Download accelerated glucose discoloration method

ACCELERATED GLUCOSE DISCOLORATION METHOD - A QUICK
TOOL FOR GLUCOSE STABILITY ASSESSMENT
Master Thesis in Analytical Chemistry, Lund University
Cristina Scret
Supervisor: Staffan Bergström, PhD, Specialist Analytical Chemistry,
Gambro Lundia AB
Abstract
The non-enzymatic browning of glucose was investigated by accelerating the glucose
degradation with or without heating glucose solutions for 1 h at 100°C with different reagents.
Evaluation of the glucose degradation was performed using two types of glucose, glucose A and
glucose B, in order to investigate the influence of the glucose manufacturing process on glucose
discoloration. The color formation was determined by measuring the UV-absorbance between
260-605 nm. The glucose degradation was also investigated by the formation of 5hydroxymethylfurfural and 2-furfural, which were determined by HPLC.
The accelerated glucose discoloration method was optimised in order to differentiate between
different glucose qualities with respect to color stability, i.e. the conditions selected were based
on maximizing the difference in glucose discoloration for glucose A and B. Direct measurement
of the absorbance difference at 350 nm, on 33 % (w/w) glucose/water solution kept at room
temperature for 1 h was found to be the optimum conditions for differentiating between glucose
A an B. Further investigation on Maillard reactions was made, and for the investigated
conditions, the reaction was found to be most accelerated for the glucose samples, 33% (w/w),
heated at 100°C for 1 hour in the presence of L-alanine.
1
Table of Content
1.
Introduction .............................................................................................................................. 3
1.1. Glucose in dialysis ........................................................................................................... 3
1.2. Glucose discoloration ....................................................................................................... 3
1.2.1.
Caramelisation .......................................................................................................... 4
1.2.2.
Maillard reaction ...................................................................................................... 4
1.2.3.
Mechanisms of nonenzymatic browning.................................................................. 5
1.3. Scope of work................................................................................................................... 5
2. Materials and Method ............................................................................................................... 6
2.1. Materials ........................................................................................................................... 6
2.2. Preparation of the glucose samples .................................................................................. 6
2.2.1.
Preparation of Caramelisation Experiments ............................................................. 6
2.2.2.
Preparation of Maillard Reaction Experiments ........................................................ 7
2.3. Ultraviolet (UV) Absorbance and Color Measurements .................................................. 8
2.4. High Pressure Liquid Chromatography (HPLC) determination of 5-HMF and 2-FA ..... 8
2.5. Method of Optimisation ................................................................................................. 10
2.6. Method of Robustness .................................................................................................... 10
3. Results and discussion ............................................................................................................ 11
3.1. Caramelisation ................................................................................................................ 11
3.1.1.
Caramelisation of glucose in the presence of different acids ................................. 11
3.1.2.
Caramelisation of glucose in the presence of water ............................................... 13
3.1.3.
Caramelisation of glucose with metal chlorides as catalyst ................................... 14
3.1.4.
Caramel colours ...................................................................................................... 15
3.2. HPLC determination of 5-HMF and 2-FA ..................................................................... 18
3.3. Maillard reaction ............................................................................................................ 19
3.4. Method of optimisation .................................................................................................. 22
3.4.1.
Temperature optimisation ...................................................................................... 22
3.4.2.
Concentration optimisation .................................................................................... 23
3.4.3.
The pH optimisation ............................................................................................... 24
3.4.4.
Time optimisation .................................................................................................. 26
3.4.5.
Heating versus no heating ...................................................................................... 27
3.5. Method Robustness ........................................................................................................ 28
3.5.1.
The robustness of the method................................................................................. 28
3.5.2.
Time ....................................................................................................................... 29
4. Conclusions ............................................................................................................................ 30
5. Acknowledgments .................................................................................................................. 30
6. References .............................................................................................................................. 30
2
1.
Introduction
1.1.
Glucose in dialysis
D-glucose is a central substance for the human metabolism since it is a source of energy and a
metabolic intermediate. Most of this energy is delivered through aerobic or anaerobic respiration.
In human metabolism glucose is critical as a precursor for the production of proteins and in lipid
metabolism.
In humans the kidneys are used to purify the blood from various salts, metabolites and waste
products and when a person's own kidneys can no longer function adequately to maintain life,
dialysis is a procedure, which is primarily used to provide an artificial replacement for some of
the kidney's normal functions. Dialysis works on the principles of the diffusion of solutes and
ultrafiltration of fluids across a membrane in order to remove the harmful wastes, extra salts and
excess of water from the blood stream. There are two main types of dialysis: haemodialysis or
peritoneal dialysis.
Haemodialysis is a method, which removes wastes and water by circulating the blood outside
the body through an external membrane device, called a dialyzer. The dialyzer contains a semipermeable membrane, where the blood flows on one side of the membrane and dialysis fluid
flows on the other side of the membrane. The purification process is controlled by the diffusion
of the salts and waste products over the membrane, and the removal, or in some cases addition of
water, so called ultrafiltration, can be controlled by the pressure difference over the membrane.
Peritoneal dialysis is a method, which removes wastes and water from the blood, inside the
body using the peritoneal membrane of the peritoneum as a natural semi-permeable membrane.
The dialysis fluid is infused into the stomach cavity, and kept there for a defined time, while the
salts and waste products are equilibrated through diffusion into the fluid, which is then replaced
with given intervals. This type of treatment is normally only given to patients with some
remaining kidney function.
Glucose is by far the carbohydrate that is used most as osmotic agent in the peritoneal and
haemodialysis fluids today.
1.2.
Glucose discoloration
The most common method of sterilisation for parental solutions of glucose is autoclaving, i.e.
heat treatment of the solution. During the sterilisation process some solutions could develop a
pale yellow color. The color formation increases with the time of heating, glucose concentration,
the presence of salts and by increased sterilisation temperature. The mechanism responsible for
this discoloration of the solution is the so called non-enzymatic browning, which is further
investigated in this work.
3
Non-enzymatic browning of glucose could cause discoloration also during production and/or
storage of various glucose solutions. Caramelisation and Maillard reaction are known as two
main types of reaction pathways of non-enzymatic browning.
1.2.1.
Caramelisation
Caramelisation involves heat treatment of monosaccharides in the absence of nitrogenous
compounds by removal of water at elevated temperature. The reaction starts the isomerisation of
the monosaccharides to low-molecular-weight (LMW) intermediates associated with aroma
compounds such as 5-hydroxymethylfurfural (5-HMF) and 2-furfural (2-FA), Figure 1, which
can undergo further condensation and polymerisation reactions to high-molecular-weight (HMW)
compounds associated with the color formation. These HMW compounds are commonly named
caramels.
Depending on processing, caramel colours can have positive or negative ionic charge.
Positively charged caramel colours are produced by reaction of ammonium with glucose and are
commonly found in soy sauces and beer, since they do not react further with proteins.
Negatively-charged caramel colours use sulphite as reactant, and are commonly found in acidic
soft drinks, (Schultz 2006).
Caramel colours have been used in a wide variety of food products and are produced by
controlled heating of carbohydrates with different reagents such as sodium hydroxide (NaOH),
sodium sulphite (Na2SO3) or ammonium chloride (NH4Cl).
There are 4 types of caramel colours:
 Caramel Class I (also known as plain or spirit caramel, prepared with NaOH)
 Caramel Class II (known as caustic sulphite caramel, prepared with sulphite containing
compounds such as Na2SO3 )
 Caramel Class III (named ammonia or beer caramel, prepared with ammonia and used in
baker’s and confectioner’s caramel)
 Caramel Class IV (known as sulphite-ammonia caramel, prepared with ammonia and
sulphite compounds and used as soft drink caramel or acid proof caramel) (Kamuf and
Nixon 2003)
Caramelisation generally requires temperatures over 200°C and a pH between 3 and 9, but the
presence of impurities in low concentration, e.g. iron ions and copper ions or other impurities,
could reduce the caramelisation temperature down to 40°C, (Coca et al. 2004).
1.2.2.
Maillard reaction
The Maillard reaction involves a complex series of reactions between monosaccharides with
amino compounds such as amino acids or proteins. Reactive intermediates are formed that are
finally degraded to important flavour components such as 5-HMF and 2-FA, which react further
4
to form brown pigments known as melanoidins. 5-HMF and 2-FA are considered to be precursors
of such polymer-compounds, (Tomasick et al. 1989).
1.2.3.
Mechanisms of nonenzymatic browning
The glucose discoloration is still not a fully understood process and the mechanisms
responsible with glucose degradation are very complex, (Ramchander et al. 1975, Martins et al.
2003 and Coca et al. 2003).
Several studies have investigated various stages of the non-enzymatic browning reaction. These
studies have shown that the rate of the reaction is strongly dependent on the concentration of
glucose, ratio and chemical nature of reactants, temperature, time of heating, and pH. The
caramelisation process is dependent on heat and is catalyzed by acids and bases (Kroh 1994). At
a pH of 2.5 and lower a considerable amount of 5-HMF is produced (Tohji et al. 2007).
Glucose discoloration by the caramelisation process was shown to be accelerated by an increase
in temperature, concentration, pH and time of heating according to Buera and co-workers (1992).
Buera and co-workers (1987), Ajandouz and others (2001), Tanaka (2005) have studied the effect
of pH on glucose discoloration. According to their studies parameters such as temperature,
concentration, time of heating and pH have an acceleratory effect on glucose discoloration.
5-Hydroxymethylfurfural
2-Furfural
Figure 1. Structures of 5-HMF and 2-FA
Proteins or other carbohydrates such as maltose and isomaltose are present in various amounts
in the raw material used when manufacturing glucose. Thus, trace levels of these compounds
could be found also as impurities in the final product. Since other factors such as impurities and
the potential use of inhibitors such as sodium bisulphite, in the purification step, may cause
changes in color formation process in the glucose solutions, the glucose manufacture process may
very well have an influence on the amount of non-enzymatic browning of the glucose. The
presence of impurities in low quality products may favour the browning reactions (Coca et al.
2004).
1.3.
Scope of work
The aim of this present work was to find an accelerated glucose discoloration method that could
be used as a quick tool for glucose stability assessment and to investigate the parameters
5
influencing the non-enzymatic browning of glucose. The accelerated glucose discoloration
method thus evaluate the quality of glucose with respect to coloration stability of solutions and
could be used to find and exclude glucose that potentially will develop a yellow color in solutions
during the manufacturing process and/or during storage of final products containing the glucose.
The glucose discoloration was studied in glucose solutions prepared from two types of glucose,
herein named glucose A and glucose B. For these two types of glucose there had previously been
observed differences in color stability. Various parameters, as described below, were investigated
in order to find the conditions were the difference was maximized for the two types of glucose.
2.
Materials and Method
2.1.
Materials
Two types of D-glucose, glucose A and glucose B, produced with different processes were
chosen as test materials. Different reagents such as: amino acids (L-lysine, L-glycine, and Lalanine), and chromium chloride were purchased from Sigma Aldrich (Steinheim, Germany),
hydrochloric acid (HCl), sulphuric acid (H2SO4), citric acid, phosphoric acid (H3PO4), sodium
sulphite, (Na2SO3), ammonium chloride,(NH4Cl) and sodium hydroxide (NaOH) were obtained
from Merck, (Darmstadt, Germany). All chemicals used were of pro analysis quality or equal.
2.2.
Preparation of the glucose samples
In order to find a quick method for assessing the glucose stability with respect to discoloration,
several parameters, were investigated.
2.2.1.
Preparation of Caramelisation Experiments
Caramelisation of glucose was tested by preparing the solutions according to the method CRA
E-20, (1987), Glucose Color Stability (Analytical Methods of the member Companies of the corn
Association, Inc), using the two types of D-glucose, glucose A and glucose B. The addition of
different reagents such as HCl, H2SO4, citric acid, H3PO4, chromium chloride, sodium sulphite,
ammonium chloride, and NaOH were investigated. The samples were heated in a Grant BT3,
block heater at various temperatures within the range of 20 to 140°C. The sample concentration,
pH and temperature were varied according to Table 1. The pH measurements were made with an
Orion pH Meter, model 290A. Where needed the pH of the solution was adjusted with HCl.
6
Table 1. Levels of the variable of the glucose aqueous sample in caramelisation process
Reagents
Water
Citric acid
Hydrochloric acid
Sulphuric acid
Phosphoric acid
Chromium chloride
Caramel classes
at lower concentrations
of reagents
Caramel class 1
Caramel class 2
Caramel class 3
Caramel class 4
Caramel classes
at higher concentrations
of reagents
Caramel class 1
Caramel class 2
Caramel class 3
Caramel class 4
Factor
Glucose concentration
pH
Temperature
Time
Glucose concentration
pH
Temperature
Time
Glucose concentration
pH
Temperature
Time
CrCl2 concentration
Glucose concentration
pH
Temperature
Time
NaOH concentration
Sodium sulphite
Ammonium chloride
Ammonium chloride+
Sodium sulphite
Glucose concentration
pH
Temperature
Time
NaOH concentration
Sodium sulphite
Ammonium chloride
Ammonium chloride+
Sodium sulphite
Units
% (w/w) 33
un-adj.*
°C
room
Hour
1
% (w/w) 20
1.5
°C
100
Hour
1
% (w/w) 33
Levels
33
un-adj.*
100
1
20
1.5
100
1
20
1.5
100
1
20
1.5
100
1
33
33
33
100
1
100
1
100
1
°C
Hour
m Molar
% (w/w)
100
1
20
33
°C
Hour
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
% (w/w)
100
1
0.5
33
33
33
0.5
0.5
33
°C
Hour
(g/l)
(g/l)
(g/l)
(g/l)
(g/l)
100
1
5
100
1
100
1
100
1
0.5
0.5
5
5
5
5
*) Un-adjusted, i.e. pH 5-6
2.2.2.
