I will then need to acquire 2cm lactase enzyme to convert the lactose into glucose and galactose. Also 8cm of alginate solution will be needed, to mix with the lactase to make the beads. In addition I will need 20cm of calcium chloride, to drop the beads into.
A glass rod will be needed to stir the alginate and lactose solution to ensure that the liquids are properly mixed.
I will then need to get three 10cm syringe. One will be needed to obtain the alginate and another one will be needed to get the lactase. Also I will need another syringe at the end of the experiment to place the beads into. I am going to use a syringe instead of a measuring cylinder or beaker, as I feel that this is smaller and so will allow me to measure more accurate definite measurements, which in turn will make the experiment fairer. Also I feel that a syringe is better as it is smaller measurements and so easier to handle
A boss clamp and a stand will be needed to hold the syringe over a beaker. I feel that using a clamp will be better than just holding the syringe over the beaker with my hands, as it more stable and the clamp will allow me to tighten the grip on the syringe. Also I will be able to control the pressing of the syringe better. A clip will ensure safety as the syringe will be firmly held so does not waver or fall.
Distilled water will be needed to wash the beads out after they have formed. I will use distilled rather than tap water because it does not have any chemicals added to it so I feel it is fairer.
A tea strainer will also be needed to strain the beads out. I feel a strainer needs to be used rather than just tipping the water out as it ensures that all the other calcium chloride is removed. Also it is safer as the beads do not come into contact with the hand.
I will then get a 220ml measuring cylinder to pour the milk in to. I will use a cylinder rather than just emptying the milk into the syringe as I could spill or pour too much in. I am going to use a small cylinder as the experiment is on a fairly small scale so I do not need large amounts.
A cut piece of nylon sheet will be required to place at the bottom of a syringe before transferring the beads into it, so that when the milk is poured in it does not flow out in large amounts. A Rubber tube will also be needed for this purpose. I will also use as hoffman clip to attach to the tubing so that I can adjust the clip to help me control the flow of milk.
At the end of the experiment I will need glucose strips to check the concentration of glucose produced /present in the solution.
Method- Before starting anything a laboratory coat must be worn. This will reduce the risk of any staining /harm of the liquids to the clothes and body. I will then wash my hands to ensure that they are clean and have no dirt on them.
Next I will obtain all equipment, so that I do not have to look for it in between the experiment and so that can carry the experiment out systematically. I will wash the beakers, cylinders and syringes and then wash/wipe them so that if any chemicals or substances are present they will be removed so as not to disrupt my experiment and results.
I will then get two syringes and collect the required amount of lactase and alginate. I will hold the syringe up to eye level and not tilt/slant it as the incorrect measurement may be obtained. I will also place a beaker on a flat surface when transferring from the syringes so that nothing is spilt.
Next I will mix the 2cm of lactase with the 8cm alginate gel solution into stirring gently with a mixing rod so that the solutions are mixed. I will then measure 20cm of calcium chloride in to a cylinder.
After the alginate solution is mixed I will collect it in a syringe filling it all up, so that I can test the maximum possible for the experiment. I will then get a clamp stand and clamp the syringe on with a boss and clamp, tightening as required. I will then place the chloride beaker under the syringe so that the solution can fall directly into it.
I will then press the syringe slowly releasing about 2cm solution each time in to the beaker. I will release the same each time so that I get the same sized beads, and so the reaction is fair. This is because bigger beads may cause longer for the lactose to be broken down. I will leave the solution in the calcium chloride for about 10 minutes to allow the beads to harden.
After 10 minutes are up I will strain the solution containing the beads into a tea strainer This is a better method than tilting the beaker as I can ensure all the liquid is out, so that no access calcium chloride remains. I will then wash the beads out with distilled water.
After I have washed the beads out I will get another syringe and cut a small piece of nylon gauze to be attached to the bottom of this. I will use another syringe as the previous one could still have some liquid which could contaminate the syringe. I will then attach a short length of rubber tubing to the end of the syringe and screw a Hoffman clip on to it. The hoffman clip will be needed to control the flow of milk.
I will remove the part of the syringe that is pressed so that the syringe is open from the top. I will then transfer the beads in to the syringe. Next I will attach a rubber tube to the end of the syringe and fasten a hoffman clip to the tube to control the flow of the milk. I will then clamp the syringe over a clean beaker, tightening and adjusting with the clamp to get the syringe stable.
I will then pour some milk into a cylinder as it could all spill if I transferred it straight from the milk bottle to the syringe. I will then transfer a little in to the syringe. After a while I will unscrew the Hoffman clip slowly to allow drops to fall into the beaker.
