RESEARCH QUESTION

How does an increase in the concentration of pectinase influence the difference in juice
production between mango variety Mangifera Indica and Mangifera Odorata?

INTRODUCTION

Macromolecular biological catalysts that speed up biochemical reactions without being altered
themselves, and are made by living cells are referred to as enzymes. Enzymes bind to active
sites, which refer to a special region on the substrate1. The physical properties of the active site
and the substrate complement each other, thus resulting in only the substrate binding to the
enzyme. Final products are released when substrates are converted into products upon being
bound to the active site2.

Present in the cell wall of terrestrial plants, pectin is a heteropolysaccharide. It is involved in

plant growth and permits primary cell wall extension and thus, is a substance beneficial to plant
substance3. The enzyme that breaks down pectin is called pectinase4. The enzyme pectinase
includes pectolyase, pectozyme, and polygalacturonase5. During fruit ripening, the enzyme
pectinase or pectinesterase breaks down pectin. During this process, as the middle lamella is
digested, the fruits become softer as their cells separate. Pectinase is used for extracting juice
from puree and is also used in the jams and wine industries6. This extraction of juice is done by
pectinase breaking down pectin. Due to its enzyme properties, pectinase is more active at
optimum temperature and pH. Before denaturation, pectinase works best at 45- 55 °C and at a pH
of 3 - 6.5.

Mango (Mangifera Indica) is the most important food crop of India which occupies almost 60%
of the total area under fruits and is also the national fruit of India7. It has been a fruit that has
been a part of my culture for over 4000 years. A few benefits of mangoes include it’s quality of

1 (Allott, and Mindorff)

2 (Sultana et al.)
3(Singh Jadaun)
4 (Khan et al.)
5 (Shah et al.)
6 (Singh et al.)

7 (Chowdhury)
cleansing the skin and treating pores, hence, leading to my interest in the fruit8. Since, mangoes
are consumed largely in the form of juice, I wanted to investigate the process through which it is
converted since, it contains a high amount of pectin and hence, is difficult to break down.

It is a fruit which extensively varies in form and size, sub-acidic or sweet and soft and juicy with
aromatics. Mango contains numerous organic acids and high amounts of sugar. Glucose, sucrose
and fructose are the primary carbohydrates in ripe mangoes with small amounts of pectin,
hemicellulose and cellulose.

There exists external factors which may affect pectinase activity: substrate concentration,
temperature (optimum is 45 to 55 °C), presence of inhibitors that can block active sites and pH
(optimum is 3.0 to 6.5). For the above, the experiment mangoes used had a pH of between 3.5 -
5.5 and the apparatus included an incubator at 50 °C.

HYPOTHESIS

Null Hypothesis: The rise in the pectinase concentration, in grams, in a 50 ml solution, will not
influence the juice production from 5g mango. Any difference that takes place is entirely due to
the effect of extraneous variables and not due to the manipulation of the independent variable.
There is no significant difference of species of mango in juice production9.

Alternative Hypothesis: The difference in juice production of 5 g of mango is influenced by the

increasing pectinase concentration in a 50 ml solution. There is a significant difference in juice
production between different species of mango.

VARIABLES

Independent Variable:

1. Pectinase concentration in the solution (%) / Amount of pectinase dissolved in 50ml of

water

● 0% (0g dissolved in 50ml of water)

● 10% (5g dissolved in 50ml of water)

8 (Ribeiro et al.)
9 (Adelakun et al.)
 ● 20% (10g dissolved in 50ml of water)

● 30% (15g dissolved in 50ml of water)

2. Different species of mango

● Mangifera Indica

● Mangifera Odorata

Dependent Variable: The difference in the amount of mango juice produced (ml)

Controlled Variable How Justification

Quantity of water (50 ml) The use of accurate measuring Accurate juice production will be
cylinders obtained through the use of equal
solution volume.

The mass of mango (5.00 g) 1 Mango pulp is the substrate thus,

Balance graduated to th of a
100
changing concentrations of substrate
gram
lead to a change in enzyme activity.

pH of Water The water used was taken from the The pH also has an impact on enzyme
same source activity, so maintaining the pH will
lead to control of extraneous
variables due to higher or lower pH.

Temperature (℃ ) Incubated at 40 °C Enzyme activity is influenced by

temperature. An increase in
temperature could lead to a rise in
enzyme activity till the optimum,
 after which it denatures.

Filter paper The filter paper used was of the This maintains the flow rate due to a
same size: Qualitative, Circle, 9cm constant thickness.
diameter

Size of the funnel For each trial, an 80mm funnel Due to equal surface area, the flow
glass was used will be equal thus leading to
comparable results.

Time 3 stopwatches were used. A change in time influences enzyme

activity.

Pectinase used For all trials, the source of pectinase This reduces inaccuracy or invalid
was common. variations in the data.

METHOD

Apparatus

1. Pectinase, total of 300g powdered pectinase

2. Mangifera Indica, total of 100g pulp

3. Mangifera Odorata, 100g in total of pulp

4. 1×Vegetable peeler

5. 1×Pair of tweezers

6. 1×Incubator

7. 2×Bowls
 8. Distilled water

9. 1×Pipette

10. 3×Stopwatches (±0.01s)

11. 8×25 ml Beakers (±0.1 ml)

12. 40×Discs of filter paper

13. 8×10 ml Measuring Cylinders (±0.1 ml)

14. 1×Grater

15. 8×100ml Beakers (±0.1 ml)

16. 6×Cuvettes

17. 1×50ml Measuring Cylinder (±0.1 ml)

18. 20×Glass Funnels

19. 1×Spatula

20. 8×20 ml Measuring Cylinders (±0.1 ml)

21. Balance graduated to the nearest 0.01g

22. 8×Glass Rods

PROCEDURE

1. Eight 25 ml beakers were named from A- H, which was repeated for each measure twice:
0, 5, 10 and 15. The 20 ml cylinders were named similarly.

2. The rind of the two mango varieties was peeled off using a vegetable peeler and then the
pulp of the different mangoes was grated into two different bowls, avoiding the core.
 3. 5g of Mangifera Indica pulp each measured into the A, B, C and D labelled 25 ml
beakers. 5g of Mangifera Odorata pulp each measured into the E, F, G and H labelled 25
ml beakers.

4. According to the percentage concentration required, the pectinase solution was prepared.
0, 5, 10 and 15 grams of pectinase was measured referring to the percentage
concentration twice in two 100 ml beakers. 40ml distilled water was poured in two 50ml
measuring cylinders.

5. A little water was added from the 50 ml measuring cylinders to the 100 ml beakers to stir
with a glass rod to mix the powder. Two 40 ml solutions were created by pouring the
contents back in the two cylinders.

6. Using a pipette and funnel, the two 40 ml solutions were divided into eight 10ml
measuring cylinders.

7. Solution of pectinase (10ml per 5g pulp) was poured in eight 25 ml beakers. In the
beakers A, B, C and D, 5 g of Mangifera Indica pulp was added in each. In the E, F, G
and H beakers, 5 g of Mangifera Odorata pulp was added in each. For 25 minutes, these
were incubated at 40 °C.

8. Post removal of the beakers from the incubator and the contents of each were poured in a
20ml measuring cylinder through filter-papered funnel.

9. At 5, 10 and 15 minutes, the production of juice from mango pulp was measured.

10. Five trials of the experiments were performed.

SAFETY

1. To avoid reaction with the skin and protect hands from enzymes, gloves were used.

2. To protect the eyes from powder and solutions used in the experiment, goggles were
used.
 3. To avoid staining of clothes, reaction with the skin or cross contamination due to
solutions spilling, a lab coat was worn.

4. All unnecessary materials cleared away from workplace.

5. Sharp cutting tools and utensils were handled with care.

6. Fragile glassware was used towards the centre of the bench with stable support.

RESULTS

Quantitative Statistics

Raw Data

Table 1- In a 50ml of solution of pectinase, volume of Mangifera Indica production of juice

from 5g pulp

In a 50ml of pectinase solution, volume of Mangifera Indica juice

produced from 5g pulp

Pectinase
Concentration
(%) 0% 10% 20% 30%
Sample
Time (minutes) Uncertainty ±0.05ml ±0.05ml ±0.05ml ±0.05ml
A 3.51 3.95 4.65 5.55
B 3.93 4.46 5.83 6.82

C 3.38 4.23 4.96 5.73

D 3.25 4.90 4.42 5.36

5 minutes E 4.20 4.86 5.64 6.39

10 minutes A 4.43 4.91 5.47 6.11
B 4.78 4.52 5.20 5.47
 C 4.62 3.98 4.98 5.34

D 4.89 4.23 5.73 5.56

E 4.93 4.74 4.82 5.62

A 5.51 4.98 5.42 5.91
B 5.3 5.1 5.2 6.3

C 5.6 5.3 5.5 6.7

D 4.8 5.7 5.9 6.9

15 minutes E 5.4 5.8 6 6.4

Table 2 - In a 50 ml pectinase solution post-filtration, volume of Mangifera Odorata productiion

of juice from 5g pulp

In a 50ml of pectinase solution, volume of Mangifera

Odorata juice produced from 5g pulp

Concentratio
n of Pectinase
(%) 0% 10% 20% 30%
Time Sample
(minutes) Uncertainty ±0.05ml ±0.05ml ±0.05ml ±0.05ml
A 2.93 3.38 3.92 4.41
B 3.05 3.45 4.51 4.32

C 2.23 3.52 4.26 4.70

D 2.65 3.17 3.63 4.92

5 minutes E 3.10 3.66 3.85 4.20

10 minutes A 3.35 3.71 3.51 4.41
B 3.52 3.62 3.70 4.66

C 3.17 3.85 3.93 4.82

 D 3.44 3.30 3.45 4.95

E 3.20 3.54 3.26 4.58

A 3.41 3.61 3.68 4.41
B 3.69 3.77 3.52 4.77

C 3.57 3.48 3.84 5.52

D 3.23 3.22 3.71 4.70

15 minutes E 3.32 3.84 4.07 4.93

Processed Data and Descriptive Statistics

Table 3- In a 50ml solution of pectinase post filtration, the mean quantity of production of juice
from 5g mango pulp

In a 50ml solution of pectinase post filtration, the mean volume of mango

juice produced from 5g mango

Mean Mangifera Indica Mangifera Odorata

Time in
minutes 5 mins 10 mins 15 mins 5 mins 10 mins 15 mins
Uncertainty ±0.05ml ±0.05ml ±0.05ml ±0.05ml ±0.05ml ±0.05ml
0% 3.62 4.68 5.32 2.88 3.30 3.40
10% 4.62 4.44 5.36 3.38 3.58 3.54
20% 4.90 5.20 5.52 3.90 3.54 3.72
30% 5.76 5.58 6.44 4.32 4.64 4.74

Table 4- In a 50ml solution of pectinase post filtration, the standard deviation of mean quantity
of mango production of juice from 5g pulp

In a 50ml solution of pectinase post filtration, the standard deviation of mean

volume of mango juice produced from 5g mango

Pectinase Mangifera Indica Mangifera Odorata

Concentration
(%)
Time (minutes) 5 mins 10 mins 15 mins 5 mins 10 mins 15 mins
0% 0.42 0.19 0.31 0.19 0.16 0.16
10% 0.36 0.40 0.39 0.19 0.19 0.24
20% 0.46 0.37 0.43 0.22 0.27 0.19
30% 0.40 0.31 0.39 0.26 0.21 0.23

Graph 1 - Mean mango juice produced using pectinase

Inferential Statistics
To derive the significance of the data from juice production of M. Indica and M. Odorata, a t-test
was conducted..
At 5 minutes, the juice production was compared for M. Indica and M. Odorata and the p-value
= 0.00265. At 10 minutes, the p-value = 0.03836 for compared juice production between the
mango varieties. Lastly, at 15 minutes, the p-value= 1.7E-05. Thus, the data obtained is
significant at p > 0.01.

CONCLUSION
The alternative hypothesis suggested that increasing pectinase concentration (in grams), in a
solution of 40 ml, will lead to a rise in production of mango juice production (in millimeters), of
5g mango pulp when filtered post-reaction. The data obtained shows the effect of pectinase on
the volume of juice produced. This was a result of higher pectinase concentrations, which aided
in digestion of pectin in mango’s cell wall. This thus allowed a higher quantity of production of
juice. Thus, the extract was liquid post filtration. This results of the experiment supported the
alternate hypothesis.

The alternate hypothesis also states that the difference in the juice production was due to the
different species of mango used. Thus, the juice production of M. Indica was significantly higher
than the juice production of M. Odorata. Therefore, the alternate hypothesis stating the
difference in juice production is due to different species is supported by the findings of the
experiment.

The overall results obtained supported the alternate hypothesis. Upon absence pectinase, the
lowest values of the quantity of production of mango juice were obtained. When the
concentration of pectinase is 30%, the quantity of the production of mango juice was the highest.
There is a general trend because the results are coherent with the alternate hypothesis. Moreover,
all results were similar, thus, if a larger volume of mango juice was used to display the
relationship, much difference will not be observed. Additionally, the alternate hypothesis was
supported by the results obtained by the t-test performed. Furthermore, when comparing the juice
produced out of M. Indica and M. Odorata, it was seen that volume of juice produced was higher
for M. Indica10.

10 (Tochi et al.)
With reference to application in real life, pectinase is used for higher juice production.
Furthermore, for commercial mango juice production, the mango variety M. Indica must be used
as it provides higher volume and concentration of juice.

EVALUATION
Since the results obtained followed a clear pattern, they are reliable. The volume of juice
produced in both the varieties is higher at 30% pectinase than 0%, 10% and 20%. The
concentration of the juice increases as concentration of pectinase increases. The
findings varied for both the varieties however, there were some variations within the results too.
In the 10% and 20% pectinase condition the difference in the juice produced was not high,
therefore, it was difficult to distinguish the correct representative and determine what may have
caused a reduced increase in the juice produced. We could stray away from the real result if one
set was to be disregarded. High degree of accuracy was suggested by the t-test which may be
unreliable.

There were numerous errors and limitations in the study. The inability of the incubator to go over
the temperature of 40 °C was one such limitation faced. Unfortunately, due to this, the activity of
pectinase at optimum temperature, which is 45-50 °C, could not be observed. Resulting from
time constraints and quantity of samples, to prepare a water bath of optimum temperature was
not possible and thus, could be a possible modification. Similarly, for the provision of a more
realistic condition, the pH of the mango pulp should have been measured to determine if the
solution of mango pulp and pectinase was more acidic or alkaline. The miscibility of the solution
was another limitation as despite several tries to stir the solution, it kept separating due to large
particles. This contributed to the uncertainty and possible inaccuracy of the findings obtained as
to whether the true effect of pectinase was observed. Moreover, all the samples had varied
amount of solute. As an example, the sample that was filled at the end must have higher
pectinase concentration due to large particles settling at the base. Thus, the pectinase
concentration would have decreased exponentially from the first to the last sample. A possible
modification to overcome this problem could be grinding the pectinase particles into fine
powder, making it easier to dissolve. Or, the miscibility of the water could be decreased by
heating it. Lastly, to mix pectinase in the water, an emulsifier could be used.
There had been a few problems with the solutions, as an example, pectinase concentrations froth
a lot result in thickness. Frequently, it was the foam could not be poured and removed
completely. This thus may lead to inaccurate readings since the foam could have blurred the
meniscus. In case the miscibility could not be corrected, then reading the data from a fixed level
can be used as a modification. The filter paper used was thick and handmade resultantly, it could
have soaked the juice produces, acting as an extraneous variable. To overcome this problem, the
brand of the filter paper could be changed to a thinner variety. Due to their ability of clearly
representing data, bar and column charts were used. Moreover, they make reading values easier
and help in establishing a correlation by viewing the representations. A tangible insight was
provided to the accuracy of the readings through the use of error bars.

The experiment provided insight into understanding of pectinase as an enzyme and thus,
generated branches of curiosity. It is important to explore the influences of enzymes such as
pectinase. Such investigations could be used to determine which enzymes are beneficial and
more efficient for extraction of juices. Such results could be useful for commercial purposes, as
it could be used to enhance products and profit. For further development to this research, it
would be interesting to investigate the influence of pectinase on all breeds of mangoes and see if
similar kinds of juice is produced. This would aid in differentiating which mango variety have
the best flavours and high quantities.
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