The Effect of 

Pectinase on the 
Production of 
Apple Juice 
 
 
 
 
 
 
SACE Registration

7 5 5 7 3 5 X 
Word Count

1492 
Introduction 
Proteins  known  as  enzymes  catalyse  reactions  allowing  host  cells  to  sustain  life.  These  can  be 
extracted  or  made  artificially  to  benefit  industrial  practices,  improving  efficiency.  Enzymes  are 
macromolecules  ​(large  molecules)  consisting  of  ​amino  acids​.  These  contain  ​polypeptide  chains​, 
which  are  amino  acid  ​monomers  held  together  by  ​peptide  bonds​.  The  sequence  of  these  amino 
acid  chains  form  the  primary  structure-  one  of  the  determining  factors  for  the  function  of  a  protein. 
Hydrogen  ​and  disulphide  bonds  further  influence  the  shape,  known  as  ​secondary  and  ​tertiary 
structures​,  respectively.  The  ​quaternary  structure  is  characterised  as  a conglomeration of multiple 
polypeptide  chains  such  as  haemoglobin,  which  contains  four  polypeptide  chains.  Once  the 
structure  of  a  protein  is  determined,  there  is  an  ​active  site  which  is  the  location  where  ​substrates 
can bind to, creating an ​enzyme-substrate complex ​(Lecornu, B & Diercks, T 2017).  
Pectin  is an enzyme used in commercial apple juice factories to break down polysaccharides (Tapre 
&  Jain,  2014).  These  are  carbohydrates  within  the  cell  walls  of  fruit  and  consist  of  monosaccharide 
units-  most  commonly  starch.  Pectinase  increases the yield of apple juice by lowering the activation 
energy  required  for  these  polysaccharides  to  be  broken  down.  Like  all  enzymes,  pectinase  has  an 
active  site  which  binds  to  the  polysaccharide  molecules’  ​substrate  to  form  an  enzyme-substrate 
complex​.  Since  more  active  sites  increase  the  capacity to catalyse reactions, more volume of apple 
juice will be produced in a shorter amount of time.  
The  aim  of  this  investigation  is  to  find a relationship between the concentration of enzymes and the 
volume of apple juice produced as a result. 
 
Deconstruction of the issue 
 
Factors  Aspects of the Question 

Types of enzymes  Types of apples  Enzyme  Method options 

concentration 

● Royal Gala  ● %  ● Powdered enzymes 

Options  Pectinase  is  the  most 
common  enzyme  used       
to  increase  the  yield 
● Fuji  ● mg/L  ● Water-based enzymes 
within  juice  factories. 
For  this  reason,   
pectinase  will  be  the  ● Granny Smith 
only enzyme tested. 

● What  type  of 

Questions to  Would  different  apple  Which  Does  efficiency  improve 
enzymes  do  juicing 
research  varieties  produce  concentrations  if  the  solution  is 
companies use? 
different juices?  should I use?  premixed? 
● What  is  the  purpose 
of this enzyme? 

Findings:  Pectinase  is  the  most  As  long  as  all  apples  %  is  the  most  It  does  not  matter  if  the 
common  enzyme  in  are  the same, it will not  effective  method,  enzymes  are  premixed. 
  
the  juicing  industry  to  make a difference. Due  recommended  by  No  findings  on  how 
aid  in  the  breakdown  to  ease  of  access,  the  the  SACE  board  for  apples  are  affected  by 
of  cell  walls  (National  Royal  Gala  apple  this  task  (South  this  were  found, 
Centre  for  variety will be used.  Australian  Certificate  however,  mixing  powder 
Biotechnology  of Education 2017).  was  supported  by 
Education 2000).  Enzyme  Stuff  as  they  will 
 be  at  the  most  active 
levels (2005). 

Possible  Number  of  species  Some  varieties  contain  Controlling  the  The  accuracy  of  mixing 
Limitations  tested-  can  the  less  juice,  however,  concentration  of  enzyme  powder  with 
findings  be  applied  to  this  will  not  impact  the  pectinase.  water  must  be  strictly 
others?  ability  to  correlate  controlled and is prone to 
results  with  real-life  errors.  
  
applications. 

Note: Highlighted sections are from 'Task 06 - Deconstruct and Design Weeds' (South Australian Certificate of
Education 2017)
 
Hypothesis 
If  the  pectinase  concentration  ​is  increased​,  the  volume  of  apple  juice  produced  will  increase  ​until 
the rate of reaction plateaus due to limited amounts of substrate.  
 
Variables 
Independent Variable 
The  independent  variable  is  altered  to  produce  a  measurable  effect.  In  this  investigation,  it  is  the 
concentration​ ​of​ the ​pectinase​, measured as a percentage (%). 
 
Dependent Variable 
The  dependent  variable  is  the  measurement  resulting  from  a  changed  independent  variable.  The 
amount of apple juice is the dependent variable, measured in millilitres (mL). 
 
Controlled Variables 

 
 
  
 
Uncontrollable variables 
Some  variables  are  not  controlled  as  they  are  caused  by  external  factors.  Although all apples were 
from  the  same  packet,  the  age  and  origin  of  each  apple  was  not  specified  and  could  impact  the 
amount of juice extracted. 
 
Materials 

● 5x Beaker  ● 60 mL 5% Pectinase 
● 3x Medium apples  ● 60 mL 10% Pectinase 
● 5x 100 mL Graduated cylinder   ● 1x Glad wrap 
● 5x Buret funnels  ● 15x Medium-width filter papers 
● 60 mL H2O  ● 1x Serrated knife 
● 60 mL 1% Pectinase  ● 1x Potato peeler 
● 60 mL 3% Pectinase  ● 1x Scales 

Method 
1. The  skin  of  each  apple  was  peeled  and  flesh  was 
separated from the core 
2. The apple segments were cut into 1 cm cubes 
3. Using  scales,  the  cubes  were  portioned  into  five  15 
gram  serves  of  apple,  weighing  with  scales  to 
ensure they are equal 

4. The  15  gram  portions  were  each  placed  a 

100  mL  beaker  with  20  mL  of  0%,  1%,  3%,  5% 
and  10%  pectinase  solution,  ensuring  the  apple 
is fully submerged (Figure 1) 
5. The  apple  and  pectinase  mixture  was 
allowed to soak for 20 minutes 
 6. Next, filter paper was placed into the buret funnels 
7. The  buret  funnels  were  then  placed  atop  the  graduated  cylinders and the apple and pectinase 
mixture was poured into this. 
8. A  timer was then started for 20 minutes to allow time for the mixture to drop any remaining fluid 
into the cylinder (Figure 2) 
9. After 20 minutes, the readings were recorded 
10. Steps 1-9 were repeated twice more as replicates to increase reliability of the data 
 
Risk Assessment 
Science  investigations  often  use  glassware  and  other  sometimes  hazardous  materials  within  their 
methods. These must be identified to ensure the risks are mitigated. 
 
Overall Hazard Assessment LOW > MEDIUM > HIGH 
 

 
Results 

Table 1: The effect of pectinase concentration on the volume of apple juice produced

Volume of Apple Juice Produced (mL)

Concentrations
% Trial 1 Trial 2 Trial 3 Average

0 17 14 17 16

1 16 14 16 15

3 16 12 17 15

5 18 17 16 16

10 18 15 18 17

 
Figure 1: the impact of pectinase concentration on the volume of apple juice produced 

Analysis of the graph 

Although  the  results  in  ​Figure  1  support  a  positive  ​linear  trendline  between  the  concentration  of 
pectinase  and  resulting  volume  of  apple  juice,  individual  results  do  not  support  this.  10%  pectinase 
appears to be the optimal concentration, however, the margin for error is large. 0%, 1% and 3% show 
much  lower  yields  of  apple  juice  than  that  of  10%  and  no  data  points  are  fitted  to  the  trendline 
alluding to low accuracy. 
 
Discussion 
The  hypothesis  that  the  inclusion  of  more  pectinase  ​would  extract  more  juice  was  supported, 
however,  it  was  not  of  significance  due  to  the  small  range  of  the  data.  This  is  because  the  data 
would plateau due to the lack of substrate available to make enzyme-substrate complexes. 
Fruit  juice  companies  have  found  there  is  a  maximum  yield  of  87%,  so  after  adding  20  mL  as  per 
methodology,  it  can  be  expected  that  the  maximum  yield  for  15  grams  of  apple  is  33.05  mL.  It 
seems  the  optimum  concentration  of  pectinase  is  40%,  shown  in  ​Figure 2 ​and supported by Demir, 
Acar,  Sarıoğlu  and  Mutlu  (2001),  however,  tests with higher concentrations of pectinase are needed 
to find where exactly the results will plateau.  

 
Figure 2: Extrapolation of pectinase concentration on the volume of apple juice produced 
A  noteworthy  observation  is  that  0%  pectinase  performed  slightly  better  than  1%  and  3%  pectinase 
solutions,  which  can  be  explained  through  diffusion.  Diffusion  refers  to  the  movement  of  ​solvent 
into  a  region  of  higher  ​solute  concentration  through  ​aquaporins​-  pores  in  the  cell  membrane. 
Osmosis  is  diffusion  that  refers  to  the  transportation  of  water  from  a  high  to  low  concentration  of 
H​2​O molecules. This occurs to keep plant cells ​turgid (full) so they can support themselves without a 
skeleton.  Since  the  cubes  of  apple  used  in  this  investigation  were  not  fresh,  the  aforementioned 
cells  may  have  become  ​flaccid  (limp  due  to  a  reduction  in  water  within  each  cell).  When  soaked in 
the  pectinase  solution  for  20  minutes,  ​osmotic pressure ​would have introduced water into the cells 
and  reduced  the  amount  of  apple  juice  able  to  be  extracted.  This  is  supported  by  ​Table  2  which 
saw  the  apples  weigh  an  average  of  11.81%  more  than  the  15  grams  when  started.  It  can  be 
hypothesised  that  10%  pectinase  and  above  contains  the  same  or  less  amount  of  water  molecules 
than  the  apple  and  does  not  introduce  water  through  osmosis,  explaining  the  increasing  volume 
created. 
Figure  2  ​shows  that  the  volume  cannot  increase  indefinitely  and  the concentration that the amount 
of  enzyme-substrate complexes would plateau at. Since more than 40% pectinase would provide an 
excess  of  active  sites,  there  would  not  be  enough  substrate  to  support  a  higher  rate  of  reaction. 
These  extra  enzymes  do  not  catalyse  reactions  and  are  wasted,  since  the  ​secondary  ​and  ​tertiary 
structure  of  the  enzymes  allow  them  to  be  reused.  The  reaction  rate  of  enzymes  and  substrate 
would stabilise, resulting in a plateau of the yield of apple juice. 
 
Table 2: Increase in weight of 3 apple samples after experiment as a percentage % of weight

0% pectinase  3% pectinase  5% pectinase  Average 

12.91 10.90 11.62 11.81 

   
Evaluation 
Sources of error

Random Errors Impact Of This Error Improvements

Inconsistent apple Not all cubes were 1 cm​3​, meaning the surface Using apple coring and slicing
cubes area was different between trials. Since more devices would yield comparable
substrate is available, results are not valid. data.

Used apples of Older apples’ cells have more flaccid cells
different ages than fresher apples, which introduces water
through osmosis when soaked. This water
does not get expelled into the cylinder, thus
the resulting volume is decreased.
This error could be omitted by
using apples of the same age
directly from an apple orchard.
Apples could be soaked prior to
the investigation to balance water
within and outside cells.

Systematic Errors Impact Of This Error Improvements

Using porous filter The filter paper soaked up much of the apple
paper juice produced. Although 20 mL of pectinase
solution was used, the maximum return was
18 mL.
An improvement to this would be
to use a mesh sieve to separate
the apple from the pectinase
solution so that no liquid is
captured.

Resolution of Since 50 mL cylinders were used, the Since 10 mL cylinders have higher
measuring resolution is accurate to 1 mL. This means no resolution, using these would
instruments decimal places were recorded, impacting increase accuracy between small
accuracy. differences in data.

A low volume of Since some apples were not fully submerged, Using 40 mL of pectinase would
pectinase solution the amount of substrate the enzymes were in have ensured the amount of apple
surrounding the contact with was reduced. This may have juice was reliable.
apples reduced the volume of apple juice produced,
limiting accuracy.

Uncontrollable Factors Impact Of This Factor

Room temperature Since enzymes perform their functions at The impact of this can be limited
different speeds depending on the by conducting the experiment an
surrounding temperature, the differing room incubator set at 25​o​, limiting the
temperature may have affected the results. impact of varying temperature.
Although the room temperature was set to
23​o​C, the nature of a science laboratory
includes heating elements, microwaves and
refrigerators which can impact this.

Origin of apples Although all apples used were from the same Sourcing apples directly from
packet, the origin of the individual apples is farms in the same conditions
not disclosed. Since soil type, weather and would reduce these risks.
fertilisation impact the amount of water
within the apple cells (​Scudellari et al., 1993), 
this  could  have  had  a  significant impact on 
the results. 
 Reliability, Accuracy and Validity 
 
Reliability 
The  reproducibility  is  low  because  of  the  inability  to gather the same data when repeated, meaning 
other  experimenters  achieved  different  results  (Appendix  A).  Additionally,  the precision of data was 
low due to low instrument resolution and apples were of different ages and origins. 
 
Accuracy 
Although  there is no ‘true value’ the data can be compared to, errors such as the use of porous filter 
paper  contribute  to  inaccuracy.  The  filter  paper  soaked  up  much  of  the  liquid,  thus  the  volume  of 
apple  juice  produced  is  unknown.  Furthermore,  growing conditions are unknown, which can impact 
water  content  (Scudellari  et  al.  1993).  Repeating  the  experiment  reduced  inaccuracies, however, as 
more replicates can better support the hypothesis. 

Validity 
The  experiment  better showed the causation between different pectinase concentrations and water 
movement  through  osmosis.  Changes  to  experimental  design  such  as  soaking  the  apples  in  water 
prior  to  soaking  them  with  pectinase  and  the  use  of  metal  sieves  may  increase  validity.  Although 
trends  regarding  the  effects  of  pectinase  concentration  in  apples and the impact of osmosis on this 
process  were  supported by literature (Tapre & Jain 2014; Lecornu & Diercks 2017), the methodology 
must be changed to increase validity. 
 
Conclusion 
The  data  supports  the  hypothesis  that  as  the  pectinase  concentration  ​is  increased​,  the  volume  of 
apple  juice  produced  will  increase​,  however,  more  data  is  needed  to  conclude  this.  Ineffective 
equipment,  flawed  methodology and uncontrollable factors such as the origin of the apples resulted 
in  invalid  and  unreliable  data.  Since  Figure  2  ​shows  the  volume  cannot  increase  indefinitely,  more 
data  points  at  a  higher  concentration  of  pectinase  will be needed to conclude where the enzymatic 
processes will plateau due to the lack of substrate. 
   
Appendix A 
 
Comparable data from a similar experiment (Hurwitt 2018)
Note: 20 mL added to match methodology

Amount of concentration Amount of juice produced

0.50% 20.4ml
1% 26ml
2% 30ml
3% 2.5ml
5% 34.5ml

 
 
   
Reference List 
Demir,  N.,  Acar,  J.,  Sarıoğlu,  K.  and  Mutlu,  M.,  2001.  The  use  of  commercial  pectinase  in  fruit  juice 
industry.  Part  3:  Immobilized  pectinase  for  mash  treatment.  Journal  of  Food  Engineering,  47(4), 
pp.275-280. 
Hurtwitt, B, 2018 ​Pectinase​ ​concentration​, Bedford Park 
Lecornu, B & Diercks, T 2017, ​Biology: Levels of Life​, vol 1. Briton Books, Adelaide 
 
Scudellari  D.,  Marangoni  B.,  Cobianchi  D.,  Faedi  W.,  Maltoni  M.L.  1993,  ​Effects  of  fertilization  on 
apple tree development, yield and fruit quality. In: Fragoso M.A.C., Van Beusichem M.L., Houwers A. 
(eds)  Optimization  of  Plant  Nutrition.  Developments  in  Plant  and  Soil  Sciences,  vol  53.  Springer, 
Dordrecht 
Tapre,  A.R.  &  Jain,  R.K.  2014,  ​Pectinases:  Enzymes  for  fruit  processing  industry​,  International  Food 
Research Journal, 21(2).  
 
   
 
Guidelines for how to address the Performance Standards in the report:

Section of the Report Requirements/Indicators Specific Feature

Introduction Relevant biological Information presented that relates specifically to the KA1
practical being investigated. The information relates to the aim of the
experiment.

Deconstruct Explores the various aspects of the problem and this links to the IAE1
(Design) aim, hypothesis and method.
Aim Indicates the purpose of the experiment KA1
Independent and dependent variables are identified.
Hypothesis Has the correct format- is not in the form of a question IAE1
Links the independent and dependent variable and is a prediction.
Method Describes how the independent variable is changed, is detailed and IAE1
describes how the dependent variable is measured.
All variables should be identified, how and why they are controlled.
Results Table has the correct format IAE2
Data is represented in an appropriate manner- all data is shown
Significant figures are correct
Graphs are drawn appropriately- axis are labelled, appropriate scale used,
title, size, correct format
Discussion Explains all the data obtained KA1,
Provides reasoning based on the data for supporting or rejecting the IAE3
hypothesis IAE4
Identifies potential sources of random, systematic and uncontrolled error.
Discusses the data’s reliability, precision, accuracy and validity
Evaluates the experimental method and suggests possible relevant
improvements to the design of the experiment
Conclusion Indicates whether the aim of the experiment has been met with respect to IAE3
the limitations already identified and restates the overall trend of the
experiment.
Safety Audit Detailed analysis of the potential risks, hazards and how they are managed IAE1
and the precautions taken in the classroom
Communication Use of appropriate biological terms and conventions KA4
Reference List Harvard Referencing Used KA4
Sources correctly cited.
Bibliography provided
Stage 2 Biology Performance Standards
- Investigation, Analysis, and Evaluation
Designs a logical, coherent, and detailed biological
A investigation.
Obtains, records, and represents data, using
appropriate conventions and formats accurately and
highly effectively.
Systematically analyses and interprets data and
evidence to formulate logical conclusions with
detailed justification.
Critically and logically evaluates procedures and their
effect on data.
Designs a well-considered and clear biological
B investigation.
Obtains, records, and represents data, using
appropriate conventions and formats mostly
accurately and effectively.
Logically analyses and interprets data and evidence
to formulate suitable conclusions with reasonable
justification.
Logically evaluates procedures and their effect on
data.
Designs a considered and generally clear biological
C investigation.
Obtains, records, and represents data, using
generally appropriate conventions and formats with
some errors but generally accurately and effectively.
Undertakes some analysis and interpretation of data
and evidence to formulate generally appropriate
conclusions with some justification.
Evaluates procedures and some of their effect on
data.
Prepares the outline of a biological investigation.
D understanding of biological concepts.
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conventions and formats inconsistently, with
occasional accuracy and effectiveness.
Describes data and undertakes some basic
interpretation to formulate a basic conclusion.
Attempts to evaluate procedures or suggest an effect
on data.
Identifies a simple procedure for a biological
E investigation.
Attempts to record and represent some data, with
limited accuracy or effectiveness.
Attempts to describe results and/or interpret data to
formulate a basic conclusion.
Acknowledges that procedures affect data.
Knowledge and Application
Demonstrates deep and broad knowledge and
understanding of a range of biological concepts.
Develops and applies biological concepts highly
effectively in new and familiar contexts.
Critically explores and understands in depth the
interaction between science and society.
Communicates knowledge and understanding of
biology coherently, with highly effective use of
appropriate terms, conventions, and representations.
Demonstrates some depth and breadth of knowledge
and understanding of a range of biological concepts.
Develops and applies biological concepts mostly
effectively in new and familiar contexts.
Logically explores and understands in some depth
the interaction between science and society.
Communicates knowledge and understanding of
biology mostly coherently, with effective use of
appropriate terms, conventions, and representations.
Demonstrates knowledge and understanding of a
general range of biological concepts.
Develops and applies biological concepts generally
effectively in new or familiar contexts.
Explores and understands aspects of the interaction
between science and society.
Communicates knowledge and understanding of
biology generally effectively, using some appropriate
terms, conventions, and representations.
Demonstrates some basic knowledge and partial
Develops and applies some biological concepts in
familiar contexts.
Partially explores and recognises aspects of the
interaction between science and society.
Communicates basic biological information, using
some appropriate terms, conventions, and/or
representations.
Demonstrates limited recognition and awareness of
biological concepts.
Attempts to develop and apply biological concepts in
familiar contexts.
Attempts to explore and identify an aspect of the
interaction between science and society.
Attempts to communicate information about biology.