Required Practicals: Food Tests and Amylase Activity

Describe the digestive system as an organ system for digestion and absorption.
Relate enzyme activity to metabolism and environmental factors like temperature and pH.
Use the 'lock and key' theory to explain enzyme action.
Recall the sites of production and action for amylase, proteases, and lipases.
Describe the breakdown of carbohydrates, proteins, and lipids into simple sugars, amino acids, and fatty acids/glycerol.
Explain the role of bile in neutralising stomach acid and emulsifying fats.

What qualitative food tests show

Food contains mixtures of biological molecules. A qualitative test identifies whether a type of molecule is present from an observable result, such as a colour change or a cloudy emulsion. It does not by itself give an exact concentration.

For a solid food, place a small sample in a mortar, add distilled water and grind it with a pestle. Stir the mixture and filter it if a clear food solution is needed. Divide the preparation between labelled test tubes so that each test uses a fresh portion. Use clean apparatus to prevent one reagent or food contaminating another.

Known positive and negative samples can be tested alongside an unknown. These controls show what positive and negative results should look like and help reveal a faulty reagent or method.

Benedict’s test for reducing sugars

Add Benedict’s solution to the food solution and heat the tube in a hot water bath. Do not heat the test tube directly in a flame. Benedict’s solution begins blue. If a reducing sugar is present, heating produces a change through green, yellow or orange to a brick-red precipitate, depending partly on how much reducing sugar is present. If none is detected, the solution remains blue.

Glucose, lactose and maltose are reducing sugars. Sucrose is a non-reducing sugar, so it does not give a positive Benedict’s test under these conditions. It is therefore more accurate to call this a test for reducing sugars, rather than for all sugars.

Iodine test for starch

Place the food sample in a well of a spotting tile or in a test tube and add a few drops of iodine solution. Iodine is orange-brown when starch is absent but becomes blue-black when starch is present. Heating is not needed.

This same test is later used to follow the disappearance of starch as amylase digests it.

Biuret test for protein

Add Biuret reagent to a fresh portion of the food solution and mix it. A negative result remains blue; a lilac or purple colour shows that protein is present. Qualitative Biuret reagent may be supplied as two solutions that are added in sequence, so the stated laboratory method should be followed.

Emulsion test for lipids

Add ethanol to a fresh food sample and shake it so that any lipid dissolves in the ethanol. Add water and mix. A milky-white or cloudy emulsion is a positive result for lipid; a clear mixture is negative.

Ethanol is highly flammable, so it must be kept away from naked flames and other ignition sources. Eye protection should be worn throughout the food tests, hot test tubes should be handled safely, and the laboratory’s risk assessment must be followed for all reagents.

Comparing food samples fairly

When comparing foods, use the same mass or volume of food, the same volume and concentration of reagent, and the same heating conditions. Label every tube clearly. Differences in sample size, heating time or reagent volume could otherwise produce different appearances even when the foods contain similar substances.

The tests identify broad groups of biological molecules, not a particular food. One food may give several positive results because foods are mixtures. For example, the presence of starch in a prepared food does not show that starch is its only nutrient.

Using amylase to investigate the effect of pH

Amylase catalyses the breakdown of starch into sugars. As the reaction proceeds, the amount of starch falls. Iodine therefore provides a way to detect when the starch has been completely digested: samples change iodine blue-black while starch remains, but leave it orange-brown once no starch is detected.

The independent variable is the pH, established using labelled buffer solutions. The dependent measurement is the time taken for starch to disappear. Temperature must be controlled with a water bath or electric heater because temperature also affects enzyme activity. The volumes and concentrations of amylase, starch and buffer must also remain constant.

Continuous-sampling method

Put one drop of iodine solution into each of a row of wells on a spotting tile.
Set a water bath to a constant temperature, commonly $35\ ^\circ\mathrm{C}$ for this method.
Measure fixed volumes of starch solution and one chosen buffer into a labelled tube. Measure a fixed volume of amylase separately.
Allow the solutions to reach the water-bath temperature. Add the amylase to the starch-buffer mixture, mix and start the stopwatch immediately.
Every 30 seconds, use a clean pipette or glass rod to transfer a small sample of the reaction mixture to the next iodine drop.
Record the time of the first sample that leaves the iodine orange-brown. This is the measured endpoint: no starch was detected in that sample.
Repeat the method at a range of pH values, using the same temperature, volumes and concentrations. Repeat trials and calculate a mean where appropriate.

