Unit: Biotechnology (AH): Food Industry: Fruit Products Title: Fruit Juice Production

TEACHER/LECTURER GUIDE
Type and purpose of activity
This experiment can be used to: provide evidence for assessment of Outcome 3 (For advice on marking Outcome 3 report, please contact the SAPS Scotland office.) develop knowledge and understanding of the use of cellulases, pectinases and amylases in the production of fruit drinks develop problem solving skills and in particular Outcome 2 PCs: (c) conclusions drawn are valid and explanations given are supported by evidence (d) experimental procedures are planned, designed and evaluated appropriately.

Background information
Fruit juice companies use a variety of different treatments and enzymes to maximise their yield. The enzymes which are used are designed to break down the cells walls within the fruits and release the liquids and the sugars which make up the fruit. Pectinases, amylases and cellulases all break down different structures of the fruit cells and so affect the extraction process in different ways. During breakdown of the fruit cells a variety of polysaccharides are found within the juice extract, these can cause the juice to become cloudy and reduce its market value. Pectinases and amylases can both break down these insoluble compounds releasing soluble sugars which clarify the juice producing a clearer, sweeter product. In this experiment students will investigate the effectiveness of these different enzymes. Note that the experimental procedure and recording of results in this practical is very straightforward. For Outcome 3 purposes, emphasis should be placed on the evaluation in the written report.

Classroom management
Students can work individually or in pairs for this experiment. The experiment will take about 1 hour to complete. The experiment can be developed further by using the Supplementary Student Information Sheet to investigate different ways of re-using the enzymes.

Supply of materials
In order to satisfy the core skill in problem solving, students will be required to identify and obtain resources required for themselves. Further advice on supply of material is given in the Technical Guide.

Extension work
1. Different fruits and vegetables can be used. 1. The enzyme data sheets can be used to identify the optimum pH and temperature of these enzymes and this can be tested through experimentation. 1. The enzymes can be immobilised and continual reuse of the enzymes can be tested (see supplementary sheet). 1. Different methods can be used to investigate re-using the enzymes (see supplementary sheet). 1. A comparison can be made between these enzymes and other amylases, pectinases and cellulases. 1. Find out the optimum combination of enzymes for maximum yield. 1. Determine the effect that reducing the concentration of enzyme has on yield.

Unit: Biotechnology (AH): Food Industry: Fruit Products Title: Fruit Juice Production

References
NCBE booklet In a jam and out of juice. ISBN: 0 7049 1373 9 Available free from NCBEs website: http://www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/juice.html or from NCBE, Science and Technology Centre, Earley Gate, University of Reading, Whiteknights, READING, RG6 6BZ. Tel: 0118 987 3743 Fax: 0118 975 0140. This experiment was produced by the SAPS Biotechnology Scotland Project. Funding for the project was provided by SAPS, Unilever and The Scottish Office. Support was also provided by Edinburgh University, Quest International, Learning and Teaching Scotland, the Higher Still Development Unit and SSERC.

Unit: Biotechnology (AH): Food Industry: Fruit Products Title: Fruit Juice Production

TECHNICAL GUIDE
Materials required
Materials required by each student/group:
one apple knife Termamyl/Pectinex/Celluclast 3 four 10 cm measuring cylinder filter papers 4 small filter funnels timer 3 four 100 cm beakers four weigh boats stirring rod 5 cuvettes (or test tubes if suitable colorimeter is used)

Materials to be shared:
balance (accurate to 0.1 g) colorimeter For the SUPPLEMENTARY part of this activity in addition to the above materials:

Materials required by each student/group

enzyme Immobilisation Instruction Cards 3 200 cm 1.5% calcium chloride 3 10 cm 4% sodium alginate enzyme solution e.g. pectinex/celluclast distilled water 3 250 cm beaker 3 10 cm syringe 3 20 cm syringe tea strainer iron fillings bar magnet in a plastic bag

Preparation of materials
An ordinary eating apple should be used or tins of Heinz apple sauce. Plastic measuring cylinders and filter funnels are best for this activity. Termamyl/Pectinex/Celluclast can be obtained from NCBE, Science and Technology Centre, Earley Gate, University of Reading, Whiteknights, READING, RG6 6BZ. Tel: 0118 987 3743 Fax: 0118 975 0140 Enzyme data sheets are automatically supplied with the order. CARE!: Avoid direct skin and eye contact, wear eye protection and gloves. Enzyme powder can cause allergies. Do not allow any spillages to dry up. Wipe up spillages immediately and rinse cloth thoroughly with water.

