Word Count: 1500

Assessment Type 1: Investigations Folio

STAGE 2 BIOLOGY

Aim: To determine the optimum and

most cost-effective pectinase
concentration for juice extraction by
investigating the effect of pectinase
concentration ( % ) on the rate of
−1
decrease in apple mass (% ∙ min ).

SACE REGISTRATION NUMBER – 203988H

 SACE REGISTRATION NUMBER – 203988H
STAGE 2 BIOLOGY
Table of Contents
Introduction............................................................................................................................................2
Background Information .................................................................................................................................. 2
Pectin .......................................................................................................................................................................................... 2
Pectinase ..................................................................................................................................................................................... 2
Enzyme Activity ........................................................................................................................................................................... 3
Commercial Pectinase Applications ............................................................................................................................................ 4
Hypothesis....................................................................................................................................................... 4
Variables ......................................................................................................................................................... 5
Independent................................................................................................................................................................................ 5
Dependent .................................................................................................................................................................................. 5
Controlled ................................................................................................................................................................................... 5
Uncontrolled ............................................................................................................................................................................... 5

Experimental Design ...............................................................................................................................6

Materials/apparatus ........................................................................................................................................ 6
Method ........................................................................................................................................................... 7
Preparation ................................................................................................................................................................................. 7
Procedure .................................................................................................................................................................................... 7
Safety/ethics ................................................................................................................................................... 8
Qualitative Observations ........................................................................................................................8
Quantitative Data...................................................................................................................................9
Sample Calculations ......................................................................................................................................... 9
Mean Initial/final Apple Mass (g) ............................................................................................................................................... 9
Mean Decrease in Apple Mass After Ten Minutes (g) ................................................................................................................ 9
Mean Percentage Decrease in Apple Mass After Ten Minutes (%) ........................................................................................... 9
Mean Rate of Decrease in Apple Mass (pectin depolymerisation rate) (% ∙ min−1) .............................................................. 10
Range of Mean Rate of Decrease in Apple Mass (% ∙ min−1) .................................................................................................. 10
Experimental Uncertainty in Mean (%) .................................................................................................................................... 10
Chordal Gradient Between Two Consecutive Pectinase Concentrations (% ∙ min−1 ∙ %−1 ) .................................................. 11
Tabular Representation .................................................................................................................................. 12
Graphical Representation ............................................................................................................................... 13
Analysis of Results ................................................................................................................................ 13
Comparison With Hypothesis ......................................................................................................................... 13
Biological Justification .................................................................................................................................... 13
Qualitative Observations vs. Quantitative Data .............................................................................................. 14
Evaluation of Experimental Design ........................................................................................................ 14
Random Error ................................................................................................................................................ 14
Systematic Error ............................................................................................................................................ 14
Comment on Reliability, Validity, Precision, and Accuracy ............................................................................... 15
Conclusion ............................................................................................................................................ 15
References ............................................................................................................................................ 16

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Introduction
Background Information
Pectin
Pectin is a heteropolysaccharide located in the middle lamella of the cell walls in terrestrial plants (see Figure
1.1) (Shoemaker, 2019). The substrate aids greatly in cellular binding, and thus play a significant role in
maintaining the cell wall’s structural integrity (The Editors of Encyclopaedia Britannica, 2023).

Figure 1.1: Diagram illustrating a section of the cell wall in a plant cell. (Villarreal, 2007)

Pectinase
Pectin depolymerisation is catalysed by the enzymatic biological protein pectinase, which exists as a natural
ripening agent within plants, fungi, and bacteria (puracy, 2020) (Martos et al., 2009). Pectinase degrades
pectin into simple monosaccharide sugars i.e., galacturonic acid (see Figure 1.2) when covalent glycosidic
bonds that holds pectin molecules together are broken (Singh, Singh and Pandey, 2019). This allows for the
release of the cell’s intracellular vacuole and cytoplasmic contents, known as ‘juice’ (Singh Jadaun, 2018).

