The effect of various ripening set-ups on the vitamin C content of M. accuminata at various stages of
ripening
1 Introduction:
Kerala , a state in the southern Part of India, is known for its flavourful banana chips and vast stretches of
banana plantations. Living in this state I was naturally curious about its various characteristics. Bananas are
characterized by its short shelf-life at tropical ambient temperature due to climacteric ripening
associated changes like rapid softening, senescence spotting, off-odour development and crown rot
disease incidence. Going through an article that I came across recently I became aware that an estimated
50 million tonnes of bananas are thrown out by stores and household based on their visual appearances.
When banana’s ripen they form several dark spots on their peel, used by many as an indicator for their
disposal1. However is this the right disposal indicator? This deeply worried me and kindled me to delve into
further research regarding this issue. I was keen on determining the ideal ripening stage of bananas where
one can reap maximum nutritional benefits. However it wasn’t practical for me to determine the entire
nutritional content of the banana at different stages of ripening. My parents often bring up the significance of
bananas as a prominent source of Vitamin C. This led me to arrive at my dependant variable, the level of
Vitamin C content in bananas. Once the dependant variable was finalised, various types of independent
variables such as different stages of ripening, the effect of various geographical locations on ripening and
ripening in different fruits were considered. Even so, I used this exploration as an opportunity to satisfy my
curiosity with respect to the efficacy of the various ripening methods. It is quite common in the local regions
to find farmers or domestic retailers, placing the harvested bananas in conditions which supposedly speeds
up the ripening process. Common methods include placing bananas in haystacks, smoking bananas in closed
drums or even placing it in rice sacks. I have always questioned the efficacy of such traditional ripening
methods. the aforementioned discussion, I formulated my research question:
How is vitamin C content in M.accuminata influenced by the various ripening set-ups like haystack,
rice bowl, paper bag with ripened fruits and smoked set-up at 3 different stages of ripening?
Through this research question I would be investigating the efficiency of various ripening conditions,
vitamin C content of M. acuminata at different stages of ripening and the rate at which vitamin C content
increases. Here ripening conditions can be considered as any and all, configurations which are believed to
enhance the rate of ripening of M. acuminata.
2 Background Research:
2.1 Vitamin C and it’s bio-synthesis:
Vitamin C is a common name given to compound known as ascorbic acid which is required for the
synthesis of collagen fibres that form a part of various tissues which includes skin and blood vessel walls.
Animals were capable of producing this vitamin in their bodies earlier. However over the years, mutations to
the GLO genes, which codes for the production of the enzyme L- gulono- 𝛄 - Lactone oxidase, made several
organisms incapable of synthesising this vitamin2. Homo sapiens (humans) are one such species who’ve lost
this ability. Thereby, humans have to opt for the intake of external dietary sources of vitamin C such as
vitamin C rich foods or supplements. Deficiency with regards to this vitamin can lead to a condition called
1
“Browning Bananas and Food Wastage.” Cosmosmagazine.com, 12 May 2022,
cosmosmagazine.com/earth/sustainability/brown-banana-computer-simulation/.
2
IB Biology Course Book: 2014 Edition: Oxford IB Diploma Program by Andrew Allott, David Mindorff
1
scurvy causing general weakness, anaemia, gum disease, and skin haemorrhages3. Vitamin C, in plants,
occurs in all cell compartments, including the cell wall, and accumulates to a level of exceeding 20 mM in
chloroplasts. Recent molecular genetic research from the ascorbate-deficient mutant of Arabidopsis
thaliana supports a biosynthetic route that uses GDP-mannose, GDP-L-galactose, L-galactose, and Lgalactono-1,4-lactone4. Vitamin C content in horticulture crops is influenced by a variety of pre- and
postharvest factors. Climate patterns and cultural customs constitute preharvest factors. The vitamin C
concentration of fruits and vegetables is also influenced by the maturity at harvest, the harvesting technique,
and the post - harvest processing environment. Significant vitamin C losses can occur during cooking and
processing processes.
The general pathway for vitamin C bio-synthesis is outlined below in figure 15.
Figure 1: Ascorbic acid biosynthesis pathway in plants and animals6.
2.2 Ethylene and its relationship with ascorbic acid bio-synthesis:
Ethylene (H2C=CH2) is a colourless, flammable gas with a sweet taste and odour that belongs to compounds
known as alkenes Petroleum and natural gas are two natural sources of ethylene. It is also a hormone that
naturally exists in plants, where it limits growth and promotes leaf fall, as well as in fruits, where it
stimulates ripening7. Ethylene and abscisic acid (ABA), two phytohormones, frequently combine to regulate
both plant growth and development processes and plant responses to stress. The ascorbic acid biosynthetic
gene VITAMIN C DEFECTIVE2 must be transcriptionally activated by the essential transcription factors
ETHYLENE-INSENSITIVE3 (EIN3) and ABA-INSENSITIVE4 (ABI4) (VTC2). Also ABA promotes the
accumulation of reactive oxygen species (highly reactive chemicals formed from diatomic oxygen) in
Arabidopsis thaliana seedlings whereas ethylene has the opposite effect 8. Furthermore, changes in the
biosynthesis of ascorbic acid validates the interactions between ethylene and ABA in controlling ROS
levels.
2.3 Musa accuminata (Banana):
3
“Scurvy: MedlinePlus Medical Encyclopedia.” Medlineplus.gov, 2016, medlineplus.gov/ency/article/000355.htm.
Smirnoff, Nicholas, and Glen L. Wheeler. “Ascorbic Acid in Plants: Biosynthesis and Function.” Critical Reviews in Biochemistry and Molecular Biology, vol. 35, no. 4, Jan. 2000, pp. 291–
314, https://doi.org/10.1080/10409230008984166.
