Conclusions - concentration affecting lipase
It is clear from the graphs, tables and statistical processes carried out that generally, increasing the volume of lipase increases the change in pH of the solution and also the rate of change of the pH of the solution. This has been proved by the increasing values of standard deviation for higher volumes of lipase, the generally larger maximum change in pH for the higher volumes of lipase and also by the shape and gradients of the graphs drawn. Although there have been anomalies, having used the t-test it can be seen that it is far more likely that the results are not down to chance, but due to the differences between the volumes of lipase being compared. The only differences between each volume of lipase are the number of enzymes and therefore active sites present in the solution. The reason for higher volumes of lipase decreasing the pH more quickly and by a larger amount can be explained by the number of enzymes present and the hydrolysis reaction taking place between the lipids present in the full fat milk.
Lipase oxidises triglycerides, using three molecules of water to break the 3 ester bonds and to produce a glycerol molecule and three fatty acids. Lipids, because of their hydrophobic fatty acid tails do not dissolve in water, and so during fat digestion in the human body, these lipids must first be emulsified by bile salts such as sodium glycocholate and sodium taurocholate. The release of bile into the duodenum is stimulated by the hormone CCK. For human digestion, the bile salts break down the fat droplets present in the lumen of the small intestine into tiny globules helping them to disperse into the watery fluids in the intestine. The lipase enzyme however is made of a globular protein, and because of the hydrophilic ‘R\' groups on the outside of its structure the enzyme can dissolve in water. In my experiment, the volume of bile salts and full fat milk remained constant, so the amount to which the globules of fat present in the full fat milk were emulsified should have remained the same for each volume of lipase. The bile salts should have enabled the lipase enzymes to act on more lipids present in the solution. As can be seen with some volumes of lipase, the pH first rose before it started to steadily fall, this may have been related to the fact that the bile salts were still emulsifying the fats. If the globules of fat were large, then their surface area available to the active site of the lipase enzymes would have been much smaller and so the rate of the reaction would have proceeded at a much slower rate.
The fact that the bile salts also contains hydrogen carbonate ions can also explain why the pH after one minute was relatively alkali, approximately varying between 7.30 - 9.73. The optimum pH for the lipase acting in the duodenum of the small intestine is approximately 7.00, so theoretically, the 5.00ml3 of lipase should have been at a slight disadvantage despite the fact that it actually produced the fastest rate of change of pH for experiment 2.
Following the emulsification of the lipids present in the full fat milk, the lipase would have then collided with the tiny globules of fat, forming enzyme-substrate complexes. The lipase would have then broken the 3 ester bond holding the triglyceride together in a hydrolysis reaction requiring three molecules of water. In the human body, the products of this reaction would most likely be converted to other things or stored in adipose tissue, rather than accumulating and creating an acidic environment. In the liver, the fatty acids can be converted to acetyl coenzyme A, which can then be fed into the Krebs cycle, while the glycogen can be phosphorylated to triose phosphate, an intermediate in glycolysis. Clearly in the test tube no such processes would be happening as there are no hepatocytes present in the solution and no respiring organisms (provided the test tube was sterile). The fatty acids and glycerol would have therefore accumulated in the test tube.



Glycerol is an alcohol and looking at its material safety data sheet (see bibliography) it should have a neutral pH. The fatty acids