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FSN 364: Food Chemistry

Non-Enzymatic Browning Reactions:
Maillard Reactions with Varying Amino Acids
Observation of Caramelization Under Different Conditions

Experiment Performed:
October 11, 2017

Alexandrea Apolinario

October 18, 2017

Food Science and Nutrition Department

College of Agriculture, Food, and Environmental Science
California Polytechnic State University, San Luis Obispo

The browning of foods, whether wanted or unwanted, play a huge role on the final
characteristics of a food. A products final taste, texture and appearance are all effected by
browning. Two major types of browning in foods[are] oxidative and non-oxidative (Brad
2013). Also known as enzymatic and non-enzymatic browning. Non-enzymatic browning
reactions are favored by heat treatments and do not require the presence of enzymes (Castro).
A Maillard reaction and caramelization both fall under the non-enzymatic browning category.
Amino acids, reducing sugars, and heat all need to be present for a maillard reaction to occur.
Whereas, for caramelization to happen, only sugar and a high heating temperature is needed. In
the cases where browning in not wanted in a food, an inhibitor such as sodium metabisulfate can
be added to prevent undesired browning from occurring.
The purpose of the following four experiments are to create and examine acid-glucose
solutions and simulate the inhibition of browning as well as recreate a browning that often occurs
in the food industry.

Materials & Methods:

Experiment #1

Glucose Solution (10%)

Amino Acids (~50mg each)
Screw-Capped Test Tubes (5)
Hot Plate
Deionized Water
250 ml Beaker
0.1M NaOH
Washing Tape
Graduated Pipette
pH Paper

Using the washi tape and the crayon, the five test tubes were labeled, each corresponding
to the amino acids that was added later. The amino acids used were phenylalanine, valine,
isoleucine, proline, cysteine, and glycine. A 250ml beaker was filled with deionized water and
placed on a hot plate and brought to a boil. With the graduated pipette, a milliliter of the glucose
was put into all five tests tube. Approximately 50 mg of each amino acid was added to their
corresponding test tube. The pH of each test tube was tested with pH paper. All the mixtures
were measured to confirm that their pH was above 7. If not, NaOH was added to raise the pH
above 7. Initial observations of the smell and color of the test tubes were recorded. The test tubes
were then sealed shut using the screw caps. Once sealed shut, the samples were placed in the
water for an hour. After the hour, the samples were cooled until they were close to room
temperature. Each test tube was opened and observations were made about the smell and
appearance of each mixture.

Experiment #2

Large screw cap test tubes (2)

Sodium Metabisulfite (0.3g)
1.0M Phosphate Buffer (pH 7.0)
Washi Tape
250ml beaker
Hot Plate
Deionized Water
Stir Rod

A 250ml beaker was filled with deionized water and brought to a 121F, using a hot plate
and a thermometer. One of the test tubes was labeled, with the crayon and washi tape, that it
would contain sodium metabisulfate. Each test tube was then filled with 15ml of phosphate
buffer, 0.75g of glucose, and 0.3g of glycine. Then, a stir rods was used to ensure that the
mixture was fully dissolved. To the pre-labeled test tube, 0.3g of sodium metabisulfate was
added and mixed in until it was thoroughly dissolved in. The screw on caps were then placed
loosely on the test tubes. The two samples were transferred to the hot water and sat in the hot
water bath for 3 minutes. Final observations, in regards to color and smell were recorded.

Experiment #3

Sugar (5g)
Aluminum weighing dishes
Draft Oven
Analytical Balance

Four aluminum weighing dishes were labeled, with a marker, with their respective time.
Each dish was labeled with a specific time: 10, 20, 30, or 60 minutes. With an analytical balance,
the trays were zeroed out and five grams of sugar was added onto the dish while on the balance.
Initial observations were made about the samples before being put into the draft oven. The four
samples were placed into the draft oven and were not removed until the appropriate time was
reached. Once sample the same was removed from the draft oven, final observations were made
about the color and smell of each sample.

25ml Beaker (2)

Nonfat Dry Milk (5g x 2)
Corn Syrup (5g x 2)
Sugar (5g x 2)
Hot Plate
Deionized Water
Analytical Balance
Washi Tape

The washi tape and crayon were first used to label one of the beakers to be able to
distinguish which beaker would contain the nonfat dry milk. Using the analytical balance, the
two 25ml beakers were then each filled with 5g of corn syrup and 5g sugar. Five grams of nonfat
dry milk was then weighed out and poured into the pre-labeled beaker. Both beakers were placed
on the hot plate and were heated simultaneously. The beakers were then heated for a few
minutes. Observations were made about each beaker and recorded.

Results & Discussion:

Table 1: Observations of Maillard Reactions Using Varying Amino Acids

Test Amino Acid Smell Color Smell Color Amount Used
Tube (Before) (Before) (After) (After) (mg)
1 Valine None Clear Strong caramel Translucent 56.5
odor. appearance
with slight
yellow tint.
2 Isoleucine None Clear Odor Translucent 57.1
reminiscent of with yellow
expired milk, a tinge and
bit musky. white
solutes on
the bottom.
3 Proline None Clear Buttered White 47.0
popcorn opaque
aroma. liquid
4 Phenylalanine None Clear Burnt rubber. Clear liquid 57.0
with yellow
tint. White
solutes at
the bottom
floating at
the top.
5 Glycine None Clear Odorless Clear 52.0
6 Cysteine None Clear Rotten Cloudy 45.1
Eggs/Sulphur liquid with
solute at
the bottom.

