How will distinctive species of berries (strawberry, blueberry, raspberry, blackberry, cranberry) have an effect on the breakdown of hydrogen peroxide in celery juice to supply the enzyme - catalase?

In my Biology IB Class, I was introduced to enzymes and how they work in different conditions in different conditions, as well as, what inhibits them to function. Regularly, it interests me how what we consume daily impacts our health. But also, how changing our specific diets can improve or deteriorate different aspects of our health. Being an athlete, it's important to consistently maintain a balanced diet, not only to maximise performance levels, but also, promote muscle repair and prevent injuries from occurring. Consequently, I started inquiring about the possible health benefits if we decided to increase our intake of flavonoids and if they have a significant impact upon increasing energy generation and blood glucose concentrations to improve performance output. Hence, I chose to use different types of berries; to explore the effects it has on the breakdown of hydrogen peroxide. Even though there are many health benefits attributed to berries, I wanted to investigate if it has any specific drawbacks as being an inhibitor of the enzyme Catalase.

Enzymes are globular proteins with a specific function that acts as a catalyst in living organisms, thus, they speed up the chemical reactions inside and outside the cell without being altered by the process itself. On the surface level, an enzyme has a region known as the active site, which is where substrate molecules bind and a chemical reaction is catalysed. However, each active site is adapted to one specific substrate, meaning one reaction can occur, this is known as the Lock and key model. As seen in Figure 1, the substrate entering the active site of the enzyme must alter itself slightly as the substrate binds into perfect contact. As a result, an enzyme-substrate complex is formed, and the products are released.

Consequently, the presence of inhibitors can occur; a molecule that disrupts the normal pathway between an enzyme and a substrate (Brent Cornell, 2016). In this experiment, flavonoids are considered to be a competitive inhibitor; they bind to the same active site. As seen in Figure 2, though the shape of the substrates are structurally and chemically similar, the inhibitor blocks the active site and prevents the development of an enzyme substrate complex, thus, no reaction occurs. There are various different types of enzymes in living organisms, but his experiment specifically focuses on the enzyme catalase found in the Celery. The presence of catalase, an antioxidant enzyme found in all living organisms, is integral for the decomposition of 2 hydrogen peroxide to 2 water molecules (H₂O) and 1 oxygen molecule (O₂). Most importantly, it protects the cells from oxidative damage carried out by reactive oxygen species and maintains an optimum level of the molecule in the cell for cellular signal processes. Inevitably, if there is an exorbitant volume of Catalase, a disease called acatalasemia, a genetic disease formed when catalase level is too low, is activated and the catalase deficiency becomes a potential threat to the cell and the living organisms. Accordingly, the methodology developed attempts to quantify the different effects between fruit species that contain flavonoids, used by 5 different types of berries: Blueberry containing 1100mg/100g dry weight, Raspberries and strawberries containing 500 mg/100g dry weight (Jiyun Lun), Cranberries containing 7.9mg/100g dry weight and blackberries containing 38.8mg/100g dry weight (Croge et al.). They were extracted and converted to liquid form to investigate how each of these inhibitors will affect the volume of oxygen being released. Celery Juice was used to display the breakdown of hydrogen peroxide as it is rich in catalase to sustain health and inhibit the enzymes of transcription factors important in inflammation. This experiment seeks to reciprocate the natural procedure of catalase reactions in the liver through controlled means, creating the optimum medium in which it naturally occurs. As a result, the breakdown of hydrogen peroxide as displayed by the celery juice that is rich in catalase to sustain health, Each of the berry juice was mixed with the celery juice to see to what extent it has inhibited the function of Catalase. If a prominent volume of O₂ is released, it indicates the berry used did not interfere significantly with the chemical action, conversely, if a minimal volume of O₂ is released, it indicates the berry used worked effectively on slowing down the chemical reaction.

