An investigation into the effect of varying pH on the yield of apple juice by pectinase.

Katie Ruck

An investigation into the effect of varying pH on the yield of apple juice by pectinase

Hypothesis

There will be an optimum pH for pectinase activity. Deviations either side of the optimum will result in decreased pectinase activity.

Biological Knowledge

Fruits are comprised mostly of unspecialised tissue called parenchyma. The parenchyma cells have thin walls made of two layers. The outermost of these, the primary cell wall, is formed from cellulose fibres called microfibrils surrounded by a matrix of pectins, hemicellulose and proteins. In unripe fruit the pectin is bound to cellulose microfibrils in the cell walls. Such pectin is insoluble and the liquid within the cells remains fluid. During ripening, pectin is partly degraded and solubilised by indigenous pectinases. The result is that the pectin becomes more soluble and its grip on the surrounding cell walls is loosened. One consequence of the partial breakdown of insoluble pectin is that it becomes soluble in water. Some of the pectin molecules are released into the juice. The presence of this polysaccharide increases its viscosity.

Pectin is a polysaccharide comprised of at least three components. The principal polysaccharide is polygalacturonic acid and this is normally combined with polygalactose and the highly branched polyarabinose. In addition, the carboxyl side groups of galacturonic acid are partly esterified with methanol residues.

Pectinase is an enzyme, a globular protein that hydrolyses pectin. It splits the bonds between the carbon 1 and the carbon 4 atoms of galacturonic acid, thus shortening the pectin chain. The addition of pectinases also breaks the link between the pectin and cellulose, releasing the pectin into the juice where it is further degraded by pectinases to increase the fluidity of the juice. Most enzymes have a characteristic pH at which they function most efficiently. This is referred to as the optimum pH.

Amino acids, from which all proteins are made, contain both acidic (COO-) and basic (NH3+) groups, and in a polypeptide chain there are many of these acidic and basic groups. Bonds between these groups maintain the secondary and tertiary structure of the enzyme. Changes in pH, i.e. presence of hydrogen ions, alter the ionic charges of the acidic and basic groups, and causes the shape of the protein to change. This may prevent the pectin (substrate) molecules fitting into the active site, reducing the number of enzyme substrate complexes formed thus reducing the activity of the pectinase enzyme and so reducing the juice yields obtained. Excess acidity (presence of H+ ions and so a low pH) together with excess alkalinity break the hydrogen bonds which give the enzyme molecules their precise three-dimensional molecular shape, so causing the pectin molecules to no longer fit into the active site, denaturing the pectinase and so causing activity to stop.

Apparatus List

1 balance accurate to 0.001grams used to determine the mass of an empty 10cm3 measuring cylinder together with the mass of the 10cm3 measuring cylinders containing apple juice. Therefore this piece of apparatus provides essential values in the flow rate calculations. It is more precise than determining the volumes of apple juice that pass into the measuring cylinders by observing the divisions printed on the glass of the apparatus.
27 pieces of filter paper, 125mm in diameter used to create cones that hold the reacting mixture of pureed apple, pectinase and buffer solution. The filter paper only allows juices to pass through.

2.7kilograms ‘Granny Smith’ apples act as a source of pectin, substrate that reacts with the chosen enzyme.

1x apple corer used to remove the pips and stalks of the apples thus ensuring that the volume of apple juice that passes into the measuring cylinder is not influenced by obstructions in the plastic funnel. This method uses as much of the apple as possible where as it would be difficult to use a knife to cut around the core without wasting rather large sections of the apple.
1x apple peeler used to remove the skin of the apples so as to eliminate any possible obstructions that would affect filtration. This method is safer than using a knife with a sharp, hazardous blade and so minimises the risk of injury.

1x plastic container used to contain any apple waste from coring, peeling and/or cutting, therefore maintaining a tidy work surface.

1x knife used to cut the apple into smaller pieces. This is done to reduce the time taken to make apple puree of the desired consistency as it is easier for the blender to manage smaller sized food substances. It also allows one to obtain as close to 100grams of apple as possible when preparing the apple puree.
1x white tile the cutting process is performed on a white tile to avoid marking the workbench. The filter funnel is placed on the white tile following the twelve-minute filtration period to prevent any solution leaking onto the work surface.
2x 500ml beaker holds the cut apple pieces until they are required therefore maintaining a tidy work surface through avoiding, leaving the apple dispersed on the workbench.

