The Effect of Temperature on Beetroot’s Cells Membrane

Introduction

Beetroot

Beetroot is one of many type of beets known as Beta Vulgaris. It can be used in cooking and drinking as it is one of the source of vitamins, minerals, nutrients and antioxidant as well including magnesium, potassium, sodium and vitamin C and betaine. Betaine in the cells serve as organic osmolytes which are synthesized or taken from the surroundings by the cells for protection against osmotic stress, drought and high temperature. It is also important for cardiovascular disease.

The high amount of nitrate content in the beetroot help to lower the blood pressure and thus,help to prevent the cardiovascular diseases. the nitrate or known as dietary nitrate in our blood is believed to be the source of ‘biological messenger nitric oxide’.

This ‘biological messenger nitricoxide’ is used by the endothelium for signalling the smooth muscle to relax. Thus, vasodilation occurs and enhance the blood flow. Other than its benefits to health, beetroot, especially the juice is used for making dyes or the food colorant as it is known for no allergic side-effects.

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Besides it is cheap. But, beetroot’s colour will change when heated so it is mainly used in confectionary, ice creams, tomato paste, jam and jellies. Beetroot dye may also be used in ink.when the bulb is older, the colour is deep crimson and the flesh is softer.

Cell Surface Membrane and Properties

Cell membrane is a structure of cell that functions as the barier separating the interior cellfrom the outside’s surrounding. This structure selectively let substances in and out of the cell.

Itis made up of lipid molecules arranged side by side with their tails facing each other forming a phospolipid bilayer

This lipid bilayer has partial-permeability for ions and polar molecules as the arrangement of hydrophilic heads and hydrophobic tails prevent the solute such as amino acids, carbohydrates, protein and ions from diffuses through it. But simple diffusion occurs for substances like water (osmosid) and glucose which doesn’t require extra helps from protein.

The model of cell membrane that is accepted widely is the ‘Fluid Mosaic Model’ by S.J.Singer and Garth Nicolson. This model of cell membrane shows that the cell membrane is described as fluid as the composition of it that consist of lipids and protein that always move sideways or literally. This is why it is said to be more fluid-like than a solid.

The protein in the phospholipid bilayer is known as transport protein which helps the transportation of substances in and out of the cell. As we know, molecules of amino acid, carbohydrate, proteins and charged ions cannot pass through the lipid bilayer by simple diffusion. Thus, the carrier protein is used to transport these substance sometimes with help of Adenosine Triphosphate (ATP).

Colorimeter

Colorimeter is a device used to measure the absorbency of substances. It is a light-sensitive device that simply measures how much colour is absorbed by objects or substances. With the help of colorimeter, we can analyse the concentration of that particular substances. This device works according to Beer Lambert’s law that state, ‘the absorption of light transmitted through a medium is directly proportional to the concentration of the medium. ‘Colorimeter works by passing through a ray of light through the solution thus measures the amount of light. The more concentrated the object or solution, the less light passing through it as more light is absorbed by the solution. The difference can be seen between the light before passing through the solution and after it passed through it. The result or the absorbance does not have a specific unit. But it is called ‘arbituary unit’ or AU.

Problem Statement

What is the effect of temperature on cell membrane and what is the relation between the result observed and the structure of cell membrane.

Hypothesis

When the beetroot cell surface membrane is exposed to high temperature, the membrane will break down causing the red pigments of the beetroot to leak out.

Variables:

• Manipulated Variable: the temperature of water bath used
• Responding Variable: the reading of the colorimeter (the absorbencyof solution)
• Fixed Variable: the type of beetroot used, the size of each sectionof beetroot, the time 30-minute time used

Apparatus:

• cork borer
• white tile
• knife
• ruler
• beaker
• boiling tubes
• forceps
• thermometer
• colorimeter
• cuvette
• measuring cylinder
• pipette
• stopwatch

Materials:

• beetroot
• distilled water
• water bath at 30 oC, 40 oC, 50 oC, 60 oC

Procedure:

1. The raw beetroot is first cut into half using a knife and lined with a white tile. Then, four sections of beetroot are cut using a cork borer. The sections then are measured to 1cm length using a ruler and put aside. Be alert when handling the beetroot as the beetroot juice can stain the clothes and skin badly.
2. The sections then are put in a beaker and rinsed with distilled water.
3. 12 boiling tubes labelled 30oC (1), 30oC(2), 30oC(3), 40oC (1), 40oC(2), 40oC(3), 50oC(1) 50oC(2), 50oC(3), 600C(1), 600C(2) and 600C(3) are prepared and 5cm3 of distilled water is added to each of the boiling tube.
4. The boiling tubes then are placed in water bath with temperature 30oC, 40oC, 50oC, and 60oC respectively for 5 minutes so the distilled water would reach the required temperature.
5. One of the sections of beetroot then placed in each boiling tube and left for 30 minutes in the water bath. The time is set using a stopwatch.
6. After 30 minutes, the beetroot sections from each of the boiling tube is taken out and the solution in the boiling tubes are shaken to disperse the dye.
7. The colorimeter is adjusted to read % absorbance.
8. 3cm3 of each dye solution is measured and added into the cuvettes separately. The cuvette then is put into the colorimeter and the reading is taken.
9. The smooth sides of the cuvette is wiped clean and dry to make sure there’s no dust, water and finger prints on it. The reading is repeated for three times to obtain a more reliable result.
10. This step is done on each of the cuvette for dye solutions.
11. All of the readings are recorded in a table and interpreted into a graph.

Results:

Discussion:

The graph tabulated shows that when the temperature is higher, the absorbency of the solutions is higher. This mean that, there are more beetroot dye leaks into the solution when the temperature is higher. From 30oC to 40oC, the line of the graph is increasing rapidly. This is because, the high temperature started to denature the proteins in the cell membrane causing the red dye to leak. The higher the temperature exposed to the cell as in to the beetroot, more kinetic energy will be gained by the red pigments inside of the beetroot cells. Thus, this will lead to diffusion out of red pigments from the internal cell of beetroot into the environment or surrounding through cell membrane. Besides, proteins are formed of amino acids coiled and attached to each other like strings. The bond between the amino acid is held by hydrogen bonds and disulphide bridge.

The hydrogen bonds however weaken when high temperature is applied and the disulphide bridge or known as SS-bond is a type of covalent bond which has a low boiling point thus, easily disrupted when exposed to high temperature. This will result the damaging of the vacuoles and leaking in the cell membrane causing the dye or red pigments inside the vacuole to leaks out or diffuse out of the cell into the surrounding. This diffusion is assist by the concentration gradient law that a substance will move from a place with high concentration to lower concentration .The enhancement is because at this temperature, more protein will be denatured and also the gaps in the cell membrane getting bigger as the red pigments of beetroot rush out of the cell into the environment or surrounding. Besides that, as we know, the cell membrane is made up of phospholipid bilayer. These lipid bilayers are consist of water-loving head and water-hating tail or simply known as hydrophilic and hydrophobic. The head (water-loving or hydrophilic is the only side that is exposed to water. When this lipid bilayer is heated or supplied heat, the bond will break down as lipid become more fluid. The water also expands and the molecules vibrate faster. This will result in disrupting the membrane. From 40oC to 60oC, the graph is increasing steadily showing that the leaking of red pigments of the beetroot cell is still happening. But, the gradient is not as steep as the gradient from 30oC to 40oC.

This is because, some of the denatured proteins are blocking the holes made at first resulting low flow –out of red pigments from the internal cell into the surrounding. In conclusion, this is all about kinetic energy. Energy is supplied to the molecules especially the red pigments of beetroot by heat. Thus, increase the rate of diffusion of the molecules into surrounding. Other than the pigments gaining energy, the cell membrane (proteins and lipid bilayers) will be damaged and denatured due to the high temperature. This, will cause the red pigments molecules that gained energy to rush out of the cell. Although the red pigments might gain a lot of energy because of the high temperature, it is the gaps between the membrane) lipid bilayer) which causes it to pass through and as the pigments move faster, it will increase the rate of diffusion resulting in faster rate of diffusion as the temperature increase.

Limitation:

Limitations are factors that you cannot control in experiments. The limitation of this experiment is the beetroot used. There are many type of beetroot and certain species of beetroot might only give out little dye as its red pigments is not as many as the other beetroot. Besides, the beetroot used is also not fresh. This might affect the result as wiltin beetroot doesn’t have so many red pigments stored in the vacuole. Other than that, there might be unseen scratch on the smooth sides of the cuvettes. As we know, the colorimeter is a very light-sensitive device. The scratches that cannot be avoided will make the result differ from what it should be as the light passing through it will be diffracted causing a wrong absorbency.