The Alternative Hypothesis (H_a) predicts that as the concentration of flavonoids increases in different species of berries, the inhibitor will become more prominent, decreasing the volume ((cm³) of oxygen (O₂) being produced. The prediction is that blueberries will have the greatest effect on preventing the breakdown of hydrogen peroxide because they are anticipated to produce the lowest volume (cm³) of oxygen ( O₂) as it has the highest concentration of flavonoids. As a result, they act similarly to mutagens, specifically these pre-oxidants act as inhibitors that prevent release of O₂ molecules. of enzymes. Consequently, Blackberries, strawberries and raspberries should produce somewhat the same volume of oxygen as they contain similar concentrations of flavonoids. Whilst, cranberries will produce the highest volume of oxygen (cm³) as it has the lowest concentration of flavonoids. Nonetheless, a Null hypothesis (H₀)can occur, whereas there is no difference in the rate of reaction when different concentrations of flavonoids are added. The topic of interest in this investigation is the impact on different species of flavonoids (Strawberry, Blueberry, Raspberry, Blackberry, Cranberry) have on the effect on the breakdown of hydrogen peroxide in Celery juice to supply the enzyme - Catalase. In numerous previous scientific studies (Skibola and Smith), flavonoids was seen as an competitive inhibitor to several enzymatic reactions, decreasing the breakdown of hydrogen peroxide.

Independent Variable -

The Different types of berries used  ( Strawberries - Fragaria anassa, Blueberries - Vaccinium sect, Raspberry - Rubus Idaeus,  Blackberries - Rubus, Cranberries - Vaccinium subg. Oxygccous). 5cc of each berry were added each trial throughout the experiment.

Dependent Variable -

The Volume of Oxygen (cm³) measured by the Gas Syringe (2 minutes)

A preliminary experiment was done to identify if there needs to be alterations to the method. It was important to determine whether tea or berries ( liquid form ) would work better as an inhibitor for the enzyme Catalase. A control variable of Celery juice ( Hydrogen peroxide ) was used to understand the effect the independent variable had on the dependent variable, as well as, to ensure the experiment results were unskewed.

• 5 different types of tea: Green tea, Black tea, English Breakfast tea, Earl Grey tea and Pure Darjeeling tea was first used in the experiment, which did trigger a reaction to occur. However, all of the tea except the Earl Grey tea hardly worked as an inhibitor, as the volume of oxygen measured in the Gas syringe system ranged from 14 - 46 cm3 which was exceedingly high in comparison to berries that ranged from 6 - 12 cm3 . As well as surpassing the control setup of 42 cm3
• In the original method, there was no recollection of the need to stir the mixture after the inhibitor was poured into the celery juice and attracted to the Gas syringe. Yet it was found in this pre-trial, that all mixtures needed to be stirred to have a noticeable impact on enzymes and thus, 5 different peers were used to stir each mixture. This would have had a slight impact on the results as it was merely impossible to control the speed and strength of each individual. Therefore, it was decided that a magnetic stirrer would be used to maintain consistency to ensure equitable results
• At first, 20cc of hydrogen peroxide and 5cc of the inhibitor was used but this ratio was found to be too disproportionate and made it difficult for the inhibitors to have an evident effect on the enzyme. Thus, it was reduced to 10cc to maintain a proportionate ratio.

• 450cc 5% Hydrogen peroxide (H₂O₂) 
• 50cc Pure (1:1 ratio, celery to water) celery (Apium graveolens)  Juice
• 10x Synrigles of 10cc (+/-0.50cc)
• 5x 250cc beakers (+/- 10.00cc)
• 5x 25cc Conical flask (+/-0.10cc)
• 5x Retort Stands
• 2x Waste Bins
• 5x 100cc Gas Syringe (+/-0.5cc)
• 1x Electronic Balance (+/- 0.005 grams)
• 1x Blender
• 1. 1x Stopwatch (+/- 0.005 seconds)
• 5x Magnetic Stirrer
• 1x Pack of Blue tack
• 80 grams Strawberries
• 80 grams Raspberry
• 80 grams Cranberry Juice
• 80 grams Blackberry Juice
• 1x Marker pen1x Marker pen
• 5x Rubber tube with band
• 200cc Distilled Water​​​​​​​

