(f) Simple experiment to investigate effect of temperature on the rate of decay of milk

Introduction

Milk naturally contains an enzyme called lipase and this breaks down fats into glycerol and fatty acids.

In the experiment you add extra lipase to speed up the decay process in milk.

You make the milk alkaline (pH >7) so that the fatty acids formed are neutralised raising the pH to >7 and this changes the colour of an indicator - this colour change is a visual marker for the reaction time.

In other words, as the milk breaks down (decays-decomposes) the pH of the milk decreases.

The experiment measures the relative rate of decay of fats in milk at different temperatures but you could adapt the experiment to keep the temperature constant and vary the concentration of lipase.

I'm not giving precise details of concentrations, and volumes quoted are just typical values.

What I do describe are the principles of the experiment and how to do it.

For an exam you need to appreciate all aspects of the experiment - design, what is needed (apparatus and chemicals), how to do it and how to process the results and draw conclusions.

 

Apparatus and chemicals needed

Thermostated water bath, test tubes, thermometer, 10 cm³ measuring cylinder

Milk,  lipase solution,  sodium carbonate solution,  phenolphthalein indicator solution

 

Investigation method (with added explanation)

For the experiment mixtures:

choose a constant volume and constant concentration of the lipase, sodium carbonate and phenolphthalein solutions,

so, for a fair test, the total volume in each experiment is the same and the only thing that varies is the temperature of the thermostated bath (see diagram).

Procedure:

Set the required temperature for the water bath and check it is constant with a thermometer.

Measure a volume of the lipase solution into a test tube, enough for several experiments.

Measure out a volume of milk in another 2nd test tube and add a few drops of phenolphthalein indicator to the milk - then add a measure of the sodium carbonate solution to this mixture.

NOTE: The solution should turn pink because the solution is alkaline and phenolphthalein turns pink above pH 10, but becomes colourless below pH 8.

Both test tubes are placed in the water bath and left to reach the ambient set temperature of the water bath.

When ready, using a calibrated dropping pipette, you measure 1 cm³ of the lipase into the milk mixture, shake gently to mix thoroughly (or stir with clean glass rod) and start the stopwatch.

The enzyme will immediately start to decompose the milk producing an acidic product (a fatty acid).

Stop the watch and measure the time taken for the pink colour of the indicator to become colourless as the alkaline sodium carbonate is neutralised by the fatty acid formed.

Repeat the experiment several times for each temperature and repeat the whole experiment at different temperatures e.g. 10, 15, 20, 25, 30, 35, 40, 45, 50^oC recording everything in a neat clear table - all the values should be recorded and the average time for each temperature too.

You can use ice cubes to cool the water bath to temperatures below room temperature, but its tricky to keep the temperature constant.

 

Results

The reciprocal of the time gives you a measure of the rate of the decay reaction e.g.

if the reaction time was 40 seconds, the rate is 1/40 = 0.025 s^-1

Using the average times and rates for each temperature, plot a graph of the rate versus temperature.

The rate of reaction is basically a measure of a fixed quantity of decay (unit of fatty acid formed) per unit time.

 

Conclusion

You should find the rate:

(i) increases at first (normal rate of reaction rule from chemistry),

(ii) goes through a maximum at the optimum temperature (typical of an enzyme)

(iii) and the rate falls away at higher temperatures as enzyme lipase protein becomes denatured.

For more details of enzyme theory see Enzymes - structure, functions, optimum conditions, investigation experiments, digestion  (gcse biology revision notes)

 

Extension to investigation

You can adapt the experiment to keep the temperature constant and vary the concentration of lipase.

You can choose a constant temperature close to the optimum e.g. 30^oC for the thermostated bath.

For the experiment mixtures:

choose a constant volume of lipase solution, BUT using different concentrations,

choose a constant concentration AND volume of sodium carbonate and phenolphthalein solution,

so, for a fair test, the total volume in each experiment is the same and the only thing that varies is the lipase concentration.

