Copy of Tamas Misky - Biology Internal Assessment

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How does the fatty acid concentration differ between 5cm³ of different types of milk, using 5% Porcine (sus) pancreatic lipase solution to metabolise lipids into fatty acids and glycerol after 5 minutes in a water bath at 40ºC, using phenolphthalein as a pH indicator to measure the percentage change in light absorption using a colorimeter (800nm)? Personal engagement: Milk is known for having a relatively high fatty acid concentration and I have been drinking a lot of milk lately due to doing a lot of sports which require a lot of energy. Fatty acids provide several vital functions in the body, including energy storage. If glucose is not available to be used for ATP synthesis, for energy, the body turns to fatty acids to power the cells. This is important for me as many times that I go to training I cannot eat any nutritious food which could fuel my body and muscle cells during training. I am a water polo player and therefore some days we swim as much as 8km a day and I need a lot of energy for that. Fatty acids have been shown to accelerate the rate at which muscles can recover during intensive physical activity and therefore help you to last longer in what you are doing 1 . This would be very important for me, therefore this was the reason I chose to investigate how the fatty acid concentrations in different kinds of milk to have an idea of which milk has the highest concentration of fatty acids, therefore, help me in my aim of lasting as long as possible at 100% each training. Another thing that this investigation could help us with is to determine which type of milk is the best to drink in order to gain weight in underdeveloped countries that lack food such as Ethiopia, as a higher fatty acid concentration in the milk means it makes it easier to gain weight. Exploration : Enzyme Background Enzymes are biological catalysts that aid in the catalysis of chemical processes. Enzymes are stated to be selective to their substrates, which indicates that they can only speed up the substrate's reactions and are not consumed in the process. Enzymes employ an induced fit model (seen on the right) 2 . This concept proposes that when the active site is exposed to a substrate, it undergoes a conformational shift to enhance binding. The active site is the area of the enzyme where the substrate may bind (picture below) 3 . When the enzyme and 3 Enzymes - Lock&Key , Online 2 Sapkota, Anupama. “Enzymes- Definition, Structure, Types, Mode of Action, Functions.” Online 1 EW;, Askew. “Role of Fat Metabolism in Exercise.” Clinics in Sports Medicine , U.S. National Library of Medicine, Online 1

substrate join together, and changes in the electron distribution in the chemical bonds of the substrate occur, which speeds up the reaction and results in the formation of products. This is accomplished by catalysis. As seen in the picture, the substrate can bond with the active site via opposing charges due to polarity and utilizing its hydrogen bonds. 4 Lipase: Lipase (porcine)is an enzyme that breaks down triglycerides (lipids), into three fatty acid molecules and glycerol using water. I will use Lipase in order to break down the lipids in the milk into fatty acids as my investigation aims to find the amount of fatty acids in the milk. This is done by a hydrolysis reaction catalyzed by lipase, breaking down the ester bonds in lipids and fats to produce fatty acids, glycerol, as well as alcohols. An ester bond can be defined as a bond formed between an alcohol group (OH) and a carboxylic acid group (COOH), and a hydrolysis reaction is the addition of water in order to break an ester bond into a Carboxylic Acid (fatty acids in this case) and an Alcohol, by the addition of a water (H 2 O) molecule. Sodium Carbonate: Sodium Carbonate is a dicarbonic salt that has alkalizing properties. For me, it is important due to its alkalinising properties in order to be able to make phenolphthalein useful to see a colour change from pink due to the alkaline environment Sodium Carbonate will create, to see through as fatty acids are created from ester bonds in lipids. Background of phenolphthalein: Phenolphthalein is an organic substance used as an indicator of acid or base. In a basic solution, it appears pinkish, while in an acidic solution, it is colourless. The pH scale ranges from 0 to 14 to identify acids and bases, with acids ranging from 0 to 6.9 and bases ranging from 7.1 to 14 5 . Phenolphthalein is colourless by nature. In alkaline conditions or bases, it changes colour to pink; and in acids, the molecule stays colourless It becomes pink around pH 8.2 and continues to develop brilliant purple in strong bases. Normally, the solution appears colourless and absorbs all colours of light, but when exposed to alkaline, it begins to block the blue colour of the light spectrum, turning the solution pink. 6 This is what we expect Sodium Carbonate to do. This colour change is important for the experiment as the milk turns from alkaline to acidic as Lipase is breaking apart the ester bonds in lipids and turning them into fatty acids, which is going to decrease the pH of the milk making the Phenolphthalein turn back to colourless. Phenolphthalein will indicate this change, and a colorimeter will measure the light absorbed; as it decreases the more acidic the greater the presence of fatty acids making the milk decrease in pH. Milk choices: I am choosing: cow’s milk, goat milk, coconut milk, almond milk and soy milk. Cow’s milk contains more or less about 3.5 - 5% lipids of its total composition and 98% of these lipids are triglycerides which are most commonly made up of 3 fatty acid chains. 7 The concentration of lipids however varies between different kinds of milk for obvious reasons like its purpose; therefore so does the concentration of fatty acids vary between these kinds of milk 7 CJ;, Jensen RG;Ferris AM;Lammi-Keefe. “The Composition of Milk Fat.” Journal of Dairy Science , U.S. National Library of Medicine, Online 6 Ranguwar, Shashank. “Phenolphthalein Indicator: Structure, Formula, Application, Uses.” Testbook Learn , Testbook Learn, Online 5 Ranguwar, Shashank. “Phenolphthalein Indicator: Structure, Formula, Application, Uses.” Testbook Learn , Testbook Learn, Online 4 Enzymes - Lock&Key , Online 2

