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

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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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Control variables: Control variable How to control it Why it needs to be controlled Temperature Through the use of the water bath I would keep a constant temperature of 40° C so that this would not affect results. If the temperature were to increase it may affect the enzyme activity of lipase and may catalyze the reaction more rapidly or more slowly if the temperature is lower. The collision theory states that the higher the temperature increases the molecules gain energy and therefore move faster and have more successful collisions so it could speed up the reaction. The time for the reaction to occur Using a stop clock and setting it go for only 5 minutes If it goes over 5 mins in one of the experiments the results may not be representative of what actually occurred in the experiment, since more reaction time may mean more enzymatic activity. The reading of the colorimeter Making sure to recalibrate the colorimeter after every use with the cuvette of distilled water. If it weren’t it could affect the way in which the other readings of the absorbance in the other kinds of milk are, this is a systematic error Volume of milk Using the syringe and making sure to measure 5cm 3 of milk every time If we have more milk in one than in other solutions it may mean that there may be more substrate, triglyceride which would result in more fatty acidsthan in others, so it wouldn't make the experiment fair or valid since the concentration of fatty acids wouldn't be representative with the results of the other kinds of milk. Volume and Concentration of Lipase Using the syringe and making sure to measure 1cm 3 of 5% lipase every time If there is more or less lipase in one or the other milk it could affect the rate at which lipids are catalyzed into fatty acids. Increasing enzyme concentration will speed up the reaction, as long as there is substrate available to bind to. Volume and concentration of Sodium Carbonate Using the syringe and making sure to measure 5cm 3 of 5% Sodium carbonate every time If there is more or less sodium carbonate in one of the other milk, they wouldn’t get the same alkaline conditions and one would be less or more alkaline making the change much more visible after adding the phenolphthalein making it unfair and not viable Volume and concentration of phenolphthalein Using the pipette to drop exactly 8 drops of 1% solution every time If there is more or less phenolphthalein in one of the milks it would cause that one has more indicator that the other which would make the change more apparent once again making one of the results unfair and unviable Age of Milk By purchasing all of the milks on the same day If some of the milk types were older than the others there may be some contamination that could contribute to certain reactions occurring faster or slower. I ensured to buy all milks at the same time and open them at the same time as well in order to eliminate this risk.. Each type of milk would be placed in different containers in order to avoid contamination too. 7

Safety precautions: Safety issue What could happen What to do if it occurs Safety precautions (preventing) Water bath 40° C The electricity of the socket could potentially be hazardous if any water comes into contact with it. Also someone could burn themselves with the water. If the water comes into contact with the skin then immediately place your hand under cold water. If water comes into contact with the socket, step away and inform the person responsible. 1. Wearing eye protection. 2. Keeping the water bath a certain distance away from the socket. 3. Wearing gloves to protect the skin. Lipase solution (5%) 15 Since 5% lipase solution is an irritant it could cause skin irritation or an allergic reaction. If it came into contact with the eyes it could cause irritation in the eyes. If it came into contact with the eyes immediately rinse it out for around 10 minutes and seek medical advice. If on the skin immediately wash off the lipase. 1. Wearing eye protection 2. Wearing gloves to avoid contact of lipase with the skin. Phenolphthalein (1%) 16 A 1% phenolphthalein solution is not too strong to cause any major harm however it may cause skin irritation and also irritation of the eyes if it gets into them If it gets in the eyes for at least 15 minutes, flush the eyes with lots of water, occasionally separating the top and lower eyelids. If it gets on the skin seek medical attention. Before reusing clothes, wash it. Take off any contaminated clothing and shoes. Wash your skin thoroughly with soap and water. 1. Wearing eye protection 2. Wearing gloves in order to avoid contact of phenolphthalein with the skin 3. Wear a lab coat in order to avoid contamination of the clothes with phenolphthalein Ethical considerations: Because everything was done under ethical conditions, no ethical concerns emerge. The experiment did not include any people or animals. Furthermore, there may be some food waste, but it isn't big enough for this experiment thanks to the little amount of milk used. Environmental Considerations: An environmental issue that I may have to make is how I would dispose of the milk because it may arrive in plastic, and so I would recycle all waste material that may be hazardous to the environment after utilising it for the experiment. I also had to greatly dilute any substances I disposed of to not harm the environment when disposed of down the sink, I have learned to proceed in this way from reading the “cleapss-waste-disposal.pdf” 17 . Analysis: Qualitative data collection There was a definite relationship between the different types of milk and having more or less translucency when Lipase was added with the milk. The Lipase had a noticeable effect on all of the milks, however, certain milks became more transparent than others such as goat milk., suggesting that fatty acid concentration does vary between the different kinds of milks. As can be observed from the difference in translucency, the fatty acid concentration does vary in different kinds of milks. Translucency is the physical characteristic of milk that allows light to flow through it. Returning to the research question, the more transparent the solution is after Lipase has been introduced, the more lipids have been broken down therefore the higher the fatty acid concentration. It is noted that the goat milk has turned almost completely back to translucent which one can assume that implies it has the highest fatty acid concentration due to the alkaline conditions created by the Sodium Carbonate indicated as pink by phenolphthalein has turned acidic due to lipase breaking down ester bonds in lipids into fatty acids that make the milk more acidic which results in the phenolphthalein indicator turning 17 Student Safety Sheets 99 Waste Disposal - CLEAPSS , Online 16 Material Safety Data Sheet - Fisher SCI . Online 15 Caymanchem.com , Online 8