In the first experiment, six glucose-amino acid solutions were produced to simulate
maillard browning reactions in the presence of heat. Each solution created had the same amount
of glucose solution to start with, however, each solution varied in the type and amount of amino
acid that was added. Table 1, which can be seen above, shows all the types of amino acids used
in the experiment. All six test tubes produced a recognizable aroma except the test tube
containing glycine. There was no visible difference between the solution before and after heat
was added. In a similar experiment preformed at the University Putra Malaysia, evaluating
Maillard reactions with specific amino acids, glycine produced sweet caramel-like aroma (Kam
Wong, Aziz & Mohamed 2008). The varying result could be due to a couple of factors; either the
test tube was not heated long enough and at a constant high temperature or the solution was not
thoroughly mixed for the reaction to properly occur. However, the remaining five test tubes
contained strong and pungent aromas and changed slightly in color. Valine and Proline were the
only two amino acids that produced the more pleasant odors. As described in Table 1, Valine
produced a caramel aroma while proline gave off a buttered popcorn odor. The remaining three
test tubes, separately containing isoleucine, phenylalanine, and cysteine, consisted of not so
pleasant aromas. Isoleucine smelled of expired milk, phenylalanine produced a burnt rubber
aroma, and cysteine produced a pungent rotten egg odor (Table 1). The aromas produced when
the glucose-amino acid solutions were heated is a result of Strecker degradation. Some of the
carbonyl derivatives [in the glucose solution]react readily with free amino acids. The result of
the degradation of the amino acids to aldehydes, ammonia, and carbon dioxide (Fennema 2008).
More specifically, the formation of certain aldehydes are why aromas are produced in a maillard
reaction. Various aldehydes are responsible for the creation of specific aromas such as the
buttered popcorn and rotten eggs.

Table 2: Inhibition of Browning

Test Tube Added Sodium Color Color
Metabisulfite (Before) (After)
1 N Clear Clear
2 Y Clear Clear

In the second experiment, two solutions were made containing a specified amount of
phosphate buffer, glucose and glycine and place in separate test tubes. The second test tube
varied from the first test tube because it contained the addition of sodium metabisulfite (an
inhibitor). The sodium metabisulfite is supposed to inhibit browning from occurring. [Ions from
the sodium metabisulfite]reacts with the aldehyde groups, forming addition compounds, and
thusinhibit Maillard browning(Fennema 2008). If the experiment was preformed correctly,
test tube one should have turned brown and test tube should have remained clear. As seen above
in Table 2, both test tubes were reported as clear and produced no odor even after heating. This
could be due to a shortage of a specific ingredient or the test tubes were not heated properly.

Table 3: Caramelization of Sugar at Varying Times

Sample Time in Oven Observations Observations
(Before) (After)
1 0 white + odorless white + odorless
2 10 white + odorless Cotton candy like odor
3 20 white + odorless Burnt sugar odor, tiny sugar clumps
4 30 white + odorless Crme brule like odor
5 60 white + odorless Very slight yellow tinge.
Light French toast odor.

In the third experiment, equal amounts of sugar were measured out and transferred to
aluminum dishes that were labeled with a specific heating time. Observations were made about
the sugar before and after the samples were placed in the draft oven. Overall the sugar samples,
remained the same in appearance, but slightly varied in odor. The aroma of the sugar deepened
as the time spent in the draft oven extended. As described in Table 3, the control sample was
odorless in contrast to the sample that stayed in the oven for 60 minutes that developed a richer
French toast like aroma. For caramelization to occur, the temperature of the oven needed to be
much higher and more time would have to be allotted for the break down the of the sugar

Table 4: Browning Reaction

Sample Nonfat Dry Milk Observations
1 Y Burnt in 3 minutes
2 N Browning and bubbling at 5 minutes after sample
1 burned

In the fourth experiment, two beakers were filled with equal amount of sugar and corn
syrup. However, the first beaker also contained five grams of nonfat dry milk powder. Both were
place on the hot plate and heated at the same time. The beaker that contained the milk powder
burned within three minutes, whereas the beaker with no nonfat dry milk didnt start browning
and bubbling for another five minutes. For caramelization to occur, a much higher temperature
needed to be reached to heat the beaker with just sugar and corn syrup for a reaction to occur.


In conclusion, the final aromas and flavors of a maillard reaction is dependent upon the
specific amino acid used in the reaction. The aldehydes produced through Strecker degradation
are was produce the aromas associated with a maillard browning reaction. Inhibitors can be used
to prevent unwanted browning in a product. For caramelization to occur, a high temperature must
be maintained for a longer amount of time. If a high enough temperature is not reached,
caramelization will not happen.

Brady JW. 2013. Carbohydrates. Introductory Food Chemistry. Cornell University Press, Ithaca.
p 205.

Castro, L. Non-Enzymatic Browning. California Polytechnic State University Department of

Food Science and Nutrition. Food Chemistry Laboratory Fall 2016. p 13.

Walstra P, Van Vliet T. 2008. Emulsion Formation. In: Damodaran S, Parkin KL, Fennema OR,
editors. Food Chemistry. 4th ed. Florida: CRC Press. p 93, 312

Wong KH, Aziz SA, Mohamed S. 2008. Sensory aroma from Maillard reaction of individual and
combinations of amino acids with glucose in acidic conditions. International Journal of Food
Science & Technology 43:15121519.