For the experiments observing the effects of temperature change on egg white denaturation, 2 egg white solutions are incubated at 60 and 80. White precipitate, milky solution and lump formation are observed in two boiling tubes after a period of time.
When egg white solution is placed in water bath above room temperature, denaturation occurs followed by coagulation. Heat disrupts hydrogen bonds inside the protein, as a result of unfolding of specific tertiary conformation of proteins. Polypeptides are reformed in different ways by interacting with other polypeptides to form coagulum. This can explain the formation of white precipitate and lump at the bottom of the boiling tubes.
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One may notice that the rate of denaturation of egg white at 80 is faster than that at 60. The higher the temperature of the water bath, the more the heat energy is supplied to protein molecules per unit time. The average kinetic energy of protein molecules increased causes them vibrate too rapid and violent. As a result, the bond disrupts faster; the rate of denaturation is increased.
When egg white solution reacts with 2 drops of 1 M acetic acid, lump and white precipitates are observed. This can be explained by the reaction between acid and egg white which causes protein denaturation by altering the electrostatic interactions. The main effect is unfolding the tertiary conformation of peptide chain, decreasing of solvation and subsequent decrease of solubility.
Sodium chloride (NaCl) solution attracts water and help protein to remain moist. Excessive amounts of NaCl solution breaks the hydrogen bonds between the amino acid strands and leads to protein denaturation. When only a few drops of NaCl solution are added, the egg white solution remains clear. It can be explained as the amount of NaCl solution is below the required amount to break hydrogen bonds. Subsequently, with the increase of NaCl solution present in the egg white solution, salt molecules readily interact with water from protein surface and remove it from the surface. The unfavourable interactions produced between protein molecules brings about the association of protein molecules. Hence, white precipitation (salt out) is observed. Nonetheless, the effect on protein denaturation by NaCl solution is not drastic and significant.
However, there is no observable change in egg white solution when distilled water is added to it. As water molecules do not interrupt bonds inside native conformation; as a result, the solution remains clear.
Egg proteins are coagulated as water-soluble albumins present in egg white are denatured to insoluble form during cooking. Precipitation and coagulation is resulted; hence, white precipitate or coagulum is observed.
Egg Custard
The watery mixture is thickened after 30 minutes of baking. The mixture changes from liquid state to semi-solid state. A gel-like structure is formed. This textural and appearance change is due to heating process.
Egg white protein, ovalbumin, and milk proteins, casein micelle and whey protein, are involved. Custards are thickened by the coagulation of ovalbumin, casein and whey proteins during baking. They denature and loss their native conformation. The unfolded polypeptides re-form in groups with new cross bonds between them. They re-combine; meanwhile, some liquid is trapped in between the networks and form a network to form a semi-solid gel.
Denaturation of Milk
Data:
pH
80ml of milk (without acid added)
6.63
48 drops of 1 M acetic acid added to 80ml of milk
5
TABLE 3.1 pH of 80ml of milk before and after adding 1 M acetic acid
24 drops of 1 M Acetic Acid
1 ml 5 M NaCl(aq)
CONTROL
Time required for incubation (minutes)
3
17
18
Appearance of the mixture after incubation
White ppt. is observed on the inner wall of the boiling
Large lump of coagulum is formed at the bottom of the mixture
White ppt. is observed on the inner wall of the boiling
Curd is observed at the bottom of the mixture
White ppt. is observed on the inner wall of the boiling
TABLE 3.2 Time required for incubation and the visual appearance of the mixture after incubation of different experimental setups and the control
Discussion:
2 layers are observed in the centrifuge tube. The top layer is a supernatant and the bottom layer is a coagulum.
Supernatant is watery; the watery texture is mainly due to the presence of water. Water and water soluble substances e.g. lactose, minerals, water-soluble vitamins, some other proteins compose this layer. Coagulum is the result of protein coagulation; hence the bottom layer consists of milk protein mainly.
There is a difference of milk fraction in the two phases as centrifugation separates layers in accordance with their mass. Substance with heavier mass i.e. protein becomes sediments and substances with lighter mass i.e. water and water soluble substances form the supernatant.
Yogurt
The appearance of the commercial yogurt is set and slightly firm. It gives a light, smooth, and fluffy mouthfeel. Sourness is also detected.
A cultured yogurt is made after two days of fermentation. The cultured yogurt exists in both liquid and semi-liquid state that the yogurt is not set and gives a watery surface. It gives a thicker mouthfeel than the commercial product; at the same time, it is grainy and rough. Sourness is also enhanced, as well as the yogurt gives a tangier taste of milk.
Milk protein denaturation have contributed to the rough texture and enhanced sourness. In the cultured sample, the sourness enhanced. The increasing number of lactose-consuming bacteria and microorganisms from surrounding trapped inside the culture during preparation are possible reasons resulted in excessive lactic acid production; hence, the yogurt is sourer than the commercial product.
The lactic acid produced by the bacteria disrupted the milk protein structure. When two casein micelles collide, they ricochet and stay intact. However, when the casein micelles are destabilized by acidic environment, micelles with rough surface due to denaturation stick together. Acidic environment is produced by lactic acid-producing bacteria. Protein denaturation followed by coagulation is resulted in acidic environment. Milk curd is observed by naked eyes. Yogurt requires an incubation period for the coagulation. During incubation, lactic acid causes the milk to curdle and decreases yogurt’s flowability. As a result, a thicker and grainy texture is tasted.
Yogurt is made by bacterial fermentation. Milk undergoes heating process, homogenisation and cooling to allow the addition of bacteria or starter culture. At favourable temperature and moisture condition, bacteria grow and release enzyme which enables to lactose fermentation to produce lactic acid. Acid plays a role in yogurt’s sour flavor.
Acid denatures milk protein molecules and resulted in coagulation. The coagulum is set and forms yogurt. Other than milk protein, fluid takes a proportion of milk. Fluid in milk is called whey. Without stabilizing whey, it separates from the yogurt after placing for a long period of time, partial shrinkage of casein protein network. Clustering of casein micelles are formed in other areas. To finish the last step of a yogurt, stabilizer e.g. gum has to be added to prevent phase separation so that whey is immobilized and retained within the yogurt. This enhances the smoothness and creaminess. Yogurt is set.
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