Protein Denaturation of Egg White and Milk: Experiment

Introduction:

Proteins are the secondary crucial food components while carbohydrates are primary ones. However, proteins must be denatured their natural structure to be unfolded before the digestion. ^{[ 1 ]}In the practical, the denaturation of egg white and milk were examined. The white of an egg is a solution of protein in water which depends upon stable interactions with the protein active groups. Factors which influence the stability of the protein may thus be expected to influence the stability of the solution.

Objectives:

The purpose of the experiment is to:

1. Examine and explain the changes in appearance of egg white that occur in different conditions in denaturation of egg white
2. Examine the phase transitions that occur in denaturation of egg and milk affected by temperature change by making egg custard
3. Examine and explain the changes in appearance of milk that occur in different conditions in denaturation of milk
4. Examine and explain the changes and differences in properties between the original sample of yoghurt and two sample with respective addition of fresh milk and boiled milk

Method:

All of the practical were followed by the procedures listed on page 11 and 12 of Laboratory Manuals & Guide 2014.

Result:

The observations are shown in the following:

The changes in appearance of egg white that occur in different conditions in denaturation of egg white

Before the treatment of different conditions, the egg white was clear yellow liquid in each of five boiling tubes.

After the treatment of different conditions, the egg white in tubes made changes in appearance at certain time in water bath or at certain number of additions shown in the table.

The phase transitions that occur in denaturation of egg and milk affected by temperature change by making egg custard

The time duration for baking at around 130℃ in the oven was 30 minutes in order to make egg custard.

Before the baking in the oven, the mixture was milky yellow liquid.

After the baking for 30 minutes, the mixture became thickened and semi-solid phase. Moreover, a gel-like structure was formed.

The changes in appearance of milk that occur in different conditions in denaturation of milk

Before the treatment of different conditions, the milk was white liquid in each of three tubes and the conical flask. The measurement of milk in pHjwas 6.39

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When 52 drops of 1M Acetic acid added into milk in conical flask, the formation of ppt occurred and the measurement in pHkwas 4.49. Since then, the three tubes that were treated to add 26 drops of 1M Acetic acid, add 1ml 5M NaCl (aq) and have no further addition were placed in the 80℃ waterbath. The time durations for changes in the three tubes were 1 minutes 22 seconds, 19 minutes and 20 minutes respectively.

After the 80℃ waterbath, the mixture in tube with addition of 26 drops of 1M Acetic acid formed large white curd. Besides, the mixture in tube with addition of 1ml 5M NaCl (aq) was observed to have white ppt. Moreover, the control tube (no further addition) remained white liquid but had very little silk-like sediment on inner wall of the tube.

Finally, the conical flask sample was taken for centrifugation for 5 minutes at 2,000rpm. After the centrifugation, the sample formed two layers: pale yellow liquid and milky sediment.

The changes and differences in properties between the original sample of yoghurt and two samples of yoghurt with respective addition of fresh milk and boiled milk

The original sample of yoghurt was light, fluffy, smooth and creamy and tasted a bit sour and the smell of milk was detected.

Two samples of yoghurt with respective addition of fresh milk and boiled milk cultured in 38℃ incubator formed two layers: firm and thickened milky gel and yellow watery surface. They are not similar to the smooth and creamy original sample of yoghurt.

Sourness was enhanced in the sample of yoghurt with fresh milk. However, sourness in the sample of yoghurt with boiled milk was similar to the original sample.

Furthermore, the sample of yoghurt with fresh milk was detected a tangier taste in comparison with the sample with boiled milk and the original sample.

Discussion:

The changes in appearance of egg white that occur in different conditions in denaturation of egg white

When egg white solution was placed in waterbath at 60℃ and 80℃, heat damaged hydrogen bonds from the connections of unfolding of tertiary conformation of proteins. Since then, there was a chance for long chain of polypeptides to interact with other polypeptides and the polypeptides were reformed to large molecular structure to form coagulum. Therefore, egg white solution in waterbath at 60℃ and 80℃ became milky gel.