The methodology developed attempts to quantify the effect of flavonoids on the breakdown of hydrogen peroxide using a Gas syringe to measure the volume of oxygen (O₂) released. Hydrogen peroxide, consisting of water and extra oxygen atoms tacked on, is broken down by the enzyme catalase to water and oxygen. This test is composed of a mixture of flavonoids (berries juice), hydrogen peroxide and catalase (celery juice). As all components are mixed together, the hydrogen peroxide reacts with the catalase, but is inhibited with the mixture of flavonoids. Therefore, it's expected that with a larger concentration of flavonoids added in the 2 minute intervals, the lesser volume of oxygen (O₂) wll be released.

• Measure 170g of strawberries using the electronic balance, and place the berries into the blender
• Measure 20cc of distilled water using a measuring cylinder and pour the water into the blender
• Start blending for 2 minutes by setting it to “smoothies” to ensure that the force of blending is identical
• Filter the juice using a strainer Repeat for all the other types of berries: blueberries, cranberries, strawberries and raspberries

• Collect 1 retort stand and place it on the counter.
• Attach the Gas syringe onto the clamp of the retort stand, adjust if necessary to ensure it's in place.
• Use the Blu Tack to secure the cork onto the tube to make it airtight. Then, attach the tube onto the rim of the Gas syringe.
• Place the magnetic stirrer plate next to the retort stand
• Measure 10cc of Hydrogen peroxide (1%) using a 50cc measuring cylinder  and then, pour the liquid into the conical flask.
• Using a syringe, extract 5cc of celery juice from the beaker. Repeat this step using a new syringe to measure 5 cc of the strawberry juice.
• Place the conical flask onto the magnetic stirrer plate. Once in position, add the magnetic onto the centre of the flask.
• Set the magnetic stirrer to half speed.
• First, add the strawberry juice from the syringe into the conical flask. Immediately after, add the celery juice from the syringe into the conical flask. The enzyme is added after the addition of an inhibitor because once the enzyme is added, the reaction would start.
• Place the cork onto the conical flask instantly after the liquid in both syringes were added to the mixture.
• Start the timer and measure the volume of oxygen produced (cm3) after 3 minutes
• Dispose the mixture into the waste bin.
• Record data on the volume of oxygen produced of the strawberry juice.
• Repeat steps 1-13 for the remaining berries: raspberries, blueberries, blackberries and cranberries. Ensure new beakers, conical flasks and syringes are used in each trial.
• Process the data and describe any trends or parents in the results. Discuss how the increasing concentration of flavonoids affects the inhibition of the breakdown of hydrogen peroxide.

Safety - Hydrogen peroxide can cause severe eye irritation, burns, and respiratory system when in contact with the skin, Therefore, goggles and lab coats must be worn when handling the acid to prevent any injuries. Additionally, the use of glass beakers and conical flasks must be handled with precaution to prevent any cuts to the conduct/peer. If an accident occurs, immediately rinse the contaminated skin and inform a teacher or supervisor.

Ethical - There were no ethical implications to be considered throughout the investigation as non living organisms were used in the procedure.

Environmental - The hydrogen peroxide used in this experiment was safely poured into waste bins to prevent pollution in the water supply. Furthermore, all berries used were disposed properly through composting, a conventional trash bin or rescuing the leftovers parts if possible. Only the minimum amount of each berries were used to avoid food wastage.

*All data are rounded to 2 significant figures

*cm³ = Volume of gas syringe

Qualitative Data - Throughout the experiment, there were many observations made on the amount of oxygen released by the reaction after being inhibited by berries (flavonoids).

