In 1857 Louis Pasteur reported that lactic fermentation, the change that sours milk, is caused by a living microorganism turning sugar into lactic acid. Many chemists of the time thought fermentation and decay were purely chemical. Around 1860 his swan-neck flask experiments showed that boiled broth stayed unspoiled as long as microorganisms from the air couldn't reach it. The popular version says the flasks settled the argument at once; in fact the debate carried on for years. But the idea at the heart of this practical comes from Pasteur's work on souring: decay is done by living things, and you can follow it by the acid they make.
This lesson covers the decay required practical for AQA GCSE Biology Topic 7 (Ecology): investigate the effect of temperature on the rate of decay of fresh milk by measuring pH change. It's a Biology-only practical, so it isn't part of Combined Science.
Decay and decomposers
Decomposers, mainly bacteria and fungi, break down dead material and waste. As they respire they return carbon to the atmosphere as carbon dioxide, and they return mineral ions to the soil. Three factors control how fast this happens:
- Temperature: warmth speeds up decomposers' enzymes, but too much heat denatures them.
- Water: decomposers need it to grow, so dry material decays slowly.
- Oxygen: most decomposers respire aerobically, so decay is faster with oxygen.
Gardeners make compost by keeping waste warm, moist and aerated, then use it as a natural fertiliser. Without oxygen, anaerobic decay produces methane, and biogas generators collect that methane to burn as a fuel.
Why the practical uses lipase
Milk left to decay naturally turns sour as bacteria make lactic acid, but that takes far longer than a lesson. AQA's handbook method speeds things up with the enzyme lipase, so the practical is a model of decay. Lipase breaks down fat into fatty acids and glycerol, and the fatty acids lower the pH.
The method
AQA's handbook method, in short:
- Put 5 cm³ of lipase solution in a boiling tube labelled "lipase".
- In a tube labelled "milk", add five drops of cresol red, 5 cm³ of milk and 7 cm³ of sodium carbonate solution. The sodium carbonate makes it alkaline, so the mixture is purple.
- Stand both tubes in a water bath at your chosen temperature and wait until they reach it.
- Add 1 cm³ of lipase to the milk tube and start the stop clock straight away.
- Stir, and record the time for the mixture to turn yellow.
- Repeat at other temperatures. AQA suggests at least five.
Cresol red is purple at about pH 8.8 and turns yellow when the pH drops below about 7.2. An earlier AQA version used phenolphthalein, which goes from pink to colourless. A pH probe and data logger can replace the indicator, so no one has to judge a colour by eye.
Variables and results
- Independent variable: temperature.
- Dependent variable: time for the colour change.
- Control variables: the volumes of milk, sodium carbonate, lipase and indicator, the lipase concentration, the type of milk and the end-point colour.
Turn each time into a rate with rate = 1 ÷ time: a shorter time means a faster rate. In AQA's technician trial, the mixture took about six minutes to change at 20 °C but under half a minute at 35 °C and 45 °C. At 55 °C and 65 °C it hadn't changed after ten minutes, most likely because the lipase had denatured.
Safety
Take care with hot water from the kettle, and follow your teacher's risk assessment. Never taste anything in a lab.
Common exam mistakes
- Saying the pH rises. It falls, because acids are made.
- Saying lipase decays milk in nature. Natural decay is done by microorganisms; lipase is the model.
- Mixing up time and rate. A longer time means a slower rate.
- Saying the enzyme is "killed". Enzymes are molecules: they are denatured.
- Forgetting to warm both tubes first. Otherwise the reaction starts below the chosen temperature.
Frequently asked questions
What is the decay required practical in GCSE Biology?
You add lipase to an alkaline mixture of milk and indicator at different temperatures and time how long the pH takes to fall. It's AQA's required practical 10, for Biology only.
Why does the pH go down in the decay practical?
Lipase breaks down fat in the milk into fatty acids, which lower the pH. In natural decay, bacteria make lactic acid instead.
Why is sodium carbonate added to the milk?
It makes the mixture alkaline at the start, so the indicator begins purple and you can see it change as acid is made.
What factors affect the rate of decay?
Temperature, water and the availability of oxygen. Warm, moist, well-aerated conditions give the fastest decay.
Try it
Say each answer before you flip the card. Then put yourself in Pasteur's place: you've shown that milk sours because of living organisms making acid. What would you have tested next?
Braineos is not affiliated with or endorsed by AQA.