All the apparatus, chemicals and method are the same for the temperature varying experiment previously described.

Your results should look something like the graph above-right.

Initially the rate of milk decay should be proportional to the enzyme concentration - as long as everything else is kept constant.

 

The background chemistry to this investigation

lipases

Lipids, like many organic molecules, only contain the elements carbon, hydrogen and oxygen.

Lipase enzymes break down lipids like natural fats and oils (triglyceride esters) into glycerol and long chain fatty acids. Lipids are NOT polymers because they are not very long chain molecules.

Enzyme reaction word equation: lipid == lipase enzymes ==> glycerol + long chain fatty acids

The sort of molecular change that takes place - details you do may not need to know for GCSE level biology.

However in GCSE chemistry you would be expected to recognise the acidic carboxylic acid group -COOH, an important 'molecular feature' in understanding this decay experiment.

See also

Enzymes - structure, functions, optimum conditions, investigation experiments, digestion  (gcse biology)  and Enzymes and Biotechnology (gcse chemistry revision notes)

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Key points about an experiment to investigate the effect of temperature on the rate of decay

Based on the syllabus-specifications for students taking the AQA, Edexcel and OCR GCSE level biology examinations (~US grades 9-10).

Here’s some detailed points about a laboratory experiment to investigate the effect of temperature on the rate of milk decay

This experiment focuses on enzyme activity and microbial growth, which influence the breakdown of organic matter.

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Investigating the Effect of Temperature on Milk Decay

Aim: To determine how different temperatures affect the rate at which milk spoils due to microbial activity and enzyme action.

Hypothesis: Higher temperatures will accelerate milk decay due to increased enzyme activity and bacterial growth, while lower temperatures will slow down decay.

Apparatus and Materials:

• Fresh whole milk (as it contains fats and proteins that decay)

• Test tubes or small bottles

• pH indicator (e.g., universal indicator or bromothymol blue)

• Data logger or pH meter (optional, for accurate pH readings)

• Thermometers

• Water baths or incubators set to different temperatures (e.g., 5°C, 20°C, 40°C, 60°C)

• Cotton wool plugs or loose lids (to minimize contamination but allow air exchange)

• Labels and markers

• Gloves and disinfectant (for hygiene)

• Stopwatch or timer

• Milk decay observation chart (optional)

Method:

1. Prepare milk samples: Pour equal amounts of milk (e.g., 10 cm³) into separate test tubes.

2. Set up temperature conditions: Place the test tubes in different environments:

   • 5°C (refrigerator or ice bath)

   • 20°C (room temperature)

   • 40°C (warm water bath/incubator)

   • 60°C (hot water bath/incubator)

3. Measure initial pH: Use a pH meter or pH indicator to record the baseline acidity of milk.

4. Leave for observation: Monitor milk samples over 48–72 hours, recording pH changes and signs of decay every 6–12 hours.

5. Observe physical changes: Record changes in color, odour, texture (curdling, separation), and gas production.

6. Measure pH again: Compare final pH readings with initial values to determine the level of decay (milk becomes more acidic as bacteria produce lactic acid).

7. Record data systematically: Use a results table to document temperature, time intervals, pH changes, and observable characteristics.

8. Repeat for accuracy: Conduct multiple trials to ensure reliable results.

Expected Results:

• Lower temperatures (5°C): Minimal decay due to slowed bacterial growth.

• Room temperature (20°C): Moderate decay; some changes in odour and texture.

• Higher temperatures (40°C): Rapid decay; significant souring, curdling, and strong odour.

• Very high temperatures (60°C): Milk may not decay as rapidly due to enzyme denaturation, but bacterial growth may vary.

Analysis and Conclusion:

• Milk decays faster at warmer temperatures, as bacteria and enzymes break down proteins and fats, producing lactic acid.

• Refrigeration slows decay by inhibiting bacterial enzyme function.

