An experiment was conducted to investigate the effect of ethylene gas on the ripening of different climacteric fruits and to determine the effects of prolonged storage of fruits in the presence of ethylene gas. The formulated research question is: ‘To what extent does the duration of storage affect the ripening effect of Banana (Musa acuminate) on the heirloom and hybrid cultivars of the tomato fruit (Solanum lycopersicum)?’
Bananas, unripen hybrid and heirloom tomatoes were obtained and treated with chlorinated water to remove microbial layers from the surfaces. Bananas were stored with the hybrid and heirloom tomatoes separately in clear disposable plastic containers and placed in a cool, dry and dark area for durations ranging from 3 to 14 days. Observations of the physical appearances of the fruits were recorded and analysed. Juice extracts were collected and tested for pH values and the concentrations of reducing sugars, using Benedict’s test. Standard solutions (5 different concentrations) of 2% glucose solution were also subjected to the Benedict’s test to derive a colour for each concentration and were applied to the actual qualitative data to deduce a trend in regards to the ripening process.
Comparing physical appearances of the tomatoes, once ripe, the heirloom tomatoes showed greater deterioration than the hybrid tomatoes, also, higher amounts of fungal growth, wrinkling of the cuticle and bruising of the fruit despite being subjected to the same conditions. From this experiment, it can be determined that the duration of storage greatly affects the ripening process of the tomatoes but the benefit of using banana as an ethylene gas producer for the ripening of the tomato cultivars is not significant.
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Introduction
The estimated levels of post-harvest losses in the Asia-Pacific region ranges from 16-50%, occurring due to the use of poor quality inputs, lack of knowledge and skill in harvesting and lack of specific infrastructure for post-harvest handling. Furthermore, local small farmers dominate the production in this region but most farmers are only concerned with production activities, showing little interest in post-harvest activities.
It would be beneficial to educate these local farmers on more efficient techniques in farming and post-harvest practices. Since technologies are expensive and not so easily accessible to rural farmers from developing countries, other simpler methods of improvisation could be explored that are within the control of these farmers. This investigation explores the use of bananas as a source of ethylene, a gas needed for ripening, and the effects of prolonged storage on the maturity rates of two different climacteric fruits. Banana is a medium-level ethylene gas producer while tomato is a low-level ethylene gas producer2 so when stored together, the banana should produce more ethylene gas in the presence of the tomatoes, accelerating the ripening process. The experiment investigated whether there was a difference in the effects of ripening on different cultivars of tomatoes, mainly, hybrid and heirloom tomatoes based on the quality parameters of reducing sugars concentration, pH and colour of the fruits.
Hence, the research question was ‘To what extent does the duration of storage affect the ripening effect of Banana (Musa acuminate) on the heirloom and hybrid cultivars of the tomato fruit (Solanum lycopersicum)?’
Literature review
Commercially used storage methods
There are various storage methods that people use to ripen fruits in different regions of the world. Utilizing effective ways of storing horticulture may aid in reducing food wastage which may be substantial, for example, the annual value of losses for produce from New South Wales, Australia alone has been estimated at over $50 million.
The various storage methods include:
1. Inducing ripening through smoking inside smoking chambers.
2. Spreading unripe fruits over paddy husks or wheat straws
3. Dipping unripe fruits into 0.1mol/dm3 Ethrel (Ethephon*) solution
4. Utilizing ripening chambers with Ethrel (Ethephon) solution and hydroxide pellets
5. Utilizing Calcium carbide which releases acetylene as an artificial ripening agent
*Ethephon is a commonly used plant growth regulator
Effect of extending the vine-holding period of ripe tomatoes
A research article on experiment was published in 2010, it investigated the ‘Changes in pH, acids, sugars and other quality parameters during the extended vine holding of ripe processing tomatoes’. In this investigation, tomatoes were left on the vine for up to 4 weeks. Though the procedure of the current experiment was slightly dissimilar to this experiment, both looked into how the ripening process had been affected which led to the differences in the compositions of the tomatoes. Anthon et al recorded pH values of tomatoes and the glucose and fructose concentration from 0 to 40 days. They found that pH values of the tomatoes had generally risen while the glucose and fructose concentration had declined with increasing maturity of the fruits after ripening. The conclusion that the researchers had arrived at was that vine holding of ripe fruit had produced negative effects on the quality, pH and titratable acidity.
