Enzymatic Browning of Apples

Apples consist of an enzyme called polyphenol oxidase (Phenolase). Phenolase has catecholase and cresolse activity, also containing little traces of copper. When apples are cut, phenolase is released from the cells and is exposed to oxygen. When phenolase reacts with oxygen, it catalyses one action of the biochemical conversion of phenolic substances. The existence of oxygen hyrdroxylates the colourless phenols, which are subsequently oxidised to coloured quinones called melanin.

( Vlasta Pilizota and Drago Subaric, 1997). The 'rust-like' reaction just occurs on the surface of the cut apples, as those cells have actually been broken and the phenolase as well as other enzymes within the cells have been launched.

When the enzymes are exposed to oxygen the result is a brown coloring forming on the surface of the apple when it has been cut. This response is referred to as enzymatic browning. SCHEMATIC DIAGRAM OF ENZYMATIC BROWNING (fig. 1).

Phenoloxidase catalysing the response.
Initial step of conversion.

Greater enzymatic browning occurs at an ideal environment for the enzyme.

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A temperature of around 37.5 degrees Celsius is understood to be the ideal temperature for enzyme conversion. Figure 2 reveals that temperatures listed below this ideal temperature level have a lower amount of activity, increasing as it ends up being closer to the optimum temperature level.

Temperatures above the optimal cause vibration within the enzyme, causing it to unfold and a denaturing of enzyme; reducing the quantity of browning. Additionally, a slightly acidic to neutral pH level between 5.0 and 7.0 is known to increase the enzymatic browning, this has a similar pattern to that displayed in fig.

2. (http://www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm).

Enzymatic browning in fruits and veggie is optimised to add colour and flavour to raisins, prunes, cocoa, tea and coffee. Nevertheless discolouration on fruits such as apples and bananas is typically an unfavorable side-effect of slicing the fruit which can reduce shelf life, decline market price and is likewise an indication of destruction of the nutrients within the fruit. (Vlasta Pilizota and Drago Subaric, 1997) To minimise the browning of fruits when sliced, reduction of required environments for optimal enzymatic browning requires to be achieved.

Temperatures extreme to the optimal enzymatic temperature of 37 degrees need to be attained, as well as pH levels contrary to the optimal phenolase pH level. A well-known method to prohibit enzymatic browning on fruit is to treat it with lemon juice and place it in the fridge. This method exploits two main properties of the natural browning process. The lemon juice increased the acidity which removes traces of copper in enzyme that turns the fruit brown.

Additionally, placing the fruit in the fridge slows the natural reaction of the browning. Commercially a variety of techniques are used to prevent the enzymatic browning of the fruits – each method exploiting a different aspect of the natural biochemical process. •Treating the fruits with acidic acids (low pH) – this removes the copper traces within the enzyme which are responsible for the function of the enzymes the turn the fruit brown •Blanching the fruit – rapidly increasing the temperature of the fruit denatures the enzyme and destroys the responsible brown colouring reactants

•Lowing the temperature of the fruit – this reduces the rate of the reaction in the fruit, however it will not stop the reaction entirely (collision theory) •Surrounding the fruit with unreactive gas such as nitrogen – this limits the necessary oxygen from reacting The collision theory links directly to the rate of browning on the apple.

Temperature of the system is easily exhibits this. As the molecules in the system heat up they move around more erectly and quickly, therefore they collide and react more frequently. This is also observed in the negative effect displayed by reducing the temperature of the system. As the temperature decreased the molecule move more slowly and have less kinetic energy therefore they collide far less frequently. Another exhibition of the collision theory in enzymatic browning is increasing the concentration of reactants.

By increasing the pressure in the system the concentration of oxygen is increased which increases the number of collisions between the phenol and the oxygen. This can be seen in the diagram below. Collision theory – low concentration of reactants vs. high concentration of reactants.

Domestically, a variety of acids/solutions can be used to prevent the browning of the apple. “Treating the apples slices in acetic and citric acid reduces the pH level of the fruit tissue and retard the phenolase. pH levels below 3.0 will severely inhibit enzyme activity”. (http://www.curriculumsupport.education.nsw.gov.au) “Enzymes generally possess metal ions at their active sites. Removal of these ions by chelating agents can therefore render enzymes inactive”. (FAO,2000) Polycarboxylic acids, such as tartaric acid, are commonly used as chelators.

