Effect of concentration of enzymes on yield of apple juice

For as long as I can remember, I have loved apple juice. I loved it so much, in fact, that my mother often tells me there was a time when I would only drink apple juice. I used to read the back of the cartons to see how many apples went into each juice drink, and I was enthused at learning that around1-2 apples were all that were needed to make the delicious drink. When I tried making my own, however, I was very disappointed by the results. I consulted the internet to find that the commercially manufactured apple juice were produced with enzymes added to them; years later, I would learn what these were in biology class. I then discovered that there were two different enzymes used for the same purpose; both pectinase and cellulase were used in order to extract more apple juice. Thus, I have elected to do a comparative analysis of the effectiveness of pectinase and cellulase in apple juice production as my area of study in my Biology internal assessment for the IB program.

To determine which enzyme, pectinase or cellulase, produces a greater yield of apple juice as their concentrations increase when added to apples, using spectrophotometry.

Pectinase and cellulase are both enzymes. Enzymes are globular proteins that function as biological catalysts. The mechanism by which the enzymes function is known as the induced fit hypothesis. According to this model, the active site of the enzymes is altered slightly to accommodate the shape of the substrate molecules, which allows for the substrate and enzyme to bind together and form an enzyme-substrate complex. After this, the substrate is released as a product and the enzyme is often reused in catalyzing the same reaction type.

Enzymes also function most effectively at optimum pH conditions and temperatures. Accordingly, in this experiment, I considered the optimum conditions for both enzymes independently of one another; both experiments were performed under different conditions. For the type of pectinase used –polygalacturunase- the optimum temperature was around 35 ̊C (according to the label on the bottle) and functioned best in acidic conditions with a pH around 5-5.5. Cellulase however, functions better at 45-50 ̊C (according to the label on the bottle, and a pH of 4.6. I, therefore, made sure to provide these exact conditions using incubators, water baths, and buffer solutions. Due to these specific requirements, there are a number of uses in industry which this exploration will evaluate.

This particular type of pectinase is known as polygalacturonase. It is used in the hydrolysis of the 1-4 glycosidic linkages between residues of galacturonic acid. Consequently, it is used to break up the underlying pectin matrix of the cell wall, producing a monosaccharide galacturonic acid, thus contributing to the process of cell wall disintegration. It, therefore, becomes easier to extract a larger volume of apple juice from the same quantity of apples because the integrity of the cell wall is compromised, so for the same pressure applied, there is a greater yield of juice.

Cellulase is primarily used in the breakdown of the largest component of the cell wall, cellulose. The product of this breakdown is usually monosaccharide but can also be poly saccharides. The reason this is important and commercially viable is because after the apples have been treated with cellulase they tend to produce a substantially larger volume of apple juice as the cell contents are no longer held in so rigidly

While both enzymes will display the same trend of a decrease in light permeability through the solution of apple juice, the apples treated with cellulase will be even more impermeable to light at 590–560 nm (yellow) as cellulase forms a larger percentage of the cell wall.

• https://www.sciencedirect.com/science/article/abs/pii/S0960308516300062
• https://www.rcsb.org/

Preparation of sodium acetate-acetate buffer for pH = 5.5

50 cm³ of 0.1 moldm^-3 ethanoic acid and 50 cm³ of 0.5 moldm^-3 aqueous solution of sodium ethanoate was mixed.

Preparation of phosphate buffer of pH = 4.6

100 cm³ of 0.1moldm^-3 aqueous solution of potassium hydrogen phthalate was mixed with 22.2 cm³ of 0.1 moldm^-3 aqueous solution of NaOH.

• First 100 cm³ of water was measured in a measuring cylinder and poured into a beaker. This was repeated 6 times, giving a total of 7 beakers for the pectinase experiment. Sodium ethanoate - ethanoic acid was then added to give the optimum pH.
• Then, 5 grams of pectinase were measured using a digital weighing scale (The pectinase was put on an aluminum sheet and the balance was corrected to 0.00) and added to the water to create a 5% pectinase solution. The same procedure was repeated to create a 10%, 15%, 20%, 25% solutions.
• The apples were then cut up into small 1cm³pieces. This was achieved by measuring 1cm by 1 cm using a scale and then precisely cutting it using a knife. They were kept on a petri dish with a cap on them so as to prevent evaporation.
• Following this, 10 pieces were put into each of the 6 jars except the first (this was done in
  order to confirm that the transmittance rate of pure water was 100%)
• Each of the beakers was incubated at the optimum temperature of 35 °C in a water bath for exactly 15 minutes.
• Then, after they had been removed, using a spatula, each of the apple pieces were systematically crushed and removed for exactly 3 minutes per beaker.
• After this, the juice samples were added to the spectrophotometer (after it had been adjusted to the yellow wavelength of 560nm) in order to determine the concentration of apple juice, and thus ascertain the effectiveness of the enzymes.
• The process above was repeated for cellulase, but the procedure was corrected to include the appropriate phosphate buffer and the appropriate temperature of 47.5°C was used.

• No animals were harmed during the experimentation.
• Minimum amount of chemicals and raw materials were used.
• All waste chemicals were exposed safely in the waste bin.

• Wear safety gloves and use protective clothing.
• No materials were ingested or exposed to skin.
• Extensive care was taken while handling the spectrophotometer so that the solution does not spill out.

The color of the juice extracted was yellow.

The above graph illustrates a linear variation between the transmittance of the extract of the sample against the % concentration of the pectinase. It is clearly observed that as the % concentration of pectinase increases, the transmittance of the solution decreases. The decrease is linear from the control value of 49.7 to 25.1; indicating that the dependence of yield of apple juice extracted is linearly dependent on the % concentration of pectinase.

The best fit linear plot follows the equation:

y =-1.278 x + 50.3

Where y= transmittance of the solution

x = % concentration of pectinase

At, x=0

y = 50.3

It indicates that the transmittance of the solution in absence of enzyme is maximum and reaches a constant value of 50.3. Thus the yield of apple juice is minimum value in absence of enzyme.

At, y =0

x = 39.35

Thus it can be theoretically claimed that the yield of apple juice will be highest and transmittance of the solution will be zero if the enzyme used has a % concentration of 39.35.

The value of correlation coefficient (R²) = 0.978 indicates a weak negative correlation between the transmittance value and the % concentration of pectinase.