How does varying the cooking temperature of superfoods affect the Vitamin C content (g) that leaches into the water after 5 minutes, as determined by iodometric titration?

In recent years, the consumption of superfoods has gained significant popularity due to their nutrient-rich composition and potential health benefits. Superfoods, such as broccoli, kale, and berries, are known for their high content of essential nutrients, including vitamin C, which plays a crucial role in tissue growth, repair, collagen production, and acting as an antioxidant. (Harvard T.H Chan, 2023) The global superfoods market experienced significant growth, driven by rising awareness of the importance of a nutritious diet for overall health. "Valued at around \( \$ 162.6 \) billion in 2022, it is anticipated to reach around \( \$ 326.3 \) billion by 2032, with a compound annual growth rate (CAGR) of \( 7.1 \% \) amid the forecast period." (MarketResearchBiz, 2023)

In the midst of the COVID-19 pandemic, many, like my own mother, developed a heightened interest in cooking and nutrition as a means to enhance the immune system and reduce health risks associated with the virus. WhatsApp forwards and anecdotal advice often played a role in shaping our understanding of healthy eating practices during this time. I found myself pondering a question that emerged from one such experience within my own household: Does prolonged cooking of superfoods result in the loss of vitamin C, as my mother had come to believe based on a popular WhatsApp-forwarded message?

I aim to investigate the scientific basis behind the claim that cooking superfoods for extended periods leads to the leaching of vitamin C into the cooking water by investigating the chemistry behind the breakdown of vitamin C at different temperatures. This research not only addresses a common belief but also holds relevance in making informed decisions about cooking methods for nutrient preservation in our daily diets. It will contribute to a broader understanding of the impact of cooking on the nutritional value of our food, particularly when preparing superfoods, which have garnered attention for their potential health benefits.

Vitamin C, also known as ascorbic acid, is an important and common nutrient in multicellular organisms. It is a water-soluble vitamin naturally present in numerous fruits and vegetables, including oranges and broccoli. The oxidation of vitamin \( C \) results in the formation of dehydroascorbic acid (DHA), which is an oxidized form of ascorbic acid. The oxidation of vitamin C can occur through a one or two-electron transfer, which terminates free radical-mediated chain reactions. (Shen et al., 2021) Below is the equation of oxidation of ascorbic acid:

\[ \mathrm{C}_{6} \mathrm{H}_{8} \mathrm{O}_{6} \rightleftharpoons \mathrm{C}_{6} \mathrm{H}_{6} \mathrm{O}_{6}{ }^{2-}+2 \mathrm{H}^{+}+2 \mathrm{e}^{-} \]

Humans need to consume vitamin \( C \) because it is an essential nutrient that the body cannot produce on its own. "Vitamin C plays a crucial role in various bodily functions, including the formation of collagen, the growth and repair of tissues, and maintenance of cartilage, bones, and teeth." (NIH, 2021) Beyond its involvement in critical biological processes, vitamin C serves as an antioxidant. Antioxidants are compounds that can counteract unstable molecules known as free radicals, which are molecular entities with unpaired electrons in atomic orbitals. Their reactivity can lead to oxidation and reduction reactions, impacting other molecules in the process. (Lobo et al., 2010) Free radicals are naturally produced within cells and by external factors. When their production overwhelms the body's natural defenses, oxidative stress occurs, causing harm to proteins, nucleic acids, and lipids. (Pham, 2008) Antioxidants interact with free radicals by neutralizing them, preventing them from causing harm. They work by donating an electron to free radicals, reducing their harmful effects on the body. (Wilson & Villines, 2017)

Vitamin C acts as an antioxidant by giving hydrogen atoms to free radicals, creating a relatively stable ascorbyl free radical. Its water-soluble nature allows it to work both inside and outside cells. Vitamin C plays three significant roles in antioxidant defense: (Traber & Pauling, 2011)

