Introduction: Enzymatic oxidation occurs when Polyphenol Oxidase comes into contact with oxygen in the air. The polyphenol oxidase, or PPO, is the catalyst in the reaction that produces the dark pigments when phenolic compounds are oxidised. Apples, especially red apples, are highly prone to enzymatic browning as they are rich in these polyphenols and phenolic compounds.
Ascorbic acid (C6H8O6), otherwise known as Vitamin C, is found in citrus fruits as well as many other fruits and vegetables and has antioxidant properties. It is abundant in lemon juice, contributing to the juice’s acidic nature and nutritional value.(Figure 1.)
pH is defined as; a figure that expresses the acidity or alkalinity of a solution on a logarithmic scale of 0 to 14, where solutions below 7 are acidic and solutions above 7 are alkaline or basic. (Figure 2.)
Its formula is determined to be;
pH=–log[H+]
Where H+ equals the hydrogen ion concentration in moles per litre (mol/L).
Controlling or slowing enzymatic browning or oxidation is of great importance to the food industry as the reaction provides detrimental effects to the attributes of colour and flavour as well as nutritional value. By investigating the effects of ascorbic acid (lemon juice) on the amount of oxidation that occurs, strategies can be implemented to keep cut or bruised apples looking fresher for longer periods. This will be especially useful in restaurants and places that require food management to increase the longevity of an apple on display. It may also increase customer satisfaction with the product.
Molecular structure of an ascorbic acid molecule (Figure 1.)
Source: Vitamin C Molecules (https://www.worldofmolecules.com/antioxidants/vitaminc.htm)
pH scale (Figure 2.)
Source: What is pH (https://www.jansanconsulting.com/ph-scale.html)
Aim: To determine which concentration of ascorbic acid (lemon juice diluted in water) is most effective in slowing or stopping the oxidation of a cut apple’s flesh.
Hypothesis: The mixture with the highest concentration of ascorbic acid will be most effective in slowing or stopping the enzymic oxidation of the apple’s flesh.
Materials:
● 4 apples per trial (red lady apples)
● Lemon Juice (bottled from coles) with a pH of 1.64
● Coles brand bottled water with a pH of 6.8
● Plastic Plates
● Knife (for cutting the apples)
● Chopping board (for cutting apples)
● 2 x 5 mL NUROFEN syringe
● 6 x 100mL cups for mixing lemon juice and water
● Timer/stopwatch (on phone)
● Camera (another phone)
● pH meter (Gardman’s soil pH tester from Bunnings)
Variables:
Independent: pH of mixture
Dependent: amount of oxidation
Controlled: type of apple, water source, lemon juice, measurement equipment, time, environment.
All apples were from the same bag, purchased at Coles on the 11th of August. The water was from Coles Brand bottled water, and all samples had a pH of 6.8. The lemon juice was sourced from bottled Coles Brand lemon juice to alleviate natural differences in the lemons. The same measurement equipment was used and where reusing equipment could interfere with results, the same model and brand of equipment was used (NUROFEN syringe). The experiment was conducted on days with similar conditions (temperature, humidity, etc.) in the same location at the same time of day.
Method:
3 mL of lemon juice was extracted using a syringe and placed in a 100 mL cup.
30 mL of water was extracted using a syringe and placed in the same cup.
The pH of the mixture was then measured using a pH meter.
Steps 1 – 3 were repeated for the dilutions of lemon juice to water, 12mL:40mL, 10mL:20mL, 15mL:15mL and 10mL:5mL.
10 mL of lemon juice was placed in the final cup.
The two cheeks of apple were cut, and then cut in half again. This was repeated until eight pieces of apple were cut.
Two mL of the first mixture (3:30) was spread over an apple piece using a syringe and placed on a plate.
This was repeated for each mixture with one piece as nothing (as a control) and one piece left over, all on different plates.
Using a stopwatch, a photo with flash was taken of each piece at 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 80 minutes, 100 minutes and 120 minutes.
The apple pieces were left over night and a finishing photo taken.
Each photo was given an integer value on an oxidation scale of 0 to 10, with 10 representing the most oxidised and 0 representing no oxidation.
The experiment was repeated 5 times
Results:
Dilutions (lemon to water):
Mixture 1 – 1:10 … has a pH of 5.2 … 3 mL lemon juice to 30 mL water
Mixture 2 – 3:10 … has a pH of 4.8 … 12 mL lemon juice to 40 mL water
Mixture 3 – 1:2 … has a pH of 4.65 … 10 mL lemon juice to 20 mL water
Mixture 4 – 1:1 … has a pH of 4.45 … 15 mL lemon juice to 15 mL water
Mixture 5 – 2:1 … has a pH of 4.35 … 10 mL lemon juice to 5 mL water
Mixture 6 – 1:0 … has a pH of 1.64 (calculated)
+ Control version (nothing)
Calculations:
The pH meter acquired does not read below 3 on the pH scale. Therefore, the pH of the lemon juice must be calculated.
pH = –log(H+)
in mol/L where H+ represents hydrogen ion concentration.
mol=6.022 ×1023
particles of a substance (known as Avogadro’s number [
NA
]
Mixture 1 = 3 mL (unknown pH) : 30 mL (pH 6.8)
This mixture had a pH reading of 5.2.
