ARTICLE IN PRESS

Lebensm.-Wiss. u.-Technol. 37 (2004) 161–170

Effect of honey types and concentration on starch gelatinization

P.J. Torleya,*, R.P.G. Rutgersb, B. D’Arcya, B.R. Bhandaria
a
School of Land and Food Sciences, The University of Queensland, Brisbane, Qld 4072, Australia
b
Division of Chemical Engineering, The University of Queensland, Brisbane, Qld 4072, Australia
Received 17 March 2003; received in revised form 13 August 2003; accepted 14 August 2003

Abstract

The complex and variable composition of honey, depending on source, season and processing, means different honey samples
could cause variation in the characteristics of the finished product. The objective of this study was to determine how the minor
components present in honey affect starch gelatinization. A Rapid Visco Analyser was used to measure changes in viscosity when
unmodified maize starch was gelatinized in a honey or model sugar solution. When honey was compared to equivalent blends of
sugars, there was an increase in starch viscosity with increasing levels of addition. However, at the same level, honey gave a lower
viscosity than the blends of sugars. Honeys from different sources (differing in pH and amylase activity) show a varied effect on
starch gelatinization, with starch viscosity increasing with addition level for six of the honeys, but decreasing with increasing
addition level for two honey samples. Varying the pH also produced variation in starch gelatinization patterns between honey types.
Between pH 3.0 and 4.0, starch viscosity was similar for all four honey types studied, while above this pH there were differences
between all honey types. As expected, starch viscosity decreased as the solution pH neared the optimum for honey amylase activity
(pH 5.3–5.6), though it did not increase as the pH moved away from the honey amylase activity optimum. Differences between
honey samples, and between honey and a model sugar mixture, in their effect on starch gelatinization was attributed to honey
amylase activity and the composition and concentration of minor organic compounds present.
Crown Copyright r 2003 Published by Elsevier Ltd. on behalf of Swiss Society of Food Science and Technology. All rights
reserved.

Keywords: Honey; Amylases; Starch; Gelatinization

1. Introduction (Mossel, 2003), with similar variation seen in other

Floral honey is a high sugar food product made by
honeybees from the nectar of flowers. The major
constituents of honey are fructose (on average 37.5 g/
100 g in 126 samples), glucose (28.1 g/100 g) and water
(18.4 g/100 g) (Mossel, 2003), with the remainder made
up of a complex mixture of mono-, di- and tri-
saccharides (Doner, 1977; Low & Sporns, 1988),
enzymes (including invertase, a-amylase, glucose oxi-
dase) and a wide variety of minor organic and inorganic
compounds (White, 1992). The composition of honey
varies widely from sample to sample depending on the
botanical origin of the nectar. For example, in a study of
15 types of Australian unifloral honeys, the average
fructose concentration varied from 32.6 to 41.7 g/100 g
*Corresponding author. Division of Chemical Engineering, The
University of Queensland, Brisbane, Qld 4072, Australia. Tel.: +61-7-
3365-3930; fax: +61-7-3365-4199.
E-mail address: p.torley@uq.edu.au (P.J. Torley).
constituents (White, 1992; Mossel, 2003).
Various studies have looked at the application of
honey as an ingredient in starch-based foods, where it is
a sweetener, flavour, humectant and source of reducing
sugars for the Maillard reaction (Clarke, 1991). These
functional properties can be attributed to the high
concentration of sugars present in honey; however, the
minor constituents can also play a role, for example, as
an antimicrobial (Bogdanov, 1997), antioxidant (Ghel-
dof, Wang, & Engeseth, 2002), antibrowning (Chen,
Mehta, Berenbaum, Zangerl, & Engeseth, 2000) or
antistaling agent (D’Arcy et al., 1999). In a study of
staling in bread, the effect of ironbark honey was
compared with a blend of sugars containing the main
sugars found in honey (D’Arcy et al., 1999). It was
shown that adding 3 g/100 g honey had an antistaling
effect compared to both the control (no added honey or
sugar) and 3 g/100 g of the sugar blend. This difference
between honey and the sugar blend indicates that the
minor constituents of honey might have contributed to

