Food Analysis

Introduction

In this kit, students will learn how to test for the presence of five components commonly found in foods. The lab is divided into five parts based upon the tests for these components: vitamin C, reducing sugars, starches, proteins and fats. After students conduct their initial tests on known substances, they can test common foods for the presence of these five components.

• Part I Vitamin C
• Part II Reducing Sugars
• Part III Starches
• Part IV Proteins
• Part V Fats 

Experiment Overview

Part I. Vitamin C Testing 
Vitamin C (ascorbic acid) is one of the important substances that contributes to the maintenance of good health. It should be included regularly in a normal diet. Ascorbic acid is water soluble and a reducing agent. In Part I of the lab, an indicator solution (dichloroindophenol) will be used to test for vitamin C. The blue indicator solution will become colorless in the presence of vitamin C. 

Part II. Reducing Sugars 
Benedict’s qualitative solution is a test reagent that reacts positively with reducing sugars. All monosaccharides and most disaccharides are reducing sugars—that is, they possess a free, or potentially free carbonyl group (C=O) that reacts to reduce Benedict’s solution. 

Part III. Starches 
Starch is a complex carbohydrate and a primary source of energy in the food we eat. Iodine is a good indicator for starch because it forms an adsorption complex with a characteristic royal blue color. The following activities demonstrate why starch must be broken down during digestion, and will allow students to test foods for the presence of starch. 

Part IV. Proteins 
Biuret solution can be used to determine the presence of protein. In this experiment, students will use biuret to test for protein in albumin and in other protein rich items. 

Part V. Fats 
Sudan III can be used to test various foods for the presence of fat.

Materials

Safety Precautions

Although vitamin C and 2,6-dichloroindophenol are not considered hazardous, students should wash their hands thoroughly after handling. Food items, once brought into a lab, are considered chemicals and, as such, should not be ingested. Benedict’s solution contains cupric sulfate and is moderately toxic and a body tissue irritant. Use insulated gloves or test tube clamps when handling the heated test tubes during the Benedict’s test procedure. Iodine solutions are irritating to eyes, irritating and mildly corrosive to skin and moderately toxic by ingestion. Biuret solution contains copper sulfate in a sodium hydroxide solution. It is corrosive to all body tissue, especially eyes. It is also moderately toxic by ingestion. Hexanes is a flammable liquid; dangerous fire risk; may be irritating to the respiratory tract: LD₅₀ 28710 mg/kg, TLV 176 mg/m³. Chemical splash goggles and chemical-resistant gloves are recommended whenever heat and glassware are used. Use insulated gloves, clamps or tongs when handling heated glassware. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All solutions from Part I. Vitamin C Testing may be disposed of down the drain according to Flinn Suggested Disposal Method #26b. The resulting mixtures from Part II. Reducing Sugars may be rinsed down the drain according to Flinn Suggested Disposal Method #26b. The resulting mixtures and solutions from Part III. Starches can all be rinsed down the drain according to Flinn Suggested Disposal Method #26b. Solutions from Part IV. Proteins should be neutralized using dilute hydrochloric acid solution and rinsed down the drain with excess water according to Flinn Suggested Disposal Method #10. The resulting materials from Part V. Fats can be disposed of by rinsing down the drain with excess water, according to Flinn Suggested Disposal Method #26b.

Prelab Preparation

Part I. Vitamin C Testing
Solutions should be prepared no more than one day in advance and refrigerated until use. Prepare the 0.025% dichloroindophenol solution by dissolving the contents of the bottle enclosed in the kit in about 500 mL of distilled or deionized water in a one-liter volumetric flask. Fill up to the one-liter mark with water. Cap and invert to mix.

Prepare a 100 mg/100 mL (0.1%) vitamin C solution by first crushing the two tablets with a mortar and pestle. Fill a one-liter volumetric flask with about 500 mL of distilled or deionized water and transfer the crushed tablets to the flask. Fill up to the one-liter mark with water. Mix thoroughly on a magnetic stirrer. Do not heat. There will be a small amount of undissolved binder material; this is not the ascorbic acid.

Part III. Starches
Activity 1: Prepare a 1% starch solution by wetting 10 g of soluble starch with a few milliliters of distilled or deionized water. Add approximately one liter of boiling water. Stir and continue heating until the starch is dissolved. Allow the solution to cool. The shelf life of a starch solution is very poor. Do not store for more than two weeks.

Activity 2: Add 50 g portions of uncooked, high-starch foods (potato, rice, dried pasta) to 400 mL of distilled or deionized water in separate 600-mL beakers. Bring the beaker contents to a boil and continue boiling for five minutes. Let cool. Pipet or decant 10–15 mL of the resulting “starch solution” from each beaker and transfer into separate test tubes or small beakers.

Part IV. Proteins
Add 100 mL of distilled water to the albumin bottle. Cap and shake to mix.

Procedure

Part I. Vitamin C Testing

1. Using a 10-mL graduated cylinder, measure out 10 mL of 0.025% dichloroindophenol solution. Transfer to one of the test tubes provided.
2. Using a Beral-type pipet, add the vitamin C solution drop by drop, counting each drop added to the test tube until the color changes from blue to the colorless/very light amber endpoint. Be sure to stir or swirl the solution after each drop is added. Record the number of drops required to turn the indicator nearly colorless. Repeat the procedure two more times to obtain more accurate results.
3. Repeat the procedure using fruit juice as the vitamin C source. Be sure to ignore the intermediate pink color; continue adding drops until the clear-amber color appears. Record number of drops for the fruit juice sample.

Part II. Reducing Sugars

1. Add 5 mL of Benedict’s solution and 8 drops of the dextrose solution to a test tube.
2. Heat a beaker of water to boiling or near boiling.
3. Place the test tube in the boiling water bath. Note any changes after three or four minutes.
4. A positive Benedict’s test can be seen by the formation of a brownish-red cuprous oxide precipitate.
5. Students can test for reducing sugars in foods. For example, place an apple slice in a mortar along with a few milliliters of distilled water. Crush the apple with a pestle and add water until a mashed apple solution is obtained. Follow steps 1–4, substituting 8 drops of the apple solution for the glucose solution. Students can test for sugar in many types of foods using these methods.

Part III. Starches

Activity 1. The Passage of Starch through a Membrane

1. Into one 600-mL beaker, place about 300 mL of distilled or deionized water and about 20 mL of iodine–potassium iodide solution. Into a second 600-mL beaker, place about 150 mL of 1% starch solution and about 150 mL of water.
2. Cut two 8- to 9-inch lengths of dialysis tubing and knot each at one end. Fill one length with iodine solution diluted 4:1 with distilled water. Fill the second with the 1% starch solution. Knot the open ends, rinse the outside with distilled water, and blot dry.
3. Place the starch dialysis bag into the beaker of iodine solution and place the iodine bag into the beaker of starch solution. Let stand, with occasional stirring (or place the beakers on a magnetic stirrer at low speed), for 15–20 minutes.
4. Record and explain your observations.

Activity 2. Testing for Starch

1. Test the starch solution samples provided by your teacher by adding one or two drops of a dilute iodine solution. (Dilute the provided iodine solution 4:1 with distilled water.)
2. Test other foodstuffs for starch using the iodine test.

Part IV. Proteins

1. Using the graduated cylinder, measure out 5 mL of the albumin solution into a test tube. Add 2.5 mL of the biuret solution.
2. Wait 30 minutes for the full color (pink to purplish) to develop.
3. Test other protein-containing foods like gelatin, beans, cheese and milk. This is done by making homogenized solutions and substituting them for the albumin solution in step 1.

Part V. Fats

Part 1. Testing Margarine for Fat

1. Add 1 teaspoon of regular margarine and 1 teaspoon of fat-free or low-fat margarine to separate evaporating dishes. Carefully melt the margarine samples on a hot plate.
2. While the margarine samples are melting, prepare the test tubes by adding 5 mL of distilled water and a few grains or particles of Sudan III to each of the test tubes.
3. Label one test tube “Regular” and the other “Fat-Free.”
4. Using Beral pipets, add 6–7 drops of melted regular margarine to one test tube and 6–7 drops of the fat-free or low-fat margarine to the other tube. The melted margarine will form a layer on top of the water in each test tube.
5. Stopper the test tubes and shake the tubes vigorously, holding the stopper with your thumb.
6. The material in the test tube containing regular margarine should turn a distinct pink color while the test tube containing the fat-free or low-fat margarine should remain yellow/white.

Part 2. Testing Salad Dressing for Fat

1. Add 12 mL of regular salad dressing and 12 mL of fat-free salad dressing to separate 100-mL beakers.
2. Add 8 mL of hexanes to each beaker and stir the contents of each beaker for one minute with the stirring rods.
3. Carefully decant the hexanes solvent from the beakers into separate evaporating dishes. Do not pour any of the salad dressing into the evaporating dishes.
4. Set the evaporating dishes in an operating fume hood or other well-ventilated area so that the hexanes solvent may evaporate quickly and safely. (This should take approximately 20–30 minutes.)
5. Once the hexanes have completely evaporated, use the spatula to sprinkle a few grains of Sudan III onto the residue left on each Petri dish.
6. Streak the Sudan III solid across the dish using a stirring rod or spatula. The Sudan III in the Petri dish from the regular dressing will dissolve in the fatty residue left, and red streaks will appear. The solid’s appearance will not change when streaked across the dish containing the residue from the fat-free dressing.

Teacher Tips

• In Part 2 of Part V. Fats, be sure to let all of the hexanes evaporate. Sudan III will dissolve in the nonpolar hexanes and if any of the hexanes solvent is present, the Sudan III will dissolve, regardless of the fat content in the food. It is best to allow the hexanes solvent to evaporate overnight.

Further Extensions

Part I. Vitamin C Testing
Try other juices, vitamin supplements or make solutions with vitamin C rich fruits or vegetables (like limes). Test other foods for the presence of vitamin C. Do any foods have vitamin C that you wouldn’t guess have vitamin C? 

Food Analysis—Testing Some Common Foods 
Now test other foods for the presence of sugars, starch, protein, fats and vitamin C. Make a table to record the results of your tests. {14117_Extensions_Table_1}

Discussion

Part I. Vitamin C Testing

Reaction

Using the vitamin C solution as a standard, the amount of vitamin C in fruit juices can be calculated. If it takes 29 drops of vitamin C solution and 77 drops of orange juice to neutralize 10 mL of dichloroindophenol solution, the calculations are as follows:

(Drops standard)(Concentration standard) = (Drops unknown)(Concentration unknown)
(29 drops) (100 mg/100 mL of vitamin C) = (77 drops) (n mg/100 mL of vitamin C)
n = 37.6 mg

Therefore the vitamin C concentration of the orange juice is 37.6 mg/100 mL.

Listed are a few juices and their ranges of vitamin C content. Vitamin C is important nutritionally. Its exact functions in the body are poorly understood. It is known to be necessary for the production of the protein collagen, which is a vital part of various connective tissues such as bone and cartilage. A deficiency in vitamin C can result in a disease known as scurvy, the symptoms of which are bleeding, spongy gums and a tendency to bruise easily. You may have heard of British soldiers historically referred to as “limeys.” The name limey was given to the sailors because during long voyages they would eat limes to prevent scurvy.

Since the body requires vitamin C on a continuing regular basis, it should be part of a daily diet. High concentrations can be found in citrus fruits, tomatoes and cabbage. Potatoes, leafy green vegetables and fresh fruits are also good sources. Nutritionists generally agree that a daily adult intake of 60–70 mg is enough to replenish normal losses and to provide a satisfactory level for cellular needs.

Part II. Reducing Sugars
Carbohydrates make up the most abundant class of organic compounds found in plants. The term “carbohydrate” is used to characterize a whole class of natural products and are named because many have a simplified molecular formula that appears to be a hydrate of carbon: (C•H₂O)_x. The basic building blocks of many carbohydrates are the simple sugars (also called saccharides) such as fructose and glucose that have the general molecular formula of C₆H₁₂O₆. Other more complex carbohydrates are simply the addition product of two, three or sometimes hundreds of sugar molecules. The common names for these compounds are disaccharides, trisaccharides, or polysaccharides. For example, sugar (sucrose) is a disaccharide consisting of one glucose and one fructose molecule.

Any sugar that has an aldehyde group is called a reducing sugar because it can be oxidized (be a reducing agent). All monosaccharides and many disaccharides are reducing sugars. Sucrose is a common example of a non-reducing sugar. A common test for identifying reducing sugars is the ability of a sugar to reduce the Benedict’s solution. Benedict’s solution consists of a copper(II) complex that oxidizes the aldehyde to a carboxylic acid and in turn is reduced to copper(I) oxide, a red precipitate. A simplified version of a typical reaction follows. Although aldoses exist mainly in the ring form, there is a small amount of the ring-open form containing the aldehyde group. As the aldehyde reacts with the Benedict’s solution, the equilibrium shifts to produce more of the ring-open form. Ketoses also, unexpectedly, react with Benedict’s because they form an isomeric aldehyde. All monosaccharides are reducing sugars because they all exist, at least partly, in the ring-open form. Disaccharides that ring-open also react positively with Benedict’s solution. Sucrose (table sugar) Sucrose does not contain an aldehyde or ketone group. It also does not ring-open. Sucrose is a disaccharide. When hydrolized, it forms glucose and fructose. It is not a reducing sugar and does not react to reduce Benedict’s solution. Glucose (dextrose) When the glucose ring opens, it forms an aldehyde that reacts with Benedict’s. Glucose is a reducing sugar.

Part III. Starches
Starch is a complex carbohydrate and a long-chain polysaccharide (polymer of glucose). It is a common energy storage molecule in plants where it typically clumps into visible grains. The most familiar sources of dietary starch are potatoes, beans (legumes) and cereal grains (corn, wheat, barley). When starch and iodine are both present in solution, they form an adsorption complex with a characteristic blue color. Since neither the starch nor the iodine is chemically altered, each can be used to indicate the presence of the other without concern for interference with potential reactions.

Activity 1 demonstrates that the starch molecules are simply too large to pass through a “biological” membrane (represented by the dialysis tubing). The implication is that starch, if unaltered, is unusable to cells—since to be usable would require the passage through the membranes of cells lining the intestines. In order to gain access to the energy stored in the starch molecule, the starch must first be broken down into di- and monosaccharides—which can pass through membranes. When starch solution is contained in the dialysis bag, we see the iodine-starch complex form only inside the bag and not in the surrounding solution. The iodine molecule passes freely through the membrane while the starch is contained within. The other beaker presents the opposite situation, and the opposite explanation.

Activity 2 simply takes advantage of the indicator complex to verify the presence of starch in common foods. High starch foods usually have a characteristic texture and are not difficult to identify.

Part IV. Proteins
Proteins are composed of one or more polypeptides and each polypeptide is composed of many amino acids regularly linked by peptide bonds into long chains. The polypeptide chains aggregate into long fibrous molecules that provide structural support or into compact globular molecules important in metabolism. When biuret solution is added to a solution containing polypeptides, the copper ions in the biuret react with the peptide bonds, producing a pinkish or purplish color.

Part V. Fats
Sudan III is a biological stain that dissolves in nonpolar solvents, such as fats and oils. It will not dissolve in polar solvents such as water. In Part I, the Sudan III dissolves in the fats in the regular margarine while it does not dissolve in the fat-free margarine. In Part II, the non-polar hexanes extract the nonpolar materials from the salad dressing. The fat-free dressing contains little or no nonpolar material while the regular dressing contains nonpolar fat. As the hexanes solvent is evaporated off, the fats are left behind, and the Sudan III will dissolve in these fats.

References

Abramoff, P.; Thomson, R. G. Laboratory Outlines in Biology—V; W. H. Freeman: New York, 1991; pp 124–127. 

Boyer, R. F. Modern Experimental Biochemistry; Addison-Wesley: Reading, MA, 1986.

Morholt, E.; Brandwein, P. F. A Sourcebook for the Biological Sciences, 3rd ed.; Harcourt Brace Jovanovich: Fort Worth, TX, 1986; pp 178–179, 241–249.