Research questions and hypotheses
Research questions
1. How is the ripening process of the heirloom and hybrid tomatoes affected by the duration of storage and the presence of the banana?
2. How do the effects of the ripening process differ between heirloom and hybrid tomatoes?
Hypotheses
1. If the two cultivars of tomatoes were stored with banana, then their ripening will be faster because of the increased amount of ethylene being made available from the banana.
2. In the presence of the banana, the heirloom tomato would receive a greater effect than the hybrid tomato due to their differences in physical attributes
Null hypothesis
1. The rate of ripening of the two cultivars of tomatoes will not be increased in to the presence of the banana as the ethylene gas produced is being used to ripen the banana.
Materials
• 100 Cavendish Bananas
• 100 Unripe Hybrid Tomato cultivar (Truss tomatoes)
• 100 Unripe Heirloom Tomato cultivar (Brandywine Tomatoes)
• 25 Clear disposable plastic containers
• 1 pH meter
• 1 Data logger
• Benedict’s reagent
• Distilled water
• Bleach
• Boiling tube
• 100ml beaker (±0.5ml)
• 10ml measuring cylinder (±0.2ml)
• 25ml measuring cylinder (±0.3ml)
• Test tube holder
• 0.111mol/dm3 2.0% Glucose (C6H12O6) Solution (0.4%, 0.8%, 1.2%, 1.6%, 2.0% concentrations)
Risk Assessment
Table 1.1 – Risk assessment
Variable/equipment
Type of Hazard
Prevention/Management
Heirloom and Hybrid tomatoes
Biological hazard – plant decay and fungal growth
Handle carefully and wash hands when exposed to bacteria/fungi. Dispose materials appropriately after experimentation.
Measuring cylinder, beaker
Personal hazard – sharp glassware may cause injuries if found broken.
Handle with caution. Report to the teacher when glassware are broken.
Electric kettle
Electrical hazard – faulty or misused machinery may become a hazard.
Keep area dry. Report to the teacher if machine is faulty and discontinue use.
Benedict’s reagent
Chemical hazard – harmful if swallowed, may cause allergic skin reactions, blood abnormalities or severe eye and skin irritation with possible burns.
Keep away from heated sources. Use gloves and safety glasses and wash with water when in contact with sensitive areas e.g. eyes.
Liquid bleach
Chemical hazard – contact with acids liberates toxic gas. Irritating to eyes and skin
Keep away from heated sources. Use gloves and safety glasses, wash with water when in contact with sensitive areas e.g. eyes.
Table 1.2 – Table of variables
Independent Variables
Dependent Variables
Duration of experimentation (3 days, 5 days, 7 days, 10 days and 14 days)
Final pH value of tomato extract
Amount of reducing sugars produced in the tomato extract.
Tomato (Heirloom & Hybrid cultivars)
Length of time for physical degradation of fruit to occur after ripening.
Table 1.3 – Table of controlled variables
Controlled Variables
Method of controlling variables
Location of experimentation
All experimentation were placed in a dark cabinet.
Sizes of tomatoes
Similarly sized tomatoes were selected mostly from the same vine.
Sizes of bananas
Similarly sized bananas were chosen mostly from the same branch
Type of bananas
Cavendish Bananas were used for the experiment
Type of tomatoes
Truss tomatoes were used for the hybrid cultivar. Brandywine Tomatoes were used for the heirloom cultivar.
Lighting
Experimentation was placed in darkness for the duration of the storage
Temperature
Experimentation was conducted in room temperature
Method
Ripening process
1. Bananas, unripe hybrid and heirloom tomatoes of similar sizes, free of observable mechanical injury, decays or blemishes were chosen.
