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 crystallize. This results in quality deterioration in terms
honey such as Acacia, Pineapple, Borneo, Gelam, of acidic taste and undesirable appearance. As the pres-
Kelulut, and Tualang honeys. These honeys can be clas- ence of osmophilic yeast and fungi in honey is inevit-
sified broadly into Apis mellifera (honey bee) honey and able during bees’ foraging process, honey needs to be
stingless bee honey. The stingless bee, Trigona spp., processed to extend its shelf life.
produces the Kelulut honey. It is a multifloral honey Industrial practices commonly use thermal pro-
stored in pots made of cerumen. In comparison with cessing to liquefy and pasteurize honey at 45  C and
the Apis mellifera honey, the stingless bee honey has 80  C, respectively (Escriche et al., 2009). However,
higher antioxidant activity, flavonoids, and polyphenol the use of high temperatures caused undesirable
content (Kek et al., 2014; Rodrı́guez-Malaver, 2013). changes in various honey properties such as decrease
Other distinctive characteristics of the stingless bee in enzymes, increase in hydroxymethylfurfural (HMF)
honeys include higher moisture content, higher elec- content, rapid loss of antioxidant activity, darkening
trical conductivity, higher acidity, higher protein con- of honey, and damage to flavor (Nagai et al., 2001;
tent, and lower diastase activity (Chuttong et al., 2016;
Kek et al., 2017). As a result of its high moisture
Department of Process and Food Engineering, Faculty of
Food Science and Technology International 0(0) 1–15
Engineering, Universiti Putra Malaysia, Selangor, Malaysia
! The Author(s) 2017 Reprints and permissions: Corresponding author:
sagepub.co.uk/journalsPermissions.nav NL Chin, Department of Process and Food Engineering, Faculty of
DOI: 10.1177/1082013217713331 Engineering, Universiti Putra Malaysia, 43400 UPM Serdang,
journals.sagepub.com/home/fst Selangor, Malaysia.
Email: [email protected]
Food Science and Technology International 0(0)

Thermosonication processing
White Jr, 1978; White et al., 1964). Ultrasonication is
one of the non-thermal technologies explored for min- An ultrasonic bath tank at 25 kHz powered by piezo-
imal processing of food owing to its ability to inactivate electric flange mounted type transducers (Branson
microorganisms and promote dehydration in foods Ultrasonics Co., Danbury, CT, USA) at 2.5 kW (Chin
(Ahmed et al., 2009; Musielak et al., 2016). In addition, et al., 2013) was fitted with heating element of 6 kW and
ultrasound treatment can preserve and improve nutri- insulation for a thermosonication effect. A test tube
tional values and organoleptic properties of food (Abid rack was suspended in the middle of the tank to hold
et al., 2013; Chaikham and Prangthip, 2015; Chemat the test tubes containing honey. Thermosonication
et al., 2011). In an ultrasound treatment, the liquid sub- treatment of honey was conducted at 45.0, 55.0, 67.5,
jected to ultrasound waves is alternately compressed 80.0, and 90.0  C where this range of temperatures
and expanded, forming microbubbles or cavities. The included the liquefaction and pasteurization process
collapse of bubbles leads to intense local heating or hot (Escriche et al., 2009; Gonnet, 1977) for 30, 50, 75,
spots, and this rapidly changes temperature and pres- 100, and 120 minutes. Water temperature was recorded
sure in its surrounding medium (Ahmed et al., 2009; using a K-type thermocouple connected to a data
Suslick, 1989). Consequently, high energy shear waves logger thermometer (CENTER 309, CENTER
and turbulence are produced in the cavitation zone Technology Corp., Taiwan). A control experiment to
(Cárcel et al., 2012) that drives physical and chemical demonstrate the effects of conventional thermal pro-
reactions in the liquid. Thermosonication is a process cessing on honey was conducted using a thermostatic
that combines heat and ultrasound waves. As ultra- water bath (WNB 22, Memmert GmbH þ Co. KG,
sound waves alone may not be very effective in destroy- Germany) with same temperatures and times.
ing microorganisms unless very high intensities are used Figure 1 illustrates the top view for the set-up of the
(Ahmed et al., 2009), thermosonication is a potential thermosonic bath system and thermostatic water bath.
alternative processing method to enhance destruction
of microorganisms. The effects of thermosonication
Quality analysis of honey
were observed on different food such as watercress
(Cruz et al., 2007), beer (Milani et al., 2016), milk Water activity. Water activity was determined using a
(Noci et al., 2009), and fruit juices (Rawson et al., water activity meter (Aqualab Series 3, Decagon
2011; Wu et al., 2008). Devices, USA) at room temperature.
The objective of this study was to integrate ultra-
sound waves and conventional heating to create a ther- Moisture content. Moisture content of honey was
mosonication effect as a technique to improve storage determined using Bogdanov et al.’s (2002) method with
quality of raw Kelulut honey as it is mostly commercia- slight modifications. The refractive index of honey was
lized in its raw or unprocessed form. The honey quality determined using a digital refractometer (Digital ABBE
attributes of water activity, moisture content, color Refractometer AR2008, A. Krüss, Germany) at 20  C. If
intensity, viscosity, HMF content, total phenolic con- temperature differed from 20  C, 0.00023 per  C was
tent (TPC), and radical scavenging activity (RSA) were added or subtracted from the refractive index. The
compared to those treated using conventional thermal refractive index was converted to moisture content by
processing. The optimum processing conditions with means of equation (1) (Sesta and Lusco, 2008)
respect to thermosonication temperature and time
were also determined using response surface method- Moisture content ð%Þ
ð1Þ
ology (RSM). ¼ ½0:2681  logðRI  1Þ=0:002243

