The action of amylase and pectinase in varying amounts when clarifying cloudy apple juice

Background information: Amylases hydrolyses glycosidic bonds in polysaccharides such as starch and glycogen and converts them to dextrins or to maltose. Different enzymes in the group work in different ways on the ?-1,4 links and the ?-1,6 links within the polysaccharides. Amyloglucosidase hydrolyses the 1,4 links and the 1.6 links at the branches in the starch molecule. Terminal glucose units are removed from the end of the chain one by one rather than giving the dextrins or maltose immediately. The debranching agent pullulanase hydrolyses the ?-1,6 links at the branching areas in the polysaccharide. Amylase is used commercially to clarify fruit juices, wines and beer by removing suspended starch.

Amylase can be found in bacteria (Bacillus spp.) and fungi (rhizopus spp., Aspergillus spp. and Streptomyces spp.). Fungal amylases are used in the clarification of fruit juices, wines and beer where they remove suspended starch. In bread making amylases can yield more sugars from the starch in flour or from barley grains. The other important commercial use is the conversion of starch to sweet glucose syrups, which are usually, used ass sweeteners in the food, bread making and brewing industry. By altering the balance between Amyloglucosidase and the fungal ?-amylase different proportions of glucose and maltose can be produced. For fermentation, a higher proportion of glucose is useful whereas in jam and confectionary production a higher proportion of maltose is useful.

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Continued conversion of the glucose produces fructose which is sweeter than sucrose and glucose is as sweeteners for food and drinks. Pectin is a substance, which helps hold plant cell walls together. It is obtained from fungi, particularly species of Aspergillus and Penicillium. Other fungal pectinases are significant in the soft rotting of fruit and vegetables, which contributes to spoilage and decay. The biggest industrial use of pectinases is in the extraction and clarification of fruit juices. Pectinases break down pectin (polysaccharides) found in the cell walls, which clarifies the juice. As the fruit ripens the plant produces proteolytic enzymes, which convert the insoluble protopectin of the unripe fruit into more soluble forms, causing the fruit to soften. When the fruit is mashed and pressed the more soluble forms of pectin enter the juice making it cloudy and causing the colour and flavour to deteriorate.

They also increase the viscosity of the juice making it difficult to obtain optimum yields. Pectinase is added to crushed fruit such as apples and grapes to reverse the effects of pectin, so increasing the yield and improving the colour. Other liquids, which contain suspended pectin material such as vinegar and wine, are also clarified with pectinases. They do this by removing some of the pectin around charged protein particles, these then settle out of the liquid in a large cluster. When fruit juices are concentrated pectinases prevent them from gelling. So, pectinase is added which means a good clear juice is obtained which retains its stability when concentrated.

For my experiment I am going to the optimum mixture of amylase and pectinase to clarify apple juice. To do this the only thing I will alter is the volume of amylase and pectinase. I will keep the volume of apple juice the same each time and I will also keep the temperature the same each time. To make sure everything is at the same temperature I will put the amylase, the pectinase and apple juice all into a water bath at the temperature I decide on after my pilot experiments. Then add the amylase and pectinase in varying volumes (e.g. 9:1 ratio, amylase to pectinase) to test tubes containing 10cm� apple juice and put them back into the water bath at the designated temperature for a period of time determined by my pilot experiments. To aim to get the most accurate and representative results I will conduct some pilot experiments to find out what is the optimum temperature for amylase and pectinase to work and for how long they need to be left at that temperature. I will also try 3 different types of apple juice. In this way I can see which type of apple juice clarifies the best. If an apple juice does not clarify then I will not use it because it would not be giving me any results to discuss. To find out how much clarification has been taken I will use a colorimeter.