2. A litre of potable water was mixed with 10ml of regular bleach to form chlorinated water solution.
3. The tomatoes were washed with cold chlorinated water to remove field heat, and reduce microbial populations from the surface.
4. The tomatoes were dried with paper towels to reduce surface moisture.
5. A hybrid tomato was enclosed in a clear disposable plastic box with a banana before being enclosed. The container was labelled ‘Hybrid3’ for the 3 days variable.
6. Step 5 was repeated for both hybrid and heirloom tomatoes for 3, 5, 7, 10 and 14 days variables and labelled accordingly.
7. The control setup was prepared with a hybrid tomato enclosed in a plastic container without a banana and labelled ‘Control3’.
8. Step 7 was repeated for both hybrid and heirloom tomatoes for 3, 5, 7, 10 and 14 days variables and labelled accordingly.
9. A control set-up containing only the banana was prepared.
10. The experiments were placed in room temperature at the same location. Observations were recorded after the duration had completed.
11. After the duration had completed, puree was extracted from each tomato and tested separately to obtain quantitative and qualitative data.
12. Steps 1-11 were repeated for 4 more trials.
Quantitative test: pH tests
1. A pH meter and data logger was used to obtain pH of the puree/juice extracted
2. The pH meter was rinsed in distilled water and placed in the ‘Hybrid3’ extract and record pH value.
3. Steps 1-2 were repeated for the following hybrid and heirloom tomato extracts for 3, 5, 7, 10 and 14 days variables and for the 4 remaining trials.
Qualitative test: benedict’s test
1. 3ml of Benedict’s reagent was measured.
2. 5ml of ‘Hybrid3’ extract was measured.
3. Both solutions were transferred into the test tube.
4. 80ml of boiled water was transferred into a 100ml beaker.
5. The test tube was placed into the beaker and swirled slowly until a colour change was observed or until 3 minutes was over.
6. Any colour change observed was recorded.
7. Steps 1-6 were repeated for respective hybrid and heirloom tomato extracts for 3, 5, 7, 10 and 14 days variables and for 4 remaining trials.
Qualitative test: benedict’s test of standard solutions of glucose solution
1. 1ml of glucose solution and 4ml of distilled water were measured separately and transfer into a test tube labelled ‘0.4%’
2. 3ml of benedict’s reagent was measured.
3. 80ml of boiled water was transferred into a 100ml beaker.
4. The test tube was placed into the beaker and swirled slowly until a colour change was observed or until 3 minutes was over.
5. Any colour change observed was recorded.
6. Steps 1-5 were repeated for 0.8% concentration with 2ml glucose solution and 3ml distilled water.
7. Steps 1-5 were repeated for 1.2% concentration with 3ml glucose solution and 2ml distilled water.
8. Steps 1-5 were repeated for 1.6% concentration with 4ml glucose solution and 1ml distilled water.
9. Steps 1-5 were repeated for 2.0% concentration with 5ml glucose solution.
Results and observations
Table 1.4 – Pre-test Initial Conditions
Type of tomato
Initial pH
Benedict’s test: colour change
Hybrid
4.3
Green
Heirloom
4.2
Yellow
Initial observations:
The colour changed from blue to green for the hybrid tomato indicating that there were only small amounts of reducing sugars present. In the heirloom tomato, the colour changed from blue to yellow showing that there were moderate amounts of sugars. This suggests that the heirloom tomato contained slightly more sugars than the hybrid tomato. The hybrid tomatoes appeared to be more turgid than the heirloom tomato, possibly due to the larger proportions of vascular bundles and radical pericarps. The external membrane of the heirloom tomato was also found to be significantly thinner than the layer of the hybrid tomato which had a layer of outer pericarp.
Figure 1.0 – Cross sections of ripe hybrid and heirloom tomatoes
Left: heirloom tomato. Right: hybrid tomato
Table 1.5 – Experimental Observations
Duration
(Days)
Hybrid Tomatoes
Heirloom tomatoes
Banana
3
No visible changes observed.
No visible changes observed.
No visible changes observed.
5
No visible changes observed.
No visible changes observed.
Small brown spots appeared on the banana skin.
7
Cuticle of the tomato was wrinkled near the stem scar and core of tomato.
The cuticle was wrinkled with streaks across the fruit.
Larger brown spots appear on the banana skin. A faint sweet scent was noticeable.
10
More wrinkles occur around tomato. Tiny fungal growth occurred randomly.
Small bruising occurs around the fruit. Fungal growth occurs randomly.
Large black spots overcrowd the banana skin. The banana had a strong sweet scent
14
Wrinkled cuticle. Large sprouts of fungal growth found.
The exocarp of tomato had torn, causing contents of the fruit to leak. Large sprouts of fungi occurred. A strong pungent scent was noticed.
The banana skin had changed to a black colour. A pungent scent was noticed.
(Left: Heirloom tomato with fungal growth after 14 days. Right: Hybrid tomato with wrinkled cuticle after 10 days)
As the ripening process progresses for both cultivars of tomatoes, it is notable that the deterioration of the physical appearance occurred typically after 7 days of storage. Bruises appeared on the fruit, possibly due to the rapid softening and fungal growth causing the fruit to decay, making the fruit no longer ‘fresh’. Despite treating the surfaces of the fruits with bleach solution to remove bacteria and field heat, fungal growth had still occurred. A possible explanation may be due to the ends of the stalk of the banana and the stem scar of the tomatoes which became possible sites for bacterial growth. This suggests that as the ethylene gas is accelerating the ripening process, the fruits may receive the effect negatively. Another notable observation would be the accelerated ripening of the banana, after 7 days, large dark brown spots had appeared and a sweet aroma was emitted. While in the control set-up, the banana that was stored alone only had small brown spots covering the skin. Hence, suggesting that the ripening of the banana had also accelerated. At 14 days, it appears that the fruits had proceeded to over-ripening which was indicated by the transition of the sweet aromas to pungent, sour scents.
Data Processing and Analysis
Analysis of Benedict’s test results:
Benedict’s test was carried out to test for the presence and amount of reducing sugars in the tomato extracts after experimentation. This was indicated by a colour change as qualitative data. To further process this data, as glucose is a reducing sugar, standard solutions of 2% Glucose Solution was tested with the Benedict’s test to derive the colour changes according to the concentrations. The concentrations were then applied to the qualitative data of the benedict’s test for the tomato extracts to find any trends of the amount of sugars in relation to the duration of the ripening process.
Table 1.6 – Table of standard glucose solution concentrations and benedict’s test colour changes
Standard solutions of 0.111mol/dm3 glucose solution (%)
Benedict’s test colour change
0.0
Blue
0.4
Green
0.8
Yellow
1.2
Orange
1.6
Dark orange
2.0
Red
After inputting the concentrations according to the colour changes for the tomato cultivars, the mean values and standard deviation of the reducing sugars concentrations were derived and graphed.
Table 1.7 – Table of the mean values and standard deviation of glucose solution concentrations of hybrid and heirloom tomatoes
Duration (Days)
Hybrid Tomato
Heirloom tomato
Mean
Standard Deviation
Mean
Standard Deviation
3
0.88
0.333
1.09
0.399
5
1.33
0.348
1.52
0.261
7
1.65
0.240
1.68
0.300
10
1.60
0.146
1.89
0.177
14
1.60
0.327
1.60
0.358
Graph 1.0 – Graph of the mean values of glucose solution concentrations of hybrid and heirloom tomatoes
Hybrid tomato:
There is a steady increase in the glucose concentration from day 3 to day 7 which suggests that, as the fruit is ripening, degradation of starch to monosaccharides, disaccharides and pectin (a gelatinous polysaccharide) solubilisation which is the disintegration of pectin occurs, resulting in increased amounts of reducing sugars. However, the graph dips slightly and plateaus from day 10 to day 14. It suggests that pectin depolymerisation is no longer occurring as much as in the beginning stages of ripening, possibly marking the completion of ripening of the fruit before proceeding to over-ripening.
