Consumption Of Fresh Fruits And Vegetables Biology Essay

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The scope of my investigation was limited on how do the concentrations of lycopene in three hybrid tomato cultivars vary during their ripening season. I was examining these changes in three cultivars in my garden: Kentaro F1, Bolzano F1 and Dasher F1 respectively. The research question was:

"How much lycopene is present and how does the quantity of lycopene vary in fresh fruits of three hybrid cultivars of tomato (S. lycopersicum): Kentaro F1, Bolzano F1 and Dasher F1, during its ripening season from 13th July to 11th September 2012?"

For the purpose of my research, method presented by Sadler et al. (1990) was modified and carried out. First of all tomato homogenates were prepared and then we extracted the lycopene from homogenates using the mixture of organic solvents and water. Finally we collected sets of quantitative data about lycopene concentration. That data were obtained with the spectrophotometric measurements of each extract.

The results show that the concentrations of lycopene are varying among cultivars of tomato (S. lycopersicum) from F1 series. Levels of lycopene in Kentaro F1 and Dasher F1 cultivars were significantly higher when compared to those of Bolzano F1 through all the experimental period. Minimums occurred in July and the maximums were, surprisingly, observed on 11th September, probably, due to extensive rainy season before 11th September - a lot of water together with the high temperatures caused cracking of tomato fruits and by that, cell walls were damaged and consequently more lycopene was released out of the cell. That leads to the conclusion, that levels of lycopene are varying trough the ripening season at different rates in different cultivars.

Word count: 269

TABLE OF CONTENTS

INTRODUCTION

Consumption of fresh fruits and vegetables is believed to have positive impact on human health. Tomatoes are one of the most commonly used vegetables in everyday European diets. Due to described fact it is not surprising that tomato plants are growing in almost every garden in our region. In our garden we planted three cultivars of hybrid tomatoes: Kentaro F1, Bolzano F1 and Dasher F1.

Recently there have been written and spoken a lot about redness of tomato fruits and its beneficial effects on human health. Recent study done by Aydemir et al. (2013) revealed that "dietary consumption of tomato products and especially the red tomato pigment lycopene has been associated with lower risk of cancer. New evidence is emerging toward metabolic pathways mediating the anti-cancer activities of lycopene". Because lycopene is frequently included in human diets as food supplement and because of its beneficial effects on human health it is worth to find out the quantity of lycopene present in different fruits and vegetables.

The main cause for redness of tomato is presence of special pigment called lycopene. Lycopene is an unsaturated straight chain hydrocarbon carotenoid which consists of 11 conjugated and 2 unconjugated double bonds and is present in many fruits and vegetables (Sadler et al., 1990). It is a fat soluble carotenoid (Sandmann, 1994) which is responsible for red colour of tomatoes, watermelons, pink grapefruits and several others fruits and vegetables. Lycopene is widely recognized for its unusually high antioxidant capacity and according to Di Mascio et al. (1989) it is known as the most efficient biological carotenoid singlet oxygen quencher, whose antioxidant capacity is twice as high as the antioxidant capacity of β-carotene.

The lycopene biosynthesis increases dramatically during the ripening process as chloroplasts undergo transformation to chromoplasts (Kirk and Tilney-Basset, 1978). Chromoplasts are non-photosynthetic plastids that accumulate carotenoids and give a bright colour to plant organs. (Bathgate et. al., 1985).

Figure 1: Absorption spectrum of lycopene in hexane extract [1] 

Figure 2: Schematic presentation of the main structural changes occurring during the chloroplast to chromoplast transition [2] 

Research by Agarval et. al in 1998 revealed, that processing of tomato increase bioavailability of lycopene by increasing the surface area available for digestion when compared to tomato in its raw form - in fresh fruit, lycopene is enclosed in the fruit tissue by the cell walls therefore only a portion of the lycopene that is present in fresh fruit is absorbed. Processing breaks down cell walls to release lycopene what results in higher yields of it (Fielding et. al., 2005).

Based on research papers I read, I decided for spectrophotometric quantification of lycopene in tomatoes (Sadler et al., 1990) as that method was the most accurate and the most rapid one. I decided for standard method, in which after extraction in solvents mixture, lycopene extract was obtained from upper - hexane layer (non-polar hexane dissolves non-polar lycopene; polar substances: water, ethanol and acetone formed a separated layer) and then its absorbance at 503 nm was measured and obtained values were further used for calculations, by which lycopene content was estimated. According to Fish et al., 2002, absorbance of lycopene at 503 nm (A503) has been selected to avoid interferences from other carotenoids present in examined tissues, as absorbance A503 is one of the characteristic absorbencies for lycopene extracted in hexane. What is more, I calculated the amount of lycopene using two types of data: experimental and theoretical and compared them. Based on that, I was able to assess the accuracy of my modified method.Cancer Flowchart

Figure 3: Possible mechanisms for the role of lycopene in human health [3] 

Research question:

How much lycopene is present and how does the quantity of lycopene vary in fresh fruits of three hybrid cultivars of tomato (S. lycopersicum): Kentaro F1, Bolzano F1 and Dasher F1, during its ripening season from 13th July to 11th September 2012?

In relation to written, the following was predicted:

H1: there will be differences in the concentrations of lycopene (mg/kg) in tomato fruits, as they are each different cultivar.

H2: one of the cultivars, Bolzano F1, produces yellow fruits. Therefore, if lycopene content is related to red colour, levels of lycopene in its fruits will be lower than in other two as the Bolzano F1 probably contains much more other carotenoids than lycopene.

H3: quantity of lycopene will rise from July to September, and then it will drop

MATERIALS AND METHOD

Plant materials and growth conditions

The measurements were carried out from July to September 2012 in the laboratories of Marifarm d.o.o. - a pharmaceutical company in Maribor. Examined cultivars were planted on my home garden in early May. The plants were protected against the sun and hail with protecting net and were watered regularly every day. This year, we planted three hybrid cultivars of tomato of F1 series respectively, as presented on Figure 2. The source of my sample is represented on Figure X bellow, where: A stands for Kentaro F1 cultivar, Japanese cocktail tomato, which produces red, sugary sweat fruits. B represents Bolzano F1 cultivar, bush tomato with yellow-orange fruits and C is for Dasher F1, which has fruity, sweet, red, crisp plum fruits.

Figure 4: Tomatoes plants, planted in my garden. Photo Alen Krajnc.

Day of harvesting

Highest temperature

(ËšC)

± 1˚C

Lowest temperature

(ËšC)

± 1˚C

Average day temperature

(ËšC)

± 1˚C

Average relative humidity (%)

± 1%

16th July

22.4

13.4

16.9

66

30th July

26.8

16.4

21

67

13th August

25.4

13.7

19.9

54

27th August

24.9

13.7

18.2

53

11th September

29.8

14.8

21.5

65

Table 1: The highest, lowest and average day temperature and average relative humidity for each day of perfuming the experiment [4] 

Five mature fruits of similar size of each cultivar were harvested every time the experiment took its place. Only mature red tomatoes were included into the investigation; selection of mature fruits was based on the "Color classification requirement in United States standards for grades of fresh tomatoes" chart, which was published by the United States Department of Agriculture, 1975 (Appendix 2).

Chemical reagents

n-Hexane, assay: ≥99% (GC)

Ethanol absolute, assay: ≥99.8% (GC)

Acetone, assay: ≥99.9%

All reagents listed above were bought from Sigma-Aldrich and were of analytical grade.

Materials and apparatus

tomato fruits of next cultivars: Kentaro F1, Bolzano F1, Dasher F1

distilled water

15 test tubes (20 mL) in stand

pipette (10ml ± 0.02mL)

pipette (15ml ± 0.06mL)

Pasteur's pipettes (5 ml and 10mL)

15 erlenmayer flasks (250mL) with stoppers

3 beakers (100mL)

glass bottle (2000mL) with stopper

measuring cylinder (1000 mL ± 5mL)

2 measuring cylinders (500 mL ± 2mL)

measuring cylinder (100mL ± 1mL)

precise balance Mettler Toledo XS20002S/M (±0.01g)

mortar and pestilence

laboratory shaker (IKA RS 10 Basic)

knife mill (Retsch Grindomix GM 200)

centrifuge (Tehtnica Železniki, LC - 320)

UV-Vis Spectrophotometer (Perklin Elmer: Lambda 25)

UV-Vis Spectroscopy cells (Perklin Elmer)

Computer with UV-Win Lab software

parafilm

knife

cutting board

Experimental procedure

Preparation of solvent needed for extraction and marking

Solvent consisted of n-Hexane, ethanol absolute and acetone in relationship 2:1:1 v/v respectively. As all three solvents are very volatile, preparing solvents just before their use is only appropriate. We usually prepared one litre of solvents mixture at once because that was enough for one series of extractions (all 5 samples from one cultivar on each measurement). The volumes of each organic solvent were measured with 500 ml measuring cylinders. The solvents were mixed in closed bottle before using it in further procedures. Solvents were prepared three times per experimental day. We marked all the beakers and test tubes appropriately with names of tomato cultivar (five of both for each cultivar).

Preparation of homogenates

Tomato fruits were harvested in the morning before performing the extraction. We labelled the bags with names of each cultivar and put 5 mature fruits of similar size into corresponding bag, in order to obtain single sample. Secondly, we prepared three beakers and labelled them with date and name of cultivar (we prepared one beaker for each cultivar). The fruits of one tomato cultivar were taken and sliced into smaller pieces on cutting board. Pieces were then transferred into homogenizer and got homogenized at 10x103 rotates per minute for three minutes. When 3 minutes passed, we transferred the homogenate into beaker. If there were still some bigger pieces present in homogenate, we used mortar to homogenize the remaining tissue manually. When homogenate was finally transferred into beakers, they were sealed with parafilm. The described procedure has been repeated for other two cultivars.

2.4.3 Weighting of tomato homogenate

Around 1.00 g of one tomato homogenate was weighed into associated 250 ml beaker (we avoided the seeds, if they were still present). We prepared 5 samples for each cultivar. The beakers were sealed then.

2.4.4 Extraction of lycopene (Sadler et al., 1990)

Using 100ml measuring cylinder we added 100 ml of mixed solvent into each beaker with tomato homogenate. The flasks were closed with stoppers and putted on laboratory shaker at 150 Mot/min and shake them for 10 minutes. As the laboratory was air conditioned on 20°C constantly, the temperature of surrounding was kept constant always, when experiment took place.

Then using 15 ml pipette we added 15 ml of distillate water to the content of the beaker to separate the phases. We were shaking the mixture at the same rate as before again for 10 minutes.

When 10 min passed, we took the beakers and gently moved them on working table. The sample was left to stand for 1 minute so that the content stopped moving. Meanwhile, marked test tubes and 15ml Pasteur's pipettes were brought to the working desk. After 1 min, we used Pasteur's pipettes to transfer around 15 ml of upper layer (coloured layer) from each beaker into corresponding test tube. We have taken special that we did not suck some water along with the upper layer. New pipettes were used for every transfer and the used ones were discarded.

Figure 3: Samples after shaking and before taking sample for centrifugation from upper (yellow coloured) layer. Photo Alen Krajnc.

The test tubes containing samples were putted into the centrifuge for 5 minutes at 3000 rpm. When centrifuging stopped, they were out and putted them into test tube stand.

2.4.5 Spectrophotometric analysis of Lycopene extracts

Firstly we turned on the computer, launched the UV-Win Lab software and turned on the spectrophotometer. Then, calibration of UV-Vis Spectrophotometer with the option "Auto-Zero" was performed and number of pararells was set (see appendix 4).

When software asked for next sample, we took out the cuvette (let us consider that we carried out all three solvent measurements), poured the content of the cuvette into beaker for waste organic solvents and washed it with around 1 cm3 of the content of test tube with the sample, that was to undergo measurement next. That "washing fluid" has been poured out. Then, using 5ml Pasteur's pipette we transferred around 2 cm3 of the same sample into it, sealed it, wiped it gently from outside (if needed) and putted it into UV-Vis Spectrophotometer. "Start Measuring" was pressed and then we were waiting until the software asked to insert next sample (samples followed automatically). The procedure was repeated for all fifteen samples at each day of measurements.

METHOD DEVELOPMENT AND PLANNING

2.5.1 In which ways was the experimental procedure modified?

After I read several articles, I decide that I will able to reach stated aim in best way by following Sadler's et al. (1990) procedure for extraction of Lycopene in tomato and measuring absorbance at 503 nm. Right after Sadler's et al. method was studied, I did a piloting experiment which revealed several deficiencies in original method, that is was I moderated Sadler's et al. method to maximize its accuracy. Formulas for calculating the amount of Lycopene in samples in mg/kg were adopted from Ravelo Perez et al., 2008.

To start with, first problem occurred at the very beginning. In original procedure, the amount of homogenate needed for extraction is pipetted into Erlenmeyer flasks using micro-pipette. Because my tomato fruits were rather small in their size, homogenization represented quite big challenge. As there were still some pieces of tomato pulp and skin in homogenate obtained after homogenization with homogenizer at highest speed, homogenates were further homogenized manually in mortar, and then transferred again into the homogenizer, until all pieces disappeared. Still, the homogenate was too dense for micro pipetting, that way I decided to follow the alternative Sadler's et al. procedure, in which instead of micro pipetting, I weighted the amount of added homogenate.

Secondly, when extraction took place, mixtures in sealed Erlenmeyer flasks were shaken for 10 minutes on laboratory shaker at 150 Mot/min to make sure that all lycopene would dissolve in solvent. As later water was added to separate the polar and non-polar layers, I decided to shake the mixture once again for the same amount of time and on the same speed as before to ensure that layers will split completely.

What is more, according to literature, Sadler's et al. (1990) extraction procedure is optimized for the amounts of lycopene typically found in undiluted juice from red tomatoes. Because I therefore used undiluted homogenate for extraction, there were still some tiny pieces present in extract that was to be used for absorbance measurements. As the presence of those tiny particles could affect the absorption, I centrifuged the extract for 5 minutes at 3000 rpm and then use just upper 3 cm of solution for measuring the absorbance.

As I wanted accurate results, I decided to make 5 parallels for each cultivar on each experimental day and 3 repeats (which were done automatically by UV-Vis Spectrometer) for each parallel. Finally I also compared my data with a calibration curve of Lycopene from literature, so that I was able to asses, whether my method was accurate or not.

2.5.2 Deriving formulas for calculating the quantity of Lycopene in mg/kg

(Adapted from: Ravelo Perez et. al, 2008)

The content of Lycopene (mg/kg) can be estimated in two ways - either based on experimental data or theoretical method, which uses calibration curve and Beer-Lamberts law respectively. For the purpose of comparing my results with literature values, I will use the calibration curve and its equation obtained from Ravelo Perez et al. (2008):

Graph 1: Calibration curve for Lycopene - absorbance at 503nm versus Lycopene concentration in hexane (mgL-1)

High correlation coefficient (R2 = 0.9999) signifies good accuracy of standard curve and thus enables us to derive the formula for calculating Lycopene content, taking into account Beer-Lambert law as mentioned above.

First of all, we have to know the value of molar extinction coefficient of Lcopene in hexane, which can be found in literature and takes value of 17.2 x 104 M-1cm-1.

Then, considering Beer-Lambert law, it follows that:

Absorbance at 503nm (A503) = É›(M-1cm-1) x b(cm) x [Lycopene concentration (M)],

Where É› is molar extinction coefficient of Lycopene in hexane and b is path-length of UV-light through cuvette.

By considering the value of É› as well as taking into account the molecular weight of Lycopene, which is 536.9 respectively and by changing units, the final equation for experimental formula of Lycopene content is:

Lycopene content (mg/kg of fresh wt.) = A503 x 31.2/g of tomato homogenate

While upper equation is based on the experimental data, which will be obtained by us, we can derive also theoretical formula by appropriate substitution in the Beer-Lambert law equation, ad what we get is that

Lycopene content (mg/ kg of fresh wt.) = (A503 - 0.0007) x 30.3/g of tomato homogenate

The use of both formulas for the same data will allow me to evaluate the Lycopene content and will enable the comparison of the experimental data with the literature values.

RESULTS

TOMATO CULTIVAR

REPETITION

AVERAGE ABSORBANCE AT 503 nm

±0.0030 nm

MASS

[grams]

± 0,01

LYCOPENE CONTENT [mg/kg fresh wt.]

KENTARO

1

0,3075

1,17

8,20

2

0,3532

1,29

8,54

3

0,3236

1,06

9,52

4

0,3072

1,02

9,40

5

0,3329

1,07

9,71

BOLZANO

1

0,0481

1,34

1,12

2

0,0477

1,52

0,98

3

0,0315

1,06

0,93

4

0,0362

1,10

1,03

5

0,0668

1,24

1,68

DASHER

1

0,3673

1,09

10,51

2

0,4321

1,31

10,29

3

0,3768

1,05

11,19

4

0,4023

1,14

11,01

5

0,4931

1,23

12,50

Table 2: Average absorbencies of Lycopene at 503 nm, masses of samples and experimentally determined amount of Lycopene present in three cultivars of hybrid tomato on 16 July 2012

Table 3: Average absorbencies of Lycopene at 503 nm, masses of samples and experimentally determined amount of Lycopene present in three cultivars of hybrid tomato on 30 July 2012

TOMATO CULTIVAR

REPETITION

AVERAGE ABSORBANCE AT 503 nm

±0.0030nm

MASS

[grams]

± 0,01

LYCOPENE CONTENT [mg/kg fresh wt.]

KENTARO

1

0,4550

1,09

13,02

2

0,4442

1,08

12,83

3

0,4203

1,03

12,73

4

0,4996

1,17

13,32

5

0,4881

1,15

13,24

BOLZANO

1

0,0448

1,04

1,34

2

0,0455

1,05

1,35

3

0,0618

1,02

1,89

4

0,0423

1,04

1,27

5

0,0450

1,03

1,36

DASHER

1

0,5458

1,03

16,53

2

0,4985

1,05

14,81

3

0,5622

1,02

17,19

4

0,5460

1,05

16,22

5

0,5714

1,11

16,06

Table 4: Average absorbencies of Lycopene at 503 nm, masses of samples and experimentally determined amount of Lycopene present in three cultivars of hybrid tomato on 13 August 2012

TOMATO CULTIVAR

REPETITION

AVERAGE ABSORBANCE AT 503 nm

±0.0030 nm

MASS

[grams]

± 0,01

LYCOPENE CONTENT [mg/kg fresh wt.]

KENTARO

1

0,5455

1,05

16,21

2

0,5636

1,04

16,90

3

0,6022

1,17

16,06

4

0,5952

1,08

17,69

5

0,5748

1,07

16,76

BOLZANO

1

0,0538

1,02

1,65

2

0,0573

1,12

1,60

3

0,0658

1,15

1,79

4

0,0558

1,05

1,66

5

0,0553

1,02

1,69

DASHER

1

0,6984

1,11

19,63

2

0,7786

1,12

21,70

3

0,6256

1,13

17,27

4

0,6897

1,11

19,39

5

0,6154

1,02

18,82

TOMATO CULTIVAR

REPETITION

AVERAGE ABSORBANCE AT 503 nm

±0.0030 nm

MASS

[grams]

± 0,01

LYCOPENE CONTENT [mg/kg fresh wt.]