0023-6438/$30.00 Crown Copyright r 2003 Published by Elsevier Ltd. on behalf of Swiss Society of Food Science and Technology. All rights reserved.
doi:10.1016/j.lwt.2003.08.002
 ARTICLE IN PRESS
162 P.J. Torley et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 161–170
its antistaling effect. There are many compounds that
can have an antistaling effect in bread, including
amylases, emulsifiers and alcohols (Pateras, 1998).
Honey can also have a negative effect on product
quality, causing a loss of viscosity of starch-based foods
due to the naturally occurring amylase found in the
honey (Babacan, Pivarnik, & Rand, 2002).
Two components of honey expected to affect starch
properties are sugars and amylase. Increasing sugar
concentrations increases the gelatinization temperature
(Ahmad & Williams, 1999), starch viscosity (Prokopo-
wich & Biliaderis, 1995) and alters the rate of starch
retrogradation (Katsuta, Nishimura, & Miura, 1992).
The type of sugar also influences starch properties,
although the order of effectiveness of different sugars
depends on the system being studied (Katsuta et al.,
1992; Prokopowich & Biliaderis, 1995; Ahmad &
Williams, 1999). Amylases hydrolyse starch, reducing
the viscosity of starch solutions (Babacan et al., 2002).
In the finished gel, the starch fragments produced by
amylases can also reduce the rate of starch retro-
gradation (Leon, Duran, & Benedito de Barber, 1997).
In addition to sugars and amylases, there are many
other compounds present in honey that may also affect
starch. A wide variety of organic compounds could
affect starch properties because of their effect on ionic
conditions (e.g. pH, ionic strength, buffering capacity),
or on some specific property of the solute (e.g. enzyme
inhibition or activation, emulsification).
The objective of this research was to determine whether
the minor components present in honey affect starch
gelatinization, by comparing honey with equivalent blends
of pure sugars and by comparing different honey types
with each other. The gelatinization (or pasting) of an
unmodified maize starch (about 26 g/100 g amylose) was
monitored with a Rapid Visco Analyser (RVA) when it
was heated in solutions containing either honey or blends
of pure sugars (fructose, glucose and maltose). Initially, a
blended honey (Light Amber) was compared to blends of
pure sugars to find out whether the minor sugars and
other components present in honey can have a significant
effect on starch gelatinization. Secondly, eight honey types
were compared to see whether the natural variation in
composition produced any differences in starch gelatini-
zation. Finally, the pH of four honey types were altered
over the range pH 3–8, which covers the range where
honey amylase is active, to see whether starch gelatiniza-
tion varied in a corresponding manner.
2. Materials and methods
2.1. Materials
A total of eight honey samples were used in this study,
six unifloral and two blended honey samples. The
samples of Light Amber blend, red gum, spotted gum,
stringybark and yapunyah honeys were donated by
Capilano Honey Ltd (Richlands, Qld, Australia).
Ironbark honey, a blend of New Zealand manuka and
Australian jellybush honeys and yellow box honey were
purchased from retail outlets in Brisbane, Australia.
The RVA studies were performed with an unmodified
maize starch donated by Penford Australia Ltd (3401C,
Lane Cove, NSW, Australia). The suppliers report that
3401C contains approximately 74 g/100 g amylopectin
and 26 g/100 g amylose. The starch sample contained
10.2 g/100 g moisture, measured by oven drying at
105
C overnight.
Fructose (>99 g/100 g), glucose (>99.5 g/100 g), su-
crose (>99.5 g/100 g) and maltose monohydrate (>90 g/
100 g, remainder glucose and maltotriose) were obtained
from Sigma Aldrich Pty Ltd (Castle Hill, NSW,
Australia). The sodium acetate trihydrate and sodium
hydroxide were analytical grade reagents. Phadebas
Amylase Test (Pharmacia Diagnostics AB, Sweden)
tablets were obtained from Australian Laboratory
Services Pty Ltd. (Sydney Markets, NSW, Australia).
The 0.1 mol/l hydrochloric acid solution was prepared
from a Volucon standard (May and Baker, Dagenham,
England). The 0.1 mol/l sodium hydroxide solution
was obtained from Australian Chemical Reagents
(Moorooka, Qld, Australia).
2.2. Methods
2.2.1. Honey pH
The pH of a honey solution (2 g of honey in 20 g of
demineralised water) was measured with an Activon AS
205 pH meter fitted with an Ionode IH20 electrode.
2.2.2. Sugar content
The concentration of fructose, glucose, sucrose and
maltose of Light Amber honey was measured using the
AOAC HPLC method 977.20 for sugar determination
(AOAC, 1996).
2.2.3. Moisture content
An Atago refractometer was used to measure the
refractive index of the honey samples, which was used to
determine the moisture content (Bogdanov, Martin, &
Lullmann, 1997). Honey samples where sugar crystals
had formed were heated in sealed containers at 60
C to
dissolve the crystals and cooled to room temperature
before their refractive index was measured.
2.2.4. Honey amylase activity
The amylase activity (a combination of a- and
