Why doesn't jelly set when fresh pineapple is used?

Basic Problem:

Jelly is made from protein, gelatin, this is dissolved in hot water and as it cools, it sets. For a childrens party it was decided to make some fruit jellies, these were made by putting sliced pineapple into the jelly before it set, the following observations were made:

oThe jelly set normally when tinned pineapple was added.

oWhen fresh, unripe pineapple was used the jelly set but was not as firm.

oWhen fresh, ripe pineapple was used, the jelly did not set at all

But why?

The reason the jelly is not setting is due to protease, which is breaking down the protein in the jelly, protease is an enzyme, and its rate of activity is affected by many things, such as temperature and concentration.

Theoretical investigation.

If the pineapple juice contains an enzyme which is stopping the jelly setting then this can be investigated by using jelly in Petri dishes with holes or wells cut into it. If some pineapple juice is added to these wells then they should get bigger as the enzyme in the pineapple juice will be breaking down the gelatin around the outside of the well, causing it to expand.

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The temperature and concentration will also affect the size of the wells as these are factors which affect an enzyme controlled reaction.

The investigation and prediction.

I am going to investigate the effect of protease concentration on the size of the wells, as it is related to the original problem. It seems apparent that the ripeness of the pineapple is related to the concentration of enzymes within it.

Perhaps this increased protease concentration is needed for seed dispersal, as pineapple is a fruit and as it ripens the concentration of protease increases. I predict the greater the protease concentration which is used then the bigger the wells in the jelly will become, this is because the enzymes are breaking down the gelatin. I also predict that there should be some degree of proportionality related to the amount of enzyme used and the area of the hole over a range.

Why enzymes cause this to happen.

Enzymes are complex protein molecules which perform actions on other molecules whilst they themselves are not used up in the reaction. Enzymes are mainly affected by temperature and pH, and the rate of the reaction is affected by the concentrations of the enzymes. Enzymes can only break or make a molecule if they come into physical contact with the substrate, this is the reason why temperature and concentration affect rate. Temperature affects rate because the increased heat gives the enzymes and substrates energy and causes them to move around more rapidly, the faster they are moving means they have more chance of colliding with each other and so if the temperature is greater then the reaction will happen faster however if the temperature is too high then the enzymes will become denatured. An increase in concentration also causes the reaction to occur faster, this is for the same reason as temperature.

Because there are more enzyme molecules then there is more chance of the substrate colliding with them, this will cause a very rapid reaction at the start and then it will gradually slow, as the substrate is being used up faster, so creating double the concentration every time the reaction occurs. Enzymes work on a lock and key mechanism where only a molecule of a certain shape will fit the enzyme, it is this specificity which makes the enzymes so effective. If the concentration or temperature are increased then there will be more substrate molecules fitting into the enzyme per second than there would otherwise be. The part that the substrate fits into is called the active site, this is the point where the enzyme has a specific shape which fits the substrate.

The reason the enzyme has this shape is because of the way it is folded, an enzyme is a long protein molecule, the way in which it is folded determines its shape and so the shape of the active site. The gelatin is fitting into the active site and is then being split up by the protease.

How exactly does the gelatin break up, and why does it turn into solution?

Gelatin is a protein, and so is made up of amino acids. It is a hydrophilic biopolymer and the reason it is broken down is because the peptide bonds between the amino acids are being hydrolysed. Gelatin is in the majority composed of the amino acids glycine, praline, and hydroxyproline. When boiling water is added to the gelatin the weak bonds are broken by the energy the hot water supplies, The helical structure falls apart, and you are left with free polypeptide chains floating about in solution. the polypeptide chains begin to re-associate and reform the tight triple helix structure. However, the chilling process is slow, and the individual strands have been widely dispersed by mixing, so the helices aren't perfectly formed. In some places, there are gaps in the helix, and in others, there is just a tangled web of polypeptide chains.

When the gelatine solution is chilled, water is trapped inside these gaps and pockets between chains. This is what causes the mixture to become solid. When the enzyme comes in contact with the gelatine it breaks down the peptide bonds between the amino acids in the gelatine chain by hydrolysing them this causes the helical structure to break down and the water to be taken in leaving you with a solution of broken chains of gelatine. The enzyme itself I have concluded to be an endo-peptidase as it breaks down bonds between acid residues in the interior of proteins.

Tinned pineapple, why does it have no effect on the gelatine?

Because it is the enzymes which are causing the gelatin to stop from setting, it is reasonable to believe that because the tinned pineapple does not stop the gelatin from setting then the enzymes must not be working properly. This is because they have become denatured, this is when the tertiary structure that gives the enzyme its specific shape is broken up, causing the enzyme to become the wrong shape, and therefore useless. The tertiary structure is broken up usually under one of two circumstances. Firstly the enzymes could be in a solution which is too acidic, ( the pH is wrong ) this acidity interferes with the hydrogen bonds in the enzymes tertiary structure, and blocks them, causing them to break up.

