Investigating the effect of varying the concentration of calcium chloride on the coagulation of milk in the process of making cheese.

Investigating the effect of varying the concentration of calcium chloride on the coagulation of milk in the process of making cheese

Nature of the problem:

To find out the optimum concentration of calcium chloride (CaCl2) needed to be used by cheese manufacturing companies, in order for the milk used to produce the greatest mass of cheese from a given amount of milk to coagulate at its best rate. So that the rennet is at its most active effect therefore achieving a more solid coagulation at the maximum time of production.

Hypothesis:

Increasing the calcium chloride concentration will increase the mass of curd produced, this is upto an optimum concentration.

Prediction:

I predict that the rate at which the milk coagulates thus mass of curd produced will increase as the concentration of calcium chloride increases. This rule will apply upto the stage where an increase in calcium chloride causes no further increase in mass of curd produced as the calcium chloride is allowing the enzyme to have its optimum activity.

Background information:

When making cheese large quantities of milk are required. Cheese is made by coagulating milk to give curds, which are then separated from the liquid, which is called Whey. The curd is then processed and matured to produce cheese.

To coagulate milk you use the addition of rennet, which is made from the lining of a calf’s stomach. Rennet contains a specific enzyme called chymosin also known as rennin. 90% of rennet is chymosin and the 10% is pepsin. The enzyme chymosin causes the particles of milk protein to join together to form a solid gel, which contains liquid called whey. The liquid whey is then drained out of the gel leaving the solid, which is called curd left behind. The pieces of curd are then pressed together forming cheese, and are then stored in rooms of controlled temperatures where the enzymes present act on the protein and fat giving cheese its distinctive cheesy taste.

The history of milk coagulants comes from accident times in Eastern Europe and western Africa. In those days people carried milk in bags, which were made of animal’s stomachs. The formation of cheese occurred literally by accident. By carrying milk in these bags made out of animal’s stomachs it resulted in the first formation of cheese.

The rennet is usually sourced from the fourth stomach of newly born calves, called abomasum. The reason being for chymosin to be present in the abomasum it that it is used to digest and absorb milk.

Chymosin breaks down milk protein casein into paracasein. This binds with calcium to form calcium paracaseinate. This helps separates out with milk fat and some water which forms a mass called curd.

Enzymes are globular proteins. They are specific and only catalyse one chemical reaction. Enzymes are catalysts because they are neither used up nor changed in any way by the reactions they catalyse. The folding of an amino acid chain forms an enzyme. When the chain is folded hydrogen bonds and disulphide bridges are formed which give the protein a stable structure.

Enzymes are specific this is because they have a particular tertiary structure. Their tertiary structure gives rise to an active site, a depression in the globular protein with a shape that is complementary to the shape of the substrate. The substrate will bind to the active site forming an enzyme substrate complex. There will be a reaction here for a brief period, after the products will be released from the active site and a new reaction will take place as the enzyme will bind to another substrate forming an enzyme substrate complex again. This is until the enzyme becomes exhausted.

The addition of calcium chloride to milk was introduced some decades ago when coagulants contained a high percentage of pepsin. This enzyme was very dependent on the calcium ion for having a good coagulant activity. Now coagulants used contain no pepsin and are much less dependent on calcium for good coagulation.
However, the addition of calcium chloride can help to reduce the amount of coagulant needed to achieve a proper set. While the addition of CaCl2 is legally allowed up to 6 oz per 1000 lb of milk, its overuse can result in short-bodied cheese.

Variables:

The term ‘variable’ means liable or capable to change. Variables are parameters that can be altered or measured in the experiment.

In an investigation there are many variables such as independent, dependent and confounding variables, which are discussed below. By being able to acknowledge which variables are which we are able to manipulate information of what is being investigated. We are also able to investigate certain variables this is done simply by controlling all the variables in the investigation apart for the one being tested. This helps makes sure you are testing the variable you wish to and no other.

The independent variables are what we are studying the effects of, the variable that is manipulated or changed and the thing that is the cause of the results. In this experiment the independent variable is the concentration of calcium chloride.

The dependant variables are what we measure in the investigation. The results on the dependant variable are dependant on how the independent variable has been manipulated. The dependant variable in this investigation is the mass of curd produced.

The other variables which can effect the results are called confounding variables and will be controlled in order for the results to be reliable. Below is a list of the factors, which can effect the investigation.