Supply of materials
It is not appropriate to provide all equipment and materials in, for example, a tray system for each student/group. Equipment and materials should be supplied in a way that students have to identify and obtain resources. Normal laboratory apparatus should not be made available in kits but should generally be available in the laboratory. Trays could be provided containing one type of specialist equipment or materials.

Unit: Biotechnology (AH): Food Industry: Fruit Products Title: Fruit Juice Production

PREPARING FOR THE ACTIVITY

Read through the Student Activity Guide and consider the following questions.

Analysis of activity
What is the aim of the activity? What is being varied in the activity? What measurements are you going to make?

Getting organised for experimental work

Collect a data sheet for the enzymes Termamyl, Pectinex and Celluclast and familiarise yourself with the action and commercial use of these enzymes. In your group decide how the activity will be managed by allocating tasks to each member. For Outcome 3 it is important that you play an active part in setting up the experiment and in collecting results.

Recording of data
Prepare tables to record your group results. You should use a ruler, correct headings and appropriate units.

Evaluation
1. How effective were the tests which you used? 2. What were the limitations of the equipment? 3. What were the sources of error? 4. What possible improvements could be made to the experiment? 5. What factors other than the enzyme used might affect the volume of juice produced? 6. What factors, other than volume of juice produced, might be important in determining effectiveness of the procedures? 7. What ideas do you have for further work? 8. What is the economic importance of the process which you are studying?

Unit: Biotechnology (AH): Food Industry: Fruit Products Title: Fruit Juice Production

STUDENT ACTIVITY GUIDE

Introduction
Fruit juice can be extracted from a wide variety of fruits. This can be done by simply squeezing the fruits but it is more common to use enzymes to increase the volume of juice produced and the speed of extraction. The enzymes which are used, both commercially and in this experiment, break down the cell walls within the fruits and release liquids and sugars. Fruit cell walls are very complex molecular structures and to get the maximum breakdown of the compounds found in them, fruit juice companies use a variety of different treatments and enzymes to maximise the yield of juice. Fruit is made up of cells linked by middle lamellae which contain insoluble proto-pectin. Pectinase breaks down the pectin chains and therefore reduces its binding action. The cell walls are composed largely of cellulose and hemicellulose and cellulases weaken the cell walls and make it easier to extract the juice. As the breakdown of the fruit cells continues, a variety of polysaccharides are found within the juice extract. These can cause the juice to become cloudy and reduce its market value. Pectinases and amylases can both break down these insoluble compounds releasing soluble sugars which clarify the juice producing a clearer, sweeter product. Different combinations of enzymes are used with each different type of fruit.

Enzymes are expensive products and clearly juice manufacturers would wish to minimise their costs by using the enzymes at their optimum conditions and therefore maximising their effectiveness and re-using the enzymes where possible. You are going to set out an investigation to look at the effectiveness of these different enzymes.

Equipment and materials

Materials required by each student/group:
one apple knife Termamyl/Pectinex/Celluclast 3 four 10 cm measuring cylinder filter paper 4 small filter funnels timer 3 four 100 cm beakers four weigh boats stirring rod 5 cuvettes (or test tubes)

Materials to be shared
balance colorimeter

Unit: Biotechnology (AH): Food Industry: Fruit Products Title: Fruit Juice Production

