Making Yoghurt

Planning (a) + (b)

Christian Kliesch

Plan an experiment to look at the effect of of milk Planning (a) Independent variable: type of milk full fat semi-skimmed skimmed milk goats milk sheep milk cats milk

LACTOBACILLUS SP.

bacteria on different types

Dependent variable: pH of the milk over time (rate of reaction) Question and thoughts Milk does not equal milk. Different species produce different kinds of milk, which are chemically different relating the percentage of fat, the protein contained (e.g. casein) and the type and amount of sugar contained. Does this affect the rate of reaction of LACTOBACILLUS SP. producing yoghurt? With the help of enzymes LACTOBACILLUS SP. is able to break down the sugars contained in milk. But enzymes are specialised on a certain type of sugar and therefore there could be differences within the reaction between the different types of milk. Furthermore it would be interesting whether the percentage of fat affects the digestion of sugar or whether the bacteria are even able to digest fat. The differences in the reaction are best represented in the rate of reaction. Of course there is no instrument to measure the rate of reaction directly, but while the bacteria breaks down the sugar it produces an acid (just as with the teeth) which leads to a change in the pH that we are able to record with pH probes. We could use a colour indicator as well, but a pH probe with data logger is far more accurate. The change in the pH causes also the clotting of the milk, as the acidic environment affects the proteins in the milk which then denaturise.

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Other Key factors that must be controlled

To have a fair test we must ensure that the following conditions are fullled: Temperature
All chemical reactions are dependant on the temperature, the movement of the particles. Generally we can say that the faster the particles move (i.e. higher temperature) the greater the possibility they collide and form/break bonds and the faster the rate of reaction. Though this is not completely true for reactions catalysed by enzymes. Every enzymes has an optimum temperature, where it works the fastest and a maximum temperature, at which it begins to denature. The hydrogen bonds break and it loses it's shape. The active side is altered and it can't work as a catalyst anymore. The rate of reaction will slow down again. Therefore we should choose a temperature close to the optimum temperature, which is for most natural enzymes at about 40 C / 313 K. Furthermore it must be ensured that all cultures have the same temperature during the test, otherwise the results would be falsied. (A shared waterbath is the best way to achieve this aim)

Time
We must take the time from the of the experiment to the end every time we measure the pH. The pH must be measured regularly, and with the closest possible distance, to ensure that we have enough sufcient and reliable data. The whole experiment must be repeated to eliminate measuring errors.

Container
The size and the shape of the containers (Boiling tubes) must remain the same during the whole experiment to ensure a fair test. Different container sizes or shapes could lead to different surface areas that would may affect the rate of reaction.

Amount of milk
Every bacteria culture must have the same amount of fodder to make the test fair and the pH measurements reliable.

Concentration of milk
Together with the amount of milk goes it's concentration. It would be the best to have to have 100% concentration of milk, to have approximately the same percentage of sugar contained. Of course every type of milk has different properties, but we must assume this has no measurable inuence, otherwise the test would become too difcult.

Amount of starter yoghurt

To every milk we must add the same amount of starter yoghurt to ensure that we add an equivalent amount of bacteria. An unequal distribution of starter yoghurt would lead to misleading results. (more bacteria faster breakdown of the sugar)

Yoghurt cultures
There are a few hundred different yoghurt cultures in the world, all with different properties and abilities. To ensure a fair test the culture must remain the same during the whole test. A further investigation may looks at different cultures, e.g. such used in sheep yoghurt. Another problem comes up with the bacteria of the outside environment. We must eliminate as much foreign bacteria as possible through sterile equipment (boiling tubes washed in boiling water) and a sterilisation of the milk (short heating over 100C) before the experiment. Otherwise the bacteria will interfere with LACTOBACILLUS SP..

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Hypothesis
I expect that the bacteria in the cows milk will be able to break down the sugar (lactose) easily and will therefore be the fastest. This is because the starter yoghurt is made of cows milk and the bacteria are used to it, i.e. have already the right enzyme. Furthermore the manufacturer of the starter yoghurt may chose a for cows milk specialised culture. I don't expect any noteworthy differences between the 3 types of cows milk (full fat, semi-skimmed, skimmed). The sugars of sheep and goats milk take probably more time, as they may contain different sugars and the bacteria may need to adjust their enzyme production. (Enzymes are usually specialised for one specic substrate, another substrate usually requires another enzyme. Because of energy saving reasons [and others] the bacteria can't afford to have all enzymes and other substances ready to use, it produces every chemical as soon as it is needed, which may takes a little while. So it is possible that we see no reaction happening for the rst minutes or that there's no reaction at all, because the bacteria is unable to produce the needed enzyme. Additionally it is possible that they clot differently as they may possess other proteins (differing acid resistance?), but as we measure the pH this will not affect our data. For the cats milk (in fact chemically modied cows milk) I expect no delay. The contained sugar mainly glucose and galactose should be easy accessible for the bacteria (as both are an intermediate product of the breakdown of lactose that the bacteria does on it's own therefore it should be able to digest both)

lactose
via lactase

maltose
via maltase

glucose
Fig 1:

galactose

glucose

glucose

A sugar gets broken down by an enzyme. LACTOBACILLUS SP. has the enzyme lactase to break down lactose (cows milk sugar), but to break down maltose (sugar in goats milk) it needs another enzyme maltase. pH 7

of milk

Fig 2: Expected time curves of the reaction. Blue are the varieties of cows milk, red the goat and sheep milk, green the cats milk. Shifts of the graph are possible, though the pH will be probably slightly below 7 to begin with.

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Planing (b) Materials and tools For each test row we need:
Tools

6 boiling tubes Syringe(s) (5cm) Measuring cylinder (25 cm) 6 pH probes + data loggers Electric waterbath (approx. 40C/313K) Watch Thermometer Electric kettle Bunsen burner, tripod and beaker with water

Chemicals:

Cows milk full fat semi-skimmed skimmed Goats milk Sheep milk Cats milk (chemically modied cows milk) natural yoghurt

(25 cm) (25 cm) (25 cm) (25 cm) (25 cm) (25 cm) (30 cm / 5 cm each)

Method

Wash the boiling tubes with boiling water Prepare 6 boiling tubes with 25 ml of milk as shown in the diagram below
Boiling tube

3) skimm ed Type of milk 1) full fat 2) semi skimm ed

4) go ats

5) sheep

6) cats

Heat the water in the beaker until it boils, place the tubes with milk into them and let them boil up shortly to kill unwanted bacteria Place them into the waterbath (40C) and let them cool down to about 40C Put one pH probe into each and record the initial pH Add 5 ml of natural yoghurt to each, stir shortly. record the pH in x intervals (e.g. every ten minutes) It would may be good to leave a delay of about 20 seconds between the adding of the yoghurt, to eliminate an overlap in the later recording process. Otherwise we'd need to record several pHs at once. Of course we must take care of the order of the tubes. Repeat the experiment at least one time to have sufcient reliable data and eliminate measuring errors!

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