Difference In Protein Structure And Composition Between Cow, Goat And Soya Bean Milk

The aim of this investigation was to explore any differences between the chemical composition of Cow, Goat and Soya Bean milk through the use of enzyme Rennin. From analysis of results of the experiment it was found that:

Procedure:

1) A water bath should be set at 37 C. Allow at least 30 minutes for temperature to stabilise. 2) 3 of the 6 beakers should be set up in preparation of the draining process which will take place later on in the experiment:

.Label each beaker with the Initials C, G and S to represent Cow, Goat and Soya Bean milk. .Place a funnel in each beaker. .Take 3 pieces of filter paper and label each with the initials C, G and S to represent Cow, Goat and Soya Bean milk. .In each funnel, insert ONE piece of labelled filter paper into respectively labelled beakers. To make the filter stay in place, wet the filter paper with DISTILLED WATER. (It is important that ONE piece of filter paper is placed in each funnel otherwise this will affect the draining rate)

3) The last of the 3 beakers should be labelled with the Initials C, G and S to represent Cow, Goat and Soya Bean milk. 4) At least 20cm3 of each milk source should be poured into their respective labelled beakers. (This is to allow for easy extraction of milk from beaker into test-tube) 5) Shake bottle containing Rennine. Using 1 of the 4 graduated pipettes provided, 3cm3 of Rennin enzyme solution should be measured and poured into 3 test-tubes each. 6) Place test-tubes containing Rennin in test-tube rack 7) The remaining of the 3 test-tubes should be labelled with the Initials C, G and S to represent Cow, Goat and Soya Bean milk. 8) Using 1 of the 3 remaining graduated pipettes, measure 10cm3 of Cow milk from respective beaker and release into respectively labelled test-tube. 9) Place test-tube in test-tube rack. 10) Using 1 of the 2 remaining graduated pipettes, measure 10cm3 of Goat milk from respective beaker and release into respectively labelled test-tube. 11) Place test-tube in test-tube rack. 12) Using the last of the graduated pipettes, measure 10cm3 of Soya bean milk from respective beaker and release into respectively labelled test-tube. (It is important to use different graduated pipettes for each milk source to avoid contamination) 13) Place test-tube in test-tube rack. 14) All 6 test-tubes (3 containing Rennin and 3 containing different milk sources) should be placed in immersible test-tube rack. (It is important that test tubes are first placed in immersible test-tube rack BEFORE the immersible test-tube rack along with the test-tubes are placed in the water bath.) 14) Place immersible test-tube rack containing 6 test-tubes into water bath and start stop watch immediately. 15) Test-tubes should be allowed to equilibrate for 15 minutes. 16) After equilibration time of 15 minutes, stop stop-watch. 17) Pour a test-tube of 3cm3 of Rennin into each test-tube containing different milk sources. (it is important that the rennin is poured INTO the milk and NOT vice-versa. This is because when equilibrated, the rennin solution is less viscous than the milk and has a greater chance of all its contents being poured successfully from the test-tube when compared with milk) 18) Start timer and make note of any significant observations noticed during reaction in Table 3 below. 19) Allow reaction time of 15 minutes. 20) After reaction time has elapsed, stop timer. 21) Pour contents of each labelled test-tube into respectively labelled beaker-funnel apparatus ( prepared in step 2). 22) Rinse each respective test tube, one at a time, with distilled water to ensure all contents of test-tube are poured into its respective labelled beaker-funnel apparatus. 23) Allow 1 hour for contents from test-tube to drain in beaker. 24) Remove filter papers containing solid coagulated milk and place in oven at temperature of 15 C 25) Allow 24 hours for filter paper containing solid coagulated milk to dry.

26) Remove from oven and weigh each of the 3 filter papers containing coagulated mass on balance and note mass. 27) Place filter papers in oven again for 1 hour. 28) Remove from oven and weigh each of the 3 filter papers containing coagulated mass on balance and note mass again. 29) Repeat steps 27-28 until mass of filter paper does not change. 30) Note the final mass of filter paper WITH coagulated mass in Table 3 Below. 31) Calculate mass of coagulated solid by subtracting mass of filter paper from mass of filter paper WITH coagulated solid. ( To obtain mass of filter paper, this may either be written on packet of filter paper OR can be obtained by calculating the mean average of 3 UNUSED filter papers.

