Aim
To study on how to prepare buffers and design an experiment to determine and design the
factor that affects Maillard reaction such as the type of sugar, pH and temperature.
Introduction
Browning is a common occurrence in food. It may yield desirable and undesirable results.
Browning can be classified into two groups which are enzymatic browning and non-
enzymatic browning. There are three types of non-enzymatic browning that usually occur in
food, namely Maillard reaction, caramelization and ascorbic acid browning. However, only
Maillard reaction is observed in this experiment. Maillard reaction is the chemical reaction
between reducing sugar, mainly D-glucose and amino groups under heated conditions.
Melanoidins is formed as a product of Maillard reaction and it contributes to the brown
colour observed in food. Maillard reaction in food can be either desirable or undesirable.
Example of desirable browning in food are caramel from milk and sugar, browning of bread
into toast and colour of coffee and chocolate. Example of undesirable Maillard reaction is
the formation of acrylamide, which is highly toxic in French fries. Rate of Maillard reaction
can be affected by several factors including temperature, pH, time, nature of reducing
sugars and nature of amino compound. The rate of Maillard reaction in this experiment is
measured by determining the absorbance value using UV-vis spectrophotometer.
UV-Vis spectrophotometry is absorption spectroscopy in the ultraviolet-visible spectral
region. This absorption spectroscopy is widely used in analytical chemistry to quantitatively
determine different analytes such as transition metal elements, organic compounds and
biological molecules. Electron can move from ground state to excited state with the
absorption of sufficient energy in the form of ultraviolet-visible light. The mechanism of UV-
Vis spectrophotometry is closely related to Beer-Lambert law which states that the
absorbance of a solution is directly proportional to the concentration of the absorbing
species in the solution and path length (Gobrecht, 2015). The concentration of absorbing
species in the solution for a fixed path length can be measured by using UV-Vis
spectrophotometry. The concentration of melanoidin molecules formed in this experiment
was determined by using UV-Vis spectrophotometry as there was only one analyte to be
measured. The wavelength used in this experiment was 430 nm.
(327 words)
Methods
Part A: Preparation of a Glucose/Glycine Model System
1. 50mL 0.5M glucose solution was prepared in the phosphate buffer. The glucose was
added a little at a time and stirred to about 30mL buffer in a beaker. The mixture
was then transferred to a volumetric flask, to be diluted to 50mL by adding buffer.
2. 50mL 0.5M glycine solution was prepared in the phosphate buffer.
3. 50mL glucose solution was mixed with 50mL glycine solution to form glucose-glycine
solution.
4. 20mL of glucose-glycine solution was transferred to each of two 50 mL beakers and
was treated as follows (one treatment per beaker):
a. The pH was adjusted to 5.0 and water was added to bring the total volume to
40mL. The pH was rechecked.
b. The pH was adjusted to 8.0 and water was added to bring the total volume to
40mL. The pH was rechecked.
50mL of 0.5M glucose solution was prepared in the
phosphate buffer
Glucose was added little at a time and stirred to
about 30mL buffer in a beaker.
The mixture was transferred into a volumetric flask
50mL of 0.5M glycine solution was prepared in the
phosphate buffer.
50mL glucose solution was mixed with 50mL glycine
solution
20mL of glucose-glycine solution was transferred
into two 50mL beakers
The pH was adjusted to 5.0 and water was added to The pH was adjusted to 8.0 and water was added to
bring the total volume to 40mL. The pH was bring the total volume to 40mL. The pH was
rechecked. rechecked.
