The Effect of Enzymes on Apple Juice Production

Background Info The purpose of this experiment was to determine which enzyme or the combination of the two enzymes makes the most apple juice from applesauce. We did his by, mixing 10 drops of an enzyme (cellulose/pectinase/both) into a few tablespoons of applesauce. We then left the rest to nature, and watched the liquid funnel out for 10 minutes. Lastly, we recorded our data.

The cell wall is a complicated structure containing both cellulose and pectin. Pectin is found in the cell walls of plants and is also specifically found concentrated near the skin and core of fruit. It is what keeps the fruit together and prevents it from getting mushy. Cellulose is an organic compound also found in the structure of plant cells, in the outer cell wall. It is basically the structure of the cell wall.

An enzyme is a biological catalyst meaning it speeds up chemical reactions in living things. Enzymes are made from amino acids and are proteins. The purpose of speeding up chemical reactions is that the cell is able to build things and take things apart quickly and efficiently. All the work carried out in a cell is carried out by enzymes. However if an enzyme is introduced to the wrong temperature, pH, or concentration, they denature, and are no longer usable. Different enzymes break down and build different things. It all depends on the shape of the enzyme.

The common term for the way enzymes work is the Lock and Key model. The Lock and Key model compares enzymes and its substrate to a lock and a key. A substrate is the biological molecule that the enzymes work on. During this process, the enzyme grabs or "locks" on to the substrate at a special area called the active site. The active site is very specially shaped and only will work on substrates that match.

Once everything is all set in place, the enzyme breaks down the substrate or combines it with something else to make something new. When this process is over, even though the substrate was changed the enzyme stays exactly the same and goes to do the whole process over again. This way the body won't waste as much energy on making enzymes over and over again when it can just make a few and save the energy to make more products instead.

The enzymes we used in this experiment had many functions. The first enzyme we used was pectinase. The function of pectinase was to break down its substrate pectin, in the applesauce so it would hopefully produce apple juice. Pectin is a polysaccharide, so by adding pectinase the units are broken down into monosaccharides. In the same way cellulase is the enzyme used to break down cellulose.

The function of cellulase in this experiment was to see if the breakdown of cellulose affected the production of apple juice. Cellulose is also a polysaccharide, and must also be broken down into single monomers. The main purpose of both these enzymes in this experiment was to see which substrate if broken down would allow the applesauce to produce more apple juice.

II. Statement of Problem and Hypothesis

Problem: What enzyme produces the most apple juice?

Hypothesis: If pectinase is added to 2 tablespoons of applesauce, then it will produce the most apple juice compared to cellulose. I know this because is also used for clarifying/producing apple juice in the first place. Also, its substrate pectin is what holds the cell wall together, therefore the enzyme that breaks it down (pectinase) must produce the most apple juice.

III. Experimental Design

Independent Variable: Enzymes (pectinase, cellulase, combination of both cellulase and pectinase)

Dependent Variable: Amount of apple juice produced

Constants:

  • Amount of applesauce
  • Stirring time • Amount of each enzyme (pectinase, cellulase, both)
  • Observing Time (or time taken for apple juice to funnel out)
  • Water temperature
  • Funnel size

Control: Water

IV. Materials and Procedures

Materials:

  • 8 tablespoons of applesauce
  • 15 drops of enzyme (pectinase)
  • 15 drops of enzyme (cellulase)
  • 4 plastic cups
  • 4 coffee filters
  • 4 funnels
  • 4 plastic spoons
  • 4 plastic beakers for mixing
  • Markers for labeling
  • 3 droppers
  • 4 stir sticks
  • 4 graduated cylinders
  • Access to tap water
  • Access to clock/timer
  • Paper towels for clean up

Procedure:

1. Assemble 4 filtration cups

▪ Roll coffee filter into funnel

▪ Place funnel on top of graduated cylinder

▪ Make sure to label each graduated cylinder with: water, cellulase, pectinase, and cellulase+pectinase

2. Assemble mixing cups:

▪ Label ALL cups with: water, cellulase, pectinase, cellulase+pectinase

3. Make sure to label all mixing spoons/droppers too.

4. Spoon 60 g (2 tablespoons) of applesauce into each of the 4 mixing cups

5. Add one of the following to each cup of applesauce (MAKE SURE TO LOOK AT YOUR LABELS):

▪ 10 drops of pectinase. Stir 3 seconds. Pour into filtration cup

▪ 10 drops of cellulase Stir 3 seconds. Pour into filtration cup

▪ 5 drops of cellulase and 5 drops of pectinase Stir 3 seconds. Pour into filtration cup

▪ 10 drops of water Stir 3 seconds. Pour into filtration cup

6. Allow mixtures to filter for 10 minutes

7. Once ten minutes are complete, remove filters and measure the juice collected in the graduated cylinder.

8. Remember to record your data in a table.

V. Results:

Observations: During the 10 minutes of filtration, I observed that the apple juice came out of the pectinase faster than the rest of the enzymes. During the last few minutes of observing, the applesauce with pectinase was still producing apple juice, only slowing down a little bit, while the others had slowed down drastically or had stopped completely. The flow of the apple juice from the pectinase was a lot smoother and clearer compared to the rest of the enzymes. Overall, the pectinase produced the most apple juice.

VI. Conclusion:

Data Analysis: In the end, my hypothesis was accepted. I believed that if pectinase was added to the apple sauce, then it would produce more juice compared to the rest of the enzymes. The enzyme pectinase was used in my hypothesis because, from some background research it was found out that pectinase is used in making juice anyway. Pectinase is responsible for the breakdown of pectin, the substance that holds a cell together. I figured that if pectin was found in the cell wall, and pectinase broke pectin down, then if pectinase was to be added to a fruit the fruit would most likely fall apart.

The results conclude that more juice was produced with an enzyme than without one. Compared to our control (water) the enzymes, on average produced more juice. The cellulase produced 3.2 mL more juice than the control, and the pectinase produced as much as 20.2 mL more! And the combination of both of the enzymes came in the middle and produced 10 mL more of apple juice than the control (water). In the end, however pectinase produced the most apple juice. At the end of the 10 minutes, pectinase (on average) produced 26.4 mL.

Compared to the cellulase, it produced 17 more milliliters of apple juice, and compared to the combination of both enzymes, it produced about 10.2 mL more. The reason we used water as our control was because water doesn't contain any enzymes in it. Because it was tap water, there are already traces of minerals and small traces of enzymes in the water, for it isn't distilled. Overall, the water did not produce more apple juice than the enzymes. The water on average produced 6.2mL of apple juice. This is because even though it was tap water, and there might have been some traces of enzymes in the water itself, it probably wasn’t enough to break down the substrate as much as the direct doses of pectinase and cellulase.

The water may have diluted whatever traces of enzymes there were in it, and therefore it may have not been as strong. From previous research we know that pH and concentration affects enzyme function, and it is possible that the water could have altered the pH level of the enzyme and the concentration of the enzyme. In order to mame the results more accurate thse results were compared to the ones of another scientist. The overall result was basically the same. In this scientist's experiment there were a few differences in the procedure.

For example, they had used a little less than 2 tablespoons of applesauce in their experiment, and they only used 5 drops of cellulase and pectinase, instead of the 2 tablespoons used our experiment and 10 drops of cellulase and pectinase. They had also not tested the production of apple juice when both cellulase and pectinase were added into the applesauce. This may have been why their numbers were a little lower. For example for pectinase they had gotten about 15.3mL of apple juice, while in our experiment we had gotten 26.4mL of apple juice. Overall however, compared to the other scientist our results were almost identical.

Validity: During the experiment, there were a few things that shouldn’t have been done and that could have affected the outcome. Things like this are known as experimental errors. During the experiment, I had used two different sized funnels, which could have allowed the juice to flow at a faster rate in the larger funnel than compared to the smaller funnel. Another experimental error was the timing. When I did the times, I didn't wait 10 minutes, however I only waited about 5, and when I say "about" I mean "about".

The waiting time was more or less estimated, and that could have affected my data because one enzyme could have gotten more time to break down its substrate than the other and vice versa. Lastly, another experimental error was the temperature of the water itself. The water was a bit warmer than usual, and as we know from our research temperature can affect the function of an enzyme. Even though the water shouldn't have really done anything, it did, and this may have been a cause of that error.

Improvements and Future Studies: A way to improve this experiment if it were to be done again or used in the future, would probably be to make the procedure a little more clear. A lot of it had unclear instructions, and the chance for error is high. Also, the materials list didn't make much sense and added a lot of extra materials which made the proicedure even more confusing because there were so many types of cups that you didn't even need and then when deciding which cup to use, it just made everything even harder. Another thing that should be changed would be to use distilled water.

Even though the tap water is "water", it still has minerals and traces of enzymes, while distilled water had absolutely nothing but water in it. Its pH is right in the middle, so that wouldn't affect the data either. However the improvement that is high priority right now, is to make the procedure more clear.

Works Cited:

  • "Background Information." In a Jam out of Juice. 1st ed. N.p.: Unilever Plc., n.d. 4-5. Print. "Cellulase."
  • Wikipedia. Wikimedia Foundation, 12 June 2012. Web. 05 Jan. 2013. "Cellulose." Wikipedia. Wikimedia Foundation, 01 Jan. 2013. Web. 05 Jan. 2013. "Pectin."
  • Wikipedia. Wikimedia Foundation, 12 Sept. 2012. Web. 05 Jan. 2013. "Pectinase."
  • Wikipedia. Wikimedia Foundation, 28 Dec. 2012. Web. 05 Jan. 2013. White, Minta. Background Info on Apple Juice Lab-Reading. N.d. Enzyme Review. Hamden High School, Hamden.
  • Apendices: (Attached) Diagram of filtration Background Info Packet In a Jam out of Juice Other scientist's apple juice lab