Effect Of Concentration Of Enzyme (Pectinase) On Volume Of Juice Extracted From Red And Green Apples

Does the volume of juice extracted from red and green apples using the enzyme pectinase depends on the concentration of pectinase used, determined using volumetric analysis?

Application of biology in food industry and other food processing process is one of the most upcoming research topic. Use of enzymes in extracting juice from food samples is something, I studied in Molecular Biology and found really interesting. During my visit to a wine factory during one of our trips, I came to know the actual use of pectinase to process wine and make it clear by removing pectin from the wine. The questions that still bothered me was why are the manufacturers so particular about maintaining specific physical conditions like temperature, pH and concentrations of enzymes during this process. I came to know from my DP Biology classes that the action or catalytic ability of enzymes depends on physical conditions like temperature, pH and even concentration of both the substrate and the enzyme. I chose this field as a topic of investigation for my Biology Internal Assessment and arrived at the research question stated above.

Enzymes are macromolecular, globular proteins that act as biological catalysts in chemical reactions without having to be a part of the reaction and lower the activation energy when the enzyme and substrate bind. They are folded and form a pocket which serves as an active site in order to allow a substrate to bind which results in a reaction. The shape of the active site is complementary to that of the substrate and forms a lock and key structure which creates specificity for the enzyme. Since enzymes and substrates are in a continuous state of motion, they collide where the substrate binds to the active site of the enzyme creating an enzyme substrate complex. When substrates are inside the active site, the products formed are as a result of catabolism, which is the breaking of bonds or anabolism, which is the formation of bonds. After the reaction takes place, the products are released. In this experiment, the enzyme used is pectinase with the substrate being pectin.

There are several factors that affect the rate of enzymatic activity namely concentration between enzymes and their substrates, temperature and pH² . This experiment will investigate the effect of different concentrations of pectinase on the production of fruit juice in red apples and green apples. As the enzyme concentration decreases, the rate of reaction will also decrease since there are not enough enzymes for substrates to bind to which inhibits a reaction to take place causing a decrease in enzymatic activity whereas when enzyme concentration increases, there will be more enzymes which will allow more substrates to bind to creating a higher number of reactions thus, increasing enzymatic activity.

Pectinase is an enzyme which break down pectin, a hetero polysaccharide present in the cell walls⁴ of fruits such as oranges and apples, and keeps the cell walls firm and rigid. As the enzyme breaks down the soluble pectin, the amylases take off starch from the juice which causes haze in the juice. Pectin gets broken down from polymers to monomers by the process of hydrolysis which are water-soluble, for example: breaking of glycosidic bonds between galacturonic acid. Pectinase is industrially used to extract juice from fruits by breaking through the cell walls, especially apples. Pectinase is found to show an optimum pH ranging from 3.5 to 9.12 and a range of temperature from 50.0^oC to 90.0^oC depending on the source of pectinase used⁵

•  “Enzymes.” Chemistry for Biologists: Enzymes, www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm. Accessed on July10, 2019 1.30 pm
• “The Effect of Substrate Concentration on Enzyme Activity.” Untitled Document, www.ucl.ac.uk/~ucbcdab/enzass/substrate.htm. Accessed on July15,2019 1.12 am
• Heena Verma1, Lokesh K Narnoliya2 and Jyoti Singh Jadaun. “Pectinase: A Useful Tool in Fruit Processing Industries.” Nutrition and Food Science , 7 Mar. 2018, doi:10.19080/NFSIJ.2018.05.555673. Accessed on August 12, 2019 7.30 am
• Vinod Joshi, et al. “Purification, Characterization of Pectinase Produced from Apple Pomace and Its Evaluation in the Fruit Juice Extraction and Clarification.” Accessed on July 21, 2019 11.45 am

There is no correlation between the volume of juice extracted from the red and green apples and the concentration of pectinase used for it. Any correlation observed is an outcome of an experimental error or by random chance.

There is a positive correlation between the volume of juice extracted from red and green apples and the concentration of pectinase used. The literature review (refer to Figure-2) supports this hypothesis.

The percentage concentration of pectinase used is the independent variable in this investigation. The pectinase solutions were prepared by dissolving pectinase (solid) in distilled water. The concentration has been measured in terms of mass percentage (mass of solute per 100 cm3 of the solvent). The solutions of pectinase used are – 10 %, 20 %, 30 %, 40 % and 50 % respectively. Digital mass balance was used to weigh the solid and the solutions were prepared in 100 cm3 volumetric flask.

