Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

INTERNATIONAL SCHOOL OF HYDERABAD

IB Biology Internal
Assessment [HL]
Pectinase Lab
Aim: To Find the Effect of Concentration of Pectinase on Apple (Malus domestica) Juice
Production

Student Name: Dhanish Bachheta

Student Number: 002347-0007
Date: 1/1/2015
Year of Submission: 2015

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

Contents
Design...................................................................................................................................................... 3
Aim ...................................................................................................................................................... 3
Research Question .............................................................................................................................. 3
Introduction ........................................................................................................................................ 3
Hypothesis........................................................................................................................................... 4
Variables ............................................................................................................................................. 4
Method ................................................................................................................................................... 6
Apparatus ............................................................................................................................................ 6
Safety .................................................................................................................................................. 6
Procedure............................................................................................................................................ 7
Data Collection and Processing ............................................................................................................... 8
For Volume using Filtration............................................................................................................. 8
Qualitative ....................................................................................................................................... 8
Quantitative .................................................................................................................................... 8
Processed Data and Descriptive Statistics ...................................................................................... 9
For Concentration using Spectrophotometry ............................................................................... 11
Quantitative .................................................................................................................................. 11
Processed Data and Descriptive Statistics .................................................................................... 11
Inferential Statistics .......................................................................................................................... 13
For Volume using Filtration........................................................................................................... 13
For Concentration using Spectrophotometry ............................................................................... 14
Conclusion ............................................................................................................................................. 15
Evaluation ............................................................................................................................................. 16
Bibliography .......................................................................................................................................... 18

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

Design
Aim
To find the effect of concentration of the enzyme pectinase on apple (Malus domestica of the
variety Jonagold) juice production

Research Question
How does an increase in the concentration of the enzyme pectinase affect the production of apple
juice from apple (Malus domestica) pulp?

Introduction
Enzymes are biological catalysts that speed up chemical reactions in the body. Pectinase is the
general term for the group of pectin-breakdown enzymes, including polygalacturonase, pectolyase
and pectozyme. Pectin is a complex set of polysaccharides and is located in the primary cell wall of
terrestrial plants. It causes plant growth and extension of the cell wall and is an extremely beneficial
plant substance. Pectin may be broken down using the group of pectic enzymes. Pectinase decreases
time taken to extract fruit juices from their pures but also has other uses in the wine and jam
industries, and in retting. Pectinase works by reducing the activation energy required to split the
pectin molecule, in effect breaking down the cell wall. It can be found in fungi such as Aspergillus
niger. The fungus produces these to break down the middle lamella of a plant so that the nutrients
of the tissues can be extracted and fungal hyphae can be embedded1. In commercial production,
such as that of wine, pectinase allows one to break down the pulp and then extract the pure flavour.
Pectinase is an enzyme and is also therefore affected by external conditions. It works well at a pH of
4.5 to 5.5 and a temperature of 45 to 55 after which it starts to denature. Pectinase improves
colourings, aromas and the clarity of juices, presss and fruit-based liquors.

There are some factors that affect pectinase activity: temperature (of which the optimum is 40 50
), pH (of which the optimum is 3.5), concentrations of the substrate and the enzyme, and the
presence of inhibitors which can invade the allosteric sites. Which is why in this experiment apples
were used which have a pH of 3.3 - 3.9 and an incubator set at 40. Furthermore, the use of
distilled water evades the possible presence of microorganisms and therefore inhibitors2.

This experiment investigated how the quantity of pectinase affected apple juice production. First,
apples were peeled and shredded apples then measured into beakers. Pectinase solutions of varying
concentrations were prepared including one of distilled water. 5.00g of the pulp was measured into
25ml beakers, the pectinase solutions were added, and incubated at 40 for 25 minutes.
Afterwards, the mixture was filtered and the juice levels were read at three 5 minute intervals.
Subsequently, a spectrophotometer was used to determine the colouring increase in the apple juice
samples, resetting it at each test using a control of distilled water.

1
(Priya & Sashi, 2014)
2
(Ceci & Lozano, 1996)

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

Hypothesis
Null Hypothesis (H0): Increasing the concentration of pectinase, in grams, in a 50ml solution, will not
have any bearing on juice production, volume or concentration, of 5g of apple when reacted with
the solution; any difference that occurs is entirely due to chance or random variables not under
control, and not due to the manipulation of the independent variable.

Alternate Hypothesis (H1): Increasing the concentration of pectinase, in grams, in a 50ml solution,
will increase juice production, in terms of volume or concentration, of 5g of apple when reacted with
the solution.

The theory behind the alternate hypothesis recognises the fact that if the concentration of pectinase
is higher, more enzymes are available to break down the pectin in the cell wall of the apple, thus
allowing a greater amount of juice to be released. Therefore, the extract should have more liquid
after filtering.

Variables
Independent Variable:

Concentration of pectinase solution (%) / Amount of pectinase (g) dissolved in 50ml of water
0% (0g dissolved in 50ml of water)
5% (2.5g dissolved in 50ml of water)
10% (5g dissolved in 50ml of water)
15% (7.5g dissolved in 50ml of water)
20% (10g dissolved in 50ml of water)
25% (12.5g dissolved in 50ml of water)

Dependent Variable:

Rate of action of pectinase on apple pulp measured as:

Volume of apple juice produced (ml)
Intensity of colour of apple juice produced

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

Controlled Variables:

Variable How Reason

Temperature () Incubated at 40 The enzyme activity varies with
temperature. As the temperature
increases the activity increases. However
beyond the optimum temperature the
enzyme activity reduces as a result of the
enzymes denaturing at the higher
temperatures.
Volume of Water Using accurate measuring Equal solution volumes will provide more
(50ml) cylinders instead of beakers accurate juice production readings.
Mass of Apple 1
Balance graduated to 100 of a The apple pulp is the substrate so an
(5.00g) gram increase in substrate concentration will
increase the enzyme activity
Equal apple pulp quantities will provide
more accurate juice production readings.
Same pH of water All water used for the experiment To keep the pH level the same so that no
was obtained from the same values could become extraneous by
source having a greater or lower pH.

Spectrophotometer Use a control sample of pure
water to reset the device before
each use and use the same
device for all readings
Same variety of All filter paper discs were of the
filter paper type: HM 2, Qualitative, Circles,
12.5cm Diameter.
Funnel Size 75mm glass funnels were used
for each
Time 6 stopwatches
One for each condition to time
the 25 minute incubation period
and also the 15 minute filtration
Source of Pectinase The Pectinase enzyme powder
was used from the same source
for all experiments involving
pectinase in this investigation.
This improves accuracy of the colour
reading
This allows same flow rates as all the discs
were the same thickness.
The surface area will be equal to allow
equal flow so all results are comparable
An increase in the time period shows
more enzyme activity hence increased
juice production.
To reduce inaccuracy or invalid variation
between the results

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

Method
Apparatus
- Pectinase, 37.5g in total of powdered enzyme (Kemphasol, CAS No. 9032-75-1)
- Distilled Water, 300ml
- 3 Jonagold Apples, 150g in total of pulp
- 1 Vegetable Peeler
- 1 Grater
- 1 Bowl (preferably rubber or plastic)
- 1 Spatula
- 5 Glass Rods
- 1 Pair of Tweezers
- 1 Pipette
- 1 Balance, graduated to the nearest 0.01g
- 1 50ml Measuring Cylinder
- 6 100ml Beakers
- 1 Incubator
- 6 Stopwatches
- 1 Spectrophotometer
- 6 Cuvettes
- 30 25ml Beakers
- 30 10ml Measuring Cylinders
- 30 20ml Measuring Cylinders
- 30 Glass Funnels (75mm in diameter)
- 30 Discs of Filter Paper (HM 2, Qualitative, Circles, 12.5cm Diameter)

Safety
- Use gloves to protect the hands from the enzyme and from cross-contamination
- Use goggles to protect eyes from the powders and solutions used in the experiment
- Wear a lab coat to avoid cross-contamination and spillages of solutions staining the clothes
or reacting with the skin

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

Procedure
1. Label five 25ml beakers with 0 and either A, B, C, D and E. Repeat for measures: 2.5, 5, 7.5,
10 and 12.5. Label the 20ml measuring cylinders in the same way.
2. Peel the rind off of the apples using the vegetable peeler and then grate the pulp into a
bowl, avoiding the stalk, core and seeds.
3. Measure 5g of apple pulp into each of the 25ml beakers. Use tweezers and spatulas to get
the accuracy of 5.00g.
4. Prepare the pectinase solution according to the concentration required. Weigh 0, 2.5, 5, 7.5,
10 or 12.5 g of Pectinase according to percentage concentration into a 100ml beaker and
pour Distilled Water into a 50ml measuring cylinder until it reaches 50ml. Add a little water
from the 50ml measuring cylinder to the 100ml beaker to dissolve the powder, while stirring
vigorously with a glass rod. Pour back into the 50ml measuring cylinder and add until the
solution is 50ml in volume. Divide into five 10ml measuring cylinders using a funnel and the
pipette.
5. Pour pectinase solution (10ml per 5g of pulp) into five 25ml beakers, stir to make sure each
shaving of apple is fully submerged. Incubate immediately at 40 for 25 minutes. Remove
and pour contents of each into a filter-papered funnel inside a 20ml measuring cylinder.
6. Measure volume of juice produced, from the meniscus, at 5, 10 and 15 minutes. Record
results.

As a further investigation a spectrophotometer can be used to measure the intensity of the

colour of the juice produced which is a measure of the concentration.

7. Discard pulp and funnels. Pour the clarified juice into 5 cuvettes, correspondingly naming
them A, B, C, D and E. Keep one cuvette named F with distilled water as a control.
8. At the 550 setting, place F into the spectrophotometer, turning the dial until a 0 reading is
obtained. Record results after testing each sample while checking accuracy in between every
sample by testing F again and adjusting the dial accordingly to required 0.
9. Repeat for all conditions.

(In this experiment, all the concentrations were tested simultaneously due to time constraints.
However, it is not necessary for all the tests to be conducted concurrently.)

10. Record the results in 6. by reading the volume from the meniscus beneath the foam, and in
8. by recording the number that appears after reading the colour analysis finishes. Plot the
results in a predesigned raw data table in an electronic spreadsheet, where the mean can be
calculated and a bar chart generated using the average and graphing functions.
11. Perform the Standard Deviation and ANOVA Statistical Tests using the functions: STDEV and
the Data Analysis ANOVA Single Factor.

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

Data Collection and Processing

For Volume using Filtration
Qualitative
When the pectinase solution was added to the apple pulp, the apple shreds rose
After the incubation period the apple pulp had oxidised to a golden-brown colour and had
sunk to the bottom
After filtration there was a clarified, coloured liquid which went from very pale yellow (0%
Pectinase) to pale yellow (5% Pectinase) to yellow (10% Pectinase) to golden (15% Pectinase)
to dark yellow (20% Pectinase) to brown-yellow (25% Pectinase)

Quantitative

Raw Data

Table 1 Volume of apple juice produced from 5g of apple pulp in 50ml of pectinase
solution after filtration
Volume (ml) of Apple Juice Produced from 5g of apple pulp in 50ml of
pectinase solution after filtration
Concentration of 0% 5% 10% 15% 20% 25%
Pectinase (%)
Time (mins) Sample | (0.05ml) (0.05ml) (0.05ml) (0.05ml) (0.05ml) (0.05ml)
Uncertainty
5 mins A 5.00 5.00 4.00 1.60 7.60 4.60
B 5.00 3.80 3.00 3.20 3.70 2.20
C 5.00 3.80 5.25 6.60 5.30 2.60
D 4.50 3.60 3.50 6.20 3.60 4.20
E 4.75 2.40 4.25 6.00 3.00 6.70
10 mins A 5.25 5.00 4.90 2.00 8.60 5.50
B 5.25 4.50 4.70 3.50 4.10 2.40
C 5.25 4.75 5.80 7.40 5.80 3.00
D 4.75 3.80 4.00 7.00 4.20 4.90
E 5.50 2.60 4.90 6.50 3.00 7.40
15 mins A 5.30 5.00 5.00 2.20 9.00 5.50
B 5.30 4.50 3.90 3.50 4.20 2.70
C 5.30 4.80 6.00 8.00 6.00 3.20
D 4.80 4.00 4.20 7.20 4.60 5.20
E 5.40 3.40 4.50 6.70 3.20 7.60

To find the juice readings, filtration was used. A funnel with a filter paper cone is placed on top of a
20ml measuring cylinder which allows a reading of the juice production at various timings. At 5
minutes the level was read; likewise at 10 and 15 minutes.

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

Processed Data and Descriptive Statistics

Table 2 Mean Volume of apple juice produced from 5g of apple pulp in 50ml of pectinase
solution after filtration
Mean Volume (ml) of apple juice produced from 5g of apple pulp in
50ml of pectinase solution after filtration (0.05ml)
Mean 5 mins 10 mins 15 mins
Uncertainty (0.05ml) (0.05ml) (0.05ml)
0% 4.85 5.20 5.22
5% 3.72 4.13 4.34
10% 4.00 4.86 4.72
15% 4.72 5.28 5.52
20% 4.64 5.14 5.40
25% 4.06 4.64 4.84

Table 3 Mean Volume of Apple Juice (ml) produced over Time (mins)
Mean Volume of Apple Juice (ml) produced over Time (mins)
Volume (ml)
Time (mins) 5 mins 10 mins 15 mins
Uncertainty (0.05ml) (0.05ml) (0.05ml)
Concentration of
Pectinase Solution (%)
0% 4.85 0.35 0.02
5% 3.72 0.41 0.21
10% 4.00 0.70 0.38
15% 4.72 0.56 0.24
20% 4.64 0.50 0.26
25% 4.06 0.58 0.20

Table 4 Standard Deviation of Mean Volume (ml) of apple juice produced from 5g of apple pulp
in 50ml of pectinase solution after filtration
Calculated using the Standard Deviation Function in Microsoft Excel (STDEV)

Standard Deviation of Mean Volume (ml) of apple juice produced from 5g of apple
pulp in 50ml of pectinase solution after filtration
Concentration of Pectinase Time 5 mins 10 mins 15 mins
Solution (%)
0 0.224 0.274 0.239
5 0.923 0.965 0.647
10 0.848 0.778 0.698
15 2.203 2.391 2.523
20 1.861 2.177 2.249
25 1.794 2.008 1.965

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

Graph 1 Graph to show Mean Juice Production using Pectinase with Standard Deviation Error
Bars

Graph to show Mean Juice Production using Pectinase with

Standard Deviation Error Bars

25

20

15 5 mins
Concentration of Pectinase (%)

10 mins

10
15 mins

0.00 1.00 2.00 3.00 4.00 5.00 6.00

Volume of Juice Produced (ml)

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

For Concentration using Spectrophotometry

Quantitative
(Absence of Qualitative data as no visual information could be obtained in this method)

Raw Data

Table 5 Spectrophotometer Readings of 5ml of Pectinase-produced Apple Juice observed at 550 nm

Spectrophotometer Readings of 5ml of Pectinase-produced Apple Juice
observed at 550 nm
Concentration of 50ml pectinase solution
Sample 0% 5% 10% 15% 20% 25%
Colour Readings of Apple Juice in arbitrary units
Uncertainty: 0.01
A 0.23 0.83 1.25 1.31 1.72 1.84
B 0.33 0.85 1.20 1.35 1.59 1.87
C 0.27 0.80 1.29 1.44 1.66 1.78
D 0.30 0.69 1.19 1.42 1.65 1.82
E 0.27 0.76 1.12 1.43 1.62 1.88

The juice samples were poured into each of the correspondingly lettered cuvettes. A control sample
F was used to set the spectrophotometer and then in the interval in between every testing of each
sample. The setting was at 550 nm and each F sample set the spectrophotometer to 0.00.

Processed Data and Descriptive Statistics

Table 6 Mean Spectrophotometer Readings in arbitrary units of 5ml of Pectinase-produced

Apple Juice observed at 550 nm
Mean Spectrophotometer Readings in arbitrary units of 5ml of Pectinase-
produced Apple Juice observed at 550 nm
Concentration 0 5 10 15 20 25
of Pectinase
Solution (%)
Uncertainty: Colour Readings of Apple Juice in arbitrary units
0.01
Mean 0.28 0.79 1.21 1.39 1.65 1.84

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 Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

Table 7 Standard Deviation of Mean Spectrophotometer Readings in arbitrary units of 5ml of

Pectinase-produced Apple Juice observed at 550 nm
Standard Deviation of Mean Spectrophotometer Readings in arbitrary units of 5ml
of Pectinase-produced Apple Juice observed at 550 nm
Concentration of Pectinase Colour Readings of Apple Juice in arbitrary units
Solution (%) Uncertainty: 0.01
i0% 0.037
i5% 0.064
i10% 0.064
i15% 0.057
i20% 0.049
i25% 0.040

Graph 2 Graph to show Change in Colour of Juice Extract using a Spectrophotometer from Water

Graph to show Change in Colour of Juice Extract using a

Spectrophotometer from Water

2.50

2.00
Colour Change in arbitrary units

1.50

1.00

0.50

0.00
Concentration i0% i5% i10% i15% i20% i25%
of Pectinase
(%)

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

Inferential Statistics
The ANOVA Test was chosen as the parametrical statistical due to its robust design, applicability for
a lab experiment and increased statistical power. However it is a rather inefficient testing
method3and local control is completely neglected.

For Volume using Filtration

Table 8 ANOVA: Single Factor Apple Juice Volume

ANOVA: Single Factor Apple Juice Volume
SUMMARY
Pectinase Count Sum Average Variance
Concentrations
0% 3 15.27 5.09 0.04
5% 3 12.19 4.06 0.09
10% 3 13.78 4.59 0.30
15% 3 15.52 5.17 0.16
20% 3 15.18 5.06 0.14
25% 3 13.54 4.51 0.16
ANOVA
Source of SS df MS F P-value Sig4
Variation
Between 2.82 5 0.56 3.67 0.03 3.10
Groups
Within Groups 1.84 12 0.15
Total 4.67 17

Sig 0.05; 5, 12 = 3.105875

However F = 3.670997

Considering the above data, the value of F is little greater than the Sig value, therefore the
significant difference was most likely caused by random factors or chance and not the Independent
Variable. This data supports the null hypothesis that there is no difference in the volume of apple
juice produced with different amount of pectinase enzyme (%).

3
Creates larger estimates of the within group [error] variance (StudyMode)
4
Also known as F critical

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

For Concentration using Spectrophotometry

Table 9 ANOVA: Single Factor For Spectrophotometer Data

SUMMARY
Pectinase Count Sum Average Variance
Concentrations
0% 5 1.40 0.28 0.00140
5% 5 3.93 0.78 0.00403
10% 5 6.05 1.21 0.00415
15% 5 6.95 1.39 0.00325
20% 5 8.24 1.64 0.00237
25% 5 9.19 1.83 0.00162

ANOVA
Source of SS df MS F P-value Sig
Variation
Between 8.30 5 1.661 592.63 2.59E-24 2.62
Groups
Within Groups 0.06 24 0.002
Total 8.37 29

Sig 0.05; 5, 24 = 2.620654

However F = 592.6373

Considering the above data, the value of F is far greater than the Sig value, therefore the significant
difference was caused by the IV and not random factors or chance.

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

Conclusion
The alternate hypothesis (H1) was that increasing the concentration of pectinase, in grams, in a 50ml
solution will increase juice production, in millilitres, of 5g of apple when reacted with the solution
and filtered. This is due to the higher availability of pectic enzymes to break down the pectin in the
cell wall of the apple, thus allowing a greater amount of juice to be released. Therefore, the extract
should have more liquid after filtering. This investigation found the Alternate Hypothesis to be
partially supported.

The data concerning the pectinase juice production shows how much the volume of juice produced
is affected by the enzyme pectinase. The high readings of juice production of the control contrast
with the general pattern (Graph 1). If one were to ignore the control group, it would be seen that
there is a general trend of a bell-shaped curve. The juice production gradually climbs higher from 5%
to 15% but then again gradually decreases towards 25%. Considering the error bars; there are
varying error levels for each time interval and category. This means that there is little consistency
between apple samples and their readings for juice production.

The spectrophotometer was used as a method to give further validity to the results. It relied on
increase in the concentration of the juice produced rather than the volume. Apple juice production
would be increased, whether it was in concentration or in volume. In accordance with the alternate
hypothesis, the expected outcome was that as the concentration of pectinase increases, the juice
produced will be more concentrated and this will be evident by greater intensity of colour. When
measured in the spectrophotometer, there will be positive increase of colour from the clarity of
distilled water.

In the spectrophotometer results, the colour change of the juice increased exponentially as the
concentration of pectinase increased. There is a positive correlation considering the upward trend.
In terms of error, there is an acceptable level of error going 0.2 plus or minus. There is not much
uncertainty in the set of results.

The alternate hypothesis was not supported by the results. In effect, the concentration of pectinase
had varying effects on the results (Graph 1). At zero pectinase, higher results were obtained than
that of 5% and 25%. There is no genuine trend because there is no coherency with the alternate
hypothesis; a bell-shaped curve was obtained whereas a positive gradient was the expected
outcome. This can be seen at the 15-minute marker where the juice production levels increased
(Graph 1), peaked, and then decreased as the concentration increased: 4.34, 5.08, 5.52, 5.40, and
4.84. However all results were similar, barely a millimetre in between each so there may not have
been as much difference if a larger volume of juice was used to display the relationship. This can be
seen by range of overall juice levels at 15 mins (termination of the experiment) was just 1.18ml. In
addition, the alternate hypothesis (H1) is similarly not supported by the ANOVA Test results (Table 8)
which differed by just 0.5.

However, when looking at the spectrophotometer results and its relationship with the alternate
hypothesis (H1) the conclusion made differs. The predicted outcome of the spectrophotometer is
supported by the results; as the concentration of pectinase increases, as does the colour change.
This can be seen from the positive gradient of the column chart indicating the positive correlation
between the independent and dependant variable as predicted (Graph 2). Numerical evidence

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

supports also (Table 6): 0.28, 0.79, 1.21, 1.39, 1.65, and 1.84. They increase exponentially. The
ANOVA Test for the spectrophotometer (Table 9) also supported the alternate hypothesis which had
a high difference between the Sig value and the F value of 590.6.

In application to real life, pectinase appears to be used more for the concentrate than a higher juice
quantity production. Considering the colour change, this indicates that there is higher concentration
of apple juice in the quantity which could be used for cordials and dilute squash drink productions.
As a colour enhancer, pectinase does a good job of intensifying the colour of the juice when more is
enzyme added. It fails however in collecting a lot of juice from a substrate.

The alternate hypothesis (H1): increasing the concentration of pectinase, in grams, in a 50ml solution
will increase juice production, in millilitres, of 5g of apple when reacted with the solution and
filtered; was partially supported.

Evaluation
The results for the juice production volume are not reliable because they do not follow a clear
pattern. The juice production is higher at 0% pectinase than 5% and 10%, and then increases beyond
the value for 0% at 15%, after which it decreases as the concentration increases. This is not a normal
pattern for an enzyme because the power of an enzyme should increase as concentration increases.

In terms of outliers, there were no distinct ones. The results varied so much. For example, at 5
minutes for 15% pectinase, the reading for A was 1.60ml, B was 3.20, C was 6.60, D was 6.20 and E
was 6.00. A and B were similar and C, D and E were also similar. Therefore, it became difficult to
distinguish which was the representative. Discarding one set could stray away from the perhaps real
result. In relation to 5 minutes for 15% pectinase, it could be that there was a higher proportion of
the pectinase in those three beakers than the other two because of the delay between adding the
pectinase and to the measuring cylinders causing the first two to get more of a diluted solution. It
was for this reason all the results were included in for data processing. In the trial of the removal of
these data the results were even less similar to the pattern causing a more pronounced bell-shaped
curve. The bell-shaped curve does not make sense because there is no scientific factor that should
make juice production less at 25% than at 15%. Increase in concentration should produce a steep
positive gradient and then level off due to quantity of the apple cell substance or percentage of juice
in the apple cells being a limiting factor. In addition, the ANOVA Test (Table 8) determined that
there was very low accuracy in the results and that their conclusions are most likely wrong.

In a collaborative scientific study by Osaka Prefecture University in Japan and the University of Ghent
in Belgium5 it was found that an increase in pectic enzymes increased fruit juice extraction of various
fruits. This is in accordance with the spectrophotometer results and supports the alternate
hypothesis and refutes the null hypothesis.

In the spectrophotometer results, a clear pattern arose which showed a pattern in accordance with
the alternate hypothesis. The ANOVA Test results (Table 9) showed a high degree of accuracy, which
help to support the accuracy of the experiment and validity of the data. However, these results may
be unreliable. The possible difference in the colour of the juice produced could be entirely due to the

5
(Sakai, Sakamoto, Hallaert, & Vandamme, 1993)

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

increase in concentration of the pectinase enzyme, which was a red colour, which therefore results
in a more intense colouring, not necessarily more concentrated juice. These results are highly
supportive but maybe entirely inaccurate.

In the experiment there were a number of limitations and errors. One such limitation was the
inability for the incubator to go above 40. This unfortunately did not show the how well the
pectinase would have worked at its optimum temperature; its optimum being 45-50. Due to the
quantity of the samples and the time constraints, it was not possible to prepare a water bath of the
appropriate temperature, otherwise that would have been the improvement. Similarly, the pH
condition of the apple pulp could have been measured using Universal Indicator. It would help to
determine whether to make the mixture of apple pulp and pectinase solution more alkaline or acidic
as the optimum pH for pectinase is 3.5. This may have given more realistic results.

Another limitation was the miscibility of the pectinase solution. The solution kept separating despite
attempts to re-mix it before addition to the apple pulp. There were large particles of pectinase
enzyme powder. This gave inaccuracy and furthermore uncertainty as to whether the true reflection
of the effect of that particular concentration was seen successfully. Furthermore, each of the 5
samples per concentration would have differing amounts of the solute; the sample that received the
10ml measuring cylinder that had had the last amount of solution from the beaker will have had
more pectinase, due to the sinking of the large particles, in comparison to the rest. This means that
from the first cylinder to the last, the pectinase concentration would have in fact decreased
exponentially. One solution is that the large particles could be ground into a fine powder before
mixing to increase the surface area for dissolving. In addition, the water could have been heated to
40 in order to increase miscibility. Similarly, an emulsifier could have been used to help the
pectinase mix better with the water.

There were some issues with the solutions; the higher concentrations of pectinase solutions frothed
a lot, causing thickness. It was often not possible to pour such thick foam and completely remove
the pectinase trapped within. The foam blurred the meniscus and reduced accuracy. Perhaps
warmer water, or an emulsifier, would have assisted in a more miscible solution. If the miscibility
cannot be improved, then reading the result from a set level can be used as the method; for
example from the topmost point of the foam.

The filter paper used was a handmade variety and was rather thick. Thus substantial amounts of
apple juice may have been soaked up by filter paper and confounded the result. Changing the filter
paper brand from handmade to a thinner type would greatly improve error.

There was also difficulty reading the level of juice produced. This was entirely due to the small
amount of liquid used in the experiment. There was not enough to liquid to compare significant
changes. It would be prudent to use increase the scale of the experiment to gain better scope.
Alternatively, a photograph can be taken using a digital camera or a smartphone at each interval,
and the data recorded after the experiment has completed.

A final improvement could have been the use of a buffer solution to control pH levels. The pH was
not controlled and the optimum was not achieved. The use of water, which was of an untested pH,
could have altered the pH in the mixture of apple pulp and pectinase; a buffer solution would have
supervised the reaction and kept it at a constant pH.

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Dhanish Bachheta ISH Pectinase Lab IB Biology Internal Assessment

Bar and column graphs were used because they allowed clear presentation of distinct categories.
Furthermore, the values are easier to read allowing a correlation to be established from viewing. The
data is also possible to read. Proportions could be presented of each time interval of the overall juice
production using the bar chart. In addition, error bars provide the viewer a tangible insight to the
accuracy of the results when determining a conclusion.

This experiment generates many branches of curiosity; it gives one the basic understanding of
pectinase to undertake further challenges. Pectinase is a group of pectic enzymes, it would be
interesting to see how the effect would be seen if the enzymes were separated, tested and
compared. This would provide insight into which enzyme is the best for juice extraction, in terms of
specification and efficiency. That would be useful in industry to enhance products and profit,
resulting in more successful company. In relation to this experiment, it would be interesting to see if
different apple breeds all produced similar types of juice if using the same enzyme; this would help
to distinguish which apples have the juice with the best flavours and quantities.

Bibliography
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Using-1119912.html

Ceci, L., & Lozano, J. (1996). Determination of enzymatic activities of commercial pectinases for the
clarification of apple juice. Planta Piloto Ingeniera de Quimica, Bah a Blanca. Retrieved from
http://www.sciencedirect.com/science/article/pii/S0308814697000885

Priya, V., & Sashi, V. (2014, March). Pectinase enzyme producing Microorganisms. P.S.G.R.
Krishnammal College for Women, Botany. Coimbatore: International Journal of Scientific and
Research Publications. Retrieved November 2014, from http://www.ijsrp.org/research-
paper-0314/ijsrp-p2758.pdf

Sakai, T., Sakamoto, T., Hallaert, J., & Vandamme, E. J. (1993). Pectin, Pectinase, and Protopectinase:
Production, Properties, and Applications. Osaka Prefecture University, Department of
Agricultural Chemistry; Biochemical and Microbial technology. Osaka: Elsevier Inc. Retrieved
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