5 IB Biology Course Book: 2014 Edition: Oxford IB Diploma Program by Andrew Allott, David Mindorff
4
6
https://www.onlinebiologynotes.com/vitamin-c-ascorbic-acid-properties-biosynthesis-biological-functions-and-deficiency/
7
ethylene | Structure, Sources, Production, Uses, & Facts | Britannica.pdf
Minorsky, Peter. “Ethylene and ABA Regulate Ascorbic Acid and Reactive Oxygen Species.” Plantae, 22 Mar. 2019, plantae.org/ethylene-and-aba-regulate-ascorbic-acid-and-reactiveoxygen-species/#:~:text=and%20ROS%20accumulation.-.
8
2
This fruit, which is also known as a banana, is a member of the family Musa accuminataceae and is one of
the most significant fruit crops in the world9. M. accuminata is cultivated in the tropics, and while it is
primarily consumed there, it is prized for its flavour, nutritional content, and year-round availability around
the world10. M. accuminata can thrive 6–7 days at 20–30 °C and 4–7 weeks in the temperature range of 10–
13 °C11. Temperatures between 14 and 20 degrees Celsius, relative humidity of 85% and 95%, and ethylene
gas concentrations between 100 and 1000 ppm are all ideal for M. accuminata ripening12.
2.4 Banana ripeness chart:
To obtain a uniform scale of measuring a M.accuminata’s ripeness, a ‘Banana ripeness chart’ was used. The
chart in figure 2 divides the ripening process into 7 different stages based on their visual and physical
characteristics13. This tool was used as standard throughout the experiment, as a means to demarcate the
various stages in the ripening process. Out of the 7 stages present in the chart, only 4 stages were used in this
investigation. The 1st stage (Raw ) was chosen as it is the beginning of the M.accuminata ripening cycle, 3rd
stage (Semi-ripe) was used to increase the breadth of observations, 6th stage (Ripe) and the 7th stage (Overripe) was chosen as it is at this stage M.accuminata ripening where it’s consumption is encouraged in
households.
Figure 2: Banana ripeness chart depicting various stages of ripening in M.accuminata
2.5 The reaction:
Through my preliminary research conducted using various websites and articles, a myriad of methods were
available to determine the vitamin C content of a solution. This included the determination of the vitamin
content through the colorimetric method, High performance Liquid Chromatography (HPLC) and a
reflectometer, a tool used to analyse different types of test which includes ascorbic acid test14. However, all
these methods were not feasible to me both economically and in terms of availability. A number of the
equipment were not available in my school.
A research paper, on the determination of vitamin C concentration by titration, published by the University
of Canterbury and various other research document validate redox titration’s capability in determining
vitamin C content of fruit samples.
Redox titration is one amongst the multitude of methods to determine the vitamin C content in a fruit. Iodine
is relatively insoluble, but this can be improved by complexing the iodine with iodide to form triiodide:
9
“Banana | Description, History, Cultivation, & Disease | Britannica.” Encyclopædia Britannica, 2019, www.britannica.com/plant/banana-plant.
“Banana | Description, History, Cultivation, & Disease | Britannica.” Encyclopædia Britannica, 2019, www.britannica.com/plant/banana-plant.
11 Murmu, Sanchita Biswas, and Hari Niwas Mishra. “Post-Harvest Shelf-Life of Banana and Guava: Mechanisms of Common Degradation Problems and Emerging Counteracting
Strategies.” Innovative Food Science & Emerging Technologies, vol. 49, Oct. 2018, pp. 20–30, https://doi.org/10.1016/j.ifset.2018.07.011.
12 frigomekanik. Banana Ripening Conditions | Blog. 4 July 2018, www.frigomekanik.com/blog/eng/banana-ripening-conditions#:~:text=The%20conditions%20for%20a%20successful.
13 Springernature.com, 2022, media.springernature.com/lw685/springer-static/image/art%3A10.1007%2Fs11694-021-010092/MediaObjects/11694_2021_1009_Fig1_HTML.png?as=webp.
14 Krishnasarma pathy. “Process for Preparation of Vitamin c and Method for Determination of Vitamin c in Tablets.” Surgery & Case Studies: Open Access Journal, vol. 1, no. 3, Lupine
Publishers, 2018, pp. 1–14, lupinepublishers.com/surgery-case-studies-journal/fulltext/process-for-preparation-of-vitamin-c-and-method-for-determination-of-vitamin-c-intablets.ID.000114.php.
10
3
𝐼2 + 𝐼 − ↔ 𝐼3 −
Triiodide oxidizes vitamin C to form dehydroascorbic acid. This reaction occurs as long as vitamin C is
available. But once there is no ascorbic acid to get oxidized (vitamin C ), then the Iodine reacts with starch
to form a bluish-black complex, which marks the end of the process15. The amount of Iodine required to
achieve the bluish black complex can be applied to the formula present in (----------) to determine the
vitamin C content of a fruit or a solution (M. accuminata in this case) .
3 Methodology:
As I probed into the research question, I understood the importance of ripening set-ups in this investigation.
There were several methods followed by to ripen fruits, in specific M.accuminata. This included placing
M.accuminata bunches in haystacks and they were often placed in closed drums with incense sticks allowed
to smoke inside. Additionally, some households also ripen fruits by placing them within a sac or bowl of rice
and sometimes even in paper bags or airtight containers with ripe fruits16. Other methods include dipping or
spraying the M.accuminata samples with 0.1 per cent ethrel solution17. However ethrel is an industrial
ripening agent which is corrosive to skin causing harm to aquatic life and soil environment. Thereby this
method was not used while the other 4 methods discussed above due to its ease of setting up and minimal
safety and environment concerns, was chosen as the ripening set-up for this investigation.
