Comparing the yields of fruit juice produced from different types of fruits using Pectinase.

Background Knowledge:

Fruits are made of a relatively simple plant cell, with thin walls made of two layers. The primary cell wall is made with cellulose fibres surrounded by a matrix of pectin, hemi cellulose and proteins. The second and inner wall is similar but contains less pectin. Between the two layers is a binding layer of pectin. The complete removal of this layer causes the tissues to fall apart. This is the reason for why fruits and vegetables become soft over a period of time. Long chains of pectin are broken down by digestive enzymes produced by bacterium such a “soft rot”.

Cellulose is also vulnerable to enzymatic attacks especially in the primary cell wall as its arranged at random compared to the stronger regular pattern form in the second cell wall. Breakdown of cellulose leads to the breakdown of cell walls and eventually their disintegration. As fruit ripens it becomes soft, there are two reasons for this softening firstly that the softening is caused by the cellulase acting on the cell walls, or pectin binding layer changing. In unripe fruit, pectin is bound to cellulose in the cell walls. This type of pectin is insoluble and the liquid within the cell remains fluid. The fluid asserts pressure on the cell walls making the fruit seem hard because of the cell walls rigidity.

Enzymes are large globular molecules of which the vast majority are made of protein.

All globular proteins have a tertiary structure, which is maintained by hydrogen bonds, ionic bonds and sometimes disulphide bonds, which are links between different parts of the polypeptide chain. Enzymes have catalytic properties; in other words, they alter the rate of reaction without themselves undergoing a permanent change. Most chemical reactions require an initial input of energy, called activation energy, to enable them to occur. Enzymes reduce the need for activation energy and so allow reactions to take place more readily and at lower temperatures than what would otherwise be necessary.

The main factors affecting the rate at which an enzyme works are;

pH level; enzyme activity is largely based upon the shape of its active site as the substrate has to match the active site for a reaction to take place. The affect of pH can alter the ionic and hydrogen bonding of the enzyme, leading to a change in the shape of the active site, therefore each individual enzyme has its optimum pH at which it works best.

Temperature; according to the kinetic theory as temperature increases molecules gain more energy resulting in their faster movement. Therefore in context with enzyme activity, the faster the movement of molecules the more often the collisions between the substrate and enzyme will occur over all increasing the rate at which the product is formed. However as before if temperature continues to rise the hydrogen and ionic bonds, which hold the enzyme in shape, will break and lead to the denaturisation of the enzyme. Therefore each enzyme has its optimum working temperature.

The concentration of either the enzyme or substrate; ...

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The concentration of either the enzyme or substrate; by having a higher concentration of substrate molecules there is a higher chance for the enzyme to collide successfully with the substrate and consequently speeding up the rate of the reaction. Increasing the concentration of enzymes may result in the rapid increase of the rate of reaction and the graph may level off very quickly as the reaction takes place as such a high speed.

Hypothesis:

My hypothesis is that a fruit with a skin and with high pectin concentration will have a higher yield of fruit juice.

Pectinase is used in the break down of pectin in fruits. Pectins are large polysaccharide molecules mainly made up of several hundred galacturonic acid residues. Pectins are well known for their ability to form gels. Pectinase breaks down the pectin in fruits. All fruits contain pectin in the skin and in lesser concentration in the pulp. The amount of pectin for each fruit varies with the type of fruit it is and at what stage of maturity the fruit is at. As the fruit matures the amount of pectin decreases.

Pectin holds the cell walls in fruits together like a "glue" and results in the formation of thick gels during pulping. The addition of Pectinase at the pulping stage increases the yield of juice as the Pectinase degrades the soluble pectin in the pulp not allowing it to form such thick gels. This allows the juice of the fruit to flow more freely and therefore resulting in a larger yield of fruit juice.

During ripening of the fruit the pectin chains begin to break down. As a result of which pectin becomes more soluble and it loosens its grip on the surrounding cell walls, so the plant tissues soften.

This suggests that it would be easier to press juice out of a mature fruit compared to a freshly ripened fruit or unripe fruits. But still there are difficulties as the pectin becomes soluble in water, some of the pectin molecules are released into the juice and so the juice becomes thick and gel like so difficult to squeeze out. However the higher the pectin concentration is in a fruit the more it will be affected by pectinase, because if the pectin concentration in a fruit is low then the juice will flow readily without adding pectinase and if pectinase is added then the difference in the yield produced will not be of a great significance. However if a large quantity of pectin is present in a fruit then a larger yield will be produced by breaking down the pectin, therefore I think that the higher the pectin concentration of the fruit the more the effect there will be on the yield of fruit juice obtained using pectinase.

