IB Biology HL Internal Assessment Scored 7 (Both Predicted and Achieved 20/24 in May 2019)
High Achiever Biology IA with the research question: What is the effect of pectinase enzyme extracted from Aspergillus Niger fungal strains on the quality and quantity of apple juices in case of apple cultiva...
Biology Internal Assessment
Study on the effect of pectinase enzyme on different varieties of apple juices
Exam session: May 2019
Word count: 3655
Background to my choice of research question:
Do you like apples, maybe apple juice? Or have you ever thought about why and how the
companies are able to gain a lot of juice out of the apples when you barely can? Well,
since I really like both apples and apple juices, and I prefer to produce the juices at home
in order to make sure that only the suitable – usually bio – apples are included, I often
think about it. In most of the cases, I find that a huge proportion of the apples are thrown
away during the process, and yet, not much juice is gained. After realising this problem
and doing a bit of research on the internet, I found out what element is missing from my
procedure but is included in the manufacturers’. This element is the usage of pectinase
enzyme. Since we have already learned about the effect of enzymes in Biology classes, it
all made sense and I started to be genuinely interested of the extent of the increase in
quantity and quality of apple juices when they are treated with the enzyme. After
establishing the topic of my investigation the next step was to choose the varieties of
apples I want to test on. Since I regularly do this for own purposes, this wasn’t very hard, I
have chosen the ones that I personally enjoy the most, which are known for high pectin
concentrations or are really different in size and colour from the previously chosen ones.
As such, I’ve picked 4 varieties from the local market. These are Fuji, Granny Smith,
Jonagold and Red Chief. My aim is to find out how their properties change when they are
treated with the enzyme at home and not in a laboratory, so even though I will conduct the
experiments in my school’s lab, I will design the method so that it will be fit to be carried
out at home too. Testing on these different cultivars of apples will help me analyse how the
pectinase enzyme affects different kinds of apples, and how the different apples react to
pectinase enzyme at the same time. Fortunately, I was familiar with the basic procedure,
so even though my initial method using the vacuum pump in my school’s laboratory did not
work out, I was able to make the juices manually. I bought the enzyme from an agricultural
shop. Eventually, I developed my research question, which is the following.
What is the effect of pectinase enzyme extracted from Aspergillus Niger fungal strains on
the quality and quantity of apple juices in case of apple cultivars Fuji, Granny Smith,
Jonagold and Red Chief?
1
,Background information: Figure 11: The structure of pectin
Pectins, which are commonly found in apples, are
heteropolysaccharides with a weight of 10-360 000 g/mol,
consisting of methyl esterified α-1-4-d-galacturonic acid with the
side chain containing various sugars, for instance, L-rhamnose, D-
galactose and L-arabinose2 , all in different concentration in case
of each pectin. Figure 23 : Pectin in plant cells
They are present in the middle lamella and primary cell wall of
most plant cells and are responsible for providing mechanical
support and flexibility to the tissues – by binding to other
organelles e. g. to cellulose and hemicellulose, and glueing them
together with the cell wall.2i They are also similar in role to collagen, which is present in
animal organisms2ii. As the plant grows and different pectinase enzymes are produced, the
pectin concentrated in the fruit is broken down mainly to D-galacturonic acid. There are a
lot of mechanisms for the reaction pathway to occur and it all depends on the type of
enzyme which is produced or added. Since in the experiment I will be adding only
polygalacturonase enzyme (PG), I’m going to focus on that mechanism. There are two
types of PG. The first is Endo-polygalacturonase (Endo PG), which is capable of splitting
the molecule in random places creating smaller molecules, this way making the tissue
softer and mushier, allowing the cells to separate from each other, thus, more liquid will
come out of the purée, and it works well in the pH range 4.5 - 6.0, and the second is Exo-
polygalacturonase (Exo PG), which is suited to cut the terminal molecule creating simple
sugar monomers, thus giving the juices a sweeter, more aromatic taste, and it works well
in a more acidic environment than the Endo PG, 3.5 - 5.0. Both of them work effectively in
temperatures below 50 ℃, that’s why I am going set up a temperature of 45 ℃. This is a
natural process, but the course of action can be sped up by adding pectinase enzyme
manually, so these favourable characteristics (more juice can be gained and it’s sweeter
and more aromatic) will come out when making the juice.
Figure 34: Reaction mechanism of polygalacturonase enzyme
,The enzyme I will use is extracted from Aspergillus Niger fungal strains, which they
produce to break down the middle lamella in plants so that they can extract nutrients from
the plant tissues and insert fungal hyphae5 .
In addition, when producing apple juice, it can be observed, that the liquid is cloudy – due
to insoluble molecules or ”cloud particles”4. These particles consist of positively charged
protein “cores” and the surrounding negative pectin molecules. Pectinase enzyme breaks
the structure of the pectin, thus the electrostatic repulsion between the negative particles
decreases, and the ”cloud particles” form bigger and bigger groups. These large groups
eventually form a precipitate which results in the juice to be more clear5. Hence,
theoretically, after the experiments, I will be able to observe that the enzyme-treated
samples are visibly clearer than the untreated ones.
protein
pectin aggregate
pectinase enzyme Figure 4: The formation of aggregates
Thus, in the inspection I will observe the change in these properties, by measuring the
volume, weight, Brix % (or in my case, the sucrose level) and the pH level* and also by
studying the transparency and the change in aroma (by smelling) of all the samples, the
ones that are treated with pectinase enzyme and the ones that are not.
*Although it is not mentioned in the literary texts, I am curious to see whether the pH of the
samples changes due to the experiment. Since I’m trying to design a method that can be
conducted at home too, I cannot use buffers to establish the optimal pH so I’m just going
to monitor it.
Hypothesis:
H 0: I. The enzyme treatment doesn’t alter the characteristics of the apple juices, very
similar values will be obtained after the experiment in both treated and untreated groups.
II. All the apple varieties and all the samples react to the treatment in almost the same
way, the experimental values are close to each other.
III. The rate of the changes in the dependent variables is not proportional to each
other.
, My personal expectations: All of the enzyme treated samples will have a higher volume,
weight and sugar concentration, will be more aromatic, more transparent and lighter in
colour compared to the untreated ones, and they will react in almost the same way. Also,
the rate in the change of the dependent variables will be highly proportional to each other.
Variables:
Independent: - the types of apples (Fuji, Granny Smith, Jonagold, Red Chief) and store-
bought juice (used as a control) on which the experiments are conducted (they differ in
colour, size, sweetness and texture in order to see a more varied range of results,
however, all of them have a pH value between 3 and 4, which sets the condition up for the
Exo PG enzyme to work properly) and the presence or lack of pectinase enzyme
Dependent: - Volume, weight and sugar (sucrose) concentration depending on the
presence or lack of pectinase enzyme (I used a measuring cylinder, an electronic mass
balance and a refractometer to measure the values of the juices, which will be represented
on tables and graphs later on) and also transparency, aroma and colour
Controlled: - time, surface area, and the temperature to which the samples were
exposed (I set the water bath instrument to 45 ℃, which is the optimal temperature for the
enzyme to work, and set the timer for 3 hours and 15 mins, and I used the same sized
beakers in case of all samples to make sure that the surface area that was in contact with
the air was constant)
Uncontrolled: - the samples’ exposure to air and light (I wasn’t able to create completely
dark nor airtight conditions so I was glad to find during my research that they weren’t a
confounding factor in the experiment, as it is not proven that they would affect the work of
the enzyme in any way)
Apparatus: Figure 5: The apples of the experiment
Materials:
• 2 Fuji apple ➜ 6 × 50 g (without core)
• 2 Granny Smith apple ➜ 6 × 50 g (without core)
• 2 Jonagold apple ➜ 6 × 50 g (without core)
• 2 Red Chief apple ➜ 6 × 50 g (without core)
• pectinase enzyme extracted from Aspergillus Niger in liquid form
• store-bought 100 % apple juice
Figure 6: ATC refractometer
4
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