KENTARO

1

0,3809

1,09

10,90

2

0,3694

1,07

10,77

3

0,3580

1,06

10,54

4

0,3421

1,02

10,46

5

0,3802

1,06

11,19

BOLZANO

1

0,0480

1,20

1,25

2

0,0458

1,01

1,41

3

0,0458

1,10

1,30

4

0,0422

1,01

1,30

5

0,0475

1,12

1,32

DASHER

1

0,4508

1,07

13,14

2

0,4887

1,08

14,11

3

0,5372

1,16

14,44

4

0,5748

1,00

17,93

5

0,5362

1,07

15,63

Table 5: Average absorbencies of Lycopene at 503 nm, masses of samples and experimentally determined amount of Lycopene present in three cultivars of local hybrid tomato on 28 August 2012

Table 6: Average absorbencies of Lycopene at 503 nm, masses of samples and experimentally determined amount of Lycopene present in three cultivars of hybrid tomato on 11 September 2012

TOMATO CULTIVAR

REPETITION

AVERAGE ABSORBANCE AT 503 nm

±0.0030 nm

MASS

[grams]

± 0,01

LYCOPENE CONTENT [mg/kg fresh wt.]

KENTARO

1

0,6734

1,08

19,45

2

0,6196

1,01

19,14

3

0,6375

1,01

19,69

4

0,5630

1,00

17,57

5

0,7565

1,18

20,00

BOLZANO

1

0,0765

1,07

2,23

2

0,0964

1,04

2,89

3

0,0861

1,03

2,61

4

0,0890

1,08

2,57

5

0,0911

1,09

2,61

DASHER

1

0,6012

1,05

17,86

2

0,5933

1,01

18,32

3

0,6137

1,02

19,53

4

0,6226

1,08

17,98

5

0,6018

1,03

18,22

Table 7: Experimentally determined average changes in the Lycopene content (mg/kg of fresh wt.) in all three cultivars of hybrid tomato from July to September and standard deviations of each examined cultivar (SD)

Date

Tomato cultivar and its SDs

Average Lycopene content

(mg/kg of fresh wt.)

16 July

30 July

13 August

28 August

11 September

KENTARO

9,07

13,01

16,72

10,77

19,17

SD (Kentaro)

0,66

0,25

0,65

0,29

0,95

BOLZANO

1,15

1,44

1,68

1,32

2,58

SD (Bolzano)

0,31

0,25

0,07

0,06

0,24

DASHER

11,10

16,16

19,36

15,05

18,38

SD (Dasher)

0,86

0,87

1,59

1,84

0,67

Table 8: Lycopene content (mg/kg) in all three cultivars of hybrid tomato from July to September calculated by using experimental (Xe) and theoretical (Xt) data (Appendix 3)

Tomato cultivar

Date of harvesting

Type of data

Average Lycopene content

(mg/kg of fresh wt.)

16 July

30 July

13 August

28 August

11 September

KENTARO

Xe

9.05

13.01

16.72

10.77

19.17

Xt

8.77

12.63

16.11

10.44

18.63

Relative error (%)

3.47

3.01

3.79

3.16

2.90

BOLZANO

Xe

1.15

1.44

1.68

1.32

2.58

Xt

1.10

1.38

1.61

1.26

2.49

Relative error (%)

4.18

4.50

4.17

4.86

3.69

DASHER

Xe

11.10

16.16

19.36

15.05

18.38

Xt

10.76

15.67

18.77

14.55

17.67

Relative error (%)

3.16

3.13

3.14

3.44

4.02

Graph 1: The changes in the average mass of Lycopene in three different tomato cultivars from July to September 2012 and ± 1 standard deviation of the data

Graph 2: Linear fit of average Lycopene content (mg/kg) in three different tomato cultivars from July to September 2012

Bolzano (best fit line equation and R2 value):

y = 0,2746x + 0,8092

R² = 0,5911

Kentaro(best fit line equation and R2 value):

y = 1,7937x + 8,3725

R² = 0,4631

Dasher(best fit line equation and R2 value):

y = 1,3452x + 11,976

R² = 0,4317

DISCUSSION AND EVALUATION

Results, as presented in graph 2 are clearly indicating that the content (mg/kg) of Lycopene is varying among cultivars of tomato (S. lycopersicum) from F1 series. That supports the first hypothesis, which states that there will be differences in Lycopene content between cultivars.

Levels of Lycopene in Kentaro F1 and Dasher F1 cultivars were significantly higher when compared to those of Bolzano F1 through all the experimental period. Bolzano F1 fruits contained, on average, 10% less Lycopene than other two cultivars. The reason for that could be the yellow colour of Bolzano's fruits. High amounts of Lycopene are closely related to presence red colour, which is occurring due to the presence of red lycopene carotenoid crystals. Bolzano F1 cultivar contains more yellow carotenoid crystals (Cao et al.; 2012) that red ones (that explains, why its fruits are coloured yellow, when mature) and thus lower yields of Lycopene are recorded. That supports the second hypothesis stating that the amount of Lycopene present in Bolzano F1 will be lower than in Kentaro F1 and Dasher F1.

We can notice some general trends, which are common to all cultivars. In first place, we can see that the lowest levels of Lycopene are occurring at the very beginning experimental period, and take values of 9.047 mg/kg for Kentaro F1, 1.146 mg/kg for Bolzano F1 and 11.10 for Dasher F1. Then the concentrations of Lycopene are rising rapidly, from 16th July to 13 August, but the rate of increase in much slower in case of Bolzano F1 - curves of other two cultivars are much stipper, therefore their increase is faster and greater and also their R2 value are smaller compared to Bolzano F1 R2 value (graph 2). After 13th August fall in levels of Lycopene in all cultivars were noticed but after that, Lycopene levels start to increase again and they reached their maximum values on 11th September. These maximum values are 19.17 mg/kg for Kentaro F1, 2.582 mg/kg for Bolzano F1 and 18, 38 mg/kg for Dasher F1 respectively. The possible explanation for sudden increase at the end of the experimental period is probably the extensive rainy season before 11th September (see appendix 1) - a lot of water together with the high temperatures (as can be seen from table 1, the highest average day temperature, 20.6°C, was measured on that date) caused that the fruits cracked, and in that was cell walls were damaged and consequently more Lycopene was released out of the cell, Fielding et. al., 2005). That means, that third hypothesis, which predicted that quantity of Lycopene will rise from July to September and then drop is rejected. At the end, the least amounts of Lycopene were still detected in Bolzano F1 fruits, and the percentage difference of it from other two cultivars increased from initial 10% to 14%.

Furthermore, what can be noticed is that levels of Lycopene were in most cases at the highest levels in Dasher F1 cultivar. However, on 11th September, the maximum lycopene value was reached by Kentaro F1. On that, the difference in Lycopene content between Kentaro F1 and Dasher F1 cultivar is not significant, as error bars are overlapping. Still, the rate of increase of Lycopene concentration, if considered from 16th July until 11th September was the highest for Kentaro F1, which rise for 10.6 mg/kg if compared to initial amount on 16th July, on the second place we can find Dasher F1, whose increase was for 7.28 mg/kg and lastly, as predicted, Bolzano F1 with rate of increase only for 1.44 mg/kg.

