The Effect of Green Tea Extract on the Hydrolysis of whole milk.

1. Introduction

1.1 Title

The Effect of Green Tea Extract on the Hydrolysis of whole milk.

1.2 Aim

The purpose of this experiment is to explore the effect of green tea on whole milk in order to understand enzyme inhibition regarding fat breakdown.

1.3 Research Question (RQ)

What is the effect of different concentrations of green tea extract (a lipase inhibitor) on the rate that lipase catalyzes the hydrolysis (or lipolysis) of whole milk through analyzing the reduction in pH of the surrounding solution in reference to how many fatty acids are produced from triglyceride molecules.

1.4 Justification/ Background Knowledge

This topic was important to me to explore because I am an avid tea drinker simply because I enjoy it, however, the habit of drinking tea is so globalized and wide-spread that I wondered if there were any other more concrete benefits to drinking tea other than the taste. Also, because there are so many different types, I figured there had to be differences between them to help people in some way. Therefore, researching the components of tea and their biological and in some cases medical properties and benefits surprised me.

Green tea extract, made from the leaves of the ‘Camellia sinensis L’ plant, is an excellent natural lipase inhibitor because of its polyphenol catechin content. Tea is made up of 30% polyphenol by weight. Polyphenols are nutrients that have “effects like reducing inflammation and helping to fight cancer.” (Gunnars, 10 Proven Benefits). Catechins are “natural antioxidants that help prevent cell damage and provide other benefits.” (Gunnars, 10 Proven Benefits). An antioxidant is a chemical that inhibits the oxidation of molecules by neutralizing free radicals thereby preventing cell damage. (Elfakhani, Thirteen Ways to Keep Free Radicals Away, And Why It’s so Important). The specific catechin EGCG is the catechin I am focusing on for my investigation. Epigallocatechin Gallate (EGCG) is one of the most powerful compounds in green tea which, because of its biological properties, is responsible for many of tea’s medicinal benefits which I found useful. EGCG is a bioactive compound that counteracts lipid metabolism/ the hydrolysis of lipids. This is what oxidizes lipids into fatty acids and glycerol.

1.5 Hypothesis

H1: As the concentration of green tea extract decreases (higher relative dilution), the rate of the lipolysis of the whole milk increases.

H0: As the concentration of the green tea extract decreases (higher relative dilution), there is no effect on the rate of lipolysis of the whole milk.

The alternative hypothesis (H1) makes sense because of a series of different effects happening in flow chart format:

Higher relative dilution leads to lower EGCG amount in green tea extract leads to less lipase inhibitor leads to more lipase action leads to more triglyceride molecules breaking down into fatty acids leads to greater reduction in pH as a measure of increased lipolysis of milk.

1.6 Variables

Independent Variable (IV): concentration of green tea extract through serial dilutions.

Dependent Variable (DV): rate of hydrolysis per unit time. This is measured by the reduction in pH (using a pH probe) from the original pH after 24 minutes.

Controlled Variables (CV):

The temperature (50°C) was kept constant through a set temperature water bath and thermometer to prevent a difference in rate of enzyme reaction. Also, the temperature is 50°C because it’s the most optimum temperature that we determined by our pilot study in that it produced the biggest range in pH.

The pressure of the reaction was kept the same and this was to prevent a change in the rate of reaction. (the atmospheric pressure of the Biology lab without alterations) (atm)

The volume of lipase solution was kept the same to prevent a change in the rate of the reaction. (3ml3). It is measured through a measuring cylinder.

The concentration of the lipase solution was kept the same to prevent a change in the rate of the reaction. (1g/cm3). This was calculated by dissolving 1g of lipase solution in 1cm3 of de-ionized water.

The time period for the hydrolytic reaction to occur was kept the same (24 minutes) and was kept by a stopwatch.

The brand of green tea as they had different amounts of EGCG which is the specific catechin the green tea that inhibits the lipase action. In addition, they had different or excessive amounts of fluoride in the lower quality brands and this is a source for error. As such, after research, I chose the most cost effective brand between cost and EGCG content, which was Lipton.

2. Materials & Methods

2.1 Pilot Study and Preliminary Experiment

Upon compiling a method to use for the experiment, I found that there were two holes in the method which could lead to inconsistency. The first was a lack of knowledge on the concentration of lipase solution to use and the second was a lack of knowledge on the optimum temperature for me to set my water bath at for the main experiment. My pilot study was determining which concentration of lipase worked best for my experiment because of the fact that my lipase came in capsules with lipase in powder form. I had 2 options of capsules is the pilot experiment was also about which brand/make of the concept molecules work together. The method to determining it was measuring the grams of lipase I needed along with putting that in a measuring cylinder of de-ionized water to find the concentration that suited my experiment the best. There was trial and error involved because I had to put different masses of lipase in different volumes of water, but in the end I settled on 1g of lipase per 1ml3 of de-ionized water. While this concentration may seem high, this was because each lipase tablet had filler. This meant that the mass of lipase tablet used wasn’t the most accurate reflection of actual lipase enzyme used. However, this is negligible to my experiment because I created this standard lipase solution that my experiment used so it was a constant. Then my preliminary experiment consisted of finding the optimum temperature of the lipase. This translated to results with the largest spread of pH reduction at a temperature.

