Article

Thermosonication and optimization of stingless

bee honey processing

KY Chong, NL Chin and YA Yusof

Abstract
The effects of thermosonication on the quality of a stingless bee honey, the Kelulut, were studied using
processing temperature from 45 to 90  C and processing time from 30 to 120 minutes. Physicochemical
properties including water activity, moisture content, color intensity, viscosity, hydroxymethylfurfural content,
total phenolic content, and radical scavenging activity were determined. Thermosonication reduced the water
activity and moisture content by 7.9% and 16.6%, respectively, compared to 3.5% and 6.9% for conventional
heating. For thermosonicated honey, color intensity increased by 68.2%, viscosity increased by 275.0%, total
phenolic content increased by 58.1%, and radical scavenging activity increased by 63.0% when compared to
its raw form. The increase of hydroxymethylfurfural to 62.46 mg/kg was still within the limits of international
standards. Optimized thermosonication conditions using response surface methodology were predicted at
90  C for 111 minutes. Thermosonication was revealed as an effective alternative technique for honey
processing.

Keywords
Honey quality, stingless bee honey, thermosonication, thermal processing, physicochemical properties
Date received: 2 March 2017; accepted: 2 May 2017

INTRODUCTION content, raw Kelulut honey is inclined to ferment and

Malaysia is a tropical country rich in various types of
honey such as Acacia, Pineapple, Borneo, Gelam,
Kelulut, and Tualang honeys. These honeys can be clas-
sified broadly into Apis mellifera (honey bee) honey and
stingless bee honey. The stingless bee, Trigona spp.,
produces the Kelulut honey. It is a multifloral honey
stored in pots made of cerumen. In comparison with
the Apis mellifera honey, the stingless bee honey has
higher antioxidant activity, flavonoids, and polyphenol
content (Kek et al., 2014; Rodrı́guez-Malaver, 2013).
Other distinctive characteristics of the stingless bee
honeys include higher moisture content, higher elec-
trical conductivity, higher acidity, higher protein con-
tent, and lower diastase activity (Chuttong et al., 2016;
Kek et al., 2017). As a result of its high moisture
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crystallize. This results in quality deterioration in terms
of acidic taste and undesirable appearance. As the pres-
ence of osmophilic yeast and fungi in honey is inevit-
able during bees’ foraging process, honey needs to be
processed to extend its shelf life.
Industrial practices commonly use thermal pro-
cessing to liquefy and pasteurize honey at 45  C and
80  C, respectively (Escriche et al., 2009). However,
the use of high temperatures caused undesirable
changes in various honey properties such as decrease
in enzymes, increase in hydroxymethylfurfural (HMF)
content, rapid loss of antioxidant activity, darkening
of honey, and damage to flavor (Nagai et al., 2001;
Department of Process and Food Engineering, Faculty of
Engineering, Universiti Putra Malaysia, Selangor, Malaysia
Corresponding author:
NL Chin, Department of Process and Food Engineering, Faculty of
Engineering, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, Malaysia.
Email: chinnl@upm.edu.my
Food Science and Technology International 0(0)
White Jr, 1978; White et al., 1964). Ultrasonication is
one of the non-thermal technologies explored for min-
imal processing of food owing to its ability to inactivate
microorganisms and promote dehydration in foods
(Ahmed et al., 2009; Musielak et al., 2016). In addition,
ultrasound treatment can preserve and improve nutri-
tional values and organoleptic properties of food (Abid
et al., 2013; Chaikham and Prangthip, 2015; Chemat
et al., 2011). In an ultrasound treatment, the liquid sub-
jected to ultrasound waves is alternately compressed
and expanded, forming microbubbles or cavities. The
collapse of bubbles leads to intense local heating or hot
spots, and this rapidly changes temperature and pres-
sure in its surrounding medium (Ahmed et al., 2009;
Suslick, 1989). Consequently, high energy shear waves
and turbulence are produced in the cavitation zone
(Cárcel et al., 2012) that drives physical and chemical
reactions in the liquid. Thermosonication is a process
that combines heat and ultrasound waves. As ultra-
sound waves alone may not be very effective in destroy-
ing microorganisms unless very high intensities are used
(Ahmed et al., 2009), thermosonication is a potential
alternative processing method to enhance destruction
of microorganisms. The effects of thermosonication
were observed on different food such as watercress
(Cruz et al., 2007), beer (Milani et al., 2016), milk
(Noci et al., 2009), and fruit juices (Rawson et al.,
2011; Wu et al., 2008).
The objective of this study was to integrate ultra-
sound waves and conventional heating to create a ther-
mosonication effect as a technique to improve storage
quality of raw Kelulut honey as it is mostly commercia-
lized in its raw or unprocessed form. The honey quality
attributes of water activity, moisture content, color
intensity, viscosity, HMF content, total phenolic con-
tent (TPC), and radical scavenging activity (RSA) were
compared to those treated using conventional thermal
processing. The optimum processing conditions with
respect to thermosonication temperature and time
were also determined using response surface method-
ology (RSM).
MATERIALS AND METHODS
Honey samples
Fresh Kelulut honey of the same batch was obtained
from a forest reserve in Teluk Intan, Perak, Malaysia
in January 2016. Freshly harvested samples were stored
at 4  C for experiments. Twenty grams of honey were
filled into test tubes with 25 mm diameter and 150 mm
height for thermal processing in the first six weeks and
thermosonication in the next six weeks. Honey was
removed from the chiller to equilibrate with room tem-
perature before experiments commenced.
2
Thermosonication processing
An ultrasonic bath tank at 25 kHz powered by piezo-
electric flange mounted type transducers (Branson
Ultrasonics Co., Danbury, CT, USA) at 2.5 kW (Chin
et al., 2013) was fitted with heating element of 6 kW and
insulation for a thermosonication effect. A test tube
rack was suspended in the middle of the tank to hold
the test tubes containing honey. Thermosonication
treatment of honey was conducted at 45.0, 55.0, 67.5,
80.0, and 90.0  C where this range of temperatures
included the liquefaction and pasteurization process
(Escriche et al., 2009; Gonnet, 1977) for 30, 50, 75,
100, and 120 minutes. Water temperature was recorded
using a K-type thermocouple connected to a data
logger thermometer (CENTER 309, CENTER
Technology Corp., Taiwan). A control experiment to
demonstrate the effects of conventional thermal pro-
cessing on honey was conducted using a thermostatic
water bath (WNB 22, Memmert GmbH þ Co. KG,
Germany) with same temperatures and times.
Figure 1 illustrates the top view for the set-up of the
thermosonic bath system and thermostatic water bath.
Quality analysis of honey
Water activity. Water activity was determined using a
water activity meter (Aqualab Series 3, Decagon
Devices, USA) at room temperature.
Moisture content. Moisture content of honey was
determined using Bogdanov et al.’s (2002) method with
slight modifications. The refractive index of honey was
determined using a digital refractometer (Digital ABBE
Refractometer AR2008, A. Krüss, Germany) at 20  C. If
temperature differed from 20  C, 0.00023 per  C was
added or subtracted from the refractive index. The
refractive index was converted to moisture content by
means of equation (1) (Sesta and Lusco, 2008)
Moisture content ð%Þ
ð1Þ
¼ ½0:2681  logðRI  1Þ=0:002243
where RI is the refractive index.
Color intensity. Color intensity of honey was deter-
mined using a UV/Visible spectrophotometer
(Ultrospec 3100 pro, Amersham Biosciences, USA) fol-
lowing methods of Beretta et al. (2005). A 50% (w/v)
honey solution was prepared with warm distilled water
(45–50  C) and filtered to remove any coarse particles.
Using distilled water as blank, the absorbance at
450 nm and 720 nm was recorded. Color intensity was
calculated using equation (2).
Color intensity ðmAUÞ ¼ A450  A720 ð2Þ