Sources of Error and Ways to Overcome:

Errors do happen in every experiment. The errors that always happen are when cutting the sections of beetroot. The size might be slightly varied when you measure the 1cm length. This will affect the surface area exposed to the surrounding and the result might be different than it should be. Besides, the part of beetroot took also must be from the same part. When the sections are taken for example, from the part that is near to the skin of the beetroot, the flesh is not juicy. So, to make it standardize and fixed, the sections should be taken from the middle part which is the thickest and juiciest as the volume of beta lain (red pigments) might differ at different parts of the beetroot. Other than that, when the beetroot is cut, there are dyes that leak out. The dye should have been washed away by soaking the section of beetroot overnight. By just washing it and simply rinse it with distilled water, the dye might still be on the surface of the beetroot. This will affect the result because the red pigments that enter the solution may come from the cuts not entirely because of the broken down cell membrane. Last but not least, the water bath heated is not consistently at the temperature required. Means that, the water baths are fluctuated. This will result in the pigments getting more or less kinetic energy that what they are supposed to get. Hence, the result is affected.

Further Study:

There are other ways to investigate the properties of cell membrane or what will affect the permeability of cell membranes. For example, the pH of the solute. The level of pH affects the pigment of the beetroot; the dye in the beetroot (betalain) diffuses out of the cell when the membrane proteins are damaged due to high acidic level of pH. The pH level is responsible for regulating the function of the protein in the cell membrane. The protein will be denatured at high alkalinity and acidity, and breaks down the lipid bilayer. For example, if we use alcohol to investigate the effect of pH on the cell membrane, more alcohol used, will result in more leakage of red pigments of beetroot into the surrounding. This is because; alcohol is a fat-solvent. Thus, it will dissolve the cell membrane is it is made up of lipids.

Safety Precautions:

A lab coat must be wear during the experiment as the beetroot juice is a good dye which can stain your skin and clothes badly. Besides, you must use all the apparatus carefully especially the cork borer and the knife to avoid any harm or injuries. While handling the test tubes with water bath, make sure you are in a suitable and safe distance with the water bath to avoid scalding or burn. When you are taking measurements such as the volume of water or the length of the beetroot, make sure you read the reading carefully. Your eyes must be at parallel with the instrument to avoid any parallax error and affect the experiment. The cuvettes also must be handled with care. Make sure you wipe the smooth sides of the cuvette and no fingerprints are left behind as it may disturb the reading of colorimeter. The reading of the colorimeter must be repeated three times to get a reliable result. Last but not least, you must wash the sections of beetroot thoroughly to make sure there are no red pigments left behind so that the result will be much more reliable

Conclusion:

In conclusion, our experiment demonstrated that exposure to high temperatures has a significant effect on the permeability of the beetroot cell membrane. As the temperature increased, the absorbency of the solutions also increased, indicating a higher release of red pigments into the surrounding solution. This phenomenon can be attributed to the denaturation of proteins in the cell membrane and the disruption of the lipid bilayer structure due to the elevated temperature.

From 30°C to 40°C, there was a rapid increase in absorbency, indicating a substantial leakage of red pigments. This is attributed to the initial denaturation of proteins and the widening of gaps in the cell membrane. However, from 40°C to 60°C, the rate of increase in absorbency became steadier, suggesting that some denatured proteins may have blocked the membrane's holes, reducing the flow of red pigments out of the cell.

Our findings support the hypothesis that high temperatures cause the breakdown of the cell membrane, resulting in increased permeability. This phenomenon is driven by the kinetic energy gained by the red pigments inside the beetroot cells, which leads to their diffusion into the surrounding environment. Additionally, the disruption of hydrogen bonds and disulfide bridges in proteins further contributes to the permeability increase.

However, it's essential to acknowledge the limitations of our experiment, such as variations in beetroot species and the freshness of the beetroot used. Future studies could explore other factors affecting cell membrane permeability, such as pH levels, to gain a more comprehensive understanding of this biological process.

Overall, this experiment provides valuable insights into the relationship between temperature and cell membrane permeability, with potential applications in various fields of biology and biotechnology.