🌏 To help support those in need, 70% of proceeds from Nail IB will go to the United Way #COVID-19 Recovery Fund, until further notice.
IB BIOLOGY SL

# Different Fruit Ripening Methods On Metabolism

UPDATED ON - 15 APR 2020

Note: The purple highlighted text has a comment attached to it

Affect of different fruit ripening methods on metabolism.

Purpose

My interest in the ripening of fruit developed from an observation that fruits bought in my local supermarket do not always ripen effectively. This stimulated me to find out more about the process of ripening in fruits. I chose nectarines as my material because they were in season and they seemed to be the worst affected by the problem of ripening.

[Comment]: The purpose is clear and the candidate justifies the choice of the research question.

Research Question

How do two different methods of fruit ripening affect the metabolism of starch to glucose in nectarines (Prunus persica) over 7 days?

[Comment]: Research question stated but it could be made more focussed by reference to the ripening methods used.

## Introduction

Glucose is one of the most important carbohydrates in biochemistry and is pivotal in the key biological processes of photosynthesis and cellular respiration. In the ripening process, starch molecules (polysaccharides) are broken down by digestive enzymes to glucose (monosaccharide). This process is made possible by the induction of ethene gas.23 Ethene gas is the biological hormone that is used in plants to stimulate key processes, for example, the germination of seeds, fruit abscission, and the ripening process. It is more readily produced by some fruit, in particular bananas and apples, and will hasten the ripening of fruit when in a contained environment, for example inside a plastic bag or box. Another method suggested is to bury the fruit in rice. It is supposed to retain the ethylene gas produced by the fruit longer.

[Comment]: Relevant background context.

This experiment aims to simulate three different ripening conditions, all of which are presumed to induce the ripening process. In the first trial, a banana will be placed with a nectarine in a closed bag. In the second, a nectarine will be placed under rice in a plastic box. Thirdly, a control whereby a nectarine is placed alone in a plastic bag will be set up as the null hypothesis, supporting the assumption that the production of ethene gas and the concentration of glucose are independent of one another. It is important that all three trials be conducted in closed environments, which favour the retention of ethene gas.

[Comment]: Good methodology. Controlled experiment.

The presence of glucose has been used in this experiment to indicate the extent to which ethene gas has affected the metabolism of starch and the concentration of simple sugars in nectarines. The detection of glucose concentration is possible through the use of a colored indicator composition of potassium permanganate (KMn04) solution and acid, in this case, sulphuric acid (H2SO4). A strong oxidizing agent, KMn04 solution is used to convert alkenes to glycols and thereby quantitatively test for the presence of unsaturated bonds within a sample. The KMn04 the solution is pink in color and its discoloring demonstrates the metabolism of starch to glucose.

[Comment]: safety needs to be considered (see page 4)

[Comment]: Does not consider the presence of other organic molecules that may get oxidized.

The time taken for the pink color to disappear is demonstrative of the concentration of glucose in the filtrate sample, e.g. the smaller the amount of time taken for the color to disappear, the higher the concentration of glucose in the sample.

## Prediction

It is expected that the nectarines exposed to the rice packaging trial will ripen the fastest. The contained environment in which they are placed will favour the retention of ethene gas around the nectarine. As a result, there will be a faster decrease in the concentration of polysaccharides (starch) and a faster increase in the concentration of monosaccharides(glucose) in this trial. The nectarines kept with the banana will also ripen faster than the control as the ethane produced by the banana will supplement that produced by the nectarines themselves.

[Comment]: This could be a typo as elsewhere “ethane” is used correctly.

## Materials

■ 36 nectarines
■ 12 bananas
■ Snap lock bags, plastic containers
■ Basmati Rice (approximately 3kg)
■ 560ml Sulphuric Acid 1M (H2S04)
■ 230ml Potassium Permanganate solution 0.01M (KMnO4)
■ Knife, cutting board, food processor, sieve
■ Stopwatch
■ Syringes - 3ml, 5ml and 10ml
■ 4x 750ml beaker (each repeat)
■ 12x 50ml beaker (each repeat)

This experiment aims to determine how ethene gas affects the concentration of glucose in nectarines. To come to a conclusion, two common methods of fruit ripening, i.e. banana packaging and rice packaging were tested together with control. The methods below correspond to these different conditions.