To maintain accuracy I tried to acquire the Yellow Cavendish variety of M.accuminata as used in the
Banana ripeness chart. However, due to its lack of availability in the local market, the “M. accuminata ×
balbisiana Colla” variety, commonly known as the “Ney Poovan” variety, was procured due to its abundant
consumption and availability in the regional market.
3.1 Variables
Independent variable:
5 independent variables were chosen to conduct the experiment. IV1, IV2, IV3 and IV4 are commonly used
methods by farmers and in certain households to ripen fruits, in specific M. accuminata. The last
independent variable was the control set-up which replicated the conventional ripening at room temperature
without the influence of any ripening methods. IV1 to IV4 was then compared to the control set-up, to
surmise the efficiency of each ripening method and how the results produced from each ripening set-ups is
different from the control set-up. M.accuminata henceforth referred to as sample below were placed in
different conditions for setting up 1 trial.
Haystack set-up
(IV1)
Rice bowl set-up
(IV2)
Smoked set-up (IV3)
Paper bag set-up
(IV4)
Control setup
Hay was spread out in a cardboard box and 3 samples were placed on it. This
was then covered using another layer of hay.
3 samples were placed within a bowl of rice.
3 sample were placed in a closed Aluminium container. 5 incense sticks were lit
per day and to generate smoke
3 samples were placed in a paper bag with a ripe guava and a ripe apple18.
3 samples were placed in the open air at room temperature.(±26° 𝐶)
Dependant variable:
15
University of Canterbury. Determination of Vitamin c Concentration by Titration (Redox Titration Using Iodine Solution). 2020, www.canterbury.ac.nz/media/documents/scienceoutreach/vitaminc_iodine.pdf.
16 “5 Quick Tips for Ripening Fruit.” Hungry Harvest, hungryharvest.net/blog/ripeningfruit#:~:text=Just%20add%20your%20fruit%20into.
17 “Vikaspedia Domains.” Vikaspedia.in, 2023, vikaspedia.in/agriculture/post-harvest-technologies/technologies-for-agri-horti-crops/technologies-for-ripening-fruits.
18 “How to Make Bananas Ripen Faster: 10 Steps (with Pictures).” WikiHow, www.wikihow.com/Make-Bananas-Ripen-Faster. Accessed 25 Aug. 2022.
4
Amount of vitamin C in M.accuminata
Controlled Variables:
Variables
Age of M.accuminata samples
In this investigation, the various ripening set-ups (IVs)
were influenced by the vitamin C content in the
samples. Redox titration was used to determine the
vitamin C content in M.accuminata.
How it was controlled
Every sample was procured from the same
M.accuminata bunch from the same vendor, to
ensure that all the samples were of the same age. All
the procured samples corresponded to stage 1 of the
ripening chart.
Weight of M.accuminata samples
A weighing scale was used weigh approximately 80
g of each sample for all experimental trials
Volume of various solutions
To maintain uniformity in the volume of solutions,
distilled water was used to dilute the solution to the
required volume (80 mL approximately)
Owing to the various experimental set-ups various factors like temperature, light and humidity could not be
regulated or monitored, hence these are the uncontrolled variable of the experiment.
3.2 Hypothesis:
H0 (Null Hypothesis): Different ripening conditions does not influence a M. accuminata vitamin C content
H1(Alternate Hypothesis): Various ripening conditions influences M. accuminata vitamin C content
3.3 Materials required:
S.No Materials
3
Burette (± 0. 1 cm3) with a
stand
Weighing scale (± 0. 005
cm3)
M. accuminata(Banana)
4
Iodine Solution
1
2
Purpose
Quantity
Used to perform titration
1
To measure the weight of M. accuminata samples and
vitamin C tablet
M. accuminata corresponding to the 1st stage of ripening
was obtained to prepare fruit extract
Used as the titrant for redox titration
1
80
---
5
Soluble Starch
Used to create a 1% starch indicator solution
--------
6
Blender
To prepare a M. accuminata sample(smooth paste)
1
7
Glass rod
To mix components
2
8
Spatula
To add and remove components
2
9
Distilled water
Used to make solutions
1.5 litre
10
Vitamin C tablet(0.250 g)
Used to prepare standard solution
1
11
Measuring cylinder(50 ml)( ±
0.5 cm3)
Used to measure the quantity of various other materials
1
5
12
Beaker(50 ml)
13
Beaker (250 ml)
Used to store fruit samples and standardized solution to
be titrated
Used for preparation of standardized solution
5
2
3.4 Protocol:
The protocol followed here has been adopted from the procedure stated in an article by ThoughtCo. titled
“Vitamin C Determination by Iodine Titration”19.
Preparation of standard vitamin C solution:
It was prepared using starch solution and vitamin C solution.
Preparation of starch solution:
 60 ml of distilled water was heated in a water bath.
 0.6g of soluble starch was measured and added to the water and was stirred constantly into
homogenous mixture
Preparing Vitamin C solution:
 A Vitamin C tablet was ground with a mortar and pestle
 0.250 g of the crushed vitamin C tablet was weighed.
 100 ml of water in a 250 ml beaker was heated in a water bath till it’s warm (±45℃).
 0.250 g of Vitamin C was added to the warm water and stirred with a glass rod till the Vitamin C
completely dissolved.
 The solution was diluted to 250 ml through the addition of distilled water.
 25 ml of the vitamin C solution was added to a 50 ml beaker.
 10 drops of the 1% starch solution was added to the vitamin C solution to prepare standard vitamin C
solution.
Redox titration of standard vitamin C solution:
 The burette was rinsed with small amounts of iodine solution.
 After rinsing, the burette was filled with iodine solution to the 0 mark.
 The aforementioned standard vitamin C solution was titrated with the titrant (Iodine solution)until
the solution achieved a light bluish-black colour which marked the end point of the titration.
 The volume of the iodine solution used to reach the endpoint was measured.
 4 more trials were performed with using the same procedure.
Titration of the M.accuminata extract:
 1 M.accuminata sample was used for the preparation of the extract.
 The peel was removed and the pulp was weighed.
 The fruit was blended in a blender until it became a smooth paste consistency.
 The amount of distilled water added to M. accuminata was equal to the weight of the M. accuminata
sample.
 This extract was then divided into 3 beakers of 25 ml each measured using a measuring cylinder.
 Each extract was titrated until the solution attained a light bluish-black colour.
 The volume of the iodine solution used to reach the endpoint was measured.
 The average of the three titrant values was then recorded as the value for one trial.
 The above protocol was used to titrate M.accuminata extracts obtained from the 1st, 3rd, 6th and 7th
stage of the ripening in each ripening set-up (IV).
19
Helmenstine, Anne Marie. “Use Iodine Titration to Determine the Amount of Vitamin c in Food.” ThoughtCo, 21 June 2019, www.thoughtco.com/vitamin-c-determination-by-iodinetitration-606322.
6


In this investigation, the M. accuminata kept in the room temperature was considered the control set
up.
4 more trials were performed using the same procedure.
To obtain initial values for the investigation, titration was performed thrice for a M.accuminata sample
corresponding to stage 1 of the banana ripeness chart using the above protocol for 1 trial. The same
procedure was repeated for 4 more trials. This gave a uniform starting vitamin C value for M.accuminata of
all IVs.
Safety, ethical and environmental considerations:
Several safety precautions were taken during the course of this investigation. Disposable latex gloves were
used while performing titration to avoid direct contact with iodine solution which is corrosive and can cause
blistering. The protocol was overseen by the lab assistant, to help avoid accidents. Only the optimum
amount of M.accuminata was used for the investigation by minimizing wastage and thoroughly following
the protocol. Furthermore, the leftover M.accuminata was disposed in the bio-degradable waste bin while
the Iodine solution remaining in the burette after titration was carefully transferred back into a glass bottle
and was stored for reuse.
Data collection and processing:
Qualitative raw data:
After careful observations, M. accuminata samples from different stages of ripening exhibited different
characteristics. These characteristics were classified based on the colour of the peel, texture of the
M.accuminata pulp and it’s odour and is presented in the table below.
Data table 1: Various characteristics exhibited by M. accuminata samples in different stages of
ripening
Stage of
ripening
Stage 1
Colour of the peel
Texture
Odour
Green, no trace of yellow
Extremely rigid,
None
colour
difficult to squeeze
Considerable amount of green
Slightly Rigid
None
Stage 3
and yellow colour
Yellow with no traces of green Soft and easy to
Emanated considerable
Stage 6
colour
squeeze
amount of odour
Yellow accompanied by
Extremely soft texture Very strong odour could be
Stage 7
numerous black spots
sensed
The exhibition of the aforesaid characteristics were observed in all the ripening conditions.
In terms of texture, M. accuminata samples placed in IV4 (smoked set-up) corresponded to Data table 1, but
in stages 3, 6 and 7 the colour of the peel and odour was beyond recognition as the peel was completely
charred (black) and had a repugnant odour.
Quantitative raw data:
The time taken by M.accuminata placed in the different ripening set-ups to transition into the various stages
of ripening as stated in the ripening chart is presented in data table 2.
7
Data table 2: Illustrates the time taken (hrs) by M.accuminata samples to transition into next stage of
ripening in different ripening set-ups
Time taken (Hours)
Stages
Haystack
Rice bowl
Smoked
Paper bag
Control
1 to 3
41
72
76
57
48
3 to 6
51
11
48
28
48
6 to 7
76
24
20
39
72
1 to 7
168
107
144
124
168
As per protocol, the M. accuminata sample placed in a ripening set-up (IV) was divided into 3 extracts, each
of them was titrated till the end point was achieved and finally the average volume of iodine used to titrate
the extracts was used as the value for one trial. 5 such trials were performed for each ripening stage in each
IV. The results are presented in the table below:
Data table 3: Table illustrating the average volume of iodine used to titrate M. accuminata extracts
obtained from each ripening set-up across 4 different stages of ripening.
Trials
1st stage
3rd stage
6th stage
7th stage
1
0.55
0.6
0.6
0.45
2
0.65
0.6
0.55
0.5
Haystack
3
4
0.6
0.55
0.55 0.6
0.5
0.6
0.55 0.55
5
0.45
0.6
0.45
0.55
1
0.55
0.5
0.45
0.4
2
0.65
0.65
0.6
0.55
Rice bowl
3
4
0.6
0.55
0.6
0.55
0.5
0.55
0.6
0.55
5
0.45
0.6
0.55
0.5
1
0.55
0.6
0.5
0.4
Incense sticks
2
3
4
0.65 0.6
0.55
0.45 0.5
0.45
0.4
0.55 0.4
0.5
0.4
0.35
Trials
1st stage
1
0.55
Paper bag
2
3
4
0.65 0.6
0.55
3rd stage
0.6
0.65
0.55
0.55
0.6
0.6
0.55
0.55
0.55
0.6
6th stage
0.65
0.55
0.5
0.55
0.5
0.55
0.55
0.5
0.55
0.6
7th stage
0.55
0.6
0.5
0.5
0.5
0.45
0.6
0.5
0.5
0.55
5
0.45
1
0.55
Control
2
3
4
0.65 0.6
0.55
5
0.45
5
0.45
0.55
0.4
0.4
Vitamin C was calculated from the above data using the formula prescribed by ThoughtCo to determine the
vitamin C in a fruit extract20.
𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑖𝑜𝑑𝑖𝑛𝑒 𝑢𝑠𝑒𝑑 𝑡𝑜 𝑡𝑖𝑡𝑟𝑎𝑡𝑒 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑐𝑖𝑡𝑎𝑚𝑖𝑛 𝐶 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑣𝑖𝑡𝑎𝑚𝑖𝑛 𝐶 𝑖𝑛 𝑡ℎ𝑒 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑣𝑖𝑡𝑎𝑚𝑖𝑛 𝐶 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑖𝑜𝑑𝑖𝑛𝑒 𝑢𝑠𝑒𝑑 𝑡𝑜 𝑡𝑖𝑡𝑟𝑎𝑡𝑒 𝑡ℎ𝑒 𝑓𝑟𝑢𝑖𝑡 𝑒𝑥𝑡𝑟𝑎𝑐𝑡
=
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑣𝑖𝑡𝑎𝑚𝑖𝑛 𝐶 𝑖𝑛 𝑓𝑟𝑢𝑖𝑡 𝑒𝑥𝑡𝑟𝑎𝑐𝑡
A new formula was derived by making changes to the aforesaid formula in accordance to this investigation.
The new formula to calculate vitamin C in M. accuminata extract is given below:
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑖𝑜𝑑𝑖𝑛𝑒 𝑢𝑠𝑒𝑑 𝑡𝑜 𝑡𝑖𝑡𝑟𝑎𝑡𝑒 𝑡ℎ𝑒 𝑀. 𝑎𝑐𝑐𝑢𝑚𝑖𝑛𝑎𝑡𝑎 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 ∗ 0.250𝑔
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑖𝑜𝑑𝑖𝑛𝑒 𝑢𝑠𝑒𝑑 𝑡𝑜 𝑡𝑖𝑡𝑟𝑎𝑡𝑒 𝑡ℎ𝑒 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑣𝑖𝑡𝑎𝑚𝑖𝑛 𝐶 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛
20
Helmenstine, Anne Marie, Ph.D. "Vitamin C Determination by Iodine Titration." ThoughtCo, Aug. 27, 2020,
thoughtco.com/vitamin-c-determination-by-iodine-titration-606322.
8
Sample calculation:
𝟎.𝟔∗𝟎.𝟐𝟓𝟎
𝟏.𝟔𝟕
= 0.082 g
The respective values were substituted in the formula and the vitamin C content in M.accuminata placed in
different set-ups were obtained which is depicted in data table 3.
Data table 4: Table illustrating the vitamin C content (g) of the M. accuminata sample in varying
ripening conditions across 4 different stages.
Haystack
Rice bowl
Smoked
Trials
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1st stage
0.082
0.097
0.090
0.082
0.067
0.082
0.097
0.090
0.082
0.067
0.082
0.097
0.090
0.082
0.067
0.090
0.090
0.082
0.090
0.090
0.075
0.097
0.090
0.082
0.090
0.090
0.067
0.075
0.067
0.082
0.090
0.082
0.075
0.090
0.067
0.067
0.090
0.075
0.082
0.082
0.075
0.060
0.082
0.060
0.060
0.067
0.075
0.082
0.082
0.082
0.060
0.082
0.090
0.082
0.075
0.060
0.075
0.060
0.052
0.060
3rd
stage
6th
stage
7th
stage
Paper bag
Control
Trials
1
2
3
4
5
1
2
3
4
5
1st stage
0.082
0.097
0.090
0.082
0.067
0.082
0.097
0.090
0.082
0.067
3rd stage
0.090
0.097
0.082
0.082
0.090
0.090
0.082
0.082
0.082
0.090
6th stage
0.097
0.082
0.075
0.082
0.075
0.082
0.082
0.075
0.082
0.090
7th stage
0.082
0.090
0.075
0.075
0.075
0.067
0.090
0.075
0.075
0.082
Furthermore the average of the vitamin C content of the samples were calculated for 5 trials of each ripening
stage in each set-up. The formula used to calculate the average is given below:
𝑆𝑢𝑚 𝑜𝑓 𝑣𝑖𝑡𝑎𝑚𝑖𝑛 𝐶 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 (𝑔) 𝑜𝑏𝑡𝑎𝑖𝑛𝑒𝑑 𝑓𝑟𝑜𝑚 5 𝑡𝑟𝑖𝑎𝑙𝑠
5
Data table 5: Table illustrating the average amount of Vitamin C (g) content present in the
M.accuminata samples in various ripening set-ups. (uncertainty-min value/2, unit, significant
figures(SF- no. of decimal places))
Ripening stages
1st stage
3rd stage
6th stage
7th stage
Haystack
0.084
0.088
0.081
0.078
Different ripening conditions
Rice Bowl
Incense sticks
Paper bag
0.084
0.084
0.084
0.087
0.076
0.088
0.079
0.067
0.082
0.078
0.061
0.079
Control
0.084
0.085
0.082
0.078
Formula used to calculate the standard deviation is as follows21:
21
“STDEV Function.” Support.microsoft.com, support.microsoft.com/en-us/office/stdev-function-51fecaaa-231e-4bbb-923033650a72c9b0.
9
∑(𝒙𝒊 − 𝒙 )𝟐
√
𝒏−𝟏
Here the following variables denote:
𝑥𝑖 - Individual values in the dataset
𝑥 - The mean of all values in the dataset
𝑛 – Number of data points
Sample standard deviation calculation using vitamin C content of M. accuminata from 3rd stage of ripening
in the Haystack setup (Data table 3):
2
2
2
2
2
√ (0.09 − 0.088) + (0.09 − 0.088) + (0.082 − 0.088) + (0.09 − 0.088) + (0.09 − 0.088)
5−1
=√
0.000052
4
= √0.000013 = 0.003
Therefore standard deviation here is 0.003
Using the values of the average vitamin C content (Data table 4) and the vitamin C content (Data table 3),
standard deviation was calculated for vitamin C content for each stage of ripening in various set-ups and the
results are presented in Data table 5.
Data table 6: Table illustrating the amount of Vitamin C(g) content present in the M. accuminata
samples under various ripening set-ups.
Standard deviation
1st stage
Haystack
0.011
Rice Bowl
0.011
Smoked
0.011
Paper bag
0.011
Control
0.011
3rd stage
0.003
0.009
0.010
0.006
0.004
6th stage
0.010
0.009
0.011
0.009
0.005
7th stage
0.007
0.011
0.008
0.007
0.009
To calculate parentage increase in the vitamin C content of M.accuminata, the following formula was
used22:
(𝑓𝑖𝑛𝑎𝑙 − 𝑖𝑛𝑖𝑡𝑖𝑎𝑙)
%𝑖𝑛𝑐𝑟𝑒𝑎𝑠𝑒 = 100 ×
|𝑖𝑛𝑖𝑡𝑖𝑎𝑙|
Here 𝑓𝑖𝑛𝑎𝑙 indicates the vitamin C content of the next ripening stage while 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 indicates the vitamin C
of the current ripening stage. To determine efficiency, the time aspect involved in ripening must be included.
According to the formula prescribed in omnicalculator.com, the percentage increase was divided by the
time taken by M.accuminata to transition to the next stage of ripening to obtain % per hour (%/hrs). The
formula was applied to the value illustrated in data table 2 and 5 and the results are tabulated below.
22
“Percentage Increase Calculator - Omni.” Omnicalculator.com, 2019, www.omnicalculator.com/math/percentage-increase.
10
Data table 7: Illustrates the percentage increase of vitamin C in M.accuminata between various stages
over time
Percentage increase over time (%/hrs)
Stages
Haystack
Rice bowl
Smoked
Paper bag
Control
0.12
0.05
-0.13
0.08
0.02
1 to 3
3 to 6
-0.16
-0.84
-0.25
-0.24
-0.07
6 to 7
-0.05
-0.05
-0.45
-0.09
-0.07
1 to 7
-0.04
-0.07
-0.19
-0.05
-0.04
Data analysis and interpretation:
Qualitative Analysis:
The colour change of the peel of M.accuminata occurs due to the breakdown of chlorophyll in the peel
resulting in the conversion of starch into simple sugars23. This causes the peel to turn yellow and the
softening of the pulp, thereby becoming sweet. Referring to data table 1, it can be said that conversion of
starch into simple sugars is the highest at stage 7 owing its qualitative characteristics and the least
conversion was observed in stage 1. The article by Science ABC on “Why do bananas change colour on
ripening” states that the levels of ethylene and the colour texture change in M.accuminata is directly
proportional. This would indicate that for each consecutive stage of the banana ripeness chart, the colour
progressively shifts from green to yellow with rising levels of ethylene.
Statistical Analysis:
In statistics, there are numerous distinct test types, such as the t-test, Z-test, chi-square test, ANOVA test,
binomial test, one sample median test and many more24. However, amongst them One-way
ANOVA(analysis of variance) test allowed the comparison of numerous group means. For this investigation,
the one- way ANOVA test aided in determining whether a statistically significant relationship existed
between Independent and dependant variable. The data used as input for the test was the average vitamin C
content of samples from the 3rd, 6th and 7th ripening stage of different IVs (Data table 5). The test was
conducted using a software called “Analysis ToolPak” which is an Add-In in Microsoft excel.
𝑯𝟎 (Null hypothesis): There is no statistically significant relationship between vitamin C content of
M.accuminata and the ripening set-ups it is placed in.
𝑯𝟏 (Alternate hypothesis): There is a statistically significant relationship between vitamin C content of
M.accuminata and the ripening set-ups it is placed in.
23
D’mello, Brendan. “Why Do Bananas Change Color When Ripening?” Science ABC, Science ABC, 22 Sept. 2015,
www.scienceabc.com/nature/bananas-change-colour-upon-ripening.html.
24
Srivastava, Anushka. “Types of Statistical Tests.” Medium, 21 July 2019, medium.com/@anushka.da3/types-of-statistical-testsb8ceb90e13b3#:~:text=There%20are%20many%20different%20types.
11
Vitamin C content of M.accuminata corresponding to stage 1 of the ripening chart in all five IVs where
excluded as they were the initial points and thereby had an absence of variance.
Figure 3 (screenshot from Microsoft Excel): Results of the one-way ANOVA test
Anova: Single Factor
SUMMARY
Groups
Haystack
Rice Bowl
Smoked
Paper bag
Control
Count
3
3
3
3
3
ANOVA
Source of Variation
Between Groups
Within Groups
SS
0.00048093
0.000282
Total
0.00076293
Sum
Average
Variance
0.247 0.08233333 2.6333E-05
0.244 0.08133333 2.4333E-05
0.204
0.068
0.000057
0.249
0.083
0.000021
0.245 0.08166667 1.2333E-05
df
MS
F
4 0.00012023 4.26359338
10 0.0000282
P-value
F crit
0.0286455 3.47804969
14
At a significance level of 0.05, there is a 5% probability of concluding there is a difference when there isn't
any25. A significance value was set to 0.05 as this is the default cut off accepted by the statical community26.