1. Regenerating the antioxidant form of Vitamin E: to reproduce vitamin E and yield the relatively stable vitamin C radical. Vitamin E is necessary because it acts as a further antioxidant. (Traber & Pauling, 2011)
2. Reducing Hydrogen Peroxide: "The enzyme ascorbate peroxidase reduces \( \mathrm{H}_{2} \mathrm{O}_{2} \) to form water \( \left(\mathrm{H}_{2} \mathrm{O}\right) \) by using ascorbate (vitamin C) as an electron donor, preventing the formation of harmful hydroxyl radicals, through the glutathione-ascorbate cycle. The detoxification of \( \mathrm{H}_{2} \mathrm{O}_{2} \) is a vital process as it can easily produce new hydroxyl radicals through the Fenton reaction." (Traber & Pauling, 2011) "This is an inorganic chemical reaction that involves the strong oxidizing nature of a mixture solution of hydrogen peroxide \( \left(\mathrm{H}_{2} \mathrm{O}_{2}\right) \) and ferrous ions \( \left(\mathrm{Fe}^{2+}\right) \)." (Burg & Meyerstein, 2012)

\[ \mathrm{Fe}^{2+}+\mathrm{H}_{2} \mathrm{O}_{2} \rightarrow \mathrm{Fe}^{3+}+\mathrm{OH} \bullet+\mathrm{OH}^{-} \]

3. Neutralizing Free Radicals: "Dehydroascorbic acid is formed by vitamin C donating high-energy electrons to free radicals. This product can be converted back into ascorbic acid through enzymatic reactions or metabolized further to release more electrons." (Traber & Pauling, 2011)

"Superfoods" is a term often used informally to describe nutrient-dense foods rich in antioxidants like vitamin C. (Olsen et al., n.d.) In this experiment, two such superfoods, carrots and beetroots, will be used to investigate how different cooking temperatures affect the leaching of vitamin C into the cooking water. Carrots and beetroots were chosen as in Kenya, both beetroots and carrots are common foods rich in vitamin C. Beetroots are easy to grow and are consistently ranked as one of the top vegetables grown in home gardens in Kenya. (Greenlife Crop Protection Africa., 2023)

"Vitamin C is an easily oxidizable and unstable compound at higher temperature conditions. At temperatures above \( 100^{\circ} \mathrm{C} \), oxygen has a greater effect on ascorbic acid (vitamin C) degradation than temperature. Vitamin C is water-soluble and easily leaches into water, which can then degrade the vitamin due to exposure to heat." (Yin et al., 2022)

Vitamin C, chemically known as ascorbic acid, is composed of four hydroxyl groups, one ester group, and a carbon-to-carbon double bond. The presence of hydroxyl groups enhances its water solubility by promoting hydrogen bonding with other molecules. This property facilitates its dissolution in aqueous environments, enabling efficient absorption and utilization within the body.

Moreover, the carbon-to-carbon double bond within the molecular structure of vitamin C contributes to its stability and reactivity. This double bond increases the enthalpy, requiring more energy to break the bonds, thereby enhancing the molecule's resistance to degradation, especially at higher temperatures.

The change in cooking temperatures when cooking superfoods does not affect the vitamin C content that leaches into the water after 5 minutes measured by an iodometric titration.

As the temperature of cooking superfoods increases, there will be a corresponding decrease in the Vitamin C content in the cooking water. This hypothesis is based on the fact that vitamin C is a water-soluble vitamin that is sensitive to heat and can easily be degraded during cooking. As superfoods are cooked, especially at elevated temperatures, the heat facilitates the breakdown of the cellular structure of these foods, causing the release of vitamin C into the cooking water and additionally the oxidation of ascorbic acid. (Lee et al., 2017) Therefore, it is expected that as the temperature of cooking superfoods increases, more vitamin \( C \) will be oxidized into the water forming dehydroascorbicacid, leading to a decrease in the nutritional value of the superfood. Additionally due to the hydroxyl groups present in vitamin \( C \) it readily dissolves in water especially at higher temperatures as higher temperatures increase the kinetic energy hence more particles achieve activation energy and cause more successful collisions. This increased kinetic energy and collisions can disrupt the structure of Vitamin C molecules, making them more likely to dissolve into the water. (Liang, 2022)

In the preliminary phase of this experiment, I conducted a few trial runs to determine the most suitable temperature ranges for the study. Initial tests were carried out at room temperature, \( 50^{\circ} \mathrm{C} \), and \( 90^{\circ} \mathrm{C} \) to assess the impact of different temperature points on Vitamin C leaching. The results from these initial trials strongly supported the alternate hypothesis, with the non-heated sample displaying the most Vitamin C leaching and the \( 90^{\circ} \mathrm{C} \) sample exhibiting the least leaching. Based on these outcomes, I made a decision to create a more systematic approach for the main experiment. I opted for a consistent \( 15^{\circ} \mathrm{C} \) interval between each temperature point, ensuring that the intervals were evenly distributed. This choice was made to enable a comprehensive analysis of the results, allowing for a more detailed evaluation of the experiment's outcomes and conclusions.