32) Note mass of coagulated solid for each milk source in Table 3 below. 33) Repeat steps 1 - 32 again until you have results for 3 repetitions.

Cow Milk Goat Milk Soya Milk Observation at 0 - 5 minutes Observation at 5 -10 minutes Observation at 10 - 15 minutes Reaction Time Mass of Filter Paper (±../ g ) Mass of Filter Paper WITH coagulated solid (±/g) Mass of coagulated solid

Table 3: Shows blank table of results.

*

Having described the procedure used in the experiment, a table of results obtained from my conduction of the experiment can be drawn. The experiment was repeated 3 times to produce 3 sets of results each showing observations noted in each repetition.

Repetition 1 Cow Milk Goat Milk Soya Milk Observation at 0 - 5 minutes No coagulation noted Specks of coagulated solid already visible on surface of test-tube seen at 2 minutes. No coagulation noted Observation at 5 -10 minutes At 5 minutes, beginning to show signs of coagulation - seen as little specks Clear liquid already visible in test-tube due to fast rate at which at which milk has coagulated No coagulation noted Observation at 10 - 15 minutes Clear liquid beginning to appear Clear liquid at bottom, coagulated mass at top of test-tube. No coagulation noted Reaction Time 15 minutes 15 minutes 15 minutes Mass of Filter Paper (±../ g ) 1.09g 1.09g 1.09g Mass of Filter Paper WITH coagulated solid (±/g) 1.67g 1.55g 1.10 Mass of coagulated solid 0.58g 0.46g 0.01

Table 4: Shows results and observations noted during repetition 1 of experiment.

Repetition 2 Cow Milk Goat Milk Soya Milk Observation at 0 - 5 minutes No coagulation noted Immediate coagulation noticed at 30 seconds No coagulation noted Observation at 5 -10 minutes Only begun to show signs of coagulation at 5 minutes Clear liquid already beginning to appear No coagulation noted Observation at 10 - 15 minutes Clear liquid beginning to appear with large clumps visible (clumps are smaller compared to those seen in Goat milk) Clear liquid with very large clumps of coagulated solid visible No coagulation noted Reaction Time 15 minutes 15 minutes 15 minutes Mass of Filter Paper (±../ g ) 1.09g 1.09g 1.09g Mass of Filter Paper WITH coagulated solid (±/g) 1.70g 1.51g 1.11g Mass of coagulated solid 0.61g 0.46g 0.02g

Table 5: Shows results and observations noted during repetition 2 of experiment.

Repetition 3 Cow Milk Goat Milk Soya Milk Observation at 0 - 5 minutes No coagulation noted Large clump seen at bottom of test-tube at 1 minute No coagulation noted Observation at 5 -10 minutes No signs of coagulation yet at 5 minutes 70% of milk appears to have coagulated No coagulation noted Observation at 10 - 15 minutes Clear liquid with small clumps of coagulated solid seen. Clear liquid at top with very large clumps of coagulated solid at bottom No coagulation noted Reaction Time 15 minutes 15 minutes 15 minutes Mass of Filter Paper (±../ g ) 1.09g 1.09g 1.09g Mass of Filter Paper WITH coagulated solid (±/g) 1.65g 1.59g 1.09g Mass of coagulated solid 0.56g 0.50g 0.00g

Table 6: Shows results and observations noted during repetition 3 of experiment.

Results from Table 4,5 & 6 can be compiled together to show the Mean average mass of coagulated solid for each milk source.

Cow Milk Goat Milk Soya Milk Mass of coagulated solid in Repetition 1 of experiment (g) 0.58g 0.46g 0.01 Mass of coagulated solid in Repetition 2 of experiment (g) 0.61g 0.46g 0.02g Mass of coagulated solid in Repetition 3 of experiment (g) 0.56g 0.50g 0.00g Mean Average mass of coagulated solid. 0.58g 0.47g 0.01g/nil

Table 7: Shows compiled results of experiment repetitions together with calculated mean mass averages.

Analysis of Results:

From Table 7, it is shown that Soya Milk had the lowest mass of coagulated solid; 0.01g while Cow Milk had the highest mass of coagulated solid; 0.58g.

