- We neutralised the filtrate we had obtained (filtrate 1) with sodium carbonate. We tested the filtrate with universal indicator after each drop of sodium carbonate in order to obtain a neutral solution, which was indicated as green on our universal indicator.
- We then boiled this filtrate 1 for 2 minutes, and filtered this solution again.
- After filtering we obtained a second filtrate (filtrate 2) and a second precipitate (precipitate 2), so that we could carry out tests on these samples.
- We carried out the Benedict’s reagent test on our filtrate in order to identify if our filtrate contained a reducing sugar.
- On our Precipitate 2, we carried out the Biuret test- and this was to identify if we had a second protein present in our sample.
- We recorder our observations of the different tests, and analysed out results.
Results
From the approach of our method we obtained 4 results from various different tests. These results were for:
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The test of protein (Biuret) on precipitate 1
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The test for fat (Emulsion test) on precipitate 1
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The test for a reducing sugar (Benedict’s) on filtrate 2
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The test for protein (Biuret) on precipitate 2
We carried out two tests on precipitate 1. The results of these were as follows.
Biuret on Precipitate 1
After adding Biuret solution to precipitate 1 and shaking we observed that the solution went lilac. This indicated that a form of protein was present in the original milk sample that we tested, as the Biuret solution changes colour.
Biuret on Precipitate 2
We observed after adding Biuret solution to Precipitate 2, which had been neutralised and boiled, a lilac colour, which was lighter than that for Precipitate 1.
Emulsion test on Precipitate 1
After producing an ethanolic mixture of the test sample and mixing it with cold water we observed white emulsion at the top of the test tube. This showed that there was fat present in the sample in the milk that we tested.
Benedict’s on Filtrate 2
After neutralising and boiling the original filtrate and carrying out the Benedict’s test, we observed an orange colour. This orange precipitate indicated that a reducing sugar was present in the milk that we tested.
Conclusions
Analyses of Biuret test on Precipitate 1 and the Biuret test on Precipitate 2
The total protein component of milk is composed of numerous specific proteins. This is evident in our test by the two Biuret tests that we carried out, one on precipitate 1, and one on precipitate 2. These two proteins contain different properties, and the main difference seems to be their solubility and the difficulty of extracting them from milk.
The primary group of milk proteins are the Caseins, these make up 80% of milk proteins. The distinguishing property of all caseins is their low solubility at pH 4.6. The common compositional factor is that caseins are conjugated proteins.
Casein is one of the most abundant organic components of milk, along with the lactose and milk fat. Individual molecules of Casein alone are not very soluble in the aqueous environment of milk. However, the casein micelle granules are maintained as a suspension in milk, and because of this the Casein micelle (multi-molecular, granular structure of different Caseins) can be separated from the rest of milk easily, e.g. centrifugation in commercial purposes, or in our case filtration.
The conformation of caseins is much like that of denatured globular proteins. The high number of residues in caseins causes particular bending of the protein chain and inhibits the formation of close-packed, ordered secondary structures. Caseins contain no disulfide bonds. As well, the lack of tertiary structure accounts for the stability of caseins against heat denaturation because there is very little structure to unfold. Without a tertiary structure there is considerable exposure of hydrophobic residues and this results in strong association reactions of the caseins and makes them insoluble in water.
In our test the initial precipitate that we obtained contained mainly Casein proteins, and this is what caused the positive response in the Biuret test. After this initial precipitation we were left with an aqueous supernatant which resembled skim milk, as most of the Casein proteins had been removed by filtration.
Our positive response for the Biuret test on Precipitate 2, meant that we could see that there were at least two different types of proteins in our milk sample.
Because the Casein is in suspension most of it was removed by our initial filtering, and so we were left with a stage of milk called whey, which was our filtrate. We neutralised this filtrate by slowly adding sodium carbonate to lower the milk pH (normally 6.6 to 6.9) to 4.6, and then we heated the neutralised filtrate.
The proteins present in our filtrate at this stage are whey proteins, and the main whey proteins in cows milk (which we used as our sample) are ß-lactoglobulin and α-lactalbumin. These globular proteins are more water soluble than Caseins and are subject to heat denaturation. This controlled acid precipitation technique enabled us to distinguish between the difference in properties between Casein and the other proteins that are present in milk. Our results show that milk contained at least two proteins, by the two positive responses to the Biuret test, one before neutralisation and hydrolisation and one before.
Emulsion test on Precipitate 1
Our response to the Emulsion test with Precipitate 1 was positive, and so this shows that milk contains at least one fat.
From our test we can see that milk is an emulsion of fat globules and a suspension of Casein micelles, all suspended in an aqueous phase which contains lactose (Benedict’s test), whey proteins, and other substances.
Benedict’s test on Filtrate 2
The positive response for our Benedict’s test on our filtrate 2, which was an strong orange colour, meant that we could conclude that milk contained a reducing sugar, after being hydrolysed.
The main carbohydrate present in milk is lactose, although milk does contain other carbohydrates but at low concentrations. Low concentrations of free glucose and free galactose are found in cow milk and milk of other species. Other carbohydrates found free in milk include amino sugars and other sugar phosphates.
Lactose is a disaccharide made up of glucose and galactose, which are both monosaccharides:
It comprises about 5% of milk. It is not as sweet as sucrose. When lactose is hydrolyzed by lactase or heating, the result is increased sweetness, and depressed freezing point. After hydrolisation in our test by heating, the glycosidic bond between glucose and galactose splits, and so there are now two reducing sugars present in our filtrate. That is why our Benedict’s test showed a positive response for a reducing sugar.
