at this scale. We will use 3 drops of phenolphthalein as it is only an indicator and so does not need to be measured very accurately and 3 drops will show the colour change clearly enough.

Bile salts are an emulsifier; they break down large fat globules into smaller ones. Lipase breaks down fat molecules into glycerol and 3 fatty acids. Lipase can only reach the outer layer of fat so I think that by breaking up the fat globules, therefore increasing the surface area we will speed up the reaction. So as we increase the concentration of bile salts I think we will increase the rate of reaction.

Fat Molecule

Lipase breaks down these bonds

Glycerol and 3 Fatty Acids

Glycerol Fatty Acids

We will measure the milk, bile salts, lipase, sodium carbonate and distilled water using calibrated syringes as these are easiest to use to measure small but accurate amounts of liquids. We will use a pipette to measure the phenolphthalein we only need the amount in each test tube to be roughly similar and a pipette is quick and easy to use.

In test tube 1 we will put no bile salts and 0.5 cm3 of distilled water, in test tube 2 we will put 0.1 cm3 of bile salts and 0.4 cm3 of distilled water, in test tube 3 we will put 0.25 cm3 of bile salts and 0.25 cm3 of distilled water, in test tube 4 we will put 0.4 cm3 of bile salts and 0.1 cm3 of distilled water, in test tube 5 we will put 0.5 cm3 of bile salts and no distilled water. This is so that we get a range making it easier to spot trends and correlations from.

We will need:

* 5 test tubes

* A test tube rack

* A pipette

* Phenolphthalein

* 2 5cm3 syringes

* 3 1cm3 syringes

* Lipase

* Full cream milk

* Bile salts

* Distilled water

* Sodium carbonate

* A stopwatch

We are setting up 5 tests to run simultaneously. This is why we need the 5 test tubes and the test tube rack. We will use the pipette to add 3 drops of phenolphthalein. This does not need to be very accurate as it is only an indicator so we can use a pipette. Phenolphthalein is pink/purple in acid and clear in alkali. We will use the syringes to add the rest of the necessary liquids. The lipase is the enzyme that will break down the fat in the milk. We will use full cream milk as it has more fat content. The milk will all be taken from the same carton to ensure that the experiment is a fair test. The bile salts are emulsifiers; they will break down the fats and so increase the surface area of the fat droplets. We will use distilled water to keep concentrations in all tubes equal. Distilled water is purer than tap water and so better for this experiment. The sodium carbonate is added to make the solution alkaline to begin with so the colour change is obvious. We will use the stopwatch to find the amount of time it takes for the solutions in the test tubes to turn cloudy white.

Test Tube 1

Test Tube 2

Test Tube 3

Test Tube 4

Test Tube 5

Lipase

0.5 cm3

0.5 cm3

0.5 cm3

0.5 cm3

0.5 cm3

Milk

2.5 cm3

2.5 cm3

2.5 cm3

2.5 cm3

2.5 cm3

Sodium Carbonate

3.5 cm3

3.5 cm3

3.5 cm3

3.5 cm3

3.5 cm3

Bile Salts

0 cm3

0.1 cm3

0.25 cm3

0.4 cm3

0.5 cm3

Water

0.5 cm3

0.4 cm3

0.25 cm3

0.1 cm3

0 cm3

Phenolphthalein

3 drops

3 drops

3 drops

3 drops

3 drops

We add the lipase or the milk last to make sure that the reaction does not start before we want it to and then we shake the test tubes vigorously to thoroughly mix up the contents. We time until the test tubes lose their pink colour. This means that enough acid has been produced in breaking down the fat to acidify the solution.

Analysing

Time Taken (s)

Rate of Reaction (s-1)

Test Tube 1

920

0.001087

Test Tube 2

790

0.001266

Test Tube 3

760

0.001316

Test Tube 4

605

0.001653

Test Tube 5

570

0.001754

The graph shows positive correlation, as the volume of bile salts is increased, the rate of reaction increases. This agrees with my prediction because as the amount of bile salts increases, the fats should be emulsified more and quicker allowing the lipase to work faster. This happens because the fat globules are broken down faster and into smaller pieces. This increases the surface area of fat the lipase can reach, speeding up the reaction.

Evaluating

From the results obtained I would say that the method we used was fairly accurate, it gave a very strong positive correlation.

One way I could have improved this experiment was to repeat the experiment 2 more times and used the average results. This would have made the results more accurate as if there were any discrepancies in one set of results the other two sets would help to even out the average. Another way I could have improved the experiment was to use 10 test tubes and spread the amount of bile salts from nothing to 1 cm3, using distilled water to keep the concentrations of fat, lipase and sodium carbonate equal. This would give us a wider range and we could see if the rate of reaction levels off when the optimum concentration of bile salts is reached.

We could test to see how temperature affects the rate of reaction when lipase digests fat. All we would need to do would be to keep the amount of bile salts and distilled water fixed. I would keep the amount of bile salts fixed at 0.5 cm3 so that the reaction does not take too long. We would not need to add any distilled water as there is water contained in the milk. We could use 5 test tubes and test at 20�C, 30�C, 37�C, 45�C and 55�C. We would use water baths to regulate the temperature in each test tube.

However the disadvantages are that a water bath is not very accurate and takes a long time to warm up. Also it is hard to make sure that the contents of the test tubes are the same temperature as the water outside the test tubes.

I would expect the test tube at 37�C to react the fastest as this is body temperature and therefore the temperature that lipase normally works at in the small intestine. At lower temperatures the enzymes have less energy and so works slower and at higher temperatures some of the enzymes become denatured and so do not work therefore the reaction is slower. If I drew a graph of temperature versus rate of reaction with temperature on the horizontal axis and rate of reaction on the vertical axis I would expect the graph to rise steeply from 20�C to 37�C and then to decline rapidly from 37�C to 55�C.

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