Using the pH formula to calculate hydrogen ion concentration;
pH= –log(H+)
5.2 = – log(H+)
H+ =10–5.2
H+ =6.31 ×10–6×NA×331000
… 1
Similarly, H+ in water;
pH= –log(H+)
6.8 = – log(H+)
H+ =10–6.8
H+ =1.58 ×10–7×NA×301000
… 2
Using 1 and 2;
let x=H+
in 3 mL of lemon juice.
x×NA×31000+1.58×10–7×NA×301000=6.31 ×10–6×NA×331000
Cancelling
NA and 1000
and solving for x.
x = 13(6.31 ×10–6×33 –1.58 ×10–7×30)
x = 6.78 ×10–5
H+ = x ×10003
H+= 2.26 ×10–2
H+ = in 3 mL of lemon juice
= 2.26 ×10–2
pH = –log(2.26 ×10–2)
= 1.65
Research expectations of lemon juice pH is approximately 2.
Using the same method on Mixture 2 (12 mL : 40 mL), gave a pH of 1.64,
Mixture 3 gave 1.65, Mixture 4 gave 1.63 and Mixture 5 gave 1.65. These values were averaged to give the pH of the lemon juice as 1.64. (Figure 3.)
(Figure 3.)
pH calculations.
Tables:
Trial 1
Time (mins)
10
20
30
40
50
60
80
100
120
Overnight
Control
2
3
4
6
7
7
8
8
8
10
pH 5.2
1
2
2
2
2
2
3
4
5
9
pH 4.8
0
0
0
1
1
1
1
1
2
6
pH 4.65
0
1
2
2
3
3
3
4
4
7
pH 4.45
0
0
1
1
1
2
2
2
3
5
pH 4.35
0
0
0
0
0
0
1
1
1
2
pH 1.65
0
0
0
0
0
0
0
0
0
1
Trial 2
Time (mins)
10
20
30
40
50
60
80
100
120
Overnight
Control
0
1
1
1
2
3
3
4
4
6
pH 5.2
0
1
1
1
1
1
1
2
3
6
pH 4.8
0
1
1
1
2
2
3
3
4
5
pH 4.65
0
1
1
1
1
1
2
2
2
5
pH 4.45
0
1
1
1
1
1
1
2
2
4
pH 4.35
0
0
0
0
0
0
0
0
0
2
pH 1.65
0
0
0
0
0
0
0
0
0
1
Trial 3
Time (mins)
10
20
30
40
50
60
80
100
120
Overnight
Control
1
1
2
2
2
3
4
5
5
9
pH 5.2
0
1
1
2
3
3
4
4
6
9
pH 4.8
0
0
0
1
1
1
3
3
3
7
pH 4.65
0
0
1
1
1
1
2
3
3
6
pH 4.45
0
0
0
0
0
1
1
2
2
5
pH 4.35
0
0
0
0
0
1
1
2
2
3
pH 1.65
0
0
0
0
0
0
0
1
1
1
Trial 4
Time (mins)
10
20
30
40
50
60
80
100
120
Overnight
Control
2
2
3
3
4
5
6
6
7
10
pH 5.2
0
0
0
0
0
0
1
1
2
4
pH 4.8
0
1
2
2
2
3
4
4
4
7
pH 4.65
0
0
0
0
1
1
2
2
2
6
pH 4.45
0
0
0
0
1
1
2
2
2
4
pH 4.35
0
0
0
0
0
0
0
0
0
1
pH 1.65
0
0
0
0
0
0
0
1
1
1
Trial 5
Time (mins)
10
20
30
40
50
60
80
100
120
Overnight
Control
1
2
2
3
4
5
5
6
7
10
pH 5.2
1
1
1
1
2
2
3
3
4
7
pH 4.8
0
0
1
1
2
3
3
3
4
6
pH 4.65
0
0
1
1
2
2
3
3
3
5
pH 4.45
0
0
0
0
1
2
2
2
2
4
pH 4.35
0
0
0
0
0
1
1
1
2
4
pH 1.65
0
0
0
0
0
0
0
0
1
2
Averages:
Time (mins)
10
20
30
40
50
60
80
100
120
Overnight
Control
1.2
1.8
2.4
3
3.8
4.6
5.2
5.8
6.2
9
pH 5.2
0.4
1
1
1.2
1.6
1.6
2.4
2.8
4
7
pH 4.8
0
0.4
0.8
1.2
1.6
2
2.8
2.8
3.4
6.2
pH 4.65
0
0.4
1
1
1.6
1.6
2.4
2.8
2.8
5.8
pH 4.45
0
0.2
0.4
0.4
0.8
1.4
1.6
2
2.2
4.4
pH 4.35
0
0
0
0
0
0.4
0.8
1
1.2
2.6
pH 1.65
0
0
0
0
0
0
0
0.2
0.4
1.2
Graphs:
Diagrams:
Discussion: Aside from one trial that gave abnormal results, the experiment proved to be generally reliable in its performance, with all other trials giving similar results. The scale used, provided an accurate and reliable way to evaluate each piece of fruit without many inconsistencies in the qualitative observations. The permanent record of the fruit at each measurement period (photos) allowed for a more accurate comparison while also reducing the variance in time taken to measure each piece of fruit at the time.
