Investigation to find out what effect changing the concentration of calcium ions has on the rate of coagulation of milk

Na3C6H5O7(aq)           +      Ca2+(aq)     →     NaCaC6H5O7(s)         +      2Na+(aq)       

Inorganic ions such as metal ions can act as cofactors. The ion combines with either the enzyme or the substrate to reduce the activation energy. These ions are called activators. Activators make the enzyme substrate complex form more easily. Ca ions are a cofactor for rennin, so they assist in forming the enzyme-substrate complex by moulding either the enzyme or substrate into a more suitable shape; in this case they mould the enzyme rennin.

Prediction

Calcium ions increase the rate at which chymosin catalyses the coagulation reaction and hydrolyses the peptide bond, therefore if there is more of it, the k-casein will be in shorter supply and so the micelles will be less stable. As a result, milk will to coagulate more, in a shorter time. Using this knowledge and the above information, I predict that the more Calcium ions present, the faster the rate of coagulation will be.

Plan

In order to observe how the concentration of calcium ions affects the rate of coagulation, I will observe how long it takes for flecks of curd to first appear on a microscope slide dipped into a sample of milk.

Safety

Preliminary Experiments

Results

Precision

1. I was checking for flecks after every 30 seconds. This was too long a time frame, as flecks could have easily formed between the 30 second intervals. I will therefore reduce the time interval to every 5 seconds, resulting in more accurate timing. Also I will stop timing after 3 minutes, as most of the concentrations would have already reacted.
2. The readings were inconsistent as all the beakers were at different stages of coagulation. Therefore it was difficult to find an exact point of coagulation.
3. The temperature of the milk was cold (11°C), which may have resulted in a slower rate of coagulation as rennin is found in the stomach of young calves, and operates at an optimum temperature of 37°C. To solve this problem I will use a water bath to regulate the temperature and also measure the temperature of the milk at regular intervals to ensure it is consistent.  

Variables

To ensure a fair test I must control several variables. These are:

• Use the same volume of milk throughout – 10cm3
• Use the same volume of sodium citrate (1cm3) and rennin (1cm3)
• Ensure the temperature throughout the experiment (37°C)
• Use the same type of milk – i.e. full fat, semi-skimmed

Apparatus

• Small beakers
• Test tubes and bungs
• 10cm3 Syringe
• 1cm3  Syringe
• Stop watch
• Microscope slides
• Full fat milk
• Rennin
• Calcium chloride
• Distilled water
• Sodium citrate
• Water bath (37°C)

Method

1. Using a syringe, add 10cm3 of milk to each of the 5 beakers
2. Again, using a syringe, add 1cm3 of Sodium Citrate to each of the 5 beakers now containing milk, this is to remove the calcium ions already present in the milk, ensuring a fair test.
3. Place the beakers in a water bath at 37°C to regulate the temperature.
4. Now prepare the Calcium Chloride solution. Using a syringe, add 10cm3 of calcium chloride to a test tube. This is 1M. To another test tube, add 7cm3 calcium chloride and 3cm3 water, to make a 0.75M concentration. Repeat this process for 0.5M, 0.25M and 0M.

5 concentrations have now been made.

5. Add the contents of one tube to a beaker and repeat for all 5 tubes.
6. Use a syringe to add 1 cm3 of rennin to the beaker containing 1M CaCl2. At the same time start the stop watch.
7. Dip the microscope slide into the beaker every 5 seconds until flecks of curd appear. At first signs of coagulation (if flecks do not appear after 180seconds, consider it as no reaction), stop the stopwatch and note the time. Carry out the same procedure for all the other concentrations.
8. Repeat the entire experiment a further 2 times to ensure the results are consistent and reliable. Finally calculate the mean time for each concentration.
9. Calculate the rate of coagulation for each concentration, by using:

Rate of Reaction =  

Analysis of results

Precision, Reliability and Validity

The main source of errors, are human errors, during timing and determining a point of coagulation for the milk samples. The timing should be accurate due to the use of a stopwatch. However having to decide on what is and what isn’t coagulation is difficult. Therefore, to ensure a higher degree of accuracy when observing the flecks and finding a definite point of coagulation, a microscope could be used to compare the flecks and determine whether or not they definitely started coagulating.  

The experiment was repeated twice and the mean was calculated. This ensures that the results obtained are as reliable as possible.

The temperature was maintained at 37°C; as rennin is an enzyme found in the stomach of young mammals, where the optimum temperature is 37°C. Maintaining this temperature ensured that rennin was provided with ideal conditions to maximise its reaction rate.

There my however be other factors that influence the rate of coagulation, such as ions. It is almost impossible to tell if all the Calcium ions have been removed. Also Chloride and sodium ions have an effect on rennin activity; therefore the presence of these may cause inaccuracies.

Boiled rennin could be used as a control, as boiling would remove all the ions and other impurities, then the samples collected could be compared to the control which may make it easier to find a coagulation point.

The rate of coagulation can be represented on the following chart.

Table to show how the rate of coagulation varies as CaCl2 concentration varies

Secondary Sources

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