Due to the subjective nature of the 'endpoint' of the solution,

[Comment]: Limitations considered

i.e. when the pink color disappears and the stop-watch is stopped, it was decided that measures should be taken to eliminate as much as possible this error. On each day of the different conditions (banana, rice, and control), 4 nectarines were pulverized and effectively, tested. The filtrate of each nectarine was tested three times. This was done to eliminate any error that might be associated with the - stirring of the solution and avoid disparity in the results.

[Comment]: Adequate number of repeats but “any error” will not be eliminated

On Day 1 of the experiment, the following was set up:

(a) one banana and one nectarine were placed into a snap-lock bag. The air inside the bag was removed and the bag was sealed
(b) one nectarine was placed into a plastic box. The container was filled with rice until the nectarine was fully covered and the box was sealed
(c) one nectarine was placed into a snap-lock bag. The air inside the bag was removed and the bag was sealed.

This was repeated in four trials for each treatment.

[Comment]: Sufficient trials during the runs considering the manipulation required in this investigation

One untreated nectarine was retained on Day 1 to establish the initial glucose levels.

[Comment]: This should have been trialled more than once.

The fruit was left for 3, 5 or 7 days in room temperature conditions. At the end of the period, the nectarines were removed and qualitative observations and measurements of the glucose levels were made in the following way.

1. The flesh of the nectarine was removed and placed into a food processor. 500ml of distilled water was then placed in the same processor and pulsed for 30 seconds. The liquid was filtered, through a sieve, into a 750ml beaker.

[Comment]: Considers factors that may influence data collection

2.     10ml of the nectarine filtrate was placed into a 50ml beaker. In addition to this, 2ml of KMn04 solution and 5ml of H2SO4 solution was added into the beaker simultaneously. The Stopwatch was started immediately. The solution was swirled in constant motion and at a constant speed.

[Comment]: Considers factors that may influence data collection

3.     When the pink color of the solution had disappeared, the stopwatch was stopped and the time taken was recorded.

This was repeated three times from the filtrate from each nectarine.

## Variables

[Comment]: Good appreciation of the controlled variables. Considers factors that may influence data collection.

 Variable Identify variable How to control variable Independent Conditions that the nectarines are exposed to, i.e. banana packaging, rick packaging, and controlled environment Dependent Time is taken for the pink color of potassium permanganate solution to disappearing (demonstrative of glucose concentration) Controlled Source and age of nectarines All the nectarines were picked on the same day and sourced from the same supplier. When chosen, it was observed that they were of similar color, size, and firmness. Source and age of bananas All the bananas were picked on the same day and sourced from the same supplier. When chosen, it was observed that they were of similar color, size and firmness. Indicator composition Remained constant. The ability of the KMnO4 solution to react with impurities meant that the same solution had to be maintained throughout trials. The same concentration of KMnO4 and H2SO4 Ensures consistency. Pour a standard solution at the beginning of the experiment and use throughout The initial concentration of glucose One nectarine was tested and used as an initial value. This value was used across all my trials. Nectarine sample The entire nectarine flesh was pulverized to a filtrate on all repeats. The judgment of endpoint The ‘end-point’ of the experiment had to be decided on. Therefore the same person had to experiment to ensure valid results. Constant temperature Temperature effects enzyme activity i.e. will affect the rate of ripening the experiment in a closed environment. Closed environment Mould and other microorganisms require oxygen to grow, therefore, restricting the amount of oxygen in samples will restrict the development of mould.

Risk Assessment

[Comment]: Risk assessment carried out.

All apparatus was labelled with relevant information (name, date class nature of materials and experiment) All unnecessary materials were cleared away from the workspace. Glassware is fragile it was used towards the centre of the bench with stable supports. Sharp cutting tools and the blender were used with care.