The colorimeter measures light intensity. So I will calibrate it using distilled water where distilled water is 10 arbitrary units, which is the clearest. The colorimeter has its own type of test tube when I calibrate the colorimeter I will mark on the test tube the position in which it lies so that any faults in the glass do not affect my results. I will also mark the 11cm� level up to which I have poured the distilled water. The amylase arrives as a powder and so I am going to mix it with water to make a 1% amylase solution. I will do one pilot experiment with everything at room temperature and when I have added the amylase and pectinase I will leave them for 15 minutes. I will repeat this using a 20-minute time interval. If I do not get results then I will do a pilot experiment using a water bath at 35?C and put the 1% amylase solution, pectinase and apple juice into the water bath for 20 minutes. Then I will add the amylase and pectinase and leave for another 20 minutes. If this is still unsuccessful then I will continue to use a water bath at 35?C and keep increasing the time intervals until I start to see some differences. Everything else will be kept the same.

Once I start the final practical the temperature will be kept the same, which is why I am using a water bath because it is more accurate as it is thermostatically controlled. The time intervals will be kept the same as I will use a stopwatch and everything will be in a test tube rack and so easy to put in and lift out of the water bath. The volumes will all be the same measure using a measuring cylinder and a syringe. The one thing, which will be changed, is the ratio of amylase to pectinase in the experiment. I will add 10cm� apple juice to a test tube. I will then add amylase and pectinase in their varying amounts to each test tube. The table below shows the different volumes of amylase and pectinase, which will be added to each test tube.

Amylase

Pectinase

Apple Juice

0.9 mm�

0.1 mm�

10 cm�

0.8 mm�

0.2 mm�

10 cm�

0.7 mm�

0.3 mm�

10 cm�

0.6 mm�

0.4 mm�

10 cm�

0.5 mm�

0.5 mm�

10 cm�

0.4 mm�

0.6 mm�

10 cm�

0.3 mm�

0.7 mm�

10 cm�

0.2 mm�

0.8 mm�

10 cm�

0.1 mm�

0.9 mm�

10 cm�

Each test tube will be labelled so I know what is in each. I think that a 1:1 ratio of amylase to pectinase will make the clearest juice because both amylase and pectinase clarify the juice. It may be possible that amylase is more efficient that pectinase or vice versa.

Equipment:

Colorimeter

10xTest tubes

Measuring cylinder

2x 100 ml beaker

Measuring balance

Amylase

Pectinase

Cloudy apple juice (3 types)

25ml syringe

2x5ml syringes

Water bath

Thermometer

Glass rod

Spatula

Test tube rack

Stopwatch

I am using 3 different types of apple juice to see from which one I get the best results. I will find this out during my pilot experiments and then I will use the one, which produces the best results in my actual experiment.

I am using a colorimeter because it measures light intensity and it is the most accurate piece of equipment available to me to measure the clarification of the apple juice. I will use the measuring cylinder to measure out the volume of distilled water to mix with the amylase. I will use one 25ml syringe to measure out 10cm� apple juice to put into each test tube. I will use one 5ml syringe to measure out the volumes of amylase to put into each test tube and I will make the plunger on it with an A so that I know it is the syringe used for amylase. This will avoid contamination. I will make the other 5ml syringe with a p on the top of the plunger where it is clearly visible and then I will use it to measure out the volumes of pectinase to add to each test tube. I am using a water bath because it can keep the water at a constant 35?C rather than a Bunsen burner and beaker full of water. I will use the glass rod to stir the 1% amylase solution to make sure all the amylase is dissolved and that there are no clumps of the powder at the bottom.

The 1% amylase solution will be made up by adding 0.1 of a gram into a beaker and then adding 0.9cm� water to the beaker and stirring. I will use the pan balance to weight out 0.1 amylase. I will do this by putting the beaker on the balance and then resetting it. By resetting the balance the weight of the beaker is taken into account and the display shows 0. Then I will use the spatula to put in 0.1 grams of amylase. I am using a metal test tube rack because it does not float in water. It means that I can rest the beakers on top of it and put the test tubes in it. This means that I can put them all into the water at the same time and remove them all at the same time. It also means there is no danger of them falling over in the water. I will use the stopwatch to time how long the experiment has been going. If I am using a water bath I will first put the apple juice, pectinase and amylase into the water bath so that they all warm up to the same temperature.