• Extreme heat may denature enzymes, reducing microbial activity despite favorable conditions for bacterial growth.

Evaluation and Improvements:

• Use more precise pH measurement tools for accurate acidity monitoring.

• Test additional temperature ranges for a wider dataset.

• Compare different types of milk (e.g., whole vs. skimmed).

• Investigate microbial growth directly using agar plates.

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Extra notes - a variation on the above experiment using fruit waste

A variation of the experiment using fruit waste, such as banana peels or apple slices, to investigate the effect of temperature on organic decay.

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Investigating the Effect of Temperature on Fruit Waste Decay

Aim: To explore how different temperatures affect the rate at which fruit waste decomposes due to microbial activity and enzymatic action.

Hypothesis: Higher temperatures will speed up fruit decay by accelerating microbial growth and enzyme activity, while lower temperatures will slow decomposition.

Apparatus and Materials:

• Fruit waste (e.g., banana peels, apple slices, orange peels)

• Glass jars or plastic containers (with breathable covers)

• Thermometers

• Water baths or incubators for different temperature settings (e.g., 5°C, 20°C, 40°C, 60°C)

• pH meter or universal indicator (to measure acidity changes)

• Gloves and disinfectant (for handling)

• Camera or notebook (to track changes over time)

• Stopwatch or timer

Method:

1. Prepare fruit samples: Cut equal-sized portions of fruit waste and place them into separate containers.

2. Set temperature conditions: Store containers at different temperatures:

   • 5°C (refrigerator or ice bath)

   • 20°C (room temperature)

   • 40°C (warm water bath/incubator)

   • 60°C (hot water bath/incubator)

3. Measure initial pH: Check the fruit’s acidity level before the experiment.

4. Observe changes over time: Monitor samples every 6–12 hours for up to 5 days, recording changes in color, texture, odour, and mold growth.

5. Measure pH again: Compare final acidity levels, as microbial activity tends to increase acidity due to fermentation processes.

6. Record decay progression: Use a results table to document observations at each temperature.

Expected Results:

• Low temperatures (5°C): Slow decay, little mold growth, minimal odour.

• Room temperature (20°C): Moderate decay with visible softening and color change.

• Warm temperatures (40°C): Faster decomposition, mold growth appears sooner, strong fermentation odour.

• Very high temperatures (60°C): Fruit may dehydrate instead of decaying normally, possibly reducing microbial activity.

Analysis and Conclusion:

• Fruit waste decomposes faster at warmer temperatures due to increased enzyme action and microbial growth.

• Low temperatures slow decomposition, preserving fruit waste longer.

• Extreme heat may inhibit microbial decomposition, depending on moisture levels.

Evaluation and Improvements:

• Test different fruit types for comparison (e.g., citrus vs. berries).

• Include humidity control to assess its impact.

• Use bacterial cultures to quantify microbial activity.

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Summary of learning objectives and key words or phrases

Be able to design, describe and interpret experiments investigating the effect of temperature on decay rate of milk and other decomposers experiments including the apparatus, chemicals,  reagents required and analysing the observations - data - results - calculations and be able to draw conclusions from experimental results.

• Revise and practical work-investigations that helped develop skills and understanding which may have included the following (which should also be revised, helps in understanding 'how science works' and context examination questions):

  • designing and carrying out an investigation to measure the rate of decay of bread by, for example, exposing cubes of bread to air before placing them in sealed Petri dishes at different temperatures and/or different moisture levels,

  • investigating the rates of decay using containers (eg thermos flasks) full of grass clippings, one with disinfectant, one with dry grass, one with wet grass and one with a composting agent.

  • If the container is sealed, a thermometer or temperature probe can be placed through a cotton wool plug to monitor the temperature

  • potato decay competition, using fresh potatoes - you decide on the environmental conditions and the rate of decay is measured over a 2 week period,

  • using a sensor and data logger to investigate carbon dioxide levels during the decay process.

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