BioChemistry

Laboratory Exercise 1
Control of Enzymatic Browning

Desired Learning Outcomes

At the end of the class, the students can:
1. Investigate the effects of enzymatic browning on various apple treatment.
2. Monitor enzymatic browning of apple slices.
3. Determine the properties of polyphenol oxidase.

Introduction
Enzymatic Browning is an oxidation reaction that occurs in some fruits and
vegetables, resulting in negative effects on color, taste, flavor, and nutritional value.
When fruits and vegetables are bruised, cut, peeled, diseased, or exposed to the air,
they form brown melanin from the oxidation of phenolic compounds by polyphenol
oxidase (PPO). Polyphenol oxidase (PPO) is a generic term for the group of
enzymes that catalyze the oxidation of phenolic compounds to produce brown color
on cut surfaces of fruits and vegetables. This (PPO) triggers the generation of dark
pigments. Particularly relevant for apples, which are rich in polyphenols and highly
susceptible to enzymatic browning.
Materials and methodology
Fresh apple slices (6 per group)
Test solutions for dipping: 0.1% Ascorbic acid, 0.1% Citric acid, 0.1% Acetic acid,
and 1.0% Acetic acid
Beaker or cup with water
Tongs/forks
Paper towels

Procedures
1. Place an untreated apple slice on a paper towel. Label the towel “Control”.
2. Using tongs (forks) dip another apple slice into one of the test solutions for 30
seconds, place it on the towel, and label the towel with the name of the solution.
3. Do not use the same tong (fork) with a different solution.
4. Repeat the same procedure for the other three solutions.
5. Soak one slice in water for 30 seconds. Place it on a towel and label the
towel “water soak”.
6. Note the time and temperature in your data table. Observe the slices every
10 minutes and record your observations. Compare your results with those obtained
by the rest of the class during class discussion.
Rewrite each materials and methods in each experiment in past tense and passive
voice. Use declarative sentence. Never use imperative sentences.
Learning Activities:
Table 1. Effect of various treatment on browning reactions of apple slices.

Name: Juvel C. Orquinaza Group #: Seat #: Score

Course: BSN 1E

Instructor: Ms. Atheena Barre Date: 11/29/2022

Report Sheet no. 1

Enzymatic Browning

Level of Browning(*)

Time (min) No Treatment Ascorbic Citric Acid Acetic Acetic Acid Water Soak
Acid (0.1%) Acid (1.0%)
Control (Distilled
(0.1%) (0.1%)
(Calamansi) (Vinegar) Water)
(Celine (Salt +
Syrup Water)
Vitamin C)

0 1 1 1 1 1 1

10 2 2 2 2 1 3

20 2 2 2 2 2 3

30 3 2 2 2 2 3

40 3 2 2 2 2 3

50 3 2 3 2 2 4

60 3 3 3 2 2 4
* Level of Browning

5 = completely dark brown

4 = fully covered light brown

3 = Half-covered light brown

2 = Slight or scant brown patches

1 = No browning present

Review Questions:
1. What causes browning when fresh fruits and some vegetable are peeled
or cut?
Fruits and vegetables contain different molecules called enzymes. This enzyme
has a particular protein that accelerates the chemical reaction and acts as a
biological catalyst. This enzyme is responsible for fruit to ripen and over-ripen.
Furthermore, they retain the enzyme trapped in their tissues. However, when fresh
fruits are peeled or cut, their polyphenol oxidase enzyme will be exposed to and
reacts with the oxygen. This reaction is called oxidation, in which the fruit undergoes
enzymatic browning.

2. What conditions enhance the browning process? Why?

Browning occurs under the conditions of the water activity, ph. level and
temperature of an apple. The enzymes range from ph. 5-9 level. If the pH level starts
increasing or decreasing away from the optional pH range, it can cause the enzyme
to alter its shape. Moreover, temperature affects the enzymes. When the
temperature rises, the peptide chain’s kinetic motion increases as well, which causes
the protein bonds to break, which can lead to the denaturation of the enzyme. The
optimal temperature for an enzyme range from 0 to 40° C.

3. How do food additives or treatment processes in use today prevent or

retard browning in fruits and vegetables?
The physical and chemical agents retard the browning of fruits and vegetables as
they inhibit the activity of an enzymatic reaction called polyphenol oxidase, which is
responsible for browning.
The followings are some food additives that slow down the browning:
1. Chemical treatments- it is used to limit the oxidation phenomenon of the fruit.
Hence, the browning of fruit may vary depending on what kind of chemical
treatment will be utilized.

 Anti-oxidant- According to Lindley (1998), anti-oxidants prevent the initiation

of browning by reacting with oxygen and other intermediate products. Thus, it
breaks the chain reaction to prevent the formation of melanin.
  Chelating Agents- According to Du, Dou & Wu (2012). PPO requires copper
ions to be active. The presence of a substance capable of binding divalent
cations reduces the enzymatic activity of PPO.

 Calcium Salts- According to Quiles, Hernando, Perez-Munuera & Lluch,

(2007); Guan & Fan, (2010); Khunpon, Uthaibutra, Faiyue & Saengnil, (2011).
The calcium salts strengthen the cell wall, and this will prevent the destruction
of cell compartments. Thus, it inhibits the contact of PPO with polyphenols in
the vacuole.

 Acidifying agents- According to Grimm, Khanal, Winkler, Knoche & Koepcke,

(2012), PPOs are sensitive to ph. Hence, fruit is naturally acidic, however, if
there is an additional acidifying agent in fruit, therefore, it will inactivate below
ph 3 level resulting in reduced PPO activity.

2. Physical Process- temperature plays a role in browning. Indeed, polyphenol

oxidase is sensitive to high temperatures. According to the report of Özel and
others (2010), the blanching of plums above 80°C inactivates polyphenol
oxidase. Whereas, in freezing temperature, there is a decrease of water for
the enzymatic reactions leading to less polyphenol oxidase as reported by
Lavelli & Caronni (2010). According to the Arrhenius law, a temperature
decrease leads to a decrease in the rate of browning reactions (Mastrocola,
Manzocco & Poiana, 1998).

4. Why do citrus juices retard browning in fresh fruits?

The acid of citrus juices lowers the ph. level of fruit. It inactivates the oxidation of
polyphenol oxidase to prevent browning.

.
Schematic Diagram
 Documentation on Enzymatic Browning of Apples

Objective:
Participants will examine the process of apples turning brown due to chemical
reactions.
Time to complete the activity: 1hr and 30 minutes.

Background:
Tons of fruits and vegetables are produced, processed, and shipped daily,
specifically to the market. However, some of it never makes it into the stores. The
reason is fruits and vegetables turn brown rapidly when they are damaged. The
primary culprits are air, moisture, light, temperature, mechanical stress (fall, cut), and
microbial growth. Furthermore, people will often reject them due to their browning
and bruises. This reaction is called Enzymatic Browning.
Enzymatic browning happens when the chemical reaction of fruit is exposed
to oxygen, resulting in browning, and may affect the taste, flavor, and nutritional
value. The enzyme responsible for browning is called polyphenol oxidase (PPO).
Due to the presence of oxygen, the PPO changes its substances to phenolic
compounds via oxidation into quinones compounds. Thus, the quinones react with
other substances to form melanin. In addition, PPO has the presence of H 2 O 2which
induces melanin. For this reason, it affects the loss of color and the modification of
taste, flavor, and nutritional properties.
It can be simplified into:
Polyphenol oxidase + O2 → Melanin (Brown color)
Apples are one of the most consumed fruits. They are rich in minerals and
vitamins. They contain high phenolic compounds that elicit protective effects against
chronic and degenerative diseases. Hence, it acts as an anti-oxidant that potentially
limits the damage to cells and tissues. The phenolic profile of an apple has five major
phenolic groups found in most cultivars: hydroxycinnamic acids, flavanols-3 and
procyanidins, anthocyanins, flavanols, and dihydrochalcones. Furthermore, phenolic
compounds may vary depending on the fruit's maturity and climatic/ environmental
conditions. Therefore, in each apple, the phenolic composition and the activity of
various enzymes are critical to understanding, as they may influence the potential for
browning.
Questions:
 Which Test solution will prevent browning the most?

Hypothesis:
If we add a slice of an apple in various five test solutions and Controlled treatment.
Then apple will act differently with different test solutions. Furthermore, time and
temperature may affect the enzymatic browning of apples.
Variables:
 Independent Variable: Slices of apple and Different Five Test Solution
 Dependent Variable: Time taken for apple slices to turn brown.
 Controlled Variable: The freshness of apple before the experiment, type of an
apple (Red Delicious), the temperature, and Amount of time monitoring the
apple slices.

Introducing the experiment:

This experiment involves dipping slices of apple in a variety of test solutions. These
include Distilled water + salt (Acetic acid), Vinegar (Acetic acid), Distilled water
(Water soak), Calamansi (Citric acid), Celine ascorbic acid syrup + distilled
water (Ascorbic acid), and Controlled Apple slice (No treatment). Lastly, slices of
red delicious apple.

Slicing an apple.
Preparing 6 Test Solutions:

Dipping the apple slices and soaking the apple slices:

Putting the apple slices on paper towel:

Parts of an apple:

Observation on Level of Browning Data:

After 10 minutes:
After 10 minutes of observation in the treatment group of apple slice that was dipped
in Distilled water (Water Soak) and Salt +water (Acetic acid), it had some slight
browning, but not as much as the Control (No treatment). The distilled water restricts
the amount of oxygen in fruit tissues, and the PPO reaction decreases. Thus, it
slows down the production of melanin in fruit. While the control apple slices with no
treatment turned light brown. Moreover, the treatment group of apple slices dipped in
the calamansi; and Celine ascorbic acid syrup + distilled water shows a sign of scant
brown patches around the apple. In comparison, vinegar shows no sign of browning
because the 4.5% ph. level of acidity inactivates the browning of enzyme.
After 20 minutes:
Observation Picture
The Vinegar (Acetic Acid) still
shows no sign of browning, even
the injured part, which we poked
using a fork.

In Calamansi (Citric acid), the poke

holes in the hypanthium flesh part
turn light brown rapidly.

Celine ascorbic acid has a slight

browning of the skin in the apple.
Thus, there is a mushy texture in
the mesocarp part.

In the Control treatment, the

endocarp (Core) of the apple
becomes light brown.
In salt+ water treatment, the apples
were pale white. The poke holes
turn light brown in hypanthium
flesh.

The Water-soak treatment has a

slight medium shade of brown in
the endocarp of the apple.
After 30 minutes:
Observation Picture
The hypanthium fresh part of the
vinegar treatment is pale white, and
the exocarp lining and endocarp
parts were turning brown.

In Calamansi (Citric acid), the poke

holes and hypanthium fresh of
apples are still the same shade and
have not darkened.

In Celine ascorbic acid, the skin of

the apple is still the same. It keeps
a mushy texture in the mesocarp
part. Furthermore, the poke holes
and exocarp lining turn light brown.
Hence, the color of the apple
started turning a bit yellow.

In the Control treatment, the

endocarp (Core) of the apple is still
the same. The apple's mesocarp
and bruises on the hypanthium
flesh turn light brown. The color of
the apple becomes a yellowish-
brown color
In salt+ water treatment, there are
no changes in appearance.

The apple in Water-soak treatment

started turning brown.
After 40 minutes:
Observation Picture
The hypanthium fresh and
mesocarp part of the vinegar
treatment is pale white, and the
exocarp lining and endocarp parts
were both deep browns.