Preparation of Maillard Reaction Experiments
Since even low amounts of amino-compounds in glucose solutions can initiate Maillard
reaction and the fact that trace levels of protein can exist in the glucose as impurities from the
manufacturing process, Maillard reaction can also contribute to the yellow color formation in
glucose products besides caramelisation.
Aqueous model systems of Maillard reaction were prepared according to the same method
described above, using the two types of D-glucose and different amino acids: L-lysine, L-glycine,
and L-alanine with concentrations, pH, temperature and time of heating as presented in Table 2.
L-lysine, L-glycine, and L-alanine are the most commonly used amino acids in testing of
Maillard reaction. The pH of the model systems was adjusted with HCl and NaOH respectively.
7
2.3.
Ultraviolet (UV) Absorbance and Color Measurements
The absorbance of the liquid samples was analyzed after letting them to cool down to ambient
temperature. Absorbance was measured based on the method CRA E-20, (1987), in 1×4 cm
cuvette, using a Perkin Elmer Lambda 20 UV/VIS Spectrophotometer. The UV spectrum from
260-605 nm was recorded for each sample, and the glucose discoloration was calculated as the
absorbance difference between 350 and 600 nm, 450 and 600 nm and as absorbance difference
between λ max and 600 nm. The subtraction of the absorbance at 600 nm was made in order to
adjust the absorbance for any potential unspecific absorbance in the sample. All samples were
measured in triplicate. The calculations of the color, absorbance difference were made using the
equation:
Color, Absorbance difference ×100=
 A x , nm
 A 600 x100
(1)
l
2.4.
High
Pressure
Liquid
determination of 5-HMF and 2-FA
Chromatography
(HPLC)
5-HMF and 2-FA determination by HPLC were made with HPLC measurements performed on
Agilent 1100 equipment, equipped with a loop injector and a UV detector. An RP C18 column, 5
µm (150×4 mm), Supelco was used for separation. The mobile phase consisted of NaH2PO4×H2O
(0.05 M) at pH 5.5 and acetonitrile. The system was calibrated for 5-HMF in the range of 0.1-2.0
ppm and 0.02-0.5 ppm for 2-FA.
8
Table 2. Levels of the variables of the samples in the Maillard model system.
Parameter
pH 1.5
pH 6
pH 9
Factor
Glucose
concentration
L-Lysine concentration
Units
%
(w/w)
M
L-Alanine concentration
M
L-Glycine concentration
pH
M
-
Temperature
Time
Glucose
concentration
°C
Hour
%
(w/w)
Levels
33
0.1
33
0.1
33
0.1
33
33
33
0.1
0.1
0.1
33
33
33
0.1
0.1
0.1
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
100
1
100
1
100
1
100
1
100
1
100
1
100
1
100
1
100
1
33
0.1
33
0.1
33
0.1
33
33
33
33
33
33
0.1
0.1
0.1
0.1
0.1
0.1
L-Lysine concentration
L-Alanine concentration
M
M
L-Glycine concentration
M
pH
Temperature
°C
Un-adj.*
Un-adj.*
Un-adj.*
100
100
100
100
100
100
100
100
100
Time
Glucose
concentration
L-Lysine concentration
Hour
%
(w/w)
M
1
1
1
1
1
1
1
1
1
33
0.1
33
0.1
33
0.1
33
33
33
33
33
33
L-Alanine concentration
L-Glycine concentration
M
M
0.1
0.1
0.1
0.1
0.1
0.1
pH
-
9
9
9
9
9
9
9
9
9
°C
Hour
100
1
100
1
100
1
100
1
100
1
100
1
100
1
100
1
100
1
Temperature
Time
*) Un-adjusted, i.e. pH 5-6
9
Un-adj.* Un-adj.*
Un-adj.*
Un-adj.* Un-adj.*
Un-adj.*
2.5.
Method of Optimisation
The method was optimised by investigating the parameters found to affect the glucose
discoloration, such as temperature, pH, concentration and heating time. The investigated levels of
these parameters are presented in Table 3. The pH of the model systems was adjusted with HCl
and NaOH respectively when needed.
Table 3. Levels of parameters, which were used to optimise the glucose color stability method.
Optimized
parameter
Time
Factors
temperature
pH
glucose concentration
time
Temperature temperature
pH
glucose concentration
time
Concentration temperature
pH
glucose concentration
time
pH
temperature
pH
glucose concentration
time
Units
°C
% (w/w)
hour
°C
% (w/w)
hour
°C
% (w/w)
hour
°C
% (w/w)
hour
room
100
100
Levels
100
100
100
100
100
Un-adj.*
Un-adj.*
Un-adj.*
Un-adj.*
Un-adj.*
Un-adj.*
Un-adj.*
Un-adj.*
33
0
70
33
0.5
80
33
0.75
90
33
1
100
33
2
110
33
4
33
8
33
24
Un-adj.*
Un-adj.*
Un-adj.*
Un-adj.*
Un-adj.*
33
1
100
33
1
100
33
1
100
33
1
100
33
1
100
Un-adj.*
Un-adj.*
Un-adj.*
Un-adj.*
Un-adj.*
20
1
100
1.5
33
1
30
1
100
3
33
1
40
1
100
5
33
1
50
1
100
7
33
1
60
1
100
9
33
1
*) Un-adjusted, i.e. pH 5-6
2.6.
Method of Robustness
The robustness of the method was tested by investigating the repeatability and reproducibility
of the method. The repeatability of the method was tested by performing three different tests of
the method and the reproducibility of the method was tested by performing three different tests of
the method by another person, at a different day. The influence of mixing time was also
investigated as a robustness parameter.
10
3.
Results and discussion
3.1.
Caramelisation
3.1.1.
acids
Caramelisation of glucose in the presence of different
The acidic hydrothermal degradation of glucose was tested using different acids, HCl, H2SO4
and H3PO4 at pH 1.5. The acidified glucose samples (33%, w/w) were heated for 1 h at 100°C.
The results are presented in Figure 2.
Normalized difference
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
At 450nm-600
At 350nm-600
ul
ph
ur
ic
ric
ho
co
se
B
_s
ho
sp
G
lu
gl
uc
os
e
_h
B
B
_p
yd
ro
ch
lo
ri
c
itr
ic
_c
B
co
se
G
lu
G
lu
co
se
ul
ph
ur
ic
_s
A
co
se
G
lu
_p
ho
s
ph
or
ic
lo
ri
c
G
lu
co
se
A
_h
A
co
se
G
lu
G
lu
co
se
A
yd
ro
ch
_c
itr
ic
At λmax nm - 600
Figure 2. The effect of four different acids on glucose discoloration in glucose solutions from the two
types of glucose, at pH 1.5, heated for 1 h at 100°C.
At the absorbance difference, 350 nm, as can be seen in Figure 2, the glucose discoloration
increases, as expected, with the amount of added acid. The effect of the four acids was in
following order: sulphuric acid< hydrochloric acid <phosphoric acid <citric acid. The pH can be
related as a measure of the concentration of H+ present in the solutions and since the pH of the
used acids was adjusted to 1.5, higher amount of the weaker acids was needed in order to achieve
pH 1.5 in solutions. The same results showed that the acidic glucose degradation was more
accelerated for glucose B compared to glucose A, i.e. glucose A is shown to be more stable with
respect to discoloration.
Schrödter (1992) and Kroh (1994) suggested that the reaction of glucose degradation in acidic
medium proceeds principally via 1, 2- or 2, 3-enolisation of the sugar and ß-elimination of water
leading to the formation of 1-3- and 4-hexuloses. The reaction scheme is presented in Figure 3
and 4.
11
Several studies on acidic thermal degradation of carbohydrates and color formation have been
performed and according to Kelly and Brown (1978) the anions of weak acids accelerate the
color formation, while the anions of strong acid had an inhibitory effect compared to the weaker
acids, which is confirmed by the obtained results. Girisuta (2006), Takeuchi et al. (2007),
reported that thermal acidic degradation of glucose leads to the formation of 5-HMF and this 5HMF formation is followed by re-hydration of 5-HMF to levulinic acid, Figure 5.
Figure 5. Glucose dehydration to 5-HMF and than 5-HMF re-hydration to levulinic acid.
At pH 1.5-2, the production of 5-HMF increases and then re-hydration of 5-HMF to levulinic
acid is accelerated by the use of strong acids since only a small amount of strong acid is needed
to catalyze this reaction, according to Takeuchi et al. (2007). This theory was confirmed by the
obtained results in this work, since the presence of strong acids such as sulphuric acid and
hydrochloric acid have a less accelerated effect on glucose discoloration compared to the weaker
acids. This effect can be explained by lower amount of 5-HMF, which is considered to be a
precursor to polymer formation. The lower amount of 5-HMF can be explained by further rehydration of 5-HMF to levulinic acid, which takes place in glucose solutions in the presence of
strong acids.
12
3.1.2.
Caramelisation of glucose in the presence of water
The results of glucose discoloration in pure glucose/water solutions heated at 100°C for 1 h
are presented in Table 4. In order investigate whether the glucose manufacture process has any
effect on the glucose discoloration, several batches from the two types of glucose were tested.
Table 4. Glucose discoloration in aqueous solutions for different batches of the two types of glucose, at
100°C for 1 h.
Glucose type
At
450nm-600nm At
350nm-600nm At
λmax-600nm
absorbance difference
absorbance difference
absorbance difference
Glucose A Batch 1
Glucose A Batch 2
Glucose A Batch 3
Glucose A Batch 4
Glucose A Batch 5
Glucose B Batch 1
Glucose B Batch 2
Glucose B Batch 3
Glucose B Batch 4
Glucose B Batch 5
0.128
0.181
0.174
0.218
0.067
0.388
0.382
0.338
0.268
0.691
0.658
0.705
0.724
0.965
0.382
1.477
1.524
1.308
1.303
2.859
6.868
5.816
6.084
6.623
4.636
7.538
8.653
7.593
8.168
11.039
In aqueous solutions, glucose is found mainly in a cyclic structure, called hemiacetal and this
structure forms the two isomers α-D-glucose and β-D-glucose. Even if the reactivity of the
aldehyde group is blocked in the hemiacetal form, the small amount of D-glucose which remains
in open-chair form (0.8%) is enough for the glucose solution to show the typical reactivity of an
aldehyde group.
According to Watanabe (2005), glucose discoloration in aqueous glucose solutions occurs, by
isomerization of glucose to fructose, followed by dehydration of both glucose and fructose to 1,6anhydroglucose (AHG) and to 5-HMF and 2- FA respectively. The reaction pathway of this
glucose degradation in water is presented in Figure 6.
13
Figure 6. Proposed reaction pathway of glucose in water, Watanabe (2005).
The results show that the color formation was more pronounced for glucose B compared to
glucose A for all the tested batches, thus an obvious difference in glucose quality with respect to
discoloration between the two types of glucose was verified.
3.1.3.
catalyst
Caramelisation of glucose with metal chlorides as
Metal chlorides have been reported to accelerate glucose degradation, and therefore the
glucose discoloration was investigated using CrCl2 as catalyst. The results are presented in Table
5. It was observed that CrCl2 gives a very strongly accelerated color formation for both types of
glucose, but no significant difference in glucose quality with respect to discoloration between the
two types of glucose could be observed. These results can be explained by when using CrCl 2 as
reagent, the reaction rate is much more accelerated and glucose degradation to 5-HMF is more
complete.
Table 5. The effect of chromium chloride as catalyst on glucose discoloration, in glucose solutions,
which were heated for 1 h at 100°C. Measurements made on samples diluted 1:100, absorbance values recalculated by this factor.
Absorbance difference
Glucose A
water/CrCl2
Glucose B
water/CrCl2
B/A Absorbance
factor
water/CrCl2
At 450nm-600nm
7.331
6.248
0.8
At 350nm-600nm
213.713
133.938
0.6
At λmax-600nm
330.259
326.595
0.9
14
Previous work in this area, found in literature, showed that using CrCl2 as reagent will lead to a
high 5-HMF conversion yield from glucose and according Zao and co-workers (2007) this can
be explained by the effect of CrCl2 on glucose mutarotation of α-glucopyranose to ßglucopyranose and then isomerisation of ß-glucopyranose to fructose see Figure 7.
Figure 7. CrCl2 catalyses the mutarotation and then isomerisation to fructose, followed by dehydration to
5-HMF, Zao and co-workers (2007).
3.1.4.
Caramel colours
The results of the investigation of glucose caramel classes by spiking glucose solutions with
NaOH (Caramel Class I), sodium sulphite, (Caramel Class II), ammonium chloride (Caramel
Class III) and with sodium sulphite and ammonium chloride (Caramel Class IV) and then heating
the samples at 100°C for 1 h, are presented in Figure 8.
15
Normalized difference
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
G
lu
co
se
A+
G
0,
lu
co
05
se
%
G
Na
A+
lu
2S
co
0,
5%
O
se
3
B+
Na
G
lu
0
2S
,0
co
O
5%
se
3
Na
B+
G
2S
lu
0
,5
co
O
%
3
se
Na
A+
2
G
SO
0,
lu
05
co
3
%
se
NH
G
A+
lu
G
4
co
0,
Cl
lu
5%
se
co
se
B+
N
G
H4
A+
0,
lu
Cl
05
co
G
0,
lu
s
0
%
e
co
5%
N
B
se
H4
+
NH
G
0,
A+
Cl
lu
5%
4C
co
0
l+
,5
se
NH
%
0
B+
,0
4C
G
NH
5%
lu
0,
l
4C
co
05
N
se
a2
l+
%
SO
0,
B+
NH
5%
3
0,
4C
Na
5%
l+
2S
µl
0,
O
05
NH
3
%
4C
Na
G
l
+
lu
2
SO
0,
co
5%
se
3
Na
A+
G
2
0,
SO
lu
05
co
3
%
se
N
G
A+
a
lu
O
co
0,
H
5%
se
B+
Na
G
O
0,
lu
H
05
co
se
%
Na
B+
O
0,
H
5%
Na
O
H
At 450nm600nm
At 350nm600nm
Figure 8. Results of glucose discoloration for glucose caramel classes, at 100°C, for 1 h.
According to these results, the glucose discoloration was accelerated for Caramel Class I and
Caramel Class II, for both types of glucose.
3.1.4.1.
Caramel Class I – Alkaline
The glucose discoloration was accelerated for Caramel Class I in alkaline conditions. No
difference with respect to discoloration could be observed for the two types of glucose since
glucose degradation is highly increased at high pH. The alkaline degradation of glucose gives
high molecular weight products, which are associated with color formation, according to Coca et
al. (2004). Monosaccharides in aqueous alkaline solutions undergo both reversible and
irreversible reactions according to De Bruijn, (1986).
3.1.4.2.
Caramel Class II – Sodium sulphite
In our experiments the addition of higher amounts of sodium sulphite gave accelerated glucose
discoloration. This was not as expected, since sodium sulphite is known to have an inhibitory
effect on glucose discoloration. However, Hoffman and others (1999) and Nursten (2005),
reported that the presence of sodium sulphite has en inhibitory effect on glucose discoloration
only in earlier stages, likely by blocking the radical precursors, glyoxal and glucoaldehyde.
Glucose discoloration was found to be more accelerated at higher concentration of sodium
sulphite in the solutions, since after the induction period, sodium sulphite functions as catalyst in
5-HMF formation from 3-deoxyglucosone.
For Caramel Class II glucose discoloration was accelerated for both types of glucose and a
significant difference between them could no longer be observed. This observation can be
explained by the differences in the manufacture process between the two types of glucose. For
16
glucose type A the addition of substances with an inhibitory effect such as sodium bisulphite is
used in the purification step, while no additions are stated for glucose type B. The extra sulphite
addition in our experiments could possibly catalyse further formation of 5-HMF as a precursor to
color formation after the induction period for glucose type A. I.e. for glucose type A the
induction step is lower compared to type B for the same amount of sulphite addition.
In order to further investigate the effect of a low level of sodium bisulphite on glucose samples,
glucose type B, was spiked with different amounts of sodium bisulphite.
The results of spectroscopic determination of color formation and results of HPLC
determination of 2-FA formation show that the addition of sodium bisulphite at lowers levels
somewhat reduces both discoloration and 2-FA formation. The results of glucose discoloration by
spectroscopic determination are presented in Figure 9, in which samples of glucose type A were
plotted together with spiked glucose type B samples. The results of 2-FA formation are presented
in Figure 10.
As the results show, the addition of 30 ppm sodium bisulphite to a glucose B solution decreases
the discoloration to the same level of discoloration as a native glucose A solution.
The results obtained from HPLC analysis of 2-FA are presented in Figure 10, were the
concentration of 2-FA was plotted against the spiked amount of sodium bisulphite. The results
presented in Figures 9 and 10 allow the conclusion that the addition of lower levels of sodium
bisulphite to the glucose type B solutions has an inhibitory effect on the 2 –FA formation and
reduces the discoloration.
Sodium bisuphite spiking
0,45
0,4
0,35
Abs 350 nm
0,3
0,25
0,2
0,15
0,1
0,05
0
Glucose A
native
Glucose B
native
Glucose B 5
ppm
Glucose B 10
ppm
Glucose B 20
ppm
Glucose B 30
ppm
Figure 9. The effect of sodium bisulphite on 5-HMF formation from glucose type B, by spectroscopic
determination.
17
2-FA formation
Concentration 2-FA (mg/l)
8,5
8
7,5
2-FA
(mg/l)
7
6,5
6
5,5
5
0
5
10
15
20
25
30
35
Spiked am ount Na-bisulphite (m g/l)
Figure 10. The effect of sodium bisulphite on 2-FA formation from glucose type B, by HPLC
determination.
3.1.4.3.
Glucose Caramel Class III and Caramel Class IV
For Caramel Class III and Caramel Class IV, no or a lower glucose discoloration could be
detected.
3.2.
HPLC determination of 5-HMF and 2-FA
The results of HPLC determination of formation of 5-HMF and 2-FA for caramelisation
process, using different reagents are presented in Table 6. It can be seen that when using CrCl2 as
reagent, the formation of 5-HMF and 2-FA was highest, for both types of glucose. When using
NaOH as reagent in the formation of Caramel Class I, 2-FA formation increases. This can be
explaining by the fact that in alkaline medium degradation of glucose occurs according to
Watanabe (2005), by isomerisation of glucose to fructose, followed by dehydration of both
glucose and fructose to 1, 6-anhydroglucose (AHG) and to 5-HMF and 2- FA respectively.
All the samples that have been subjected to heat did, however, produce some color, varying
from pale yellow to brown. The results of glucose discoloration in water, Caramel Class III and
Caramel Class IV, show that the heated samples, in which no visible yellow color could be
observed, can be explained by different reactivity of the used reagents.
When using strong reagents such as NaOH and CrCl2, the glucose discoloration was
accelerated so strongly for both types of glucose so that no significant difference in glucose
quality between the two types could be observed any more. This can be explained by when using
these reagents as catalysts the reaction of glucose degradation is more complete.
18
Table 6. HPLC evaluation of 5-HMF and 2-FA in the caramelisation process.
Glucose sample
5-HMF(ppm) 2-FA(ppm) Absorbance difference at 350nm
Supplier A/water
<0.1
<0.02
0.382
Supplier B/water
<0.1
<0.02
1.303
Supplier A Caramel Class I*
4.7
3.08
588
Supplier B Caramel Class I*
5.6
3.28
574
Supplier A Caramel Class II* <0.1
<0.02
615
Supplier B Caramel Class II* <0.1
<0.02
609
Supplier A Caramel Class III* 0.2
<0.02
0.454
Supplier B Caramel Class III* 0.2
<0.02
1.358
Supplier A Caramel Class IV* <0.1
<0.02
14.9
Supplier B Caramel Class IV* <0.1
<0.02
14.6
0.27
214
Supplier A/CrCl2**
186.6
Supplier B/CrCl2**
204.7
0.28
*) Samples of Caramel Classes were diluted 1:10.
**) Samples of glucose containing CrCl2 were diluted 1:100
3.3.