Another way of the enzymes becoming denatured is if the temperature is too high, when this happens the enzymes are given too much energy, and with all molecules they vibrate and move faster. If the temperature is too hot, and the enzymes are given too much energy then the bonds holding the enzyme together are overpowered by the amount of energy. During the canning process one of these two things must happen, and that is the reason why the canned pineapple has no effect.

Method.

Making the jelly and cutting the holes:

Performing the experiment will involve filling Petri dishes with two times concentrated jelly solution, making sure to get equal amounts in each dish, so that the surface area in contact with the enzymes does not differentiate. Then letting them set. It is preferable to do all the dishes from the same mixture as to not vary the concentration of gelatin. The measuring of the solution will be done by using a measuring cylinder. After the jellies have set the holes must be cut with cork borers, the position of the hole is not entirely relevant, unless the hole is too close to the side of the Petri dish.

Preliminary experiment:

The preliminary was set up as described above, various methods such as scooping and cutting were tried in which to remove the disk from the jelly, however it was found that they were too inaccurate and that a cork borer was much better, as was round, and provided the right cutting action. It was decided to use the most concentrated solution of protease, which was 20%, and water to produce the widest range of difference. The size of the hole was found to take five millilitres of solution and not spill over the top due to surface tension.

This was most effectively measured by using a syringe as a measuring cylinder was not accurate enough for such a small amount of solution. It was found that after four days the 20% concentration had reached the edge of the dish and so it was decided that the dishes should be checked after 2 days, incidentally, the water had almost no effect on the jelly whatsoever.

Adding the solution to the hole:

The different concentrated solutions will be added into the hole, five milliliters has been decided for this as it is just under the rim of the jelly as to prevent spillage on to the surface. These will be measured by using a syringe, as it is more accurate for small quantities of liquid rather than a measuring cylinder. As it is virtually impossible to add all the enzymes at once the lowest concentrations will be done first, so creating less difference in the time between those and the higher concentrations. The disks will then be left, and will be checked everyday until a sufficient difference has been noted, they will all be left in the same place as to prevent temperature variances.

Key factors:

The key factors which will affect the results are the temperature at which the investigation is conducted, the concentration of the gelatin, and of the solutions. Also the size of the hole, and the amount of time which the disks are left for. These factors will all have to be controlled.

Independent variable:

This is the concentration of the protease solution itself, the protease is diluted from a concentrate supplied by the National Center for Biotechnological Education, the solutions will all be made up to 50ml, so for instance a solution of 20% concentration would have 10ml of protease solution and 40ml of water, which would make the overall solution 50ml, but the concentration would be 20%. These volumes are present in the table of results.

Dependant variable:

The finished product will be the disks with jelly, and the hole, containing the enzymes and broken down gelatin. The rate at which the reaction has progressed is expressed by the size of the hole, and more precisely the area of the hole, to measure the area of the hole this scheme was devised:

The jelly is placed on the OHP, and then an image of the hole is projected onto the board this greatly increases the size of the hole, a scale of 4:1 in fact, it also allows the hole to be easily drawn around, as graph paper is placed on the board and the perimeter of the hole is drawn around on to it. A scale of 1:4 is acceptable, more accuracy is not needed. The perimeter is marked by a large black line which encircles the hole, this is caused by the surface tension as the fluid meets the side of the jelly, it has been decided to draw where the black line meets the jelly, as it is the same for every disk and hole, then this ensures a degree of consistency within the results.

To ensure that the position of the Petri dish is the same every time it will be drawn around on the OHP and on the board also. The squares on the graph paper will be counted and used as a numerical value rather than a measurement. It has been decided to count more than half squares as full squares, and less than half as no square, however if there are enough less than half squares which would be representative of one full square then they will be counted.

Rejected Ideas:

Among these were immersing the finished dish in water and then comparing the difference with that of a dish with just the hole cut out. However to do this the vessel in which the dish would be placed would have to be so big that the difference in the level of the water would be almost immeasurable. Next was the idea of measuring the mass of the dish after it had been washed out, and comparing it with that of the disk with just the hole in. this is a fairly good idea, however it is very difficult to determine whether all of the solution has been removed, whether any of the water used to wash it out has been left in, or whether there may be slight differences in the weight of the Petri dishes.

Control:

A control was set up to make sure that no other factors were present in causing the reaction other than the enzymes, the control just consists of a disk with the hole cut out, and water in place of a solution.

Tinned pineapple:

This was set up the same as the other experiments. It is the only real juice used, as it can not be reproduced as a solution.

References:

Advanced Biology principles and applications, C J Clegg and D G Mackean

www.howstuffworks.com

Cambridge advanced sciences Mammalian physiology and behaviour