Experiment 3 : Identification Of Food Constituents In Milk

Main objective: To identify the food constituents in milk

Experiment 3(A)

Objective: To observe the movement of milk K1 and K2 in copper (II) sulphate solution.

Materials: Milk K1, milk K2 and copper (II) sulphate solution

Apparatus: Test tubes, droppers, test tube rack and small label

Methods:

1. A clean test tube was filled until 1cm from the top with copper (II) sulphate solution.
2. A small amount of milk K1 was sucked into a dropper and the dropper was lowered into the copper (II) sulphate solution.
3. Milk K1 was gently released into the copper (II) solution drop by drop.
4. The dropper was withdrawn slowly without disturbing the copper (II) sulphate solution.
5. The movement of milk K1 was observed carefully and noted.
6. Steps 1 – 5 were repeated using a new, fresh copper (II) sulphate solution, new clean test tube, new clean dropper and sample milk K2.
7. The observations on the movement of milk K2 was recorded and the differences in the behavior of the drops K1 and K2.

Observations:

Conclusion: Both milk K1 and K2 have colloidal substances inside it.

Experiment 3(B)

Objective: To test for fat content in milk

Materials: Cold water, dye (K3), milk K1 and K2

Apparatus: Dropper, test tubes, cork and magnifying lens

Methods:

1. 1 cm³ of each type of milk, K1 and K2 was measured and separated into 2 clean, dry test tubes respectively. The test tubes were labeled appropriately.
2. 5 drops of dye, K3 was added to each test tube and was shaken gently to be mixed.
3. Both test tubes were filled with cold water that was provided in the lab until ¾ full.
4. A bung or cork was placed in each test tube and the tubes were shook vigorously.
5. The contents of the tubes were examined carefully using the magnifying lens:

1. Immediately after shaking and
2. After ten minutes

6. All the observations were recorded.

Results:

Conclusion: Milk K1 is fat free whereas milk K2 contains a little amount of fat.

Experiment 3(C)

Objective: To compare the concentration of reducing sugar and protein in milk K1 and K2.

Materials:

Milk K1 and K2, Benedict’s reagent, potassium hydroxide solution, copper (II) sulphate solution and water.

Apparatus: Test tubes, cork, test tube rack, test tube holder, dropper, water bath incubator and stopwatch.

Methods : 3C(i) Reducing sugar test

1. The water bath was brought up to boiling point and then turned down the source of heat but the temperature must be maintained.
2. 2cm³ of the milk K1 was mixed with 2cm³ of Benedict’s reagent in a test tube.
3. The test tube was placed in water bath for 5 minutes and shake occasionally.
4. The colour changed was observed and recorded.
5. Steps 1-4 were repeated with milk K2. All the observations were recorded.

Methods : 3C(ii) Protein test

1. 2cmᵌ of 10% potassium hydroxide solution was added to 2cmᵌ of the K1 being tested and the tube was shook to mix the contents.
2. 0.5% copper sulphate solution was added a drop at a time, the tube was shake continuously.
3. Procedures 1 and 2 were repeated using K2.
4. All the observations were recorded.

Results:

Conclusion: The reducing sugar content in milk K1 is lesser than that of milk K2 whereas the protein content of protein in milk K1 is more than that of milk K2.

Discussion:

     Experiment 3(A) shows that both the milk K1 and milk K2 contain colloidal suspension substances when it is released in copper (II) sulphate solution. Milk is an emulsified colloid of liquid butterfat globules dispersed within a water-based solution. A colloid is a substance microscopically dispersed evenly throughout another substance (Britannica Online Encyclopedia, 2009). It's particles can be seen when you put light in it. Milk contains a series of lipid globules surrounded by an outer coating in a proteinous structure called a micelle. This micelle shields the lipids from separation by providing an electronegative and consequently hydrophilic covering around the lipids. The electronegativity also prevents the micelles from sticking together and forming large grains. However, these micelles are still reasonably-large particles which are not dissolved in the surrounding water, so the substance is a colloid.

     The more rapid descent of the milk K2 suggests it has a higher density than the milk K1. Therefore, milk K1 is less dense as it contains a lot more lipids (fat). The fat is dispersed throughout the milk in globules. These globules have a lower specific gravity and cause the milk K1 to fall slower than the milk K2.

