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The effect of various temperatures on the membrane permeability of Beta Vulgaris, more commonly known as the red beet, will be investigated in this experiment. Using seven different samples, each treated to a different temperature, it was possible to compare how temperature effects betacyanin secretion, which is not released under normal conditions. The amount of betacyanin pigments released was determined using light spectrophotometry at a wavelength of 475nm. It was found that an increase in temperature is related to the amount of betacyanin pigments which pass through the membrane. For example, at the temperatures 22°C, 60°C and 100°C the values of absorbance were 0.0558, 1.285 and 1.401 respectively. This trend reinforces the belief that increases in temperature and the amount of betacyanin that is able to pass through the membrane is directly proportional because the membrane fluidity increases.
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Chemical structure of betanin, the most prevalent betacyanin in Beta Vulgaris (Sepúlveda-Jiménez et al., 2004)
Belonging to the Chenopodiaceae family, Beta Vulgaris, or more commonly known as the red beet is a root vegetable (Rhodes, 2008), and is red in color, due primarily to the presence of betacyanin (Czapski et al., 1988). Although there are different types of betacyanins, the main betacyanin of the red beet is betanin, which is present in high concentrations (Sepúlveda-Jiménez, 2004). The stability of Betacyanin is susceptible to a number of factors, such as: temperature, pH, oxygen, light, water activity and certain metal ions (Czapski et al., 1988). These factors account for the amount of betacyanin released, as under normal conditions it cannot pass through the selectively permeable plasma membrane.
The plasma membrane of prokaryotic cells is a selectively permeable membrane composed of an amphipathic phospholipid bilayer with embedded lipids, proteins and carbohydrates. It is described as selectively permeable because certain things can pass through the membrane without being impeded by the phospholipid bilayer, while other substances are completely blocked from passing through the membrane. These membranes must remain fluid in order to work properly. As temperature decreases a membrane becomes decreasingly permeable until the point where it finally solidifies, causing the membrane to rupture. However, as temperature increases, the membrane becomes too fluid, as the channel and carrier proteins embedded in the membrane start to deform; causing more substances to leak and pass through the membrane (Reese et al., 2011).
In this experiment, we observed the effect that various temperatures had on the membrane permeability of Beta Vulgaris. Since an increase in temperature causes the membrane of prokaryotic cells to become more permeable, along with increasing the rate at which molecules diffuse, it is expected that an increase in temperature will cause more betacyanin to pass through the membrane. As temperature decreases, the membrane permeability is also expected to decrease, until the point where the membrane ruptures, allowing the contents to flow freely out of the cell (Reese et al., 2011). The primary objective of this experiment was to investigate that effect that the different temperatures had on the membrane permeability of Beta Vulgaris.
Six uniform cylinders of a diameter of 1.0cm and a length of 3.0cm were cut using a cork borer. These cylinders of red beet root were placed under running cold water and rinsed for approximately 5 minutes. A previously frozen (-20°C) sample of beet root was inspected to ensure a length of 3.0cm and then thawed to room temperature. These seven samples of beet root were then put in a solution of 10mL of distilled water. Then, one solution of Beta Vulgaris was placed in the fridge at a temperature of 3°C, the previously frozen sample along with another fresh sample were left at room (22°C) temperature and four samples were placed in water baths of 40°C, 60°C, 76°C and 100°C. These solutions were left to incubate at the test temperatures for fifteen minutes. Once finished their incubation period, the solutions were transferred into fresh cuvettes, extracting the Beta Vulgaris core in the process. Following this, a SpectroVis Plus spectrophotometer by Vernier, using Logger Pro 3.8.4, was used to determine the absorbance of each sample at 475nm. This process was then repeated four times (Mitchell et al., 2012).
First, to analyze this data, the data was compiled into tabular form. Following this, the mean absorbance of each treatment was calculated in order to account for the different value of absorbance in each trial. Using the average value of absorbance, we were then able to calculate the standard deviation for each trial. As the data collected was sub dividable, it was deemed to be continuous. Therefore, a line graph was produced with standard deviation error bars (Mitchell et al., 2012).
Among the different temperatures in which the beet cylinders were treated, variation observed in values of absorbance was expected. As seen in Figure 1, the highest absorbance value was 1.604, observed at a temperature of -22°C. Comparatively, the lowest value of 0.0558 was seen at a temperature of 3°C. Three points of interest can be seen in the graph. The first occurs in the frozen sample where the absorbance is the highest value on the graph. Second, the absorbance readings at 3°C and 22°C were extremely close, 0.0558 and 0.0588 respectively. Finally, the absorbance reading at 100°C does not follow the increasing trend of absorbance value established from temperatures 3°C to 76°C. The value, 1.401, was in fact lower than that of 76°C (1.438) but greater than the value observed at 60°C. It can be noted that a general trend can be established. As the temperature of Beta Vulgaris increased, the absorbance and therefore the amount of betacyanin, also increased. However, the frozen and 100°C samples did not seem to follow this trend.
Figure 1. The effect of seven different temperatures on the absorbance of Beta Vulgaris, calculated using light spectrophotometry at a wavelength of 475nm.
As the betacyanin pigments present in Beta Vulgaris are hydrophilic and require storage in a vacuole (Mukundan et al., 1998), it is crucial that some sort of treatment be applied to the beet root in order to ease the release of the pigments. In this case, the temperature was changed in order to make the membrane of the red beet more permeable to the release of betacyanin. However, there are more efficient ways to increase the loss of pigment. As stated by Czapski (1998), an increase in pH would have a greater effect in the changes of colour attributes, while temperature would have a smaller effect. Therefore, if pH had been varied in this experiment rather than the temperature, it would have been possible to increase the pigment loss by Beta Vulgaris.
Variation in the results can be the consequence of many factors; such as the age of the beet root sample, cores from different beets were used and the amount of time the samples were treated at the test temperatures. The age of the beet would have played a large role as the proteins in the sample could already be broken down before the experiments are performed, thereby decreasing the amount of betacyanin that could be released. Furthermore, throughout the different trials, cores from different beets were used. These cores contained different concentrations of betacyanin, therefore affected the amount of betacyanin which passed through the membrane. Finally, the amount of time the samples were treated was also an important factor. These solutions were supposed to be treated for fifteen minutes; however, if left under treatment for more time, the amount of betacyanin secreted by the Beta Vulgaris would increase.
It can be concluded that as temperature increases above 3°C, the amount of pigment, betacyanin, which was initially unable to pass through the membrane, released is proportional to the increase in temperature. This is caused by the membrane becoming too fluid while the channel and carrier proteins embedded in the membrane start to deform (Reese et al., 2011), causing leakage through the membrane. In another similar experiment, the researchers concluded that the amount of betacyanin released was proportional to an increase in temperature (Thimmaraju et al., 2002); however, only the change between 40°C, 45°C and 50°C Beta Vulgaris samples was studied. In the case of the frozen sample, the result can be explained in terms of the cell membrane; when frozen, the membrane of the cell ruptures (Roquebert and Bury, 1993). This results in the betacyanin passing through the membrane with relative ease.
This experiment established the general trend that as temperature increases, the amount of betacyanin which passes through the membrane also increases. Although, two points of interest occur at -22°C and 100°C, which did not follow this trend. At -22°C the membrane ruptured (Roquebert and Bury, 1993), which allows the pigment to be released freely. While at 100°C a declining trend is established as the samples lost their viability (Thimmaraju et al., 2002). Further research in the area of the membrane permeability of Beta Vulgaris should focus on the effects that pH has on the amount of betacyanin released, comparing these results to those which have undergone temperature treatments.
Czapski, J., Maksymiuk, M., & Grajek, W. (1998). Analysis of biodenitrification conditions of red beet juice using the response surface method. Journal of Agricultural and Food Chemistry, 46(11), 4702-4705
Mitchell, G, Roe, G., Beaulieu, G., and Creasey, D., Brand, D., Lisson, P., Marx R., and Metacalfe, R. (2012). Biology 190A Laboratory Manual. Department of Biology, University of Victoria, Victoria, B.C.
Mukundan, U., Bhide, V., Singh, G., & Curtis, W. (1998). pH-mediated release of betalains from transformed root cultures of beta vulgaris L. Applied Microbiology and Biotechnology, 50(2), 241-245.
Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., & Jackson, R. B. (2011). Campbell Biology (9th ed.). San Francisco, California: Benjamin Cummings.
Roquebert, M. F., & Bury, E. (1993). Effect of freezing and thawing on cell membranes of lentinus edodes, the shiitake mushroom. World Journal of Microbiology and Biotechnology, 9(6), 641-647. doi: 10.1007/BF00369571
Rhodes, D. (2008, January). HORT410 – Vegetable Crops. Horticulture and Landscape Architecture – Purdue University. Retrieved October 12, 2012, from http://www.hort.purdue.edu/rhodcv/hort410/spina/sp00001.htm
Sepúlveda-Jiménez, G., Rueda-Benítez, P., Porta, H., & Rocha-Sosa, M. (2004). Betacyanin synthesis in red beet (beta vulgaris) leaves induced by wounding and bacterial infiltration is preceded by an oxidative burst. Physiological and Molecular Plant Pathology, 64(3), 125-133.
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Thimmaraju, R., Bhagyalakshmi, N., Narayan, M. S., & Ravishankar, G. A. (2003). Kinetics of pigment release from hairy root cultures of beta vulgaris under the influence of pH, sonication, temperature and oxygen stress. Process Biochemistry, 38(7), 1069-1076.
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