Affect of concentration of calcium ions on the coagulation of milk

Introduction

Milk contains proteins, the majority of which being caseins; there are main types of casein molecules. Three of which readily precipitate with calcium. The fourth type of casein is kappa casein, which does not precipitate with calcium. Kappa casein keeps the other types of casein from coagulating, so the milk remains soluble. The rennin (enzyme) interacts with the kappa casein (substrate), breaking it down into para-kappa-casein (product). Now that the kappa casein has been broken down, the other casein molecules and new para-kappa-casein molecules precipitate with calcium, and milk, fat and some water combines forming a curd.

Prediction

As the concentration of calcium ions is increased, the time taken for flecks of curd to appear will decrease; therefore the rate of the reaction will increase. In this investigation, the rate of the reaction refers to how long it takes for the product to be formed from the enzyme and substrate, and the product to react with calcium. This will happen because if there is a greater concentration of calcium ions, there will be more collisions between the para-kappa-casein, other casein molecules and calcium, meaning the curd will be formed more quickly.

Variables/control factors

In order to be a fair and scientifically valid investigation, various factors need to be controlled:

In this investigation, rennin is the enzyme and the protein casein is the substrate. Increasing the concentration of the enzyme also increases the rate of an enzyme and substrate reaction. This aim of this investigation is to find the affects of the calcium concentration on the rate of coagulation, so the enzyme concentration must remain constant throughout, so only the concentration of calcium ions affects the results.

Temperature affects enzyme activity, so the temperature will also need to remain constant throughout the investigation.

The volumes of the milk, rennin, calcium chloride solution, sodium citrate solution will all remain constant.

The independent variable in this investigation will be the concentration of the calcium ions. By varying the concentration and analysing the results we can see if there is any link between the concentration of calcium ions and rate of coagulation.

The dependant variable in this investigation is the time taken for the milk to coagulate.

Risk assessment/safety

Potential hazards in this procedure ...

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Temperature affects enzyme activity, so the temperature will also need to remain constant throughout the investigation.

The volumes of the milk, rennin, calcium chloride solution, sodium citrate solution will all remain constant.

The dependant variable in this investigation is the time taken for the milk to coagulate.

Risk assessment/safety

Potential hazards in this procedure include the chemicals being used. Both calcium chloride and sodium citrate can act as an irritant to eyes and skin. They can also be harmful if swallowed or inhaled. Gloves and lab coats must be worn throughout the experiment. Another possible hazard is broken glass. Be careful with glassware, if any glass is broken don’t attempt to tidy it up yourself, ask a lab technician or teacher to do it for you. Tie up long hair and clean up any spillages immediately.

Method

The following method will be used as, from preliminary work it I found it to be the most accurate way to carry out the investigation. Instead of using a microscope slide to judge the time taken to coagulate, I decided it was more effective to put the milk solution into a boiling tube with a bung in. By doing this the solution can be easily mixed, and flecks of curd are very easy to observe on the inside of the glass as soon as the solutions are mixed. The water baths will be used and set at a temperature of 40ºC as this is around the optimum temperature for the enzyme rennin, and the milk, rennin solution, calcium chloride solutions and sodium citrate solution will be placed in and allowed to equilibrate to the temperature.

The only source of calcium ions needs to be from the calcium chloride, so the concentration will be as accurate as possible. Because there are already calcium ions in the milk, these will need to be removed by using sodium citrate.

The volumes of the solutions that will be used each time are as followed.

10cm3 of milk
1cm3 of rennin
1cm3 of sodium citrate solution
1cm3 of each concentration of calcium chloride solution

The procedure for each concentration of calcium chloride will be repeated thee times in order to determine an accurate average value of time.

Apparatus

I have chosen the following equipment in order to carry out this investigation as accurately as possible. The syringes enable the volumes to be measured precisely to 0.1cm3 and the water baths allow the temperature to remain constant throughout the experiment.

Rennin solution
Milk
Sodium citrate solution
1.0mol dm-3 calcium chloride solution
Hydrostatic water baths
Distilled water
1.00cm3 syringe
5.00cm3 syringe
10.0 cm3 syringe
Boiling tubes
Large beaker
Six 250cm3 beakers
Stopwatch

Preparation

We have been provided with only one concentration of calcium chloride, so this will need to be diluted to make different concentrations. The dilution factor and amount of calcium chloride required is calculated by the following equations:

Dilution factor = Original concentration

New Concentration

Volume of CaCl2 required = Dilute solution volume

Dilution factor

For example:

Dilution factor = Original concentration = 1.0 mol dm-3 = 2.5

New Concentration 0.4 mol dm-3

Volume of CaCl2 required = Dilute solution volume = 10cm3 = 4cm3

Dilution factor 2.5

Because the procedure is going to be repeated three times, with 1cm3 of each concentration of calcium chloride being used, 10cm3 of each concentration will be made up.

Listed in the table below, are the amounts of calcium chloride and distilled water required to make 10cm3 of the stated concentrations. The concentration 0.0 mol dm-3 will be included as a control.

For the first concentration, measure 10cm3 of calcium chloride and 0cm3 of distilled water into a small beaker. Label this 1.0 mol dm-3, and place it into the water bath (set at 40ºC) to equilibrate to the temperature.
Repeat step 1 for each concentration of calcium chloride, using suitable size syringes.
Pour about 200cm3 of milk and 20cm3 of rennin solution and sodium citrate solution into separate beakers, and also place these into the water bath and allow them to equilibrate.

Procedure

Measure 10cm3 of the milk from the water bath into a boiling tube, and add 1.0cm3 of the first concentration of calcium chloride and 1.0cm3 of sodium citrate to remove any calcium ions already present in the milk.
Using a syringe, add 1.0cm3 of rennin to the boiling tube and immediately start the stopwatch.
Swirl the tube to mix the solution, with as much as possible in the water to maintain a constant temperature. As soon as any flecks appear, stop the stopwatch and record the time in a table similar to the one below.
Pour the waste solutions into a large beaker for disposal.
Repeat steps 1-4 twice more with the same concentration and calculate the average time.
Repeat steps 1-5 with each concentration of calcium chloride.

Analysis

Upon completion of the experiment, once results have been recorded in the table, they can be plotted on a graph to see if they agree with the prediction. Plot a graph of average time (x-axis) against concentration (y-axis) to see if there is any trend or relationship between time and concentration of calcium ions.

References

http://www.vivo.colostate.edu/hbooks/pathphys/digestion/stomach/rennin.html

Title - Chymosin (Rennin) and the Coagulation of Milk Date visited - 6/05/08.

http://www.vegsoc.org/info/cheese.html

Title - Manufacture Date visited - 6/5/08.

Title - Properties of Rennin Coagulation Date visited - 8/5/08.