2.2 Materials/Apparatus

- 15ml3 of whole milk

- 3ml3 of phenolphthalein

- 9ml3 sodium carbonate

- 5 Burettes

- 1 test tube stirrer

- 1 Pipette filler

- 5 5ml3 syringes and 5 3ml3 syringes

- 7 test tubes

- 1 test tube rack

- 1 water bath at lipase optimum temperature (50°C)

- 1 Thermometer

- 1 Electronic pH probe + attachment that measures data

- “Sparkvue” app on mobile phone

- 114ml3 lipase solution

- Green tea extract at different concentrations from serial dilution of it (100%, 50%, 25%, 12.5%, 6.25%, 3.125% which is using the relativity chart) (measured as percentages of full strength)

2.3 Method

Preliminary experiment:

1. Preheat the water bath to the temperature you are testing.

2. Measure out the sodium carbonate (NaCO3), phenolphthalein and whole milk using clean burettes, a pipette filler and syringes. I used phenolphthalein as a hopeful qualitative measurement for the change in pH because it can change color from pink to colorless.

3. Mix them all in a single test tube and place in a water bath that has reached the temperature you are testing.

4. In a separate test tube, measure out the volume of lipase needed for the trial of the primary experiment.

5. Pour the lipase into the test tube with the other substances.

6. Record the pH change in the test tube by using an electronic pH probe with the measuring attachment and the “Sparkvue” app. Record my results every minute for 10 minutes.

7. Repeat for each temperature ( body temperature which is 37°C and 50°C)

Main experiment:

1. Set water bath to the optimum temperature (in my case 50°C)

2. Add the milk to the test tubes along with the phenolphthalein and put them in the water bath. Stir them so they aren’t in layers in the test tube using the stirrer.

3. Measure out the lipase 1g/ml3

4. Prepare the green tea extract to get the highest amount of EGCG out of it by steeping it in hot water for 10 minutes (Fitzgerald, How to Get the Most EGCG from Your Green Tea). Then let it cool to room temperature.

5. Then dilute it through the process of serial dilution (1)

6. Add the lipase and a set amount of green tea extract (3ml3) at the same time and start the stopwatch. Do this in 2 minute time intervals using the stopwatch so you end up creating a chain of reactions happening and it’s a flow process. Also, measure the initial pH.

7. After 24 minutes, the first one will be done so record the pH change afterwards and do this for each one.

8. Repeat steps 2-4 for each concentration of green tea extract (there are 6 concentrations as percentages of full strength: 100%, 50%, 25%, 12.5%, 6.25%, 3.125%) 6 times in order to get 36 sets of results of the reduction in pH after 24 minutes.

(1): Serial dilution is the step by step process by which you dilute a solution. Serial dilution works in determining relative and accurate concentrations because at each step, the “dilution factor is constant, resulting in a geometric progression of the concentration in a logarithmic fashion.” (Wikipedia, Serial Dilution) For my experiment. it was 100%, 50%, 25%, 12.5%, 6.25%, 3.125% as I diluted each by half each time.

To the left is a picture of my setup as taken 14/12/2018 and it has my solution that I used in my experiment which contained the milk and phenolphthalein.

2.4 Precautions and Considerations

Safety considerations: Be sure to be careful when measuring your results in the hot water bath as the water will be hot. Also, open the container so that the steam goes away from your face so you won’t burn yourself.

Ethical considerations: Tea is occasionally known to be produced unethically and with harmful effects to the environment, so in an effort to be most ethically conscientious, try to use a tea brand that was sourced ethically.

Environmental considerations: There were no major environmental considerations to be taken into account. However, you can compost tea bags so to recycle the green tea bags that you used would be the most environmentally conscientious way to go about this experiment.

3. Data

3.1 Raw Data for pilot study and preliminary experiment (including uncertainties)

The highlighted values are the anomalous values. The whole of the first dilution (50%) seemed to be an anomalous result substantiated by the trend hypothesized and seen in the processed data as the average “change” increasing as the dilutions got smaller however, with the 1st dilution, the change decreased first.

Processed Data:

Data Analysis

I used several formulas in processing my experiments including the change in pH calculation, mean and standard deviation.

Change in pH calculation, calculated in excel:

starting pH - ending pH

Mean, calculated in excel:

Standard Deviation, calculated in excel:

In addition, through observing my graph, I can deduce that there is a strong negative correlation between the two variables and that the relationship is not linear. The spread in the results is very large because the standard deviations and error bars on the graph are large for the 12.5% and 6.25% strengths.

4. Discussion

4.1 Conclusions

Through my data, I’ve deduced that there is a strong negative correlation between the two variables and as such to reject the null hypothesis (H0) stating that there isn’t a correlation between them. As the % strength decreases, the pH change increases. the I accept the alternate hypothesis (H1) which states that “as the concentration of green tea extract decreases (higher relative dilution), the rate of the lipolysis of the whole milk increases.” This is because there is less EGCG lipase inhibitor in the green tea extract but the same amount of lipase to work on. Therefore, there is more fatty acids and glycerol produced by the triglycerides. The data shows that as the green tea extract % strength EGCG concentration goes down, the more fatty acids are produced, as shown by the reduced pH.

My results are supported and substantiated by the more advanced study on Green tea and EGCG done by the King Khalid University Hospital in Saudi Arabia, published on April 17, 2014. (Al Salafi, Rokia et al., Does Green Tea Help…) It’s in relation to fat oxidation as well. What EGCG does more specifically than mentioned above is decrease BMI and body weight because of its ability to increase postprandial thermogenesis and fat oxidation. Fat oxidation or fatty acid metabolism is the catabolic process by which triglycerides break down into fatty acids and glycerol in generating energy. (Wikipedia, Fatty Acid Metabolism) In my case, these triglycerides were in the whole milk and I chose whole milk over other types because of its high fat content. These fatty acids are important in doing things like being components of the phospholipids in the phospholipid bilayer for cell membranes.

The reliability of my data is not the best upon looking at my graph because the standard deviation error bars overlap. Therefore, there is a possibility that the curve shown on the graph is due to chance. However, this is because of the sample size of my data. It is clear that the amounts of each material that I used were very small and they were too small when trying to find more reliable data. I would want to increase my sample size because if I had used more of each material, the uncertainty and standard deviation error bars wouldn’t be as significant in determining the reliability of my data, which would in turn give me a more reliable and accurate curve of data.

4.2 Evaluation

There were some anomalous results, however, for the most part, they were so sparse so it’s unlikely to have been a systemic error. I think that there are several ways to improve on the experiment. The first is to have bigger volumes of materials used as that reduces errors and their significance in the reliability of my data. Also, I would have more repeats as this makes my results more reliable and also helps to pick out anomalous results. In addition, I would like to take the results over a longer time period so I can observe the change in pH on a larger scale. Also, it will be easier to tell anomalous results and comment on the accuracy of the results as the data for the results will be larger and not within decimals of each other. In addition, the size of the test tube was a problem because with too thin test tubes, putting in the pH probe and data logger became difficult in that the solution inside always ran over and into the water bath. To combat this procedural error, all that is necessary is to simply use a bigger and wider test tube. In addition, it was difficult to keep the temperature controlled inside the water bath because the length of the pH probe sticking out meant that the lid had to be partially off, letting out heat. Also, other procedural errors that could have been improved upon was the methodical cleaning of the pH probe after each reading in order to ensure it’s been reset. Leaving some of the last experiment’s residue on the probe meant that part of the reading would be taken from the last experiment again, which could skew some results. However, these could and were fixed throughout the experiment as I encountered them but a more efficient process would be helpful.

It would also be interesting to use other brands of tea to see if a clearer correlation could be determined from them, for example, the more expensive tea brands with more EGCG content. Also, because of my trend and standard deviation error bars, it would be interesting to see if it is real or due to chance, and all of the ways in improving the experiment would help to further understand that.

Overall, however, there is good reliability of data because of the 6 repeats for each data set resulting in 36 readings and this allowed us to see that there was relatively good concordance of the results.

References

1. Al-salafi, Rokia, et al. “Does Green Tea Help to Fight against Obesity? An Overview of the Epidemiological Reports.” Austin Journal of Nutrition and Food Sciences, 17 Apr. 2014, austinpublishinggroup.com/clinical-medicine/fulltext/ajcm-v1-id1011.php.

2. Elfakhani, Manal. “Thirteen Ways to Keep Free Radicals Away, and Why It's so Important.” Medical Xpress - Medical Research Advances and Health News, Medical Xpress, 22 Dec. 2016, medicalxpress.com/news/2016-12-thirteen-ways-free-radicals-important.html.

3. “Fatty Acid Metabolism.” Wikipedia, Wikimedia Foundation, 16 Jan. 2019, en.wikipedia.org/wiki/Fatty_acid_metabolism.

4. Gunnars, Kris. “10 Proven Benefits of Green Tea.” Healthline, Healthline Media, 17 Jan. 2018, www.healthline.com/nutrition/top-10-evidence-based-health-benefits-of-green-tea#section1

5. “How to Get the Most EGCG from Your Green Tea.” Kara Fitzgerald ND Naturopathic Doctor, 12 June 2017, www.drkarafitzgerald.com/2017/06/12/get-egcg-green-tea/.

6. “Serial Dilution.” Wikipedia, Wikimedia Foundation, 18 Jan. 2019, en.wikipedia.org/wiki/Serial dilution.