Heating Experiment
1. 10mL aliquots of the solutions listed below was transferred to test tubes. The tubes
were capped loosely and labelled with a permanent marker.
a. Glucose-glycine, pH 5
b. Glucose-glycine, pH 8
c. Fructose-glycine, pH 5
d. Fructose-glycine, pH 8
e. Sucrose-glycine, pH 5
f. Sucrose-glycine, pH 8
g. Lactose-glycine, pH 5
h. Lactose-glycine, pH 8
i. Sorbitol-glycine, pH 5
j. Sorbitol-glycine, pH 8
k. Glucose, pH 5
l. Glucose, pH 8
m. Fructose, pH 5
n. Fructose, pH 8
o. Sucrose, pH 5
p. Sucrose, pH 8
q. Lactose, pH 5
r. Lactose, pH 8
s. Glycine, pH 5
t. Glycine, pH 8
u. Sorbitol, pH 5
v. Sorbitol, pH 8
2. A boiling water bath was prepared (Approximately 300mL of water was filled into a
1L beaker) by adding a few boiling chips and bringing to a boil on a hot plate. All
tubes were placed in a boiling water bath for 30 minutes. The tubes were transferred
to a beaker of tap water to cool.
10mL aliquots of the solutions listed in the lab manual was
transferred to test tubes. The tubes were capped loosely
and labelled with a permanent marker.
Boiling water bath was prepared. A few boiling chips was
added and bringing to a boil on a hot plate.
All tubes were placed in the boiling water bath for 30
minutes.
The tubes were transferred to a beaker of tap water to
cool.
Part B: Measurement of Extent of Browning
1. The pH in each tube was measured after the tubes had cooled.
2. Spectrophotometer was turned on and it was allowed to warm up. The wavelength
selector was turned to 430nm. Water was used to set zero absorbance.
3. The absorbance of each solution was measured. The darker solutions were diluted
with water to keep them on scale. The absorbance of the diluted solution was
multiplied by dilution factor to calculate the absorbance of the original undiluted
solutions.
The pH in each tube was measured after the
tubes had cooled
Spectrophotometer was turned on and the
wavelength sector was turned to 430nm
Water was used to set zero absorbance
The absorbance of each solution was measured
Part C: Determination of the Effect of Multiple Processing Parameters on the
Extent of Maillard Browning
1. 30mL of 0.25M glucose solution was diluted to 50mL by adding phosphate buffer
solution.
2. 30mL of 0.25M glycine solution was diluted to 50mL by adding phosphate buffer
solution.
3. The two solutions were mixed and 10mL of the glucose-glycine solution was
transferred into two test tubes each.
4. The two test tubes were labelled as test tube A and test tube B.
5. Test tube A was kept at room temperature for 30 minutes while test tube B was kept
in a boiling water bath for 30 minutes.
6. The tubes were then let to cool down in a beaker of cold water.
7. The absorbance of test tube A and test tube B were measured at wavelength of
430nm.
Results and Calculations
Part A: Preparation of a Glucose/Glycine Model System
Table 1: The rechecked pH value of glucose-glycine model after addition of water for
adjusted pH of 5 and 8
Adjusted pH pH pH
(before addition of (after addition of
water) water)
5 5.11 5.32
8 8.05 8.06
Part B: Measurement of Extent of Browning
Table 2: The absorbance reading of twenty-two sugar solution with pH and dilution factor
No. Solution pH pH Abs. at 430nm Dilution Abs. in
(before (after factor undiluted
heating) heating) solution
1 Glucose-glycine 5 5.06 0.018 - 0.018
2 Glucose-glycine 8 7.42 0.673 - 0.673
3 Fructose-glycine 5 5.04 0.162 - 0.162
4 Fructose-glycine 8 7.39 0.195 10 1.954
5 Sucrose-glycine 5 5.49 0.002 - 0.002
6 Sucrose-glycine 8 7.95 0.000 - 0.000
7 Lactose-glycine 5 5.06 0.033 - 0.033
8 Lactose-glycine 8 7.44 0.597 - 0.597
9 Sorbitol-glycine 5 5.10 0.016 - 0.016
10 Sorbitol-glycine 8 8.03 0.012 - 0.012
11 Glucose 5 4.97 0.024 - 0.024
12 Glucose 8 8.12 0.194 - 0.194
13 Fructose 5 4.44 0.114 - 0.114
14 Fructose 8 7.62 0.289 - 0.289
15 Sucrose 5 4.86 0.010 - 0.010
16 Sucrose 8 8.24 0.002 - 0.002
17 Lactose 5 5.09 0.026 - 0.026
18 Lactose 8 7.67 0.266 - 0.266
19 Glycine 5 5.12 0.012 - 0.012
20 Glycine 8 8.00 0.020 - 0.020
21 Sorbitol (stable 5 4.82 0.013 - 0.013
to heat & pH)
22 Sorbitol 8 8.33 0.001 - 0.001
Part C: Determination of the Effect of Multiple Processing Parameters on the
Extent of Maillard Browning
Table 3: The absorbance value of glucose-glycine solution treated with two different
temperatures.