134
Maillard reaction
The contribution of Maillard reaction to non-enzymatic browning of glucose was tested in
three different aqueous model systems, using two types of D-glucose and different amino acids:
L-lysine, L-glycine, and L-alanine. The effect of pH on Maillard reaction was also tested. The
effect of the amino acids on non-enzymatic browning in glucose solutions at pH 1.5 (HCl) and 9
(NaOH) and in the absence of amino acids is presented in Figures 11 and 12.
19
Normalized difference Maillard Reaction
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
At 450nm-600nm
G
lu
co
se
G
lu
co A/L
se
ys
G
in
A/
lu
e/
A
co
Na
la
se
ni
ne OH
A/
G
lu
/N
co Gly
aO
ci
se
G
H
ne
lu
/N
co B/L
aO
se
ys
G
H
in
B
lu
e/
co /Al
Na
a
se
n
O
H
B/ ine
/N
G
G
ly
aO
lu
c
co
H
in
se
G
e/
lu
Na
A
co
/
O
se Lys
H
G
i
A
n
lu
e
/
Al
/
co
an HC
se
l
in
G
A
e/
lu
/G
H
co
l
Cl
se ycin
G
lu
e
B
/H
co
/
Cl
se Lys
G
i
n
B
lu
e
/
A
/H
co
la
C
se
n
l
B/ ine
/
G
H
ly
Cl
G
ci
lu
co ne
se /HC
G
l
A
lu
co /Na
O
se
H
B/
G
Na
lu
co
O
H
se
G
A/
lu
H
co
se C l
B/
HC
l
At 350nm-600nm
Figure 11. Effect of different amino acids on glucose discoloration, at pH 1.5 and 9 of glucose solutions
heated for 1 h at 100°C.
The glucose discoloration increased in the presence of amino acids. The acceleration effect of
amino acids on glucose discoloration was in following order: lysine > glycine >alanine. Glucose
discoloration increased, as expected with increased pH, shown in Figures 11 and 12. This is
consisted with the results reported by Weismann (1993).
Normalized difference
100%
80%
60%
At 450nm-600nm
40%
At 350nm-600nm
20%
0%
Glucose
A/Lysine
Glucose Glucose
A/Alanine A/Glycine
Glucose Glucose
B/Lysine B/Alanine
Glucose
B/Glycine
Glucose
A/water
Glucose
B/water
Figure 12. Effect of different amino acids on glucose discoloration, at pH 6 of glucose solutions heated
for 1 h at 100°C.
20
The glucose discoloration was more accelerated for glucose type B compared to glucose type A
and therefore the same difference in glucose quality between the two types of glucose could be
observed in the Maillard reaction as well as for the caramelisation process.
The results of HPLC determination of 5-HMF and 2-FA formation in Maillard reaction are
presented in Table 7. The HPLC determination of 5-HMF and 2-FA confirmed the difference in
color formation for the two types of glucose obtained by spectrophotometric determination. The
same difference in stability of the glucose between the two types could be confirmed at pH 6 and
9, since 5-HMF was detected only in the glucose solutions containing glucose type B. For
glucose type A at the same conditions of reactions, no formation of 5-HMF and 2-FA could be
detected.
At pH 1.5, 5-HMF formation was detectable for both types of glucose, but more accelerated for
glucose type B. This difference in 5-HMF formation between the two types of glucose can only
be explained by the better quality of glucose A with respect to stability.
Table 7. HPLC evaluation of 5-HMF and 2-FA formation in Maillard reaction.
Glucose sample
Supplier A Lysine + water
Supplier A Alanine + water
Supplier A Glycine + water
Supplier B Lysine + water
Supplier B Alanine + water
Supplier B Glycine + water
Supplier A Lysine + NaOH
Supplier A Alanine + NaOH
Supplier A Glycine + NaOH
Supplier B Lysine + NaOH
Supplier B Alanine + NaOH
Supplier B Glycine + NaOH
Supplier A Lysine + HCl
Supplier A Alanine + HCl
Supplier A Glycine + HCl
Supplier B Lysine + HCl
Supplier B Alanine + HCl
Supplier B Glycine + HCl
5-HMF(ppm)
<0.1
<0.1
<0.1
0.2
0.1
0.1
<0.1
<0.1
<0.1
0.1
0.1
0.1
0.2
0.3
0.1
0.9
0.6
0.8
21
2-FA(ppm)
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
<0.02
3.4.
Method of optimisation
Optimisation of the accelerated glucose discoloration method was made by testing the
parameters, which affect non-enzymatic browning of glucose such as temperature, pH,
concentration and time and the results are presented in Figures 13, 15, 17, and 19. In order to find
the optimum parameters of accelerated glucose discoloration method, the difference in quality of
glucose with respect to discoloration between the two types of glucose was taken in to
consideration and results are presented in Figures 14, 16, 18 and, 20.
3.4.1.
Temperature optimisation
The largest difference in glucose quality with respect to discoloration between the two types of
glucose was obtained at 90°C, Figures 13 and 14, which seems to be the optimum temperature in
the glucose discoloration assessment. The decrease in absorbance difference at 350 nm for 90100°C was confirmed by additional experiments, but remains unexplained. However, at the same
time the difference between the two types of glucose was increased, which is beneficial for
assessing the relative glucose quality with respect to discoloration.
Temperature optimization
1,2
1
Absorbance
0,8
Glucose A
0,6
Glucose B
0,4
0,2
0
75
80
85
90
95
100
105
Temperature ( °C)
Figure 13. Effect of temperature on glucose discoloration for 1 h heating of glucose samples (33%w/w)
an absorbance difference of 350 nm.
22
Glucos e B/Glucos e A Abs orbance Factor
4,5
4
3,5
Absorbance
3
2,5
B/A Abs. Factor
2
1,5
1
0,5
0
60
70
80
90
100
110
120
Te m pe rature (°C)
Figure 14. Absorbance factor between the two types of glucose in temperature optimisation at an
absorbance difference of 350nm.
3.4.2.
Concentration optimisation
The largest difference in glucose quality with respect to discoloration between the two types of
glucose was obtained at 40 % (w/w) glucose concentration, Figures 15 and 16, which seems to be
the optimum concentration in glucose discoloration assessment. Even a higher glucose
concentration might have an inhibitory effect on glucose discoloration, according to Kjellstrand
et al. (2001).
Concentration optimization
2
1,8
1,6
Absorbance
1,4
1,2
Glucose A
1
Glucose B
0,8
0,6
0,4
0,2
0
10
20
30
40
50
60
70
Concentration (% w/w)
Figure 15. Effect of concentration on glucose discoloration, at an absorbance difference of 350 nm, at
100°C for 1 h.
23
Glucose B/Glucose A Absorbance Factor
6
Absorbance
5
4
3
B/A Abs Factor
2
1
0
10
20
30
40
50
60
70
Concentration % (w /w )
Figure 16. Absorbance factor between the two types of glucose in concentration optimisation at an
absorbance difference of 350 nm.
3.4.3.