Outline method:

From previous experiments I know the importance of a control for each fruit sample i.e. replacing the 2cm3 of pectinase with 2cm3 of distilled water. This will enable the results to illustrate that the result was influenced solely by the presence of pectinase. Measure the volume of fruit juice produced by the presence of pectinase and the control. Calculate difference in volume produced, the difference should illustrate the effect of the pectinase if the difference is significant than pectinase does have an influence upon the volume of fruit juice from the same volume of fruit puree.

Key variables:

The temperature of the surrounding area in which the enzymes are working at have to be kept constant.

The concentration of Pectinase has to be kept constant for all tests.

The amount of fruit on which the Pectinase is working on has to be kept constant.

Using a volume of 40g of different fruit purees, so that the enzyme Pectinase has an equal amount of surface area to work on.

For each experiment use an equal amount of 2cm³ of 1% Pectinase solution.

Use a consistent temperature in the water bath at 45°C to keep the surrounding atmospheric temperature at the optimum level for the Pectinase to work at. (ref: bibliography1)

Incubate the two test tubes in the water bath for 2 minutes to equilibrate temperatures.

Time over a total period of 30 minutes.

Filter the fruit juice substrate through filter paper and measure the amount of fluid produced.

Repeat each experiment 3 times at the least to enable more reliable results.

As a control experiment add 2cm ³ of distilled water in place of the 2cm³ Pectinase solution to act as a control for the fruits. This will be able to conform that the excess fruit juice yield produced is caused by the use of Pectinase.

For each fruit tested upon a control will be produced and therefore the difference between the controls juice yield and experiments juice yield will show the increase in the yield by using Pectinase.

Risk assessment:

When using Pectinase solution at 1% because of the danger that some may splash into the eye and because enzymes breakdown substances they will attack the eye, so it is essential that when carrying out experiments to wear goggles. If in the case that the enzyme is splashed into the eye the eye should be washed out under a gentle running tap of cold water and seek medical attention as soon as possible.

Wash any spillages of enzyme solution from the skin with water as soon as possible, wash skin with running cold water, if irritation persists or a rash forms then seek medical attention.

Juice prepared in this way should NOT be consumed. The proportion of enzyme used is far greater than that used in commercial production.

Pupils should take care when they are handling knives to chop the apple and using the blender if they are making the puree themselves. Enzyme spills should be wiped up promptly and the area washed with water.

Care should be taken when using the water bath, as using electrical equipment in the presence of water can be dangerous, dry hands before inserting the plug, do not put hands into water bath encase the temperature is high.

Pilot Experiment:

The pilot studies will be used to make sure that the amount of fruit puree and amount of pectin is efficient enough to produce a measurable result and give results within my intended time span of 30 minutes.

To test this the intended method will be put to the test and results will be recorded.

Also I need to check whether the fruit puree alone will produce a significant fruit produce, as I consider that the fruit puree will require some water to gain a better consistency for the enzyme to work on. Therefore I will carry out two pilot studies one to check if the method considered is appropriate and will produce a analysable set of results and then a further pilot study to see how the consistency of the fruit puree effects the results.

Results Of Pilot Test:

1st pilot study testing how the consistency of the fruit puree (apple puree used for pilot study) affects the yield of fruit juice produced:

Looking at the results obtained I can see that the amount of fruit juice produced by making the puree more consistent makes a large difference to the amount of fruit juice produced. This is because the enzyme has a better consistency to work upon compared to the largely solid puree before hand. Also with the better consistency we found that when after adding the pectin the test tube was shook the two substance mixed much better whilst when using pure fruit puree it was quite thick and solid and did not mix with the pectinase very well.

2nd pilot study carried out a single trial using fruit puree with 1cm3 of water added to every 1g of fruit used in the sample. Using 40g of fruit sample and 2cm3 pectinase and water bath temperature of 45oC. This would test if the method was effeicient in producing analysable results.

In conclusion the method has to be altered so as to allow a constant value of distilled water of 1cm3 of distilled water for every 1g of fruit puree in the sample in total of 40cm3 of distilled water as the sample of puree to be used will be 40g and 2cm3 of pectinase will be efficient and 30 minutes incubation and 15 minutes filteration will also be efficient. The temperature will be kept constant at 45oC.

Apparatus: