Investigation on Protein Denaturation

By y. c. pong Introduction: When you heat an egg, the egg white clump together and turned white. It is because the protein in egg white undergoes denaturation, the cross linkage(the hydrogen bonds, ionic bonds and disulphride bonds) which maintain the protein shape destructed, so protein lose its tertiary conformation. This denaturing process is very important, because before protein can be used in digestion they must be unfolded. Part A: denaturation of egg white Aim: To examine the factors on the effect of denaturation of egg white.
Principle: As protein denaturation can be cause by several factors such as temperature, pH, salt concentration. In this experiment, we are examining how these factors affect the denaturation of protein. We use egg white, which is actually a solution of protein in water in this experiment. After the egg white had been dilute, the egg white solution can be put in 60? C and 80? C water bath to test for how temperature affects denaturation. It can be record by the time need for the first change of appearance.
To find out how pH of affect denaturation, we can add dropwise the actetic acid to the egg white solution. Beside, the NaCl can also be add dropwise to egg white, to test for how salt concentration cause denaturation. Count the number of drop of solution added for an appearance change to occur. Observation: |60 ? C water bath |After 15 minutes, a pale yellow | | |semi-solid formed | |80 ?

C water bath |After 2 minutes 30 seconds, a white jelly| | |like solid formed. | |1M acetic acid |After 10 drops of acid added, the | | |solution become pale yellow and clumping | | |of egg white occurs | |5M NaCl |After 27 drops of NaCl added, the | | |solution become yellow and clumping f | | |egg white occurs | |Add equal volume of water |No observable change | Discussion: Protein? s three dimension conformation is held by the interaction between its amino acids. This interaction included hydrogen bonds, ionic bonds and disulphride bonds. But in some extreme condition, such as high temperature, extreme pH, high salt concentration, the protein will be denaturate. It is because the cross linkage had been broken, the secondary and tertiary structure are alter.
The protein now has primary structure as peptide bonds are strong enough not to be destructed. In our experiment, we observed that the egg white clump together after denaturation (either by heat, pH or salt concentration). It is because after the normal structure is destructed, some new bonding may be formed between proteins. They can have a big clump of proteins hooked together. That is the coagulation. In coagulation process, the egg white turned from transparent to a cloudy solution, because as the protein clump together, there are no much space for light the pass through, so it looks more cloudy.
Besides, in 80 ? C boiling tube, the egg white turned white while at 60 ? C the egg white only clump to a yellow semi-solid. It is because at higher temperature more kinetic energy is provided for the molecules to vibrate, so the bondings are disrupted more greatly. So it can be concluded that a high temperature will have a stronger effect on denaturation. Part B: egg custard Aim: To measure the phrase transitions that occurs in denaturation of egg and milk as affected by temperature change. Principle:
Egg and milk are the main ingredients of an egg custard, through baking an egg custard, we can examine the phase transition of transition of denaturation of egg and milk as affected by temperature. Result: After baking the egg custard for 30 minutes, the egg custard set to a yellow smooth gel. Discussion: The egg custard is at liquid phase before baking, and it turn to solid phase after baking. It shows that phase transitions of liquid to solid had occur in the denaturation process of egg and milk as affect by high temperature. The egg custard has a smooth and spongy texture.
In the experiment, our egg custard is made of egg, milk, sugar only. Through applying of heat and manipulation, custards can become a viscous and semi-rigid gel, denaturation of the egg protein, causes a clumping of proteins, is primarily contribute for the thickening of the custard. Milk, although not as important as egg, but calcium ions present in the milk are also needed in order to form thicker custard. Sugar is also important to the viscosity and gel strength of custards. Sugar tends to increase the denaturation temperature of the egg proteins resulting in less stiff custard.
The concentration of egg protein is proportional to the viscosity or gel strength of the custard. With increasing concentration, a custard sauce becomes more viscous and the gel strength of a baked custard firmer. A custard heated slowly begins to thicken at a lower temperature, thickens gradually over a wider temperature range and result in a smoother texture. Slow cooking can be achieved by placing the custard mixture in a water bath to warm to certain temperature before put into the oven which has been skipped in our experiment
Part C: denaturation of milk Aim: To examine the factors on the effect of denaturation of milk. Principle: Milk protein, similar with egg white, will also undergo denaturation in extreme condition. So acid and NaCl is added to milk respectively, to observe the changes and examine how pH and salt concentration affect denaturation. Then, they are place in a 80 ? C water bath, also a control tube is tube is put in water bath, to examine the effect of temperature on denaturation. Observation: original milk pH= 6. 63 1 M acetic acid |60 drops of 1 m acid had been added | | |The pH=4. 67 | | |There are white ppt formed. | |Half quantity of 1 M acetic acid added |30 drops of 1M acid added there are no | | |observable change. | | |And after heat for 2 minutes, there are | | |white precipitate formed. |1 ml of 5M NaCl |No observable changes soon after addition| | |of NaCl. There are little white | | |precipitate after heat for 8 minutes. | |control |No observable changes even after 20 | | |minutes | |Centrifugation tube |3 layers formed.
The upper layer is | | |translucent, the middle one is white and | | |the bottom layer is white precipitate. | Discussion: There are 3 layers present in the centrifugation tube after centrifugation. The upper layer is the acetic acid we added, the middle layer is the milky serum, the bottom layer is the precipitate formed bring out by the addition of acid. There are 2 major components of milk, casein and serum .
The casein contains phosphorus and will coagulate or precipitate at about pH 4. 6. The serum proteins do not contain phosphorus, and these proteins remain in solution in milk at pH 4. 6. So, when acetic acid added the pH drop to 4. 69 the casein precipitate so white precipitate is seen. The addition of NaCl only cause a little precipitation, shows decrease in pH has a greater effect on milk denaturation than increase in salt concentration. There are no observable changes in control because the protein are not yet denaturated in 80 ? C.
Part D: yoghurt Aim: To experience the making of yoghurt and the theory of formation of yoghurt. Principle: As natural yoghurt is made from fresh milk without other ingredient added, it can be made by simply add a little yoghurt and milk together and incubate in 38 ? C incubator, which is a suitable temperature to make yoghurt. Yoghurt can be available for tasting after 1-2 days. Results: All milk turned into yoghurt, the sample is no longer liquid but is in semi-solid. The yoghurt is a bit sour than the original sample. Discussion:
Yoghurt is made by fresh milk, but often some specific bacteria strains are introduce into the milk. The bacteria ingest natural milk sugars and released lactic acid as a waste product. The acid decrease the pH and cause the denaturation of milk protein during which the cross linkage between protein break and protein uncoiled. Then, the protein will tangle into a solid mass, yoghurt formed In our experiment, we mixed the 50 ml milk with 5 ml yoghurt to made the sample yoghurt instead of milk only . the 5 ml yoghurt is to provide the bacteria.
Only a small amount of live yoghurt can is needed to inoculate a new batch of yoghurt because the bacteria can reproduce and multiple themselves during the yoghurt making process. Besides the incubator’s temperature is 38 ? C because it is the optimum temperature of bacteria to reproduce. The yoghurt newly made is more sour than the original sample. It may be due to the long incubation hours, as we had kept it over 48 hours. The bacteria ingest a higher % of milk sugar and release more lactic acid. To stop the incubation we can place the yoghurt in a refrigerator.

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