• For Blueberries, as soon as the magnetic stirrer was on and both celery juice and blueberry juice were added to the hydrogen peroxide, it immediately changed the colour from transparent to a dark blue and brownish colour. The solution also began to slightly foam.
• For Raspberries and Cranberries, as soon as the magnetic stirrer was on and both celery juice and raspberries/cranberries juice were added to the hydrogen peroxide, it immediately changed the colour from transparent to maroon and brownish colour. The solution also began to foam.
• For Strawberries, as soon as the magnetic stirrer was on and both celery juice and strawberries juice were added to the hydrogen peroxide, it immediately changed the colour from transparent to a pinkish and brown colour. The solution also began to foam.
• For the control, as soon as the magnetic stir and the celery juice were added to the hydrogen peroxide, it immediately turned into a light green colour, with a large amount of foam

In all trials, there was never any distinctive smell that was released from the solution besides the smell of the berries. The foam foamed in all trials was created because celery contains an enzyme called catalase, as it comes into contact with hydrogen peroxide (H202), this reaction happens so quickly, the bubbles formed are pure oxygen bubbles created by catalase. In general, there was an inverse relationship because as the concentration of flavonoids increased, the amount of foam decreased.

Decision for uncertainties -  The uncertainty for the volume of oxygen released +/- 0.5cc, was used as the gas syringe used in the experiment changed by 1, not more or less, meaning, the volume oxygen released changed by 1, therefore it was unable to measure the volume of 17.6cc of 22.3 as an example. Therefore, this unit was kept constant throughout the mean and standard deviation,

*all data rounded to 0 decimal places

*The error bars represent the standard deviation for the different types of berries

T-Test - As the investigation's main objective is to determine if there’s a correlation between both variables, a t-test was used to determine the significance between two groups. In this experiment, the significance was determined on a 95% confidence interval.

*There were other overlapping errors but it was not calculated because it was prominent that the T-Test value was P< 0.05. *Rounded to 5 significant figures

he alternate hypothesis (H1) stated: as the concentration of flavonoids increases in different species of berries (strawberry, blueberry, raspberry, blackberry, cranberry), the inhibitor will become more prominent in restricting the breakdown of hydrogen peroxide by the enzyme catalase. As illustrated by the processed data and graph above, the results are mostly in accordance with the alternate hypothesis indicating a strong inverse relationship between the concentration of flavonoids and the volume of oxygen (O2)  released by the enzyme reaction. As shown on the graph, all the data was according to the hypothesis with the controlled variable expected to release the greatest amount of oxygen at a mean volume of 45cm2  because there's no inhibition on the catalase enzyme, thus it proceeds to decompose the hydrogen peroxide (H202) into oxygen and water.  Additionally, cranberries released a mean volume of 33cm3 oxygen molecules, this was anticipated because it states in the background hypothesis the concentration of flavonoids is 7.9mg/100g per dry weight. The qualitative data also indicates both these claims, with the fairly large amount of foam generated indicating pure oxygen bubbles being created by the catalase.

The hypothesis expected both Raspberries and Cranberries to have relatively similar results because they both have the same flavonoid concentration of 500 mg/100g per dry weight (Jiyun Lun, 2020). The results contradict this claim as illustrated in the graph, raspberries were proven to inhibit the enzymatic activity of the hydrogen peroxide by releasing a mean volume of 15cm3 of oxygen molecules. Whilst, strawberries worked sufficient enough as an inhibitor, releasing a mean volume of 21cm3 of oxygen molecules. But when taking into consideration that both fruits have the sameflavonoid concentration. it was more ineffective. On the other hand, blueberries and blackberries had somewhat unexpected results. Whilst having the lowest mean volume of oxygen molecules at 15cm3 for blueberries and 6cm3 for blackberries. These results differ from the prediction made in the Alternate hypothesis, suggesting that blueberries were supposed to inhibit the hydrogen peroxide greater because they had the highest concentration of flavonoids of 1100mg/100g in contrast to blackberries (Jiyun Lun) containing 38.8mg/100g per dry weight (Croge et al.). This may be explained through the biological variation of both fruits in terms of age as fruits undergo significant changes during ripening. Although they were kept in the same conditions, the raspberries had an earlier harvest date. Because of heat, oxygen and light, the nutrients (flavonoids) are lost because enzyme activity is slowed down and decreases the synthesis pathway of flavonoids. However, it's possible that the human errors made when starting the experiment, injecting the juice into the Hydrogen peroxide and turning on the magnetic stirrer contributed to these results. The reaction time and preciseness of measurement of both the berry juice and celery juice could also have been inaccurate. Likely, with a partner, these errors could be eliminated and blueberries will release a smaller volume of oxygen molecules (cm3)