In Calamansi (Citric acid), the poke

holes and hypanthium fresh of
apples are still the same shade,
and the color of the apple has not
darkened.

In Celine ascorbic acid, the skin of

the apple is still the same. It keeps
a mushy texture in the mesocarp
part. Furthermore, the poke holes
and exocarp lining are a light brown
shade. Hence, the color of the
apple is a bit yellowish-brown.

In the Control treatment, the

endocarp (Core) of the apple is still
the same. The apple's mesocarp
and bruises on the hypanthium
flesh turn a medium brown shade.
The color of the apple becomes a
90% yellowish-brown color and a
10% light brown shade.
In salt+ water treatment, there are
no changes in appearance. The
two poke holes turn light brown.

The apple in the Water-soak

treatment is still the same. The skin
of the apple is a brown shade.
After 50 minutes:
Observation Picture
The hypanthium fresh and
mesocarp part of the vinegar
treatment is white, and the exocarp
lining and endocarp parts were
both deep browns.

In Calamansi (Citric acid), the poke

holes are deep brown shade, and
hypanthium fresh is still light brown.

In Celine ascorbic acid, the

exocarp (skin) of the apple is still
the same. It keeps a mushy texture
in the mesocarp part. Furthermore,
the poke holes and exocarp lining
are in deep brown shade. Hence,
the color of the apple is a bit
yellowish-brown.

In the Control treatment, the

endocarp (Core) of the apple is still
the same. The apple's mesocarp
and bruises on the hypanthium
flesh turn a medium brown shade.
The color of the apple becomes a
90% yellowish-brown color and a
10% light brown shade.
In salt+ water treatment, there are
no changes in appearance. The
two poke holes turn deep brown.

The apple in the Water-soak

treatment is fully light brown.
After 60 minutes:
Observation Picture
The hypanthium fresh and
mesocarp part of the vinegar
treatment is white, and the exocarp
lining and endocarp parts were
both deep browns. There are
brown linings around in hypanthium
fresh.

In Calamansi (Citric acid), the poke

holes are a deep brown shade, and
hypanthium fresh is still light brown.
There are a lot of spotted patches.

In Celine ascorbic acid, the skin of

the apple is still the same. It keeps
a mushy texture in the mesocarp
part. Furthermore, the poke holes
and exocarp lining are in deep
brown shade. Hence, the color of
the apple is a bit yellowish-brown.

In the Control treatment, the

endocarp (Core) of the apple is still
the same. The apple's mesocarp
and bruises on the hypanthium
flesh turn a medium brown shade.
The color of the apple becomes a
90% yellowish-brown color and a
10% light brown shade.

In salt+ water treatment, there are

no changes in appearance. The
two poke holes turn deep brown.
 The apple in the Water-soak
treatment is fully light brown. The
exocarp lining becomes medium
brown.

In Conclusion:
The hypothesis was accurate since the apple browning was differently
affected by various test solutions and controlled treatments. In conclusion, the acetic
acid found in vinegar reduces the browning by lowering the ph. level of an apple
below 3.0, and it inhibits the polyphenol oxidase enzyme. Although vinegar is
effective, it is not commonly used as a fresh preservative because it dramatically
changes the taste of an apple due to its sour and pungent smell. Furthermore, our
second option will be the calamansi; the citric acid also lowers the ph. level of the
apple. It is an easy way to prevent browning and does not negatively affect the taste.
Lemon juice is commonly citric acid used to preserve apples because it has a
chelating agent that causes metal ions to react with other molecules to form a ring
structure. Citric acid inhibits the polyphenol oxidase enzyme by binding up metal ions
in the enzyme. Therefore, it inhibits enzymatic browning. Lastly, salt + water, known
as acetic acid, slows down the browning because chloride ions in the salt inhibit the
polyphenol oxidase (PPO) enzymes, and the water shields the cut-off enzyme from
the air, which contains oxygen.

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