The action of amylase and pectinase in varying amounts when clarifying cloudy apple juice.

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The action of amylase and pectinase in varying amounts when clarifying cloudy apple juice.

Background information: Amylases hydrolyses glycosidic bonds in polysaccharides such as starch and glycogen and converts them to dextrins or to maltose. Different enzymes in the group work in different ways on the α-1,4 links and the α-1,6 links within the polysaccharides. Amyloglucosidase hydrolyses the 1,4 links and the 1.6 links at the branches in the starch molecule. Terminal glucose units are removed from the end of the chain one by one rather than giving the dextrins or maltose immediately. The debranching agent pullulanase hydrolyses the α-1,6 links at the branching areas in the polysaccharide. Amylase is used commercially to clarify fruit juices, wines and beer by removing suspended starch. Amylase can be found in bacteria (Bacillus spp.) and fungi (rhizopus spp., Aspergillus spp. and Streptomyces spp.). Fungal amylases are used in the clarification of fruit juices, wines and beer where they remove suspended starch. In bread making amylases can yield more sugars from the starch in flour or from barley grains. The other important commercial use is the conversion of starch to sweet glucose syrups, which are usually, used ass sweeteners in the food, bread making and brewing industry. By altering the balance between Amyloglucosidase and the fungal α-amylase different proportions of glucose and maltose can be produced. For fermentation, a higher proportion of glucose is useful whereas in jam and confectionary production a higher proportion of maltose is useful. Continued conversion of the glucose produces fructose which is sweeter than sucrose and glucose is as sweeteners for food and drinks. Pectin is a substance, which helps hold plant cell walls together. It is obtained from fungi, particularly species of Aspergillus and Penicillium. Other fungal pectinases are significant in the soft rotting of fruit and vegetables, which contributes to spoilage and decay. The biggest industrial use of pectinases is in the extraction and clarification of fruit juices. Pectinases break down pectin (polysaccharides) found in the cell walls, which clarifies the juice.  As the fruit ripens the plant produces proteolytic enzymes, which convert the insoluble protopectin of the unripe fruit into more soluble forms, causing the fruit to soften. When the fruit is mashed and pressed the more soluble forms of pectin enter the juice making it cloudy and causing the colour and flavour to deteriorate. They also increase the viscosity of the juice making it difficult to obtain optimum yields. Pectinase is added to crushed fruit such as apples and grapes to reverse the effects of pectin, so increasing the yield and improving the colour. Other liquids, which contain suspended pectin material such as vinegar and wine, are also clarified with pectinases. They do this by removing some of the pectin around charged protein particles, these then settle out of the liquid in a large cluster. When fruit juices are concentrated pectinases prevent them from gelling. So, pectinase is added which means a good clear juice is obtained which retains its stability when concentrated.

For my experiment I am going to the optimum mixture of amylase and pectinase to clarify apple juice. To do this the only thing I will alter is the volume of amylase and pectinase. I will keep the volume of apple juice the same each time and I will also keep the temperature the same each time. To make sure everything is at the same temperature I will put the amylase, the pectinase and apple juice all into a water bath at the temperature I decide on after my pilot experiments. Then add the amylase and pectinase in varying volumes (e.g. 9:1 ratio, amylase to pectinase) to test tubes containing 10cm³ apple juice and put them back into the water bath at the designated temperature for a period of time determined by my pilot experiments. To aim to get the most accurate and representative results I will conduct some pilot experiments to find out what is the optimum temperature for amylase and pectinase to work and for how long they need to be left at that temperature. I will also try 3 different types of apple juice. In this way I can see which type of apple juice clarifies the best. If an apple juice does not clarify then I will not use it because it would not be giving me any results to discuss. To find out how much clarification has been taken I will use a colorimeter. The colorimeter measures light intensity. So I will calibrate it using distilled water where distilled water is 10 arbitrary units, which is the clearest. The colorimeter has its own type of test tube when I calibrate the colorimeter I will mark on the test tube the position in which it lies so that any faults in the glass do not affect my results. I will also mark the 11cm³ level up to which I have poured the distilled water. The amylase arrives as a powder and so I am going to mix it with water to make a 1% amylase solution. I will do one pilot experiment with everything at room temperature and when I have added the amylase and pectinase I will leave them for 15 minutes. I will repeat this using a 20-minute time interval. If I do not get results then I will do a pilot experiment using a water bath at 35°C and put the 1% amylase solution, pectinase and apple juice into the water bath for 20 minutes. Then I will add the amylase and pectinase and leave for another 20 minutes. If this is still unsuccessful then I will continue to use a water bath at 35°C and keep increasing the time intervals until I start to see some differences. Everything else will be kept the same. Once I start the final practical the temperature will be kept the same, which is why I am using a water bath because it is more accurate as it is thermostatically controlled. The time intervals will be kept the same as I will use a stopwatch and everything will be in a test tube rack and so easy to put in and lift out of the water bath. The volumes will all be the same measure using a measuring cylinder and a syringe. The one thing, which will be changed, is the ratio of amylase to pectinase in the experiment. I will add 10cm³ apple juice to a test tube. I will then add amylase and pectinase in their varying amounts to each test tube. The table below shows the different volumes of amylase and pectinase, which will be added to each test tube.

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Each test tube will be labelled so I know what is in each. I think that a 1:1 ratio of amylase to pectinase will make the clearest juice because both amylase and pectinase clarify the juice. It may be possible that amylase is more efficient that pectinase or vice versa.

Equipment:

Colorimeter

10xTest tubes

Measuring cylinder

2x 100 ml beaker

Measuring balance

Amylase

Pectinase

Cloudy apple juice (3 types)

25ml syringe

2x5ml syringes

Water bath

Thermometer

Glass rod

Spatula

Test tube rack

Stopwatch

I am using 3 different types of apple juice ...

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