b-amylase activity) of the honey samples was measured
using the Phadebas method described by Bogdanov et al.
(1997). In this assay, the amylase activity is determined
by measuring the release of a blue dye from an insoluble
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cross-linked starch when incubated with a honey ingredients. In Pure Sugar Blend 1 (PSB1), the unknown
solution at 40
C for 15 min. The absorbance of the blue material was replaced with water (Table 1). In this case,
dye at 620 nm was measured (Jasco V-550 UV-Vis the unknown material was treated as a diluent, with only
Spectrophotometer and Spectra Manager for Windows the principle sugars assumed to have a significant effect
version 1.30.00; Jasco Corporation, Tokyo, Japan) and on starch gelatinization. In Pure Sugar Blend 2 (PSB2),
expressed as diastase number using the equation given the unknown material was replaced with additional
by Bogdanov et al. (1997). sugars in the same proportions as Light Amber honey
(Table 1). This maintained the same overall solids
2.2.5. RVA analysis content, with the unknown material assumed to have
The starch gelatinization studies were performed the same effect on starch gelatinization as sugars. If the
using a Rapid Visco Analyser RVA 4 (Newport effect of Light Amber honey on starch lies between the
Scientific Pty Ltd., Warriewood, Australia) controlled two model honeys (PSB1 and PSB2), the minor
by Thermocline for Windows version 2.2 (Newport components can be treated as having a similar effect
Scientific Pty Ltd., Warriewood, Australia). A 3.00 g to sugars on starch gelatinization, while if it is
sample of starch was weighed into a sample test canister substantially higher or lower greater care would be
and 25.00 g of liquid was added. The RVA impeller was required when using honey as a substitute for sugar in
jogged up and down at least ten times to suspend the starch-based foods.
starch in the liquid. The RVA canister and impeller To reduce the viscosity of the honey, and to simplify
were then positioned in the RVA and the trial started. dissolving the sugars in the PSB1 and PSB2 samples,
The test profile involved stirring at 960 rpm for 10 s at stock solutions were prepared. The honey stock solution
50
C, stirring at 160 rpm for 50 s at 50
C (stirring was was prepared by dissolving honey in demineralised
maintained at 160 rpm in all subsequent stages of the water (Table 2). The model honey sugar blends (PSB1
test), heating from 50
C to 95
C in 225 s, holding at and PSB2) were prepared by dissolving the appropriate
95
C for 300 s, cooling from 95
C to 50
C in 285 s and blend of sugars (Table 2) in demineralised water. For
holding at 50
C for 300 s. The total test time was 1170 s each RVA trial, the appropriate stock solution (Light
(19.5 min). Amber honey, PSB1 or PSB2) was further diluted with
The RVA curve was analysed using Thermocline for
Windows to determine the pasting temperature (tem- Table 1
The composition of Light Amber honey determined by HPLC and the
perature of the initial increase in viscosity), peak
proportions of sugars used to make up two model honey sugar blends
viscosity (maximum viscosity during heating to and
holding at 95
C), peak temperature (temperature at Light Amber PSB1 PSB2
peak viscosity), trough viscosity (minimum viscosity Honey (g/100 g) (g/100 g)
(g/100 g)a
that occurs after the peak viscosity) and final viscosity
(viscosity at the end of the test profile). Breakdown Fructose 38.7 38.7 46.5
(difference between peak viscosity and trough viscosity) Glucose 28.2 28.2 33.9
Maltose 2.5 2.5 3.0
and setback (difference between final viscosity and
Sucrose —b — —
trough viscosity) were calculated. Unknown 14.0 —c —d
Water 16.6 30.6 16.6
2.3. Experimental designs a
Determined by HPLC analysis.
b
Less than 0.1 g/100 g sucrose detected in Light Amber honey.
2.3.1. Experiment 1: Light Amber honey and pure sugar c
Unknown material in Light Amber honey replaced with extra
blends water.
d
The objective of this experiment was to see what effect Unknown material in Light Amber honey replaced with a blend of
the sugars present.
the ‘‘unknown’’ material in honey (e.g. sugars present at
low concentrations, enzymes, organic compounds)
(Mossel, 2003; White, 1992) have on starch gelatiniza- Table 2
tion. In this experiment, Light Amber honey was The stock solution formulation for Light Amber honey and two model
compared with two model honeys (prepared from a honey sugar blends
blend of the principle sugars found in Light Amber
Stock solution formulation (g/100 g)
honey) over a range of addition levels (0, 5, 7.5, 10, 12.5,
15 and 20 g/100 g). The two factor experimental design Light Amber honey PSB1 PSB2
was replicated three times, giving a total of 63 RVA Light Amber honey 33.3 — —
trials. Fructose — 25.8 20.8
When Light Amber honey is compared to a model Glucose — 18.8 15.2
honey prepared from a mixture of sugars, the unknown Maltose — 1.7 1.3
Water 66.7 53.7 62.6
material in the honey must be replaced with one or more
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164 P.J. Torley et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 161–170

Table 3
The formulation of RVA samples for different addition levels for Light Amber honey and two model honey sugar blends

Addition level (g/100 g) Light Amber (g/100 g) PSB1 (g/100 g) PSB2 (g/100 g)

Honey solids Water Sugar blend solids Water Sugar blend solids Water

0 0.0 90.4 0.0 90.4 0.0 90.4

5 4.2 86.2 3.5 86.9 4.2 86.2
7.5 6.3 84.1 5.2 85.2 6.3 84.1
10 8.3 82.0 6.9 83.4 8.3 82.0
12.5 10.4 80.0 8.7 81.7 10.4 80.0
15 12.5 77.9 10.4 80.0 12.5 77.9
20 16.7 73.7 13.9 76.5 16.7 73.7

demineralised water to produce the target honey or
sugar blend level of addition in the sample canister
(Table 3). At the same level of addition, the PSB1
sample contained the same level of fructose, glucose and
maltose as the Light Amber honey sample, while the
PSB2 sample contained the same total solids as the
Light Amber honey sample.
2.3.2. Experiment 2: comparison of honey types
The objective of this experiment was to compare the
effect of different honey types on starch gelatinization.
The composition of the minor components of honey
varies widely depending on floral source, and this
variation could cause differences in the effect of honey
on starch gelatinization. In this experiment, eight honey
samples (ironbark, Light Amber blend, manuka and
jellybush blend, red gum, stringybark, spotted gum,
yapunyah and yellow box) were compared at four levels
of addition. Each combination of conditions was
repeated three times. A total of 96 RVA trials were
performed.
Since the eight honey samples have a substantial
range of moisture contents (15.9–20.2 g/100 g), their
moisture contents were adjusted during sample prepara-
tion to the equivalent of 16.6 g/100 g moisture (the level
in Light Amber honey). At the same level of addition, all
RVA samples contained the same concentration of
honey solids (2.08, 4.17, 8.34, 12.51 g/100 g honey solids;
equivalent to adding 2.5, 5, 10 and 15 g/100 g of a honey
containing 16.6 g/100 g moisture). The results are pre-
sented as though all honey types contained 16.6 g/100 g
moisture to allow easier comparison with Experiment 1.
In Experiment 2, the method used for sample
preparation and RVA measurement were the same as
those described in Experiment 1 for honey, except that
the stock solutions contained about 35 g/100 g honey
and 65 g/100 g demineralised water.
2.3.3. Experiment 3: pH and honey type
The objective of this experiment was to compare
different honey types over a pH range from about 3 to
about 7. Different honey types naturally differ in pH,
and pH affects the properties of minor components in
honey, such as enzyme activity and the disassociation of
organic acids. Differences between honey types could be
a result of a combination of these affects. In this
experiment, the effect of four honey types (Light Amber
blend, stringybark, spotted gum and yellow box) on
starch gelatinization was studied from about pH 3.0 to
about pH 7.5.
A 12.51 g/100 g honey solids addition level (equivalent
to 15 g/100 g Light Amber honey containing 16.6 g/100 g
moisture) was used in this experiment. Honey stock
solutions were prepared by mixing honey (approxi-
mately 35 g/100 g) and demineralised water (approxi-
mately 65 g/100 g). To obtain a 12.51 g/100 g honey
solids addition level, 16.34 g of honey stock solution was
mixed with demineralised water and either 0.1 mol/l
NaOH or 0.1 mol/l HCl to give a total weight of 35.00 g.
The pH of this solution was measured, and then 25.00 g
was added to the 3.00 g starch in the RVA sample
canister, giving a suspension containing 12.5 g/100 g
honey solids, 9.6 g/100 g starch solids and 77.9 g/100 g
moisture.
2.3.4. Statistical analysis
All experimental data were analysed using the
ANOVA and Regression data analysis tools in Micro-
soft Excel 2000 (Microsoft Corporation, Washington,
USA).
Unless otherwise noted in the text, a Po0:05 level is
used where values are described as being significantly
different.
3. Results
3.1. Honey characteristics
The moisture content, pH and amylase activity of the
eight honey samples used in this study are presented in
Table 4. The moisture content of the honey samples was
within the range of 13.2–20.2 g/100 g previously found
for Australian honeys (Chandler, Fenwick, Orlova, &
Reynolds, 1974). The pH values of the honey samples
varied from 3.95 to 5.03, and were similar to the average
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P.J. Torley et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 161–170 165