(a) 4 5
2 2
1
3
635 mm
Figure 1. Top view schematic diagram of the experimental set-up for (a) thermosonication and (b) conventional thermal
processing: (1) side ultrasonic generator, (2) bottom ultrasonic generator, (3) steel rack containing test tubes, (4) heaters,
(5) insulation for ultrasonic tank, and (6) test tube holder.
where A450 and A720 are the absorbance at 450 nm and
720 nm, respectively.
Flow behaviour. A rheometer (AR-G2, TA
Instruments, New Castle, USA) with a 60-mm diameter
cone and plate geometry (30 mm truncation gap, one-
degree cone angle) was used to determine the dynamic
viscosity of honey. Honey samples were not preheated
as temperature would affect viscosity readings. Steady-
state measurements were performed at 20  C, with a
shear rate range of 1–1000 s1 in ascending ramp. The
temperature was controlled by a circulating water
system. Dynamic viscosity was obtained from the aver-
age of 20 points. Data were analyzed using its accom-
panying computer software, Rheology Advantage Data
Analysis Version 5.7.
HMF content. HMF content was determined using
White’s spectrophotometric method (White,
1979). Five grams of honey samples was diluted up to
50 mL with distilled water after addition of 0.5 mL of
Carrez I solution (consisting 15 g of potassium hexacya-
noferrate (II), K4Fe(CN)6.3H2O in 100 mL water) and
0.5 mL of Carrez solution II (consisting 30 g of zinc
acetate, Zn(CH3.COO)2.2H2O in 100 mL of water).
The solution was then filtered and the first 10 mL of
the filtrate was rejected. Aliquots of 5 mL each were
transferred to two test tubes. Five milliliters of distilled
water was added to the first tube as sample and
another 5 mL of 0.2% (w/v) sodium bisulphite solution
was added to the second test tube as reference.
Absorbance of sample solution was determined against
reference solution at 284 nm and 336 nm in quartz
Chong et al.
(b)
6
235 mm
595 mm
1
320 mm
cuvettes. HMF was determined using equation (3)
(White, 1979).
HMF ðmg=kg honeyÞ
ð3Þ
¼ ðA284  A336 Þ  149:7  5=W
where A284 and A336 denote the absorbance at 284 nm
and 336 nm, respectively, the factor 149.7 is a theoret-
ical value linked to the molar extinction coefficient
of HMF at 284 nm, and W is the weight of honey
sample in g.
Total phenolic content. TPC was determined using the
Folin-Ciocalteu spectrophotometric method (Singleton
et al., 1999). Minor modification was made by diluting
honey to 0.05 g/mL with distilled water. One milliliter
of honey solution was mixed with 5 mL of Folin-
Ciocalteu reagent (diluted 1:10 with distilled water).
After 5 minutes of incubation, 4 mL of 7.5% (w/v)
aqueous sodium carbonate solution was added. The
mixture was well mixed and incubated for 2 hours in
dark at room temperature. The absorbance of the mix-
ture was determined at 765 nm using distilled water as
blank. A standard calibration curve was plotted using
gallic acid with concentration range from 10 to 100 mg/
mL. TPC was expressed in mg of gallic acid equivalent
(GAE) per kg of honey. Measurements were performed
in triplicates.
Radical scavenging activity. Radical scavenging activ-
ity was determined by using 2,2-diphenyl-1-picrylhy-
drazyl (DPPH) as a stable radical purchased from
Sigma-Aldrich (USA). Combining the method of
3
Food Science and Technology International 0(0)

