Investigate the effect of bile salt concentration on the digestion of milk by the enzyme lipase.

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The effect of bile salt concentration on the digestion of milk by lipase   Seham Akkad 13I

Biology Coursework

Investigation into the effect of bile salt concentration on the digestion of milk by lipase

Seham Akkad 13I

Skill P - Planning


Investigate the effect of bile salt concentration on the digestion of milk by the enzyme lipase.

Background knowledge:


In humans about 1000 cm3 of bile is produced each day. In the body, bile is synthesised in the liver, from cholesterol, by hepatocytes. The bile is then concentrated and stored in the gall bladder ready to be secreted into the bile ducts, travelling into the small intestine making the conditions alkaline.


Bile salts have two main roles; emulsification of dietary lipids and transport of lipids in a water soluble environment. The emulsification of lipids involves breaking down the large fat globules into smaller droplets increasing the surface area to volume ratio, as well as allowing lipases to access the lipids inside the droplets. As bile salts are made from cholesterol, the are involved in the transport of lipids around the body in the form of micelles, were the hydrophilic areas point outwards and the hydrophobic areas point inwards encasing lipids such as triglycerides, and allowing them to be transported. Bile salts also allow the transport of lipid soluble vitamins in the bloodstream.




Cholesterol in the bile interacts with the bile salts to form water soluble particles. If the bile contains too much cholesterol or not enough water, excess cholesterol can precipitate in the gall bladder forming gallstones. Bile pigments, such as bilirubin and biliverdin, have no known function, however, give bile its distinctive colour. Bile also contains hydrogencarbonate ions, which are alkaline and help neutralise the acidic chyme that has just come from the stomach. I will use sodium hydrogen carbonate instead of sodium carbonate to make the milk alkaline. The reason for this is I am trying to mimic the conditions found in the small intestine in the human body.

Bile also contains products from the breakdown of red blood cells, haem and globin. The haem part contains the haem prosthetic group found in haemoglobin, the iron central complex, and a yellowish greenish compound called bilirubin. The bilirubin is an excretory product from the liver. The globin contains the polypeptide chain from the haemoglobin molecule. This protein can be hydrolysed into its constituent amino acids, which can later be used for protein synthesis.



Lipases are a group of water soluble enzymes that work to catalyse the hydrolysis of the fatty acid ester bonds found in water insoluble lipids. However, as the reaction is reversible the enzyme is also able to catalyse the reverse reaction; the esterification of glycerol and fatty acids into triglycerides.

Most lipases are formed in the pancreas, and then secreted into the ileum. In the small intestine lipases are adsorbed by the glycoprotein on the surface of the epithelial cells of the villi. This allows digestion to occur efficiently near to the area where the products need to be absorbed.

As lipases are soluble and work on insoluble substrates, they must work in emulsions at the oil-water interface. However, as the substrate is in equilibrium with the emulsion and oil state, the rate of reaction actually depends on the amount of substrate present in the emulsion state, which depends on the concentration of oil. Additionally, products that have already been hydrolysed may build up at the oil-water interface and so reduce the rate of reaction. Lipases sometimes need to be activated. A way to overcome both these problems would be to use an emulsifying agent, such as bile salts, to increase the surface area at the interface.

Lipases are enzymes. Enzymes are protein molecules which speed up the rate of reaction, however are not used up in the process. They can be defined as biological catalysts. Nearly all reactions which take place in a living organism take place with an enzyme present.

Enzymes are made of a specific sequence of amino acids which are coded by in the DNA. This specific sequence makes up the enzymes primary structure. The secondary structure is made when the peptide group of the amino acids interact to form hydrogen bonds between the molecules. There are three types of folding that occur; alpha helix, beta pleated sheet or a random shape. The tertiary structure is the enzymes 3D structure and occurs when numerous bonds form between the R-groups on the amino acids. Such bonds include disulphide bridges, ionic interactions, hydrogen bonds and hydrophobic interactions. The tertiary shape of the enzyme is of utmost importance as it dictates the shape of the active site of the enzyme.  