where RI is the refractive index.

MATERIALS AND METHODS
Color intensity. Color intensity of honey was deter-
Honey samples
mined using a UV/Visible spectrophotometer
Fresh Kelulut honey of the same batch was obtained (Ultrospec 3100 pro, Amersham Biosciences, USA) fol-
from a forest reserve in Teluk Intan, Perak, Malaysia lowing methods of Beretta et al. (2005). A 50% (w/v)
in January 2016. Freshly harvested samples were stored honey solution was prepared with warm distilled water
at 4  C for experiments. Twenty grams of honey were (45–50  C) and filtered to remove any coarse particles.
filled into test tubes with 25 mm diameter and 150 mm Using distilled water as blank, the absorbance at
height for thermal processing in the first six weeks and 450 nm and 720 nm was recorded. Color intensity was
thermosonication in the next six weeks. Honey was calculated using equation (2).
removed from the chiller to equilibrate with room tem-
perature before experiments commenced. Color intensity ðmAUÞ ¼ A450  A720 ð2Þ

2
 Chong et al.

(a) 4 5 (b)
6

235 mm
595 mm
2 2 1
1
320 mm
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 cuvettes. HMF was determined using equation (3)
720 nm, respectively. (White, 1979).

Flow behaviour. A rheometer (AR-G2, TA HMF ðmg=kg honeyÞ

Instruments, New Castle, USA) with a 60-mm diameter ð3Þ
cone and plate geometry (30 mm truncation gap, one-
¼ ðA284  A336 Þ  149:7  5=W
degree cone angle) was used to determine the dynamic
viscosity of honey. Honey samples were not preheated where A284 and A336 denote the absorbance at 284 nm
as temperature would affect viscosity readings. Steady- and 336 nm, respectively, the factor 149.7 is a theoret-
state measurements were performed at 20  C, with a ical value linked to the molar extinction coefficient
shear rate range of 1–1000 s1 in ascending ramp. The of HMF at 284 nm, and W is the weight of honey
temperature was controlled by a circulating water sample in g.
system. Dynamic viscosity was obtained from the aver-
age of 20 points. Data were analyzed using its accom- Total phenolic content. TPC was determined using the
panying computer software, Rheology Advantage Data Folin-Ciocalteu spectrophotometric method (Singleton
Analysis Version 5.7. 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-
HMF content. HMF content was determined using Ciocalteu reagent (diluted 1:10 with distilled water).
White’s spectrophotometric method (White, After 5 minutes of incubation, 4 mL of 7.5% (w/v)
1979). Five grams of honey samples was diluted up to aqueous sodium carbonate solution was added. The
50 mL with distilled water after addition of 0.5 mL of mixture was well mixed and incubated for 2 hours in
Carrez I solution (consisting 15 g of potassium hexacya- dark at room temperature. The absorbance of the mix-
noferrate (II), K4Fe(CN)6.3H2O in 100 mL water) and ture was determined at 765 nm using distilled water as
0.5 mL of Carrez solution II (consisting 30 g of zinc blank. A standard calibration curve was plotted using
acetate, Zn(CH3.COO)2.2H2O in 100 mL of water). gallic acid with concentration range from 10 to 100 mg/
The solution was then filtered and the first 10 mL of mL. TPC was expressed in mg of gallic acid equivalent
the filtrate was rejected. Aliquots of 5 mL each were (GAE) per kg of honey. Measurements were performed
transferred to two test tubes. Five milliliters of distilled in triplicates.
water was added to the first tube as sample and
another 5 mL of 0.2% (w/v) sodium bisulphite solution Radical scavenging activity. Radical scavenging activ-
was added to the second test tube as reference. ity was determined by using 2,2-diphenyl-1-picrylhy-
Absorbance of sample solution was determined against drazyl (DPPH) as a stable radical purchased from
reference solution at 284 nm and 336 nm in quartz Sigma-Aldrich (USA). Combining the method of

