Effect Of Australian And Malaysian Originated Daucus Carota On The Concentration Of β-Carotene.

To what extent does the Australian and Malaysian originated carrots (Daucus carota) affect the concentrations of β-carotene present by extracting β-carotene from the Daucus carota from using solvent extraction and measuring the absorbance of β-carotene using a UV Spectrophotometer, followed by calculating the concentration of β-carotene present in both origins of Daucus carota?

The consumption of vegetables over the years has been increasing tremendously due to the myriad of people changing their lifestyles in order to lead healthier lives. Furthermore, the consumption of organically-produced foods has been drastically increasing due to the attention it obtains from social media. Thus, there is a paradigm shift in terms of food choices as now, more people are consuming healthier foods. According to a survey done by The Research Institute of Organic Agriculture and the International Federation of Organic Agriculture Movements in 2015, it showed that the growth in the market for organic foods (vegetables) had increased almost five-fold since 1999 [15.2 bn $USD] to 2013 [72.0 bn $USD] (Willer). This statistic supports the fact that the consumption of vegetables is ever growing.

Australian farms predominantly grow their vegetables organically. This means that about 25 pesticides are only allowed to be used to produce organic foods as compared to conventional production in Malaysia where over 900 pesticides can be used (Roseboro). Additionally, This is also another contributing factor to why so many people prefer organically-produced food over conventionally grown foods as it is a healthier option. However, the price allocated to organically-produced foods is considerably higher than conventionally grown foods. I wanted to see whether the concentration  β-carotene correlated with the origin of the Daucus carota (CABI) itself. Moreover, I wanted to know why my parents would rather buy carrots from a specific origin such as Australia, even when given the high price?

β-carotene is the orange pigment which is found in photosynthetic organisms. It is instrumental  in photosynthesis, and is an accessory pigment in the light-dependent reaction. It is also an antenna pigment due to its properties, enabling it to form protein complexes such as photosystems that absorb photons of light (Andrew Allot).  β-carotene’s main function is to essentially defend the plant from molecular oxidation from singlet oxygen which are produced from chlorophyll triplet states. Singlet oxygen is a highly unstable molecule produced during photosynthesis and can lead to oxidation or isomerisation of the plant. Oxidation or isomerisation could hinder the process of photosynthesis in the plant.

UV-Spectrophotometry will be used to determine the concentration of  β-carotene present in the Daucus carota. It essentially measures the absorbance of a particular solution in order to determine the concentration of  β-carotene. Based on the absorption spectrum of  β-carotene,  β-carotene absorbs the most light around 410nm-490nm(Evens). Hence, the colour region would be calibrated to the green-blue colour region according to the respective wavelength chosen, in this case being 450nm. Once the absorbance of  β-carotene is determined, the concentration of  β-carotene can be calculated using

\(\frac{A×V(ml)\times10^4}{A^{1\%}_ {1cm}\times \ P\ (g)}\)

A refers the absorbance used [450nm], V refers to the volume of β-carotene extracted. P refers to the weight of Daucus carota used and \(A^{1\%}_ {1cm}\)  represents the β-carotene coefficient [2500] (Cucurbita Moschata Duch).

Organic farming uses a higher concentration of potassium/K⁺ ions as compared to conventional farming methods (Fess ID, Benedito). The increase in K⁺ ions correlates with the increase in ATP [Adenine tri-phosphate] formation during aerobic respiration in the plant. More ATP corresponds to a higher rate of hypertrophy, which increases the size of the organelles. Therefore, having an enlarged cell would allow for a higher concentration of β-carotene to occupy the thylakoid membranes of the chloroplasts (Bogacz-Radomska, Harasym). Furthermore, a study indicated that photosynthetic rate can also be affected by the concentration of metal ions present in the air (Tarek Houri, Yara Khirallah, et al). This would lead to the decline in the concentration of β-carotene as metal ions and primary pollutants attack the chloroplast, leading to the destruction of its organelles (Sewelam, et al). Since β-carotene is located in the thylakoid membrane, it would also be adversely affected. Metal pollution is determined to be higher in Malaysia than in Australia (Bernhard A, et al), (Laidlaw, et al). Therefore, this leads to the hypothesis.

H₁:The Australian Daucus carota will contain a higher concentration of β-carotene as compared to Malaysian Daucus carota.

H₀: There is no statistical significance between the concentrations of β-carotene between the Malaysian and Australian originated Daucus Carota.

In order to obtain the concentration of the β-carotene, the process will be needed to be split into 3 separate sections. The first process requires the extraction of carotene from the carrot. This is followed by measuring the absorbance of the carotene. Finally, the absorbance of all the values will be used to calculate the concentration of β-carotene. A t-test is then used in order to evaluate the accuracy of the data (Rodriguez-Amaya).

• Measure 10.00 grams of Australian Daucus carota using an electronic balance.
• Place 10.00 grams of Australian Daucus carota into a mortar.
• Start to crush the Daucus carota into tiny pieces using the pestle.
• Once the Australian Daucus carota has been crushed, pour 25.0 cm³ of acetone using a 25.0 cm³ measuring cylinder into the mortar and further crush the Australian Daucus carota.
• After a few minutes, pour 25.0 cm³ of hexane using a 25.0 cm³ measuring cylinder into each mortar and start to further crush the carrots.
• After 10.0 minutes, pour an additional 30 cm³ of acetone using a 50 cm³ measuring cylinder into the mortar and start to crush again. At this point, the Australian Daucus carota should be close to being completely macerated.
• Stop crushing the Australian Daucus carota once an orange solution is finally formed in the mortar.
• Use a 50 cm³ gas syringe, transfer the solution from the mortar into separate 500 cm³ separating funnels. The solutions in the separating funnels have to be filtered through a filter paper first. This is to get rid of the macerated Australian Daucus carota.
• Next, prepare 100 cm³ of 10% sodium chloride solution by diluting 10.00 grams of pure solidified sodium chloride followed by adding distilled water until it reaches the 100 cm³ mark on the 100 cm³ measuring cylinder.
• Pour 100 cm³ of distilled water into each separating funnel and shake the mixture.
• This is then followed by adding 100 cm³ of 10% sodium chloride solution to the separating funnel. The NaCl salt is added to the mixture to allow for the cells to rupture and release β-carotene.
• Shake the separating funnel vigorously and wait for approximately 5.0 minutes for the liquids to settle and separate. Remember to open the stopcock while shaking to release pressure in the flask. After shaking, 2 layers should be observed. The top layer is the supernatant containing β-carotene.
• Open the stopcock to allow the bottom layer of the mixture containing water and sodium chloride to flow out into a measuring cylinder and close the stopcock once the supernatant is only left in the separating funnel.
• The supernatant remaining in the separating funnel contains the β-carotene (Rebecca)
• Repeat steps 1 to 14 for the Malaysian originated Daucus carota

• Once the β-carotene has been extracted, use a UV Spectrophotometer at a wavelength of 450 nm (Lucia). to determine the absorbance of the β-carotene. Do this for both solutions from their respective separating funnels.
• Once the absorbance of the carotenoids is determined, use the formula to calculate the 

concentration of β-carotene,\(\frac{A×V(ml)\times10^4}{A^{1\%}_{1cm} × P \ (g)}\).

Where A: Absorbance reading found at 450nm

V: Total extracted volume of β-carotene in mL

\(A^{1\%}_{1cm}\) β-carotene coefficient at 2500

P: Sample weight in grams

A T-test will also be used. This is to determine the statistical significance as well as determine whether there is a significant difference in terms of their β-carotene concentrations.


The formula is: t = \(\frac{(x_1-x_2)}{\sqrt{\frac{(s_1)^2}{n_1}+\frac{(s_2)^2}{n_2}}}.\) x₁ and x₂ is the mean of the sample sizes of each origin of Daucus carota(Australian and Malaysian). s₁ and s₂ refer to the standard deviation of each sample while n is the sample size of each origin.

• Since the use of a knife is prevalent and it has a sharp edge, take caution whilst cutting the carrots.
• Hexane is an organic solvent, thus short-term exposure could cause nausea, drowsiness and even unconsciousness. Long-term exposure could potentially cause neurotoxic damage (MSDS).
• The use of a lab coat was ubiquitous as hexane and acetone could easily spill causing irritation or even corrosion of the skin.
• Use the fume hood when dealing with chemicals like hexane as they are carcinogenic  (MSDS).

• The carrots that were not used for the experiment can be composted.
• A minimal volume of chemicals were used to prevent any further wastage. However, this was done through trial and error to ensure the most volume of carotenoids with the least volume of solvents used.

• Hexane once again has to be disposed properly as if it is mishandled and thrown into the sink, it could potentially end up in bodies of water and this could indirectly affect marine life. One way to tackle this problem is by disposing the hexane into an organic waste bottle.

• As hexane and acetone were added, it became easier to crush the Daucus carota with a pestle.
• After the maceration, the solution possessed a dark orange colour.
• As the solution was filtered through a filter paper, the Daucus carota pieces were observed as the residue.
• When the sodium chloride solution together with water was added into the separating funnel, the mixture was shaken vigorously for 5.0 minutes. Every time the stopcock was opened, a loud hissing sound was made.
• After shaking the separating funnel and letting the mixture settle, 2 distinguishable layers had formed that were separated by a white mucus layer that separated the 2 layers. 
• The extracted β-carotene possessed a dark orange colour.

Sample Calculation: [Trial 1 of Australian originated Daucus carota] To determine the concentration of β-carotene in 10.00 grams of Daucus carota.

Using the equation,\(\frac{A\times V(ml)\times10^4}{A^{1\%}_{1cm}\times P\ (g)}.\)

Where A: Absorbance reading found at 450nm

V: Total extracted volume of β-carotene in mL

\(A^{1\%}_{1cm}\) β-carotene coefficient at 2500

P: Sample weight in grams

• Mass = \(\frac{2.469\times3.5\times10^4}{2500\times10}\) 
  = 3.457µg/g

• Convert µg/g to g.
  3.457×\(\frac{1}{10^4}\)

= 0.00034566 g
≈0.000346 g

• Convert mass of Daucus carota to concentration using C=\(\frac{m}{V}\)

Concentration of β-carotene = \(\frac{0.000344566}{(\frac{10}{1000})}\)

= 0.035 gdm^-3

^{T-test to determine statistical difference between differing originated Daucus Carota}

^{t = \(\frac{(0.036390-0.0313215)}{\sqrt{\frac{(0.003)^2}{10}+\frac{(0.005)^2}{10}}}\)}

t = 8.2535

To determine the degrees of freedom(df) for t-test:
 

(Sum of sample size of both origins) – 2
 

= 18

\(\bigg(\frac{0.01}{10}+\frac{0.5}{25}+\frac{0.5}{25}+\frac{0.5}{30}+\frac{0.01}{10}+\frac{1}{100}+\frac{1}{100}+\frac{0.005}{2.599}\bigg)\)×100

=8.058%

≈8.06%

The figure above shows a bar graph indicating the concentration of -carotene against the origin of Daucus carota. From the bar chart, the higher mean concentration of β-carotene is from the Australian originated Daucus carota of 0.036 gdm^-3, while the Malaysian originated Daucus carota had a lower mean β-carotene concentration of 0.031 gdm^-3. Therefore, Australian originated Daucus carota have a higher β-carotene concentration than Malaysian originated Daucus carota. Furthermore, Australian originated Daucus carota had a higher mean absorbance value at 2.599 ABS than Malaysian originated carrots with a mean absorbance of 2.238 ABS. From this, we can conclude that Australian originated carrots contained a higher concentration of β-carotene as compared to Malaysian originated carrots. In terms of their standard deviation, the standard deviation of the Australian originated carrots is lower at 0.002815 as compared to Malaysian originated carrots with a standard deviation of 0.005453. This indicates that Malaysian originated Daucus carota concentrations have a wider spread. Therefore, it is less reliable and this makes the experiment less precise, as the Malaysian originated Daucus carota’s concentration is spread over a wider range. After the 33% of the mean and the standard deviation was calculated, the standard deviation was lower than the 33% mean. This indicates that the data is reliable. Additionally, based on the graph, the error bars that were present were large and this indicated that there was a weak correlation. Additionally,  the data collected was more variable from the mean itself. In terms of the data’s significance, it could not be deduced from the graph itself, as a T-test would need to be conducted. However, the error bars overlapped. This could indicate that the difference in concentrations between the Australian and Malaysian Daucus carota were not significant. However, this cannot be confirmed until a  T-test is carried out. Therefore, the size of the error bars did not make the conclusion that Australian originated Daucus carota has a higher concentration of β-carotene than Malaysian originated Daucus carota difficult to reach. Moving on to the t-test, the degrees of freedom [d_f] was determined to be 18.

Furthermore, in Biology, the significance value is usually taken as 5% [0.05]. From the table, it was determined that the critical T-value was 2.101. The calculated T-value was determined to be 8.255. Since the calculated T-value was higher than the critical T-value, it indicated that the difference between the mean concentrations of Australian and Malaysian originated Daucus carota does have a significant statistical difference. This contradicts with the error bars in the graph, as from the graph it seems as if the difference in their respective concentrations are not statistically significant. However, from the t-test, the conclusion is drawn that there is in fact a statistical difference  between the concentrations of Australian and Malaysian Daucus carota.

Going back to my Research Question, “To what extent does the Australian and Malaysian originated carrots (Daucus carota) affect the concentrations of β-carotene present by extracting β-carotene from the Daucus carota from both origins using a separating funnel and measuring its absorbance using a UV Spectrophotometer, followed by calculating the concentration of β-carotene present in both Daucus carota?”. In conclusion, the Australian originated Daucus carota indeed did have a higher concentration of β-carotene that the Malaysian originated Daucus carota. This supports the H₁ hypothesis, which states that Australian originated ​Daucus Carota ​would possess a higher concentration of β-carotene as it possesses a lower concentration of primary pollutants as well as a higher concentration of K⁺ ions in the soil. Moreover, my data rejects the null hypothesis [H⁰], which states that there would be no statistical significance between the difference in the concentrations of β-carotene between the Australian and Malaysian Daucus carota. The evidence supporting the hypothesis is seen in ​Graph 1​, where it shows that the Australian originated ​Daucus carota c​contains a higher mean β-carotene concentration (0.036390 gdm^-3​) than the Malaysian originated ​Daucus Carota ​(0.031325 gdm^-3​). The null hypothesis was contradicted by a t-test which showed that the calculated T-value (8.255) was higher than the critical T-value (2.101), which showed that there was a significant statistical difference in terms of the different concentrations of β-carotene. The differing concentrations of β-carotene does answer our research question, by showing that differing origins does affect the concentration of β-carotene present in the ​Daucus carota due to external factors affecting the production of β-caroten such as the quality of soil and the concentration of primary pollutants in the atmosphere.​ Although there is not any scientific data published that specifically shows how Australian and Malaysian originated Daucus carota affect the concentrations of β-carotene. The absorbance readings were collected at 450nm as usually, carotenoids are measured at λ​max ranging from 410nm to 490nm (ResearchGate). This indicates that the wavelength in which β-carotene i Thus, I had decided to take the average wavelength, being 450nm, where the absorption of β-carotene would be the maximum. The percentage error was calculated by

\(\frac{[Experimental\ Value-Literature\ Value]}{Literature\ Value}\)×100

There was no specific literature value regarding the concentration of β-carotene in the Australian and Malaysian originated ​Daucus ​carota. However, an article (ResearchGate) had indicated that the concentration of β-carotene ranged from 3.20 mg/kg all the way to 170.00 mg/kg (Tanveer Ahmad). Thus the average was taken in order to calculate the percentage error. Since our experiment determined the concentration in grams, the concentration of β-carotene was multiplied by 1000. This made the average concentration of β-carotene 36.40 g/kg in Australian originated carrots and 31.30 g/kg in Malaysian originated carrots. The percentage error was determined to be -56.4% and -62.5% in Australian and Malaysian originated carrots respectively. The negative value indicates that the experimental value was lower than the literature value. The average percentage uncertainty of the experiment was also calculated to be 8.06% and 8.09% for the Australian and Malaysian originated Daucus carota as seen in the sample calculation for the Australian originated Daucus carota. The difference in the percentage uncertainties is due to the lower average absorbances observed in Malaysian originated carrots. Causing the increase in percentage uncertainty. Since the percentage error is higher than the percentage uncertainty, it indicates that systematic errors were tantamount in this experiment as compared to random errors.

The experiment did possess a very low percentage uncertainty of about 8%, which was calculated from the sample calculation. Furthermore, the error bars on the graph indicates a lower possibility of error occurring. Additionally, the concentration β-carotene was only obtained from the middle of the storage root of the Daucus carota. This was to prevent any uncertainty, as different parts of the carrot may contain different concentrations of β-carotene, thus affecting the accuracy of the data. Moreover, the experiment was conducted rapidly. This is to prevent the oxidation or isomerisation of the carotenoids. Another strength was how the experiment was adapted in order to yield the most volume of β-carotene with wasting the least volume of reagents. At first, the volume of hexane used was 5 cm³​ ​. However, this did not yield a sufficient volume of β-carotene as it would not be enough to fill up a cuvette. On the other hand, the volume of hexane used was 50 cm³​. This yielded too much β-carotene which meant that a large volume of hexane and acetone was wasted. And this contradicted with one of the ethical considerations. Thus, the volume had been adjusted to 25 cm³​. Furthermore, β-carotene is susceptible to changes in heat, light intensity, humidity as well as molecular oxygen. Since the experiment was carried out in an open environment, it could cause the β-carotene go oxidise and could even isomerase (Yahia). This would cause the concentration of β-carotene to deteriorate. This affects the precision of the data, as this would mean that the concentration of β-carotene would always be different even if it were taken from the same part of the Daucus carota. Therefore, the experiment was planned out first and the experiment was conducted in a darker environment, such as a fume cupboard. This would prevent light causing any decomposition of the β-carotene.

Instead of having the origin of Daucus carota as the independent variable, the effect of temperature on the concentration of β-carotene can also be investigated.. Additionally, the deterioration of β-carotene can also be investigated. This will be achieved by measuring the initial concentration of β-carotene, followed by heating it to different temperatures and calculating the concentration of β-carotene after heating.

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