Lipids and Fatty acids: Lipids are not only essential components of the cell membrane, that play important roles in energy storage, insulation, and vitamin absorption. Soy milk is most abundant lipids are phospholipids, followed by triacylglycerols and glycolipids. Soy milk contains predominantly polyunsaturated fatty acids, such as linoleic and linolenic acid, which are important for the proper functioning of the human nervous system. Goat milk is rich in medium-chain fatty acids, which are easier to digest and absorb than the long-chain fatty acids found in for example cow's milk. It also contains high levels of conjugated linoleic acid 8 which is a polyunsaturated omega-6 fatty acid. Coconut milk is unique in that it is high in saturated fatty acids, specifically lauric acid. It also contains medium-chain fatty acids, which are easily absorbed and converted into energy by the body. Cow's milk 9 is composed of approximately 98% triacylglycerols, which are predominantly made up of long-chain fatty acids. Which takes a long time to break down by lipase. These fatty acids, such as palmitic and stearic acid, have been associated with an increased risk of heart disease. However, cow's milk also contains small amounts of conjugated linoleic acid and omega-3 fatty acids, which have been shown to have health benefits. Finally almond milk although having a low fat content, mostly contains monounsaturated and polyunsaturated fats, which are regarded as healthy fats. Colorimeter: Lightwave absorption may be measured using a colorimeter. The change in the intensity of electromagnetic radiation in the wavelength visible to the eye segment of the spectrum following transmission or reflection by an object or solution is measured during colour measurement. Because the amount and colour of light absorbed or transmitted relies on the parameters of the solution, including the number of particles in it, such a test can aid in determining the concentration of substances within a certain solution 10 . I'd be comparing the wavelengths in the milk before and after adding the Lipase. It would be pink due to the Sodium Carbonate making milk alkaline and the phenolphthalein indicator working together turning the milk pink. Assuming that the Lipase would catalyse the lipids in the milk, the solution would become transparent after a specific time period, giving me quantitative data from which I could estimate which milk allows for the maximum breakdown of lipids into fatty acids. Because the pink colour of phenolphthalein has a maximum absorption at around 550–570nm, the light absorbed by a phenolphthalein indicator moving from pink to colorless would be measured using a colorimeter calibrated to 800nm. Hence, a colorimeter with a wavelength of 800 nm would be appropriate as it can detect the changes in the absorption of light by the solution in order to precisely quantify the colour shift from pink to colourless. This is due to the colorimeter's reduced sensitivity to any pink colour that might still be present in the solution and its capacity to measure the change in absorbance brought on by the acidic solution's addition more precisely than it would otherwise. 11 Aim: I aim to investigate which milk out of the five I will be looking at has the highest concentration of fatty acids within them. I will use 5 different kinds of milk and repeat the experiment 5 times in order to ensure it is repeatable 11 “UV-Vis Spectroscopy: Principle, Strengths and Limitations and Applications.” Analysis & Separations from Technology Networks , Online 10 “Colorimeter.” Colorimeter - an Overview, Online 9 Paszczyk, Beata, and Elżbieta Tońska. “Fatty Acid Content, Lipid Quality Indices, and Mineral Composition of Cow Milk and Yogurts Produced with Different Starter Cultures Enriched with Bifidobacterium Bifidum.” 8 “What Is Linoleic Acid?” Zero Acre Farms: Meet Cultured Oil, Online 3

Research questions: Determine how the fatty acid concentration differs between 5cm³ of different types of milk, when catalysed by 1cm³ of 5% lipase and added 7 cm³ of 0.05mol dm-3 Sodium Carbonate solution along with 8 drops of 5% phenolphthalein as the pH indicator after 5 minutes in a water bath at 40C, through measuring light absorption using a colorimeter before and after the addition of Lipase. Hypothesis: The alternate (H1) hypothesis is that animal milks are expected to have the highest concentration of fatty acids due to their longer unsaturated lipid chains that will turn more ester bonds into fatty acids, while plant milks are expected to have the least due to their typically lower unsaturated lipid chains and calorie content, used for internal processes. The null (H0) hypothesis, however, is that the concentration of fatty acids is independent of the types of milks. Apparatus: Equipment Absolute uncertainty Relative uncertainty % uncertainty 5cm³ syringes x 5 to measure 5cm³ milk +/-0.05cm³ 0.05/5.00 = 0.01cm³ 0.01x100 = 1% 2cm³ syringes x 5 to measure 1cm³ Lipase +/-0.05cm³ 0.05/1.00 = 0.05cm³ 0.05x100 = 5% Stop clock +/-0.01ms 0.01/5.00(milliseconds) = 0.002ms 0.002x100 = 0.2% 26 cuvettes N/A N/A N/A Colorimeter +/-0.001 L mol -1 cm -1 N/A See below in quantitative data collection Porcine (Sus) Lipase solution (5%) 5.00g Lipase: +/-0.05g 100ml water: +/-0.05ml 5.00g Lipase: 0.05/5 = 0.01g 100 ml water: 0.05/100 = 0.0005ml 5.00g Lipase: 0.05 x 100 = 5% 100 ml water: 0.0005x100 = 0.05% Sodium Carbonate solution (0.05mol dm -3 ) 5.00mol Sodium Carbonate: +/-0.05g 100ml water: +/-0.05ml 5.00g Sodium Carbonate: 0.05/5 = 0.01g 100 ml water: 0.05/100 = 0.0005ml 5.00g Sodium Carbonate: 0.05 x 100 = 5% 100 ml water: 0.0005x100 = 0.05% Phenolphthalein (1%) 1.00g Phenolphthalein: +/-0.01g 100ml water: +/-0.05ml 1.00g Phenolphthalein: 0.01/5 = 0.002g 100 ml water: 0.05/100 = 0.0005ml 1.00g Phenolphthalein: 0.002 x 100 = 0.2% 100 ml water: 0.0005x100 = 0.05% 5 different milks of 5cm³ +/-0.05cm³ 0.05/5.00 = 0.01cm³ 0.01x100 = 1% Distilled water measured using a syringe +/- 0.05cm³ 0.05/2.00 = 0.025cm³ 0.025x100 = 2.5% Test Tube N/A N/A N/A Water Bath +/-0.01° C 0.01/40 °C = 0.00025°C 0.00025x100 = 0.025% Thermometer +/-0.05°C N/A N/A Test tube rack N/A N/A N/A 4