Table 4 increases the viscosity of gelatinized corn starch

The origin and characteristics of the eight honey samples analysed in
this study
Honey type Moisture pH
content
(g/100 g)
Ironbark 16.7 4.09
Light Ambera 16.6 4.20
Manuka and jellybush 18.6 3.95
Red gum 15.9 4.12
Spotted gum 20.2 5.03
Stringybark 17.0 4.36
Yapunyah 16.3 4.35
Yellow box 16.2 3.97
a
Honey types in this blend are unknown.
pH values reported by Mossel (2003), where the same
floral varieties were analysed. Spotted gum honey, which
was not analysed by Mossel (2003), had an exceptionally
high pH compared to the other honeys being studied,
though well below the highest value reported for
Australian Eucalyptus honey of pH 6.32 (Chandler
et al., 1974). The average pH of the spotted gum samples
analysed by Chandler et al. (1974) was 4.31. The
amylase activity, expressed as diastase number, of
the honey samples fell in the range 5.0–39.1. Two of
the values (stringybark and spotted gum) were excep-
tionally high (Table 4) compared to the other samples in
the study, though within the range reported for
Australian honeys of 9–44 (Chandler et al., 1974) and
American honeys of 2–62 (White, 1992).
The concentration of fructose, glucose, sucrose and
maltose in the Light Amber honey sample was
determined by HPLC (Table 1). Generally, the levels
of sugars found corresponded to the average levels
reported by Mossel (2003), except for sucrose, which
was present at a very low level.
3.2. Experiment 1: Light Amber honey and pure sugar
blends
The sugar source (Light Amber, PSB1 and PSB2) and
level of addition both affected starch gelatinization
properties (Fig. 1); however, there was a significant
interaction between sugar source and level of addition
for only some RVA parameters (pasting temperature
and peak, final and setback viscosities). Since the
effect of sugar source and level of addition produced a
similar pattern of results for final and setback viscosi-
ties, only the results for peak and final viscosities are
presented in Fig. 1.
Not surprisingly, there were significant differences
between PSB1 and PSB2 for many treatment combina-
tions, particularly at higher levels of addition (Fig. 1).
When the starch concentration is maintained at a
constant level, increasing the sugar concentration
(Schirmer, Toledo, & Reyes, 1986), and the differences
reflect the effect of the higher concentration of sugars
Diastase present in PSB2 samples than PSB1 samples (Fig. 1).
number The difference in concentration between PSB1 and PSB2
had a smaller effect on the pasting temperature
11.9 (significant difference only at 20 g/100 g level of addi-
16.3 tion) or peak viscosity temperature (only a significant
5.0 difference as a main effect, not as an interaction between
17.1
concentration and sugar source).
26.4
39.1 When Light Amber is compared to the pure sugar
14.3 blends it is apparent that the minor components might
11.5 have a significant effect on starch gelatinization. When
the unknown material in Light Amber was replaced by
pure sugars (comparison between Light Amber and
PSB2), Light Amber gave a lower viscosity than PSB2
for all of the RVA viscosity parameters (Fig. 1). At all
addition levels when compared to PSB2, Light Amber
had a significantly lower peak viscosity, final viscosity
(except at 12.5 g/100 g) and setback viscosity. By
contrast, Light Amber had a consistently higher pasting
temperature than PSB2 at all levels of addition, though
the difference was only significant at the 10 g/100 g
addition level.
There was a more complicated relationship when the
unknown material was replaced with water (comparison
between Light Amber and PSB1). Light Amber viscosity
parameters were significantly lower than for PSB1 at
lower concentrations (peak and final viscosities at 7.5 g/
100 g addition level; setback viscosity at 5–12.5 g/100 g),
but significantly higher at higher concentrations (peak
viscosity at 15 g/100 g level and final and setback
viscosities at 20 g/100 g level). As with PSB2, the pasting
temperature of Light Amber was consistently higher
than PSB1, though the difference was only significant at
the 20 g/100 g level of addition.
The only RVA parameter where Light Amber fell
between PSB1 and PSB2 was breakdown. While both
sugar source and addition level were highly significant
(Po0:0001) as main effects, their interaction was not
significant (P ¼ 0:1501). There was a significant differ-
ence in breakdown between Light Amber and PSB2, and
PSB1 and PSB2.
The unknown material present in Light Amber honey
is a complex mixture including minor di- and tri-
saccharides, organic acids, enzymes, ash and phenolic
compounds (White, 1992; Gheldof et al., 2002). If the
unknown material had little effect on starch gelatiniza-
tion, Light Amber would be expected to lie between
PSB1 and PSB2, as was seen with breakdown (Fig. 1).
However, for most RVA parameters Light Amber was
significantly lower than both PSB1 and PSB2, particu-
larly at lower levels of addition. Thus, the composition
and concentration of the unknown material is high
enough that they can affect the whole starch gelatiniza-
tion process, from initial gelatinization of the starch
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5500 5500

5000 5000

Final Viscosity (cP)

Peak Viscosity (cP)
4500 4500

4000 4000

3500 3500

3000 3000

2500 2500
0 5 10 15 20 0 5 10 15 20
Level of Addition (g/100 g) Level of Addition (g/100 g)

2400 85

Pasting Temperature ( C)
2200

o
Breakdown (cP)

2000
80
1800

1600
75
1400

1200

1000 70
0 5 10 15 20 0 5 10 15 20
Level of Addition (g/100 g) Level of Addition (g/100 g)
Fig. 1. The effect of the level of addition of Light Amber honey or blends of pure sugars equivalent to honey on the gelatinization of maize starch.
Least significant difference (P ¼ 0:05 level): peak viscosity, 78 cP; final viscosity, 195 cP; breakdown, 102 cP; pasting temperature, 1.3
C. Legend:
, Light Amber honey; &, PSB1; n, PSB2.