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Food Science and Technology International 0(0)

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

the raw honey sample having average water activity of This may be a result of honey fermentation at moderate
0.807 and 0.795. temperatures. It is known that ultrasonication has the
The water activity values for Kelulut honey reduced potential to inactivate microbes and yeast. However,
substantially compared to its non-treated form despite the inactivation is influenced by temperature, ultra-
still exceeding the level of 0.6, where below 0.6, there sound frequency and power, exposure time, type of
will be no microbial proliferation (Fontana Jr et al., microorganism, volume of food to be processed, and
2008). Australian stingless bee honey recorded an aver- composition of the food. Bermúdez-Aguirre and
age water activity of 0.74. In contrast, water activity of Barbosa-Cánovas (2012) showed that low temperatures
other types of honey was lower. Greek honeydew and such as 40  C did not have any effect on yeast inactiva-
floral honey ranged from 0.528 to 0.663 (Lazaridou tion in fruit juices but at 60  C, highest inactivation was
et al., 2004), while Slovenian honeydew honey and achieved. Thermosonication of apple juice from 20 to
floral honey’s mean water activity were 0.528 and 60  C resulted in decreasing yeast count with increasing
0.521, respectively (Abramovič et al., 2008). Figure 3 temperature but only achieved complete yeast inactiva-
shows that moisture content was significantly reduced tion at 60  C (Abid et al., 2014). Similar results were
by 16.6% to 25.9% moisture using thermosonication as demonstrated by Saccharomyces cerevisiae in orange
compared to the conventional thermal processing by juice (Ganesan et al., 2015). Thus, there is a possibility
6.9% to 28.8% moisture. of yeast presence at 45  C and 55  C. Yeast ferments
The relatively high moisture content of raw stingless glucose, fructose, and sucrose strongly to produce
bee honey is a common phenomenon and poses prob- carbon dioxide, alcohol, and small quantities of acid
lems to its storage and shelf life. Besides Kelulut from (Marvin et al., 1931). At these temperatures, the alco-
Malaysia, stingless bee honey from other countries such hol and acid could not evaporate and this may contrib-
as Thailand (31%) (Chuttong et al., 2016), Venezuela ute to increment in moisture. Another possibility of the
(14.8% to 30.2%) (Vit et al., 1994), Brazil (23.4 to moisture increase is from thermal degradation of sugars
33.1%) (Silva et al., 2013), and Ecuador (34.1%) by Maillard reaction. This complex reaction involving
(Guerrini et al., 2009) were reported to have high mois- different compounds and pathways produces water as a
ture contents. The higher reduction of water during byproduct at its initial condensation stage. This reac-
thermosonication treatment can be attributed to mech- tion varies according to product composition and pro-
anical effects of collapsing microbubbles which increase cessing conditions. There were many sugar systems
mass transfer. Intense shock wave is produced as honey studied over a wide range of temperatures, but none
rapidly rushes into the volume previously occupied by was reported for honey yet (Turkmen et al., 2006; van
the bubble, resulting in enormous shear forces (Mason, Boekel, 2001). Caramelization is excluded as a reason
1991). At moderate temperatures of 45  C and 55  C, for the increase in moisture as the two main monosac-
water activity and moisture content increased. charides in honey, glucose and fructose, melt at

(a) 34 (b) 34

33 33
32 32
Moisture content (%)

Moisture content (%)

31 31
30 30

29 29
28 28

27 27
30 min 30 min
26 50 min 26 50 min
75 min 75 min
25 100 min 25 100 min
120 min 120 min
24 24
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 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.

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Food Science and Technology International 0(0)

temperatures of 165  C and 120  C, respectively (Örsi, cavitation effects during sonication where chemical
1973). As the processed Kelulut honey surpassed reactions are accelerated and diffusion rates increased
the maximum moisture content allowed of 20% (Sala et al., 1995; Tiwari et al., 2008a). The lightness in
(Alimentarius, 2001) and water activity of 0.6, it is honey color using thermosonication at 45  C and 55  C
favorable for osmophillic yeasts and fungi growth. As is similar to other sonication studies. Rawson et al.
the maximum moisture content limit of 20% referred to (2011) found that thermosonication of watermelon
honey from Apis mellifera species, Vit et al. (2004) pro- juice at 35  C and 45  C resulted in a significant increase
posed that the moisture content for stingless bee honey of lightness value. Tiwari et al. (2010) reported lightness
should be raised to 30%. Both thermosonication and in red grape juice when compared to the control for all
thermal processing reduced moisture content to less treatment times at constant temperature of 25  C. The
than 30% with note of higher efficiency in the thermo- initial increase in lightness value was attributed to the
sonication method. 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
Color intensity
values of sonicated orange juice increased with 40%
Figure 4(a) shows that there was a steady increase in the amplitude level and then decreased when the levels
color intensity of honey that was subjected to thermoso- were increased beyond 40%. Lightness was mainly
nication at higher temperatures of 67.5, 80.0, and 90  C, affected by sonication time and was most likely because
with the darkest reading at 765 mAU. Thermosonication of increased exposure of color pigments in orange juice
of honey at lower temperatures of 45  C and 55  C to high shear forces from collapsing bubbles. Zhao
has reduced its color intensity which was also more et al. (2006) reported a carotenoid pigment, (all-
intense with longer thermosonification time. E)-astaxanthin, that was degraded to unidentified col-
Honey color is an important characteristic as it is orless compounds when an ultrasonic probe system was
linked to consumer acceptance of the product. In used and the degradation was higher with increased
Germany, Austria, and Switzerland, consumers espe- treatment time and ultrasonic power. However,
cially appreciated dark honeydew honeys (Bogdanov, Chaikham et al. (2016) reported that sonicated
et al., 2004). Dark honey is also beneficial as antioxi- honeys are darker when treated at 40% and 80% amp-
dant activities of Malaysian honey were reported to litude levels for 30 min. The browning index was also
increase with its color intensity as indicated by strong found to be higher when compared to control honey.
positive correlations (Kek et al., 2014; Moniruzzaman For the thermal processing, Figure 4(b) illustrates
et al., 2013). that color intensity increased up to 67.6% where the
The darker color change of thermosonicated honeys darkest honey observed was 783  4 mAU, at 90  C
is mostly attributed to both simultaneous heat and for 120 min. Honey became darker after both

(a) 800 (b) 800

30 min 30 min
50 min 50 min
75 min 75 min
100 min 100 min
700 120 min 700 120 min
Color intensity (mAU)

Color intensity (mAU)

600 600

500 500

400 400

300 300
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 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.