P- value illustrate probability that one would have discovered a specific set of observations if the null
hypothesis were true. If the p-value is below the significance level, then it is an evidence against 𝐻0 and
lower the p-value, the stronger the statistical relationship. The one-way ANOVA test generated a p-value of
0.0286455 which is lower than the 0.05. Thereby, this rejects 𝐻0 and accepts 𝐻1 , statistically establishing
that the ripening set-ups influence the vitamin C content in M.accuminata. Furthermore if an F test were to
be performed, then it is observed that the F value (4.26359338) is greater than the F critical value
(3.47804969), indicating that the variances in the average vitamin C content is not equal and H1 is the best
hypothesis27.
Graphical Analysis:
Average vitamin C content (g)
0,100
0,090
0,080
0,070
0,060
0,050
0,040
0,030
0,020
0,010
0,000
1st stage
Haystack
3rd stage
Rice Bowl
Incense sticks
6th stage
Paper bag
7th stage
Control
25
Frost, Jim. “Significance Level - Statistics by Jim.” Statistics by Jim, 2019, statisticsbyjim.com/glossary/significance-level/.
“Why 0.05? Two Examples That Put Students in the Role of Decision Maker | Statistics Teacher.” Www.statisticsteacher.org, www.statisticsteacher.org/2017/01/05/why-0-05-twoexamples-that-put-students-in-the-role-of-decision-maker/#:~:text=The%20significance%20level%20defines%20how. Accessed 7 Jan. 2023.
27 Mahbobi, Mohammad, and Thomas K. Tiemann. “Chapter 6. F-Test and One-Way ANOVA.” Opentextbc.ca, Dec. 2015, opentextbc.ca/introductorybusinessstatistics/chapter/f-testand-one-way-anova-2/#:~:text=Compare%20it%20to%20the%20samples.
26
12
Graph 1: Compares the average vitamin C content of different M. accuminata samples corresponding
to stage 1,3,6 and 7 of the ripening cycle in various ripening set-ups.
Vitamin C is the highest at the 3rd stage of ripening for all set-ups, except the smoked set-up. The vitamin C
content of M.accuminata samples of the smoked set-up follows a declining trend with the highest vitamin C
content at stage 1 and keeps declining through stages 3 and 6 and finally achieves the least vitamin C
content at stage 7. This could be caused due to the heat produced by the burning incense sticks as vitamin C
can be destroyed by heat and light28. Samples from all IVs exhibits the lowest vitamin C content at the 7th
stage. This can be explained by an article by Gregory Tucker published in the frontier for young minds,
where it was revealed that ethylene increases drastically during ripening which according to section 2.2,
suppresses the vitamin C biosynthesis pathway29.
0,014
Standard deviation
0,012
0,010
0,008
0,006
0,004
0,002
0,000
1st stage
Haystack
3rd stage
Rice Bowl
Smoked
6th stage
Paper bag
7th stage
Control
Graph 2: Compares the standard deviation for the Vitamin C content of different M. accuminata
samples corresponding to stage 1,3,6 and 7 of the ripening cycle under various ripening conditions.
Percentage increase per hour
(%/hrs)
Evidently, the 1st stage being the initial point, samples from all IVs have the same standard deviation.
Samples in the 1st stage, the smoked set-up in 6th stage and the rice bowl set-up in 7th stage have the highest
standard deviation of 0.011. This can be rooted form various sources like instrumental errors caused by the
weighing scale or burette, human errors during titration of M.accuminata extracts or even observational
errors occurred while recording values. The sample from the 3rd stage of the haystack set-up has the least
standard deviation indicating the greatest accuracy. In spite of this, the high standard deviation values in the
other stages of ripening question the reliability of the experiment.
0,20
0,00
-0,20
-0,40
-0,60
-0,80
-1,00
1 to 3
Haystack
28
29
3 to 6
Rice bowl
Smoked
6 to 7
Paper bag
1 to 7
Control
Harvard T.H. Chan. “Vitamin C.” The Nutrition Source, Mar. 2020, www.hsph.harvard.edu/nutritionsource/vitamin-c/.
Moirangthem, Kamaljit, and Gregory Tucker. “How Do Fruits Ripen?” Frontiers for Young Minds, vol. 6, Apr. 2018, https://doi.org/10.3389/frym.2018.00016.
13
Graph 3: Line graph comparing the percentage increase of vitamin C content between ripening stages
1-3, 3-6, 6-7 and 1- 7 in different IVs over a period of time
Graph 3 was used to determine the most efficient ripening set-up. Efficiency is at its peak when the
M.accuminata` samples yields the highest vitamin C content within short time periods. The samples placed
in the haystack set-up has the highest percentage increase per hour of 0.12 %/hrs. This implies that the
period between stages 1 and 3 exhibited the greatest increase in vitamin C content over 41 hours, the
shortest period. The samples between the 3rd and 6th stage of the rice bowl set-up has highest negative
percentage increase of -0.84%/hrs. Implying not only slow rate of ripening but also a decrease in the vitamin
C content. The rate of vitamin C increase is positive only between stages 1-3. Referring to 3.2, Ethylene
content has an inverse relation to the levels of vitamin C in the fruit sample.