Solution Treatment Absorbance
Glucose-glycine Room temperature, 25 °C 0.027
Boiling water bath, 100 °C 0.673
Discussion
In part A, the pH of the glucose-glycine model slightly increases when water was added to it.
pH measures the concentration of hydrogen ions in a sample. When water was added to the
model, the concentration of hydrogen ions decreases. As for that, the pH of the glucose-
glycine model slightly increases as pH value is inversely proportional to the concentration of
hydrogen ions.
In part B, fructose-glycine model has the highest absorbance value for both pH 5 and pH 8
which was 0.162 and 1.954 respectively. Fructose-glycine model at pH 8 was diluted with a
factor of 10 and the absorbance value was taken again. The absorbance value for fructose-
glycine model after dilution was 0.195. The absorbance value at pH 5 were followed by
lactose-glycine model and glucose-glycine model after fructose-glycine model. Meanwhile,
fructose-glycine model has the highest absorbance value at pH 8, then followed by glucose-
glycine model and lactose-glycine model. The other sugar solutions have relatively low
absorbance value as they are non-reducing sugar while glucose, lactose and fructose are
reducing sugars. Presence of free aldehyde group in the reducing sugars enables it to react
with amine group of amino acids. Maillard reaction will occur and melanoidins is formed as
the product of browning.
In part C, glucose-glycine model which was kept in the boiling water bath for 30 minutes has
higher absorbance value than the glucose-glycine model which was kept at room
temperature for 30 minutes. This is because the rate of Maillard reaction increases with the
increase of temperature. When the glucose-glycine model was being heated up, the free
aldehyde group of glucose tend to collide with the amine group of glycine more frequently
and thus more effective collisions can be produced which can lead to faster formation of
melanoidins molecules as the product. As to conclude, higher temperature cause more
effective collisions and faster formation of melanoidins.
Some errors might encounter in performing this experiment. One of the errors that might
be encountered was contamination of test tubes. When contaminated test tubes were used
in performing the experiment, the pH of the sugar solutions might alter and affect the rate
of Maillard reaction. Therefore, the absorbance value obtained might be inaccurate.
Besides, leaking of water into the test tubes while heating the sugar solutions in boiling
water bath might also lead to inaccuracy of absorbance value obtained. Leaking of water
into the test tubes will decrease the concentration of sugar solutions in the test tubes.
Therefore, the absorbance value obtained might be lower than the expected absorbance
value. In addition, scratches on the cuvette used for UV-vis spectroscopy analysis also might
affect the result of the experiments. Scratches on the cuvette might cause refraction of light
during the analysis was done and caused the absorbance values of the tests to alter. As to
overcome this, a new cuvette should be used to perform UV-vis analysis and should be
washed thoroughly with distilled water before being used.
Conclusion
As a conclusion, high temperature can increase the rate of non-enzymatic browning.
Presence of reducing sugars such as glucose and fructose is essential for Maillard reaction to
occur. Non-reducing sugars such as sucrose and sorbitol will not undergo Maillard reaction.
Presence of free aldehyde group in reducing sugars to react with the amine group in glycine
enable it to undergo Maillard reaction while free aldehyde group is absence in non-reducing
sugar, thus non-reducing sugar is unable to undergo Maillard reaction. The rate of
enzymatic browning reaction is also affected by external factors such as pH and
temperature. An increase in the pH will accelerates the enzymatic browning as the higher
pH causes the amino acid not protonated, therefore amino group can react with aldehyde
group in reducing sugar and undergo Maillard reaction. Besides, an increase in temperature
also can accelerate the enzymatic browning as the higher temperature increase the
aromatic characteristics in high and low molecular products. Formation of melanoidin
contributes to the brown colour formation differs between the tubes that keep on room
temperature and the tubes that kept in a boiling water bath.
Problem Set
1. 1L 1/15 M phosphate buffer was prepared by mixing 587mL of 1/15 M Na2HPO4 with
413mL of 1/15 M KH2PO4.
1/15M KH2PO4
Molecular weight of KH2PO4 = 39.098 + 2(1.008) + 30.974 + 4(16)
= 136.088 g/mol
Concentration of KH2PO4 = 1/15 mol/L
No of mol in 1L = 1/15 x 1
= 1/15 mol
Mass of KH2PO4 needed for 1L = 1/15 x 136.088
= 9.0725g
Mass of KH2PO4 needed for 413mL = (9.0725/1000) x 413
= 3.7469g
1/15M Na2HPO4
Molecular weight of Na2HPO4 = 2(22.99) + 1.008 + 30.974 + 4(16)
= 141.962g/mol
Concentration of Na2HPO4 = 1/15 mol/L
No of mol in 1L = 1/15 x 1
= 1/15 mol
Mass of Na2HPO4 needed for 1L = 1/15 x 141.962
= 9.4641g
Mass of Na2HPO4 needed for 587mL = (9.4641/1000) x 413
= 5.5554g
1/15M pH 7.0 phosphate buffer
3.7469g of crystalline KH2PO4 and 5.5554g of crystalline Na2HPO4 were weighed and
transferred into 1L volumetric flask. Water was added to the calibration mark to
make 1/15M pH 7.0 phosphate buffer.
2. For glucose,
Concentration of glucose = 0.25M
No of mol in 100mL = 0.25 mol/L x 0.1L
= 0.025 mol
Mass of glucose needed = 0.025 x 198.17
= 4.9543g
For glycine,
Concentration of glycine = 0.25M
No of mol in 100mL = 0.25 mol/L x 0.1L
= 0.025 mol
Mass of glycine needed = 0.025 x 75.1
= 1.8775g
4.9543g of glucose and 1.8775g of glycine were required to prepare 100mL of
solution containing 0.25M glucose and 0.25M glycine in water. 4.9543g of glucose
and 1.8775g of glycine were weighed and transferred into a 100mL volumetric flask
and water was added up to the calibration mark.
Questions
1. The absorbance value of glucose-glycine solution at pH 5 was 0.018 while the
absorbance value of glucose-glycine solution at pH 8 was 0.673. However, the
absorbance value of sucrose-glycine solution at pH 5 was 0.002 while the absorbance
value of sucrose-glycine solution at pH 8 before dilution was 0.000. Glucose-glycine
model produce more browning than sucrose-glycine model as glucose is a reducing
sugar while sucrose is a non-reducing sugar. Maillard reaction is a chemical reaction
between reducing sugar and amino acids under heated conditions. Maillard reaction
can only occur with the presence of reducing sugar. Reducing sugar, glucose can
react spontaneously with protein, glycine and thus Maillard reaction occur. On the
other hand, no significant browning was observed in the sucrose-glycine model due
to the absence of reducing sugars and thus Maillard reaction cannot occur in this
model.
The absorbance value of glucose-glycine solution at pH 5 was 0.018 while the
absorbance value of glucose-glycine solution at pH 8 was 0.673. However, the
absorbance value of sorbitol-glycine solution at pH 5 was 0.016 while the absorbance
value of sorbitol-glycine solution at pH 8 before dilution was 0.012. No significant
browning was observed in sorbitol-glucose model due to the presence of sorbitol, a
glucose alcohol in the model. Glucose alcohol will delay the Maillard reaction and
causing the absorbance value to be relatively low as the heating time for Maillard
reaction to occur is insufficient. Glucose alcohol molecules have the similar chemical
structure to glucose molecules but the aldehyde group in glucose which is essential
for Maillard reaction is replaced by alcohol group (Vaclavik & Christian, 2008).