The amounts of Lycopene in examined cultivars of tomato were calculated by two formulas, derived from Ravelo Perez et al. (2008) - formula concerning the experimental data and the theoretical one. Table 7 shows Lycopene content of all samples that were analysed by the use of both - experimental and theoretical data. By considering theoretical value, we were able to calculate the relative errors. As can be noticed from described table, all relative errors are below 5% thus we can conclude, that difference in equations are not significant, that the results are comparable. In other words, our experiment design was enough accurate and precise, that we obtain reliable and comparable data. The quality and the reliability of data are also indicated by error bars, which are representing ± one standard deviations, which are presented in table 6. Small standard deviations which are on average 0.56 for Kentaro F1, 0.19 for Bolzano F1 and 1.16 for Dasher F1 are indicating that values are spread closely around corresponding average Lycopene concentrations. As they, in majority are not overlapping, that signifies that there are significant differences between the Lycopene contents of three cultivars and therefore small standard deviation are suggesting, that we obtained reliable and confidante data. The most precise and reliable data were obtained at Bolzano cultivar (it has the smallest value for standard deviation). The reason for that it probably the fact, that the fruits of Bolzano F1 were in general 2-3 times greater compared to fruits from other two cultivars, and therefore homogenization was more efficient, and that provided better homogenate for Lycopene extraction. In some cases, the overall masses of Kentaro F1 and Dasher F1 fruits of tomatoes resulted in unsuccessful (uncompleted) homogenization. This condition resulted in slightly higher standard deviations.

Also, what should be considered at the end is the quality of the method. UV-Vis Spectrophotometric analysis was proved to be accurate method for quantifying the amount of Lycopene in tomatoes. One of its greatest advantages, when compared to other methods mentioned in articles is that it requires low quantities of both - sample and organic solvents and its relatively fast. Based on written and considering the final result of my research, I would say that the method I used was appropriate for providing reliable answer on my research question. However, one of the weaknesses is probably the differences in masses of homogenates that were used for extractions. If I was to investigate again, I would suggest deciding for smaller range, for example to weight 1.00 ±0.01g of homogenate, so that better control of that independent variable would be assured.

CONCLUSION

It was proved that tomato fruits contain Lycopene and that its quantity is changing during the ripening season. As predicted, we extracted Lycopene from all three cultivars, but its concentrations in mg/kg are varying among all three examined cultivars during ripening season. Minimums occurred in July and were 9.047 mg/kg for Kentaro F1, 1.146 mg/kg for Bolzano F1 and 11.10 for Dasher F1 respectively. The maximums were observed on 11th September: 19.17 mg/kg for Kentaro F1, 2.582 mg/kg for Bolzano F1 and 18, 38 mg/kg for Dasher F1 respectively. Small standard deviations and mostly (except in one case) non-overlapping error bars are indicating that there are statistical significant differences in Lycopene content in-between cultivars. That leads to the conclusion, that levels of Lycopene are varying trough out the ripening season at different rates in different cultivars.

As a result of performing my experiment, another idea comes to my mind. Maximum which occurred at the very end of the ripening season was, as written, were probably result of fact, that even more cell walls were damaged (even those, that were not damaged yet after homogenization) and consequently more Lycopene was released. Therefore by, for example, cooking, we could get also that Lycopene that is still trapped inside the cells and by taking into account also that Lycopene results would be more accurate. It would be interesting to compare Lycopene extraction yields when obtained from cooked tomatoes or from fresh tomatoes.

What is more, it would be rather interesting to check what is the amount of ß-carotene present in examined cultivars; maybe there is a correlation between levels of Lycopene and ß-carotene. Last but not least, we could maybe investigate and check, whether the presence of Lycopene in some way inhibit the growth and development of cancer as tissue cultures by pouring extracted Lycopene over them and by using of different cultivars, we could estimate, which cultivar would the most successful one. Lastly, it would be interesting to make our own calibration curve using standard Lycopene and then compare the results obtained, when yield of Lycopene would be estimated using our calibration curve and when the data obtained from spectrophotometric analysis of Lycopene extracts.

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