The essentially nil value of 0.01g obtained for Soya Milk will first be addressed. This can be explained by the specific nature of enzymes in general; enzymes are shape specific and will only work/act on equally shape specific substrates. In the same way, Rennin will only act on Kappa-Casein (K-casein) , a type of protein which can be found in all Milks. However, Soya Milk is not "pure milk".

Soya Milk is made from Soya Beans. An outline of the preparation process of Soya Milk is shown below:

1 Soya Beans are soaked for a minimum of 3 hours 2 Skin of Beans are removed by kneading the beans 3 Soaked Soya Beans are then ground with water to produce a paste. 4 The paste mixture is then sieved; the liquid collected from this process is what is known as "Soy Milk". 5 The "Soy Milk" is then boiled.

In the presence of Rennin enzyme, Soy Milk will NOT produce any coagulated solid because Soy Beans originally do not have the protein Kappa-Casein present in them. As mentioned before, enzymes are shape and substrate specific; this therefore makes it impossible for Rennin to act on a k-casein containing micelle structure that is simply NOT present in Soy Milk. This is the reason for the nil value obtained for Soy Milk during the experiment.

On the other hand, Cow and Goat Milk react with Rennin because they both have Casein Micelle structures containing k-casein protein on the exterior. From the results in Table 7, two assumptions explaining the difference in coagulated mass between Cow and Goat milk can be made.

Assumption 1: The first of these assumptions states that each individual Casein Micelle molecule/structure in Cow Milk is slightly larger than those present in Goat milk. This supposed larger Casein Micelle structure of Cow Milk will contain more Alpha (s1), Alpha (s2) and ?-Casein proteins - as introduced in Page 2. This means that on the rupturing of the Casein Micelle structure by Rennin, more of this Alpha and Beta Caseins will precipitate on exposure to calcium and form a greater mass of coagulated solid.

Assumption 2: The second of these assumptions states that Cow and Goat milk may have about the same sized Casein Micelle structures; however, a greater proportion of Cow Milk Micelle structures will be composed of Alpha (s1), Alpha (s2) and ?-Casein proteins compared to Goat milk. This also means that on the rupturing of the Casein Micelle structure by Rennin, more of this Alpha and Beta Caseins will precipitate on exposure to calcium and form a greater mass of coagulated solid.

Both assumptions have the common theme of Alpha (s1), Alpha (s2) and ?-Casein proteins being responsible for the greater mass of coagulated solid in Goat milk. However, there is strong evidence to support this theory of more of Alpha (s1), Alpha (s2) and ?-Casein proteins being present in Cow milk than Goat milk. . Various research has shown that people's preference to Goat milk rather than Cow milk lies in their dairy allergy to Cow milk-not to be confused with lactose intolerance. Alpha s-1 and beta casein proteins have been linked to being the cause of dairy allergies in sufferers.Furthermore, it has also been explained and suggested that the trace and sometimes lack of alpha s-1 and beta casein proteins in Goat milk helps to reduce this dairy allergy. In conclusion, this supports my theory that there is less Alpha s-1 and beta casein proteins in Goat milk than in Cow milk.

* Nevertheless, none of these assumptions explain why Goat Milk reacts quickest with Rennin - this is shown in observations made in Tables 4, 5 & 6; Goat milk is the first to show signs of coagulation in ALL 3 repetitions. *

Conclusion :

An explanation or theory must now be suggested which will take into account; the faster reacting Goat Milk and the greater yielding coagulated mass of Cow milk:

The difference in chemical composition in Cow and Goat Milk lies in their individual Casein Micelle structures. Cow Milk has a slightly larger Micelle structure than Goat milk. This explains the greater yield in coagulated mass in Cow milk because there is more Alpha s-1 and Beta casein proteins present in each Cow milk Micelle molecule compared to Goat milk. It is these Alpha s-1 and beta casein proteins that precipitate on contact with calcium forming the "aggregates" - these proteins are responsible for the mass of coagulated solid yielded.

The larger micelle structures of Cow milk also accounts for the slower reaction rate of Cow milk with Rennin compared to Goat milk. Due to the smaller micelles present in Goat milk, the K-casein protein on the outside of individual micelles is broken down at a faster rate compared to those of Cow milk; it takes longer to rupture the micelle structures of cow milk. This explains why Goat milk shows signs of coagulation first. "