Chaikham and Prangthip (2015) and Molyneux (2004),
0.5 g of honey was added with 10 mL of methanol. The
solution was centrifuged at 4000 r/min for 15 min
(Universal 320, Hettich, USA). Two milliliters of super-
natant were mixed with 2 mL of 0.1 mM DPPH radical
methanol solution. The mixture was mixed well and
incubated in the dark for 30 min at room temperature.
Absorbance was immediately measured at 517 nm using
a UV/Vis spectrophotometer. Methanol was used as
blank and a control was prepared using 2 mL methanol
instead of the honey solution. The radical scavenging
activity was calculated using equation (4)
DPPH radical scavenging activity ð%Þ
ð4Þ
¼ ½1  ðAs =Ac Þ  100%
where As and Ac are the absorbance of sample and
control, respectively.
Experimental design for optimization
A comprehensive design of experiment was made to
encompass both full factorial experiments and RSM
test conditions. Response surface methodology was
used to optimize the processing conditions using
Minitab Statistical Software (Version 16, Minitab
Inc., USA). A face-centered central composite design
was used to evaluate the effect of two independent vari-
ables, T (temperature) and t (time) on seven response
variables, Y1–Y7, namely water activity, moisture con-
tent, color intensity, viscosity, HMF content, TPC, and
RSA of honey. The independent variable levels studied
for optimization were temperature, namely 45.0, 67.5,
90.0  C and time, 30, 75, 120 min. The design consisted
of 13 sets of test conditions including five replicates of
the center point. The behavior of the response surface
was fitted using second-order polynomial model equa-
tion (5)
Yi ¼ 0 þ 1 T þ 2 t þ 11 T2 þ 22 t2 þ 12 Tt ð5Þ
where Yi is the predicted response, 0 is a constant
coefficient, 1 and 2 are coefficients for linear effect
terms, 11 and 22 are coefficients for quadratic terms,
and 12 is the coefficient for interaction terms. T and t
are the uncoded independent variables.
Statistical analysis
The experiments were performed in triplicates, and
results were expressed as mean  standard error.
Error bars in graphs are standard error of means.
One-way analysis of variance (ANOVA) was used to
test for significant difference between means using
Tukey’s multiple comparison test. Two-way ANOVA
was used to test significant effects of the model.
RESULTS AND DISCUSSION
Water activity and moisture content
Figure 2 shows that the maximum thermosonication
and thermal processing of 90  C for 120 minutes
reduced water activity to 0.743 and 0.767, respectively.
It was a reduction of 7.9% and 3.5% prior to thermo-
sonication and thermal processing, respectively from

(a) 0.820 (b) 0.820

0.800 0.800

0.780 0.780
Water activity

Water activity

0.760 0.760

0.740 30 min 0.740 30 min

50 min 50 min
75 min 75 min
100 min 100 min
120 min 120 min
0.720 0.720
45.0 55.0 67.5 80.0 90.0 45.0 55.0 67.5 80.0 90.0
Temperature (°C) Temperature (°C)

Figure 2. Effect of temperature and time on water activity using (a) thermosonication and (b) thermal processing. Black
dashed line indicates values for raw honey. Values on and beyond the gray dotted lines indicate significant differences
(P < 0.05) compared to raw honey.