Then I will mix them. If I did not do this and mixed them and put them straight into the water bath they wouldn’t have time to reach the correct temperature because they are stored in a fridge and so are cold. If the time interval is 15 minutes then I will put them into the water bath for 15 minutes and then mix them and replace them into the water bath for another 15 minutes. In between experiments I will wash out all my equipment thoroughly so that no contamination occurs and for safety reasons. The syringes will all be clearly labelled and will not be used for any other things to avoid contamination.

The apparatus was set up as shown in the diagram below.

Risk assessment: Amylase can be an irritant so I will wear plastic goggles to protect my eyes and use a spatula when handling it and take care. If I spill any of it I will wash it off as soon as possible. When I put it into the beaker I will make sure the beaker is clearly labelled and I will also label the test tubes containing it. I will avoid inhaling it by being sensible around it. When handling pectinase I will wear eye protection. If I get any of it in my eyes I will wash it out. If I get any of it on my skin I will wash it off as soon as possible. I will avoid ingesting it but if I were to do so I would rinse my mouth and throat with water and drink water. If it were to catch fire I would use water or foam to put it out. As pectinase arrives in solution it is dust free. However dust can occur if it is inappropriately handled. Therefore if I spill it I will remove it immediately to avoid dust formation. I will make sure there is sufficient ventilation and wash any contaminated clothing. The safety sheet that comes with the pectinase is in the appendix at the back.

Number of readings

Once I have completed my pilot experiments and decided on what temperature and time interval to use I will do the experiment three times. Each time I will repeat the experiment exactly as I did the first one. I am repeating the experiment because then if I get some anomalous results I have another 2 sets to check them against. Once I have gained my results I will put them into a table like this.

Experiment 1

Colorimeter reading (arbitrary units)

Experiment 2 Colorimeter reading (arbitrary units)

Experiment 3 Colorimeter reading (arbitrary units)

0.9-0.1

0.8-0.2

0.7-0.3

0.6-0.4

0.5-0.5

0.4-0.6

0.3-0.7

0.2-0.8

0.1-0.9

What will be measured

I will be using a colorimeter to measure how much light passes through the apple juice. The information, which comes with the colorimeter, is in the appendix. The diagram below shows how it does this.

Pilot Experiment Results

To start with I decided that I would see which apple juice gave me the best results. I decided to do it at room temperature because I knew at that temperature amylase and pectinase work (although it is not their optimum temperature). I decided to use a time of 30 minutes because I thought that would be long enough for some clarification to happen. I set the colorimeter up with distilled water at 0 arbitrary units. This meant that 0 was the clearest it could be. So, the cloudier it was the higher the arbitrary units. The results are shown in the table below.

Tesco Apple Juice

Colorimeter reading (arbitrary units)

Copella Apple Juice

Colorimeter reading (arbitrary units)

Tesco English Apple Juice

Colorimeter reading (arbitrary units)

0.9-0.1

6.2

10

7.2

0.8-0.2

6.1

10

7.4

0.7-0.3

6.2

10

7.3

0.6-0.4

10

7.4

0.5-0.5

10

7.6

0.4-0.6

6.2

10

7.5

0.3-0.7

6.2

10

7.5

0.2-0.8

6.2

10

7.4

0.1-0.9

6.2

10

7.3

The results for these pilot experiments were not encouraging. During the experiment involving Tesco Apple Juice I spilled the 0.6-0.4 and 0.5-0.5 test tubes and so could not get results for them. The copella apple juice did not clarify at all. The Tesco English Apple juice did have some variation in the results. This is why I choose to continue the rest of my pilot experiments using Tesco English Apple juice.

I decided to see if maybe the apple juice was too cloudy. So I made up 50%distilled water and 50% apple juice solutions and did the experiment as normal at room temperature leaving 30 minutes for the reaction to take place. The table below shows my results for that.