Use a fresh portion of iodine for every sample. The iodine belongs on the spotting tile, not in the main reaction tube, because adding it to the reaction mixture would interfere with the investigation.

Continuous-sampling method, shown schematically and not to scale. Fixed volumes of starch solution and one pH buffer are held in a thermostatically controlled 35 °C water bath. Amylase is added and mixed, and timing starts immediately. Every 30 seconds, a small reaction sample is transferred to a fresh iodine drop in the next spotting-tile well. In this sequence, samples through 120 seconds are blue-black, indicating starch remains; the first orange-brown sample at 150 seconds is the measured endpoint, with no starch detected. Iodine stays on the tile and is never added to the reaction tube. Repeat with different buffer pH values while keeping temperature, solution volumes and concentrations, and sampling interval constant. — Continuous sampling: test a small sample with a fresh iodine drop every 30 seconds and record the first orange-brown result as the endpoint.

Converting completion time into reaction rate

A short completion time means starch was digested quickly, whereas a long time means it was digested slowly. A useful comparative rate is

\text{rate} = \frac{1}{\text{time taken}}

If time is measured in seconds, the unit of this rate is $\mathrm{s^{-1}}$ . Plot pH on the horizontal axis and rate on the vertical axis. The highest rate identifies the optimum pH under the conditions used. Activity usually decreases on either side of the optimum because pH changes can alter bonding within the enzyme and therefore the shape of its active site; at sufficiently unsuitable pH, the enzyme may become denatured.

Results can depend on the source and condition of the amylase, so an optimum should be concluded from the evidence rather than assumed. Amylase and starch solutions should be fresh and the enzyme should be checked beforehand so that the endpoint occurs in a practical length of time.

Evaluating the endpoint

Sampling every 30 seconds gives an endpoint only to the nearest sampling interval. If one sample is blue-black at 60 seconds and the next is orange-brown at 90 seconds, the starch disappeared sometime between them. Sampling more frequently would improve time resolution, although it would make the method harder to perform consistently.

The judgement of an iodine colour can also be subjective. Equal iodine drops, equal sample sizes, a white background and repeated trials improve consistency. Separate pipettes or thorough rinsing help prevent iodine, buffer or enzyme being carried into the wrong solution.

Food-test recall

Substance tested	Method	Positive result	Negative result
Reducing sugar	Add Benedict’s solution; heat in a water bath	Green, yellow, orange or brick-red precipitate	Blue
Starch	Add iodine solution	Blue-black	Orange-brown
Protein	Add Biuret reagent	Lilac or purple	Blue
Lipid	Shake with ethanol, then add water	Cloudy white emulsion	Clear mixture

Prepare solid foods by grinding with distilled water and filtering where necessary. Use a fresh portion for each test.

Amylase and pH method

Put iodine drops into a spotting tile.
Mix fixed volumes and concentrations of starch, buffer and amylase.
Start timing when amylase is added.
Keep temperature constant with a water bath or electric heater.

For each food test, state the reagent, any required treatment and the positive result. For Benedict’s test, include heating in a water bath.

Do not say that Benedict’s solution tests for every sugar: it detects reducing sugars. Sucrose does not give a positive result unless it is first broken down.

In the amylase investigation, iodine is used to test samples removed from the reaction mixture; it is not added to the main reaction tube.

When comparing pH values, use rate, $1/t$ , rather than time alone. A shorter digestion time means a faster reaction.

If asked for control variables, name a variable and how it is controlled: for example, keep temperature constant using a water bath and use the same volumes and concentrations of starch and amylase.

The first orange-brown iodine result means that no starch was detected in that sample. It does not mean that the iodine has been broken down by amylase.

A 30-second sampling interval limits the precision of the endpoint because starch may have disappeared at any time since the previous sample.

A substance that produces an observable change indicating whether a particular type of chemical is present.

A sugar, such as glucose, lactose or maltose, that gives a positive result with Benedict’s solution when heated.

A cloudy mixture of tiny droplets of one liquid dispersed through another liquid.

The stage at which the measured reaction is judged to be complete; here, it is the first sample in which iodine detects no starch.

A solution that resists changes in pH and therefore keeps the reaction mixture at a chosen pH.

Removing and testing small samples repeatedly while a reaction is taking place.

The factor deliberately changed during an investigation; in the amylase practical, this is pH.

A factor kept constant so that it does not affect the comparison between results.