Biomolecule Test

Testing for Biomolecules in Food

We can test for lots of different biomolecules in foods. The main molecules we usually test for are proteins, fats (lipids), and carbohydrates (sugars and starch). The tests we use to look for the presence of these foods are called qualitative tests.

Let's think about flame tests in chemistry. A specific colour might tell us if a particular metal is present (e.g. a yellow flame often indicates sodium is the metal present), but it cannot tell us how many grams of the metal is present. Similarly, the tests that we use to identify the presence of biomolecules in food only show us whether the molecule is present or not, not how many grams of the molecule is present.

Testing for Proteins

We can test for proteins by using a biuret solution. Biuret comes in two solutions, often named biuret A and biuret B. These two solutions are copper (II) sulphate and either potassium hydroxide or sodium hydroxide. When we add these two solutions together, we get biuret A; we then add potassium sodium tartrate and have biuret B.

Now that we understand the biuret solutions, let's have a look at the method behind testing for proteins;

1. Add 1 cm³ of biuret solution A to the food solution

2. Mix the two liquids

3. Add 1 cm³ of biuret solution B and shake OR add 1 cm³ of biuret solution B carefully down the side of the test tube to form two layers

The qualitative tests we are discussing can either have positive or negative results. A positive result shows us that the molecule we tested for is present in the solution. A negative result tells us that the molecule is not present in the substance. Have a look below at the positive and negative tests when using biuret solution to test for proteins:

• Positive test - colour change from blue to purple or a purple ring forming between the two layers.
• Negative test - colour remains blue, and no purple ring formed.

Testing for Fats/Lipids

The terms fats and lipids are often used interchangeably, so testing for lipids/fats will require the same test. We can use two tests to test for the presence of lipids/fats in a substance. The two tests that we can use are;

• Sudan III Test

• Emulsion Test

Let's look at the Sudan III test first. Sudan III is a dye that causes a colour change in fats.

Here's how we carry out the test;

1. Add equal amounts of the food in question, and water to a test tube

2. Add a few drops of Sudan III solution to the test tube

3. Shake the test tube

A positive Sudan III will result in a red-stained layer on the water's surface.

A negative Sudan III will result in no red-stained layer forming.

Now let's have a look at the emulsion test:

1. Crush up the food in question and add it to a test tube

2. Add ethanol to the test tube

3. Take the liquid from the test tube, and pour it into a second test tube containing water

A positive emulsion test causes a white emulsion to appear in the second test tube.

A negative emulsion test leads to no emulsion being formed. Instead, a colourless solution remains.

Remember, when discussing the formation of an emulsion, we still have to include the word from. It is a colour change from a colourless solution to a white emulsion.

Biomolecule Test for Reducing Sugar

Sugars are carbohydrates. We can have two different types of sugars, either reducing or non-reducing. The tests that we use to test for the presence of reducing sugars and non-reducing sugars are slightly different, and we also have a completely different test for starch too. Let's look at each of these tests and work out when we need to use each.

Reducing Sugar

Examples of reducing sugars are;

• glucose

• fructose

• lactose

• maltose

To test for reducing sugars, we use Benedict's solution. Benedict's solution is a complex mixture of sodium carbonate, sodium citrate, and copper (II) sulfate pentahydrate.

Let's have a look at how we can use Benedict's solution to test for reducing sugars;

1. Add Benedict's solution to the substance in question

2. Heat the solution in a water bath

A positive test for reducing sugars using Benedict's solution shows a colour change over time. This includes a change of green, yellow, orange, red or brown - depending on how much glucose is present. The solution changes from blue to brick red if we look at the overall colour change. If enough reducing sugar is present, a brick red precipitate (ppt) is formed.

In the Benedict's test, whether a precipitate is formed depends on how much reducing sugar is present. This makes the test semi-quantitative because the more reducing sugar there is, the greater the likelihood that a brick red (red/brown) predicate will be formed.

Non-Reducing Sugar

Non-reducing sugars do not reduce other compounds/substances. Examples of non-reducing sugars are;

• Sucrose

• Trehalose

Let's have a look at how we can test for non-reducing sugars;

1. Add dilute hydrochloric acid (HCl) to the substance in question

2. Heat in a water bath at boiling point

3. Neutralise the solution with sodium hydrogen carbonate

   Using a suitable indicator (such as litmus paper), identify when the solution has been neutralised, then add more sodium hydrogen carbonate as the conditions need to be alkaline for the Benedict’s test to work.

4. Then carry out Benedict’s test as normal

   You can see this test in full in our 'reducing sugars' section!

5. If a colour change occurs from blue to a brick red precipitate, a non-reducing sugar is present.

If we know that our substance is sugar, but we are not sure whether it is a reducing or non-reducing sugar, we can simply carry out a Benedict's test to determine which type of sugar is present. If the sugar causes a colour change with Benedict's without the addition of sodium hydrogen carbonate or dilute hydrochloric acid, then it is a reducing sugar. If it only has a colour change after the addition of these substances, then it is a non-reducing sugar.

Starch

Starch is another type of carbohydrate, like the reducing sugars and non-reducing sugars we have already discussed. However, we use a different solution to test for starch, as starch is too complex and large of a sugar.

As mentioned in the deep dive above, we have to use iodine to test for starch. Let's have a look at how to use iodine to test for starch;

1. Add the substance in question to a solution (for example, potatoes)

2. Add drops of iodine to the food in question

A positive result for starch will result in a colour change from yellow/brown to blue/black.

A negative result remains yellow/brown, with no colour change being observed.

Testing for Biomolecules in the Lab

When we test for biomolecules in a laboratory, for example, when testing for these different molecules in school practicals, there are a few safety precautions that we have to follow. Let's look at some of the safeguards we must take and why we must take them.

Let's look at some of the substances discussed above, the potential risks of using them, and how we can reduce those risks as much as possible to carry out a safe biomolecule test in the lab.

Table showing the hazards and precautions associated with different food tests: