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.

The above graph illustrates a polynomial 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 exponential from the control value of 49.7 to 21.3; indicating that the dependence of yield of apple juice extracted is exponentially dependent on the % concentration of pectinase.

The best fit polynomial plot follows the equation:

y = 0.037 x² – 2.178 x + 49.81

Where y= transmittance of the solution x = % concentration of pectinase

Calculation of maxima & minima

y = 0.037x² -2.178x + 49.81

dy/dx = 0.074 x – 2.178

d²y/dx² = 0.074

Since, d²y/dx² is negative, the curve has a minima but not a maxima At minima,

dy/dx = 0

0.074 x – 2.178 = 0

x = 2.178 / 0.174 = 29.43 9

This indicates that the transmittance of the solution will have minimum value if the concentration of the enzyme used is 29.43%. It clearly indicates and coherently proves that the yield of apple juice extracted will have maximum value when the concentration of the enzyme used is 29.43%.

The value of correlation coefficient(R²) 0.999 indicates a strong negative correlation between the transmittance value and the % concentration of cellulase.

Analysis of mean value and standard deviation:

The above column graph indicates and analyses the mean value of transmittance of the controlled sample and other enzymes. The transmittance value is highest for the control indicating that the yields of apple juice have minimum value for it. The mean values in enzymes are significantly different from the mean value of the control. It indicates that the yield of apple juice is highly influenced by the presence of enzymes. Among the enzymes-pectinase and cellulase, the transmittance is higher for pectinase and lower for cellulase, indicating that the yield of apple juice is more with cellulase than pectinase.

Null hypotheses

There is no significant difference between the two class of enzymes-pectinase and cellulase. Any difference found is due to random or sampling error.

Alternate hypotheses

There is significant difference between the two class of enzymes-pectinase and cellulase. Any difference found is not due to random or sampling error.

Critical T value = ( X₁-X₂ ) / [ ( S₁) ² /N₁ + (S₂) ²/N₂ ] ^1/2 = ( 34.47 – 29.57) / [ (8.79)²/5 + (8.24)²/5] ^1⁄2 = 0.9094

Probability value (p) = 1.383 (20% significance level)

It means that the null hypotheses can be rejected and the alternate hypotheses may be accepted; there is a significant difference between the enzyme activity of pectinase and cellulase.

The main aim of the investigation was to answer the research question:

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

• As illustrated from the data collected and coherently processed, use of enzymes pectinase and cellulase has a major impact on the yield of juice extracted from the samples of apples. It is seen that pectinase yields lower amount of juice than cellulase.
• The yield of apple juice increases with increase in % concentration of enzymes for both pectinase and cellulase, as indicated by the linear and exponential decrease in the value of transmittance respectively.
• The optimum levels of the enzymes to be used to obtain the maximum yield of apple juice are 39.35 % and 29.43% respectively. The yield of apple juice extracted increases linearly with the increase in % concentration for pectinase and exponentially for cellulase.
• The apple juice is extracted from the samples of apples by the decomposition of proteins in the apples or cellulase from the cell wall of the apples. The transmittance measured indicates the amount/quantity of the protein, fibers or cellulase in the samples. With the use of enzymes, these biomolecules are degraded or chemically hydrolysed which in turn increases the yield of the juice produced or the amount of it. As the mass of the biomolecules decreases, the solution becomes more yellow and thus more light is absorbed and hence the transmittance decreases.

The experiment was conducted in an incubator and it is claimed that optimum levels of pH and temperature has been maintained but any check or monitoring to ensure the same has not been done.

The same experiment if repeated can be done with other sorts of similar enzymes.

The investigation can be conducted at other values of temperature.

Similar sort of study with other types of fruits along with apples would lead to a more detailed report and valuable analysis.

Journals

https://www.agriculturejournals.cz/publicFiles/136826.pdf Plant Cell Wall Degrading Enzymes, Pectinase and Cellulase, in the Digestive System of the Red Palm Weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae) Mohammad VATANPARAST1 , Vahid HOSSEININAVEH1 , Mohammad GHADAMYARI 2 and Seyede MINOO SAJJADIAN1 1 Department of Plant Protection, College of Agriculture, University of Tehran, Karaj, Iran; 2 Department of Plant Protection, College of Agriculture, University of Guilan, Guilan, Iran

https://www.sciencedirect.com/science/article/pii/S0032959204001219 Effects of cellulase and pectinase concentrations on the colour yield of enzyme extracted plant carotenoids Author links open overlay panelİnciÇinar Department of Food Engineering, Kahramanmaraş Sütçtü İmam University, Kahramanmaraş 46060, Turkey Received 11 September 2003, Accepted 25 February 2004, Available online 9 June 2004.

http://www.arpnjournals.com/jabs/research_papers/rp_2010/jabs_0910_211.pdf REGULATION OF CELLULASE AND PECTINASE ACTIVITIES IN CHERRY TOMATO (Lycopersicon esculentum MILL var. cerasiforme) FRUIT BY USE OF 1-METHYLCYCLOPROPENE Arnold M. Opiyo1 and Tie-Jin Ying2 1 Department of Crops, Horticulture and Soils, Egerton University, Kenya 2 Department of Food Science and Nutrition, Zhejiang University, Hangzhou, China

Websites

https://dalspace.library.dal.ca/bitstream/handle/10222/73500/Wang-Jingyi-MSC-AGRI-December- 2017.pdf?sequence=5

https://www.tandfonline.com/doi/abs/10.1080/03235400500094100?src=recsys&journalCode=ga pp20

Videos

https://www.youtube.com/watch?v=Zh7rY0o7Fxc Pectinase & Cellulase compared to Water

https://www.youtube.com/watch?v=yFLoCneDFms MB202 enzyme technology video assignment -- What does cellulase do –

Books

Enzymes and Enzyme Activity: Structure, Biology and Clinical Significance (Protein Biochemistry, Synthesis, Structure and Cellular Functions: Microbiology Research Advances)UK ed. Edition by Eva M. Lashinski (Editor)