## Electrical apparatus

The connections of the balance, magnetic stirrer and blender, were kept away from running water and trailing cables were avoid spills were cleaned up.

## Chemicals

Sulphuric acid is corrosive and toxic. KMnO4 is a powerful oxidizer and can cause fires. Eye protection, gloves and lab jacket were worn when handling these chemicals.

## Results

[Comment]: Good qualitative observations, Concise & unambiguous table

## Table showing the observations of the three methods on the ripening process

 Banana Rice Control Day 1 One nectarine was used for all of the trials to ensure that the initial concentration of all the repeats was constant. All nectarines on Day 1 where firm, white/yellow and had no visible mould on their surfaces. Day 3 Nectarines 1 and 4 showed signs of developing mould. The bananas of these nectarines were discoloring and condensation was visible inside the snap lock bags. Nectarines were 90-100% covered by the rice. There was minimal condensation inside the box. No mould present. No mould. No condensation. White/yellow.Firm. Day 5 All the nectarines were softer. Signs of mould. White residue on nectarine 4.The flesh was noticeably darker. Condensation inside the bag. All nectarines were moldy, with nectarines 2 and 4 showing the largest mold colonies. White residue. Condensation inside the box. Nectarines were mostly covered by rice, one nectarine was only 75% covered. No mold. Minimal condensation. Pinkish in color. Day 7 All nectarines are at least partially covered by mold and are emitting white residue. All nectarines at least 9.0% covered in mold. The flesh is a deep brown color. White residue. Pink and white. No mold. 'Bruising' patches (soft spots on surface).

[Comment]: This looks a bit more precise than is possible.
[Comment]: Relevant quantitative data collected Comm: Concise, unambiguous & conventions respected

## Table showing the amount of time taken for the pink color of the potassium permanganate solution to disappear

[Comment]: Appropriate, successful processing. Uncertainties considered
[Comment]: Processing can be followed. Correct notation and conventions used.
 Day Time for KMnO4 colouration to disappear / s ± 0.05s Banana Rice Control 1 3 5 7 1 3 5 7 1 3 5 7 Trial 1 76.23 52.37 47.00 33.03 76.23 56.09 30.57 56.78 76.23 54.33 47.13 36.96 52.98 48.87 34.31 54.59 31.00 57.23 54.67 46.98 36.78 52.66 47.96 35.97 54.35 30.76 57.13 55.13 47.96 35.98 Trial 2 54.34 48.28 44.53 50.50 30.19 25.19 54.78 46.56 37.23 55.65 47.88 45.66 49.86 28.20 26.63 54.65 46.78 37.65 54.23 48.53 45.17 50.06 29.37 24.78 55.07 46.99 37.98 Trial 3 54.75 47.76 44.27 48.98 29.22 26.78 55.02 47.12 36.87 54.17 48.22 43.18 49.43 30.45 25.87 55.34 47.56 36.45 54.23 47.89 44.73 49.56 30.76 26.98 54.69 47.32 36.22 Trial 4 53.98 45.66 38.97 56.33 28.25 57.43 54.79 46.98 36.87 54.37 46.76 39.24 57.19 27.91 56.91 54.99 47.51 36.98 54.21 46.23 39.58 56.74 27.65 56.50 55.34 47.35 36.56 Mean 76.23 54.00 47.59 40.72 76.23 52.81 29.53 41.52 76.23 54.90 47.19 36.88 St Dev 0.00 0.91 0.97 4.50 0.00 3.32 1.25 16.18 0.00 0.30 0.38 0.56

N.B There is only one value for Day 1 as only one nectarine was used to test for the initial concentration of glucose. This value was used as the initial value (Day 1 value) for all of the subsequent trials.

[Comment]: Concise presentation of processed data. Processing can be followed (color-coded R2 values). Unambiguous (title, the key used, color code used). Correct notation and conventions used. Appropriate processing. R2 values. Uncertainties presented as a trend line, error bars, and R2 values

## t-test

The data for the banana treatment and the control does not show much difference for the time taken except after 7 days. The control looks as though it has a higher glucose content than the banana treatment on Day 7. I decided to see if this difference was significant.