4

the raw honey sample having average water activity of
0.807 and 0.795.
The water activity values for Kelulut honey reduced
substantially compared to its non-treated form despite
still exceeding the level of 0.6, where below 0.6, there
will be no microbial proliferation (Fontana Jr et al.,
2008). Australian stingless bee honey recorded an aver-
age water activity of 0.74. In contrast, water activity of
other types of honey was lower. Greek honeydew and
floral honey ranged from 0.528 to 0.663 (Lazaridou
et al., 2004), while Slovenian honeydew honey and
floral honey’s mean water activity were 0.528 and
0.521, respectively (Abramovič et al., 2008). Figure 3
shows that moisture content was significantly reduced
by 16.6% to 25.9% moisture using thermosonication as
compared to the conventional thermal processing by
6.9% to 28.8% moisture.
The relatively high moisture content of raw stingless
bee honey is a common phenomenon and poses prob-
lems to its storage and shelf life. Besides Kelulut from
Malaysia, stingless bee honey from other countries such
as Thailand (31%) (Chuttong et al., 2016), Venezuela
(14.8% to 30.2%) (Vit et al., 1994), Brazil (23.4 to
33.1%) (Silva et al., 2013), and Ecuador (34.1%)
(Guerrini et al., 2009) were reported to have high mois-
ture contents. The higher reduction of water during
thermosonication treatment can be attributed to mech-
anical effects of collapsing microbubbles which increase
mass transfer. Intense shock wave is produced as honey
rapidly rushes into the volume previously occupied by
the bubble, resulting in enormous shear forces (Mason,
1991). At moderate temperatures of 45  C and 55  C,
water activity and moisture content increased.
(a) 34
33
32
Moisture content (%)
31
30
29
28
27
30 min
26 50 min
75 min
25 100 min
120 min
24
45.0 55.0 67.5 80.0
Temperature (°C)
Figure 3. Effect of temperature and time on moisture content using (a) thermosonication and (b) thermal processing.
Black dashed line indicates values for raw honey. Values on and beyond the gray dotted lines indicate significant
differences (P < 0.05) compared to raw honey.
Chong et al.
This may be a result of honey fermentation at moderate
temperatures. It is known that ultrasonication has the
potential to inactivate microbes and yeast. However,
the inactivation is influenced by temperature, ultra-
sound frequency and power, exposure time, type of
microorganism, volume of food to be processed, and
composition of the food. Bermúdez-Aguirre and
Barbosa-Cánovas (2012) showed that low temperatures
such as 40  C did not have any effect on yeast inactiva-
tion in fruit juices but at 60  C, highest inactivation was
achieved. Thermosonication of apple juice from 20 to
60  C resulted in decreasing yeast count with increasing
temperature but only achieved complete yeast inactiva-
tion at 60  C (Abid et al., 2014). Similar results were
demonstrated by Saccharomyces cerevisiae in orange
juice (Ganesan et al., 2015). Thus, there is a possibility
of yeast presence at 45  C and 55  C. Yeast ferments
glucose, fructose, and sucrose strongly to produce
carbon dioxide, alcohol, and small quantities of acid
(Marvin et al., 1931). At these temperatures, the alco-
hol and acid could not evaporate and this may contrib-
ute to increment in moisture. Another possibility of the
moisture increase is from thermal degradation of sugars
by Maillard reaction. This complex reaction involving
different compounds and pathways produces water as a
byproduct at its initial condensation stage. This reac-
tion varies according to product composition and pro-
cessing conditions. There were many sugar systems
studied over a wide range of temperatures, but none
was reported for honey yet (Turkmen et al., 2006; van
Boekel, 2001). Caramelization is excluded as a reason
for the increase in moisture as the two main monosac-
charides in honey, glucose and fructose, melt at
(b) 34
33
32
Moisture content (%)
31
30
29
28
27
30 min
26 50 min
75 min
25 100 min
120 min
24
90.0 45.0 55.0 67.5 80.0 90.0
Temperature (°C)
5
Food Science and Technology International 0(0)
temperatures of 165  C and 120  C, respectively (Örsi,
1973). As the processed Kelulut honey surpassed
the maximum moisture content allowed of 20%
(Alimentarius, 2001) and water activity of 0.6, it is
favorable for osmophillic yeasts and fungi growth. As
the maximum moisture content limit of 20% referred to
honey from Apis mellifera species, Vit et al. (2004) pro-
posed that the moisture content for stingless bee honey
should be raised to 30%. Both thermosonication and
thermal processing reduced moisture content to less
than 30% with note of higher efficiency in the thermo-
sonication method.
Color intensity
Figure 4(a) shows that there was a steady increase in the
color intensity of honey that was subjected to thermoso-
nication at higher temperatures of 67.5, 80.0, and 90  C,
with the darkest reading at 765 mAU. Thermosonication
of honey at lower temperatures of 45  C and 55  C
has reduced its color intensity which was also more
intense with longer thermosonification time.
Honey color is an important characteristic as it is
linked to consumer acceptance of the product. In
Germany, Austria, and Switzerland, consumers espe-
cially appreciated dark honeydew honeys (Bogdanov,
et al., 2004). Dark honey is also beneficial as antioxi-
dant activities of Malaysian honey were reported to
increase with its color intensity as indicated by strong
positive correlations (Kek et al., 2014; Moniruzzaman
et al., 2013).
The darker color change of thermosonicated honeys
is mostly attributed to both simultaneous heat and
(a) 800
30 min
50 min
75 min
100 min
700 120 min
Color intensity (mAU)
600
500
400
300
45.0 55.0 67.5 80.0
Temperature (°C)
Figure 4. Effect of temperature and time on color intensity using (a) thermosonication and (b) thermal processing. Black
dashed line indicates values for raw honey. Values on and beyond the gray dotted lines indicate significant differences
(P < 0.05) compared to raw honey.
6
cavitation effects during sonication where chemical
reactions are accelerated and diffusion rates increased
(Sala et al., 1995; Tiwari et al., 2008a). The lightness in
honey color using thermosonication at 45  C and 55  C
is similar to other sonication studies. Rawson et al.
(2011) found that thermosonication of watermelon
juice at 35  C and 45  C resulted in a significant increase
of lightness value. Tiwari et al. (2010) reported lightness
in red grape juice when compared to the control for all
treatment times at constant temperature of 25  C. The
initial increase in lightness value was attributed to the
partial precipitation of unstable suspended particles fol-
lowed by a decrease as a result of oxidative darkening.
Similarly, Tiwari et al. (2008) found that lightness
values of sonicated orange juice increased with 40%
amplitude level and then decreased when the levels
were increased beyond 40%. Lightness was mainly
affected by sonication time and was most likely because
of increased exposure of color pigments in orange juice
to high shear forces from collapsing bubbles. Zhao
et al. (2006) reported a carotenoid pigment, (all-
E)-astaxanthin, that was degraded to unidentified col-
orless compounds when an ultrasonic probe system was
used and the degradation was higher with increased
treatment time and ultrasonic power. However,
Chaikham et al. (2016) reported that sonicated
honeys are darker when treated at 40% and 80% amp-
litude levels for 30 min. The browning index was also
found to be higher when compared to control honey.
For the thermal processing, Figure 4(b) illustrates
that color intensity increased up to 67.6% where the
darkest honey observed was 783  4 mAU, at 90  C
for 120 min. Honey became darker after both
(b) 800
30 min
50 min
75 min
100 min
700 120 min
Color intensity (mAU)
600
500
400
300
90.0 45.0 55.0 67.5 80.0 90.0
Temperature (°C)

