Title Macromolecules&Food and Diet Analysis

Objective 1. To define monosaccharide, disaccharide, and polysaccharide and give examples of each. 2. To name the monosaccharide components of sucrose and starch. 3. To describe the test that indicates the presence of most small sugars. 4. To describe the test that indicated the presence of starch. 5. To define hydrolysis and give an example of the hydrolysis of carbohydrate. 6. To define the protein and lipid and give examples of each. 7. To describe the test that indicates the presence of protein and lipids.

Introduction Living organisms have the potential to carry out the life processes of nutrition, movement, growth, reproduction, respiration, sensitivity and excretion. Biochemistry is the study of chemical elements found in living systems, and how these elements combine to form molecules and collections of molecules which carry out the biological functions and behaviors that we associate with life. Many molecules present in biological cells are very large by the standard of inorganic and organic chemistry. Three major classes of natural polymeric macromolecules are found in biological cells: the nucleic acids, proteins, and polysaccharides. Lipids are also considered a major class of biomolecules, but because they are not polymeric macromolecules. The major classes of macromolecules participate in a complex array of biological processes, such as storing of genetic information(nucleic acid), catalyzing biochemical reactions(enzymes) and holding cells and organism together (structural of protein and carbohydrate). They perform all the functions of life.

Part 1: Carbohydrates Carbohydrates include both sugars and the polymers of sugars and it contain only carbon, oxygen, and hydrogen. Carbohydrates are the most abundant members of the

large and important class of biomolecules. The carbohydrates are probably best known for their roles in energy metabolism. The simplest carbohydrates are the monosaccharides or single sugars, also known as simple sugars. Disaccharides are double sugars, consisting of two monosaccharides joined by a condensation reaction. The carbohydrates that are macromolecules are polysaccharides, polymers composed of many sugar building blocks.

Investigation 1: Monosaccharides and Disaccharides The monosaccharides are white, crystalline solids that contain a single aldehyde or ketone functional group. They are subdivided into two classes: aldoses and ketoses- on the basis of whether they are aldehydes or ketones. They are also classified as a triose, tetrose, pentose, hexose, or heptose on the basis of whether they contain three, four, five, six, or seven carbon atoms. Monosaccharides generally have molecular formulas that are some multiple of the unit CH2O. Glucose, the most common monosaccharide, is of the central importance in the chemistry life. Glucose and fructose have the same formula: C6H12O6. Glucose is the sugar with the highest concentration in the bloodstream; fructose is found in fruit and honey. Monosaccharides serve as fuel molecules for immediate use by organisms. A disaccharide consists of two monosaccharides joined by a glycosidic linkage, a covalent bond formed between two monosaccharides by dehydration (condensation) process. For example, maltose is a disaccharide formed by the linking of two molecules of glucose. Also known as malt sugar, maltose is an ingredient used in brewing beer. Sucrose also is a type of disaccharide sugar, which is table sugar. Its two monomers are glucose and fructose. Lactose, the sugars present in the products of milk, in this case, a glucose molecule joined to a galactose molecule. Benedicts reagent is generally used to test for the presence of reducing sugars. This includes all the monosaccharides and disaccharides except sucrose. Sucrose do not react with the Benedicts reagent due to it prevent the glucose isomerizing to aldehyde, so it is a non-reducing sugar. If the result obtained is red precipitate, or green precipitate, or yellow precipitate, it is positive result. While the solution remains clear, this show the negative result.

Investigation 2: Starch Carbohydrate are found in the form of polysaccharides, which is a long chain that joined by the glycosidic bond. Starch, is a carbohydrate consisting of a large number of glucose units joined together by glycosidic bonds. Plants store glucose as the polysaccharide starch. The cereal grains (wheat, rice, corn, oats, and barley) as well as tubers such as potatoes are rich in starch. During photosynthesis, plants use light energy to produce glucose from carbon dioxide. The glucose is stored mainly in the form of starch granules, in plastids such as chloroplasts and especially amyloplasts. Animal consume starch to provide energy for the body. They were store glucose in the form of glycogen, which is another type of polysaccharides. Glycogen is the storage form of glucose in animals and humans which is analogous to the starch in plants. Glycogen is synthesized and stored mainly in the liver and the muscles.