Independent variable:
\( > \) Cooking temperatures \( \left({ }^{\circ} \mathrm{C}\right) \pm 0.05 \): the temperature at which the superfoods (beetroot and carrot) were cooked in the water was the independent variable as it changed increasingly from \( 29.00^{\circ} \mathrm{C}, 40.00^{\circ} \mathrm{C}, 55.00^{\circ} \mathrm{C} \), \( 70.00^{\circ} \mathrm{C}, 85.00^{\circ} \mathrm{C}, 100.00^{\circ} \mathrm{C}\left(29.00^{\circ} \mathrm{C}\right. \) is the controlled temperature \( ) \). The solutions were heated on a bunsen burner set up and each solution was heated for 5 minutes in their respective temperature after which they were allowed to cool for 5 minutes before testing.

Dependent variable:
\( > \) Volume of iodine solution \( (\mathrm{ml}) \pm 0.05 \): the volume of the iodine solution used during the titration is the dependent variable. This was measured using a burette and reading from the bottom of the meniscus.

In order to assess the Vitamin C content in the water following the exposure of vegetables to varying temperatures, an iodometric titration reaction will be conducted. In this process, the ascorbic acid present in the solution interacts with iodine, leading to the formation of dehydroascorbic acid. The endpoint of the titration is signaled by a blue-black color, due to the iodine-starch complex. This occurs once all the ascorbic acid within the solution has been completely used up in the chemical reaction. Below are the equations for this process:

\[ \mathrm{C}_{6} \mathrm{H}_{8} \mathrm{O}_{6}+\mathrm{I}_{2} \rightarrow 2 \mathrm{HI}+\mathrm{C}_{6} \mathrm{H}_{6} \mathrm{O}_{6}^{2-} \quad \mathrm{I}_{2}+ \text{ starch } \rightarrow \text{ starch-iodine complex } \]

1. Set up the apparatus as shown in figure 3.
2. Weigh 20 g of the chosen vegetable cutting them into small even cubes.
3. Measure 200 ml of distilled water using a 100 ml measuring cylinder twice, transfer the water in a 200 ml beaker.
4. Transfer the vegetables in the water beaker.
5. Heat the vegetable and water until it reaches a temperature of \( 40^{\circ} \mathrm{C} \) and maintain this temperature for 5 minutes.
6. Remove the beaker from the heat source and use forceps to extract the vegetable pieces. Allow the water to cool for 5 minutes.
7. Using a pipette, transfer 25 ml of the heated vegetable water into a conical flask.
8. Measure 2 ml of starch solution using a 10 ml measuring cylinder and add it to the conical flask. Ensure thorough mixing.
9. Titrate the solution against the iodine solution, monitoring the color change. Stop titration when the solution completely changes color from the original color to a blue-black color. Be sure to read the results from the bottom of the meniscus.
10. Perform the experiment until three concordant results are obtained (within 0.1 ml of each other).

a. Repeat steps 3-10 for each of the desired temperature points \( \left(55^{\circ} \mathrm{C}, 70^{\circ} \mathrm{C}, 85^{\circ} \mathrm{C}, 100^{\circ} \mathrm{C}\right) \) and both vegetables.
b. Repeat steps 3-10 without heating the water for the control experiment for both vegetables.

Environment:

Iodine solution has negative impacts due to it being highly toxic on the environment hence when disposing it it is important to dilute it before safely pouring it down the drain. (EDVOTEK, 2024)

Ethics:

As the superfoods are food items and being used solely for experimentation purposes. This use can be seen as wasteful as the food items are not ingested and are used for experimentation. To avoid this waste the boiled superfoods and remaining ones will be left in the school garden to decompose naturally in comparison to throwing them away.

Raw Data: