A Comparison of the Concentration of Lycopene in Australian and Italian Canned Tomatoes

A Comparison of the Concentration of Lycopene in Australian and Italian Canned Tomatoes

Jacob Hill – St Ives High School

Lycopene, a carotenoid commonly found in tomatoes and other red fruits has powerful antioxidant properties that combat free radicals in the body. This study aimed to determine if there is a difference in the concentration of lycopene in tinned tomatoes from different regions, namely, Australia and Italy. Lycopene concentration can be measured using colourimetry after it has been dissolved into a solution of hexane:BHT:acetone. This study revealed that the lycopene concentration of canned tomatoes from Italy and Australia was not significantly different with a P value of 0.768. Consuming foods and meals made with tomatoes sourced locally or from Italy will provide the same amount of lycopene and thus the same antioxidant properties and health benefits.

LITERATURE REVIEW

Antioxidants are a group of organic compounds that promote health in the human body by counteracting free radicals. Free radicals are compounds with unpaired valence electrons. Their unpaired valence electrons, give them the ability to either donate or accept a proton making them highly reactive (Martemucci, et al., 2022).

Free Radicals are created in the body from metabolic processes, usually they are beneficial, helping with the destruction of pathogens. However, free radicals also enter the body from external sources such as, smoking, x-rays, air pollutants and ozone. This can cause an excess of free radicals and this imbalance causes oxidative stress resulting in damage to the membranes and contents of cells. The action of free radicals has been linked to heart disease, increased risk of stroke, diabetes, cancer, macular degeneration and general acceleration of ageing (Lerner & Lerner, 2014 and Ashok, 1999).

Lycopene (C40H56) (Figure 1) is a long chain hydrocarbon molecule classified as a carotenoid. Carotenoids are a class of red pigments with antioxidant properties that are found in many fruits and vegetables. Lycopene has the greatest antioxidant ability of the carotenoids and is one of the major carotenoids found in foods (Zhou, et al., 2016). Consuming lycopene has been linked to reduced risk of cancers, particularly prostate cancer and decreased oxidisation of cholesterol (Górecka, et al., 2020 and NHMRC 2017).

Figure 1; Chemical structures of Lycopene and β-carotene

(Gumus & Turker, 2014)

The National Health and Medical Research Council, (NHMRC, 2017) recognises the antioxidant benefits of lycopene, however, they do not give a level of recommended intake. Other studies suggest that intakes between 8–21mg per day appear to be most beneficial (Petre, 2018). The greatest dietary sources of lycopene include red fruits and vegetables such as tomatoes, watermelon, carrots, pink guava, papaya and dried apricots. Unfortunately, Australian studies reveal that only 3.7% of children from the ages of 14-18 met their dietary requirements for vegetables in 2015 (AIHW, 2022). There is evidence that up to 80% of dietary lycopene comes not from fresh fruit and vegetables but from processed tomato products used in foods such as pizza and pasta (Zhou, et al., 2016 and Górecka, et al., 2020).

With the majority of dietary lycopene coming from processed tomato products it raises questions regarding the lycopene content in processed tomatoes. This study will investigate the difference in concentration of lycopene levels in processed tomato products originating from different areas. Góreka, et al. (2020) states that the concentration of lycopene in tomatoes correlates to the time that the tomatoes spend in the sun. Australian consumers can generally access both Australian and Italian tomatoes which are readily available in Australian supermarkets. Tomatoes are a summer crop, since Italy has comparatively longer days in summer than Australia, this could signify a greater lycopene concentration in Italian tomatoes over Australian. There are many other factors that could affect the lycopene content in addition to sunlight such as the manufacturing processes and time taken to transport to the supermarkets. It is not known if this creates a significant difference between the lycopene content of these products (Górecka, et al., 2020).

A review of the literature revealed multiple studies with methods for extracting and measuring lycopene from tomato products using colourimetry. Colourimetry studies were very similar across all studies with minor differences regarding mass of product tested, volumes of solution used, and time taken to stir the solution.

A 2:1:1 Hexane:Ethanol:Acetone (solvent solution) mixture is needed to dissolve the lycopene and separate it from the tomato. After stirring, adding water causes the solution to separate into polar and non-polar layers. The top, coloured, layer containing non-polar molecules dissolved in hexane is then analysed using colourimetry at 503nm (Alda, et al., 2009; Adejo, et al., 2015; Fish, 2002; Fish, et al., 2003; Suwanaruang, 2016; Anthon & Barret, 2007; de Montemas, 2020).

To prevent the oxidation of lycopene, some studies dissolved butylated hydroxytoluene (BHT) in ethanol prior to its addition into the solvent solution. BHT is a synthetic antioxidant commonly used as a preservative because it oxidises preferentially to other compounds. This prevents the lycopene in solution from being oxidised (Anthon & Barret, 2007).

Lycopene will oxidise by photo-oxidation in the presence of light (MartínezHernández, et al., 2016). In food products, oxidation of lycopene is undesirable as it reduces the oxidative ability of the food and results in loss of beneficial antioxidant properties. Fish, (2002) states that the absorbance of lycopene in solution decreases at 1% per hour when stored in amber vials under fluorescent lighting. This provides evidence of the constant degradation of lycopene in light and provides incentive to use of BHT as a preservative.

Lycopene is not the only red pigmented carotenoid present in tomatoes. βcarotene is a prevalent antioxidant having the same chemical formula of C40H56 and a similar absorbance spectra to lycopene. β-carotene is the second most prevalent carotenoid in fresh tomatoes having a concentration of 15% relative to lycopene (Anthon & Barret, 2007).

Lycopene and β-carotene have slightly differing absorbance spectra. Testing the absorbance at 503nm and then again at 444nm enables the concentration of Lycopene and β-carotene to be calculated using the Beer-Lamberts law. This equation states there is a linear relationship between the concentration and the absorbance of the solution. To calculate absorbance, the extinction coefficient is needed. This is the amount of light absorbed by a compound at a specific wavelength per concentration (MolL-1) per optical path length (cm) (Anthon & Barret, 2007).

When considering both the absorbance of lycopene and β-carotene, the following equations are needed.

Equation 1. Where ε is the extinction coefficient, A is the absorbance, C is the concentration and optical path length is omitted because in a colourimeter this is 1. Subscript L is used to signify the concentration and extinction coefficients of lycopene and β for β-carotene. Subscript 444 and 503 to signify the wavelengths of light the values correspond to.

These equations are rearranged into equation 2 which will be used to calculate concentration from the absorbance values gathered.

Equation 2. Relating the absorbance values and extinction coefficients to find concentration of lycopene

SCIENTIFIC RESEARCH QUESTION

Do cans of tomatoes from different regions contain similar concentrations of lycopene.

SCIENTIFIC HYPOTHESIS

There is negligible difference in the concentration of lycopene in Italian and Australian canned tomatoes available to Australian consumers.

Preparation of solvent

A solution of 2:1:1 Hexane:BHT:Acetone (solvent solution), was used in the extraction of lycopene. The BHT solution was created as a 0.5%(w/v) BHT in ethanol solution. 20 ml of freshly made solvent solution was added to three 25ml conical flasks.

Preparation of Tomatoes

A tin of pre-chilled diced tomato was opened and half was placed into a beaker and blended using a handheld blender. Approximately one gram of tomato was weighed and placed into each conical flask.

Extraction of lycopene

Conical flasks were covered with plastic wrap to decrease loss of solvent by evaporation. Each sample was stirred for 15 minutes using magnetic stir bars. carotenoids were dissolved into the solvent solution. 10 mL of water was then added into the solution where two distinct layers formed.

Colourimetry

Colourimetry was conducted using PASCO PS-2600 spectrometer and standardised quartz cuvettes. The colourimeter was calibrated using hexane. The top layer of each solution was tested for absorbance at 444 and 503nm and was tested twice at each wavelength. The concentration of lycopene was calculated using Equation 2.

RESULTS

Ten tins of tomatoes were tested, with three samples were taken from each tin. Each sample was then run through the colorimeter twice to reduce the impact of random error in the data. For each test the concentration of lycopene was calculated. Outliers were considered as any values 2 standard deviations away from the mean and were removed. Results from each tin were then averaged for statistical testing Table 1.

t-Test: Two-Sample Assuming Equal Variances

Table 1. t-test results

An f test was performed with α value of 0.05 that gave a p value of 0.1218425 (shown in appendix) showing an insignificant difference in the variance of data sets. Two tailed t-test was preformed to investigate a difference in means of the two data sets. As shown in Table 1 it produced a P value of 0.768 which is an insignificant difference between results

Figure 2. Graphical representation of the five-number summary of concentration of Lycopene in Australian and Italian tins of diced tomatoes. The Average concentration of lycopene in milligrams of lycopene per kilogram of tomato for Australian tomatoes was 21.633 and 21.447 for Italian.

Figure 3. Concentration of lycopene vs mass of tomato mass

There was an inverse relationship between the calculated concentration of lycopene and the mass of tomato used in testing (Figure 3).

DISCUSSION

This report aimed to determine if there is a significant difference in the concentration of lycopene in canned tomatoes from Australia and Italy. This investigation provides insight into the differences, or lack thereof between concentrations of lycopene in Australian and Italian canned tomatoes. Due to its antioxidant abilities, lycopene is an important molecule and diced tomatoes with higher concentration of lycopene are more desirable.

The results revealed that to a confidence interval of 95%, the calculated P value of 0.768 was much greater that the 0.05 needed to indicate a significant difference. This supports the hypothesis that there was no significant difference in the concentration of lycopene in Australian and Italian canned tomatoes.

The results also revealed an inverse relationship between concentration of lycopene and mass of tomato (Figure 3) which was an unexpected trend. Since the mass of the tomato is considered when calculating the concentration of lycopene per kg of tomato, it was anticipated that this gradient would trend near zero. This trend may be a result of time stirring solution, which was kept consistent at 15 minutes or until colouration of tomato was lost. Lycopene from samples of tomato with a higher mass may not have been fully dissolved into solution when stirring was completed.

A number of processes were employed to ensure data validity. Due to the volatility, fresh solvent was used regularly through the experiment to ensure that components were in their required concentrations.

Repetitive testing of the same cans of tomato and averaging of data collected served to remove the impact of random error on the experiment and increase its reliability.

All equipment used in this study was rinsed three times in a dishwasher without the use of detergent which would act as an emulsifying agent, preventing the separation of the solution into layers as is required in the method.

Multiple studies have been conducted on lycopene levels in fresh tomatoes and there is a wide variation in results are as follows; 6.6 – 490 mg/kg (Fish, et al., 2003); 120mg/kg (Alda, et al., 2009); 29.4mg/kg (Górecka, et al., 2020); 104.699mg/kg (Suwanaruang, 2016); 25 – 6700 mg/kg (Martínez-Hernández, et al., 2016). The results that have been collected in this experiment at 21.632 and 21.447 mg/kg are consistently lower than a number of the studies described in literature. This may be worth investigating, to determine if tinned tomatoes have a lower concentration of lycopene than fresh tomatoes. This difference could also be indicative of systematic error present in the method. However, systematic error that may have occurred in the experiment has little bearing on the conclusion as the hypothesis of the experiment was to investigate a difference and as long as random error was removed from the method, then conclusions drawn from the experiment are valid.

Drawbacks were encountered with the software that was used because of its inability to select specific wavelengths. However, this did not impact the overall results of the study and only resulted in increasing the time taken for the absorbance to be measured.

An extension of this study could further investigate the trend that arose in the concentration of lycopene and the mass of tomato tested. This would involve testing smaller and larger masses of tomato to investigate if the trend continued. Suwanaruang, (2016) used 0.001g of tomato and 8 mL of solvent solution which is 0.000125 g of tomato per mL of solvent compared to the 0.05g/ml used in this study. Testing with ratios such as this may yield a more consistent result for the concentration of lycopene. However, due to limitations in the accuracy of scales available, a mass of 1g was chosen as to increase the number of significant figures. As lycopene is soluble in hexane to 1g/L (PubChem, 2022), and initial testing with 1g of tomato showed a mass of dissolved lycopene in 20mL of hexane to be 2.187 x10^-5g. or 0.0010935 g/L solubility wasn’t perceived as an issue.

Variations on this study could investigate the concentration of lycopene in different tomato products that are commonly found in a western diet and compare them to the concentration of a fresh tomato. The results of an investigation of this idea would depend of the amount of processing that was done, largely the removal of water which makes up a majority of a tomatoes mass. This would be a further extension of this study and an opportunity to not only study one ingredient but to study whole foods and extrapolate to average quantities consumed by teenagers, and consider which meals would give greater lycopene content and thus greater antioxidant benefits.

CONCLUSION

This report investigated whether there was a difference in lycopene concentration between Australian and Italian diced canned tomatoes. The results of this experiment support the hypothesis stating that there is an insignificant difference in lycopene concentration. There was a low random error in the experiment allowing for the above conclusion to be drawn irrespective of the presence of systematic error because of its equal affect to both Australian and Italian tomato tests.

Irrespective of any systematic error that was present reducing the accuracy, effecting the concentration of lycopene in both the Australiana and Italian tomatoes. the conclusion that has been drawn from the experiment is valid because of the removal of random error from the experimental method. For the average Australian consumer, purchasing either locally grown or Italian tomatoes will provide the same lycopene intake and associated health benefits of reduction in risk of cancer, stroke and diabetes. There may be other factors that influence consumers’ product choice such as supporting locally grown produce, farmers and industry and reducing the environmental impact of transport of tomatoes grown overseas. However, Australian consumers can be reassured that both the Australian and Italian tomatoes on their local supermarket shelves are equal in their lycopene concentrations and thus health benefits.

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AIHW, 2022. Australia's health 2018, Fruit and vegetable intake - Australian Institute of Health and Welfare. [Online] Available at: https://www.aihw.gov.au/reports/australia s-health/australias-health2018/contents/indicators-of-australiashealth/fruit-and-vegetable-intake [Accessed January 2022].

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APPENDIX

F-Test Two-Sample for Variances

Statistical analysis of variance using an f test

Collection of data collected from each can of tomatoes after averaging of raw data

Data after calculation of concentration and removal of outliers

Image of absorbance of the top layer of solution showing the slight inaccuracy in the software.