The type of sample can also be considered as an independent variable for this investigation as two different varieties of apples were used – red apple and green apple.

The volume of clear juice obtained from the mesh of the fruits after adding pectinase to it is the dependent variable of this investigation. The volume was measured using a graduated measuring cylinder. Data were reported at a confidence level of ± 0.5 cm3.

• Temperature of samples
  - The extraction of juice is an enzyme catalysed reaction. The rate and yield of this process depends on the temperature at which it is carried out. The temperature was maintained constant at 60.00C as the optimum temperature of pectinase ranges from 50.0 ⁰C to 90.0^oC A water bath was used for this purpose.
• Volume of water
  For a fair comparison, the volume of pectinase solution added to the sample must also be kept constant. 50.0 cm³ of pectinase solution was added in all trials using a graduated measuring cylinder.
• Time
  The volume of juice extracted depends on the duration for which the pectin in the mesh of the fruit samples are in contact with the enzyme. Longer the duration, more the volume of juice extracted from it (until all the enzyme has been used up). Thus, the time was kept constant at 20 minutes for each trials and monitored using a stop-watch.
• Concentration of substrate
  Rate of enzyme catalysed reactions depends on the concentration of substrate used. In this investigation, the fruit sample is the substrate and thus the quantity of substrate used was kept constant at 5.00 g in all trials using a digital mass balance.

The required volume of pectinase was was transferred to a 100 cm³ clean and dry volumetric flask and distilled water was added to it using a graduated measuring cylinder.

• 2 red apples were taken and cut them into fine pieces using a knife.
• The cut pieces were put into a blender
• The blender was started and stopped when puree was formed.
•  5.00 ± 0.01 g of the puree was weighed using a spatula, watch glass and a digital mass balance.
•  The weighed puree was transferred to a 100 cm³ glass beaker using a spatula.
•  75.00 cm³ of 0.00% pectinase solution (controlled) was added to it using a 100.0cm³ graduated measuring cylinder.
•  The beaker was placed on the water bath and the temperature of the water bath was set at 60.0^oC The beaker was placed there for 20.00 ± 0.01 minutes and the time was monitored using a stop-watch.
•  The content of the beaker was then filtered using a filter paper and a funnel and the filtrate was collected in a 100.0 cm³ graduated measuring cylinder.
•  The volume of the filtrate was noted down.
•  All of the above steps were repeated for four more times to collect data in five sets.
•  All of the above steps were repeated for other solutions of 20%, 30%, 40% and 50% solutions of pectinase.
•  All of the above steps were repeated for green apples.

• The apple extract turned red rose in colour on addition of pectinase solution.
• The colour of the filtrate (juice) collected was different for the different strengths of pectinase solution. The colour was pale yellow for 10%, 20% and 30% pectinase solution while golden yellow to brownish yellow for 40% and 50% pectinase solution.
• The intensity of the colour of the filtrate increases as the mass percentage of the pectinase solution added was increased.

Sample calculation

For 10.00% pectinase solution,

Average= \(\frac{16.00+16.00+16.00+17.00+16.00}{5}\)= 16.20 ± 0.50 cm³

Standard deviation

S.D = \(\frac{(16.00-16.20)^2 +(16.00-16.20)^2+(16.00-16.20)^2+(17.00-16.20)^2+(16.00-16.20)^2}{5}\)= 0.45

Error propagation

For 10.00 % pectinase solution

Average volume of juice extracted (x) = 16.20 ± 0.50 cm³

Absolute uncertainty (∆x) = ±0.50

Fractional uncertainty = \(\frac{∆x}{x}=\frac{0.50}{16.20}\)= 0.0309

Percentage uncertainty = \(\frac{∆x}{x}\) X 100 = 0.0309 =3.09

The graph above represents the variation of volume of juice extracted from red apples against the concentration (in volume percentage) of pectinase solution. The volume of juice obtained is plotted along the y axes as it is the independent variable and volume percentage is plotted along the x axes as it is the dependent variable. The reading at 0.00% pectinase is the data for the controlled set while the other points indicates the dependence of volume of juice on volume percentage of pectinase used. There is a significant difference of the volume of juice extracted from 7.80 cm³ to 16.20 cm³ as the concentration of pectinase increases from 0.0% to 10.0%. There is a sharp increase in the volume of juice from 16.2 cm³ to 51.8cm³ as the volume percentage increases from 10 % to 20%. The data after this depicts a gradual increase from 51.8cm3 to 54.2 cm3 as the volume percentage increases from 20% to 40%. The values at 50% and 60% stays constant at 54.2 cm3 . Thus, in general we can claim that the volume of juice extracted increases on use of pectinase and a gradual increase is observed as the volume percentage increases from 20% to 40% while becomes constant after that. Beyond the value of 40% (50% and 60%), the volume percentage of pectinase becomes a limiting factor and the volume of juice extracted does not depend on it anymore.

The graph above represents the variation of volume of juice extracted from red apples against the concentration (in volume percentage) of pectinase solution. The volume of juice obtained is plotted along the y axes as it is the independent variable and volume percentage is plotted along the x axes as it is the dependent variable. The reading at 0.00% pectinase is the data for the controlled set while the other points indicates the dependence of volume of juice on volume percentage of pectinase used. There is a significant difference of the volume of juice extracted from 5.20 cm³ to 20.60 cm³ as the concentration of pectinase increases from 0.0% to 10.0%. There is a sharp increase in the volume of juice from 20.60 cm³ to 33.4 cm³ as the volume percentage increases from 10 % to 20%. The data after this depicts a gradual increase from 33.4 cm³ to 62.6 cm³ as the volume percentage increases from 20% to 40%. The values at 50% and 60% stays constant at 63.4 cm³ . Thus, in general we can claim that the volume of juice extracted increases on use of pectinase and a gradual increase is observed as the volume percentage increases from 20% to 40% while becomes constant after that. Beyond the value of 40% (50% and 60%), the volume percentage of pectinase becomes a limiting factor and the volume of juice extracted does not depend on it anymore.

The above bar graph compares the average volume of juice extracted for both green and red apples against the volume percentage of the pectinase solution used. It is clear from the above graph that at the pectinase concentration being 10.0%, 30.0%, 40.0%,50.0% and 60.0%, the volume of juice extracted from green apples is much greater than that from red apples. Hence, if we ignore the controlled variable and the value at 20.0%, we can claim that for the same concentration of pectinase the volume of juice extracted from green apple will be greater than that from red apples. The difference between the volume of juice between green and red apple is much greater in case of the pectinase solution being 40.0%,50.0% and 60.0%. In reference to graph-1 and graph-2 , we have noted that in case of both green and red apples the volume of juice reaches maxima at 40.0% and becomes constant after that. Hence, we may claim that, the maxima value of volume of juice extracted is much more in case of green apples than in case of red apples.

Is there any correlation between the volume of juice extracted and the volume percentage of pectinase solution used?

Both graph-1 and graph-2 were plotted using Ms-Excel and the magnitude of correlation coefficient (R² ) was calculated using the More Trendline option. The value of R² obtained for the red apple and the green apple are 0.906 and 0.974 respectively. This value confirms that in both the cases there exists a weak positive correlation between the volume of juice extracted and the volume percentage of pectinase used. The correlation is relatively stronger in case of green apples than that in case of red apples as confirmed by the higher value of R² for it.

To study that if there is any significant difference between the volume of juice extracted from red apple and green apple or not, one way ANOVA Test at a significance level (α) of 0.05 was performed.

Null hypothesis(H0): There is no significant difference between the volume of juice from red apple and green apples

Alternate hypothesis(H1): There is a significant difference between the volume of juice from red apple and green apples

Total number of values(N) = 7 + 7 =14

Number of values under each category (n) = 7

Number of variables (a) = 2

df  _between  =  a -1 =2-1=1 

df  _within =  N-a = 14-2=12

df  _total  = N-1 =14-1 =13

Decision rule Critical value will be at (1,12). The critical value is 4.74

Test statistic

SS  between = \(\frac{(237)^2+(242.40)^2}{7}\) - \(\frac{(237+242.40)^2}{14}\)= 2.08

SS  within = \(\sum(y)^2\) - \(\frac{(237)^2 + (242.40)^2}{7}\)= 8027.85

SS  total = \(\sum(y)^2 \) -  \(\frac{(237) + (242.40)^2}{14}\) = 8029.93

MS between = \(\)\(\frac{2.08}{1}\) = 2.08

MS within = \(\frac{8027.85}{12}\) = 668.98

F = \(\frac{2.08}{668.98}\) = 0.003

As F (0.003) is smaller than the critical value (4.74), we cannot reject the null hypothesis. Thus, the alternate hypothesis is rejected. Any significant difference between the volume of juice extracted from red apple and green apple is merely an outcome of an error or by random chance.

The basic aim of the investigation was to answer the research question-

Does the volume of juice extracted from red and green apples using the enzyme pectinase depends on the concentration of pectinase used, determined using volumetric analysis?

• In general we can claim that the volume of juice extracted increases on use of pectinase and a gradual increase is observed as the volume percentage increases from 20% to 40% while becomes constant after that. Beyond the value of 40% (50% and 60%), the volume percentage of pectinase becomes a limiting factor and the volume of juice extracted does not depend on it anymore. Analysis of both Graph-1 and Graph-2 supports this.
• In reference to graph-1 and graph-2 , we have noted that in case of both green and red apples the volume of juice reaches maxima at 40.0% and becomes constant after that. Hence, we may claim that, the maxima value of volume of juice extracted is much more in case of green apples than in case of red apples. (Refer to Graph-3).
• The value of correlation coefficient R² (red apple-0.906 and green apple-0.974 ) confirms that in both the cases there exists a weak positive correlation between the volume of juice extracted and the volume percentage of pectinase used. The correlation is relatively stronger in case of green apples than that in case of red apples as confirmed by the higher value of R² for it.
• As confirmed by ANOVA test, there is no significant difference between the volume of juice extracted from red and green apple.
• The qualitative observation (darkening of colour from pale yellow to brownish yellow) confirms the fact that the volume of juice extracted increases with the increase of pectinase concentration.

• The standard deviation values are minimal and thus indicates the preciseness of the values collected.
• The methodology of the investigation done is repeatable and reliable.
• Conclusions made are supported by graphical analysis.
• Statistical tests are used to establish and investigate the correlations.

• The uncertainty of apparatus used especially the graduated measuring cylinder is a major source of random error in the investigation. Use of more precise apparatus like burette instead of it would have been better. To optimise this, data has been collected in five trials and average values are considered.
• The optimum pH and temperature of pectinase depends on the source of pectinase. The investigation has been conducted at a temperature of 60.0^oC and we cannot sure about whether it was the optimum temperature or not as the source of pectinase used is unknown. The pH was not maintained at the optimum level. A buffer solution could have been used for this.
• The filter paper used in the investigation might interfere with the volume of juice extracted as there is a high chance that part of the juice formed could have been absorbed by the filter paper used.

Apple juice develops color because of the presence of a flavonoid pigment. I would like to vary the pH at which the pectinase treatment of apple juice is done and measure the quantity of the pigment in the juice using spectrophotometry. This would enable me to establish a quantitative relationship between the pH at which enzyme treatment is done during the making of juice and the yield of it. This can provide a more accurate and reliable value of optimum pH. The same investigation can be done with temperature as well.

“Brent Cornell.” BioNinja, ib.bioninja.com.au/standard-level/topic-2-molecular-biology/25- enzymes/enzyme-catalysis.html. Accessed on August 7, 2019 11.30 am

“The Effect of Substrate Concentration on Enzyme Activity.” Untitled Document, www.ucl.ac.uk/~ucbcdab/enzass/substrate.htm. Accessed on July15,2019 1.12 am

“Enzymes.” Chemistry for Biologists: Enzymes,http://www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm. Accessed on July10, 2019 1.30 pm

Heena Verma1, Lokesh K Narnoliya2 and Jyoti Singh Jadaun. “Pectinase: A Useful Tool in Fruit Processing Industries.” Nutrition and Food Science , 7 Mar. 2018, doi:10.19080/NFSIJ.2018.05.555673. Accessed on August 12, 2019 7.30 am

Vinod Joshi, et al. “Purification, Characterization of Pectinase Produced from Apple Pomace and Its Evaluation in the Fruit Juice Extraction and Clarification.” Accessed on July 21, 2019 11.45 am