9x plastic pipettes used to transfer a small volume of buffer solution onto narrow range indicator paper, rather than simply pouring the solution onto the paper, where it is likely to spill onto the workbench.
narrow range indicator paper and colour chart used to verify that the buffer solutions provided are indeed of the correct labelled pH. This is more suitable than the addition of an indicator solution to a sample of the pH buffer that would have to have been transferred from the source to a separate beaker.
1x 1ml graduated pipette used to transfer precise 1ml volumes of 100% fungal pectinase into a 50ml beaker. This piece of apparatus allows easier transfers than a measuring cylinder and also reduces the risk of transfer losses. Graduated pipettes are more precise than other measuring apparatus including syringes and the available measuring cylinders.
75cm3 of pH1, 2, 3, 4, 5, 6, 7, 8 and 9.5 phosphate buffer solution used to create varying pH conditions for the reaction to occur without interfering with the reactions itself, as other substance such as acid solutions would do if used to alter pH.

3x 25cm3 measuring cylinder used to measure the required volumes of buffer solutions. Measuring cylinders are more precise than the other available measuring apparatus of 25cm3 including syringes.
2x glass rod used for stirring and so ensures that pectinase comes into contact with the pH buffer solutions and that the pureed apple and pectinase react. A glass rod is utilised as the consistency of the pureed apple means that swirling the beaker will not ensure a reaction between all the apple and pectinase. Swirling the buffer and pectinase solution, as a means of mixing is likely to induce ...

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3x 25cm3 measuring cylinder used to measure the required volumes of buffer solutions. Measuring cylinders are more precise than the other available measuring apparatus of 25cm3 including syringes.
2x glass rod used for stirring and so ensures that pectinase comes into contact with the pH buffer solutions and that the pureed apple and pectinase react. A glass rod is utilised as the consistency of the pureed apple means that swirling the beaker will not ensure a reaction between all the apple and pectinase. Swirling the buffer and pectinase solution, as a means of mixing is likely to induce greater losses than if a glass rod is used to stir the substances.
4x 100ml beaker contains the apple pieces that are being weighed. This is more practical than placing each individual apple chunk on the balance. Also contains the pureed apple, pectinase and buffer solution during the seven-minute reaction period. This allows the blender container to be reused to produce the apple puree required for the next repeat or next pH to be tested.
1 digital stop clock accurate to 0.01 seconds used to record the blending, reaction, stirring and filtration time periods throughout the experiment.
1 plastic blender container contains the substances to be blended, i.e. apple pieces, pectinase and pH buffer solution. The shape of the container ensures that little substance is released to the surroundings during blending. A plastic blender container is more appropriate than using a glass beaker to hold the substances during the blending process as a glass beaker is likely to crack.
1 handheld blender due to the relatively small volumes of the substances to be blended it was more appropriate to use a handheld blender as opposed to a regular food blender, where the blades would not act on all the apple pieces because the level of the substances was below that of the blades.
3x 50ml beaker contains 25grams of the pureed apple (pectinase and buffer solution) before it is transferred into the plastic funnel. If a measuring cylinder were used to transfer 25cm3 of the pureed apple into the funnel it would be challenging to remove all of the 25cm3 from the apparatus (that has a very small cross sectional area). Therefore, using a beaker and spatula greatly reduces transfer losses and makes it easier to maintain consistency.
1 spatula used to transfer the 25grams of the pureed apple from the blender into the beaker, and then into the filter funnel. Using a spatula helps to reduce transfer losses. It can move more substance than a glass rod for example.
30ml 100% NCBE fungal pectinase a suitable and relatively safe enzyme that reacts with the chosen substrate, i.e. catalyses pectin hydrolysis.
1 mercury thermometer with 0.1oC markings used to monitor the temperature of the environment and so control one variable of the experiment.

Plan

Reset the balance so that the display reads 0grams. Place an empty 10cm3 measuring cylinder on to the balance and record the mass.
Create a cone using a piece of filter paper, before placing this in a plastic funnel. This funnel should be centrally positioned above a 10cm3 measuring cylinder.
Use an apple corer to remove the pips and stalks of the apples. Discard the cores into a plastic container. Remove the skin with an apple peeler, putting any waste into the plastic container. Use a knife to cut the apple into smaller pieces. The process should be performed on a white tile. Place the apple chunks into a 500ml beaker.

Before proceeding, use a plastic pipette to draw up a small volume, i.e. 0.2ml of the phosphate buffer solution. Transfer this onto narrow range indicator paper before comparing with the colour chart, to determine the pH being tested.

Use a 1ml graduated pipette to draw up 1ml of 100% fungal pectinase. Transfer this into a 50ml beaker and add 25cm3 of pH3 buffer solution, before starting the clock. This volume of phosphate buffer should be measured using a 25cm3 measuring cylinder. Ensure that readings are taken at eye level so as to avoid parallax error. Leave the pectinase solution to equilibrate to the conditions for five minutes. During this time period ensure that all pectinase comes into contact with the pH3 buffer solution, by stirring at a rate of one revolution per second, using a glass rod. Continue this process for twenty seconds.
During the ‘equilibration’ period, place a 100ml beaker onto the balance and press the tare button so that only the mass of apple will be shown on the display. Weigh 100grams of the apple pieces. If necessary, use the knife to obtain smaller pieces in order to achieve exactly 100grams. Place the 100grams of apple into the blender container.
After the five minutes has elapsed, stop and reset the digital clock before adding the beaker of pectinase and buffer solution to the apple pieces. Blend for one minute using a handheld blender. This time period should be again be measured using a digital stop clock and will achieve apple puree of a moderate consistency. The clock should be started immediately when the pectinase comes into contact with the pieces of apple. After one minute, use the spatula to transfer all substance in the blender container into a 100ml beaker.
Leave the pureed apple and pectinase to react for a further seven minutes. Stir continuously using a glass rod for ten seconds at one-minute intervals, at a rate of one revolution per second. Using the digital stop clock should monitor these time periods. During the reaction time, place a 50ml beaker onto the balance and press the tare button so that only the mass of apple puree and pectinase will be shown on the display. After seven minutes has elapsed, stop and reset the clock, whilst using a spatula to transfer 25grams of the pureed apple into the beaker.

Transfer the apple puree and pectinase contained in the beaker into the plastic filter funnel suspended over the 10cm3 measuring cylinder. Start the stop clock and allow for a twelve-minute filtration period.

After this time has elapsed, remove the funnel before placing it on the white tile. The digital clock should then be stopped and reset.
Ensure that the balance reads 0grams before placing the measuring cylinder on to the balance and recording the mass. In order to determine the volume of juice contained in the measuring cylinder, evaluate the following calculation:

mass of 10cm3 measuring cylinder containing apple juice (grams) - mass of an empty 10cm3 measuring cylinder (grams) = mass of apple juice (grams)

Assuming that 1gram of solution is equivalent to 1cm3 of solution, the mass of apple juice found using the above equation could be directly converted to volume of apple juice in cm3. For a fair comparison of pectinase activity to be made the flow rate will be calculated for each repeat at each pH tested, by evaluating the following calculation:

volume of juice in measuring cylinder (cm3)= flow rate (cm3/seconds)

filtration time (seconds)

This procedure should be repeated three times for each of the nine pHs tested ranging from pH1 to 9.5, ensuring that the blender container together with the blades are washed thoroughly with water and dried between each pH tested and that both the filter paper and measuring cylinder are replaced.

Diagram

Variables

The independent variable of the experiment will be pH. This will be altered using nine different pH phosphate buffer solutions. PH paper will be used to verify that the buffer solution is indeed of the correct pH before reacting the pectinase and buffer solutions. In order to produce reliable results, other variables that may affect pectinase activity need to be controlled.

As the concentration of pectinase increases, the number of active sites will also increase. Provided that there is an excess of substrate molecules, the rate of reaction increases in proportion to the concentration of the enzyme, thus resulting in a higher yield of apple juice passing through the measuring cylinder for the pH being tested. Therefore, the concentration of pectinase will be kept constant at 100%. On each repeat, 1ml of pectinase will be taken from the same sample/beaker so as to maintain consistency.

The volume of apple puree placed into the plastic filter funnel will be kept constant throughout the investigation to provide roughly equal volumes and therefore concentration of pectin for each repeat at each pH. Increasing the concentration of pectin, will increase the rate of reaction, providing not all active sites are in use, because the more pectin molecules there are in solution, the more enzyme substrate complexes form. This will cause increased masses of apple juice and so increased flow rates to be obtained at the pH being tested, as an increased rate of reaction will result in more hydrolysis of pectin molecules. This remains true up to a point where all active sites are occupied and the volume of pectinase becomes the limiting factor causing no further increases in the rate of reaction. To ensure that the volume of apple puree remains constant throughout, a balance accurate to 0.001grams will be used to weigh 25grams of the blender solution (containing pureed apple pieces together with pectinase and buffer solution) to which 1ml pectinase will be added. Although it is challenging to exclude any variation in pectin concentration and volume, the same type of apples (green ‘Granny Smith’ apples) of approximately equal mass (100grams) will be used and when each batch of apple puree is produced equal proportions of apple pieces and liquid will be blended i.e. 100grams apple and 25cm3 liquid.

Fluctuations in temperature will affect the activity of the pectinase. As temperature increases, the kinetic energy of the pectin and pectinase molecules increases and so resulting in increased movement. The faster these molecules move, the more frequently they will collide with each other, increasing the number of enzyme-substrate complexes formed and so increasing the rate of reaction. An increased rate of reaction would result in more pectin hydrolysis in a given time, so leading to an increased rate of juice flow. Although highly unlikely in this experiment, substantial increases in temperature would cause the atoms within the pectinase molecules to vibrate. This breaks the hydrogen bonds holding the molecules in their shape and so altering the three-dimensional shape of the pectinase molecules to such an extent that their active sites no longer fit the pectin molecules. This would denature the pectinase and result in lower rates than normal of juice flow. The temperature of the environment will be monitored using a mercury thermometer with 0.1oC markings. Readings will be taken at the beginning middle and end of the three-hour investigation period. To prevent fluctuations, all repeats will be performed in the same environment (i.e. laboratory), on the same day and away from any radiators or sunny windows.

The time period allowed for a reaction to occur between the pectin contained in the puree and pectinase, before the substance is transferred into the filter funnel, will remain consistent for a fair comparison of pectinase activity to be made. An increased length of time would allow more enzyme-substrate complexes to form and so more hydrolysis of pectin to occur thus resulting in a greater yield of juice passing into the measuring cylinder in the given filtration time. The time allowed for filtration must also be invariable for the same reason. A digital stop clock, accurate to 0.01 seconds, will be used to ensure that this is so. The stop clock will be started immediately when the pectinase and buffer solution comes into contact with the apple pieces, and stopped after eight minutes, when the solution will then be transferred into the filter funnel suspended over the measuring cylinder. The apparatus will be reset, and restarted as the solution comes into contact with the filter paper. After a twelve-minute filtration period the clock will be stopped. A blending time of one minute will be measuring with the digital stop clock. This time period will remain constant for each batch of apple puree produced.

In order to make a reliable comparison of pectinase activity at varying pHs the mixing procedure must be consistent throughout the experiment. To ensure that this is so, a glass rod will be used, following the addition of buffer solution to the pectinase, stirring at a rate of one revolution per second. This process will be continued for twenty seconds. During the seven minute reaction time following blending, the pureed apple and pectinase will be stirred continuously using a glass rod for ten seconds at one-minute intervals, at a rate of one revolution per second.

The volumes of the reacting substances (i.e. pectinase and phosphate buffer solutions) will need to be invariable for a comparison of enzyme activity to be made. 1ml graduated pipettes will be used to obtain 1ml of pectinase and measuring cylinders accurate to 5664, will be utilised to obtain 25cm3 buffer solution. The former are highly precise pieces of apparatus that will ensure the volumes of reacting substances remain constant.

It is important to ensure that filter paper of the same thickness is used and that the diameter of the funnel does not change during the course of the experiment. Alterations in these two factors would directly affect the rate at which juice flows into the measuring cylinder. Each time a cone is constructed filter paper 125mm in diameter will be used and plastic funnels of 8mm in diameter will be utilized. The same sized measuring cylinders (i.e. 10cm3) with the same cross sectional area, will be employed to maintain consistency and allow a comparison of flow rates to be made.

Risk Assessment

Pectinase is an irritant and potential allergen, and should therefore be handled so as to minimise contact or inhalation. It can cause asthma and/or irritate the membranes of the eyes and nose. If the solution gets in your eyes, flood the eye with gently running tap water for ten minutes and seek medical attention. If spilt on skin or clothes, remove the contaminated clothing. Flood the area with water and wash thoroughly with soap and cold water. If spilt in the laboratory, scoop up as much solid as possible before washing the area with detergent and water.
Be cautious when handling the mercury thermometer. If spilt in the laboratory; use a good syringe or a ‘pooter’ connected to a vacuum pump via a trap to suck up the bulk of large spills. Mop up the remainder with a hot paste of 1:1 mixture of Calcium hydroxide and flower of sulphur in water. Remove any contaminated clothing and carefully wash the affected area.
A number of the buffer solutions to be used contain sodium hydrogen carbonate. This substance is irritating to eyes, skin and the respiratory system. Although there are minimal hazards associated with the usage of this substance, it would be harmful if ingested in quantity. If swallowed, give plenty of water to the subject and seek medical attention. If the substance gets in the eyes, flood the eye with gently running water for ten minutes and seek medical attention. If spilt on skin or clothes, remove the contaminated clothing, wash off skin with plenty of water and soak the contaminated clothing before rinsing repeatedly. Should the substance be spilt in the laboratory, scoop up as much solid as possible. Add mineral absorbent and scoop up into a bucket. Rinse the area of spill or mop thoroughly. Wear protective clothing i.e. lab coat, and safety goggles, due to the nature of the chemicals being handled.
Making the apple puree is a hazardous procedure and all apparatus used should be managed with care. Always core away from you, ensuring that your hand is not covering the receiving side so as to avoid injury. When using the knife, cut downwards onto the white tile and avoid contact with the blade.
Ensure that hands are kept away from the blades of the blender and that no solution surrounds the electric socket in use in order to reduce the risk of experiencing an electric shock. When not in use turn off the blender from the mains supply.
When placing the bulb on to the graduate pipette ensure that it is twisted on without applying force to avoid the glass from shattering. Do not position the glass tubing of the graduated pipette toward you when applying the bulb, so as to avoid any injury.
Do not position the apparatus close to the edge of the workbench as it is made of glass and if knocked off of the table, it will break.