Heirloom tomato:
There is a similar steady increase in the glucose concentration from day 3 to day 10 but a sharper decline from day 10 to day 14. The graph suggests that the progressive ripening process has resulted in an increased generation of reducing sugars. The sharp decline may be due to over-maturation of the fruit as the reducing sugars are disintegrating, the excess ethylene gas may have caused depolymerisation which includes dissolution of middle lamella, reducing cell adhesion. As bacterial and fungal growth had occurred after 10 days, the lack of sugars in the tomato might be because bacteria were feeding on the sugars for respiration. It is also notable that the glucose concentrations of the heirloom tomato are consistently higher than those of the hybrid tomatoes. It has been observed that the hybrid tomato had a thicker outer cuticle layer and higher concentration of vascular bundles and radical pericarps compared to the heirloom tomatoes. This may have increased its permeability to ethylene gas.
Hence, the heirloom tomatoes had a greater concentration of reducing sugars.
Mean and standard deviation
The mean pH values and standard deviation of both tomatoes across all 5 trials are calculated and graphed, according to the time variable.
Table 1.8 – Mean of pH values and standard deviation of hybrid and heirloom tomatoes
Duration (Days)
Hybrid Tomato
Heirloom tomato
Mean (pH)
Standard Deviation
Mean (pH)
Standard Deviation
3
4.77
0.226
4.62
0.229
5
4.81
0.153
4.77
0.107
7
4.74
0.120
4.57
0.169
10
4.64
0.071
4.51
0.081
14
4.45
0.136
4.33
0.144
Graph 1.1 – Mean pH values of hybrid and heirloom tomatoes across 5 trials
For the hybrid tomato, the pH increased slightly from day 3 to day 5 but steadily decreased from 5 to 10 days before a sharper decrease from 10 to 14 days. For the heirloom tomato, the graph increased sharply from 3 to 5 days before another steep decreased from 5 to 7 days and narrower decreases from 7 to 14 days. The findings suggest that in the early stages of the ripening process, the acidity decreased due to the starch degradation. But as the fruits approach completion of the ripening process or the beginning stages of over-ripening, the acidity decreased, possibly due to the pectin depolymerisation as pectin is rich in galacturonic acid.
t-test and data analysis
The t-test for one sample is used when wanting to compare one measurement variable with a theoretical explanation of what the mean should be under the null hypothesis. It tests whether the mean of the measurement variable is different from the null expectation. The means of the pH values of the actual set-up were compared with the means of the control set-up according to the duration the experiment was subjected to. It is executed with the assumption that the one-sample t-test is not at all sensitive to the non-normality. The t-test values are calculated with Excel using two-tails.
Table 1.9 – Tabulations of t-test results using the control set-up for the comparison (Hybrid tomato)
Duration (days)
Hybrid tomato
Control
Hybrid
3
4.50
4.77
5
4.70
4.81
7
4.82
4.74
10
4.72
4.64
14
4.38
4.45
T-test value (P-value)
0.59
Table 2.0 – Tabulations of t-test results using the control set-up for the comparison (Heirloom tomato)
Duration (days)
Heirloom tomato
Control
Hybrid
3
4.44
4.62
5
4.62
4.77
7
4.78
4.57
10
4.70
4.51
14
4.54
4.33
T-test value (P-value)
0.56
For the hybrid tomato, a P-value of 0.59 was calculated, which means that there is a 59% chance that the difference in the means is due to random chance or 41% chance that the difference between the two samples is due to the variable (presence of banana as an ethylene gas producer) being investigated. While for the heirloom tomato, a P-value of 0.56 was calculated, meaning that there is a 56% chance that the difference is due to random chance while 44% is due to the variable being investigated.