Temperature at which the experiment will be carried out has to be maintained and kept constant this is to enable a fair test. The temperature will be maintained at 30oc by using a thermostatically controlled water bath. If the temperature was to be increased from 30oc which is the temperature being used in the experiment the activity of the solutions will increase causing the rate at which the milk will coagulate to increase, and visa versa if the temperature was to be decreased the rate at which the milk would coagulate would also decrease.

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Concentration of rennin has to be kept constant. If the concentration is altered the results collected will not just measure the effect of calcium chloride but also the effect of rennin concentration on the coagulation of milk. By increasing the concentration of rennin the milk will coagulate more quickly. I say this because there will be more active sites available from the enzyme which will bind to more substrates therefore forming more enzyme substrate complexes thus the rate at which the milk will coagulate will increase or visa versa if the rennin concentration was to decrease the rate at which the milk would coagulate would not be as effective as if the concentration was to be greater. This is because the number of active sites would decrease therefore the enzyme would become saturated quickly. For the same reason above the volume of rennin used in the experiment will also be kept constant.

The time the solutions CaCl2, rennet and milk will be left to react will be kept constant at 420 seconds (7minutes), this is so that they are all investigated under the same conditions. If the solutions were left to react for times that differed the mass of curd produced would be different. This is because some of the solutions would react more than others therefore producing more curd.

Only one type of milk will be used in this investigation. This is because for example goat’s milk in comparison to cow’s milk will coagulate at a different rate. Therefore by using different types of milk the rate at which the milk coagulates will be different which will cause the results to change according to which type of milk is being used. This is why only full fat cows milk is to be used in this investigation. The volume of milk used in the experiment will also be kept constant this is to initiate a fair test.

The time the solutions rennin, calcium chloride and milk, which will all be mixed, is to be filtered will also be kept at a constant of 900 seconds (15minutes). This is to allow a time long enough for the curd and solution to filter and also an equal and constant time for the solutions to react and produce curd which will help initiate a fair test when the experiment is repeated.

The time of incubation in the water bath will also be kept constant. I will incubate the boiling tubes containing the three solutions separately for 420 seconds (7minutes) prior to mixing the contents all together this is to make sure all the solutions were at a constant temperature of 30oc before mixing. This will keep a fair test.

The volume of calcium chloride used in the experiment will also be kept constant this will also help keep a fair test.

Apparatus:

Boiling tubes x3 – these will contain the three solutions calcium chloride, rennin and full fat cow’s milk.

Thermostatically controlled water bath- used to keep the solutions used at a constant temperature of 30oc enabling a fair test.

Measuring cylinder x2- this will be used to measure the volumes of the solutions accurately.

Beaker x5- 4 of these beakers will be used to contain the different concentrations of calcium chloride used in the experiment. These beakers will be labelled with a permanent marker so that no human errors are made during the experiment i.e. mistaking the concentrations for each other causing an error in results recorded. The fifth beaker will be used to collect the whey, which passes through the filter paper when the solution containing curd and whey are filtered.

Filter funnel and filter paper- used to separate (filter) the whey from the curd produced so that the curd can be weighed.

Digital weighing scales- used to measure the mass of curd produced giving a precise figure.

Test tube rack- this will be used to hold all the test tubes being used till they are placed in the water bath

Digital stopwatch- to measure the 420 seconds (7minutes) for which the test tubes will be placed in the water bath for equilibration and then the 420 seconds (7minutes) where the calcium chloride and milk solution are added to the rennin solution. By using a digital stopwatch the time measured will be done with precision.

Glass rod x1- this will be used to mix the milk, calcium chloride and rennet when placed in one boiling tube

Method:

28cm3 of full fat cow’s milk will be placed in a boiling tube, which will be measured out using a measuring cylinder.

Mix 1cm3 of chymosin solution with 99cm3 of distilled water to form the 1% chymosin solution needed for the experiments.

6cm3 of chymosin will be measured out and will be place in a boiling tube.

I will measure 4cm3 of calcium chloride of the selected concentration in a measuring cylinder.

Place the boiling tubes containing the rennin, full fat cow’s milk and calcium chloride separately in a water bath at constant temperature 30oc for 420 seconds.

This enables a fair test for each of the experiments carried out, as all the solutions will be kept at a constant temperature of 30oc.