There are no ethical implications of the investigation.

Preliminary Work

Preliminary work allowed me to come to a number of conclusions regarding the experimental design. It was found that the maximum volume of juice did not exceed 5cm3 for any of the pHs tested. I therefore decided that it was unnecessary to use a 20cm3 measuring cylinder to collect the apple juice and that a 10cm3 measuring cylinder would more appropriate. After a twelve-minute filtration period, differences in juice volume existed between the two pHs (pH3 and pH9.5) tested, however it was impossible to determine the exact volumes simply by observation, due to the absence of division lines at the base of the measuring cylinder. The flow rate will therefore be calculated by a previously described alternative method.

Performing preliminary work determined the blending time needed to produce apple puree of an appropriate consistency. Originally, 100grams of apple chunks and 25cm3 of pH3 buffer solution together with 1ml pectinase was blended for three minutes, creating a runny solution. Should this have been used in the investigation, it would be hard to determine whether the solution passing into the measuring cylinder was due to the action of pectinase on pectin or rather a result of the exceedingly low starting viscosity of the solution. However, a blending period of one minute achieved a solution of moderate viscosity, whilst ensuring that all apple chunks had been blended.

The actual blending procedure was formulated through carrying out a pilot study. Originally the handheld blender was used to puree 50grams of apple. However, it was found that this mass was greatly reduced during the blending process because losses occurred to the blades for example. Therefore a greater mass of apple was blended. It was found that unless a volume of solution was added to the apple pieces, the substance would attach to the blades or become fixed to the bottom of the blender. After observing the viscosity of the solution obtained from adding 50cm3 pH3 buffer solution with 1ml of pectinase to 100grams of apple, it was decided that 25cm3 of phosphate buffer and 1ml 100% fungal pectinase, was a sufficient volume to achieve a solution of moderate viscosity.

Whilst carrying out a pilot study, I found that a white tile acted as a suitable cutting surface. It meant that the workbench remained unmarked and I did not have to hold the apple whilst cutting, which would create a high risk of injury. By running through the procedure I found it was beneficial to use a spatula to transfer 25grams of the pureed apple into the beaker as doing this would minimise spillages.

During the pilot study it was found that a twelve-minute filtration period produced significant variation for manageable and therefore comparable flow rates to be calculated. After twelve minutes, the volume of juice obtained when pectinase reacted in pH3 conditions was approximately 0.8ml and roughly 0.2ml of juice was obtained in pH9 conditions (Please note that these are only approximate values and were measured by using a 1ml graduated pipette to draw up the juice that passed into the measuring cylinders. Therefore the values are likely to be influenced by the presence of air bubbles within the graduated pipette and transfer losses).

The only available fungal pectinase was of 100% concentration. In order to slow down the reaction rate thus allowing us to see a difference between the flow rates obtained at different pHs, a small enzyme volume of 1ml pectinase was added to the pureed apple. This volume resulted in sufficient variation between pHs, therefore allowing a conclusion to be formulated.

The shape of the predicted graph resembles that of a bell, therefore it was decided that more than five pHs (i.e. nine) should be tested in order to formulate a valid conclusion regarding the effect of pH variation on pectinase activity. The pHs to be tested range from pH1 to pH9.5 therefore enabling the activity of pectinase to be observed in acidic, neutral and alkaline conditions. A larger number of acidic pH phosphate buffers are being used because in the pilot study (though values were inaccurate) a greater volume of juice passed into the measuring cylinder when pectinase reacted in pH3 conditions as opposed to pH9 conditions, thus illustrating that the optimum pH for enzyme activity is acidic. Also, the product information sheet for the pectinase enzyme solution being utilised states that relative pectinase activity increases as pH4.5 is approached again indicating that pectinase has an acidic optimum pH.