Additionally, placing the sliced apple in a water bath will restrict the amount of oxygen in contact with the phenolase – temporarily inhibiting the melanin formation. Heating also reduces the enzymatic browning by inactivating the phenolase. However as heating cooks the fruit this is not a viable solution to the browning for fruit being served fresh. AIM

To monitor the effects of altered pH solutions and temperatures on enzymatic browning in apple slices. HYPOTHESIS
It was hypothesised that apples slices drenched in each of the acids (citric, tartaric and acetic) would experience significantly less browning than the untreated apples; due to the decreased pH levels caused by the acetic and citric acids or the removal of metal ions in the enzymes, the phenolase would be restricted, unlike the untreated slices.

It was also hypothesised that the apples at the optimal enzymatic temperature (37 degrees) would result in far greater browning than those at any other temperature. Additionally, apples far greater than this temperature would experience no browning, and slices below this temperature would experience minimal browning. This is due to the ‘slowing down’ of the enzyme at temperatures below this optimum and the predictable denaturing of enzymes above this temperature.

It was further hypothesised that the apple slices drenched in water would have limited browning at the begging of the test but then begin to slowly brown after a period of time. This is due to the initial restriction of oxygen to react with the phenolase, but is later subsided by the evaporation of the water, or the traces of oxygen found within the water.

It was lastly hypothesised that the Red Delicious apples and the Granny Smith apples would undergo the same rate of browning. However the Red Delicious apples were expected to begin at a higher level of browning than the Granny Smith due to the natural colour of the flesh.

MATERIALS
Red Delicious apples
Granny Smith apples
Citric acid
Tartaric acid
Acetic acid
Sharp knife
Blunt knife
Petri dishes
Paper towel
Gloves
Incubator
Fridge
Scale
100ml beaker
Stirring rod
Measuring cylinder

METHOD
Diluting acids
1. Scales, beaker, tartaric acid, citric acid, acetic acid, string rod and measuring cylinder were collected 2. Beaker was placed on the scale
3. Scale was zeroed
4. 2mg of tartaric acid was placed in the beaker
5. Using the measuring cylinder 98ml of diluted water was measured

6. The water was added to the beaker
7. The solution was stirred using the stirring rod until all of the acid crystals were dissolved

8. Steps 2-8 were repeated for each acid

Creating visual scale
1. 1 red delicious apple, 1 Granny smith apple, knife, petri dishes, camera were gathered 2. Using the knife the apples were cut in half
3. The cuts of apple were placed on a petri dish
4. As the apples browned photos were taken of each piece
5. When ‘maximum’ browning was reached photos were ordered from lightest to darkest

Testing enzymatic browning of the apples at various temperature whilst treated with various solutions – 27 degrees
1. Red Delicious apples, Granny Smith apples, knife, petri dishes and diluted solutions were collected 2. 2 Red Delicious apples were cut in half
3. Cut apples were saturated with tartaric acid solution
4. 3 halves of the saturated apples were placed skin side down on petri dishes 5. Steps 2-3 were repeated for each acid
6. Steps 2-5 were repeated for the Granny Smith apples
7. Level of browning was recorded for each apple slice every 10 minutes

– 8 degrees
1. Red Delicious apples, Granny Smith apples, knife, petri dishes and diluted solutions were collected 2. 2 Red Delicious apples were cut in half
3. Cut apples were saturated with tartaric acid solution
4. 3 halves of the saturated apples were placed skin side down on petri dishes 5. Petri dishes were placed in refrigerator set to 8 degrees 6. Steps 2-3 were repeated for each acid
7. Steps 2-5 were repeated for the Granny Smith apples
8. Level of browning was recorded for each apple slice every 10 minutes

– 37 degrees
1. Red Delicious apples, Granny Smith apples, knife, petri dishes and diluted solutions were collected 2. 2 Red Delicious apples were cut in half
3. Cut apples were saturated with tartaric acid solution
4. 3 halves of the saturated apples were placed skin side down on petri dishes 5. Petri dishes were placed in incubator set to 37 degrees
6. Steps 2-3 were repeated for each acid
7. Steps 2-5 were repeated for the Granny Smith apples
8. Level of browning was recorded for each apple slice every 10 minutes

– 60 degrees
1. Red Delicious apples, Granny Smith apples, knife, petri dishes and diluted solutions were collected 2. 2 Red Delicious apples were cut in half
3. Cut apples were saturated with tartaric acid solution
4. 3 halves of the saturated apples were placed skin side down on petri dishes 5. Petri dishes were placed in incubator set to 60 degrees
6. Steps 2-3 were repeated for each acid
7. Steps 2-5 were repeated for the Granny Smith apples
8. Level of browning was recorded for each apple slice every 10 minutes

RESULTS
Enzymatic browning at 60° Celsius
Table 1, Average Enzymatic Browning of apples at 60° Celsius
REDGREEN
ControlCitricTartaricAceticdH2OControlCitricTartaricAceticdH2O 011.21.21100000
1011.21.21.21000.200
201.21.31.31.3100.30.20.20
301.21.31.31.31.30.30.30.50.20.5
401.31.81.61.61.30.50.60.60.30.6
501.6221.61.60.80.80.80.50.6
601.62.22.321.61.211.20.61.3

Moderate/ high levels of browning occurred to all apples at this temperature. Apples treated with citric and tartaric acid had the highest level of browning throughout the entire test. Apples drenched in distilled H2O had the least amount of browning.

Low/ moderate level of browning occurred with minimal browning in the first 20 minutes. The tartaric acid and distilled water solutions had the highest level of browning, acetic acid had the lowest amount of browning.

Enzymatic browning at 37° Celsius
Table 2, Average Enzymatic Browning of apples at 37 ° Celsius
REDGREEN
ControlCitricTartaricAceticdH2OControlCitricTartaricAceticdH2O 01111100000
101111.2100000
201.21.21.21.31.20.20000
301.21.71.31.51.50.20000
401.51.71.31.81.60.50.2000
501.61.71.71.81.80.60.200.20
601.61.81.7220.80.200.20.2

Moderate/ high levels of browning occurred to all apples at this temperature. Apples treated with acetic acid and distilled water acid had the highest level of browning throughout the test. There was no constant lowest/highest level of browning solution for the entire test.

Low/ moderate level of browning occurred with minimal browning in the first 40 minutes of testing. The control apple had significantly more browning than all other apples. The tartaric acid apples had no browning.

Enzymatic browning at 22° Celsius

Table 3, Average Enzymatic browning of apples at 22° Celsius
REDGREEN
ControlCitricTartaricAceticdH2OControlCitricTartaricAceticdH2O 01111100.2000
101111100.2000
201111100.2000
3011.311100.2000
401.2311100.2000
501.2311.31.600.2000
601.21.611.31.600.2000

Moderate browning occurred throughout the entire test. The browning did not increase for any of the tests until 30minutes. Citric acid had the most browning overall with the other solutions remaining fairly constant.

Minimal to no browning occurred in all of these tests. Browning seen in the citric test can be seen as an anomaly; possibly due to browning that had occurred prior to the test. Enzymatic browning at 8° Celsius

Table 4, Average Enzymatic browning of apples at 22° Celsius
REDGREEN
ControlCitricTartaricAceticdH2OControlCitricTartaricAceticdH2O 0111.21100000
10111.11100000
201.211.211.20.200.200
301.21.21.21.21.20.200.200
401.31.21.21.21.20.200.200
501.31.21.31.31.50.500.300
601.21.31.31.70.800.3000

Moderate/ high levels of browning occurred to all apples at this temperature. Apples treated with tartaric acid began browning first but apples drenched in distilled water had the highest level of browning at the end of the test. Citric acid treated apples had the least amount of browning in the test.

Minimal browning occurred to these apples. Distilled water, acetic acid and citric acid treated apples had no browning. The control apples in this test showed the most browning.

DISCUSSION
The purpose of this investigation was to determine the degree of enzymatic browning occurring to sliced apples of different species, at different temperatures and treated with different solutions. The investigation explored some of the common storage methods used when attempting to prohibit browning. Initial predictions of the browning were that at extreme temperatures (8°C and 60°C) little browning would occur; as the enzyme would be inactivated when it became too hot, and the rate of collisions would be slow and highly unsuccessful as it became too cold.

It was also expected that at temperatures around 37°C enzymatic browning would be at its most; as this the optimal enzyme temperature. Significant observations during the test were: overall, far less browning than expected, generally, the Red delicious apples browned more than the Granny smith apples, levels of browning according to temperature did not occur as hypothesised or as expected via research obtained. It can be seen in figures 3-10, that the average level of browning for the Granny Smith in all tests at 60 minutes were far less than the Red Delicious Apples - this can also be seen in figure

11. As seen in tables 1-4, at 0 minutes all the Red delicious apples started the test with a 1 on the scale, however, the Granny smith apples began the test with a level of 0 browning. This is due to the natural fresh colour difference between the two species of apples.

When creating the scale this had to be considered as a single scale was required for a comparison that could be used for the two species. It was found during testing that the Red delicious apples had more surface moisture after a length of time compared to the Granny Smith. The Granny Smith apples had dried out and shrivelled within 40 minutes of the test.