After the 420 seconds pour the solution, which contains the calcium chloride into the milk, and the rennin solution into the boiling tube containing milk and calcium chloride.

The solution will be left for 420 seconds to incubate, and then the product of the solution will be filtered.

I will filter the solution, which is called whey and contains curd for 900 seconds (15minutes).

The filtrate (product) collected in the filter paper will be weighed and the results will be recorded in a suitable results table.

The different concentrations of calcium chloride will be repeated three times. This will bring precision and accuracy to the results collected. The following concentrations of calcium chloride will be used in the investigation 0.01moldm-3, 0.02moldm-3, 0.03moldm-3, 0.05moldm-3, 0.07moldm-3 and 0.1moldm-3.

These concentrations will be made by using a 0.1M calcium chloride solution and distilled water. These two solutions will be mixed together to give the concentrations needed. For example for a solution of calcium chloride of 0.03moldm-3 14cm3 of distilled water (H2o) will be mixed with 6cm3 of calcium chloride solution of concentration 0.1M. By mixing these two solutions together 0.03moldm-3 of calcium chloride will be produced.

I will also use a control in my investigation. This will enable me to verify by conducting a parallel experiment in which the variable being investigated in this case calcium chloride concentration, is compared with a standard, which is the control. In place of calcium chloride I will use distilled water for the control, this will be the standard of my investigation allowing me to compare my results to the control.

The concentration of rennit I will be using will be 1 percent (1ml3 of rennit to every 99ml3 of distilled water).

Risk assessment:

There are many things that could occur by accident in a laboratory and this is the reason why you should always know where the exits, medical box and eye rinses are kept in the laboratory.

When glass objects are being used great care should be taken that nothing breaks as broken glass may lead to various injuries, which could be very serious. If by any chance a glass object does break a member of staff should be clearly informed and everyone in the laboratory should be aware and told to stay clear of this area till it is cleaned up and all the glass is removed. The glass, which is broken, should be picked up using dustpan and placed in a bin designed for glass disposal (sharps bin).

Gloves should be used when dealing with chemicals, and after use they should be disposed off in an appropriate manner. The inhalation of enzymes must be avoided, as it is an irritant. Any spillages of rennin or calcium chloride should be washed thoroughly with soap and water. If any rennin or calcium chloride were to be accidentally taken in, you would seek for medical attention immediately.

Food or drink should not enter the lab. And under no circumstances be eaten or drunk in the laboratory.

Generally when in a laboratory, there should be a correct manner of behaviour, and a certain level of maturity.

Full fat cows milk is of low risk which means care should be taken when using the milk but it is not hazardous whereas when using calcium chloride an extreme amount of care should be taken as it is an irritant. It is irritating to the eyes, skin and respiratory system. To prevent any hazards occurring with calcium chloride and other solutions used in the investigation eye protection (goggles) and gloves should be worn.

If there are any splashes eyes should be washed immediately using eye wash which are kept in bottles on the lab walls.

Pilot study:

A pilot study is a small-scale experiment designed to decide how to carry out the actual full-scale experiment you are going to undertake. By carrying out a pilot study you can test for the actual experiment, testing if the experiment works and if anything needs to be altered. This saves time and money, as a pilot study is only a small-scale study.

The objective of me carrying out a pilot study was to show that the volume of milk, rennet and calcium chloride were all suitable and adapted to the full-scale experiments I was going to carry out. I would be able to recognise if the volumes were suitable, as I would obtain a good set of results from the experiments carried out in my pilot study. I also studied the times of incubation and filtration as this would also help make my investigation successful by giving me suitable results. The pilot study was also used to establish which volume of calcium chloride to use for each concentration with the volume of milk.

The pilot study, which I carried out, was similar to the experiment described above. The difference to the above experiment was that the concentrations of calcium chloride were kept constant at 0.03moldm-3 whereas the volume of calcium chloride used was altered to establish which volume to use with each of the calcium chloride concentrations using volumes of 2cm3 and 4cm3 investigating which volume would coagulate the best.

Set up a water bath to temperature 30oc. Make a solution of 0.03moldm-3 of calcium chloride by adding 14cm3 of distilled water to 6cm3 of 0.1M calcium chloride solution and place in a boiling tube.

Measure out 2cm3 of 0.03moldm-3 calcium chloride solution into a boiling tube using a measuring cylinder.