50:50 water Tesco English Apple Juice

0.9-0.1

3.2

0.8-0.2

2.7

0.7-0.3

2.6

0.6-0.4

2.65

0.5-0.5

2.7

0.4-0.6

2.8

0.3-0.7

2.75

0.2-0.8

2.8

0.1-0.9

2.55

When looking at this and the other experiment involving Tesco English Apple Juice then both had results, which didn’t seem to be in any particular order. I decided not to continue using 50:50 water-apple juice solutions because it did not seem to make a huge difference except that they started off clearer. I decided that at room temperature I was not getting the results I needed. Room temperature was about ?C so I decided to use 35?C water bath because in “Molecules and Cells” by J.Adds, E.Larkcom and R.Miller it states that the optimum temperature for pectinase was 35?C however the optimum temperature for ?amylase is 95?C. I decided to continue with the use of 35?C because over 35?C then the pectinase enzyme would denature, however, below 95?C the ?amylase enzyme woul not denature but it would not be as effective. I decided at first to keep with the 30-minute time.

However, because everything needed to be warmed up to 35?C I decided that I would put the apple juice into the test tubes. Put the 1% amylase solution into a beaker and the pectinase into another beaker and then put them into a water bath for 15 minutes. This would allow them all to reach the same temperature. Then after 15 minutes I would take them out and mix them and then put them back in for another 15 minutes. This would mean that the enzymes had reached their optimum temperature for working and then had been giving 15 minutes to work. My results for this are shown below.

15 minute time intervals

Colorimeter reading (arbitrary units)

0.9-0.1

2.65

0.8-0.2

2.55

0.7-0.3

2.55

0.6-0.4

2.55

0.5-0.5

2.6

0.4-0.6

2.65

0.3-0.7

2.6

0.2-0.8

2.8

0.1-0.9

2.8

The results shown here showed me that there was not that much clarification going on. However, it did seem to suggest that a larger proportion of amylase was needed for maximum clarification. I decided to repeat the experiment but this time using 20-minute time intervals with an overall time of 40 minutes. The results are show below.

20 minute time intervals

Colorimeter reading (arbitrary units)

0.9-0.1

2.8

0.8-0.2

2.5

0.7-0.3

2.5

0.6-0.4

2.65

0.5-0.5

2.7

0.4-0.6

2.65

0.3-0.7

2.7

0.2-0.8

2.75

0.1-0.9

2.8

Apart from the 0.9-0.1 the results seem to show a similar trend to those of 15-minute time intervals with more amylase giving more clarity. I decided to use 25-minute intervals and the results for these are shown below.

25 minute time intervals

Colorimeter reading (arbitrary units)

0.9-0.1

2.9

0.8-0.2

2.8

0.7-0.3

2.75

0.6-0.4

2.6

0.5-0.5

2.7

0.4-0.6

2.7

0.3-0.7

2.7

0.2-0.8

2.75

0.1-0.9

2.9

These results seemed to show that an equal mix of amylase and pectinase was best. Looking back on the result I got for 15 and then 20 minutes I decided that I would use 25 minutes as my time interval and 35?C as my temperature because at 35?C with 25 minute time intervals I got the best results which showed a trend.

Results

Below is my table of results for my three experiments.

Experiment 1

Colorimeter reading (arbitrary units)

Experiment 2

Colorimeter reading (arbitrary units)

Experiment 3

Colorimeter reading (arbitrary units)

0.9-0.1

2.9

2.9

6.4

0.8-0.2

2.8

2.8

6.6

0.7-0.3

2.75

2.7

6.4

0.6-0.4

2.6

2.6

6.4

0.5-0.5

2.7

2.7

6.6

0.4-0.6

2.7

2.7

6.4

0.3-0.7

2.7

2.75

6.4

0.2-0.8

2.75

2.75

6.4

0.1-0.9

2.9

2.9

6.4

Below is a graph of my results.

