What effect changing the concentration of calcium ions has on the rate of coagulation of milk

Aim: Plan an investigation to find out what effect changing the concentration of calcium ions has on the rate of coagulation of milk.

Prediction: I predict that as the concentrations of calcium ions is increased; the rate of milk coagulation will also increase, but at a certain point the calcium ions will stop affecting the rate of coagulation, as the amount of Rennin the calcium can bind to will be kept constant.

Explanation: Rennin is an enzyme; enzymes help lower the activation energy required for a reaction, but remain unchanged at the end. They work by forming an Enzyme Substrate complex, in which the substrate binds with the active site of the enzyme and the substrate will be broken down. The rate at which an enzyme works depends mainly on 4 factors:

* Enzyme Concentration – The more of the enzyme, the more molecules that can be broken down (up to a certain point).

* Substrate concentration – As this increases the initial rate of creation increases up to a certain point, called Vmax, at which the enzyme is working at its fastest possible rate.

* Temperature – The greater the temperature the more energy both the substrate and enzymes have, and so there is a higher probability of forming an enzyme-substrate complex, however, if the temperature is too high, the enzyme will be denatured and can no longer function. As Rennin is used in the body, it should have an optimum temperature around 37oC.

* pH – pH is a measure of hydrogen ions, which can interact with the r group of enzymes and so affect there structure, most enzymes work best around a neutral pH.

Milk proteins are made up of 80% [1] molecules called casein which can be split into 4 groups, Alpha-s1, Alpha-s2, Beta and Kappa. Unlike the other proteins K-Caesin is soluble, meaning that it will not form a solid substance and therefore will not coagulate. Rennin is an enzyme which converts K-Caesin into an insoluble molecule called para-kappa-casein (or paracasein) by breaking the peptide bond between the phenylalanine and methionine in K-Caesin[2], and in this way will allow milk to form a solid curd, this is coagulation. Calcium ions are enzyme activators, which will join to rennin and allow it to do more work, usually by making the active site of the enzyme fit the substrate better, or by lowering the activation energy required for the reaction.

Apparatus: 0.1 moldm-3 Rennin Solution, Milk, Sodium citrate solution, 1moldm-3 calcium chloride solution, measuring cylinder, glass beakers, syringe, distilled water (to prevent impurities such as Fluorine), light microscope and slides, stop clock.

Diagram:

Method:

* Setup apparatus as shown in the diagram.

* Add 25cm3 of milk to a beaker, with 2.5cm3 of Sodium Citrate.

* Add 25cm3 of milk to a beaker, with 2.5cm3 of Sodium Citrate, and to this add 3cm3 of the rennin, and add 3cm3 of distilled water; this will be used as a control.

* Heat the milk solution and rennin, separately to 37oC.

* Simultaneously start the stop clock, and add 3cm3 of the rennin solution and 3cm3 of Calcium Chloride at the selected concentration, maintaining the temperature throughout 37oC.

* Dip the slide in the milk at 5 second intervals until flecks are observed, record the time taken.

* Repeat the experiment with same concentration of calcium ions 3 times and calculate the average time taken, record the results in a table like the one shown below.

Calcium Chloride Concentration

Coagulation time – 1

Coagulation time – 2

Coagulation time – 3

Coagulation time – Average

0 – Water

0.2 moldm-3

0.4 moldm-3

0.6 moldm-3

0.8 moldm-3

1.0 moldm-3

To make sure this is a fair test we will use the same amount of solutions (the control variables) which are:

* Rennin Solution – 3 cm3

* Milk – 25 cm3

* Sodium Citrate – 5 cm3

* Calcium Chloride – 3 cm3

And only change the dependant variable, which is the concentration of Calcium ions.

Accuracy:

* To make the results as accurate as possible, we will repeat the experiment 3 times per concentration of calcium ions and take the average.

* We will measure all substances at eye level to prevent a parallax error, keeping the results as precise as possible.

* The light microscope will be pre-adjusted before we start the experiment, so the milk solution can be viewed straight away.

* Graduated pipettes will be used instead of syringes to keep all measurements as precise as possible, although this may be affected by the tolerance of the pipette used: “The single volume pipette is usually more accurate, with an error of � 0.1 or 0.2 mL” [5].

* The accuracy may also be affected by the purity/grade of solutions used e.g. “Calcium Chloride Powder Anhydrous: Purity: 90%min – 92%min – 94%min”[4]

* Using water as a control to see if the milk naturally coagulates at a fast rate/at all.

Safety:

* As with any experiment, goggles and a lab coat will be worn at all times.

* Care must be taken with all chemicals used; Calcium Chloride and Sodium Citrate can cause irritation to eyes and skin [5] [6].

* The experiment will be done in a well ventilated area to prevent inhalation of any chemicals.

* Care must be taken when using glass equipment such as graduated pipettes and microscope slides, if any glass is broken it must be cleaned away immediately.

Preliminary Work

In the preliminary work, I first test the coagulation rate using:

* 50cm3 of milk.

* 3cm3 of 0.1 moldm-3 Rennin solution.

* 3cm3 of 0.2 moldm-3 Calcium Chloride solution.

The mixture did not coagulate after 3 minutes and so we halved the amount of milk to 25 cm3 and used 1moldm3 Calcium Chloride solution, this coagulated after 64 seconds, because of this I will now be using cm3 of milk, and to speed up the rate at which Rennin works, we will heat the solution to 37oC in a water bath, as well as this I found the flecks are easily noticeable with the eye, and so a microscope is not necessary.