The standard deviation identifies how to spread the recorded data from the average or expected value. It is represented in the error bars on the data point in the bar graph. The greatest standard deviation is at 6 for raspberries, exemplifying that majority of the data recorded as far away from the mean value. Strawberries also had a relatively large standard deviation of 4. This can potentially mean for both berries there were some consequential errors made in this independent variable. The standard deviation for Blackberries and cranberries had the smallest size error par and the same because they were the first two berries being tested, promoting there may have been fewer human errors made. Moreover, Blueberries and the control variable were also the same with a standard deviation of 2.

Outliers - Although no anomalies had been detected in the data besides the Raspberries due to human error as mentioned above because the variation is negligible to the average. There are some outliers in Trial 3 of the strawberries, all having unusually small volumes of oxygen released compared to the rest of the trials. As noted, this was due to a human error of mixing up the cranberry syringe with the blueberry syringe, mixing both juices. Therefore, because blueberries contain more flavonoids, they increased the flavonoids concentration and inhibited the catalase activity more.

It can be concluded that the results obtained support the alternative hypothesis and suggest that there is an inverse relationship between the concentration of flavonoids and the mean amount of oxygen released (cm3) for all berries. In comparison to the Null hypothesis which states there were no changes. There is conclusive evidence that blueberries had the greatest concentration of flavonoids and thus, are an inhibiting factor of the enzyme catalase by producing the least volume of oxygen molecules. Research has shown that computer inhibitors disrupt the normal pathway between an enzyme (catalase) and a substrate (hydrogen peroxide), limiting the decomposition of the substrate into water and oxygen molecules. Additionally, according to Pubmed (Skibola and Smith), the abstracted state aligned with the conclusion made in this experiment, that the inhibition is caused by the formation of hydrogen bonds between catalase and flavonoids. Hence, the greater the concentration of flavonoids, the less effect the inhibition has on the enzyme. However, the difference between the experiments was that Pudmed focused on the relationship between the molecule structure and the degree of inhibition.

Observing that higher concentrations of flavonoids may result in the lower inhibition of the enzyme catalase may institute that people should adjust their diet to consumer berries that have higher concentrations, such as blueberries and blackberries as investigated in this experiment. As flavonoids are an antioxidant, they may help your body remove hydrogen peroxide at a more efficient rate. Therefore they help protect cells, prevent damage related to ageing and chronic conditions, and most importantly it helps maintain an optimum level for the molecule in the cell for cellular significant processes to function.

Strength - Aside from finding all the berries in the local supermarket, all materials used are relatively easily obtained from any standard laboratory. The highly controlled lab environment helped minimise the risk of contamination and increased the accuracy of the data collected, forming a strong inverse relationship between both variables. The wide range of berries (blueberry, raspberry, blackberry, strawberry, cranberry) and multiple controls (ie. duration, correct volume/concentration, time, temperature) kept consistent, it contributed to more precise results further supporting the conclusion and making the data more reliable. Additionally, having 8 trials per berry helped reduce the standard deviation, whilst enabling more comparable results, increased precision and increased the confidence in the data.

As flavonoids are a common antioxidant found in various types of berries, it would be useful to extend the research into a broader range of berries instead of simply the most common ones: blueberry, raspberry, strawberry, blackberry, cranberries. Moreover, there have been limited attempts made at researching the relationship between flavonoids as an inhibitor for the enzyme catalase, this extended research could enable a better understanding of the benefits of flavonoids on our body, thus by increasing the range it helps determine which berries are likely better/worse impactful for our health.

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