Glycosidic
bonds

Galacturonic
acid monomer

Figure 1.2: Diagram illustrating the galacturonic acid monomers within pectin held together by glycosidic bonds. (Edgar181, 2006;
Health Jade Team, 2018)
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Enzyme Activity
The enzyme-substrate binding between pectinase and pectin is correspondent with the induced fit model
(see Figure 1.3). However, due to their non-complimentary nature, the pectinase active site must undergo a
conformational change in shape to induce a tighter and more specific fit with pectin (Khan, 2015).

This allows pressure to be exerted on pectin, providing an alternate metabolic pathway of decreased
activation energy for the breakdown of pectin to be catalysed (see Figure 1.4) (Byjus, 2020).

Pectin
Other
(substrate) Galacturonic
products
acid monomer
Pectinase
Pectinase Pectin
(enzyme)
active site Conformational depolymerises
change in shape

Imperfect fit,
not exactly complimentary
Figure 1.3: Diagram illustrating the reaction between the enzyme pectinase and the substrate pectin as per the induced-fit model
of enzyme-substrate binding. (The Science Hive, 2021)

Factors that affect the rate of enzyme-catalysed reactions includes the reactant’s pH, concentration, and
temperature (AP Biology, 2023). By increasing pectinase concentration, the availability of active sites for
enzyme-substrate complexes to form would also increase, leading to an increase in depolymerisation rate.
However, as substrate concentration becomes a limiting factor (all pectin molecules has been
depolymerised), a saturation point is reached and the reaction plateaus to a constant (see Figure 1.5).

Point of saturation

Enzyme Concentration

Figure 1.4: Graphical representation of the differences in Figure 1.5: Graphical representation of the point of saturation on the
activation energy of metabolic reactions catalysed and relationship between enzyme concentration and the rate of reaction
uncatalyzed by enzyme. (NAU, 2019) over time. (Nagwa, 2023)

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Commercial Pectinase Applications
In addition to extracting fruit juice, cloudiness caused by the heterogenous mixture of juice and pulp can be
clarified by further breaking down the pectin within the pulp, drastically increasing both visual and textural
appeal (Sakai et al., 1993). Hence, its significance in the juice industry cannot be understated.

However, pectinase solutions at increasing concentrations would require increasing costs and effort to
produce, rendering the saturation point indicated in Figure 1.5 as the optimum and most cost-effective
pectinase concentration for maximising juice yield.

Hypothesis
If pectinase concentration (%) is increased, then the rate of decrease in apple mass (% ∙ min−1) will also
increase at a constant rate initially, but eventually plateau once the saturation point is reached.

This increase is linear initially as active site availability increases, allowing for more enzyme-substrate
complexes to form, thus catalysing reaction. However, the plateau occurs due to the finite quantity of pectin
molecules, where a saturation point is reached once the substrate concentration becomes a limiting factor,
as demonstrated in Figure 1.5.

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Variables
Independent
Table 1.1: Independent Variable
Variable Method of variation
Pectinase solution Measurements were taken from solutions of 0.5, 1, 1.5, 2 and 4% pectinase
concentration (%) concentration.

Dependent
Table 1.2: Dependent Variable
Variable Method of measurement
Rate of decrease in The initial and final apple mass was measured over a ten-minute period, and its
apple mass (% ∙ min−𝟏 ) percentage decrease per minute was calculated.

Controlled
Table 1.3: Controlled Variables
Variable Method of control Significance
Apple surface All apple slices were Apple surface area directly distorts the availability of surface
area produced using the same pectinase active sites for pectin molecules to collide and
mandolin to ensure bind for enzyme-substrate complex formations, affecting
uniform thickness. The depolymerisation rate.
same cookie-cutter was
used to ensure equal
radius in each slice.
Pectinase 4 mL of pectinase solution The quantity of active sites is also dictated by the volume of
volume were used across all trials. equal-concentration pectinase solution, where
increasing/decreasing volumes would introduce more/fewer
active sites within reactions respectively, affecting
depolymerisation rate.
Reactant Reactants were incubated Depolymerisation rate is directly reduced at both high and
temperature to 40℃ both prior to and low temperatures. High temperatures cause proteins such as
during the conduction of pectinase to lose its three-dimensional structure due to
this experiment, where all thermal denaturing, hindering its catalytic function. Low
reactions are undertaken temperatures result in the less molecules possessing enough
within this incubator. kinetic energy to react, altering depolymerisation rate.

Uncontrolled
Table 1.4: Uncontrolled Variable
Variable Reason for uncontrollability
pH of The acidity or alkalinity of pectinase is solely dependent on its source; therefore, solution
pectinase concentrations would also have different pH values. Varying pH values would affect
solution enzyme activity in enzyme-catalysed reactions such as depolymerisation. The optimum pH
of pectinase obtained from Aspergillus niger is 5.0 (Khatri et al., 2015), where its
performance is maximised.

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Experimental Design
Materials/apparatus

Electronic
Silicon ice-
weighing balance
Mandolin Beaker cube tray
(50 mL) Pipette
pump

Permanent marker Plastic forceps

Circular
cutting tool
Graduated
pipette (50 mL)
Handguard
Granny Smith
apple
Heavy duty gloves

Figure 2.1: Photograph of the materials/apparatus laid-out on a laboratory bench excluding the pectinase solutions as they are
stored in the incubator to maintain their temperature. (By author, 2023)

Table 2.1: Chemical and Materials/apparatus

Chemical Volume (mL) Material/apparatus Quantity (#)
Pectinase solutions (0.5, 20 Beaker (50 mL) 1
1, 1.5, 2, and 4% Circular cutting tool 1
concentration)
Cling wrap As required
Electronic weighing balance 1
Tap water (control) 20 Graduated pipette (10 mL) 1
Granny Smith apple 3
Heavy-duty gloves 1
Ice-cube tray (6 slots) 6
Tap water (for rinsing) As required Incubator (set to 40℃) 1
Mandolin with handguard 1
Paper towel As required
Pipette pump 1
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Method
Preparation
Step 1. The incubator was preheated to 40℃.
Step 2. One ice cube tray was labelled as the control (0%), and the rest were labelled with one of the five
different concentration levels (0.5, 1, 1.5, 2, 4%). Each compartment was also labelled with numbers 1 to 6.

Procedure
𝟎. 𝟓% Pectinase Concentration
Step 3. The mandolin tool with its handguard were used to
cut the apple into slices of approximately 3 mm thickness.
Step 4. The circular cutting tool was used to cut out one
circular disks from one piece of apple slice.
Step 5. The electronic weighing balance was tared before
the mass of the circular disk was weighed and recorded
(initial apple mass).
Step 6. The apple slices were immediately placed into a
compartment of the 0.5% pectinase concentration ice-
cube tray (Figure 2.2).
Step 7. Procedural steps 3 to 6 were repeated six more
times to fill the rest of the compartments and produce Figure 2.2: Photograph of apple disks placed in each
more trials. compartment of the ice-cube tray. (By author, 2023)
Step 8. The graduated pipette was attached to the pipette
pump and rinsed with tap water.
Step 9. The 50 mL beaker was filled with 0.5% pectinase
concentration solution.
Step 10. 4 mL of 0.5% pectinase concentration solution
from the beaker was collected and transferred to each
compartment in the chronological order of which each
apple disk was placed inside.
Step 11. Cling wrap was laid over the ice cube tray to cover
all compartments.
Step 12. The completed reaction chamber is transported
to the incubator, where a ten-minute timer is initiated as
the incubator door is closed (Figure 2.3).
Figure 2.3: Photograph of the ice-cube trays being
Step 13. The ice cube tray was removed from the incubator placed inside the incubator. (By author, 2023)
after ten minutes and transported back to the laboratory
bench.
Step 14. The mass of each apple disc was measured and recorded using the same electronic weighing balance
(final apple mass).

𝟎*, 𝟎. 𝟓, 𝟏, 𝟏. 𝟓, 𝟐, and 𝟒% Pectinase Concentrations

Step 15. Procedural steps 3 to 14 were repeated five more times using the 0, 0.5, 1, 1.5, 2, and 4%
pectinase concentration solutions.

*Tap water was implemented to demonstrate that the apple’s submergence under aqueous solutions does
not attribute to the weight loss experienced by the apple. Thus, 0% pectinase concentration was merely
used as a control and not a legitimate “pectinase concentration” to be measured.
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Safety/ethics
Table 2.2: Safety Considerations
Consideration Risk Precaution
Glass apparatus If shattered can produce broken General caution was taken when handling
glass pieces with sharp edges glassware, and closed footwear was worn at
that can cause cuts and injuries. all times. All equipment (including glassware)
were placed at the centre of the laboratory
bench to minimise the risk of cylindrical
glassware falling off (see Figure 2.1)
Mandolin cutting tool Misuse and accidental slips can Gloves and handguards were used at all times
cause mild to severe cuts on to minimise the risk of accidental slips and
finger that may lead to bleeding injuries. Apples were also disposed of once
and possible infections. the core is reached to minimise the distance
between hand and blade.
Pectinase solution Inhalation and physical contact All group members equipped personal
with pectinase solutions protective equipment (PPE) prior to and
(especially at higher throughout the practical. Laboratory coats
concentrations) can cause mild were worn to prevent contact with the
irritation and/or allergic pectinase solution, safety glasses were worn
reactions. to prevent accidental splashes from coming
into contact with eyes, and facemasks were
worn to reduce inhalation of pectinase. All
measurements of pectinase solutions were
also conducted below eye level.

Table 2.3: Ethical Considerations

Consideration Resolution
Data/result integrity All data collected from this experiment have been honestly recorded without the
fabrication of any result.
Information/source All information collected from their relative sources has been appropriately
acknowledgement acknowledge using in-text Harvard referencing.
Source of pectinase All pectinase used throughout the experiment was sourced ethically from the
fungus Aspergillus niger.
Wastage and disposal Whole apples and pectinase solution were used sparingly under the condition
of used materials that safety and result quality are maintained. All used pectinase solution and
apple slices were disposed of ethically in a compost bin.

Qualitative Observations
The trials conducted using both 2 and 4% pectinase concentrations have left substantial residual fluid within
each compartment after ten minutes inside the incubator. Contrastingly, the other previous pectinase
concentration solutions (0.5, 1, and 1.5%) has remained significantly less residual fluid over ten minutes,
suggesting that less mass has been lost from the apple disc.

As the experiment progressed and the concentration of pectinase used increased, the circular apple discs
became increasingly fragile, signifying degradation. Marginal discolouration was also observed, indicating
potential polyphenol oxidation from the apples.

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Quantitative Data
Sample Calculations
Although only the initial and final masses of the apple discs were recorded throughout the experiment, this
data can be used to calculate more useful values to assist with visualisation.

Mean Initial/final Apple Mass (g)

Since the initial/final apple mass (g) is the dependent variable being measured, its mean over the six trials
conducted for each concentration can be evaluated to increase the reliability of results*. This can be given
by the arithmetic mean formula:
𝑛
1
𝐴 = ∑ 𝑎𝑖
𝑛
𝑖=1
where 𝐴 = mean apple mass,
𝑛 = number of trials,
and 𝑎𝑖 = trial values.

i.e.: the mean initial apple mass (g) at 0.5% pectinase concentration over the six trials can be given by:
1 6.54
𝐴initial = (1.15 + 1.18 + 0.900 + 1.14 + 1.13 + 1.04) = = 1.09 g
(6) 6

i.e.: the mean final apple mass (g) at 0.5% pectinase concentration over the six trials can be given by:
1 5.84 73
𝐴final = (1.05 + 1.06 + 0.790 + 1.00 + 1.01 + 0.93) = = ≈ 0.97 g
(6) 6 75

*Due to some inexact rounded values, any further calculations involving the mean initial/final apple mass
should only use the exact values of the mean when necessary.

It was observed (excluding the control group which is not officially measured) that all apple discs exposed to
pectinase undergone a loss in mass. Therefore, for the sakes of simplicity, the decrease in mass is used for
further calculations instead of its additive inverse, difference in mass.

Mean Decrease in Apple Mass After Ten Minutes (g)

The volume of apple juice extracted per unit time is directly proportional to the decrease in apple mass.
Hence, the decrease in apple mass (g) can be calculated by subtracting the initial apple mass (g) from the
final apple mass (g), such that:
decrease in apple mass (g) = initial apple mass (g) − final apple mass (g)

i.e.: the mean decrease in apple mass (g) at 0.5% pectinase concentration can be given by:
5.84 7
mean decrease in apple mass = 1.09 − = ≈ 0.117 g
6 60

Mean Percentage Decrease in Apple Mass After Ten Minutes (%)

Due to the varying conditions of each apple disc (pectin concentration, size/mass etc.), the percentage
decrease in apple mass after ten minutes (%) is more reliable and representative of the rate of pectin
depolymerisation and juice extraction. This can be evaluated through the percentage decrease formula:
initial mass (g) − final mass (g)
percentage decrease in apple mass (%) = × 100
initial mass (g)
decrease in apple mass (g)
percentage decrease in apple mass (%) = × 100
initial mass (g)
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i.e.: the mean percentage decrease in apple mass (g) at 0.5% pectinase concentration after ten minutes can
be given by:
7
(60)
mean percentage decrease in apple mass after ten minutes = × 100
(1.09)
3500
mean percentage decrease in apple mass after ten minutes = ≈ 10.7%
327

Mean Rate of Decrease in Apple Mass (pectin depolymerisation rate) (% ∙ min−1 )

The percentage decrease in apple mass after ten minutes can be divided by the reaction duration, resulting
in the rate of decrease in apple mass (% ∙ min−1 ), which is also illustrative of pectin depolymerisation rate.
Hence:
percentage decrease in apple mass (%)
rate of decrease in apple mass (% ∙ min−1 ) =
reaction duration (min)

i.e.: the mean rate of percentage decrease in apple mass (% ∙ min−1) at 0.500% pectinase concentration
can be given by:
3500
( 327 )
mean rate of decrease in apple mass =
(10)
350
mean rate of decrease in apple mass = ≈ 1.07% ∙ min−1
327

Range of Mean Rate of Decrease in Apple Mass (% ∙ min−1)

The range of a set of data can be calculated to determine the severity of the dataset’s spread. The can be
calculated by subtracting the lowest value from the highest value of the six trials for each pectinase
concentration. Hence, this can be expressed as:
range of rate of decrease in apple mass (% ∙ min−1 ) = highest value − lowest value

i.e.: the range of the mean rate of decrease in apple mass over the six trials conducted for 0.5% pectinase
concentration can be given by:
range of mean rate of decrease in apple mass = 12.3 − 8.7
range of mean rate of decrease in apple mass = 3.6% ∙ min−1

Experimental Uncertainty in Mean (%)

The values of experimental uncertainty in mean indicates the dispersion of the data and the range of which
the true value of the mean rate of decrease in apple mass may lie for each pectinase concentration, allowing
for the reliability of the mean to be assessed.

Uncertainty values less than 1% are by convention corrected to one significant figure. Furthermore, the
number of significant figures that the mean value should be rounded to is also dependent on its experimental
uncertainty (Gaines, 2023). Thus, in the scope of this experiment, mean values are rounded to three
significant figures by convention. Hence, the experimental uncertainty in mean can be given by:
range of data (% ∙ min−1 )
uncertainty (%) =
number of trials

i.e.: the experimental uncertainty in mean for 0.5% pectinase concentration can be given by:
3.6
uncertainty = = 0.6%
6

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Chordal Gradient Between Two Consecutive Pectinase Concentrations (% ∙ min−1 ∙ %−1)
Due to the graph’s plateauing nature, the gradient of increasing pectinase concentrations is deduced to
decrease. While this decrease in slope is quantifiable by evaluating the instantaneous rate of change using
calculus, simply calculating the chordal gradient between two consecutive pectinase concentrations is
sufficient to demonstrate the existence of the plateau. Thus, this can be calculated via the slope formula:
𝑦2 − 𝑦1
𝑚=
𝑥2 − 𝑥1

where 𝑚 is the chordal gradient between two consecutive pectinase concentrations,

𝑥1 and 𝑦1 is the lower pectinase concentration and rate of decrease in apple mass respectively,
and 𝑥2 and 𝑦2 is the higher pectinase concentration and rate of decrease in apple mass respectively.

Hence, the unit for the chordal gradient of mean rate of decrease in apple mass between two consecutive
pectinase concentrations can be evaluated similarly, such that:
unit 𝑦 % ∙ min−1
unit 𝑚 = =
unit 𝑥 %

Although the % unit appears to cancel out, they are still distinct in nature, representing different entity of
values, with one being percentage change in mass and the other being pectinase concentration.

Since the two units cannot be treated equivalently, the units for the gradient of a chord between two
consecutive pectinase concentrations is derived to be:
unit 𝑚 = % ∙ min−1 ∙ %−1
𝑦2 − 𝑦1
∴ 𝑚 (% ∙ min−1 ∙ %−1 ) =
𝑥2 − 𝑥1

i.e.: the gradient of the chord between 0.5 and 1% pectinase concentrations (% ∙ min−1 ∙ %−1 ) can be given
by:
10.76 6.46
( 6 )−( 6 )
𝑚=
(1) − (0.5)
10.76 6.46
( 6 )−( 6 )
𝑚=
(1) − (0.5)
4.3
𝑚= ≈ 1.43% ∙ min−1 ∙ %−1
3

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Tabular Representation
Table 4.1: Data table displaying pectinase concentration (%); trial number (#); initial and final apple
mass (g); individual and mean rate of decrease in apple mass (% ∙ min−𝟏 ), all corrected to their
respective conventional significant figures (Kirkup, 1996).
Rate of Decrease Mean Rate of Decrease
Pectinase Initial Final Mean
Trial in Apple Mass in Apple Mass
concentration Apple Apple Uncertainty
(#) (depolymerisation (depolymerisation rate)
(%) Mass (g) Mass (g) (%)
rate) (% ∙ min−𝟏) (% ∙ min−𝟏)
1 1.15 1.05 0.87
2 1.18 1.06 1.02
3 0.90 0.79 1.22
0.5% 1.07 ±0.6
4 1.14 1.00 1.23
5 1.13 1.01 1.06
6 1.04 0.93 1.06
1 1.08 0.88 1.85
2 1.23 1.03 1.63
3 1.05 0.87 1.71
1% 1.79 ±0.5
4 1.14 0.93 1.84
5 1.03 0.83 1.94
6 1.17 0.96 1.79
1 0.95 0.74 2.21
2 1.23 0.94 2.36
3 1.00 0.78 2.20
1.5% 2.32 ±0.4
4 1.04 0.79 2.40
5 1.00 0.77 2.30
6 1.12 0.85 2.41
1 0.89 0.67 2.47
2 1.04 0.78 2.50
3 0.87 0.64 2.64
2% 2.52 ±0.5
4 1.19 0.87 2.69
5 1.07 0.81 2.43
6 1.17 0.89 2.39
1 1.13 0.83 2.65
2 1.11 0.81 2.70
3 1.09 0.82 2.48
4% 2.58 ±0.4
4 0.94 0.71 2.45
5 1.17 0.87 2.56
6 0.99 0.73 2.63

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Graphical Representation
The direct relationship between pectinase concentration and mean rate of decrease in apple mass
(depolymerisation rate) can also be graphically visualised.

The Effect of Pectinase Concentration (%) on the Mean Rate of Decrease

in Apple Mass (%∙min-1) During Pectin Depolymerisation
3.00
Mean Rate of Decrease in Aapple Mass (%∙min-1)

2.50

2.00

1.50

1.00

0.50

0.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
Pectinase Concentration (%)

Figure 4.1: Graph illustrating the effect of pectinase concentration (%) on the mean rate of decrease in apple mass (% ∙ min−1 )
across the five pectinase concentrations experimented.

Analysis of Results
Comparison With Hypothesis
Figure 4.1 illustrates that as pectinase concentration increases, the mean rate of decrease in apple mass
(depolymerisation rate) also increases, establishing an initial linearised relationship. The largest and smallest
value of pectinase concentration explored is also respectively equivalent to the largest and smallest value of
the depolymerisation rate obtained.

The applied trendline appears to plateau at higher pectinase concentrations, losing its initial linear
relationship. This can also be identified through the decrease in chordal gradient (1.43% ∙ min−1 ∙ %−1
between 0.5 and 1% vs. 0.03 between 2 and 4% pectinase concentrations).

Thus, the shape and nature of the graph (initial linear relationship followed by a subsequent plateau)
perfectly corresponds with the expected theoretical relationship modelled in Figure 1.5, supporting the
hypothesis.

Biological Justification
Increasing pectinase concentration would increase availability of active sites for pectin to bind to when
volume of pectinase is kept constant. This also increases enzyme-substrate complex formation rate, and thus
pectin depolymerisation rate. A plateau is approached as pectin availability becomes a limiting factor at
higher concentrations, and hence can be biologically justified.

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Qualitative Observations vs. Quantitative Data
The quantitative data is supported by qualitative observations, in which volume of residual fluid at greater
enzyme concentrations with greater depolymerisation rate is also observed to be higher.

Evaluation of Experimental Design

Random Error
Random errors are unpredictable experimental errors that introduces scatter and are directly affected by
the precision of measurements taken during the experiment, reducing result reliability.
Table 5.1: Elaboration of random errors with their respective mitigation methods.
Error Elaboration Mitigation Method
Heterogeneous Pectinase distributions in heterogeneous solutions are Homogenising
Solution inconsistent, leading to varying pectinase concentrations in solutions with
different areas of the solution. Thus, independent trials may magnetic stirrer.
yield altering quantities of active sites in different trials,
affecting depolymerisation rate, reducing precision.
Meniscus Incorrect identification of the meniscus introduces varying Utilising a stationary
Misidentification angular differences in where measurements were taken from. observer (camera)
This causes inconsistent pectinase volumes used and thus and placing light
active site availability for each trial fluctuating absorbing materials
depolymerisation rate, diminishing precision. (black paper) behind
meniscus.
Temperature Due to the repeated opening and closing of the incubator door, Ensuring incubator
Fluctuations probability fluctuations from the assumed temperature of 40℃ remains sealed for
increased. Had this fluctuation occurred, kinetic energy the duration of the
possessed by reactant molecules would also alter and thus experiment.
affect rate of reaction, reducing precision.

Systematic Error
Systematic errors are consistent or proportional distortions in results between the observed and true value
of a variable being examined that cannot be mitigated through trial repetitions, diminishing data validity.
Table 5.2: Elaboration of systematic errors with their respective mitigation methods.
Error Elaboration Mitigation Method
Electronic Poor electronic balance calibration may display inaccurate Checking readings
Balance readings of apple mass, altering all data collected by a on balance against
consistent factor, reducing accuracy and thus validity of results. known-mass objects
(calibration weights)
for inaccuracies.
Solution Inaccurate pectinase concentrations differing from their Repeating trials with
Preparation assumed values would alter pectinase availability for enzyme- different solutions of
substrate complex formations, and thus depolymerisation rate. ‘equal’
This would manipulate all trials involved by a consistent factor, concentration for
affecting accuracy and thus validity of data. comparison.

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Comment on Reliability, Validity, Precision, and Accuracy
Table 5.3: Comments on Reliability, Validity, Precision, and Accuracy
Categories Comments
Reliability • The mean of six individual trials for each pectinase concentration was evaluated to
increase reliability.
Validity • The relationship between the dependent and independent variable was successfully
established, significantly increasing construct validity.
• The implementation of a control to ensure fluid loss was not a result water present due
to osmosis also increases validity.
Precision • The results remained highly precise, as evident in the marginal ±0.6% and lower
uncertainties.
Accuracy • Due to the absence of the true values, the experiment’s accuracy cannot be determined.
• Figure 3.1’s geometric similarity with the hypothesised relationship modelled in Figure
1.5 suggests that a degree of accuracy was present within the data.

Conclusion
The effect of pectinase concentration on the enzymatic rate of pectin depolymerisation has been confirmed
by submerging apple discs into solutions of varying pectinase concentrations (%) and calculating the rate of
decrease in apple mass (% ∙ min−1). The hypothesised trend in Figure 1.5 is supported by the highly precise
and therefore reliable graphical representation of the experimental results indicated by the minimal
uncertainty across all means. Though the marginal rate of decrease in apple mass between 2 and 4%
pectinase concentration indicates that the optimal concentration had most likely been reached, further
trialling of intermediate pectinase concentration is required should the exact value of the optimum pectinase
concentration for juice extraction wish to be obtained. However, the data and results yielded by this practical
report does indicate that any pectinase concentrations around the 2% region would be plentiful for juice
extraction, resolving the aim.

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