Justification of materials used: Because the uncertainties are so little, the apparatus will have little influence on my results. There were material modifications after preliminary testing, which needed extra cuvettes to guarantee there was no contamination between experiments and a change in the concentration of the lipase which in the preliminary was too small being only 1%; this change proved to be vital as it was what caused the whole experiment to work. Preliminary testing: The preliminary testing allowed me to test each type of milk I chose to use such as almond, coconut, soy, goat, and cow milk. This was very helpful as change could be seen in the cow’s milk which was expected to have the greatest amount of fatty acids after adding the 1% concentration lipase solution but for the other milks, it was not doing anything. This prompted me to try out different concentrations of Lipase and after thinking it through I chose to use the 5% concentration Lipase solution because that would no doubt have an effect on each of the milks, and it did. I was able to establish that all control variables were the same and that the process was reproducible by 5 repetitions, in addition to the low standard deviation supporting this. I also examined the qualitative data to guarantee that the translucency was seen not just through the colourimeter. Furthermore, I investigated what temperature was optimal for the Lipase I was using and discovered that 40° C 12 was the best to use since this is its optimum temperature and hence indicates more successful collisions therefore catalysis was occurring. I also tested multiple timings to determine that 5 minutes was the ideal time for the reaction to occur, so I tried 3 minutes with very little change, 4 minutes with still little change, and 5 minutes with both qualitative and quantitative change, prompting me to use 5 minutes as the time I will wait. Independent variable: My independent variable will be the different types of milk; I will use 5 different types of milk (almond milk, coconut milk, soy milk, goat milk, and cow milk) to see whether there is a link between the types of milk and the concentration of fatty acids via the breakdown of Lipids by Lipase. I'll next perform the experiment five times to ensure that the experiment is reproducible. Dependent variable: The dependent variable is the difference in milk translucency, which is measured as the quantitative data by the mean change as well as the mean % change before and after the addition, before and after the addition of lipase. The difference is calculated to determine the breakdown of lipase into fatty acids, using a colorimeter. As a result, the lower the fatty acid content, the greater the light absorption and the more acidic the solution. Qualitative data: The qualitative data would determine with the average eye whether the solutions has become transparent or white. This would obviously be more biased towards my own perspective of what transparent or white would be. Quantitative data: This is my colorimeter calculation, calculating the translucency of the milk before and after adding Lipase to my milk, which has previously had sodium carbonate and phenolphthalein added to render it pink. 12 Importer. “Temperature's Effect on Enzyme Activity.” Online 5

Method: 1. Get 5% Lipase solution prepared by a laboratory technician 2. Get 5% Sodium Carbonate solution prepared by a laboratory technician 3. Get 1% Phenolphthalein solution prepared by a laboratory technician 4. Calibrating the colorimeter 13 : 1. Slide the lid of the Colorimeter open to reveal the cuvette slot. 2. Set to wavelength 800nm 3. Insert a cuvette, filled with distilled water. 4. Press the CAL button on the Colorimeter to begin the calibration process. 1. Preheat the water bath to 40° C. 14 2. Place a test tube rack into the water bath 3. Insert the Lipase, Sodium, Phenolphthalein solutions and milk into the water bath until they are all at 40°C. 4. Using different 5cm 3 syringes put 5cm 3 of each milk being tested for into separate test tubes and place them into a test tube rack 5. Using a different and clean 5cm 3 syringe measure out 7cm 3 of the Sodium Carbonate solution for each milk and put it into the milks 6. Using a pipette drop, 8 drops of phenolphthalein into each of the milks 7. Using a pipette fill a cuvette of each of the milks and put them into the colorimeter before adding the lipase 8. Recalibrate the colorimeter using the cuvette with distilled water in it 9. Using the 2cm 3 syringe measure out 1cm 3 of Lipase solution for each of the 5 milks 10. Add the 1cm 3 of Lipase to each milk in the test tube and immediately start the stopwatch 11. After 5 minutes remove the milk being tested from the water bath and put it in a cuvette and measure its absorbance rate using the colorimeter. 12. Repeat steps 4-11 with a different milk 13. Calculate the difference between the absorbance rates of each milk before and after the addition of Lipase 14. Recalibrate the colorimeter using the cuvette with distilled water in it 15. Rinse the cuvettes and the syringes and repeat steps 4-13 above 4 more times so there are 5 repeats for each milk 14 Importer. “Practical Biology.” Investigating Effect of Temperature on the Activity of Lipase, Online. 13 Colorimeter User Manual, Online 6

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