processing methods as a result of non-enzymatic
browning from Maillard reaction. Turkmen et al.
(2006) reported that both temperature and time had a
large effect on brown pigment formation in honey
during heat treatment. Figure 4(b) also suggests that
thermal processing at low to medium temperatures
can minimize color changes.
Viscosity
Figure 5 shows that both the thermosonicated and ther-
mally processed honey had higher viscosities compared
to raw honey. The change in viscosity was fairly small
at 45  C to 80  C, but at 90  C, viscosity increase was
noticeable.
The small change in viscosity may be as a result of
high water content at lower temperatures where less
evaporation is taking place. Abu-Jdayil et al. (2002)
found that the incremental percentage in viscosity
decreased as water content in fresh honey increased.
Besides water content, colloidal matters and proteins
present in honey are also known to affect its viscosity.
At high temperatures, denaturation of proteins
increases water uptake at the expense of free unbound
water present in the honey matrix. This decrease in free
unbound water content leads to an increase in honey
viscosity (Abu-Jdayil et al., 2002). These findings are in
contrast to the study from Chaikham et al. (2016) who
reported that the dynamic viscosities of honey
decreased after thermal processing at 90  C and ultra-
sonication at 40% and 80% amplitude levels for
30 min. It was deduced that this decline was mainly
because of melting of sugar crystals. For this study
(a) 0.70
30 min
50 min
0.60 75 min
100 min
120 min
0.50
Viscosity (Pa.s)
0.40
0.30
0.20
0.10
0.00
45.0 55.0 67.5 80.0
Temperature (°C)
Figure 5. Effect of temperature and time on viscosity using (a) thermosonication and (b) thermal processing. Black
dashed line indicates values for raw honey. Values on and beyond the gray dotted lines indicate significant differences
(P < 0.05) compared to raw honey.
Chong et al.
on Kelulut honey, its viscosity was fairly consistent at
lower temperatures processing but increased greatly at
90  C, particularly for thermosonicated honey. Hence,
it is recommended to use 90  C if concentrated honey is
desired.
HMF content
Figure 6 shows no HMF detected for raw and pro-
cessed Kelulut honey for both thermosonicated and
thermally processed below 67.5  C/75 min. Beyond
this combination, HMF values increased with tempera-
ture and time for both methods.
HMF is a commonly used parameter to evaluate
honey overheating and ageing. The absence of HMF
in raw stingless bee honey may be due to its higher
fructose content, higher water activity, and higher acid-
ity as compared to the Apis mellifera honey (Biluca
et al., 2014) which slows down Maillard reaction.
Some of the HMF values and origins for stingless bee
honeys are 15 mg/kg (Ecuador) (Guerrini et al., 2009),
8.7 mg/kg (Thailand) (Chuttong et al., 2016), and less
than 20 mg/kg (Venezuela) (Vit et al., 1994).
As there was a strong correlation between storage
duration and HMF concentrations reported in
Malaysian honeys (Khalil et al., 2010), HMF content
was measured prior to both processing methods to
eliminate storage effects because of a lapse of six
weeks’ storage between the thermosonication and con-
ventional heating treatments. This lapse had some
slight effects shown in Kelulut honey’s properties
except for HMF content, which remained undetected.
HMF is formed as an intermediate product of
(b) 0.70
30 min
50 min
0.60 75 min
100 min
120 min
0.50
Viscosity (Pa.s)
0.40
0.30
0.20
0.10
0.00
90.0 45.0 55.0 67.5 80.0 90.0
Temperature (°C)
7
Food Science and Technology International 0(0)

(a) 70 (b) 70
30 min 30 min
50 min 50 min
75 min 75 min
60 60
Hydoxymethy furfural content (mg/kg)

Hydoxymethy furfural content (mg/kg)

100 min 100 min
120 min 120 min
50 50

40 40

30 30

20 20

10 10

0 0
45.0 55.0 67.5 80.0 90.0 45.0 55.0 67.5 80.0 90.0
Temperature (°C) Temperature (°C)

Figure 6. Effect of temperature and time on hydroxymethylfurfural content using (a) thermosonication and (b) thermal
processing. Values on and beyond the gray dotted lines indicate significant differences (P < 0.05) compared to raw
honey.