The pH optimisation
The largest difference in glucose quality with respect to discoloration between the two types of
glucose was at pH 7, Figures 17 and 18, which seems to be the optimum in the assessment of
glucose discoloration.
pH optimization
50
45
40
Absorbance
35
30
Glucose A
25
Glucose B
20
15
10
5
0
0
2
4
6
pH
24
8
10
pH optimization
2
1,8
1,6
Absorbance
1,4
1,2
Glucose A
1
Glucose B
0,8
0,6
0,4
0,2
0
0
2
4
6
8
10
pH
Figure 17. Effect of pH on glucose discoloration at an absorbance difference of 350 nm, by heating of
glucose samples (33% w/w) at 100°C for 1 h. The lower graph shows pH 1.5-7 in a larger scale
Glucose B/Glucose A Absorbance Factor
3
Absorbance factor
2,5
2
B/A Abs. Factor
1,5
1
0,5
0
0
2
4
6
8
10
pH
Figure 18. Absorbance factor between the two types of glucose in pH optimisation at an absorbance
difference of 350 nm.
25
3.4.4.
Time optimisation
The largest difference in glucose quality with respect to discoloration between the two types
of glucose was obtained when heating the glucose sample at 90°C for 1 h and without heating of
glucose samples at time of 0 h, Figures 19 and 20, which seem to be the optimum times in
accelerated glucose discoloration method.
Different times at ambient temperature were further investigated, and showed that results had a
larger variation when the samples were measured directly after apparent dissolution of the
glucose i.e. time 0. The repeatability was significantly improved when allowing the samples to
dissolve thoroughly for 1 h prior to the absorbance determination at ambient temperature.
Time optimization
12
10
Absorbance
8
Glucos e A
6
Glucos e B
4
2
0
0
5
10
15
20
25
30
Time (hours)
Figure 19. Effect of time of heating on glucose discoloration at an absorbance difference of 350nm by
heating of glucose sampes (33% w/w) at 100°C.
26
Glucose B/Glucose A Absorbance Factor
5
4,5
4
Absorbance
3,5
3
B/A Abs. Factor
2,5
2
1,5
1
0,5
0
0
5
10
15
20
25
Tim e (hours)
Figure 20. Absorbance factor between the two types of glucose in time optimisation at an absorbance
difference of 350 nm.
3.4.5.
Heating versus no heating
Since a larger difference in quality of glucose with respect to discoloration between the two
types of glucose was obtained without heating of the glucose samples, more tests were performed
in order to find the optimum time and temperature. The results are presented in Figures 21 and
22.
Glucose discoloration before heat
0,22
0,17
Absorbance
A water Day1
0,12
B water Day 2
A water Day 2
B water Day 2
A water Day 3
0,07
B water Day 3
0,02
260
310
360
410
460
510
560
610
-0,03
Wavelength (nm)
Figure 21. The effect of water on glucose discoloration before heating of glucose sample (33%, w/w), in
three different days.
27
Glucose discoloration after heat
0,5
0,45
0,4
A water Day 1
B water Day 1
0,35
Absorbance
A water Day 2
0,3
B water Day 2
A water Day 3
0,25
B water Day 3
0,2
0,15
0,1
0,05
0
260
310
360
410
460
510
560
610
Wavelength (nm)
Figure 22. The effect of water on glucose discoloration after heating of glucose sample (33%, w/w) for 1
h, in three different days.
According to the obtained results in the experiments with or without heating of the glucose
samples, the color formation was more accelerated for glucose type B compared to glucose type
A and therefore an obvious difference in glucose quality with respect to discoloration between
the two types of glucose could be observed. Since a larger difference in discoloration between
the two types of glucose could be observed when mixing glucose with water, without heating of
the samples, (Figure 21), and the optimum temperature for the glucose discoloration method was
found to be at ambient temperature.
According to the obtained results, based on the difference in quality between the two types of
glucose, the optimum conditions for accelerated glucose discoloration method as a tool for
glucose stability assessment were found to be in aqueous glucose samples 33% (w/w), kept at
room temperature for 1 h.
3.5.
3.5.1.
Method Robustness
The robustness of the method
The robustness of the method was tested by investigating the repeatability and reproducibility of
the chosen method and the results are presented in Table 8.
28
Table 8. Robustness test of the accelerated glucose discoloration method in two different days, each day
represent an average of three tests.
Tests
Absorbance
difference
Glucose Type A
Type
A
RSD%
Glucose Type B
Type B
RSD %
Day 1
At 450nm-600nm
0.072
3.5
0.244
2.1
At 350nm-600nm
0.244
6.1
1.106
1.0
At λmaxnm-600nm
1.575
1.4
4.788
1.1
At 450nm-600nm
0.064
13.6
0.248
2.3
At 350nm-600nm
0.241
6.3
1.106
0.3
At λmaxnm-600nm
1.571
3.0
4.651
0.4
At 450nm-600nm
0.068
10.5
0.246
2.2
At 350nm-600nm
0.242
5.6
1.106
0.7
At λmaxnm-600nm
1.573
2.1
4.720
1.8
Day 2
Day 1-Day 2
According to above the results, the tested method is repeatable and reproducible.
3.5.2.
Time
The influence of time on the glucose discoloration at ambient temperature was tested and the
results are presented in Table 9. No influence of time on color formation in glucose sample could
be observed at ambient temperature. The method is robust at ambient temperature with respect to
time.
Table 9. Effect of time in glucose solutions at room temperature.
Time
Glucose A
Glucose B
B/A Absorbance factor
0,75
0.203
1.009
5.0
1
0.237
1.068
4.5
2
0.236
1.149
4.9
4
0.223
1.104
4.9
6
0.225
1.077
4.8
24
0.198
1.078
5.5
48
0.233
0.974
4.2
29
4.
Conclusions
The optimum conditions for assessing the glucose stability with respect to discoloration was
found to be when mixing glucose with water, 33% (w/w), keep the samples at ambient
temperature for 1 h before determination of the color, absorbance difference at 350 nm by
UV/Vis spectrophotometer in a 4 cm cuvette.
For Maillard reaction, based on the difference between the two types of glucose, the, optimum
conditions were found to be the glucose-L-alanine model system, when heating the samples (33%
glucose, w/w) at 100°C for 1 h.
The stability of glucose with respect to discoloration and 5-HMF formation was proven to be
better for glucose type A compared to glucose type B.
The results showed that glucose manufacturing process plays an important roll in non-enzymatic
browning of glucose.
The proposed glucose discoloration method could be used as a quick tool to separate glucose that
potentially will develop yellow color in the manufacturing process and/or during storage of final
products containing glucose.
5.
Acknowledgments
The author is grateful to Gambro Lundia AB Sweden for the financial support for this project and
would like to specially thank specialist Staffan Bergström for all his support and work in the
present study as well as to the personal at the Analytical Chemistry Group at Gambro Lundia AB.
6.
References
Analytical methods of the Members companies of the Corn Refiners Association, Inc. CRA E20 Glucose color stability (1989)
Ajandouz, Tchiakpe, Dalle, Benita and Puigserver, Effects of pH on Caramelization and
Maillard reaction kinetics in fructose-lysine model systems, Journal of Food and Science, Vol.
66, No. 7, p. 926-931, (2001)
Bostan and Boyacioglu, Kinetics of nonenzymatic color development in glucose syrup during
storage, Food Chemistry, Vol. 60, No. 4, p. 581-585, (1997)
Buera, Resnik, and Petriela, Relative browning rates of glucose and fructose in high water
activity systems as function of amino compound concentration, Journal of Food ad Science, Vol.
51, No.4, p. 1073-1074, (1992)
30
Buera, Chirife, Resnik and Wetzler, Nonenzymatic browning in liquid model systems of high
water activity: Kinetics of color changes due to Maillard’s reaction between different single
sugars and glycine and comparison with Caramelization browning, Journal of Food Science Vol.
52, p. 1063-1067, (1987)
Coca, Garcia, Gonzales, Pena, Garcia, Study of coloured components formed in sugar beet
processing, Food Chemistry Vol. 86, p. 421-433, (2004)
De Bruijn, Monosaccharides in aqueous alkaline medium: Isomerisation, degradation and
oligomerisation, Starch, Vol. 39, No. 1, p. 23-28, (1987)
Erixon, Wieslander, Linden, Carlsson, Forsbäck, Svensson, Jonsson and Kjellstrand, How to
avoid glucose degradation products in peritoneal dialysis fluids, Peritoneal Dialysis International,
Vol. 26, p. 490-497, (2006)
Fournier, Colorimetric Quantification of Carbohydrates, Current Protocols in Food
Analytical Chemistry, E1.1.1-E1.1.8, (2001)
Girisuta, Janssen, Heeres, Green Chemicals: A Kinetic Study on the Conversion of Glucose
to Levulinic Acid, Chemical Engineering Research Design Vol. 84, No. 5, p. 339-349, (2006)
Holladay, Zao, Brown and Zhang, Metal Chlorides in ionic liquid solvents convert sugar to 5Hydroxymethylfurfural, Science, Vol. 316, No. 5831 p. 1597-1600, (2007)
Kelly and Brown Thermal decomposition and color formation in aqueous sucrose solutions.
Sugar Technology Reviews, Vol. 6, No. 1, p. 1-48, (1978)
Kamuf, Nixon, Parker, and Barnum, Overview of Caramel Colours, American Association of
Cereal Chemists, Inc., Vol. 48, No. 2, (2003)
Kroh, Caramelization in Food and Beverage, Food Chemistry Vol. 51, p. 373-379, (1994)
Kjellstrand, Martinson, Wieslander, Kjellstrand, Jeppsson, Svensson, Jäkelid, Linden and
Olsson, Degradation in peritoneal fluids may be avoided by using low pH and high glucose
concentration, Peritoneal Dialysis International, Vol.21, p. 338-344, (2001)
Li, Zhang and Zao, Direct conversion of glucose and cellulose to 5-hydroxymethylfurfural in
ionic liquid under microwave irradiation, Tetrahedron Letters Vol. 50, No. 38, p. 5403-5405,
(2009)
Licht, Shaw, Smith, Mendoza, Orr and Myers, Development of specifications for caramel
colures, Food Chemical Toxicology, Vol. 30, No. 5, p. 359-363, (1992)
Lim, Kwak, Lee, and Lee, Effect of antibrowning agents on browning and intermediate
formation in the glucose-glutamic acid model, Journal of Food Science, Vol. 75, No.8, p. 678683, (2010)
Lee and Nagy, Relative reactivity’s of sugar in the formation of 5-hydroxymethylfurfural in
sugar-catalyst model systems, Journal of Food Processing and Preservation Vol. 14, p. 171-178,
(1990)
Lin, Peng, Zhang, Zhuang, Zhang and Gong, Catalytic conversion of cellulose to levulinic
acid by metal chlorides, Molecules, Vol. 15, No. 8, p. 5258-5272, (2010)
Moliner, Leshkov and Davis, Tin-containing zeolites are highly active catalysts for the
isomerisation of glucose in water, Proceedings of the National Academy of Science of the United
States of America, Vol. 107, No. 14, p. 6164-6168, (2010)
Meikle, Carbohydrates in food science, Food chemistry, p. 264-269, (2007)
Martins, Martinus, Boekel, Melanoidins extinction coefficient in the glucose /glycine
Maillard reaction, Food Chemistry, Vol. 83, No. 1, p. 135-142, (2003)
Neil, Carbohydrate Chemistry: A review of the literature, Volume 14, Vol. 1, Royal Society
of Chemistry, (1980)
31
Nursten, The Maillard reaction chemistry, biochemistry and implications, Royal Society of
Chemistry, (2005)
Phongkanpai, Benjakul and Tanaka, Effect of pH on anti-oxidative activity and other
characteristics of caramelisation products, Journal of Food Biochemistry Vol. 30, No. 2, p. 174–
186, (2006)
Pieschetsrieder, Chemistry of Glucose and Biochemical Pathways of Biological Interest,
Peritoneal Dialysis International, Vol. 20, No.20, p. 26-30, (2000)
Porretta, Nonenzymatic browning of tomato products, Food Chemistry, Vol. 40, No. 3, p. 323335, (1990)
Ramchander and Feather, Studies on the mechanism of color formation in glucose syrups,
Journal of Paper, Vol. 52, No. 6857, p. 167-173, (1975)
Schultz, Caramel color, Food Product Design, (2006)
Schrödter, Heat-induced decomposition of disaccharide Amadori compounds in quasi-waterfree reaction conditions, Vol. 194, No. 3, p. 216-221, (1992)
Takeuchi, Jin, Tohji, Enomoto, Acid catalytic hydrothermal conversion of carbohydrate
biomass into useful substances, Journal of Mater Science Vol. 43, No.7, p. 2472-2475, (2008)
Tomasick, Palanski, and Wiejak, The thermal decomposition of carbohydrates Part 1: The
decomposition of mono- di and oligosaccharides, Advanced Carbohydrate Chemistry
Biochemistry Vol. 47, p. 203-270, (1989)
Weissman, Kopelman and Mizrahi, A kinetic model for accelerated tests of Maillard browning
in liquid model system, Journal of Food Processing and Preservation Vol. 17, No. 6, p. 445-470,
(1993)
Watanabe, Aizawa, Iida, Taku, Levy, Sue and Inomata, Glucose reactions with acid and base
catalysts in hot compressed water at 473K, Carbohydrate research Vol. 340, No. 12, p. 19251930, (2005)
Witowsky, Bender, Gahl, Frei and Jörres, Glucose degradation products and peritoneal
membrane function, Peritoneal Dialysis International, Vol. 21, Peritoneal Dialysis International,
Vol. 21, No. 2, p. 201-205 (2000)
32