     Sudan red test is a test for fat. Sudan red, dye K3 is a fat-soluble dye that stains lipids red. Using Sudan red can show the amount and the location of lipids. (n.a, n.d) ().  When lipids are in an aqueous suspension, the dye will colour them red. Milk K1 shows no red spot suggest that milk K1 is fat free while milk K2 which shows some red spot suggest that it contains a little amount of lipid inside it.

     In a normal benedict’s test, a red precipitate should form in the presence of reducing sugars. Benedict's reagent is made from anhydrous sodium carbonate, sodium citrate and copper(II) sulfate pentahydrate. Once added to the test solution, reducing sugars reduce the blue copper sulphate from the Benedict's solution to a red brown copper sulphide, which is seen as the precipitate and is responsible for the color change. After mixing K1 and K2 with Benedict’s reagent in each test tube, they were left in water bath for 5 minutes. Each solution change to orange colour after 5 minutes but the result of K2 appears darker orange than K1. The change in colour of Benedict’s test is determined by the amount of reducing sugar present and the colour change will progress from green, yellow, orange, red and then a dark red or brown. The results suggest that milk K2 has higher concentration of reducing sugar compare to milk K1. At the same time, the main type of reducing sugar tested in the milk K1 and K2 should be lactose as lactose is the main carbohydrate content present in milk.

     From the experiment, milk K1 should have higher protein content compare to that of milk K2. The Biuret Reagent is made of potassium hydroxide and copper sulphate. The blue reagent turns violet in the presence of proteins, and changes to purple when combined with short-chain polypeptides.  In this test for proteins there is a positive reaction between the copper ions and the amino groups in the peptide bonds. The biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper (II) ion forms violet-coloured coordination complexes in an alkaline solution. Several variants on the test have been developed. The Biuret reaction can be used to assay the concentration of proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the colour, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law. Despite its name, the reagent does not in fact contain biuret ((H2N-CO-)2NH). The test is so named because it also gives a positive reaction to the peptide-like bonds in the biuret molecule (n.a, n.d)  

     The largest structures in the fluid portion of the milk are "casein micelles": aggregates of several thousand protein molecules with superficial resemblance to a surfactant micelle, bonded with the help of nanometer-scale particles of calcium phosphate. Each casein micelle is roughly spherical and about a tenth of a micrometer across. There are four different types of casein proteins: αs1-, αs2-, β-, and κ-caseins. Collectively, they make up around 76–86% of the protein in milk, by weight. Most of the casein proteins are bound into the micelles (Fox, P. F., 1995).Milk contains dozens of other types of proteins beside the caseins including enzymes. These other proteins are more water-soluble than the caseins and do not form larger structures. Because they proteins remain suspended in the whey left behind when the caseins coagulate into curds, they are collectively known as whey proteins. Whey proteins make up approximately 20% of the protein in milk, by weight. Lactoglobulin is the most common whey protein by a large margin (McGee and Harold, 2004).

Conclusion: Milk K1 is fat free and contains reducing sugar and a higher amount of protein content whereas milk K2 contains fat, high amount of reducing sugar but a lower amount of protein content.

References :

• William H. Bowen and Ruth A. Lawrence,2005. Available from : < date: 25-5-2012]
• Frank O’ Mahony,March 1988. Available from:  <http://wiki.answers.com/Q/Why_is_milk_a_colloid#ixzz1vxEI8Uhi> [Accessed date:25-5-2012]
• Britannica Online Encyclopedia,2009.Available from: <http://en.wikipedia.org/wiki/Biuret_test#cite_note-0>[Accessed date:26-5-2012]
• Fox,P.F,1995.Available from: <http://www.ilri.org/InfoServ/Webpub/fulldocs/ilca_manual4/Factors.htm#TopOfPage> [Accessed date:24-5-2012]
• MC Gee and Harold,2004. Available from: <http://www.seplessons.org/node/362> [Accessed date: 25-5-2012]
• N.a,n,d.Available from: <http://www.writework.com/essay/experiment-write-up-including-method-and-discussion-testin>[Accessed date: 25-5-2012]