## Alternative Hypothesis = There is a difference between the results for the banana treatment and the control on Day 7 t-test equation                                                      t=   X1 - X2

$\sqrt[]{\frac{S_1}{n_1}}+\frac{S_2}{n_2}$

tcalc = 2.93 For p = 0.05 using a two-tailed test tcrit = 2.07

[Comment]: Processing can be followed

Therefore there is a significant difference the alternative hypothesis has retained the null a hypothesis is rejected. However, this difference is not great, it is only significant-top = 0.01

[Comment]: Processing successful

## Glucose calibration

 Glucose / % Time is taken / s ± 0.05s 1 280.00 2 194.00 3 150.00 4 126.00 5 115.00 6 105.00 7 96.00 8 91.00 9 87.00 10 83.00

Unfortunately, the data obtained was outside of the range of the standard curve so curve could not be used to obtain an estimate of the glucose content of the filtrate.

## Error and Limitations

[Comment]: Uncertainties considered

It was acknowledged that the method for this experiment contained certain flaws and that the results obtained from the trials were subject to error. Error-reducing methods were implemented where possible.

Uncertainties were accounted for and are recorded below:

 Identify uncertainty Degree of uncertainty Stopwatch Reaction time + 0.05 s 3ml syringe + 0.1ml 5ml syringe + 0.1ml 10ml syringe + 0.2ml Beakers + 1.0ml

Because the glassware used in the experiment was not altered from trial to trial, the level of uncertainty in each trial would have remained constant. Care was taken to measure exact values, for example, the amount of water added to the food processor and the volume of sulphuric acid, potassium permanganate solution and nectarine filtrate added to each trial. The stopwatch would have caused the greatest amount of uncertainty in the method as it relied on the reaction time of the person conducting the experiment. While the observer was constant

[Comment]: Considers uncertainties and their impact.

However, it is not so much the experimenter’s reaction time as the ability to judge when the fading pink color has disappeared that will influence the results.

throughout all of the trials, several different factors could have affected how quickly a stopwatch was started/stopped and subsequently, the time that was recorded. In improving a method, the 'end-point' could be objectively tested for using colorimetric methods. A standard solution could be passed through the colorimeter and the time is taken for the solution to reach a certain percentage of light absorption recorded. Each trial would be tested similarly.

[Comment]: Sensible realistic improvement

Potassium Permanganate, which was used as the indicator solution for this experiment is a strong oxidizing agent. With the ability to convert alkenes to glycols and thereby detect the presence of unsaturated bonds in a solution, the potassium permanganate could have reacted with impurities in the nectarine filtrate. In such a case, this would have affected the results considerably as the time taken for the pink color of the potassium permanganate solution to disappearing might not have been just testing for glucose. Thus the person experimenting was in reality testing for another variable, the metabolism of impurities in the filtrate,

[Comment]: Logical suggested an improvement. It will be specific to falling starch levels rather than rising glucose levels.

which had not been accounted for in the method. In order to reduce this error, another indicator solution, which does not react with impurities to the same extent as potassium permanganate, could be used, for example, iodine solution. Deep blue in color, iodine solution detects the presence of starch in biological samples. Recognizing that starch hydrolyzes into glucose molecules, iodine could be used to show the concentration of starch in the nectarine filtrate, diminishes with the ripeness of the fruit. Alternatively, a specific glucose test such as that used by diabetics could be used.

In the method, it was decided that each fruit should be tested three times, i.e. the time it took for the pink color of potassium permanganate solution to disappear when placed with the filtrate was tested three times using a constant solution. Due to the subjective nature of the 'end-point' test, where we look for a change in color to indicate the metabolism of carbohydrates to glucose, testing each solution three times limited any error that might be associated with the stirring of the solution and minimized the possibility of outliers in my results. Each repeat was independent of one another, i.e. the nectarines from Day 3 and Day 5 trials had no relation to one another. A variety of different factors, which were not accounted for in this experiment and which could have been present in the repeats, for example, the presence of pesticides and artificial ripening agents, or former exposure to ethene gas, could have influenced the results. In effect, this meant that the method relied on commonalities between all of the nectarines in determining a relationship between the production of ethene gas and glucose concentration. The standard deviations remain reasonable except for the rice

[Comment]: Considers reliability and its impact.

packaging treatment on Day 7. In general, the standard deviation increased with the duration of the ripening. This might be expected as the fruits will vary at slightly different rates.

The abscission zone, or the region the closest to the stem of the fruit, has been shown to contain higher concentrations of glucose5. In order to minimize this factor, when pulverizing the

[Comment]: Considers accepted scientific theory but the discussion could have referred back to the context set in the introduction.

nectarines into a filtrate, the person conducting the experiment made use of all of the flesh of all the nectarines. This meant that the variation of glucose concentration within the fruit would remain constant throughout the experiment.

The biodegradation process, whereby microbes chemically digest materials, was one of the largest sources of error in this experiment. Mould, which develops as a result of an excess of moisture in an environment, was observed on all nectarines in the banana and rice trials after Day 5. The extent to which the propagation of mold had on the results can be seen in the calculated standard deviation values for the rice packaging trial. Day 7, in particular, had a massive standard deviation (16.18s), indicating that there was an enormous spread of data.

[Comment]: Evaluates the reliability of the data.
[Comment]: Relative impact of uncertainty considered.

Furthermore, because chance was a major factor in these results, they are not reliable and could probably not be reproduced again. The reproduction of microorganisms is affected by temperature. Therefore, the maintenance of a constant and relatively low (around 15°C) the temperature would restrict the development of microorganism reproduction without significantly affecting the temperature required by the ripening process (remembering that the enzymes involved in the conversion of polysaccharides to monosaccharides work within a specific and narrow temperature range). The contamination of the fruit by microbes might be reduced by making sure the fruit is thoroughly cleaned on its surface before use. A sterilization solution might be used.

Certain measures were taken to achieve environmental controls, for example, temperature and exposure to light. The experiment was conducted in room temperature conditions, with the temperature of the laboratory being recorded twice each day. It was observed that

[Comment]: These data could have been presented earlier.

the temperature fluctuated between 28°C and 29.5°C during the day. No recordings were taken between 3 pm and 8 am, there would have been great variation at night; however, this could not be controlled by the observer due to practical reasons, ideally, the experiment would be left in a consistently controlled environment, for example, an incubator, where a constant temperature could be maintained.

[Comment]: Suggests a realistic improvement.

The standard reference curve for glucose concentration that was produced proved to be irrelevant for the data. The data obtained was outside of the range of the standard curve. It was not possible to extrapolate the standard curve to cover the range of outcomes and therefore to infer the glucose concentration arising from the experimental trials. A calibration curve using higher concentrations of glucose would have to be reproduced.

Due to time constraints, each trial was only repeated four times. In order to be able to draw concrete conclusions, 20 repeats would be required. This was taken into account when processing the results and it was acknowledged that any conclusions drawn from this experiment may or may not be wholly accurate.

## Evaluation and Conclusion

It was hypothesized that the nectarines exposed to the rice-packaging trial would contain the highest concentration of glucose. It was thought that the rice would be conducive to the retention of ethane gas produced by the nectarines themselves around the fruit, hastening the

[Comment]: Another typo?

ripening process and increasing the rate at which starch metabolized to glucose. Also, the rice and nectarine were stored in a container from which air had not been removed. By contrast, the air had been removed from the plastic bags containing the fruit from the other two trials. It is possible that the higher concentration of oxygen in the box would have helped promote the metabolic process and the propagation of mold.