Discussion and conclusion:
The one-way ANOVA test rejected the null hypothesis and provided evidence suggesting that the ripening
set-ups influenced the vitamin C content in M.accuminata. Graph 2 revealed high standard deviation values,
except those samples from stage 3. Although, this questions the reliability of the investigation, these
standard deviation values are not significant enough to scrutinize the experiment. Graph 1 revealed that
vitamin C content of M.accuminata follows a declining trend from stage 3 onwards and experiences the
highest average vitamin C content at stage 3. Therefore, consuming M.accuminata at stage 3 of the banana
ripeness chart would yield the maximum vitamin C benefits to the consumer. Graph 3 which analyzed the
percentage increase of vitamin C content over a period revealed that the highest percentage increase over the
shortest period of time was between stages 1 and 3. Furthermore, the qualitative observations conveyed that
the M.accuminata is relatively more sweet and flaccid at stage 6 and 7, making it more convenient and
appealing to consumers and producers who sell them.
Overall, the smoked set- up is the least effective ripening set- up. Substantiated by the negative percentage
increase values, unrecognizable qualitative characteristics, and the least average vitamin C content. On the
contrary, the samples placed in the haystack set-up had the least standard deviation, highest average vitamin
C content as well as the highest percentage increase, making it the best ripening set- up. Ideally,
M.accuminata should be consumed at stage 3 but due to the rigidity of the pulp, consumption is difficult.
Therefore, it is recommended for consumers to consume M.accuminata at earlier stages of ripening stated in
the banana ripeness chart for more vitamin C.
To answer the research question, M.accuminata is influenced by various ripening set-ups like haystack, rice
bowl, paper bag with ripened fruits and smoked set-up at 3 different stages of ripening.
Evaluation:
Strengths:
I carried out five trials for each titration which enabled me to acquire a plethora of data for each ripening setups. This let me to sidetrack a substantial number of random errors which could have questioned the
accuracy of the experiment. Moreover, the values obtained in the processed data were rounded off to 3
decimal places to improve the accuracy of the calculations. Additionally, the iodometric redox titration is a
reliable method to achieve accurate results as iodine reacts easily with organic materials30.
Weaknesses and suggested improvements:
A great number of potential errors revolve around the subjective nature of the experiment.
30
“Advantages and Disadvantages of Experiments - Cardiovascular Science -.” StuDocu, www.studocu.com/en-gb/document/birmingham-city-university/cardiovascularscience/advantages-and-disadvantages-of-experiments/12020688.
14
During titration, when the Iodine solution was added to the M. accuminata extract, the color change to
bluish black seemed to be visible only after 30- 40 seconds due to the presence of the pulp. This delay in the
appearance of the color change interfered with the determination of the endpoint of the titration. Moreover,
the colour change of the extract occurred in various shades of blue and black. Thereby, establishing the right
shade of bluish black colour and using that as a standard throughout the experiment was a strenuous task.
The M.accuminata ripeness chart that was used as a standard throughout the experiment, was based on the
“M.accuminata from AAA cultivar group”, commonly known as yellow Cavendish. This is variety usually
obtained in the American markets. However, due to absence of the aforementioned variety in the local
markets, the “M.accuminata × balbisiana Colla” variety, commonly known as the “Ney Poovan” variety,
was purchased, and used. Therefore, numerous discrepancies might have occurred in the qualitative
observations made based on this ripeness chart. In certain areas assumptions had to be made which could
have increased the uncertainty due to subjectivity.
The incorrect calibration of the electronic weighing scale could have been another conceivable cause of
systematic error. The M.accuminata pulp was frequently weighed and distilled water was added
proportionate to the weight of the pulp. Any incorrect measurement would have caused the M.accuminata
concentration in the extracts to be off, resulting varied chemical responses during titration. This error could
have been avoided by making sure the weighing scale was calibrated correctly in the first place.
Furthermore, the burette used for the titration was subjected to an error of ± 0. 1 cm3. This could have led to
the procurement of erroneous values. Since the vitamin C content is directly proportional to the amount of
iodine used to titrate a sample, the slightest change in the iodine volume could have fiddled with the vitamin
C content obtained and other experimental outcomes. This can could have been avoided by maintaining the
appropriate eye level during measurement and filling the burette back to 0 at the beginning of each titration
to avoid misreading.
Limitations and further scope of investigation:
As previously mentioned the Ney poovan variety was chosen due to its availability and extensive
consumption in the local market. However commercial Indian M.accuminata varieties includes
Dwarf cavendish, Robusta, Nendran, Red Banana, Ardharpuri, Basrai, Karpuravalli, and Rasthali31. The
investigation might have yielded varied results in terms of the standard deviation values and the time taken
by the sample to ripen which would have in turn affected the values obtained from the percentage increase
per hour in the vitamin C content. An article by Stacey Anderson titled “How to Test Vitamin C in Fruits at
Home” elaborates on a protocol which includes adding predetermined quantity of starch solution to the fruit
extract. This however is different form the protocol used in this investigation, where instead of a starch
solution, the natural starch present in the extract was relied upon.
The investigation could have explored an entirely different dimension if it had delved into other plausible
independent variable such as the effect of various geographical locations on vitamin C content of
M.accuminata or the effect of ripening on vitamin C of a fruit other than M.accuminata. Similarly, the
investigation quantified and analysed the effect of ripening on the vitamin C content of M.accuminata while
ignoring other nutrients present in it. Therefore the investigation could have explored the effect of ripening
on starch or potassium content of M.accuminata.
31Google.com,
2023,
www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&cad=rja&uact=8&ved=2ahUKEwjwjPPA8sP6AhU9BbcAHV47CDkQFnoECAMQAw&url=https%3A%2F%2Fwww.abcfruits
.net%2Fbanana-varieties-production-and-season-in-india%2F&usg=AOvVaw2ikZr5tRWNhQA3eSwCvH4g.
15