Instructions
1. Cut up the apple into small pieces. 2. Divide up the chopped apple into roughly 4 equal parts and use the balance to weigh each part to equalise accurately and record the weight. 3. Place each quarter into a beaker and add 2 cm of pectinase to one beaker, 2 cm of cellulase to 3 3 another, 2 cm of amylase to a third and 2 cm of distilled water to the last beaker. CARE!: Avoid direct skin and eye contact, wear eye protection and gloves. Enzyme powder can cause allergies. Do not allow any spillages to dry up. Wipe up spillages immediately and rinse cloth thoroughly with water. 4. Stir each beaker and leave for 5 minutes. 5. Place beaker contents into a funnel and filter the apple juice into the measuring cylinders. 6. Record the volume of juice every 2 minutes and the final volume of juice. 7. Take a sample of each juice and measure its clarity by using a colorimeter. 8. Weigh the waste apple from each extraction. 9. Record your results in a table with suitable headings.
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Unit: Biotechnology (AH): Food Industry: Fruit Products Title: Fruit Juice Production

SUPPLEMENTARY STUDENT INFORMATION

Re-using enzymes Introduction
You are going to try immobilising the cellulase or the pectinase enzymes, or a combination of the two, to see if you can extract the enzymes from the waste apple and re-use them. You will probably already be familiar with the technique of enzyme immobilisation. If you cannot remember how to immobilise enzymes collect an Enzyme Immobilisation Instruction Card. Ideally you should work in a group of about 4 people and share out the 4 tasks shown in the following table.

Additional materials required

1.5% calcium chloride 4% sodium alginate distilled water 3 250 cm beaker 3 10 cm syringe 3 20 cm syringe tea strainer iron filings bar magnet in a plastic bag Enzyme/s Immobilise the enzyme/s Use the immobilised beads to extract juice and then remove the beads from the waste fruit and re-use them Compare your results with the results from the initial experiment. Enzyme/s plus air bubbles Immobilise the enzyme/s using a 20 cm syringe adding air bubbles to the enzyme/alginate mixture by passing it in and out of the syringe before placing into calcium chloride Use the immobilised beads to extract the juice from the fruit Place the waste apple into a beaker of water and see if the beads float and are easier to remove from the waste fruit Reuse the beads Compare your results with the results from the initial experiment. Enzyme/s and iron filings Add 3 g of iron filings to the enzyme/s Immobilise the enzyme/s and iron filings Use the immobilised beads to extract the juice from the fruit Place the waste apple into a beaker of water and use a magnet to try to remove the beads from the apple Reuse the beads Compare your results with the results from the initial experiment. Enzyme/s plus air bubbles and iron fillings Add 3 g of iron filings to the enzyme/s 3 Immobilise the enzyme/s and iron filings using a 20 cm syringe adding air bubbles to the enzyme/alginate mixture by passing it in and out of the syringe before placing into calcium chloride Use the immobilised beads to extract the juice from the fruit Place the waste apple into a beaker of water and use a magnet to try to remove the beads from the apple Reuse the beads Compare your results with the results from the initial experiment.
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Now work as a team to discuss the results of the 4 different experiments and further work which could be done to find out more about the action of these enzymes.

Unit: Biotechnology (AH): Food Industry: Fruit Products Title: Fruit Juice Production

ENZYME IMMOBILISATION INSTRUCTION CARD

Introduction
Enzymes such as pectinase are often used in industrial processes. At the end of the process the enzyme is often mixed up with the product and cannot be easily separated from it. Immobilisation is a method which traps the enzymes in a bead which can be more easily separated from the product. The method also means that it may be possible to reuse the enzyme once it has been separated from the product. This method is often used in a continuous flow system.

Equipment and materials

Materials required by each student/group
4% sodium alginate 1.5% calcium chloride enzyme solution e.g. pectinase distilled water 3 250 cm beaker 3 10 cm syringe tea strainer

Instructions
The enzyme is mixed with sodium alginate and then fixed in a solution of calcium chloride. 1. Draw up 3 cm of enzyme solution into a syringe (if you are using a combination treatment use 1.5 cm of each enzyme). 2. 3. 4. 5. Add 7 cm of sodium alginate to the same syringe and mix thoroughly in the syringe. Gently drop by drop release the contents of the syringe to a beaker containing calcium chloride solution and leave for 5 minutes to set. Place the beads in a small tea strainer and thoroughly rinse them in distilled water. Now place the beads in the apple puree, leave for 5 minutes and then place in the filter funnel.
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