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The atypical results of Trial 2 and Trial 4, pH 5.2, show that there were other variables that altered the results of the experiment. These variables were outside the control of the experiment as it lacked the equipment and technology to properly control those elements. The dissimilarities in results most likely emanated from the variation of cuts i the apple slices, where some pieces had bruising or core in the centre, and the amount of apple’s juice that was released.
Through the calculations done to find the pH of the straight lemon juice, it was found that the comparative readings of the pH meter were reliable but inaccurate. The calculated pH of the lemon juice in each lemon juice mixture were within 0.02 of each other (1.63 to 1.65), but the average was outside of the researched expectation of the approximate range of lemon juice pH (pH 2 to 2.7). Therefore, there is a fair degree of equipment error and inaccuracy in the meter. As the pH meter used was a soil pH meter from Bunnings, the inaccuracy is not surprising, and could be eliminated through the use of pH strips, or a professional liquid pH meter.
This experiment is valid, as it accurately reflects the claims it supports. The method and its results demonstrates the correlation between the concentration of ascorbic acid the amount of oxidation. The results support the hypothesis that the more acidic mixtures would be most effective. This is displayed through the graph that clearly shows the large difference in oxidation levels between the apple piece with straight lemon juice (Mixture 6), compared to that of the control or Mixture 1 (1:10, pH 5.2).
Conclusion: The experiment aimed to determine which concentration of ascorbic acid, or lemon juice diluted with water, would delay the enzymatic oxidation process of an apple’s flesh, most effectively. The hypothesis was, that the mixtures with the highest ratio of lemon juice to water would have the largest impact in slowing the browning, which is supported by the results that state that the most acidic mixtures (highest concentration) were most effective in keeping the apples looking fresh. The apple pieces with straight lemon juice on average had the same amount of oxidation on their flesh overnight, as the control experiments did after ten minutes.
To further test the effectiveness of ascorbic acid against oxidation, one would have to remove the variables of differences in the apples themselves. The outliers in this experiment most likely stemmed from natural differences in the way the apple pieces were cut and the amount of apple juice that was released from the slice. Apples also have ascorbic acid in their juice, which may have altered results, where an apple slice released more juice from a cut than others. To alleviate any discrepancies between trials, the apple slices must be cut in the same way, to avoid different surface areas to be covered by the juice. With the equipment and technologies available, the precision of cuts was not viable in this experiment.
Bibliography:
Ali, H.M., El-Gizawy, A.M., El-Bassiouny, R.E.I. and Saleh, M.A. (2014). Browning inhibition mechanisms by cysteine, ascorbic acid and citric acid, and identifying PPO-catechol-cysteine reaction products. Journal of Food Science and Technology. [online] Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4444905/.
Deane, J.A. (1973). Lange’s Handbook of Chemistry. 11th ed. p.Section 5, pg 66.
Hacişevkđ, A. (2009). AN OVERVIEW OF ASCORBIC ACID BIOCHEMISTRY ASKORBĐK ASĐT BĐYOKĐMYASINA BĐR BAKIŞ. Ankara Ecz. Fak. Derg. J. Fac. Pharm, Ankara, [online] 38(3), pp.233–255. Available at: https://pdfs.semanticscholar.org/31df/ad1b9c3b1467e4b68a3fba4e6065927ee15f.pdf [Accessed 11 Aug. 2019].
Holderbaum, D.F., Kon, T., Kudo, T. and Guerra, M.P. (2010). Enzymatic Browning, Polyphenol Oxidase Activity, and Polyphenols in Four Apple Cultivars: Dynamics during Fruit Development. HortScience, [online] 45(8), pp.1150–1154. Available at: https://journals.ashs.org/hortsci/view/journals/hortsci/45/8/article-p1150.xml [Accessed 8 Aug. 2019].
Perry, R.H. and Chilton, C.H. (1973). Chemical engineers’ handbook. 5th ed. New York (Osv.): Mcgraw-Hill.
PubChem (2019). Ascorbic acid. [online] Nih.gov. Available at: https://pubchem.ncbi.nlm.nih.gov/compound/Ascorbic-acid [Accessed 2 Aug. 2019].
Walker, J.R.. (1963). STUDIES ON THE ENZYMATIC BROWNING OF APPLES II. PROPERTIES OF APPLE POLYPHENOLOXIDASE. [online] Available at: http://www.publish.csiro.au/bi/pdf/bi9640360 [Accessed 2 Aug. 2019].
Weast, R.C. (1975). CRC handbook of chemistry and physics : a ready-reference book of chemical and physical data. 56th ed. Cleveland, Ohio: The Chemical Rubber, Cop, p.133.
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