Why did the colour leak out of cooked beetroot?

Name: Eldhose Skaria Biology Core Practical Candidate no: 2440

Aim:

The objective of this investigation is to find out, “why does the colour leak out of the cooked beetroot?” and the aim of this is to investigate the effect of temperature on cell membrane. This experiment allows us to see the permeability of the membrane structure of beetroot. Beetroot contains red pigments called betalains, located within the cell vacuole. Normally, the pigment cannot pass through membranes but they leak out when the beetroot is boiled. And therefore by observing the beetroot under different temperature, I will be able to determine the effect of temperature on cell membranes.

Hypothesis:

As the temperature increases, so does the percentage absorbance of the beetroot pigments.

Fair testing:

Definition:

‘Fairness’ implies that the outcome of the activity truly depends on what is being investigated, and is not being distorted by other external factors. Therefore a fair test is one where all the variables are kept constant or the same, except the variable that is being investigated. A variable is anything that can change and which may influence the outcome of the investigation.

List of variables:

The different variables are:

The amount of distilled water
The temperature of the water
The length of the beetroot. – 1 cm in length

Variables that needs to be controlled (independent variable)

The variables that needs top be controlled under experimental procedure are as follows:

The amount of distilled water – 5cm3
The temperature of the water ---- 00C, 100C, 200C, 300C, 400C, 500C,600C, 700C

Dependant variable:

Amount of die solution.
Surface area of the beetroot.

Statement of fairness:

We are going to investigate the effect temperature has on the permeability of the membrane and there by transporting substances across the membrane. And also we will look the method of transport of substances around the body.

Theory:

Beetroot contains red pigments called betalains, located with in the cell vacuole; normally the pigments cannot pass through cell membrane they leak out when the beetroot is cooked. In petals they presumably attract pollinating insects and may be present in seeds/fruits to encourage birds to eat them and so disperse the seeds. Man has selected for colour in beetroot, both because it is more attractive but also because it may well be linked to genes for flavour too. There is no indication that they have any protective function (e.g. against UV light or insect/fungal/viral attack).

Effect of heat:

When you heat a beetroot, you disrupt the cell membranes. A biological membrane is made of a phospholipid bilayer. These are formed because the phospholipids that make it up have a polar "water-loving" (hydrophilic) head and a “water-hating” (hydrophobic) tail. The tails pack together, exposing only the polar heads to the water. The most effective way of doing this is to create two blankets one top of the other, with the fatty acid tails towards each other. This is the phospholipid bilayer. In a cell they form sacks. One goes all around the cell (the plasma membrane); others may form vacuoles (such as the tonoplast). Yet others may be like stacks of half empty bags (the endothelial reticulum, which is also continuous with the nuclear envelope. In these lipid seas, there will be a number of proteins in various degrees of submersion. Some span all the bilayer, thus being exposed on both sides. Others just drift on either of its surfaces. Typically, you will find that about 70% of a cell membrane is protein. The water around and within the compartments formed by the phospholipid bilayers are also packed with protein made in the cytoplasm.

Betalains:

Betalains are alkaloid pigments that are found in some families of plants belonging to the order Caryophyllales, but in no other plants. Little is known about the role of betalains.

Commercial uses of beetroot:

Beetroot pigment is used commercially as a food dye. It changes colour when heated so can only be used in ice-cream, sweets and other confectionary, but it is both cheap and has no known allergic side-effects. Beetroot itself, of course, is a common salad ingredient – when cooked, vinegar is added to the water to lower the pH.

...

This is a preview of the whole essay

Betalains:

Betalains are alkaloid pigments that are found in some families of plants belonging to the order Caryophyllales, but in no other plants. Little is known about the role of betalains.

Commercial uses of beetroot:

Types of transports across cell membrane:

Diffusion: this is the movement of molecules or ions from an area of high concentration in to an area of low concentration. Diffusion will continue until the substance spread evenly throughout the whole volume. Small uncharged particles diffuse across the cell membrane, passing between the lipid molecules as they move down the concentration gradient. Small molecules like oxygen and carbon dioxide can rapidly diffuse across the membrane.

Osmosis: Osmosis = the diffusion of water across a semi-permeable membrane

Plasma membrane permeable to water but not to solute

Solute = dissolved particle
Solvent = liquid medium in which particles may be dissolved

Water moves from solution with lower concentration of dissolved particles to solution with higher concentration of dissolved particles
Water moves from dilute solution to concentrated solution
Osmotic potential is the total of all dissolved particles

Equipments needed:

You need

Raw beetroot
Size 4 cork borer
White tile
Knife
Ruler
Water baths at 0, 10, 20, 30, 40, 50, 60, 70 °C
Plastic beaker, about 250 cm3
2 boiling tube racks
Crushed ice
8 boiling tubes
Thermometer (one per water bath)
Colorimeter
Cuvettes
Stop clock
Distilled water
Pipette for measuring 2 cm3
Small measuring cylinders

Variables to be controlled:

The variables that need to be controlled are as follows:

The temperature of the water- this needs to be controlled manually because any variation in this would affect my conclusions.
The amount of distilled water added must also e controlled as a miscalculation of this would affect my outcome.

Method:

1 Cut sections from a single beetroot using a size 4 cork borer. Cut eight, 1 cm length slices from these sections. Be careful not to spill beetroot juice on your skin or clothing as it will stain very badly.

