Decay required practical

Aim: To investigate how temperature affects the rate of decay of fresh milk by measuring the time for lipase to lower the pH.

Variables

• Independent: Temperature of the water bath.
• Dependent: Time taken for the phenolphthalein indicator to turn from pink to colourless.
• Control: Volume and type of milk (same fat content); Volume and concentration of lipase; Volume of sodium carbonate solution; Number of drops of phenolphthalein

Equipment

• Full-fat milk
• Lipase solution
• Sodium carbonate solution
• Phenolphthalein indicator in a dropping bottle
• Test tubes and rack
• Beakers and water baths at a range of temperatures (e.g. 10, 20, 30, 40, 50 °C)
• Thermometer
• Measuring cylinders or syringes
• Stopwatch
• Glass rod

Method

1. Put five drops of phenolphthalein into a test tube.
2. Add 5 cm³ of milk and 7 cm³ of sodium carbonate solution; the mixture turns pink because it is alkaline.
3. Put the tube, together with a separate tube containing 1 cm³ of lipase, into a water bath at the first temperature.
4. Wait until both tubes reach the water bath temperature, checking with a thermometer.
5. Pour the lipase into the milk mixture, stir with a glass rod and start the stopwatch.
6. Stop the stopwatch when the pink colour disappears and record the time.
7. Repeat the experiment at each of the other temperatures.
8. Repeat the whole set three times and calculate a mean time for each temperature.

Safety

• Wear eye protection; sodium carbonate is an irritant and phenolphthalein is dissolved in flammable ethanol.
• Lipase can cause allergic reactions, so avoid skin contact and wash off spills.
• Do not drink the milk and take care with hot water baths.

Results and calculations

Record the time for the colour change at each temperature and calculate means. Work out rate = 1000 ÷ time (s^-1) and plot rate against temperature to show how temperature affects the rate of decay, identifying the temperature with the fastest rate.

Common mistakes and improvements

• The end point is hard to judge by eye; compare against a tube of milk mixture with no lipase, view against a white background, or use a pH probe or data logger.
• Tubes not at the right temperature before mixing; leave them in the water bath until a thermometer confirms the temperature.
• Water bath temperature drifting during the experiment; use an electric thermostatically controlled water bath.
• Too few temperatures to find the optimum clearly; test more temperatures, especially near the fastest rate.

Exam tips

• Explain what happens: lipase breaks down fats in milk into fatty acids and glycerol, the fatty acids lower the pH, and the indicator loses its pink colour.
• Explain the effect of temperature: rate increases as molecules gain kinetic energy and collide more, then falls above the optimum as lipase is denatured.
• Link the practical to decay by microorganisms and to why food is kept in fridges and freezers.
• Calculate rate from time, plot the graph, and suggest improvements such as using a pH meter for an objective end point.