Conclusion
To answer the research question, ‘To what extent does the duration of storage affect the ripening relationship between Banana (Musa acuminate) and the heirloom and hybrid cultivars of the tomato fruit (Solanum lycopersicum)?’, the longer the duration of storage, the more the tomato cultivars and the banana deteriorate in physical appearance after ripening. Based on the pH values and Benedict ’s test data, the ripening of heirloom tomato was faster than the ripening of hybrid tomato. The pH increased from 0 to 5 days but decreased after 7 days, it suggests that the fruits had reached full maturity after approximately 5 days. The longest duration that the tomatoes typically stay in ‘fresh quality’ would be 7 days after ripening has progressed. It was observed that at 10 and 14 days, fungal growth and plant decay occurred, compromising the quality of the tomatoes and this is especially apparent in the heirloom tomatoes compared to the hybrid tomatoes. When compared to the control set-up of each tomato cultivars, it was observed that the adverse effects on the physical attributes of the tomato cultivars, especially the heirloom tomato, and the banana had occurred earlier at 7 and 10 days whereas in the control set-up, the adverse effects occurred later at 10 and 14 days.
Analysing the T-test p-values calculated, the hypothesis cannot be supported as the probabilities of both tomato cultivars being affected by the presence of the banana and the durations of storage, in terms of pH values, exceed 50%. Therefore, the t-test values support the null hypothesis that the banana had little to no effect on the tomatoes as there was high probability that it was due to random chance. Though the t-test values do not support the hypothesis, using the experimental observations made previously with comparison to the control set-ups, it is evident that the heirloom tomatoes in overall, had received a greater effect from the ripening process in the presence of the banana and the respective time periods. It was shown that the heirloom tomatoes had lower mean pH values but higher mean reducing sugars concentrations compared to the hybrid tomatoes. Therefore, the longer the duration of storage, the more adverse the effects on the tomato cultivars, particularly the heirloom tomato. The results of the experiment suggest that the optimum duration of storage time would be 7 days, any longer durations result in plant decay and fungal growth as the fruits proceed to over-ripening. In conclusion, the duration of storage greatly influences the ripening process of the tomato cultivars. But the presence of banana did not create much significance in the ripening of the tomato cultivars, hence, cannot determine the ripening relationship between the banana and the tomatoes.
Table 2.1 – Table of Limitations and suggestions for improvements
Variable
Limitations
Suggestions for improvement
Method of storage
This experiment has only utilized one method of storage; the use of disposable transparent containers that were placed in a dark cabinet.
Other storage methods mentioned in the Literature Review could be investigated to explore the effectiveness and the effect on the ripening of fruits.
Method of deriving data
pH test and benedict’s test were the only tests used.
Total Soluble Solids (TSS) and acidity ratio of the pulp through the reduction of Fehling’s solution could be used to further the investigation and determine the sugar content of the tomato pulp. Other quality parameters could also be explored, such as pectin solubility, lycopene and total pectin.
Selection of climacteric fruits
The experiment specifically used two different cultivars of the tomato fruit, hybrid and heirloom, and bananas.
Different climacteric and non-climacteric fruits could be used to further investigate the differences or similarities between different fruits due to the varied levels of ethylene gas produced by each fruit.
Quality of fruit extract collected
The extracts had been refrigerated for some time before being tested. This may have altered the quality or ‘freshness’ of the extract to a certain degree.
Quantitative and qualitative tests used to derive data could be done as soon as the tomato juice has been extracted.
Standard glucose solution concentrations
As the numerical values of the standard glucose concentrations were applied to qualitative data of the tomato extracts, it is assuming that the tomato extracts have the same concentrations of reducing sugars as the standard glucose solutions according to the Benedict’s test colour change
To derive greater accuracy of the composition of the reducing sugars in the tomato extracts, supernatants can be prepared from the extracts and tested for individual sugars using enzyme kits.