28cm3 of full fat cow’s milk for 2cm3 of calcium chloride will be placed in a boiling tube, which will be measured out using a measuring cylinder, Whereas for 4cm3 of calcium chloride 26cm3 of full fat cows milk will be used.

Now place the boiling tubes containing the rennin, full fat cow’s milk and calcium chloride separately in a water bath at constant temperature 30oc for 420 seconds.

After the 420 seconds pour the solution, which contains the calcium chloride into the milk, and the rennin solution into the boiling tube containing milk and calcium chloride.

The solution will be left for 420 seconds and then the product of the solution will be filtered.

I will filter the solution, which is called whey and contains curd for 900 seconds (15minutes).

The filtrate (product) collected in the filter paper will be weighed and the results will be recorded.

I will carry out the same proceedings for both the volumes (2cm3 and 4cm3) of calcium chloride and record the results and present then in the results table below.

As you can conclude by analysing the results from the results table 4cm3 of 0.03moldm3 of calcium chloride coagulated the best in comparison to 2cm3 of calcium chloride of the same concentration.

By analysing these results I have chosen to use a volume of 4cm3 when carrying out my experiments investigating the effect of varying the concentration of calcium chloride on the coagulation of milk in the process of making cheese.

After carrying out the pilot study there was no need for me to alter anything from the method I had already used as the experiment that I carried out went smoothly and no problems occurred.

Main Method:

28cm3 of full fat cow’s milk was placed in a boiling tube, and was measured out using a measuring cylinder.

I mixed 1cm3 of chymosin solution with 99cm3 of distilled water to form the 1% chymosin solution needed for the experiments.

6cm3 of chymosin was also measured out and placed in a boiling tube.

I then measured 4cm3 of calcium chloride of the selected concentration in a measuring cylinder.

The boiling tubes containing rennin, full fat cow’s milk and calcium chloride were then separately placed in a water bath of constant temperature 30oc for 420 seconds.

After the 420 seconds I poured the solution, which contained the calcium chloride solution into the milk, and the rennin solution into the boiling tube containing milk and calcium chloride.

The solutions were left to incubate for 420 seconds, and then filtered.

The solution was then filtered, for 900 seconds (15minutes).

The filtrate (product), which was collected in the filter paper, was weighed and the results were recorded in a suitable tabulated results table.

The different concentrations of calcium chloride were then repeated three times. This was to bring precision and accuracy to the results I had collected. The following concentrations of calcium chloride were the one’s I used in the investigation 0.01moldm-3, 0.02moldm-3, 0.03moldm-3, 0.05moldm-3, 0.07moldm-3 and 0.1moldm-3.

These concentrations I made by using a 0.1M calcium chloride solution and distilled water. These two solutions were mixed together to give the concentrations I needed. For example for a solution of calcium chloride of 0.03moldm-3 14cm3 of distilled water (H2o) was mixed with 6cm3 of calcium chloride solution of concentration 0.1M. By mixing these two solutions 0.03moldm-3 of calcium chloride were produced.

I also used a control in my investigation. This was to enable me to verify by conducting a parallel experiment in which the variable being investigated in this case calcium chloride concentration, was compared with a standard, which is the control. In place of calcium chloride I used distilled water for the control, this is the standard of my investigation allowing me to compare my results to the control.

The concentration of rennet I used was 1 percent (1ml3 of rennet to every 99ml3 of distilled water).

Results:

Below are four tables, which represent the results for the 3 tests I carried out, and a results table, which represents the average of my results.

The results for the 1st test:

The results for the 2nd test:

The results for the 3rd test:

Below is a results table representing the average mass of curd produced for each concentration used in the experiment:

The results table below represents the rate at which the curd coagulated. I calculated the rate of coagulation by using the formula: -

Rate of coagulation of milk= Mass of curd/ Time of incubation

To calculate the rate of coagulation of milk I only used the time of which the solutions were incubated together in the boiling tube. This was because when filtering some of the enzyme would drain through the filter paper used, therefore this would mean that the enzyme concentration would no longer be constant once the solution was placed in the filter funnel. This is the reason why I had chosen to use the time of incubation to calculate the rate of coagulation. The time of incubation was used for all the experiments carried out in this investigation to calculate the rate of coagulation of milk.

