19 minute read
Concentration of Lycopene in Different Varieties of Tomatoes
Charlie Scholefield
Barker College
Tomatoes are an important source of antioxidants, involved in reducing the risk of many chronic diseases such as cancer. This is because they contain a large number of carotenoids. Lycopene is one of these, an organic compound that gives red fruits and vegetables their bright pigment. This report will outline the effect of different tomato varieties on the concentration of lycopene extracted. Data was collected on three different varieties: Truss, Roma, and Cherry tomatoes, and analysed with an ANOVA and a Post-
Hoc Tukey test to determine whether or not there was a significant difference in the means of the three groups. The results supported the hypothesis, suggesting that there is a significant difference in the concentration of lycopene from different varieties of tomatoes.
Literature review
Lycopene (C40H56) is a naturally occurring organic compound found in many red fruits and vegetables such as tomatoes, watermelons, pink grapefruits, red carrots, and papayas (May, 2020). It is the molecule that gives these compounds their bright red pigment. It is a longchain hydrocarbon molecule with alternating single and double carbon-carbon bonds, as seen in figure 1, referred to as a conjugated structure (de Montemas, 2020). This makes it part of a group of molecules called carotenoids, which give colour to red, yellow, or orange plant parts. β-carotene, also found in tomatoes, is another member of this group.
Free Radicals
Free radicals are molecules capable of independent existence that contain at least one unpaired electron (Agarwal et al., 2014), which they can either donate or accept. This makes them both oxidants and reductants, causing them to be highly reactive (Lobo et al., 2010). They can be produced through normal metabolic processes, or exposure to external sources such as Xrays, ozone, cigarette smoking, air pollutants, and industrial chemicals (Lobo et al., 2010). When functioning normally, free radicals are useful in helping fight off pathogens, protecting the body from infections. However, when there is an imbalance between free radicals and antioxidants, excess free radicals can cause oxidative stress. This means they begin to react with fatty tissue, proteins, and DNA (Agarwal and Rao, 2000) causing chain reactions that slowly damage cells. This can lead to harmful, chronic diseases such as atherosclerosis, asthma, diabetes, and cancer.
Antioxidants
Antioxidants are extremely beneficial in the way they prevent excess oxidation of free radicals from occurring. They effectively balance out the oxidative stress caused, preventing major problems from occurring in the body. This makes it important to maximise antioxidant consumption. Carotenoids are known for their antioxidant properties in humans. This means they can prevent slow damage to cells that occur as a result of excess free radicals. This is important in preventing harmful chronic diseases, such as cancer. Lycopene is one of the most potent antioxidants, often referred to as a free radical scavenger (Fish, Perkins-Veazie and Collins, 2002). Because of its structure, lycopene (and other antioxidants) can easily react with free radicals, essentially ‘cleaning up’ the number of them in the body when there is an excess. This helps prevent oxidative stress damage caused to cells, therefore reducing the risk of diseases like cancer. In particular, cancer is one of the leading causes of death in the western world. Lifestyle and diet are considered as major risk factors, with about 50% of cancers related to diet (Agarwal and Rao, 2000), resulting in 35% of cancer mortalities (Williams, 1999). Producers will therefore often look to increase lycopene concentration in their tomatoes, because of the antioxidant property the compound holds. Tomatoes with higher levels of lycopene have greater health benefits, appealing to consumers as a better choice. Tomatoes are one of the most widely grown vegetables on Earth, second to potatoes (Baliyan and Rao, 2013),
Figure 1: Chemical structure of lycopene.
meaning it is important to maximise the health benefits from their consumption. On top of this, studies have found that fresh tomatoes account for 50% of a person’s total lycopene intake (Rao, Waseem and Agarwal, 1998).
Other Reports
Many other reports have investigated ways to maximise the concentration of lycopene in tomatoes. Common variables include the storage temperature and the use of processed tomato products. It has been found that processed tomato products, such as tomato paste, contain higher amounts of lycopene than fresh tomatoes (Gärtner, Stahl and Sies, 1997). It was found that, on a dry weight basis, cherry tomatoes contained 124.0 mg of lycopene per 100 g, while tomato paste contained 204.6 mg of lycopene per 100 g (Toma et al., 2008). This suggests processing tomatoes leads to an increase in lycopene levels, possibly due to increased concentration from water loss (Story et al., 2010)and/or the use of heat and oils in cooking.
There have also been many studies into the proposed health benefits of lycopene, such that it improves male fertility. Trials have been conducted reporting improvements in sperm parameters and pregnancy rates with the daily supplementation of lycopene for 3-12 months (Agarwal et al., 2014). Furthermore, lycopene levels in the body have been found to be inversely related to the incidence of cancers, including breast cancer and prostate cancer (Agarwal and Rao, 2000). Its proposed prevention of prostate cancer is extremely important, since it is the most common cancer and the fifth driving reason for death in men (Soares et al., 2019).
Lycopene content is predicted to be affected by variety of tomatoes since it has been found in studies that there is a relationship between the amount of lycopene and redness of tomatoes (Toma et al., 2008).
Extraction of Lycopene
A majority of methods used for lycopene extraction are not possible in a school lab, as they are time consuming, expensive, and use hazardous organic solvents (Davis, Fish and Perkins-Veazie, 2003). An example of this is high performance liquid chromatography, HPLC. This is a technique used that separates and identifies each component in a mixture, making it easier to single out and measure the amount of lycopene.
The methodology in this report involves the use of colourimetry to analyse a sample of tomato puree dissolved in a solvent mixture. Colourimetry works by emitting a specific wavelength of light through a solution. The compound absorbs some light and the light transmitted is measured and related to absorbance. The Beer Lambert law (Figure 2) states that the light absorbed is proportional to the concentration of the compound absorbing the light, making it possible to calculate the concentration of lycopene in solution.
Figure 2: The Beer Lambert Law
Problems with this method involve the inability to accurately calculate the concentration of lycopene, because of interference of other carotenoids, such as βcarotene, in the colourimetry process. The light absorption spectrum for β-carotene is shifted slightly more to the blue end than lycopene, because of small differences in molecular shape (May, 2020). The closeness of each molecule’s respective spectra makes it important to choose a wavelength of light that not only absorbs the most lycopene, but the one with the least interference. This can be seen through the graph in Figure 3.
Figure 3: Light absorption spectra for lycopene and βcarotene (Source: de Sousa, 2014)
Lycopene has three absorbance peaks, at 444, 471, and 503 nm. The peak at 503 nm is the most effective since the absorbance of β-carotene (and other carotenoids) at this wavelength is relatively low, leading to minimal interference. A wavelength of 500 nm was chosen for this experiment, as this is the closest setting to match the peak shown.
What will this report involve?
This report will investigate the change in lycopene concentration across different varieties of tomatoes. This will be carried out with a more simple, accessible method than previous reports have used, allowing also for the testing of this method’s reliability for future use.
A large amount of literature is focused on the effect of processing on lycopene concentration and the health benefits of lycopene. It is therefore important to join these together in research, by studying the most efficient ways to boost lycopene concentration for human consumption. One of these is the growing of different varieties of tomatoes, something that is simple for producers to implement.
This report therefore aims to find any difference in concentration of lycopene from different tomato varieties. Education on the importance of tomato variety in human consumption and agricultural production can improve the amount of lycopene in the western diet, reducing the risk of diseases such as cancer.
Scientific research question
How does the specific variety of tomato affect the concentration of lycopene extracted?
Scientific hypothesis
That the variety of tomato affects the concentration of lycopene extracted.
Methodology
Preparation of Chemicals
A solvent mixture of 2:1:1 Hexane: BHT: acetone was used to extract the lycopene, and it was made as follows:
Butylated hydroxytoluene (BHT) (0.10 g, 0.0454 mol) was dissolved in 200 mL of absolute ethanol. The resulting solution was protected from light by covering the bottle with aluminium foil.
BHT in ethanol (100 mL) was combined with acetone (100 mL) and hexane (200 mL) to produce the solvent mixture.
Preparation of Tomatoes
It is important to note that the following procedure was carried out with the lights turned off and the blinds closed, to minimise light exposure.
Three groups of tomatoes were made, each containing roughly 300 g of a different variety of tomato. The groups were selected as follows: • Truss Tomatoes • Roma Tomatoes • Cherry Tomatoes Tomatoes in group 1 were blended in a Thermomix to form a uniform puree. This was placed into a 500 mL Schott bottle and covered with aluminium foil to protect the sample from light. This bottle was marked with the group number. These steps were repeated for each group, resulting in three separate Schott bottles and three varieties of puree.
Extraction of Lycopene
Tomato puree (0.6 g) was accurately weighed into a 20 mL volumetric flask on an analytical balance. This was repeated five times for each variety, resulting in fifteen flasks with puree. Each flask was filled to the 20 mL mark with the Hexane: BHT: acetone solvent mixture using a clean, dry, glass pipette.
Each stoppered flask was shaken briefly, and magnetic stirrer bars were added. The mixtures were stirred magnetically for fifteen minutes. The flasks were then filled to the top with distilled water and stirred for an additional five minutes. Finally, the flasks were shaken and allowed to stand until two distinct layers were visible. This was made up of a coloured organic layer on top and a colourless aqueous layer underneath.
Colourimetry
A colourimeter with Spark Vue data logging software was used to determine the absorbance of each sample. A small sample of the Hexane: BHT: acetone solvent mixture was placed into a clean, quartz cuvette. This was used to calibrate the colourimeter at a wavelength of 500 nm.
The coloured, organic layer from the top of each flask was removed with a plastic pipette and used to fill a cuvette. This was placed into the colourimeter and the absorbance at 500 nm was recorded.
These absorbance values were converted into lycopene concentration using the Beer Lambert Law.
The absorbance value was calculated through the colourimeter. The molar absorption coefficient for lycopene is 17.2∗104 ��������−1 ∗���������������� −1 (Fish et al., 2002). The optical path length was the length of the cuvette, in this case 1 cm.
Using this law, an equation was constructed (Equation 1) to calculate the milligrams of lycopene per gram of tissue:
(1)
Results
Table 1: Absorbance values for each sample
Variety A B C D E
Truss 0.419 0.422 0.394 0.393 0.389
Roma 0.732 0.662 0.626 0.609 0.604
Cherry 0.348 0.280 0.310 0.286 0.305
Table 2: Concentration of lycopene in each sample (mg/g tissue)
Variety A B C D E
Truss 21.788 21.944 20.488 20.436 20.228
Roma 38.064 34.424 32.552 31.668 31.408
Cherry 18.096 14.560 16.120 14.872 15.860
Table 3: Mean and standard deviation for each variety.
Variety Mean concentration (mg/100 g) Standard deviation
Truss 20.9768 0.8194
Roma 33.6232 2.7495
Cherry 15.9016 1.3897
Mean concentration of lycopene (mg/100 g) Mean concentration of lycopene for each variety
40 33.6232
30
20 20.9768
15.9016
10
0
Truss Roma Cherry Variety of Tomato
Figure 4: Bar graph representing mean concentration of lycopene for each tomato variety.
H0 = There is not a significant difference in the average concentration of lycopene in each variety. HA = There is a significant difference in the average concentration of lycopene in each variety.
Table 4: ANOVA test output for comparing the concentration of lycopene extracted from different varieties.
Truss Roma Cherry
Average 20.9768 33.6232 15.9016
Standard Deviation 0.8194 2.7495 1.3897
F statistic 122.93912
Alpha Value 0.05
P value < 0.00001
Analysis p < 0.05, significant difference in means
Table 5: Post-Hoc Tukey test p-values for comparing the mean concentration of lycopene extracted from each variety.
Treatment Pairs Tukey HSD Q statistic Tukey HSD pvalue
Tukey HSD analysis
1:2 15.36 < 0.000001 Significant (*p < 0.05) 1:3 6.17 0.0247 Significant (*p < 0.05) 2:3 21.53 < 0.000001 Significant (*p < 0.05)
Discussion
Analysis of results
The extraction of lycopene from each variety of tomato showed quite concise results. From inspection, the mean concentration of lycopene was different between each variety. Roma tomatoes were found to have the highest lycopene concentration, followed by Truss tomatoes, and lastly Cherry tomatoes. The low standard deviation values for each group supported these results, indicating a low variance in the data points. This suggests there is a difference in the concentration of lycopene between different tomato varieties, but further analysis was required to determine this.
An ANOVA test was conducted to determine whether or not there was a significant difference between the means. This also involved a Post-Hoc Tukey test, which indicated, if any, which two groups had a significant difference. ANOVA results generated a p-value < 0.00001, which was less than the alpha value of 0.05. This allowed for the rejection of the null hypothesis, supporting the alternate hypothesis that there was a significant difference in the average concentration of lycopene in each variety. The Post-Hoc Tukey test also indicated a significant difference in concentration between all 3 groups, each pair showing a p-value of less than the alpha value of 0.05.
This research agrees with previous reports, which also suggested different concentrations of lycopene in different tomato varieties. In 2008, Toma et al. also found that on a dry weight basis Roma tomatoes contained the highest amount of lycopene, while Truss tomatoes contained the least. This compares very similarly to the results of this report.
These results could be due to a range of factors. Each variety of tomato is grown under different conditions, and the shape and size differ significantly. To gain a further understanding on the topic much more research could be done into the specific elements of the varieties that lead to this difference in lycopene concentration, as the results show this is quite a significant amount.
One research area is the concentration difference of lycopene in the skin of the tomato versus the flesh. This could have a major impact between varieties, since a key difference between each is the size and shape. Cherry tomatoes are generally smaller, and therefore have a higher ratio of skin to flesh than larger tomatoes. This could be experimented through similar methods outlined in this report.
Furthermore, the growing conditions of each variety may have an effect on the lycopene concentration. This includes things such as irrigation, access to sunlight, location, and time until harvest – all which fluctuate with the growth of different varieties. Processing methods could also severely impact the lycopene content, including time from farm to supermarket, packaging, and additional treatments.
It has been found that one way to maximise lycopene concentration is the processing of raw varieties into products such as tomato paste and tomato juice. This had been investigated by many reports, indicating that this is because of the high heat conditions in some processing methods. Also, the interactions between lycopene and fats enhance its bioavailability, suggesting cooking processes using oils to form tomato sauces and paste are what lead to this increase (Soares et al., 2019).
Although exposure to temperature was not studied in this report, this research suggests lycopene concentration is also affected by the variety of tomato, indicating that this should be considered when forming commercial tomato products. It is therefore important for producers to consider the type of tomato they are growing, especially if they are aiming at maximising the antioxidant properties of their produce. This is also an important factor for consumers to consider when aiming at increasing their antioxidant intake as part of achieving a more balanced diet.
Although this can be an important thing to consider in some cases, it is often recommended to consume any variety of tomato for its antioxidant properties, and this research still indicates that all tomatoes possess high levels of lycopene.
Possible sources of error
Although the methodology produced clear and concise results, there were some potential sources of error that could be minimised in further research. It was fairly difficult to properly blend the tomatoes, leaving moderately sized pieces of skin in the mixture instead of a uniform puree. This was found to be quite problematic, since it was difficult to take a small sample of each puree to test that was a true reflection of the tomato. For example, some groups had flakes of skin while others had none. There was, however, a large effort in ensuring each group’s puree was similar in consistency, and multiple samples were taken to reduce the impact of this error margin.
Since the tomatoes were store bought, it was difficult to know whether all variables were fully controlled. Differences in lycopene concentration therefore could be due to other factors, such as the time harvested, or processing and packaging methods. It would have been ideal to grow the tomatoes for this experiment, but time was a major issue. Therefore, for future experimentation, to improve reliability of results the tomatoes should be grown instead of purchased.
Furthermore, the colourimetry method of extraction posed some sources of error that were ultimately unavoidable. This mainly involved the interference from other carotenoids, such as β-carotene in the absorbance of light. To minimise this impact, a wavelength of 500 nm was chosen, aimed at maximising the absorbance potential of lycopene and minimising interference from other compounds. This should not significantly impact the difference in means between varieties though, since all samples were tested at the same wavelength. This reflection of concentration would not be entirely accurate, compared to HPLC methods. It was, however, important to apply this method, since more advanced HPLC methods are impossible to do in a school lab, as the equipment is expensive and not easily accessible.
It could therefore be very beneficial for this research to be conducted with a more advanced method, aiming at refining and confirming the results found.
Areas of further research
This report provides a starting point for further research to be conducted, as there are many improvements that could be made to ensure all other variables are controlled. It does, however, suggest a difference in concentration exists, allowing for more research to be built off this. More research could also be conducted on why this relationship exists, such as an investigation of the effect of growing conditions on tomato lycopene, or the difference in concentration of lycopene in the skin versus the flesh of the tomato.
Also, research could be conducted in investigating the effect of different variables on the concentration of lycopene in tomatoes. One example is a further look at processed tomato products, gaining a deeper understanding why they contain higher concentrations of lycopene.
Conclusion
This report has investigated the effect of different varieties of tomato on the concentration of lycopene extracted. Three groups were created, each consisting of
either Truss, Roma, or Cherry tomato varieties. These were each mixed with a solvent Hexane: BHT: acetone mixture, and absorbance at 500 nm was recorded using a colourimeter. These absorbance values were converted into concentration of lycopene using Beer Lambert’s Law. The data analysis involved an ANOVA test to compare whether any of the mean concentrations were significantly different, followed up with a Post-Hoc Tukey test to determine which groups were different. The results of this showed all groups had significantly different mean concentrations of lycopene, leading to the acceptance of the hypothesis that the variety of tomato affects the concentration of lycopene extracted.
Acknowledgements
I would like to thank Dr Katie Terrett for her assistance in formulating an idea, help in setting up the method, guidance throughout the data collection process, and for her support in writing and editing my report. Also, thanks to James Wilson, for suggesting valuable edits and supporting me in my data collection.
References
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