Finally I will get a glucose test strip and dip it in to the milk. I will then wait a few seconds to allow the glucose to absorb on to it and then read the % of glucose present in the lactase treated milk against a colour scale. I will then wash my hands to remove any substances that may have come into contact with my hands.
I will use the following measurements in the experiment:
The measurements I am going use are fairly small amounts, this will allow me to work more accurately without having to measure a lot.
Fair testing- To make sure that the test is fair so that reliable results are obtained the following will need to be ensured:
- All equipment is clean before starting the experiments. And if essential wash and then dried. This is important as the equipments could have chemicals/substances from prior use which could affect the investigation.
- A different syringe is used to obtain the Calcium chloride and the Sodium alginate solutions, as otherwise it could transfer particles from one to another and corrupt the substances which in turn could affect the experiment.
- The experiment is carried out on a flat surface so that the correct measurements are transferred, and so that none is spilt.
- In between the experiments the solutions are moved as least as possible to avoid speeding up a reaction involuntarily
- The experiment is carried out on a flat surface so that the right amount of solution is transferred in to the beakers/syringes/cylinder. Also to avoid an accident so that nothing is spilt
- Unwanted equipment is put away so that the desktop I am working on is clear so that a risk of an accident is minimised. Also if there are any spillages/accidents they are cleaned or reported straight away.
Safety- Safety is very important in any scientific experiment and needs to be taken into account. A laboratory coat must be worn at all times to reduce the risk of chemicals getting to the clothes or skin. The equipment especially the glassware must be handled with caution to avoid any spillages/breakages, and if there is any they must be cleaned up or reported straight away to avoid further accidents and disruptions. Loose hair/scarves must be tied back so that it does not get in the way of the experiment and create a hazard. The desktop which is being worked on must be clear and clean at all times and only the apparatus needed should be present, also will work on a flat surface to reduce risk of an accident. I will not eat or drink whilst doing the experiment as the product could contaminate the milk or the products could get contaminated.
Ethical issues- In the experiment I will be using ingredients which could be consumed. However they are going to be used in an experiment and so I must make sure no milk should be drunk before or after it is treated and that beads should not be put into the mouth either.
Results- Attached below is the glucose strip and colour chart showing the colour obtained by dipping the strip in to the milk at the end of the experiment.
The colour is an indicator of how much glucose was present/obtained at the end.
Conclusion- The result obtained was as predicted, and proves that at the end glucose was obtained from the lactose. This is because the enzyme lactase broke down the lactose in milk into its two single sugar units.
The diagram above shows the process in a simplified version. The enzyme in this case was the lactase and the substrate was lactose. The lactose binds to the active site of the lactase where it is broken down and the products-glucose and galactose are released.
Although the investigation was proved very successful and the desired outcome was obtained I acknowledge that there could still have been many errors in the procedure.
When leaving the beads to harden I did not use a stop clock, but just used a watch to keep a track on time. I could have used a clock to make sure that the time was more accurate.
When transferring the beads in to the tea strainer and then in to the syringe I used my hands. I realise that I could have pressed a bit hard and slightly altered the shape of the beads. Using a plastic spoon would have been better.
Although the immobilised enzyme has many advantages over the free ones I understand that it could have had a few disadvantages, such as;
- Some enzymes may have undergone denaturisation in the immobilising process.
- By immobilising the enzyme the reaction kinetic of the enzyme could have been reduced.
- Because the enzyme is immobilised diffusion of larger enzyme molecules may have been difficult, so rate of immobilisation might have been slower.
Evaluation-I feel that the method used was apt, however to make the results more certain and reliable things could be changed or improved.
Although the experiment gave the desired results, it was only carried out once. To obtain a more reliable results it cold have been carried out at least one more time to see if there was any variation. Therefore I cannot check the precision as I only carried it out once.
During the experiment I did not wear goggles. I realise that there could have been a health risk of any of the solutions/acids coming in contact with the eyes.
I did not specifically measure the amount of milk I poured over the beads and how long it stayed over. I feel that I should have as I could have then tested the effect of the production of glucose depending on the amount and time allowed for the milk to flow over the beads.
When leaving the beads to harden a stop clock could have been used to give more accurate timing. And when transferring the beads instead of using my hands I could have used something such as a spoon.
To obtain a far more accurate reading, a data logger would be an option to use. This would contain a sensor that detects the % of glucose present. It would be far more accurate than human interpretation.
To see if my result was reliable and to see if the same result was obtained I could have tried other ways of immobilising the enzyme such as carrier binding-which binds water to insoluble carriers, or entrapping the enzyme in a matrix.
Bibliography-http://www.rpi.edu/dept/chem-eng/Biotech-Environ/Biotech-Environ/IMMOB/methods.htm
http://www.rpi.edu/dept/chem-eng/Biotech-Environ/FUNDAMNT/lactose.gif