2. The reaction rate of Maillard reaction can be affected by the pH of the system. As
the pH increases, the reaction rate of Maillard reaction will also increases as it
depends on the rate at which sugar in ring form is converted into reducible open
chain form. Lesser amino acids are being protonated at high pH which lead to an
increase rate of Maillard reaction as more free amino groups are made available.
Thus, the pH of the system affects the rate of Maillard reaction as pH is related to
the protonation of amino groups (Ajandouz et. al., 2001).
The optimum pH for Maillard reaction falls in the range pH 6 – pH 9. An increase in
pH will accelerates the formation of melanoidin as the number of unprotonated
amino group increases with increasing pH. Therefore, rate of Maillard reaction
increases.
3. Non-reducing sugars should be used to manufacture a formulated product. Absence
of free aldehyde group in the non-reducing sugars which is required to react with the
amine group in protein prevent Maillard reaction to occur. Non-reducing sugars such
as sucrose and sorbitol have their aldehyde functional groups changed to other
functional groups. Sorbitol acts as humectants that increase the water holding
capacity of the food and thus delay the Maillard reaction.
Heat and pH should be regulated as to reduce Maillard reaction. The formulated
food products should not be heated too long as persistent heating will cause
caramelization of sugars to occur. Besides, pH of the formulated food samples
should be kept low to promote the protonation of amino acid so that no free amine
groups are available to react with free aldehyde group in reducing sugars.
The formulated food product should be kept in a condition with high pH to ensure
the amine group is not protonated. Besides, the formulated food products also
should be kept in a cool area to prevent Maillard reaction to occur in the food
packages.
4. There are desirable and undesirable non-enzymatic browning in food systems. One
of the examples of desirable non-enzymatic browning is enhancing appearance and
flavours of foods such as browning of bread into toast, flavour of roast meat and
production of caramel. Meanwhile, an example of undesirable non-enzymatic
browning in food systems is the production of acrylamide in French fries.
Part C: Determination of the Effect of Multiple Processing Parameters on the
Extent of Maillard Browning
1. The parameter chosen for the design experiment is temperature.
Figure 1: Flow chart of designed experiment using temperature as the chosen
parameter.
The expected absorbance value obtained from the designed experiment are test
tube A has lower absorbance than test tube B. Higher temperature will increase the
rate of Maillard browning and thus having higher absorbance value. Increase in
temperature leads to an increase in the aromatic characteristics in high and low
molecular products. The structure of melanoidin which contribute to the brown
colour formation synthesized at high temperature is different from those that form
in room temperature. This is because melanoidin structure form at higher
temperature has different aliphatic carbons and lesser carbon-carbon double bond
in its structure (Benzing-Purdie et. al., 1985).
2. Control experiment is required for this experiment to be used as guide to determine
whether the experimental result was accurate or not. The control turns brown as it
contains glucose, which is a reducing sugar. Free aldehyde in the reducing sugar will
react with amine groups of protein which cause non-enzymatic browning to occur.
References
1. Ajandouz, E., Tchiakpe, L., Ore, F., Benajiba, A. and Puigserver, A. (2001). Effects of
pH on Caramelization and Maillard Reaction Kinetics in Fructose-Lysine Model
Systems. Journal of Food Science, 66(7), 99. pp. 926 – 931.
2. Benzing-Purdie, L., Ripmeester, J. and Ratcliffe, C. (1985). Effects of temperature on
Maillard reaction products. Journal of Agricultural and Food Chemistry, 33(1), pp. 31
– 33.
3. Gobrecht, A., Bendoula, R., Roger, J. and Bellon Maurel, V. (2015). Combining linear
polarization spectroscopy and the Representative Layer Theory to measure the Beer-
Lambert law absorbance of highly scattering materials. Analytical Chimica Acta, 853,
pp. 486 – 494.
4. Vaclavik, V. and Christian, E. (2008). Essentials of Food Science. 3rd ed. New York. NY:
Springer Science+Business Media, LLC, pp. 33 – 67.