Investigation 3: Hydrolysis of Carbohydrates Hydrolysis is a chemical reaction during which molecules of water are split into hydrogen cations and hydroxide anions in the process of a chemical mechanism. In the process of hydrolysis carbohydrates just can involve disaccharides and polysaccharides. Hydrolysis of sucrose will produce out glucose and fructose. For starch, the product will be glucose. HCl was used in the process of hydrolysis of sucrose, this is due to sucrose is non-reducing sugar, so HCl is used to break the glycosidic linkage between fructose and glucose. Hydrolysis of starch just added in water the glucose component will produced out.

Part 2: Proteins Protein consists of one or more polypeptides folded and coiled into specific conformations. Polypeptides are the polymers of amino acids. When two amino acids are positioned that carboxyl group of one is adjacent to the amino group of the other, an enzyme can cause them to join by catalyzing s dehydration reaction, with the removal of one water molecule, the resulting bond is peptide bond.

Proteins are very important to the molecules in our cells. Each protein having it own functions in the body. Some proteins are involved in the structural support, while others are involved in bodily movement, or in defense against germs. Biuret test was used to test for the presence of peptide bond in which potassium hydroxide is added to the test solution, followed by the careful addition of drops of 1% copper(II) sulphate solution. A violet colour indicates a positive result. The intensity relate to the number of the peptide bond presence.

Part 3: Lipids A lipid is a fat-soluble molecule. To put it another way, lipids are insoluble in water but soluble in at least one organic solvent. Lipids are used by organisms for energy storage, as a signaling molecule (steroid hormones), and as a structural component of cell membranes. Lipid digestion occurs primarily in the small intestine. Bile acids play their first critical role in lipid assimilation by promoting emulsification. It breaks the lipid globules into smaller droplet. The pancreatic enzymes break the large lipid molecules into smaller size, so that the body can easier to absorb the entire lipid. Material Part 1: Carbohydrates Investigation 1: Monosaccharides and Disaccharides y y y y y Benedicts reagent 1% solution of glucose, fructose, lactose, sucrose, and starch Test tubed Beaker Hot plate

Investigation 2: Starch y y y Starch solution Iodine reagent Test tubes

Investigation 3: Hydrolysis of Carbohydrates

y y y y y y y y

Starch solution Sucrose solution 2N HCl solution Benedicts reagent Iodine reagent Test tubes Pipettes Water bath

Part 2: Proteins y y y y 1% egg albumin Concentrated KOH 0.5% CuSO4 Test tubes

Part 3: Lipids y y y Brown paper Vegetable oil Water

Method Part 1: Carbohydrates Investigation 1: Monosaccharides and Disaccharides 1. A boiling water bath was made by filling a beaker about half full of water and was heated on a hot plate. 2. 1ml of 1% glucose solution and 5ml of Benedicts solution were placed in a test tube 3. A control was prepared. 4. The two tubes were placed in boiling water for 2-3 minutes. 5. The colour of the solution was observed and precipitate formed was noted. 6. The experiment was repeated by test with 1% solution of fructose, lactose, sucrose, starch, potatoes, soft drinks, onions, instant noodles and soya beans. 7. The results were recorded in a table. Investigation 2: Starch

1. A starch solution was prepared by mixing thoroughly 2g of starch with 10ml of water and then 200ml of boiling water was poured into the mixture. 2. Two test tubes were got, 1 and 2 was labeled. 3. A few ml of starch solution were put in tube 1.This is positive control. 4. Tube 2 is negative control. 5. A few drops of iodine were put into each tube. 6. The observation was recorded. 7. The experiment was repeated by test with potatoes, soft drinks, onions, instant noodles and soya beans. 8. The results were recorded in a table. Investigation 3: Hydrolysis of Carbohydrates 1. Eight test tubes were got and is labeled with 1 to 8.The test tubes were lined up in order in a test tube rack. 2. Two large test tubes were got and labeled with starch and sucrose. An empty beaker was used as a test tube holder if the test tubes dont fit in the rack. 3. 6ml starch solution and 3ml 2N HCL were pipetted into the tube that labeled starch. 4. 5ml sucrose solution and 1ml 2N HCL were pipetted into the tube that labeled sucrose. 5. The tubes were gently swirled and the contents were mixed. Sampling: 6. 1ml of solution was draw from the sucrose tube and put in tube 1. 7. 1ml of solution was draw from the starch tube by using a different pipette and put in tube 3. 8. An additional ml of solution was draw from the starch tube and put in tube 4. 9. The extra-large starch and sucrose tube were placed in boiling water bath. 10. 1ml of solution was draw from the sucrose tube after 2-3minutes and put in tube 2. The step for sucrose solution is finished and the tube is removed from water bath. 11. 1ml of solution from starch tube was draw into tube 5 after 5 minutes. 12. A second ml of starch was put in tube 6. 13. Steps 11 and 12 were repeated after 10 minutes, the solution were put in tube 7 and 8. Testing for starch and sugar:

14. 5ml of Benedicts reagent is added to tube 1, 2, 3, 5, and 7.These tubes were placed in the boiling water bath for 5minutes. 15. 3 to 4 drops of iodine reagent were added to tubes 4, 6 and 8. 16. The tubes were removed from the water bath and 5minutes is waited for them to cool. The results were recorded. Part 2: Proteins 1. Two test tubes were got and labeled with 1 and 2. 2. 3ml of 1% egg albumin was put into Tube 1.(Tube 2 is the control) 3. An equal volume of concentrated KOH(~20%) was added to both tubes andmixed thoroughly. 4. 1ml of 0.5% CuSO4 was added and mixed. 5. The colour in each tube after 2 minutes was recorded. 6. The experiment was repeated by test with potatoes, soft drinks, onions, instant noodles and soya beans. 7. The results were recorded in a table. Part 3: Lipids 1. A small square of brown paper was got. Oil was wrote on one half and water was wrote on other one half. 2. A tiny drop of vegetable oil was put on the half of the paper labeled oil. The oil was rubbed gently with fingertip. 3. A tiny drop of water was put on the half of the paper labeled water as a negative control. The water was rubbed gently with different fingertip to avoid contamination. 4. The spots were allowed to dry. 5. The paper was hold to the light when the spots were dry. 6. The observations were recorded. 7. The experiment was repeated by test with potatoes, soft drinks, onions, instant noodles and soya beans. 8. The results were recorded in a table.

Result Part 1: Carbohydrates Investigation 1: Monosaccharides and Disaccharides Sugars Glucose Observation(s) Benedict's solution form a thin layer of blue colouration above the viscous brick-red precipitate formed. Fructose Benedict's solution form a thin layer of blue colouration above the viscous reddish-orange precipitate formed. Benedicts solution form a thin layer of blue colouration Lactose above the less viscous brownish-orange precipitate formed. Sucrose Light blue transparent Benedicts solutions remain unchanged. Starch Light blue transparent Benedicts solutions remain unchanged. Distilled water Light blue transparent Benedicts solutions remain unchanged. Reducing sugar presence Reducing sugar absence Reducing sugar absence Reducing sugar absence Present/Absent Reducing sugar presence Reducing sugar presence

Food samples Potato

Observation(s)

Present/Absent

Light blue transparent solution turns green opaque solution.

Reduce sugar presence Reduce sugar presence Reduce sugar presence Reduce sugar absence

Soft drink (Sprite) Onion

Light blue transparent solution turns orange opaque solution. Light blue transparent solution turns yellow transparent solution.

Instant noodles Soya bean

Light blue transparent solutions remain unchanged.

Light blue transparent solutions remain unchanged.

Reduce sugar absence

Investigation 2: Starch Solution Observation(s) Present/Absent Starch presence

Starch solution Brownish-yellow transparent solution turns dark blue opaque solution. Distilled water Yellow transparent solutions remain unchanged.

Starch absence

Food samples Potato

Observation(s) Yellow transparent solution turns dark blue opaque solution.

Present/Absent Starch presence

Soft drink (Sprite) Onion

Yellow transparent solutions remain unchanged.

Starch absence

Yellow transparent solutions remain unchanged.

Starch absence Starch presence

Instant noodles Yellow transparent solution turns dark blue opaque solution. Soya bean Yellow transparent solutions remain unchanged.

Starch absence

Investigation 3: Hydrolysis of Carbohydrates Tube number Sucrose 1 Time(min) Benedicts solution 0 Blue solution remained Iodine reagent 2 2-3 Orange 3 0 No 4 0 Starch 5 5 No reaction 6 5 7 15 No reaction 8 15

solution reaction formed Dark green solution with black precipitate formed.

Dark blue opaque solution formed.

Dark blue opaque solution formed.

Part 2: Proteins Solution 1% egg albumin Observation(s) The light blue transparent solution turns violet transparent solution. A little amount of blue precipitate form at the bottom of the violet transparent solution. Distilled water Blue precipitate form at the bottom of the colourless Protein absence solution. Present/Absent Protein presence

Food samples Potato

Observation(s) The potato remains blue colour.

Present/Absent Protein absence Proteinabsence

Soft drink (Sprite) Light blue transparent solutions remain unchanged. Onion Light blue transparent solutions remain unchanged. Instant noodles Light blue transparent solutions remain unchanged. Soya bean The light blur transparent solution turns to violet transparent solution. A little amount of blue precipitate form at the bottom of the violet transparent solution.

Protein absence

Protein absence

Proteinpresence

Part 3: Lipids Solution Vegetables oil Observation(s) The brown paper was translucent whereby light can pass through the brown paper. Distilled water The brown paper is opaque. Lipid absence Present/Absent Lipid presence

Food samples Potato

Observation(s) The brown paper is opaque.

Present/Absent Lipid absence Lipid absence Lipid absence Lipid presence

Soft drink (Sprite) The brown paper is opaque. Onion Instant noodles The brown paper is opaque. The brown paper was translucent whereby light can pass through the brown paper. Soya bean The brown paper is opaque.

Lipid absence

Discussion In the first part experiment, Benedicts solution was used to determine the presence of reducing sugar in glucose, fructose, lactose, sucrose, starch, and distilled water. From the theory, most of the small sugars contain reducing sugar. The experiment show that glucose, fructose, and lactose having the presence of reducing sugar inside. This is because after the Benedicts reagent added, the colour changing was happened. The colour of the product is almost in brick-red precipitate form. A colour change would signify the presence of small sugar. The common disaccharides lactose are directly detected by the Benedicts reagent, because it contains a glucose with a free reducing aldehyde moiety, after isomerization. Sucrose also is a disaccharide sugar, it contain two sugars (glucose and fructose) joined by their glycosidic bond. While it does not change in the colour after the Benedicts reagent is added in, this is because the glycosidic bond in such a way to prevent the glucose isomerizing to aldehyde. (Diamond, 2011). Therefore, sucrose is classified to non-reducing sugar. Starch having the same result with the sucrose, which is the blue colour, remain unchanged. Starch does not react with the Benedicts reagent is due to the relatively small number of reducing sugar moieties, which occur only at the ends of carbohydrate chains. In the experiment, distilled water act as a constant, so it does not react with the Benedicts reagent and given out a negative result. There have some of the food have been tested by the Benedicts reagent, which to indicate the presence of the reducing sugar. After the adding of the Benedicts solution, potato, soft drink, and onion give a positive result. This means that there was a changing

in colour during the reaction. This shown that reducing sugar also present in potato, soft drink, and onion. The intensity of the colour represents the number of glucose in the solution. Therefore, soft drink contains large amount of glucose present when it is compared with the potato and onion. The blue colour was remained in the instant noodle and soya bean, which mean both of them without the presence of reducing sugar. In the second experiment, iodine reagent was used to test for the presence of starch in the starch solution and distilled water. Starch is polysaccharides with have a large number of glucose which connected with glycosidic bond. (Reece, 2008). The reaction is due to the formation of polyiodide chains from the reaction of starch and iodine. The amylose in starch forms helices where iodine molecules assemble, forming a dark blue or black color. (Starch-iodine test, 2008). Distilled water was used as a constant, so that the colour remain in yellow colour, does not have any reaction occur with the iodine reagent. From the food analysis, only potato and instant noodle contain the presence of starch. Both of them have the same result show with the starch solution. The colour of the solution turns to dark blue colour after the iodine reagent is added. The presence of starch in potato and instant noodle show that both of this food provides highly energy for our human body. The soft drink, onion, and soya bean do not turns the colour (remain in yellow colour) when the iodine is added. Three of them give a negative result and prove that they do not contain the nutrient of starch. After the hydrolysis of sucrose and starch, sucrose can react with the Benedicts solution only in tube 2 and do not in tube 1, while starch remains the same which is unable to react with the Benedicts solution.The sucrose in tube 2 was put into the water bath to let the reaction become faster, and theHCl solution was added into the sucrose solution before it react the Benedicts solution. The function of HCl solution is to break the glycosidic bond between the two small sugars (glucose and fructose). Therefore, the Benedicts solution can react with the glucose inside. The HCl solution is unable to break the glycosidic bond of the starch. This is because the bond is very strong and only the used of enzyme to hydrolyze starch. The enzyme that used to break down or hydrolyze the starch into smaller sugar is amylase. (Ganong, 2009). All the starch having reaction

with the iodine reagent to turns the colour to dark blue. This shows that HCl solution does not break the strong glycosidic bond. Therefore, starch does not react with Benedicts solution and just can react with the iodine reagent. In the fourth experiment, is to indicate the presence of protein in the 1% of egg albumin and distilled water. In the same case, distilled water is used as a constant. So that after the CuSO4 added in, the colours of the solution remain in blue colour. (Bailey, 2011). KOH was used in this experiment to react with the egg albumin and the KOH function is to break the peptide bond in the protein. After the CuSO4 added in, the amino acid will react with the Cu2+,this causes the form of the blue colour complex. The biuret test is used for the quantitative photometrical determination of total protein concentration. The intensity of the color produced in the biuret reaction is proportional to the number of peptide bonds participating in the reaction. Therefore, the egg albumin consists of a lot peptide bond, so the colour of the result is in violet colour precipitate. (Becker, 2009) In the food analyze, only soya bean contain protein inside. It gives a positive result after the CuSO4is added in, the colour turns to violet. Others food (potato, soft drink, onion, and instant noodle) without the protein inside from the experiment have done. Actually instant noodle have protein contain, but the protein level is very low, therefore the colour of the solution cannot view obviously. In the last part experiment which is the test to determine the presence of lipids in the solution. As usual, distilled water is acted as a constant and it does not change the brown paper to translucent when view under the light. The vegetables oil contains lipids, this is because it turns the brown paper to translucent and it can view under the light. This is the test to indicate the presence of lipid in any solution. Only instant noodle make the brown paper to translucent when view under the light among the other 4 types of food. Potato, soft drink, onion, and soya bean does not contain lipids because it give a negative result which is do not translucent when view under the light.

Conclusion Monosaccharide is a carbohydrate that has a single carbonyl group and two or more hydroxyl groups. Disaccharide is two monosaccharide molecules linked by a glycosidic bond. Polysaccharide is several or many monosaccharides linked by glycosidic bonds. Example of monosaccharide is glucose, fructose and galactose; for disaccharide which has maltose, sucrose and lactose; for polysaccharide which consist starch, glycogen, and cellulose. The monosaccharide components of sucrose consist of glucose and fructose. While the monosaccharide component of starch is just glucose, it is made up by two or more glucose. Benedicts reagent was used to test for the presence of most of the small sugars. Brick-red precipitate will formed if the result is positive, while if it is a negative result then the blue colour solution remains unchanged. Iodine reagent was used to test the presence of starch. When the result is positive, the solution will turns to dark blue colour. In the other hand, it will maintain brown colour when it is a negative result. Hydrolysis is a chemical process that lyses or splits molecules by the addition of water. If the sucrose undergoes the process hydrolysis without adding the HCl, this will give a negative result. This is because sucrose is a non-reducing sugar. The glycosidic bond needs to break by the HCl then only the Benedicts solution can react with the small sugar. It will show positive result when it turns to brick-red precipitate. For starch, due to the glycosidic bond is strong, so HCl do not have the ability to break the bond.So at do not show positive result when Benedicts solution was used in the starch solution. Protein is a biopolymer composed of amino acid monomer units. Biuret test was used to determine the presence of protein in the solution. The KOH solution was added in the protein to break the peptide bond in the protein, the CuSO4 is used to let amino acid to react. If the solution turns to purple or violet, this indicates the solution contain protein. While the solution remain blue colour mean it is a negative result. Lipid is a class of biological compounds characterized by high solubility in organic solvents but low solubility in water. If the solution contains lipid, the brown paper will show transparent when the brown paper view under the light. This is the easiest test among the three macromolecules.

References 1. Bailey, R. (2011). Protein Function. Retrieved July 25, 2011, from About.com Biology: http://biology.about.com/od/molecularbiology/a/aa101904a.html

2. Sucrase. (2011). In Encyclopdia Britannica. Retrieved from http://www.britannica.com/EBchecked/topic/571354/sucrase 3. Boyer, R. (2006). Concepts In Biochemistry (3rded). John Wiley (Asia): Wiley Asia Student Edition 4. Becker,W.M., Kleinsmith,L.J.,&Hardin,J. (2009), The world of the cell. (7th ed.), San Francisco: Pearson/Benjamin Cummings. 5. Campbell,N.A.,Reece,J.B.,&Mitchell,L.G.(2008), Biology. (8th ed.), San Francisco: Pearson/Benjamin Cummings.