PRACTICAL- 04

ENZYMATIC AND NON- ENZYMATIC BROWNING

Introduction
Browning is a common occurrence in foods. Melanins and other chemicals are responsible for the
brown color. Melanins may be synthesized by enzymatic or non-enzymatic (Maillard) reactions.
Browning is undesirable in some cases while desirable in others. The goal in handling food is to
inhibit undesirable browning reactions as much as possible and to control desirable browning
reactions.

Enzymatic Browning
It is a natural phenomenon that occurs widely in many fruits and vegetables. When fruits and
vegetables are bruised, cut, peeled, diseased, or exposed to any abnormal conditions, they darken
rapidly on exposure to air as a result of the formation of brown melanins from the oxidation of
phenolic compounds. Polyphenol oxidase (PPO) present in most fruits and vegetables, and some
seafood, is responsible for enzymatic browning. In addition to PPO, the presence of peroxidase, a
similar oxidative enzyme, may initiate enzymatic browning of fruits and vegetables. It occurs
during various procedures used in the food industry: deep-freezing, dehydro freezing, freeze-drying,
hot-air drying, etc. In practice, there are three approaches to prevention of this browning: varietal
selection, physical methods, and chemical inhibitors.

Methods of Prevention of Enzymatic browning

Enzymatic browning is a result of processes involved in the production of pigments from
enzymatically oxidized phenolic compounds of natural origin. Sulfites
form inactive complexes with enzymes or combine with breakdown products to form stable
complexes, thus inhibiting enzyme-induced formation of abnormal colors in fruits and vegetables
during processing and storage. Sulfite dips are used to control discoloration due to enzymic
browning in frozen stored fruits and vegetables, as food enzymes are not destroyed by freezing. The
development of white specks during storage in prawns can be controlled with the use of 10% salt
and 0.04% sodium metabisulfite solution without loss of nutrients. The formulations based on 4-
hexyl-resorcinol and sulfides can delay the appearance of melanosis in prawns by inhibiting
polyphenol oxidase activity. Commercial sulfides can inhibit luminescent bacterial growth.
Deep-frozen fruits and vegetables are considered in detail, since enzymatic browning is most
difficult to resolve in these products. Also, the preventive methods used during industrial drying
and freeze-drying, and even canning and bottling.
These physical methods work by the use of high or low temperature, by removing oxygen, and
irradiation. They work by destroying the enzymes, slowing down the enzyme activity or by oxygen
removal. While chemical methods include the use of antioxidants, acidifying agents and/or
chelating agents. Also, a new way to fight browning is also starting to gain a following—genetic
engineering.
 Picture.1- Enzymatic Browning
Procedure
 Prepare the following solutions before slicing the apples:
a) Ascorbic Acid: Add 15 g of commercial ascorbic acid to one cup of water
b) Dilute Sugar Syrup: In a 1-quart saucepan, dissolve 100g of sugar in I cup of water. Bring
to boiling. Cool before adding slices.
c) Keep 1 cup of cold water, ready for use.

 Once the solutions are ready, cut the apples into quarters and core. Cut each quarter into 4
slices. Use 4 slices of each variety, and perform the following treatments immediately.
a) Arrange 4 slices of each variety on plates. Do not cover. Allow to remain exposed to air for
1 hour before evaluation.
b) Dip the slices from each variety into the ascorbic acid solution immediately after slicing.
Remove slices from solution after each side of slice is properly wetted. Arrange on plate. Do
not cover. Allow to remain exposed to air for one hour, before evaluation.
c) Immerse the slices in dilute sugar syrup until evaluation.
d) Immerse slices in cold water until evaluation.

 Evaluation: Compare the appearance (surface browning) by the various treatments.

Non - Enzymatic Browning

Non-enzymatic browning is a chemical process that produces a brown color in foods without the
activity of enzymes. The two main forms of non-enzymatic browning are caramelization and the
Maillard reaction. Both vary in reaction rate as a function of water activity.
Caramelization is a non-enzymatic reaction that occurs when carbohydrates or sugars in food are
heated. It is the process of removal of water from a sugar followed by isomerization and
polymerization steps.
The Maillard reaction is a chemical reaction between an amino acid and a reducing sugar, usually
requiring the addition of heat. Like caramelization, it is a form of non-enzymatic browning. The
reactive carbonyl group of the sugar interacts with the nucleophilic amino group of the amino acid,
and interesting but poorly characterized odor and flavor molecules result. This process accelerates
in an alkaline environment because the amino groups do not neutralize. This reaction is the basis of
the flavoring industry, since the type of amino acid determines the resulting flavor. In the process,
hundreds of different flavor compounds are created. These compounds in turn break down to form
yet more new flavor compounds, and so on. Each type of food has a very distinctive set of flavour
compounds that are formed during the Maillard reaction.
 Picture.2 - Maillard Reaction Picture.3 - Caramelization

OBSERVATIONS:
Table 1 - Enzymatic Browning in apples and potatoes subjected to various treatments.

Treatment Browning, raw apples Browning, raw potatoes

a. Exposed to air Browning Browning
b. Ascorbic acid solution Slight browning No browning
c. Dilute sugar syrup No browning No browning
d. Cold water Sligh browning No browning

Fig 1 – Enzymatic Browning in Fig 2 – Enzymatic Browning in

apple potato
Discussion:

Fresh fruit and vegetables normally keep enzymes trapped in their tissues.  However, when the
fruit is sliced, or squashed, or when the fruit or vegetable begins to break down with age, the
enzymes come into contact with oxygen in the air. This causes the fruit to turn brown.

The apples and potatoes in contact with oxygen leads to melanin or brown colour formation seen in
(A) while a coating of ascorbic acid, which is a known antioxidant doesn’t let oxygen react with
apples and potato. Sugar syrup forms a coating on the apples and potato and doesn’t let the oxygen
react with the phenols present in the substrate, this can be applied for storing cut fruits. Cold water
control showed slight browning in apples and no browning in potatoes as apple has high
concentration of phenols and phenolase, the reaction rate is higher leading to more browning in
comparison to potato.
Non-Enzymatic Browning
Procedure

Formula:
Cake Flour 117 g
Baking Powder (double action) 4.5 g
Salt 2.25 g
Vanilla 3 ml
Milk 119 ml
Shortening 62 g
Sugar 150 g
Egg 54 g

 Note weight of empty pan. Prepare pan by oiling and lining the bottom of the pan with wax
paper. Use a 19 x 9x 9-5 cm pan if available, otherwise use 0.375 g of batter / ml capacity of the
pan, rather than the specified 300 g).
 Pre-heat oven to 190°C (374° F).
 Sift together the flour, baking powder and salt.
 Add vanilla to milk.
 Cream the shortening, using the medium high speed of the mixer, for 1 minute; Add sugar
continually for 1 minute; continue creaming for 90 seconds, using medium high speed.
 Add egg gradually and continue creaming for 1 minute. Stop the mixer and scrape the sides of
the mixing bowl thoroughly to the bottom.
 Add one-third of the flour mixture and one-third of the milk and mix for 45 seconds, using low
speed. Repeat twice until all the flour is blended. Blend the batter for 45 seconds at medium-
high speed.
 Weigh 300 g of batter into a loaf pan and note the initial height of the batter, using vernier
 calipers (the height measurements can then be used to estimate per cent increase in volume).
 Bake at 374°F until the cake springs back under the pressure of a finger (about 30 minutes).
Note baking time. Cool at least 10 minutes on a rack before removing from pan. Measure the
final height of the cake using vernier calipers. Measure the weight of the cake to calculate the
specific gravity (weight/volume).
 Use the rest of the batter to calculate the specific gravity. Perform line spread test, by using a
1/4 cup (metal) full batter, and observe spread for 10 minutes (at intervals of 2 minutes).

Variations:
1. Control Cake: prepared from the basic formula.
2. Fructose: Substitute 100 g of sucrose with 100 g of fructose.
3. Twice the amount of sugar: Prepare the cake using the basic formula but increase sugar
content to 200g.
4. Sugar substitute: Use the following sugar replacers at the recommended levels:
Splenda (10g splenda for every 100g of sucrose)

Optional
5. Lower baking temperature: Bake the cake at 163°C (325° F), instead of 190°C
6. Higher baking temperature: Bake the cake at 218°C (425° F), instead of 190°C

Evaluation:
Objective
1. Volume: Determine the volume of each cake. Since the cakes will also be used for subjective
testing, wrap tightly in a single layer of plastic wrap before testing.
2. Appearance: The appearance of the cake can be recorded by obtaining its photocopy. These
provide a record of the actual shape and size of a slice, and give some information regarding grain
size. The method is more accurate with light colored products than with dark colored products like
chocolate cake. The photocopy can be made by placing a sheet of clear plastic film on the glass
plate of the photocopy machine, placing the sample on the film and placing a white paper on top of
the sample. Notations of significant characteristics of each sample should be made on the copy.

Subjective
The cakes made with different variations will have some distinct, but different effects on exterior
appearance, texture, color, flavor and tenderness of the cakes. Evaluate these aspects carefully.

Optional
3. Hardness / Tenderness: Use Penetrometer to test the tenderness of the cake. The penetration
probe of the Texture Analyzer can also be used for this test.
 OBSERVATIONS:

Table 2 - Objective Evaluation of shortened cakes.

Variable Batter Cake
Line Specific Initial Final Inc. Final Dec. Volum Densit Hard
spread gravity height height volum weight in e y ness
(cm) (kg/m3) (cm) (cm) e (%) (g) weight (cm3) (Wt/V
(g) ol)
(g/ml)
Control 7.25 0.81 0.69 1.39 0.7 382 85 - 0.59 -
Fructose 7 1.0 0.95 1.49 0.54 432 54 - 0.8 -
Sugar 2x 8 1.02 1.11 1.06 -0.04 239 18 - 0.95 -
Splenda 9 0.3 0.91 1.9 1.9 123 66 - 0.74 -
High 8 0.73 1.54 1.32 0.22 265 45 - 1.03 -
temperature
Low 8 0.73 1.54 0.99 0.45 284 19 - 1.03 -
temperature

CALCULATIONS:
1. Variable – Control
Line-spread = 7+8+7+7/4 = 7.25 cm
Specific gravity = Density of the batter/Reference density of the water
Mass of the batter = 467 g
Volume of the batter = 570 ml
Reference density of the water = 1 g/ml
Specific gravity = 467/570 = 0.81/1
= 0.81 kg/m3
Increased volume = 1.39 – 0.69
= 0.7%
Decreased weight = 467-382
= 82 g
Density of the cake = 382g/640ml
= 0.59 g/ml
 2. Variable – Fructose
Increased volume = 0.95 – 1.49
= 0.54%
3. Variable – Sugar 2x
Increased volume = 1.11 – 1.06
= -0.04%
4. Variable – Splenda
Line-spread = 9+9+9+9/4 = 9 cm
Specific gravity = Density of the batter/Reference density of the water
Mass of the batter = 189 g
Volume of the batter = 165 ml
Specific gravity = 189/165 = 0.3/1
= 0.3 kg/m3
Increased volume = 0.91 – 1.9
= 1.9%
Decreased weight = 189 - 123
= 66 g
5. Variable – High temperature
Increased volume = 1.54 – 1.32
= 0.22%
6. Variable – Low temperature
Increased volume = 1.54 – 0.99
= 0.45%
OBSERVATIONS:

Table 3 - Subjective Evaluation of shortened Cakes - Hedonic

Cake type Colour of Colour of Tenderness Aroma Flavor Overall eating

Surface interior experience

Control 1 1 1 1 1 1

Fructose 3 5 5 4 4 5

Sugar 2X 6 4 7 8 9 8

Splenda 4 3 9 9 7 7
 High 8 5 7 5 4 9
temperatur
e

Low 8 6 8 9 8 9
temperatur
e

Characteristics –
 Colour of Surface
 Colour of Interior
 Aroma
 Tenderness
 Flavor
 Overall eating experience (mouthfeel)
Scale -
• 1 = Like extremely
• 2 = Like very much
• 3 = Like moderately
• 4 = Like slightly
• 5 = Neither like nor dislike
• 6 = Dislike slightly
• 7 = Dislike moderately
• 8 = Dislike very much
• 9 = Dislike extremely

Fig 3 – Shortened cakes – Fig 4 – Viscosity of Control

Control variable. variable.
 Fig 5 – Shortened cakes – Fig 6 – Viscosity of Fructose
Fructose variable. variable.

Fig 7 – Shortened cakes –

Sugar 2X variable.

Fig 8 – Shortened cakes – High Fig 9 – Shortened cakes – Low

temperature (218 degree C) temperature.
 Fig 10 – Shortened cakes – Fig 11 – Viscosity of Splenda
Splenda. variable.

Discussion:
 Four cakes with a different type of sweeteners like white sugar, fructose, sugar 2× and
Splenda with variation in temperature were compared. The cake made with standard recipe
and sugar as sweetener was rated 1 for all the aspects making it the most likable and ideal
sample.
 The cake made with fructose had a color score of 3 for surface and 5 for interior, which
means the color was not desirable and showed more browning due to high browning rate of
fructose in comparison to sucrose. The tenderness of the cake got a score of 5 which
indicates that it wasn’t as soft as the control and had a hard consistency which might be due
to caramelization of fructose. The taste and overall experience were also lesser than the
control but the participants didn’t dislike it.
 The cake made with sugar 2× had gotten a score of 6 for surface color while the interiors
was given 4 and the level of brown was high on the surface and less in the interiors which
showed that the surface in direct heat showed more browning. The tenderness of the cake
was given 7 which indicates moderate dislike which might be due to high sugar content and
high level of caramelization.
 The cake made with Splenda, which is a sugar substitute got a score of 4 for color of surface
and 3 for interior which indicates it was liked moderately. The degree of browning was low
in comparison to fructose and 2× sugar but more than control. This shows that Splenda can
be used as a efficient browning agent in cakes. The tenderness of this cake was given a 9
which indicates a extreme dislike. This might be due to the structural property sugar
provides to the cake which is compromised due to presence of Splenda. The aroma and
overall acceptance are also low for this cake.
 High temperature used to bake cake can lead to higher degree of browning and make the
cake denser while low temperature can reduce the degree of browning and make the cake
chewier and more undesirable and affect the overall acceptance and flavor of the cake
negatively.
 Laboratory Exercise No. 3

1. Summary of principles and applications of enzymatic and non-enzymatic browning.

Browning is a common color change seen in food during pre-preparation, processing, or storage of
food. It occurs in varying degrees in some food material. The color produced range from cream or
pale yellow to dark brown or black, depending on the food item and the extent of the reaction.
Browning reactions may be desirable or undesirable. In some food, the brown color and flavor
developed during browning is highly desirable and associated with a delicious, highly acceptable,
and quality product. Browning reactions contribute to the aroma, flavor and color of the product
such as the browning crust of bread, and all baked goods, potato chips, roasted nuts, roasted coffee
beans and many other processed foods.

Enzymic browning

 Fresh fruit and vegetables normally keep enzymes trapped in their tissues.  However, when
the fruit is sliced, or squashed, or when the fruit or vegetable begins to break down with age,
the enzymes come into contact with oxygen in the air. This causes the fruit to turn brown.
 Enzymic browning is an oxidation reaction that takes place in some foods, mostly fruit and
vegetables, causing the food to turn brown. 

 Oxygen in the air can cause sliced fruit to brown, a process called enzymic
browning. Phenols and the enzyme phenolase are found in the cells of the fruit, and when
these are exposed to oxygen in the air, for example through slicing, the oxygen causes a
reaction.  The phenolase changes the phenols into melanin, which has a brown colour.

Application:

 Developing colour and flavour in coffee, cocoa beans, and tea.

 Developing colour and flavour in dried fruit such as figs and raisins.

 Enzymatic browning is undesirable and leads to wastage of food. To stop the oxidative
reaction, the phenolase enzymes need to be denatured.  This could be done by using heat and
acids.

Non-Enzymatic Browning
1. Maillard Reaction– it is a protein-sugar interaction. It leads to the development of brown colour
in a mixture containing amino acids and reducing sugars. It is also called carbonyl-amine reaction.
The brown colour formed contributes to the aroma, flavour, and colour of many ready to eat cereals,
baked food, malted barley etc. various factors which affect this reaction are-type of sugar- the more
percentage of reducing sugar, the darker the crust in the bakery products, type of amino acid,
temperature, moisture.
Maillard reaction also leads to undesirable changes like unfavourable brown colour eg. In dried
milk. Detrimental flavour changes eg. Burnt flavour.
Caramelization- when sugar is heated at high temperature (160 deg C) in the absence of water and
amino acids, it turns brown and this sugar-sugar interaction is known as caramelization. Sugar is
broken down into a number of compounds due to intense heat. (sucrose caramel + acid)
Development of caramel flavour and colour in caramel custard is a desirable change, whereas
uncontrolled heating of sugar can lead to objectionable burnt colour and flavour.

3. Reaction of oxidation products-

Ascorbic acid browning- Ascorbic acid present in fruits undergo oxidation with the formation of a
compound which produces a brown pigment and causes discolouration. This type of browning is
seen in preserves. Low storage temp can help in retarding the reaction.
Lipid browning-many fatty foods during processing and storage exhibit browning which can be
attributed to the reaction of lipid oxidation products with amino acids, amines and proteins
Applications:
1.Development of caramel flavor and color in caramel custard
2The crust in the bakery products
3Ascorbic acid browning- prevention of browning and other oxidative reactions in food products 
4.Lipid browning- white fish containing PUFA and high concentration of protein shows
discolouration in muscle during frozen storage.

2. Summarize your results of the experiments, both objective and subjective for differences in
different treatment methods:
Was it as expected to occur or different? Discuss why you see differences in your results.
a. for enzymatic browning with 4 variation apples.
b. for non-enzymatic browning with 3 different cake variations?
a) Yes, the expected results were observed which can be explained by:

Fresh fruit and vegetables normally keep enzymes trapped in their tissues.  However, when the fruit
is sliced, or squashed, or when the fruit or vegetable begins to break down with age, the enzymes
come into contact with oxygen in the air. This causes the fruit to turn brown. The apples and
potatoes in contact with oxygen leads to melanin or brown colour formation seen in (a) while a
coating of ascorbic acid, which is a known antioxidant doesn’t let oxygen react with apples and
potato. Sugar syrup forms a coating on the apples and potato and doesn’t let the oxygen react with
the phenols present in the substrate, this can be applied for storing cut fruits. Cold water control
showed slight browning in apples and no browning in potatoes as apple has high concentration of
phenols and phenolase, the reaction rate is higher leading to more browning in comparison to
potato.
b) Four cakes with a different type of sweeteners like white sugar, fructose, sugar 2× and Splenda
with variation in temperature were compared. The cake made with standard recipe and sugar as
sweetener was rated 1 for all the aspects making it the most likable and ideal sample.
Cake made with fructose had a color score of 3 for surface and 5 for interior, which means the color
was not desirable and showed more browning due to high browning rate of fructose in comparison
to sucrose. The tenderness of the cake got a score of 5 which indicates that it wasn’t as soft as the
control and had a hard consistency which might be due to caramelization of fructose. The taste and
overall experience were also lesser than the control but the participants didn’t dislike it.
The cake made with sugar 2× had gotten a score of 6 for surface color while the interiors was given
4 and the level of brown was high on the surface and less in the interiors which showed that the
surface in direct heat showed more browning. The tenderness of the cake was given 7 which
indicates moderate dislike which might be due to high sugar content and high level of
caramelization.
The cake made with Splenda, which is a sugar substitute got a score of 4 for color of surface and 3
for interior which indicates it was liked moderately. The degree of browning was low in comparison
to fructose and 2× sugar but more than control. This shows that Splenda can be used as a efficient
browning agent in cakes. The tenderness of this cake was given a 9 which indicates a extreme
dislike. This might be due to the structural property sugar provides to the cake which is
compromised due to presence of Splenda. The aroma and overall acceptance are also low for this
cake.
High temperature used to bake cake can lead to higher degree of browning and make the cake
denser while low temperature can reduce the degree of browning and make the cake chewier and
more undesirable and affect the overall acceptance and flavor of the cake negatively.

References

 Types of browning (enzymatic and non-enzymatic). hmhub. (2020, January 4). Retrieved
February 8, 2023, from https://hmhub.in/types-browning-enzymatic-non-enzymatic/
 Bharate, S. S., & Bharate, S. B. (2014, October). Non-enzymatic browning in citrus juice:
Chemical markers, their detection and ways to improve product quality. Journal of food
science and technology. Retrieved February 8, 2023, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4190239/#:~:text=Ascorbic%20acid%20is
%20used%20for,oxygen%20in%20polyphenol%20oxidase%20reactions.