Bananas are used in both traditional and industrial situations to induce the ripening of fruit, due to their ethene-producing characteristics. This assertion, however, cannot be seen in the results. Whilst the bananas might have produced a small amount of ethene, on Day 7 of the experiment the control trial had a higher concentration of glucose though the results are not very different from the banana treatment though this difference is significant according to the t- test carried out on these data. The fact that the nectarines placed into plastic bags individually ripened at a faster rate than the nectarines that were placed with the bananas points to two possible conclusions. Firstly, methodological error meant that the conditions in which the bananas were placed were not conducive to the production of ethene. Or, secondly, that the nectarines used in the control trial were affected by factors that were not accounted for in this experiment, for example, they contained higher concentrations of glucose at the beginning of the

[Comment]:Identifies weakness but needs to suggest improvements.

experiment.

On Day 5 of the experiment, the banana and controlled trials continued to increase their glucose concentrations at a similar rate, albeit slower than the rate increase from Day 1 to Day 3. The rice packaging trial, however, had continued to increase its glucose concentration at the same rate, demonstrating a linear relationship between the concentration of glucose (y-axis) and time (x-axis). All of the nectarines subjected to these conditions were moldy and were secreting a white residue. This was not the case with the nectarines in the banana and controlled trials, which showed little to no mold. One can deduce that it was the presence of mold that caused a sharp increase in glucose concentration. The enzymes from the mold are probably hydrolyzing the starch of the nectarines.

As the nectarines in the banana and controlled trials continued to increase their glucose concentrations from Day 5 to Day 7, the nectarines in the rice packaging trial began to decrease in glucose concentration. Probably consumed by the microbes. At the same time, it was observed that all of the nectarines in this trial had become increasingly moldy -all were at least 90% covered in mold - and that all nectarines were secreting a white residue. One possible conclusion that can be drawn from this observation is that there exists a 'threshold' whereby the increased glucose concentration is counteracted by the increasing development of mold colonies. As large starch molecules are metabolized there will be a rise in the concentration of glucose.

This process develops parallel to the growth of mold and bacterial colonies, which will feed off the increasing concentration of simple sugars and 'spoil' the fruit. From the results obtained in this experiment, it can be seen that the glucose concentration corresponding to the 29.53 seconds it took for the pink color of the potassium permanganate solution to disappear is the highest attainable concentration of glucose. After this, the amount of glucose consumed by the microbial colonies outnumbers the amount of glucose being produced by the hydrolysis of starch, and thus a decrease in glucose concentration can be observed. As seen in all three of the trials, the development of mold before this 'threshold' does not have a significant effect on the increased glucose concentration.

The only differentiating factor that could be observed in this experiment was the removal of air (oxygen) from the plastic bags. On Day 5, the controlled and banana trials possessed relatively similar glucose concentrations and in both of these trials, the air had been removed. Therefore it is unlikely that methane gas produced by the banana was a significant factor in the conversion of starch to glucose. In the rice trial, where the air was not removed from the box, the glucose concentration was significantly higher. The hypothesis that the presence of rice caused the ethene to be concentrated around the fruit does not hold up as ethene gas would equally have been retained around the fruit in the control trial. It is more likely that it was the presence of air, and oxygen, in particular, that promoted both the growth of mould and the higher glucose concentration.

[Comment]: Valid conclusion
[Comment]: Implications considered

All of the trials produced more or less the same outcome (the final values all lay within a 4 second period except Day 7 of the rice treatment). Qualitatively, all of the nectarines were observed as being rotten and covered in mold. The large standard deviations that were calculated from these results emphasized the widespread data around these three points and demonstrated the unreliability of the data on Day 7 of the rice treatment. The R2 values remain high for the control and banana treatment remains high but the rice treatment R2 is lower, reflecting the problems with these fruits.

As the nectarines were observed as being covered in mold and at this stage, it was likely that other significant chemical reactions were taking place within the fruits. The rice packaging trial had a standard deviation of 17.8 seconds, producing an error bar that encompassed all of the experimental results of the other trials (see Figure 1). The results of the experiment are in part due to processes that were not initially anticipated.

[Comment]: The candidate is getting a bit repetitive here.