Determining the effect of changing the concentration of calcium ions on the rate of coagulation of milk

Coagulation is essentially the formation of a gel by destabilizing the casein micelles causing them to aggregate and form a network which partially immobilizes the water and traps the fat globules in the newly formed matrix. This may be accomplished with enzymes. * Ref 1

Rennin is an enzyme produced in the stomach of mammals, particularly young ones, to curdle milk, preventing the rapid flow through the digestive system without time for digesting the available proteins. The casein proteins found in milk are the substrate. This will be the enzyme used to coagulate milk in my experiment.

Calcium activates rennin through allosteric regulation; the active site of rennin is not naturally specific to the shape of the substrate in milk, but with Calcium as an allosteric activator, the active site is altered to the correct shape. This allows the reaction to be catalysed.

Prediction

I predict that as the concentration of calcium ions increase, the rate of coagulation will also increase. I suspect this because calcium ions activate the enzyme, and so a lack of calcium ions will cause less catalytic activity, thus lowering the rate of reaction.

Preliminary Work

I performed a small-scale version of the experiment to see the general outcome. In this experiment, I removed all the calcium ions from milk with sodium citrate, and then mixed with the enzyme rennin and calcium chloride at 1.0 mol dm-3 concentration on a slide. The time taken for the first flecks of curd, the clear sign of coagulation, took place within seconds making it hard to accurately judge the time. This was originally in a water bath at 38.6˚C (standard bovine body temperature * Ref 2), a pH level of 4.0 that was attained by using pH buffer tablets.

Factors kept constant to ensure fair testing

Type of milk – I shall use full fat cows milk; full fat because it will produce the thickest curd possible as the majority of proteins are in it, and be easiest to recognise.

pH of solution – In young calves, rennin is produced in the abomasums, where the pH level ranges from 3.8 to 4.2. * Ref 3

Temperature – As I have found that at 38.6˚C coagulation only takes a few seconds, I shall lower this to 20˚C for my final experiment, so that the coagulation may take longer to occur and ...

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Factors kept constant to ensure fair testing

Type of milk – I shall use full fat cows milk; full fat because it will produce the thickest curd possible as the majority of proteins are in it, and be easiest to recognise.

pH of solution – In young calves, rennin is produced in the abomasums, where the pH level ranges from 3.8 to 4.2. * Ref 3

Temperature – As I have found that at 38.6˚C coagulation only takes a few seconds, I shall lower this to 20˚C for my final experiment, so that the coagulation may take longer to occur and be easier to distinguish. * Ref 4

Volume of milk, enzyme and CaCl2 solution.

By adding sodium citrate to milk, the calcium ions are removed from the milk, so even in the presence of rennin it would not coagulate. In doing so, I can fully control the concentration of calcium ions present in the milk, by adding the afterwards in the form of Calcium Chloride, a 1.0 mol dm-3 solution. I will dilute the CaCl2 with distilled water to achieve the desired concentration of calcium ions. By measuring the time taken for coagulation to begin, I can work out the rate and determine which concentration of calcium ions gives the fastest rate.

When I add the sodium citrate, I will shake well and leave for 10 minutes to allow for all the calcium ions to be taken up, forming the insoluble precipitate calcium citrate; this can be filtered out and removed from the solution, but as the calcium ions are no longer available to the enzyme, it isn’t necessary.

I will also include a reaction between the milk, devoid of calcium ions, and the rennin with distilled water instead of calcium chloride; essentially, this is my control.

Precision

By repeating the experiment, I can produce average results and avoid anomalies.

I am using the pH level of a bovine abomasums, and was intending to use the temperature, due to the fact this is where the enzyme is secreted and so these are the optimum conditions for it to work – by replicating this, the enzyme should work efficiently.

By using a control, it will show that no instant coagulation occurs without the presence of calcium ions.

Keeping all other factors aside from CaCl2 concentration constant prevents unwanted anomalous results.

Safety Precautions

Calcium Chloride is an irritant, and therefore when handling the solution and performing the experiment, goggles should be worn and care should be taken so that it does not come into contact with the skin.

Apparatus

1x 50 ml Beaker 30 cm3 Whole Milk

3x 5 ml syringe 3 cm3 Sodium Citrate

1x 10 ml syringe pH 4 Buffer tablet

36x test tube 20 cm3 Rennin

Water bath 30 cm3 CaCl2 at 1.0 mol dm-3

3x 1 ml pipette 30 cm3 distilled water

5x microscope slide

1x stopwatch

1x thermometer

Method

Measure out 30 cm3 of whole milk into a beaker, and add 3 cm3 of sodium citrate. Stir well and leave for 10 minutes.
Add pH buffer tablet to milk to attain a pH level of 4.0.
Add 20 cm3 of rennin to a test tube.
Distribute CaCl2 1.0 mol dm-3 and distilled water into 6 test tubes, as directed below:

Test tube A – 10 cm3 of CaCl2, 0 cm3 of water.

Test tube B – 8 cm3 of CaCl2, 2 cm3 of water.

Test tube C – 6 cm3 of CaCl2, 4 cm3 of water.

Test tube D – 4 cm3 of CaCl2, 6 cm3 of water.

Test tube E – 2 cm3 of CaCl2, 8 cm3 of water.

Test tube F – 0 cm3 of CaCl2, 10 cm3 of water.

Place the milk, enzyme and CaCl2 solutions into a water bath at 20˚C and allow all solutions to reach that temperature.
Using a graduated pipette, add 2 ml of milk, 2 ml of rennin and 2 ml of CaCl2 from test tube A to a clean test tube. Shake well and start the timer.
Every 5 seconds, use the pipette to take one drop and place it on a microscope slide to check for curd flecks. When they appear, stop the timer.
Follow stages 1-4 for each CaCl2 solution.
Repeat 4 times.

I would record the results gained in the table displayed below, and they can also be plotted in a graph to show the correlation; I have included my estimation of the graph, showing the increase in rate, as the concentration of calcium ions increases.