2 Place the slices in a beaker of distilled water. Leave overnight to wash away excess dye.

3 Next day, place eight labelled boiling tubes each containing 5 cm3 distilled water into water baths at 0 °C, 10 °C, 20 °C, 30 °C, 40 °C, 50 °C, 60 °C and 70 °C. Leave for 5 minutes until the water reaches the required temperature. Place one of the beetroot sections into each of the boiling tubes. Leave for 30 minutes in the water baths.

4 Remove beetroot sections; shake the water/solution to disperse the dye.

5 Switch on the colorimeter and set it to read % absorbance.

6 Set the filter dial to the blue/green filter.

7 Using a pipette accurately, measure 2 cm3 distilled water into a cuvette. Place the cuvette into the colorimeter, making sure that the light is shining through the smooth sides.

8 Adjust the colorimeter to read 0 absorbance for clear water. Do not alter the setting again during the experiment.

9 Place 2 cm3 of the dye solution into a colorimeter cuvette and take a reading for absorbency. Repeat the readings for all the temperatures.

10 Present your results in an appropriate way.

Prediction:

I predict that as we increase the temperature of the water solution more pigment will leak out. The increase in temperature will disrupt the cell structure of vacuole of the beetroot and therefore the red pigment will leak out of it because of the increase in kinetic energy that the molecules have. And I also predict the movement of the leakage of the pigment will be by diffusion.

Risk assessment:

For this experiment I will need to take care when using certain equipments for example, I should take care when I use the knife to cut the beetroot, otherwise I could cut my hand. Another safety measure is to use gloves as beetroot stains your hand. I should also take care when using a cork borer.

If a piece of equipment say for an example glassware is broken; then I will need to notify my instructor and needs to be dealt with immediately. Also long hairs need to tied back and lab coat must be worn at all times.

Results:

These are the results which I obtained after taking the reading in the calorie meter. These results shows the red pigment that has been leaked out at different temperature if water and the concentration of the red pigment. By plotting this information on a line graph I will be able to determine the effect of temperature on the cell membrane.

This graph shows the increase in percentage absorbency rate. It shows a regular increase from 0°C to 70°C.

Analysis:

From the graph shows above I can see clearly that as we increase the temperature of the water, then the concentration of the pigment that has been leaked out of the beetroot cell is much greater than the one at lower temperature. This can be seen from the table or from the graph. For example the percentage absorbency at 30oC was 1.06 %but at 60oC the amount of the pigment that has been leaked increased by a factor of 11/2 to 2. This is the percentage increase in the concentration of the pigment that has been leaked put of the solution.

From 0°C to 30°C there is a gradual increase of absorbency which shows that as the temperature increases it is denaturing the cell wall and cell membrane and allowing the beetroot pigment to leek out into the distilled water. As soon as the sample of beetroot is placed into the distilled water diffusion occurs naturally. Diffusion is the net movement of molecules from a region of high concentration (beetroot sample) to a region of low concentration (distilled water).

From 40°C upwards there is a sudden increase of absorbency, this is because the optimum temperature of which enzymes and cells can work at is 37°C and as soon as you go above this temperature it will cause the enzymes and cells to denature and cease from working as effectively. Even if you then lower the temperature again the cell will not regain its original shape.

When you heat something you give it energy. Molecules start to spin and vibrate faster. So the heat energy acts as activation energy for the particles to move around. The water will expand too. This will have a disruptive effect on any membrane in its way. To make things worse, lipids become more fluid as temperature goes up so the membranes become more fragile.

Proteins are remarkable machines: they're formed of coiled and folded strings of amino-acids, held together by hydrogen bonds and disulphide bridges. If you heat them too much, they will untangle and break apart (vibrations again). When this happens to the proteins spanning a lipid membrane, they will form holes that will destroy the delicate structure. Now, any pigments in the innermost compartment will spill out.

Conclusion:

In conclusion to my experiment, I have worked and carried out all the procedures according the plan and followed it safely through out to get accurate readings form the calorimeter. And the results which I obtained proves my theory and my prediction which was that as we increase the temperature of the water the amount of the pigment that has been lost out of the beetroot cell membrane will be high, as this is proven by the graph which I obtained as I inputted the information on to excel to produce a graph (line graph). All in all my experiment has been successful and the end results were accurate.

Evaluation

Although my experiment was successful there could have been errors mainly man-made errors which could have influenced my results. On of these sort of errors that could have happened was when we timed the for how long the beetroot solution will have to be in the water, another could have happened when measuring 5cm3 or 1 cm of water and the length of the beetroot. To control all this man made errors, the best way to obtain precise readings is by taking the reading from the calorimeter more than once after taking out the beetroot from the test tube. So basically, I repeated my experiment twice for accuracy and there were no anomalies.

But still there are many things that could have affected my results and these are as follows:

The length of beetroot cut out – if a larger length was cut, more of the pigment would have been released increasing the percentage absorbance.
The temperature of the water was not constant as it wasn’t left for the specified 30 minutes.

To further improve my results or to have more confidence in my conclusion I could have done the following:

I could have taken measurements in shorter temperature intervals for example instead of measuring from 10◦C, 20◦C, 30◦C ---- I could have used 0◦C, 5◦C, 10◦C and so on… with an interval of 5◦C.
I also could have done the same experiment perhaps with another plant to see if the relationship between the temperature and its effect on cell membranes are the same.
The experiment can be modified – using different apparatus such as a more accurate colorimeter, which measures light at narrow range of frequencies.