Graph 1 shows more clearly the sudden drop in cloudiness towards the more equal mixtures of amylase and pectinase. Graph 2 shows the results of experiment 3 plotted on it as well. Experiment 3 produced some anomalous results, which I shall analyse in my conclusion. The table below is a seummary table of experiments 1 and 2. I have not included experiment 3 in this table because of it’s anomalous results and so it would give me extremities not reflecting my true results. Hence for 0.9:0.1 the average would become 4.066666666�

Amylase:Pectinase ratio

Summary table of experiments 1 &2

0.9:0.1

2.9

0.8:0.2

2.8

0.7:0.3

2.725

0.6:0.4

2.6

0.5:0.5

2.7

0.4:0.6

2.7

0.3:0.7

2.725

0.2:0.8

2.75

0.1:0.9

2.9

Conclusion

The ration which clarified the apple juice best was the 0.6cm� amylase to 0.4cm� pectinase. I think this is because the apple juice already had some pectinase added to it to increase the yield of the juice whilst still keeping some of the cloudiness. If the apple juice had been cloudy because of a mixture of pectin and starch the best mixture of pectinase and amylase would have been a 50:50 ratio. This is because if the was more amylase in the mixture it would have cleared the starch but it would not have cleared the pectin. The same goes for if there was more amylase in the mixture, however, a 50:50 mixture of amylase and pectinase would have cleared both starch and pectin.

Evaluation

I thought that I would be seeing a huge difference in clarity of the apple juice before and after. However, this was not so. This could have been for a number of reasons. Many different enzymes are added as part of the clarification process. It is possible that amylase and pectinase had already been added. Pectinase is used to increase the yield of fruit juice from crushed fruit so that may be a reason why pectinase had been added. If I had used some different enzymes, such as protease then I might have found that the clarification process was much quicker and there was a visible result.

I do not know what the reason for the anomalous results in experiment 3 could be. They might have been contaminated in someway or the colorimeter may not have be calibrated properly. To improve my experiment I could spend more time on the pilot experiments trying different apple juices. I could also try different temperatures. I could have tried using a stronger amylase solution such as a 2% or 10% amylase solution which may have clarified the apple juice more. If I had found out what solution the pectinase was (e.g.1% pectinase solution) I could have made the amylase up to that figure as well so they were both equal. Because I used the same equipment for each experiment, although I washed them out contamination could still have occurred. A way to avoid contamination could have been to use boiling water to wash the equipment. When using the colorimeter it came with 3 small test tubes. I decided to use only one of these test tubes. This was because if there were any faults in the glass of any of the test tubes it would have affected the results. I also marked on the test tube the spot where it faced the spot on the colorimeter.

Each time I made sure these dots aligned up to each other. However, because I used the same test tube contamination could easily have occurred if I did not wash it out properly. When I used the colorimeter to examine the apple juice the other test tubes of apple juice were left next to the colorimeter. This means that they had extra time for the enzymes to work. To avoid this I would have had to stagger putting them all into the water bath and then getting them out. In my experiments I did not check that the pH was constant. If for some reason the pH had changed it would have affected enzyme activity. This could help to explain the anomalous results in experiment 3. For another experiment to ensure the pH was kept the same I could use a pH buffer. The enzyme amylase works best at a pH of 7.0 and the enzyme pectinase works best at a pH of 5.5.

As amylase works at an optimum temperature of 95?C then using it at 35?C would mean my results would be affected by it’s inactivity. So, to alter this I would have to make slight changes to a future experiment. I would suggest heating the pectinase at 35?C and the amylase at 95?C in separate water baths. Then I would add pectinase to apple juice at 35?C and leave it for 30 minutes. Then I would add amylase at 95?C to the same apple juice at 95?C and leave it for 30 minutes. I could do this using the different volumes of enzymes. For other future experiments I would use a pH buffer, to get an optimum at which they would both work I would suggest a pH buffer of 6.0. I would also stagger my experiments, for example, putting the 9:1 test tube into the water bath first and taking it out first with a time gap sufficient enough to test its clarity using the colorimeter and be able to get the next test tube out of the water bath on time.

I used distilled water to set up the colorimeter, I could have gained more accurate readings if I had used pure apple juice to set it up. The problem with using distilled water is that it is already clear, I calibrated the colorimeter so that distilled water was at 0, 0 being the most clear. If I had used apple juice and set that at 10, 10 being the most cloudy the following results I had got would have had a larger difference because compared to apple juice before the experiment they had cleared. So for a following experiment I could set the colorimeter up using pure apple juice.

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