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EXPERIMENT 9: ENZYMATIC BROWNING OF FRUITS/VEGETABLES.

Introduction:
The purpose of this experiment is to monitor the level of enzymatic browning of apple
slices. Apples contain an enzyme called polyphenol oxidase (phenolase). In the presence of
oxygen from the air, this enzyme catalyses the formation of brown pigments called melanins.
Treatment of the apple slices with ascorbic acid, citric acid, or acetic acid will reduce the
level of browning. The reduction in browning is dependent on the type of substance and its
concentration. Soaking in water alone will temporarily reduce the level of browning by
restricting the amount of oxygen in contact with the apple slice. When fruits or vegetables are
peeled or cut, enzymes contained in the plant cells are released. In the presence of oxygen
from the air, the enzyme phenolase catalyses one step in the biochemical conversion of plant
phenolic compounds to form brown pigments known as melanins. This reaction, called
enzymatic browning, occurs readily at warm temperatures when the pH is between 5.0 and
7.0. The presence of iron or copper can increase the rate of reaction. This can be easily
observed when fruit is cut with a rusty knife or mixed in a copper bowl. Bruising or other
injury to the plant tissue disrupts the arrangement of chemicals within the cells and allows
these chemicals to make contact with oxygen. This may lead to browning of uncooked fruits.
Enzymatic browning can be a significant problem, limiting the shelf life of many fruits and
vegetables which have had little heat applied during processing. However, enzymatic
browning is not always a defect. The browning reaction contributes to the desirable colour
and flavour of raisins, prunes, coffee, tea, and cocoa.

Objective:
The monitor the level of enzymatic browning of apple slices.

Materials/Apparatus:
Fresh fruit/Vegetable, (Apple)
1% ascorbic acid
1% citric acid
1% acetic acid
2% acetic acid
Beaker or cup
Paper towel
Tong
Glove
Knife
Stopwatch or timer
Procedure:
1. The fruit (apple) was slice into 6 pieces with same size.
2. The unheated fruit slice was place on a paper towel. The towel was label as control.
3. Another apple slice was dip into one of the test solution (1% ascorbic acid) using tong
for 10 minutes.
4. The tong was rinse and repeat the same procedure for the other three solutions (1%
citric acid, 1% acetic acid, 2% acetic acid).
5. The one apple slice was soak in water for 10 minutes, the apple slice was place it on a
paper towel and label the towel water soak.
6. The time and level of browning in table was recorded. The slices every 10 minutes for
2 hours and record your observations.

Results:
Level of Browning

Time (min)

Untreated
(control)

Ascorbic
acid (1%)

Citric acid
(1%)

10

20

30

40

50

60

70

80

90

100

110

120

Key
5 = Completely dark brown
4 = Fully covered light brown
3 = Half-covered light brown
2 = Slight or scant brown patches
1 = No browning present

Discussion:

Acetic acid Acetic acid


(1%)
(2%)

Water
soaked

In this experiment, we need to monitor the level of enzymatic browning of apple


slices. Enzymatic browning is a chemical process, involving polyphenol oxidase, catechol
oxidase, and other enzymes that create melanins and benzoquinone from natural phenols,
resulting in a brown color. In general, enzymatic browning requires exposure to oxygen, for
example the browning that occurs when an apple is cut.
Several substances have been used in the food industry to prevent browning of fruits
and vegetables. Sulfites prevent browning by releasing sulfite ions, which prevent melanin
formation. The Australia New Zealand Food Standards Code permits the presence of sodium
or potassium sulfites in apples for manufacturing purposes to a level of 200mg/kg (ppm).
Sulfites are not allowed in foods in Australia unless expressly permitted by the Foods
Standards Code.
Next, ascorbic acid (vitamin C) acts as an antioxidant. Oxygen is more likely to react
with the ascorbic acid, rather than with the phenolic compounds in the fruit or vegetable.
Browning does not proceed until most of the ascorbic acid is used up in the reaction. Citric
acid and acetic acid lower the pH of the fruit tissue to retard the action of the phenolase. If the
pH is reduced below 3.0, the activity of the phenolase will be severely inhibited. Placing
fresh fruit in a water bath will temporarily inhibit the browning reaction, since water restricts
the amount of oxygen in contact with fruit tissues. Heating also prevents browning by
inactivating the phenolase. Since heat also cooks the fruit, heating cannot be used as a
treatment for fruits which will be served fresh.
There are some apples seem to brown faster than others. This is because all plant
tissues contain polyphenol oxidase (PPO), however, the level of PPO activity and
concentration of substrate is different each other. The phenolic compounds can vary between
varieties of fruits. In addition, a tissue's PPO level can vary depending on growing conditions
and fruit maturity. One approach the food industry employs to prevent enzymatic browning is
to select fruit varieties that are less susceptible to discoloration either due to lower PPO
activity or lower substrate concentration. In the home kitchen enzymatic browning can be
prevented by either reducing PPO oxidation activity or lowering the amount of substrate to
which the enzyme can bind. Coating freshly cut apples in sugar or syrup can reduce oxygen
diffusion and thus slow the browning reaction.

Conclusion:

In conclusion, recent researches have shown that the browning of apples can be
prevented or inhibited using a combination of combinations, either natural products or their
derivatives that are food additives. Various type combinations of enzymatic inhibitors,
reducing agents and anti-microbial compounds containing Ca2+ were tested. They all slowed
down microbial decay and changes in organic acids and sugars during storage. It was also
found that irradiating gamma rays could reduce rate of browning of apple juice and increase
anti-oxidant activity. This is found to have importance especially in improving the shelf-life
of food. More evidently, fresh-cut fruits could now be preserved longer without the need of
special packaging.

Questions:
1.

What causes browning when fresh fruits and some vegetables are peeled or cut?
The browning is caused by the conversion of phenolic compounds to melanins
catalysed by the enzyme polyphenol oxidase.

2.

What conditions promote the browning process? Why?


Oxygen is required for the reaction to occur. Browning increases with increasing
temperature until the polyphenol oxidase is inactivated. The optimal pH for the
browning reaction is between 5.0 and 7.0. If the pH is significantly below 3.0, the
enzyme will be inactivated. Metal ions such as iron and copper can increase the rate
of the reaction.

3.

How do food additives or treatment processes in use today prevent or retard browning
in fruits and vegetables?
Sulfites will inhibit melanin formation. Ascorbic acid (vitamin C) will prevent
oxygen from reacting with the polyphenol oxidase. Citric acid and acetic acid will
lower the pH, which decreases the enzyme activity. Citric acid also ties up copper
ions and prevents them from participating in the reaction. Heating process inactivates
the polyphenol oxidase.

4.

Why do citrus juices retard browning in fresh fruits?


Because ascorbic acid and citric acid are present in the fruit juices and they lower the
pH which may reduce the activity of the enzyme.

References:
1. http://www.sciencedaily.com/releases/1999/01/990113074812.htm.com
2. http://www.nps.ars.usda.gov/publications/publications.htm.com.?
SEQ_NO_115=122938
3. Polyphenol Oxidase". Worthington Enzyme Manual. Retrieved 13 September 2011.
4. Mayer, AM (November 2006). "Polyphenol oxidases in plants and fungi: Going
places? A review". Phytochemistry 67 (21):
23182331. doi:10.1016/j.phytochem.2006.08.006.PMID 16973188.
5. Questions and Answers: Okanagan Specialty Fruits Non-Browning Apple (Events
GD743 and GS784)" (PDF). APHIS. Retrieved 18 March 2014.
6. Milkovich, M. "Non-browning apples cause controversy" Fruit Growers News. April
29, 2011.

References:
1. http://www.sciencedaily.com/releases/1999/01/990113074812.htm
2. http://www.nps.ars.usda.gov/publications/publications.htm?SEQ_NO_115=122938

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