Analysing evidence and drawing conclusions:

The two graphs I have drawn represent the average mass of curd produced with concentrations, which vary of calcium chloride and also the rate at which the curd coagulated with different calcium chloride concentration.

The graph, which represents the average mass of curd produced with different calcium chloride concentrations, has a general trend. This trend shows that as the calcium chloride concentration increases the average mass of curd also increases. This trend only follows to a point where the concentration of calcium chloride is 0.03moldm-3, here 31.73g of curd was produced thereafter this point as the concentration of calcium chloride increased the average mass of curd produced decreased. You can read this from the graphs which represent the results as at 0.05moldm-3 of calcium chloride the average mass of curd produced was 25.50g but for the 0.07moldm-3 solution of calcium chloride 31.29g of curd was produced. I can say for 0.05moldm-3 of calcium chloride I have an anomalous result by looking at the graph I have drawn representing the average mass of curd produced, as the result does not show a trend or follow the trend it is expected to. I would have expected the mass of curd produced to continue to increase in mass or stay constant for the 0.05moldm-3 solution of calcium chloride as the result for 0.07moldm-3 of calcium chloride solution produced a mass, which was close to the mass the 0.03moldm-3 solution of calcium chloride. This showed that there was a trend in my graph but I had an anomalous result, which can been seen on my graph.

By looking at my graph I can say that the 0.03moldm-3 of calcium chloride was the optimum concentration used as it produced the greatest average mass of curd compared to the concentrations I used.

The graph shows that for a concentration of 0.00moldm-3 0.37g of curd were produced this was the control used in my experiment. Instead of using calcium chloride distilled water was used for this experiment. This showed that by not using any calcium chloride curd was still produced, but by increasing the calcium chloride concentration the mass of curd produced would increase, this was upto the optimum concentration of 0.03moldm-3, by increasing the concentration after this point the mass of curd produced would decrease.

The solution of 0.01moldm-3 of calcium chloride did not produce much more curd than the control, it produced 0.53g only, an average of 16g more curd than the control.

Evaluation:

By looking at the results plotted on my graph it is shown that variability is not very small. This information is gathered from the error bars drawn on my graph which represents the average mass of curd produced.

The variability in my results was small but could have been decreased if the experiments were repeated more as the more repeats I do the less the variation would have been. The reason I was not able to repeat my experiment more than twice was because my time was limited which withdrew me from repeating my results more than three times.

When I was measuring the volumes of the solutions I used a measuring cylinder but if I had used a pipette instead to measure the volumes I would have decreased the variability in the error bars on my graph. This is because by using a pipette to measure the volumes of the solutions I used in a measuring cylinder it would have increased the accuracy instead of using only a measuring cylinder to measure the volumes.

When I filtered the solutions of calcium chloride, milk and rennet there was still a trace of the solutions left in the test tubes and small solids, which were stuck to the sides of the boiling tube. I could have used a set volume of distilled water to rinse the boiling tube which would have made sure al the solutions had been filtered and used in the experiment. By keeping the volume of distilled water constant it would have been practical to use it as the test would have still been kept fair but more precise as all the solutions would have been used therefor no substrates or enzymes would be left in the boiling tubes. This would help decrease the variability in my results, as it would bring more accuracy and precision to my results.

For a further experiment I could use more individualistic concentrations. By doing this I would bring more precision and also accuracy to my results. By having tested individualistic concentrations of calcium chloride I would be able to plot more points on my graph. This would be of great use to my analysis as I would be able to find a more detailed trend in my results and be able to spot any anomalous results I have not already. I would mainly investigate concentrations in between large gaps I have on my graph, for example there is a large difference in the average mass of curd produce for 0.01moldm-3 and 0.02moldm-3, therefor I would use a concentration of 0.015 which would give me a point on the graph showing what happens between the two points.

Acknowledgements

I would like to thank all the science department for all the support and guidance they gave me when I was carrying out my experiments.

Firstly I would like to thank Miss Welfare. Miss Welfare made it possible for me to carry out all my experiments as she ordered all the chemicals and equipment I required for my experiments and made sure they were ready in the laboratory for me to use on the day I was carrying out my experiments.

I would also like to thank Mrs Lassman and Mrs Chatterjee who made it possible for everyone to carry out their experiments by coming into college in her own time and offering guidance to anyone who was in need of it as well as supervising the students as the experiments were carried out.