Characterize of organic macromolecules, which are carbohydrates, protein, and lipid, that present food.

2. Two boiling tubes were prepared with label it with starch and sucrose, the boiling

     tubes were placed in a beaker as a holder.

3. 6 ml of starch solution and 3 ml of 2N HCL was added into the starch labeled boiling tube.

4. 5ml of sucrose solution and 1 ml of 2N HCL was added into the sucrose labeled boiling tube.

5. Two boiling tubes swirled gently, to mix the solution.

6. 1 ml of sucrose solution from the boiling tube was added in Tube 1.

7. 1 ml of starch solution from the boiling tube was added in Tube 3 and Tube 4 respectively.

8. The boiling tubes were placed in the water bath for 5 minutes.

9. After 5 minutes, 1 ml of sucrose solution from the boiling tube was added in Tube 2.

10. Sucrose solution in the water bath been removed from water bath.

11. After 5 minutes, 1 ml of starch solution from the boiling tube was added in Tube 5 and Tube 6

       respectively.

12. After 10 minutes, 1 ml of starch solution from the boiling tube was added in Tube 7 and

      Tube 8 respectively.

13. 5 ml of Benedict’s reagent added into Tube 1, 2, 3, 5, and 7 respectively, and the test tubes

      were placed in water bath for 5 minutes.

14. 3 drops of iodine reagent was added into Tube 4, 6, and 8.

15. After 5 minutes, Tube 1, 2, 3, 5, and 7 removed from the water bath, the test tubes were left

       on test tube rack for 1 minute to settled down the solution.

16. Observation was done on Tube 1 to Tube 8, and all result was recorded in a table.

Part 2: Protein         

1. Two test tubes were prepared with label on it respectively.
2. 3 ml of 1% egg albumin solution was added in the first test tube.
3. A negative control was prepared for the second test tube.
4. 2 ml of concentrated KOH added into both test tubes respectively.
5. 1ml of 0.5% CuSO4 slowly added into both test tubes respectively.
6. Two boiling tubes swirled gently, to mix the solution.
7. After 2 minutes, observation had been done on respective test tubes.
8. Each sample prepared was repeated with the 1-7 methods.
9. All result was recorded in a table.

Part 3: Lipid test

1. A brown paper was prepared, and divided it into 8 parts, each part labeled respectively.
2. A drop of vegetable oil was dropped on the “oil” part on the brown paper.
3. A negative control was prepared on the “water” part on the brown paper.
4. The drop of solution was left to dry it.
5. When the drop was dry, the paper was hold under the light.
6. Observation been done on each drop of solution.
7. Each sample prepared was repeated with the 1-6 methods.
8. All result was recorded in a table.

Results:

Part 1: Carbohydrate

1.1: Benedict’s test

Table 1: The observation on Benedict’s test in different solution.

1.2: Iodine test

Table 2: The observation of Iodine test in different solution.        

        

1.3: Hydrolysis of Carbohydrates        

Table 3: The observation on hydrolysis of sucrose and starch.

Part 2: Protein

Table 4: The observation of Biuret test in different solution.

Part 3: Lipid

Table 5: The observation of lipid test on brown paper in different material.

Discussion:

Part 1: Carbohydrate

  1.1: Benedict’s test

In this experiment Benedict’s reagent was used to test the presence of reducing sugar in a particular solution or food, if the solution (or food) shows the presence of precipitate in yellow, green, yellowish red, or red colour, it give a positive result on the test. Whereas, if the solution (or food) showed no changes from the initial colour (blue colour), it give a negative result in the test.

Benedict’s reagent is a clear blue colour reagent, the blue colour showed in the reagent indicate the presence of cupric copper ion, Cu2+. When boiling the reducing sugar with Benedict’s reagent, the reducing sugar will reduce the cupric copper ion,Cu2+ and oxidized it became, Cu2O, which the Cu2O is a substance insoluble in water form precipitate in the test, and give a positive result.

According to Table 1, Glucose gives a positive result in the test, because glucose is a monosaccharide with aldehyde structure which indicates it as an aldose, which known as a reducing sugar. While, fructose also gives a positive result, because fructose is isomer of glucose, which it is a monosaccharide with ketone structure which indicates it as a ketose, and then it needed to undergo isomerism, became aldose, then only known as a reducing sugar. For disaccharide, lactose gives positive result; because two monomer of lactose are aldehyde, and the monomers are linked by glycosidic bond, so through the structure of lactose, lactose is consider as an aldose, which is a reducing sugar. But for Sucrose, it gives a negative result; because the sucrose has aldose monomer and also ketose monomer, the monomers are link by the glycosidic bond. Because of the glycosidic bond, the ketose cannot isomerize to form aldose, so sucrose is not considered as a reducing sugar. For starch, it is a polysaccharide which is the complex sugar, which is large number of glucose joint together by glycosidic bond. So, it did not consider as a reducing sugar, so it is impossible to get positive result in the Benedict’s test., unless the starch was being hydrolyzed. Where distilled water is a negative control in this test, so, it showed a negative result. The food sample, which is apple juice, potatoes juice, soya bean, soft drink, onion juice, and instant noodle give a positive result in the test, because all food sample form red and yellow precipitate, that indicate the Cu2O form, reducing sugar presence. Those form yellow precipitate have low concentration of reducing sugar then those that form red precipitate.

1.2: Iodine test

This experiment Iodine reagent was used to determined the presence of starch in the solution (or food). When iodine reagent added in the starch presence solution, the iodine ion will integrate in the polysaccharide chain and result in a dark blue colour, the positive result showed. Whereas when the solution showed yellow colour, which is the iodine reagent initial colour, the negative result showed. According to Table 2, for the starch solution, the iodine test showed positive result, because the starch solution itself definitely contain of starch molecules. As for the distilled water, it serves as a negative control of this experiment, so, it showed a negative result. While, for the food sample, only the potato juice and instant noodle showed positive result which indicates the presence of starch, the rest all showed a negative result. But the potato juice and instant noodle did not showed the same colour, because the initial colour of the food sample will affect the final colour of the test.

1.3: Hydrolysis of Carbohydrates

        This experiment use Benedict’s reagent and Iodine’s reagent to test the hydrolysis of sucrose and starch by using hydrochloric acid. For Benedict’s test, the positive result for the theoretically should be showed in test tube 2 for Benedict’s test of sucrose and 7 for  Benedict’s test of starch. For the starch Iodine test, the test tube 4, and 6 should gives positive result, and the test tube 8 should give a negative result.

According to Table 3, the Benedict’s test of sucrose, test tube 2 showed positive result in the Benedict’s test, the reason of the test tube 2 will showed the positive result but not test tube 1 is, the sucrose need to boiled for few minutes in order to hydrolyze the disaccharide into monomer (fructose and glucose), then the monomer formed only will give positive result. For the Benedict’s test of starch, test tubes 3, and 5 showed a negative result, indicate that no presence of starch, but the test tube 7 showed unexpected negative result, that test tube 7 are absence of reducing sugar, which mean that the starch in the tube 7 lack of time to hydrolyze starch (polysaccharide) to simple reducing sugar. From Table 3, the test tube 4 and 6 get the expected positive result, which determined that at the first 5 minutes boiling, the starch did not hydrolyze into monomer and dimmer, iodine test still give positive result. But test tube 8, it showed unexpected result, that test tube 8 still presence of starch, because lack of time to hydrolyze starch.

Part 2: Protein

        Protein was tested by using the 0.5% CuSO4 also known as Biuret reagent. The Cu2+ o f the reagent will fuse with the peptide bond between the amino acid, to produce a violet colour to indicate the presence of protein. If the Biuret test shows negative result, a solution with light blue colour formed. From the Table 4, the 1% egg albumin showed a positive result, because it form dark blue colour as the final colour, which is a colour near with the violet colour. Same as previous, the distilled water also serves as a negative control that should give a negative result. For the food sample, all food samples show a positive result that indicates the presence of protein, except for apple juice.  The final colour of the food sample, showed different from violet, which because the initial colour of the food sample will affect the final colour of the test.

Part 3: Lipid

        Lipid was test by using brown paper, and divided into 8 parts which each part have different droplet of solution (or juice). The positive result showed when the paper was dry and holds under the light, and the droplet became transparent, if the paper doesn’t have any changes on the paper, it showed negative result. The oil droplet showed a positive result which the whole droplet became transparent, distilled water was also act as negative control showed no changes on the paper. For the food sample, only apple juice, onion juice, and the instant noodle show slightly positive result, because when observing the paper, there is slightly transparence in outer part of the droplet. The other food sample droplets, which showed no change on the paper, negative result showed.

Conclusion:

1. The Benedict’s test only able to determined the reducing sugar in a given solution, which show  precipitate at the bottom part of the solution, the precipitate can be yellow, green, yellowish red, or red colour that all this colour of precipitate indicate the positive result of the test.
2. The Iodine test are able to determined the starch compound in a given solution, which show a dark blue solution that indicate the positive result of the test.
3. When added the non-reducing sugar, sucrose with hydrochloric acid, and heated for few minutes, sucrose able to hydrolyze to form simply reducing sugar, fructose and glucose that able to detect by Benedict’s test.
4. When added the polysaccharide, starch with hydrochloric acid, and heated for a longer time (longer then the time needed to hydrolyze sucrose), starch able to hydrolyze to form simple reducing sugar, that able to detect by Benedict’s test.
5. The Biuret test able to determined the presence of protein in a given solution, which show dark blue solution that indicate the positive result of the test.
6. The lipid test by using brown paper, able to determined the presence of lipid in a given solution, which the changes the transparency of  brown paper indicate the positive result of the test.
7. The apple juice provided was tested, then found out that apple juice consist of carbohydrate, and also little amount of lipid, did not consists of protein and starch.
8. The potatoes juice provided was tested, then found out that potatoes juice consist of carbohydrate, starch, protein, did not consist of lipid.
9. The soya bean provided was tested, then found out the soya bean consist of carbohydrate, and protein, did not consist of lipid and starch.
10. The soft drink provided was tested, then found out the soft drink consist of carbohydrate, and protein, did not consist of lipid and starch.
11. The onion juice provided was tested, then found out the onion juice consist of carbohydrate, protein and slightly lipid, did not consist of starch.
12. The instant noodle provided was tested, then found out that instant noodle consist of carbohydrate, starch, protein, and slightly lipid.

Reference:

1. Eldra PS, Linda RB, Diana WM, 2005 Biology 8th edition, Thomson Brooks/Cole
2. Taylor DJ, 1997 Biological Science, Cambridge: Cambridge University Press
3. Stanton, Rosemary, 1995 Rosemary Station’s complete book of food and nutrition, East Rosevill, N.S.W.: Simon & Schuster.
4. 2004 A Dictionary of Biology, Oxford: Oxford University Press