As seen in figure 12, when the catecholase and Cresolase as exposed to oxygen, they reacts to form the browning pigments as well as traces of H2O. The lack of moisture on the Granny Smith apples suggests that this reaction is not occurring, or is not occurring sufficiently. This was also determined as no browning was observed on the cut apple. Commercially, enzymatic browning on apples reduces the shelf life, so this is highly undesirable. It is suggested that the apples were treated with an enzymatic prohibitor, before purchase, therefore disallowing the apples to brown.

Figure 11 illustrates that on average the highest level of browning occurred to apples stored at 60°C. This was followed by 37°C, then 8°C, then with the lowest level of browning on average occurred at 22°C. These results suggest that to minimise enzymatic browning the apples should be stored at 22°C. At a temperature as high as 60°C, the enzyme should have been inactivated, and enzymatic browning should have been minimal or not occurring at all, however, results suggested that this was not the case.

This unusual browning was investigated further and it was hypothesised that the browning occurring was not due to enzymatic browning, but instead the flesh of the apple began to cook. Furthermore, it was expected that at the optimal enzymatic temperature (37°C) the highest level of browning would occur. Again, this hypothesis was not supported by the results.

Results obtained and graphed in figure 11, indicate that the highest level of enzymatic browning occurred in the acetic acid and distilled water solutions. Comparatively, on average the apples treated with tartaric acid solution had underwent the lowest amount of browning overall. It is evident that the most effective solution to prohibit the activation the polyphenol oxidase is tartaric acid. It was also observed that rate of browning did not have a constant trend across all tests. The hypothesis made that apples treated in the distilled water would not undergo browning until sometime into the test could not be confirmed as the results were highly varied across each of the tests.

Although an abundance of results were obtained numerous inconsistencies, anomalies and contradictions were prevalent in the results. The collision theory states that as the temperature decreases, less successful collision will occur. This is due to the lowered kinetic energy at these temperatures. Results obtained contradict with this theory; as levels of browning for apples at 8°C was higher than those the a higher temperature of those at 22°C. Figure 8 displayed highly unexpected results, as only the apple slice treated in citric acid had any signs of browning.

Though, this browning was constant from 0 minutes, suggestion that initial browning was present on the apple and no further browning occurred. Results also determined the level of browning to have occurred as follows: 60°C  37°  8°C  22°C. However, this should have occurred 37°C  60°  22°C  8°C. Inconsistencies and anomalies such as the ones indicated by the results maybe have been due to a range of factors. Firstly, varying temperatures in each environment may have had some effect. Due to the nature of the environment that the test was performed in incubators and fridges were not left closed for the entire period of the test.

This may have increased or reduced the temperature of the area. Furthermore, the room temperature value was only checked once during the entire few weeks of the testing. This temperature had potential to shift up to 6 degrees at any time during the test. Additionally, preparation of apples may have been varied. Time of soaking for each apple slice in the solutions was not recorded; this may have resulted in additional soaking for some apples compared to other.

This would have increased/decreased the saturation level of some of the apples slices which would have varied the potential for inactivation of te polyphenol oxidase within the apple slices. The accuracy and possible consistency of the results may be improved with several alterations the experiment. As it was expected that the apples may have been treated with preservatives prior to purchase, and this had a significant impact on the browning of the Granny Smith apples, it is suggested that with further testing organic apples should be purchased for the test.

Moreover, an improved regulation of experimental environment is highly recommended. This would include more frequent checks of temperatures that the apples are being stored in. Additionally, humidity or experimental area may have had an effect on browning but this was not investigated thoroughly. It is proposed that regulation of humidity should be tested in further testing.

Furthermore, possible variable testing of humidity and its direct effects on the enzymatic browning could be investigated. Also, monitoring of knife used is suggested. If rust is present on the knife it is likely to promote more browning on the apples, additionally, a blunt or serrated knife will break more of the apple’s cell, therefore releasing more enzymes to react with the oxygen. Finally, a consistent and monitored saturation time is advised to improve the fairness of the test.

CONCLUSION
In conclusion, the effects of a range of temperatures and solutions and their effects on enzymatic browning in apples has been investigated, therefore the aim was completed. Some of the hypothesises made were challenged by the results but verification and scrutiny rendered the results abnormal and the hypothesises correct. Other hypothesise were found to be true.

Many improvements were suggested to improve accuracy of results and to obtain more justified and expected results. Overall it was found that the most effective method to prohibit the enzymatic browning occurring on sliced apples is storage at a cooler temperature, and in a solution of tartaric acid.