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 Chong et al.

processing methods as a result of non-enzymatic on Kelulut honey, its viscosity was fairly consistent at
browning from Maillard reaction. Turkmen et al. lower temperatures processing but increased greatly at
(2006) reported that both temperature and time had a 90  C, particularly for thermosonicated honey. Hence,
large effect on brown pigment formation in honey it is recommended to use 90  C if concentrated honey is
during heat treatment. Figure 4(b) also suggests that desired.
thermal processing at low to medium temperatures
can minimize color changes.
HMF content
Figure 6 shows no HMF detected for raw and pro-
Viscosity
cessed Kelulut honey for both thermosonicated and
Figure 5 shows that both the thermosonicated and ther- thermally processed below 67.5  C/75 min. Beyond
mally processed honey had higher viscosities compared this combination, HMF values increased with tempera-
to raw honey. The change in viscosity was fairly small ture and time for both methods.
at 45  C to 80  C, but at 90  C, viscosity increase was HMF is a commonly used parameter to evaluate
noticeable. honey overheating and ageing. The absence of HMF
The small change in viscosity may be as a result of in raw stingless bee honey may be due to its higher
high water content at lower temperatures where less fructose content, higher water activity, and higher acid-
evaporation is taking place. Abu-Jdayil et al. (2002) ity as compared to the Apis mellifera honey (Biluca
found that the incremental percentage in viscosity et al., 2014) which slows down Maillard reaction.
decreased as water content in fresh honey increased. Some of the HMF values and origins for stingless bee
Besides water content, colloidal matters and proteins honeys are 15 mg/kg (Ecuador) (Guerrini et al., 2009),
present in honey are also known to affect its viscosity. 8.7 mg/kg (Thailand) (Chuttong et al., 2016), and less
At high temperatures, denaturation of proteins than 20 mg/kg (Venezuela) (Vit et al., 1994).
increases water uptake at the expense of free unbound As there was a strong correlation between storage
water present in the honey matrix. This decrease in free duration and HMF concentrations reported in
unbound water content leads to an increase in honey Malaysian honeys (Khalil et al., 2010), HMF content
viscosity (Abu-Jdayil et al., 2002). These findings are in was measured prior to both processing methods to
contrast to the study from Chaikham et al. (2016) who eliminate storage effects because of a lapse of six
reported that the dynamic viscosities of honey weeks’ storage between the thermosonication and con-
decreased after thermal processing at 90  C and ultra- ventional heating treatments. This lapse had some
sonication at 40% and 80% amplitude levels for slight effects shown in Kelulut honey’s properties
30 min. It was deduced that this decline was mainly except for HMF content, which remained undetected.
because of melting of sugar crystals. For this study HMF is formed as an intermediate product of

(a) 0.70 (b) 0.70

30 min 30 min
50 min 50 min
0.60 75 min 0.60 75 min
100 min 100 min
120 min 120 min
0.50 0.50
Viscosity (Pa.s)

Viscosity (Pa.s)

0.40 0.40

0.30 0.30

0.20 0.20

0.10 0.10

0.00 0.00
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 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.

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

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

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 Chong et al.

CONCLUSION content in stingless bee honey by CE before and after ther-

It is commonly believed that honey degrades during mal treatment. Food Chemistry 159: 244–249.
processing. In this paper, it was demonstrated that Bogdanov S, Martin P and Lullmann C. (2002). Harmonised
honey processing, specifically, thermosonication Methods of the International Honey Commission. Liebefeld,
FAM: Swiss Bee Research Centre.
enhanced its quality in terms of water activity, moisture
Bogdanov S, Ruoff K and Persano Oddo L. (2004). Physico-
content, color intensity, viscosity, total phenolic con- chemical methods for the characterisation of unifloral
tent, and radical scavenging activity. Although HMF honeys: A review. Apidologie 35(Suppl 1): 4–17.
content was high in thermosonicated honey, the value Capuano E and Fogliano V. (2011). Acrylamide and 5-hydro-
was still within the limits of international standards. xymethylfurfural (HMF): A review on metabolism, tox-
Using response surface methodology, the processing icity, occurrence in food and mitigation strategies. LWT
conditions for optimum honey quality based on overall – Food Science and Technology 44(4): 793–810.
attributes for thermosonication is at 90  C for 111 min. Cárcel JA, Garcı́a-Pérez JV, Benedito J and Mulet A. (2012).
Further research could be conducted by integrating Food process innovation through new technologies: Use
high-temperature short-time heating with ultrasound of ultrasound. Journal of Food Engineering 110(2):
waves to enhance honey quality. 200–207.
Chaikham P, Kemsawasd V and Apichartsrangkoon A.
(2016). Effects of conventional and ultrasound treatments
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