acid-catalyzed dehydration reaction of hexose and/or 352.73 mg GAE/kg honey (Moniruzzaman et al.,
by Maillard reaction (Fallico et al., 2008). In Maillard 2013). Gelam and coconut honey recorded a phenolic
reaction, the free amino group, usually from an amino content of 21.4 and 15.6 mg GAE/kg honey, respect-
acid, condenses with the carbonyl of a reducing sugar, ively (Aljadi and Kamaruddin, 2004). Figure 7 also
namely glucose and fructose present in honey. Since shows that the TPC of thermosonicated and thermally
water is removed during condensation, the reaction is processed honey in this study increased and ranged
favored by dehydration conditions (Hoseney, 1984). As from 490.18 to 750.75 mg GAE/kg and 473.51 to
moisture is reduced during heating, this leads to an 684.27 mg GAE/kg, respectively.
increase in HMF. Figure 6 indicates the maximum These processed honeys had higher TPC than raw
HMF values obtained for thermosonicated and ther- honey. This increase of TPC may be attributed to
mally processed honey were 62.46 mg/kg and Maillard reaction products (MRPs). When food is
42.40 mg/kg, respectively. The HMF increase in ther- exposed to heat, it undergoes Maillard reaction which
mosonicated honey was likely due to the enhanced leads to the formation of various brown melanoidins
Maillard reaction by ultrasound effects as Guan et al. (Nicoli et al., 1997). It is also reported that the loss of
(2011) found such phenomena at ultrasound intensity natural antioxidants in heated food can be minimized
higher than 15.29 W/cm2 in glycin-glucose solution. It is or compensated by the formation of non-nutrient anti-
important to control HMF levels as HMF can be cyto- oxidants such as the MRPs. Similar studies reported a
toxic, mutagenic, carcinogenic, and genotoxic linear increase of antioxidant in commercial floral
(Capuano and Fogliano, 2011). Despite increased honeys at 50  C and 60  C for a heating period of 12
HMF values, the maximum HMF values of treated days, but increased logarithmically at 70  C (Turkmen
honey was still within the limit provided by inter- et al., 2006). This is in agreement with the findings of
national standards of 80 mg/kg for honey originating Chaikham and Prangthip (2015) who reported that by
from countries with tropical ambient temperatures using thermal processing, an increasing trend of TPC
(Alimentarius, 2001). was observed in longan flower-honey at 50  C and
70  C, but a decreasing trend was noticed for 100  C.
As shown in Figure 7(a) and (b), all thermosonicated
Total phenolic content and radical
honey recorded higher TPC values as compared to ther-
scavenging activity
mally processed honeys. Further analysis shows that
Figure 7 shows that raw Kelulut honey had a TPC value thermal processing caused a larger incremental percent-
of 474.95  4.33 and 443.58  4.37 mg GAE/kg honey, age in TPC compared to thermosonication, except for
prior to thermosonication and thermal processing, heating conditions at 90  C, where samples treated with
respectively. These values are still higher than other 30 min of thermosonication showed an increase of
Malaysian honeys such as Acacia, Pineapple, Borneo, 22.07% in TPC compared to thermal treatment with
and Tualang honeys, which ranged from 186.70 to 20.85% increase. The low TPC increment may be

8
 Chong et al.

(a) 800 (b) 800

30 min 30 min
50 min 50 min
750 75 min 750 75 min

Total phenolic content (mg GAE//kg)

Total phenolic content (mg GAE//kg)

100 min 100 min
120 min 120 min
700 700

650 650

600 600

550 550

500 500

450 450

400 400
45.0 55.0 67.5 80.0 90.0 45.0 55.0 67.5 80.0 90.0
Temperature (°C) Temperature (°C)

Figure 7. Effect of temperature and time on total phenolic content using (a) thermosonication and (b) thermal
processing. Black dashed line indicates values for raw honey. Values on and beyond the gray dotted lines indicate
significant differences (P < 0.05) compared to raw honey.

(a) 100 (b) 100

30 min 30 min
50 min 50 min
75 min 75 min
90 100 min 90 100 min
120 min 120 min

80 80
DPPH Inhibition (%)

DPPH Inhibition (%)

70 70

60 60

50 50

40 40
45.0 55.0 67.5 80.0 90.0 45.0 55.0 67.5 80.0 90.0
Temperature (°C) Temperature (°C)

Figure 8. Effect of temperature and time on DPPH inhibition using (a) thermosonication and (b) thermal processing.
Black dashed line indicates values for raw honey. Values on and beyond the gray dotted lines indicate significant
differences (P < 0.05) compared to raw honey.

caused by cushioning effect. At high temperatures, pronounced when thermosonication at lower tempera-
vapor pressure inside the bubbles increases, thus creat- tures was carried out. This may be another reason of
ing a cushioning effect which produces less effective the lower TPC increment as enzymes in thermosoni-
collapses (Suslick, 1989). Experiments at temperature cated honey could be inactive. In another study
near to the solvent’s, in this case, water’s boiling involving ultrasonic treatment of honey, an increase
point which generates more bubbles concurrently, in phenols was attributed to the disintegration of
would act as a barrier to sound transmission and sub- pollen as a result of ultrasonic stimulation which
sequently dampen ultrasound intensity (Mason, 1991). releases enzymes and proteins into the honey
Additionally, Raviyan et al. (2005) who studied the (Chaikham and Prangthip, 2015). Other antioxidative
inactivation of tomato pectinmethylesterase (PME), effects of ultrasonic processing were documented for
an endogenous pectic enzyme in tomato cell walls, various foods such as grape by-products where its
found that an increase of PME inactivation was more TPC increased by approximately twice than the control

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Food Science and Technology International 0(0)

Table 1. Regression coefficients, R2, adjusted R2, and P or probability values for honey quality-dependent variables for
thermosonication

Radical
Colour HMF Total phenolic scavenging
Moisture intensity content content activity Viscosity
Coefficient Water activity content (%) (mAU) (mg/kg) (mg GAE/kg) (% inhibition) (Pa.s)

b0 0.702461 21.4714 623.289 149.416 1015.44 169.333 0.977027

b1 0.003315 0.2698 7.624 4.477 17.97 3.318 0.023069
b2 0.000539ns 0.0681 2.724 0.825 1.33ns 0.318ns 0.006005
b11 0.000024 0.0018 0.078 0.031 0.14 0.025 0.000153
b22 0.000000ns 0.0001ns 0.002ns 0.001ns 0.00ns 0.001ns 0.000010ns
b12 0.000011 0.0010 0.051 0.014 0.04 0.002ns 0.000090
R2 0.9602 0.9591 0.9940 0.9357 0.9773 0.8942 0.9279
R2(adj) 0.9319 0.9299 0.9897 0.8899 0.9611 0.8187 0.8764
P (lack of fit) 0.059ns 0.005** 0.205ns 0.000** 0.099ns 0.693ns 0.000**
P (overall 0.000** 0.000** 0.000** 0.000** 0.000** 0.003** 0.001**
model)
b0 is the estimated constant regression coefficient.
b1 and 2 are the estimated linear regression coefficient of heating temperature and time, respectively.
b11 and 22 are the estimated quadratic regression coefficient of heating temperature and time, respectively.
b12 is the estimated interaction regression coefficient of heating temperature and time.
*P < 0.05, **P < 0.01; ns: not significant when P > 0.05

Table 2. P-values and F values obtained from two-way ANOVA for thermosonicated honey

Responses Factors Temperature Time Interaction

Water activity P 0.000 0.000 0.000

F 390.160 74.960 20.140
Moisture content P 0.000 0.000 0.000
F 304.780 58.760 18.790
Color intensity P 0.000 0.000 0.000
F 1120.370 22.330 20.780
Viscosity P 0.000 0.000 0.000
F 96.53 26.09 11.480
Hydroxymethylfurfural content P 0.000 0.000 0.000
F 6907.470 1091.240 674.710
Total phenolic content P 0.000 0.000 0.000
F 295.170 30.490 9.250
Radical scavenging activity P 0.000 0.000 0.018
F 269.73 11.790 2.180
Note: All values are significant at P < 0.05.

when treated with ultrasonic waves at 35 kHz at 70  C
(Corrales et al., 2008). A significant increase in total
phenolics was also observed with sonicated apple
juice at 20  C compared to non-sonicated samples
(Abid et al., 2013).
Figure 8 shows the effect of thermosonication and
thermal processing on Kelulut honey’s radical scaven-
ging activity. All the treatments stimulated an increase
in radical scavenging activities compared to raw honey.
The maximum DPPH inhibition for thermosonication
and thermal treatment increased by 63.0% and 72.5%,
respectively, at 90  C for 120 min.
Similar to honey’s total phenolic content, the
increase in radical scavenging activity can be associated
with thermal extraction and disintegration of pollen.
Likewise, Chaikham and Prangthip (2015) reported

10
 Chong et al.

(a) (b)

Moisture content (%)

Water activity
0.80
30
0.78
28
0.76 120 120
0.74 90 26 90
60 Time (min) 60 Time (min)
40 50 40 50
60 70 80 30 60 70 80 30
90 90
Temperature (°C) Temperature (°C)

(c) (d)
Colour intensity (mAU)

Viscosity (Pa.s)
0.5
700
600 0.4
500 0.3
120 120
400 0.2
90 90
40 50 60 Time (min) 60 Time (min)
60 70 80 40 50
30 60 70 80 30
90 90
Temperature (°C) Temperature (°C)

(e) (f)
TPC (mg GAE/kg)

60
HMF content

700
40
600
20
120 120
0 500 90
90
60 Time (min) 60 Time (min)
40 50 40 50 30
60 70 80 30 60 70 80
90 90
Temperature (°C) Temperature (°C)

(g)

80
RSA (%)

70
120
60
90
60 Time (min)
40 50
60 70 80 30
90
Temperature (°C)

Figure 9. Effects of temperature and time on (a) water activity, (b) moisture content, (c) colour intensity, (d) viscosity,
(e) hydroxymethylfurfural content, (f) total phenolic content, and (g) DPPH radical scavenging activity for thermosonicated
honey.

Response surface regression analysis

a rise in DPPH inhibition using ultrasonication on
honey. DPPH inhibition showed little improvement Table 1 lists the coefficients for the response surface
when thermosonication was used at 55  C. In another equations in uncoded units with indication of signifi-
work by Chaikham et al. (2016), there was no specific cance through its P-value. The regression models for
trend on DPPH inhibition when amplitudes of ultra- honey quality properties were fitted adequately with
sound waves were increased. This may be caused by second-order polynomial model. All the response
interference of carotenoid pigments in honey at the models are statistically significant at P < 0.05 with
wavelength used (Alvarez-Suarez et al., 2009). acceptable R2 values which were more than 0.7.
Nevertheless, there was a high positive correlation, Granato et al. (2010) stated that value of R2  0.7 is
0.801 (P < 0.05) between DPPH inhibition and TPC considered good for sensory, colorimetric, and physico-
of thermosonicated Kelulut honey. chemical results.