Besides, the time duration for change to occur at 80℃ was much shorter than that at 60℃. That meant the rate of denaturation of egg white at 80℃ was faster than that at 60℃. The reason was that the higher the temperature of the water bath, the more the heat energy was supplied to protein molecules per unit time. The higher kinetic energy of protein molecules vibrated more rapidly and violently. Therefore, the bonding disrupts faster and the rate of denaturation increased.

When egg white solution was added with 22 drops of 1M Acetic acid, acid reacted with egg white to alter the electrostatic interactions and resulted in unfolding the tertiary conformation of peptide chain. It decreased the solubility of the egg white, and thus, formed silk-like sediment in the middle of three layers.

When egg white solution was added with 110 drops of 5M NaCl (aq), excessive amount of NaCl (aq) easily attracted water from protein surface and removed it from the surface. The unfavourable interactions produced between protein molecules leaded to the connection of protein molecules. Hence, egg white solution with 110 drops of 5M NaCl (aq) became silk-like white sediment in yellow egg white.

However, there was no observable change in egg white solution with additions of equal of volume of distilled water. As water didn’t react with the egg white solution, the solution remained clear yellow liquid.

The phase transitions that occur in denaturation of egg and milk affected by temperature change by making egg custard

Ovalbumin, from egg white protein, casein micelle and whey protein, from milk proteins, were involved. The mixture was thickened by the condensation of these three proteins during baking. They denatured by heat and became unfolded polypeptides with the chance to interact with other polypeptides and the polypeptides were reformed to large molecular structure. Meanwhile, the water was trapped into the network of polypeptides. Thus, the mixture became semi-solid phase or a gel-like structure.

The changes in appearance of milk that occur in different conditions in denaturation of milk

In the centrifuge tube, the top layer was pale yellow liquid and the bottom layer was milky gel. Pale yellow liquid contained water and water soluble substances such as water-soluble vitamins, minerals, lactose from milk. On the other hand, milky gel contained milk protein mainly.

There was a difference between the milk conditions because of different mass of substances. Milk protein had large molecular structure and heavier mass. It formed in the bottom layer of the tube. By contrast, water and water soluble substances had smaller molecular structure and lighter mass. It formed in the top layer of the tube.

The changes and differences in properties between the original sample of yoghurt and two samples of yoghurt with respective addition of fresh milk and boiled milk

During the formation of yoghurt, the lactose-consuming bacteria and micro-organisms from air trapped inside the culture at favourable temperature and moisture condition grew and released enzyme to lactose fermentation to produce lactic acid that disrupted the milk protein such as casein micelles.^[2] When casein micelles were destabilized by acidic environment, due to denaturation, micelles stuck together. Therefore, coagulation happened in acidic environment. Two sample of yoghurt formed firm and thickened milky curd.

The sample of yoghurt with fresh milk was detected a tangier taste and more sourness in comparison with the sample with boiled milk. The amount of lactose-consuming bacteria in sample of yoghurt with fresh milk was much higher than that with boiled milk. It was because the fresh milk did not involve a process of boiling that killed bacteria at boiling temperature. High amount of bacteria produced excessive lactic acid. Thus, the sample of yoghurt with fresh milk tasted tangy sourness.

To make commercial yoghurt, the first step is to denature boiled milk protein molecules by acidic environment was required and the coagulation was resulted in to form coagulum. Since then, a proportion of milk is taken to fluid called whey. Stabilizer such as starch has to be added to prevent whey separation so that whey is immobilized and retained within the yoghurt. This increases the smoothness and creaminess.

References:

1. Protein, wikipedia 2014 http://en.wikipedia.org/wiki/Protein
2. Production of yoghurt, The Dairy Council http://www.milk.co.uk/page.aspx?intPageID=81
3. http://www.hsph.harvard.edu/nutritionsource/what-should-you-eat/protein/
4. http://en.wikipedia.org/wiki/Egg_white
5. http://www.ukessays.com/essays/biology/denaturation-of-egg-white-data-biology-essay.php