Alpha amino acid                        Peptide linkage -CONH-

Every type of enzyme can only work on one variety of substrate. This is because the substrate has to fit perfectly into the active site of the enzyme. The substrate binds with the enzyme forming the enzyme-substrate complex. This is called the lock and key mechanism. Alternatively, the substrate may be a slightly different shape and because the enzyme contains some flexibility it may be induced to change its shape slightly to fit in the molecule. However, an enzyme is specific for its substrate and only works to catalyse that reaction. In this case the enzyme is lipase, produced in the pancreas, and the substrate is lipids and triglycerides. The optimum pH and temperature for lipase activity is 8.0 - 9.0 pH and 30°C – 40°C respectively.

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A substrate bonds to the active site, and the enzyme works on catalysing the reaction. This can either be the splitting of the substrate into numerous products, or of the two pieces of the substrate molecule joining together. Interaction between the R groups of the enzyme and substrate can promote the breaking or formation of bonds, ultimately helping the enzyme’s main job of catalysing the reaction.  


Milk is an emulsion that is full of nutrients that is secreted by a female mammal. An emulsion is two liquids or a liquid and a solid that are not soluble in each other and so form droplets.  Milk is slightly acidic. It is also a buffer which resists small changes in pH. The main constituents of milk are water, unsaturated lipids, proteins, lactose, vitamins and minerals. Lipids contained in milk are naturally arranged in fat globules due to the insolubility of lipids in water. This emulsion can be broken down by shaking or stirring.

The lipids found in milk are triglycerides. Triglycerides are esters of three fatty acid chains linked to a glycerol molecule by three ester bonds. When the molecule is hydrolysed in digestion, the molecule is split into its constituents and is therefore acidic due to the presence of carboxylic acid group.




Digestion is the breaking down of large macromolecule polymers into smaller micro monomers which are then absorbed by the body. The body is then able to rebuild these small molecules into larger more complex ones such as proteins and lipids which are needed in the body. The body digests food by mechanical and chemical methods. The mechanical movement of the mouth and teeth as well as the churning of the stomach are both examples of mechanical digestion. Chemical digestion in the body occurs with the aid of enzymes that help catalyse hydrolysis. Chemical digestion occurs in the mouth, stomach and small intestine.


In a reaction, increasing the temperature of the reactants usually increases the rate of reaction. This can be explained using the molecular kinetic theory. Temperature is proportional to the kinetic energy in a closed system; therefore increasing the temperature increases the kinetic energy of the molecules and subsequently the rate of reaction. This is because as the molecules travel faster they are more likely to collide with other molecules and they are also more likely to have the activation energy required to initiate the reaction.

However, when the reaction is catalysed by an enzyme, the rate of reaction often peaks at the optimum temperature and then dramatically falls. The reason for this is because even though the molecules have more energy, the enzyme is denatured. As the temperature increases, the vibrational energy also increases. This affects the secondary and tertiary structures bonds, weakening them and eventually breaking them. The weakest bonds break first such as hydrogen bonds, and the stronger bonds break later. This is why enzyme activity drops quickly but not immediately as the enzyme is denatured gradually. The breaking of the bonds alters the shape of the enzyme and thus the shape of the active site. This means that substrates can no longer bind to the enzyme and so the reaction cannot take place.


pH has a huge influence of the rate of reaction of an enzyme controlled reaction, as it affects the stability of the tertiary structure of the enzyme. pH affects the tertiary structure, specifically the ionic interactions. If there is a large change in pH some of the charged groups charges change, e.g. at low pH: COO- + H+ changes to COOH and at high pH: NH3+ changes to NH2 + H+. This means that the ionic interaction no longer exists and so the tertiary structure breaks and the 3D structure alters and thus the active site changes. This means that substrates can no longer bind to the enzyme and so the reaction cannot take place.

Substrate Concentration:

Increasing the substrate concentration increases the rate of reaction. This is because the substrate is more likely to collide with an enzyme and have the activation energy needed to produce the products. However, after a certain point, increasing the substrate concentration no longer increases the velocity of the reaction. This is because all the enzymes present have already formed the enzyme-substrate complex and so in essence there are no free active sites available for the catalysis of the increased substrate. The rate of reaction has reached its maximum value (Vmax).

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Competitive inhibitors compete with the substrate and bind to the active site of the enzyme and therefore do not allow the substrate to be converted into its products. This also reduces the rate of reaction. Non-competitive inhibitors bind to the allosteric site of an enzyme and change the active site slightly; therefore the substrate can still bind to the active site. However, it cannot be changed into its products until the inhibitor has moved. This has a detrimental effect on the rate of reaction.

Reversible inhibitors only bind to the enzyme for a limited time and ...

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