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Food Science and Technology International 0(0)

Table 3. Process variables, responses optimization, desirability, and experimental value for the responses at optimum
condition for thermosonicated honey

Desirability Experimental Deviation

Variables Goal Lower Target Upper Predicted (d) (mean  SD) (%)

Optimized point for chemical properties: 90 C/63 min. D ¼ 0.6513

HMF content (mg/kg) Minimum 0.00 0.00 62.46 25.67 0.5890 21.38  0.24 16.7
TPC (mg GAE/kg) Maximum 490.18 750.75 750.75 653.73 0.6277 626.92  19.35 4.1
RSA (% inhibition) Maximum 50.74 83.56 83.56 75.26 0.7472 73.95  2.00 1.7

Optimized point for physical properties: 90 C/120 min. D ¼ 0.9337

Water activity Minimum 0.743 0.743 0.806 0.747 0.9307 0.759  0.003 1.6
Moisture content (%) Minimum 25.905 25.905 30.817 26.298 0.9199 27.921  0.062 6.2
Colour intensity (mAU) Maximum 372 765 765 771 1.0000 813  5 5.4
Viscosity (Pa.s) Maximum 0.1643 0.5794 0.5794 0.5328 0.8876 0.3831  0.0350 28.1

Optimized point for overall properties: 90 C/111 min. D ¼ 0.7030

Water activity Minimum 0.743 0.743 0.806 0.752 0.8624 0.770  0.008 2.4
Moisture content (%) Minimum 25.905 25.905 30.817 26.778 0.8223 28.204  0.890 5.3
Colour intensity (mAU) Maximum 372 765 765 758 0.9812 768  16 1.3
Viscosity (Pa.s) Maximum 0.1643 0.5794 0.5794 0.4939 0.7941 0.5410  0.1410 9.5
HMF content (mg/kg) Minimum 0.00 0.00 62.46 51.48 0.1758 53.70  0.53 4.3
TPC (mg GAE/kg) maximum 490.18 750.75 750.75 728.39 0.9142 707.92  24.84 2.8
RSA (% inhibition) Maximum 50.74 83.56 83.56 82.10 0.9555 78.40  2.16 4.5

HMF: hydroxymethylfurfural; TPC: total phenolic content; RSA: radical scavenging activity.

Although there was significant lack of fit for
responses for moisture content, HMF content, and vis-
cosity, Table 2 which included all the data from the full
factorial experiments, shows that temperature and time
significantly affected all seven responses when analyzed
using two-way ANOVA.
The combined effect of temperature and time on all
responses is illustrated by three-dimensional surface
plots in Figure 9. For thermosonicated and thermally
processed honey, water activity and moisture content
decreased with temperature and time. For color inten-
sity, HMF content, TPC, and RSA, the values
increased with temperature and time.
Optimization of honey quality
Optimum conditions for honey processing were deter-
mined by specifying the goal for each response. It was
targeted to obtain minimum water activity, moisture
content, HMF content and maximum color intensity,
viscosity, TPC, and radical scavenging activity.
Maximizing color intensity reflects higher antioxidant
content (Frankel et al., 1998), while maximizing viscos-
ity was intended to facilitate transportation and distri-
bution of honey in concentrated forms so that its
weight and volume can be reduced. The weight factor
and importance of all responses in this study were set to
be one to ensure that all responses have the same influ-
ence on the composite desirability. Table 3 shows three
optimized solutions for each treatment generated for
different purposes, which were chemical properties,
physical properties, and overall properties. This
was performed because of a contradiction between
HMF content and other properties. At higher tem-
peratures and longer heating time, HMF content
increased. Nevertheless, phenols and antioxidant activ-
ities needs to be maintained as high as possible. For
optimization of overall properties, the response HMF
had very low individual desirability which was 0.1758
for thermosonication. The desirability values indicate
the satisfaction of the combined goals for the
responses set. The composite desirability (D) and indi-
vidual desirability (d) are shown for each solution.
For thermosonication, the optimized solution for
chemical properties, physical properties, and overall
properties were 90  C at 63 min, 120 min, and 111 min,
respectively.
Table 3 also shows the model verification results for
optimized solutions. The percentage deviation between
experimental values and predicted values was less than
10%, except for responses of HMF content and viscos-
ity. This variation may be a result of sensitivity of
these properties to the time required to perform the
experiments.

12

CONCLUSION
It is commonly believed that honey degrades during
processing. In this paper, it was demonstrated that
honey processing, specifically, thermosonication
enhanced its quality in terms of water activity, moisture
content, color intensity, viscosity, total phenolic con-
tent, and radical scavenging activity. Although HMF
content was high in thermosonicated honey, the value
was still within the limits of international standards.
Using response surface methodology, the processing
conditions for optimum honey quality based on overall
attributes for thermosonication is at 90  C for 111 min.
Further research could be conducted by integrating
high-temperature short-time heating with ultrasound
waves to enhance honey quality.
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