see through from pink, hence the more acidic and see through the milk become the more fatty acids can be found in it. Quantitative data collection Example calculations: I will demonstrate the calculations that I used with the quantitive information I got from soy milk Mean change = ((-0.089)+(-0.084)+(-0.133)+(-0.143)+(-0.084))/5 = -0.107 Standard deviation (-6.836)-(-8.155)=1.319; 1.319 2 =1.740 (-6.422)-(-8.155)=1.733; 1.733 2 =3.003 (-10.153)-(-8.155)=-1.998; -1.998 2 =3.992 (-11.051)-(-8.155)=-2.896; -2.896 2 =8.387 (-6.316)-(-8.155)=1.839; 1.839 2 =3.382 ((1.740+3.003+3.992+8.387+3.382)/5) ½ =0.0226 9

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The bar chart on the x-axis above shows from left to right going from Soy milk, Goat milk, Coconut milk, Almond milk, and Cow milk the mean difference in absorption after their lipids have been catalysed by the Lipase. The error bars show the standard deviation from the mean and the y-axis shows the mean change in values of nm. The standard deviation is varied for each bar to show that the findings are not identical and won’t ever be the same. Although the standard deviation isn't particularly great in this example, the largest value is for the almond milk which was 0.067 L mol-1 cm-1 since the standard deviation from the mean is measured in the same base units as the dependent variable. This may theoretically suggest that a specific figure is impacted by other factors such as a higher concentration of substrate in some cuvettes than others, however, this isn't very substantial as is evident from the difference in absorption, which is around 0.121. Statistical analysis: 10

I used ANOVA testing for my statistical analysis to evaluate the difference between the means of each data set. Because my data was discontinuous, I utilised it to assess whether or not there was a statistical difference between the absorptions in milk. This may then be used to evaluate if a statistically significant difference exists between the means of unrelated data groupings. This will next assess if it was by accident or due to varied fatty acid concentrations in each. StatPlus was used to do my ANOVA test. I chose a one-way ANOVA test since my independent variable is categorical (various types of milk) and my dependent variable is quantitative. In order for this test to be significant, the p-value must be 0.05 (5%) and the F value must be bigger than the F critical value. Due to the p-value being less than 0.05 (p = 0.0248 3s.f.) we must reject the null hypothesis (H0) and accept the alternative (H1) meaning that there is a statistically significant difference between the fatty acids concentration in the milks Conclusion: To summarise, from the observations of the qualitative data the absorption and translucency varied in the presence of Lipase for different kinds of milk. This is demonstrated by the qualitative results acquired. It was less pink so the light was able to pass through in goat milk, suggesting that Lipase had catalysed lipids more efficiently inside this into fatty acids. This is due to the indicator phenolphthalein turning from pink in alkaline conditions before any ester bonds in lipids were broken down to colourless after lipase was added and the indicator phenolphthalein turned to see-through. It is appropriate to conclude from the ANOVA testing that the results are statistically significant, with the p = 0.0248 (3s.f.) being less than 0.05, and that there are different concentrations of fatty acids within each of the milk types, as it shows a 95% certainty that there is a statistically significant difference in the milks. Because there were varying quantities of substrate for Lipase to catalyse, there may be variances in absorbance, eventually supporting the qualitative findings of the different translucencies. The quantitative data also support the hypothesis as according to the colorimeter almond milk has a higher concentration of fatty acid. Why goat milk has the highest concentration of fatty acids in it can be explained by goat milk containing smaller fat globules than cow milk. Lipase is an enzyme that breaks down fats, and because the fat globules in goat milk are smaller, they are easier for lipase to break down, making the lipase in goat milk easier to digest. Additionally, goat milk contains a higher proportion of medium-chain fatty acids (MCFAs) compared to cow milk. MCFAs are shorter in length than long-chain fatty acids (LCFAs), which are more prevalent in cow milk. MCFAs are broken down more quickly and efficiently by lipase which is why in the 5-minute period much more fatty acids were created from the ester bonds in lipids by lipase. 18 18 “Goat Milk vs Cow Milk.” Summerhill Goat Dairy , 18 Aug. 2021, Online 11

Evaluation: Strengths: This experiment concentrated on performing 5 repetitions of each type of milk and so can be regarded as more dependable and repeatable in terms of results; moreover, because an average of these overall results was determined, it can be considered more reliable overall. The preliminary testing in this inquiry allowed me to improve the investigation and guarantee that I could control any external elements, such as determining the optimal temperature for Lipase and the right concentrations of the enzyme, as well as the indicators. This helped me to decrease the number of uncertainties and make my results as accurate as possible. Because of the tiny uncertainties, they had little impact on my results. A low level of uncertainty indicates that the measurement findings are more precise. Data is intended to provide a reliable outcome of measurement plus the uncertainty, which generally defines a range of values within which they expect this "true value" to fall. As a result, the lower the uncertainties, the smaller the range, and hence the more accurate the readings. My equipment's minimal uncertainties, such as the +/- 0.01° C uncertainty of the digital water bath, provide improved precision of a value. Another strength is that 33% of my mean change is significantly higher than my standard deviation. For instance, in soy milk, 33% of my mean change is 0.0352 and my standard deviation is 0.0226. This indicates a high level of precision and repeatability in my experiment. The standard deviation measures the variability of your data, while the mean change measures the average magnitude of the effect you are investigating. When 33% of the mean change is much larger than the standard deviation, it suggests that the effect is consistent and reproducible across multiple trials or experiments. This is important because it means that the results of my investigation are reliable and can be confidently replicated by other researchers or in future experiments. Additionally, having a precise and repeatable experiment helps to reduce the possibility of random error or chance of influencing your results, which strengthens the validity of my findings. Limitations: Although I tried to regularise the procedure as much as possible, when using the stopwatch, I was not always as efficient in removing the mixture from the water bath at exactly 5 minutes, which could have had an effect on the overall hydrolysis of the lipids because some of the test tubes might have had more time to be broken down by the lipase. This human 12

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error might have affected the reaction time next time and an alarm a few seconds before allowing me to prepare myself could eliminate this potential limitation of my experiment. Improvements: A potential improvement that could be made to the experiment is denaturing the enzyme so it stops working after the 5 minutes have passed. I would do this by putting the test tubes into the other water bath that is at a very high temperature which would immediately denature Lipase and stop the reaction from going on. This would increase the accuracy of my results due to stopping the Lipase acting after the 5 minutes have passed. I could also extend the length of the experiment in order to let the lipase turn all the lipids in every type of milk into fatty acids in order to make it fairer as in goat milk lipase is turned by lipase faster into fatty acids. I could have also calculated the rate of enzyme activity for lipase, which would have been influenced by a variety of factors. These include the concentrations of both the enzyme and substrate, as well as the pH of the reaction system. The temperature at which the reaction takes place is another important factor, as is any presence of inhibitors or activators in milk or other substances that are added to it. The rate of reaction for lipase can be tracked by monitoring either substrate consumption or product production over time. Additionally, free fatty acids can be detected via colorimetric or titration testing. Extension: To expand upon this investigation, it would be beneficial to explore how the age of milk impacts the concentrations of fatty acids and the rate at which Lipase can break them down. Research that has already been done on this subject suggests that as milk matures the fatty acid concentration increases due to a decrease in the variety of proteins. Therefore, we can conclude that mature milk should contain higher calories, therefore, energy content than fresh milk. This extension regarding the investigation of how the age of milk affects these factors, we can gain a deeper understanding of the nutritional properties of milk at different stages of maturity. This information could have practical implications for the dairy industry, as well as for consumers who seek to make informed choices about the nutritional content of the milk they consume. 13

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● Ranguwar, Shashank. “Phenolphthalein Indicator: Structure, Formula, Application, Uses.” Testbook Learn, Testbook Learn, 14 Sept. 2022, https://testbook.com/learn/chemistry-phenolphthalein/ ● Sapkota, Anupama. “Enzymes- Definition, Structure, Types, Mode of Action, Functions.” The Biology Notes, 10 Sept. 2022, https://thebiologynotes.com/enzymes/ ● Student Safety Sheets 99 Waste Disposal - CLEAPSS. Online 10.02.2023. https://science.cleapss.org.uk/Resource/SSS099-Waste-disposal.pdf ● “UV-Vis Spectroscopy: Principle, Strengths and Limitations and Applications.” Analysis & Separations from Technology Networks, https://www.technologynetworks.com/analysis/articles/uv-vis-spectroscopy-principle-s trengths-and-limitations-and-applications-349865 ● “What Is Linoleic Acid?” Zero Acre Farms: Meet Cultured Oil , https://www.zeroacre.com/blog/linoleic-acid-facts ● Importer. “Practical Biology.” Investigating Effect of Temperature on the Activity of Lipase, https://practicalbiology.org/bio-molecules/factors-affecting-enzyme-activity/investigati ng-effect-of-temperature-on-the-activity-of-lipase 15

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