(pasting temperature) to the increase in viscosity on Six of the eight honey types studied produced similar
cooling (final and setback viscosities). effects on starch gelatinization. Increasing levels of
The composition of honey varies widely, with addition of ironbark, Light Amber, manuka and jelly-
substantial variation seen in the levels of the major bush, red gum, yapunyah and yellow box samples all
components (fructose, glucose, sucrose, maltose and gave similar changes in RVA viscosity and temperature
water) as well as the minor components present (White, parameters (Fig. 2), though there were some significant
1992; Gheldof et al., 2002). Depending on the origin of differences between honey types particularly at the
the effect of honey on starch gelatinization (in addition higher levels of addition. In general, these six honeys
to the established effect of the major sugars present), showed an increase in all viscosity parameters with
different honey types may produce similar or very increasing levels of addition.
different effects on starch gelatinization properties. The other honey types (stringybark and spotted gum)
showed a very different response to increasing levels of
3.3. Experiment 2: comparison of honey types addition. Stringybark and spotted gum gave atypical
results, when compared to either a blend of pure sugars
Different honey types have large effects on starch (Fig. 1) or the other honey types investigated (Fig. 2). At
gelatinization properties over a range of levels of addition levels of 5 g/100 g or higher, stringybark and
addition (Fig. 2). There was a highly significant spotted gum gave significantly lower peak, final and
interaction between honey type and level of addition setback viscosities, and peak viscosity temperature
for pasting temperature (P ¼ 0:0062) and very highly values than the other honey types studied. Notably,
significant interactions for all other RVA parameters each incremental increase in level of spotted gum honey
(Po0:0001). Since the effect of honey type and level of produced a significant decrease in final and setback
addition produced a similar pattern of results for viscosities.
trough, final and setback viscosities, only the results Regression analysis showed that there was a signifi-
for final viscosity are presented in Fig. 2. cant negative correlation between amylase activity and
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P.J. Torley et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 161–170 167

5000 5000

4500 4500

Final Viscosity (cP)

Peak Viscosity (cP)
4000 4000

3500 3500

3000 3000

2500 2500

2000 2000
0 5 10 15 0 5 10 15
Level of Addition (g/100 g) Level of Addition (g/100 g)

2400 100

Peak Visocosity Temp. ( C)

2200

o
Breakdown (cP)

2000
95
1800

1600
90
1400

1200

1000 85
0 5 10 15 0 5 10 15
Level of Addition (g/100 g) Level of Addition (g/100 g)
Fig. 2. The effect of the level of addition and honey type on the gelatinization of maize starch. Least significant difference (P ¼ 0:05 level): peak
viscosity, 73 cP; final viscosity, 149 cP; breakdown, 103 cP; peak viscosity temperature, 0.7
C. Legend: , control; B, ironbark; J, Light Amber; &,
manuka and jellybush; m, red gum; ’, stringybark; ~, spotted gum; n, yapunyah;  , yellow box.

peak viscosity (P ¼ 0:0172; R ¼ 0:80), as well as honey type. All honey types had similar effects on
amylase activity and peak viscosity temperature peak, trough, final and setback viscosities up to about
(P ¼ 0:0295; R ¼ 0:76). That is, as amylase activity pH 4.0 (Fig. 3, results for trough viscosity not shown).
increased peak viscosity and peak viscosity temperature Between about pH 4.0 and 5.0, the peak, trough,
decreased. Examination of the residuals found that final and setback viscosities all decreased with in-
the data from the spotted gum sample stood out as creasing pH, though the extent of the decrease in
having very large residuals for most RVA parameters. viscosity varied with honey type. Above pH 5.0,
When spotted gum honey data were excluded from peak, trough, final and setback viscosities all remained
the analysis, there was a significant negative correla- largely constant with increasing pH, though the actual
tion between amylase activity and peak viscosity viscosity differed substantially between the different
(P ¼ 0:0007; R ¼ 0:96), peak viscosity temperature honey types.
(P ¼ 0:0034; R ¼ 0:92), final viscosity (P ¼ 0:0028; Breakdown also showed variation with honey type
R ¼ 0:93) and setback viscosity (P ¼ 0:0262; and pH; however, the response was opposite, with, for
R ¼ 0:81). Similar correlations were also found example, stringybark honey producing the highest
between pH and the RVA parameters. Since the amylase breakdown, but lowest peak, trough and final viscosities
activity and pH values of the honey samples used in this (Fig. 3).
study were highly positively correlated with each other Pasting temperature was not affected by either honey
(P ¼ 0:0007; R ¼ 0:93), it is not possible to separate type or pH (results not shown), while variation in pH
their effects on starch gelatinization. produced marked differences in peak temperature
depending on honey type (Fig. 3), with the trends
3.4. Experiment 3: pH and honey type following a broadly similar pattern to the viscosity
parameters.
The effect of varying the pH of the honey solution on When the correlation between RVA parameters and
starch gelatinization varied markedly depending on the amylase activity was examined at about the pH
 ARTICLE IN PRESS
168 P.J. Torley et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 161–170

5000 5000

4500

Final Viscosity (cP)

Peak Viscosity (cP)
4500
4000

3500
4000
3000

2500
3500
2000

3000 1500
2.5 3.5 4.5 5.5 6.5 7.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5
pH pH

2500 100

Peak Viscosity Temperature ( C)

o
2300
Breakdown (cP)

95
2100

1900
90

1700

1500 85
2.5 3.5 4.5 5.5 6.5 7.5 8.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5
pH pH
Fig. 3. The effect of pH and honey type on the gelatinization of maize starch. Legend:  , Light Amber; &, stringybark ; n, spotted gum; B, yellow
box.

5000 100 positive correlation between amylase activity and break-

down viscosity (P ¼ 0:0360; R ¼ 0:96).
Peak Viscosity Temperature (oC)

4000

95
Viscosity (cP)

3000 4. Discussion

The effect of honey on starch gelatinization may be

2000
1000
0 85
5 10 15 20 25
Diastase Number
Fig. 4. The relationship between diastase number and RVA char-
acteristics for Light Amber, stringybark, spotted gum and yellow box
honeys. Legend: n, peak viscosity; B, peak viscosity temperature; &,
final viscosity; J, setback;  , breakdown.
optimum (pH 5.3–5.6; Babacan et al., 2002) for honey
amylase activity (Fig. 4), significant negative correla-
tions were found for peak, final and setback viscosities,
and for peak viscosity temperature (P in the range
0.0087–0.0478, R in the range 0.95 to –0.99) and a
due to a number of factors including the concentration
90
of added sugars, amylase activity and the minor
constituents present in the honey. Increasing the
concentration of sugars in the heating medium produced
an increase in starch viscosity and pasting temperature
(Fig. 1). However, the effect of Light Amber honey
30 35 40 45 differed significantly from those of the blend of pure
sugars, indicating that the effect of honey on starch
gelatinization is not simply a result of adding sugar.
When the effect of different honey types on starch
gelatinization was examined, large differences between
honey types were observed. Six of the eight honey types
examined had similar effects on starch gelatinization,
while two (stringybark and spotted gum) showed
marked differences, in particular lowering peak, final
and setback viscosities, and peak viscosity temperature
(Fig. 2). The high positive correlation between honey
pH and amylase activity (R ¼ 0:93) meant that their
effects on starch gelatinization could not be isolated.
 ARTICLE IN PRESS
P.J. Torley et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 161–170
When the pH of the honey solutions was varied by
adding NaOH or HCl, the effect on starch gelatinization
depended on honey type and the pH range being studied
(Fig. 3). Increasing the pH from about 3.0 to 4.0 resulted
in an increase in starch viscosity, while further increas-
ing the pH from 4.0 to 5.5 resulted in a decrease in
starch viscosity. Once the minimum starch viscosity level
was achieved at about pH 5.5, further increases in pH
did not see any increase in starch viscosity.
The effect of pH on starch viscosity may be a result of
the combination of starch viscosity increasing naturally
with increasing pH (Freeman & Verr, 1972) and honey
amylase activity (Babacan et al., 2002). The viscosity of
maize starch cooked to 95
C increases with increasing
pH to a maximum at pH 5–6 (Freeman & Verr, 1972).
The honey amylase activity is at a maximum at pH 5.3–
5.6, with no amylase activity at pH 3.6, and approxi-
mately 24% of peak amylase activity at pH 7.1
(Babacan et al., 2002). Up to pH 4.0, the increase in
viscosity with increasing pH may dominate over the
decrease in viscosity produced by the relatively low
honey amylase activity. Conversely, above pH 4.0, the
decrease in viscosity caused by increasing honey amylase
activity may dominate over the increase in starch
viscosity produced by an increase in pH.
Above pH 5.5, changes in viscosity do not seem
consistent with an explanation based on honey amylase
activity (Babacan et al., 2002) and the effect of pH on
starch viscosity (Freeman & Verr, 1972). An increase in
pH should lead to a decrease in honey amylase activity,
resulting in the viscosity of the four honey types (Light
Amber, spotted gum, stringybark, yellow box) conver-
ging, in the corollary of the divergence between honey
types seen between pH 4.0 and 5.5. The reason that the
viscosity of the four honey types remains constant above
pH 5.5 is not clear. Since the pH of the honey solution
was adjusted before it was added to the starch, the
action of amylase on starch during the time taken to
adjust the pH can be discounted. However, variation in
the heating medium pH has been shown to have a
complex effect on maize starch gelatinization depend-
ing on the type of starch and temperature (Freeman &
Verr, 1972; Schirmer et al., 1986), and limited manu-
facturers’ trials with the type of maize starch used in
this study have also shown that pH affects its vis-
cosity during gelatinization (Wilkinson, 2003, personal
communication).
Variation in honey type, pH and level of addition had
a similar effect on peak viscosity temperature as they did
on peak and final viscosities (Figs. 2 and 3). As observed
for peak and final viscosities, six of the eight honey types
studied had similar peak viscosity temperature, while
stringybark and spotted gum showed markedly lower
peak viscosity temperatures (Fig. 2). When the pH of the
four honey types was adjusted in Experiment 3, a similar
pattern of changes was produced in peak viscosity
169
temperature and peak and final viscosities (Fig. 3). The
peak viscosity represents the point where the increase in
viscosity produced by the swelling of the starch granule
and leaching of amylose from the granule, is balanced
by the loss of viscosity caused by granule rupture
(BeMiller & Whistler, 1996). The decrease in peak
viscosity temperature could be a result of the honey
amylase activity attacking the amylopectin in the starch
granule surface (Franco, Ciacco, & Tavares, 1998),
making it more susceptible to rupture, causing the
viscosity to reach a maximum at a lower temperature.
Part of the effect of pH on starch gelatinization may
also be explained by differences in the composition and
concentration of acids present in different honey types.
Titration curves show differences between the four
honey types, with greater buffering capacity at lower
pH (Fig. 5). Low-molecular-weight organic compounds
affect starch gelatinization (D’Appolonia, 1972; Deffen-
baugh & Walker, 1990; Singh, Singh, & Saxena, 2002),
increasing or decreasing starch viscosity. Possibly, the
exact composition of the organic compounds present, as
well as the charge on the acids, contributed in
combination with amylase activity to the differences
between honey type, and the difference between the
blend of pure sugars and Light Amber honey. Phenolic
compounds present in the honey (Andrade, Ferreres,
Gil, & Tomas-Barberan, 1997) may also modulate the
properties of the honey amylase (Rohn, Rawel, & Kroll,
2002). Further studies with amylase inhibitors would
help clarify how honey affects starch gelatinization, in
particular the absence of an increase in starch viscosity
above pH 5.5.
4
NaOH or HCl Addition (g/100 g)
2
0
-2
-4
-6
-8
-10
2.5 3.5 4.5 5.5 6.5 7.5 8.5
pH
Fig. 5. The effect of adding 0.1 mol/l NaOH (positive values) or
0.1 mol/l HCl (negative values) on the pH of four honey solutions.
Legend:  , Light Amber; &, stringybark; n, spotted gum; B, yellow
box.
 ARTICLE IN PRESS
170 P.J. Torley et al. / Lebensm.-Wiss. u.-Technol. 37 (2004) 161–170
5. Conclusions
This study shows that the unknown material present
in honey (e.g. minor sugars, enzymes, organic acids)
affect the gelatinization of unmodified maize starch,
with the effect of the minor components depending on
the honey type, addition level and pH. The importance
of the unknown material in honey was demonstrated
when Light Amber honey was compared to mixtures of
the main sugars found in honey (fructose, glucose and
maltose). Light Amber honey responded in a different
manner (Fig. 1) indicating that the unknown material
(14.0 g/100 g of the Light Amber honey sample)
influenced starch gelatinization. When eight honey types
were compared, large differences between honey types
were observed, in particular spotted gum and stringy-
bark honey produced large reductions in starch peak,
final and setback viscosities compared to the other
honey types studied (Fig. 2). This is consistent with the
unknown material affecting starch gelatinization, since
the composition of the unknown material of honey
varies between honey types.
It appears that the amylase activity present in honey is
responsible for some of the differences in starch
gelatinization properties, with significant correlations
found between amylase activity and RVA viscosity
parameters. However, the effect of the unknown
material is not straightforward, since increasing the
pH of the honey samples from pH 4.0 to 5.5 resulted in a
reduction in starch viscosity, consistent with the increase
in honey diastase activity over this pH range. Above pH
5.5, honey amylase activity decreases; however, there
was no corresponding increase in starch viscosity. This
may be due to other unknown material present in honey
affecting starch gelatinization or amylase activity.
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