CAPE Food Tests.

This note looks at Specific Objective 1.9 of the CAPE Unit 1 Biology Syllabus, which asks us to carry out tests for reducing and non-reducing sugars, starch, lipids and proteins, using Benedict’s test, KI/I2 test, the emulsion test, and Biuret test. Additionally, we are asked to investigate and compare quantitatively reducing sugars and starch.

This note will build on some of our previous knowledge gained from studying food tests at the CSEC level (see CSEC food tests in a previous Edukatte note.)

Reducing Sugars:

Some examples of reducing sugars include both glucose and fructose and refers to these sugars having free aldehyde or ketone groups which can reduce Cu2+ ions (found in Benedict’s Solution) to copper (I) oxide (CuO).

The following method can be used:

1.      Place crushed food sample in a boiling tube and add about 2cm3 Benedict’s Solution.

2.      Heat in a water bath at about 80°C for 5 minutes.

3.      Observe the change in colour.

Negative Result- no change in colour observed as the solution remains blue.

Positive Result- Colours are based on the amount of reducing sugar present.

This test may be referred to as semi-quantitative, as it partly accounts for the quantity of reducing sugar present. The reduction of these Cu2+ ions will result in the following colour change:

·        Green- low level of reducing sugar present.

·        Yellow- moderate level of reducing sugar present.

·        Orange- high level of reducing sugar present

·        Brick-Red- very high level of reducing sugar present.

Non-Reducing Sugars

This experiment is a combination of two parts- turning the non-reducing sugar components into reducing sugar components, and then if a non-reducing sugar was present, the reducing sugar components now obtained can be used in a previously described reducing sugar test. Note that in the end, if no colour change is observed and the solution does remain blue, this is indicative that there is no reducing sugar present.

The overall experiment can be seen below:

1.      Place crushed food sample in a boiling tube.

2.      Add 2cm3 dilute HCl for hydrolysis of non-reducing sugar components into reducing sugar components (monosaccharides).

3.      Speed up the hydrolysis reaction by heating up the mixture in a water bath.

4.      Add a small amount of sodium hydroxide, NaOH, to neutralise the solution.

5.      Now add about 2cm3 Benedict’s Solution.

6.      Heat in a water bath at about 80°C for 5 minutes.

7.      Observe the change in colour, the same as in the reducing sugar test:

·        Green- low level of reducing sugar present.

·        Yellow- moderate level of reducing sugar present.

·        Orange- high level of reducing sugar present

·        Brick-Red- very high level of reducing sugar present.

Explanation:
Take the example of the non-reducing sugar of sucrose. Non-reducing sugars, like sucrose, contain a glycosidic bond. To break this bond, we are required to heat the solution with dilute HCl, and then neutralise the solution after with sodium hydroxide. 
Using the example of sucrose, this now yields two reducing sugars- glucose and fructose, and the remainder of the test can be carried out following the method of the reducing sugar test.

Starch (The KI/I2 Test)

1.      Add a crushed food sample to the test tube.

2.      Add a few drops of iodine solution (also called Lugol’s Iodine).

Positive Result- colour change from brownish colour iodine solution to blue-black solution.

Negative Result- no colour change, i.e. brownish colour remains.

Explanation:
Earlier in Module 1 of CAPE Unit 1 Biology, you would have learnt that starch is composed of both amylose (a coiled structure) and amylopectin. When iodine dissolved in a potassium iodide (KI solution) interacts with the helical structure of starch, a shift occurs in the wavelength of light absorbed by starch, and results in a visible colour change, allowing for the characteristic blue-black confirmatory colour, indicating the formation of a complex being formed by the interaction of amylose and iodine.

The Semi-Quantitative Nature of the KI/I2 Test:
Similar to the reducing sugars test, this test is semi-quantitative as the shade of blue obtained can indicate the level of starch present in the solution:

• No starch- The solution does not turn blue, instead brownish iodine colour will remain.
• Low starch content- Pale blue colour observed.
• Moderate starch content- A darker shade of blue is seen, compared to the pale blue colour observed in a low starch content.
• High starch content- The solution turns deep blue-black.

Fats- The Ethanol Emulsion Test

1.      Add a crushed food sample to a test tube.

2.      Add a small amount of ethanol, equivalent to the amount of crushed food sample present.

3.      Shake the test tube vigorously (important for proper dissolving to occur).

4.      Add an equal volume of water to the mixture.

Positive Result- a cloudy white solution is observed, indicating that fats are present.

Negative Result- No cloudy white solution observed, indicating that fats are not present.

Explanation:

Lipids are hydrophobic and thus do not dissolve in water, but do dissolve in ethanol. Hence, when ethanol is added at first, the lipids dissolve in the ethanol, but as water is added, an emulsion is formed as the lipid molecules begin to dissociate, which scatters light, and hence makes the solution appear cloudy white. This only happens if a lipid is present, and thus this cloudy white solution appearing, is indicative of such.

Test for Proteins (Biuret Test)

Steps:

1.      Obtain a crushed food sample.

2.      Place 2cm3 of this crushed food sample into a test tube.

3.      Add 2cm3 of Biuret Solution to the test tube.

Positive Result- Colour change from blue to purple, meaning that proteins are present.

Negative Result- No colour change observed (blue solution remains), meaning that proteins are not present.

Explanation:

You would have previously learnt that proteins contain peptide linkages. Biuret Solution, which is used in this test, contains Copper II (Cu2+) ions that form a complex with these peptide bonds, and thus produces its characteristic colour change.