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ORIGINAL RESEARCH ARTICLE Physicochemical properties, antioxidant activity
and stability of spray-dried propolis
Felipe C. da Silva1, Carmen S. Favaro-Trindade1*, Severino M. de Alencar2, Marcelo Thomazini1
and Julio C C. Balieiro1
1 Universidade de São Paulo - Faculdade de Zootecnia e Engenharia de Alimentos. Av. Duque de Caxias Norte, 225. CP 23. CEP: 13535 900, Pirassununga –SP, Brazil. 2 Universidade de São Paulo - Escola Superior de Agricultura “Luiz de Queiroz”, Av. Pádua Dias, 11. CEP: 13418900, Piracicaba –SP, Brazil. Received 03 September 2010, accepted subject to revision 11 January 2011, accepted for publication 14 February 2010. *Corresponding author: Email: carmenft@usp.br
Summary The aim of this study was to prepare and characterise alcohol-free propolis powder obtained using the spray-drying technique. The inlet air
temperature that caused the least loss of phenolic compounds during spray drying was determined. A temperature of 120ºC was selected,
and a sprayed dried sample of propolis was obtained. The sample was evaluated for moisture, water activity, hygroscopicity, solubility,
thermal behaviour, morphology, stability of flavonoids and antioxidant activity whilst stored for 120 days at a temperature under 25oC. The
sample displayed low hygroscopicity and water solubility. Its flavonoids remained stable during the storage period; however, the sample
presented a tendency to agglomerate and it did not present a well-defined glass transition range. The spray drying process caused significant
loss of phenolic compounds (approximately 30%) but antioxidant properties were retained. Thus, it was concluded that the spray-drying
dehydration process may be used to obtain alcohol-free propolis powder that possesses antioxidant activity and stable flavonoids during
storage.
Keywords: dehydration, propolis, spray drying, hygroscopicity, flavonoids, storage, solubility.
Journal of ApiProduct and ApiMedical Science 3 (2): 94 - 100 (2011) © IBRA 2011 DOI 10.3896/IBRA.4.03.2.05
Introduction Propolis is a resin with variable colour and consistency, and it is
collected by bees from different parts of plants, such as flower buds
and resin exudates (Ghisalberti, 1979). Propolis extract has been
utilised in popular medicine since 300 B.C. (Banskota et al., 1998),
and its worldwide consumption is approximately 700-800 tons per
year (Da Silva et. al., 2006).
Propolis presents a strong and characteristic smell due to its
volatile phenolic acid fraction, strong adhesive properties and
complex chemistry (55% of resins and balsams, 30% of wax, 10%
of volatile oils and around 5% of pollen), as well as mechanical
impurities (Thomson, 1990; Banskota et al., 2001).
Due to its colour, in the Brazilian southeast it is known as
green propolis, with Baccharis dracunculifolia (Asteraceae) the most
prominent plant source (Marcucci et al., 1996; Park et al., 2002;
Alencar et al., 2005). Some of the main compounds found in green
propolis are caffeic, ferulic, p-coumaric and cinnamic acids (Bankova
et al., 2000; Funari et al., 2007).
Propolis is known for its antimicrobial, anaesthetic, anti-
inflammatory, healing (Bankova, 1989; Ghisalberti, 1979),
antioxidant (Kumazawa et al., 2004; Alencar et al., 2007) and
anticancer properties (Duarte et al., 2006), which allow its use not
only in pharmaceutics but also in the food sector as a natural
additive.
Because it is suspected that synthetic compounds that are
added to food may be toxic, there is an increasing interest in
searching for natural products to replace them. Some industries such
as those related to food additives, pharmaceutics and cosmetics have
been searching for bioactive compounds that are obtained from
natural products. Antioxidant compounds from propolis may improve
the shelf-life of products, particularly in the food industry, by slowing
down the lipid peroxidation process, which is one of the most
important causes of food deterioration during storage (Halliwell,
S% =
1999). In this context, obtaining natural extracts in powder form,
(such as propolis), may be of value in pharmaceutical formulations
such in the production of pills and capsules (Marquele et al. 2006).
Natural extracts might also be useful in the food industry as
ingredients of functional food formulas.
Marquele et al. (2006) dried propolis extracts using a spray-
drying method and noted that after atomisation under various inlet
air temperatures there were variable losses of total phenolics and
flavonoids from 45.1 % to 54.9 % and 30.6 % to 40.8 %,
respectively. Subsequently, Souza et al. (2007) dried propolis by
spray-drying and concluded that the propolis obtained showed a
similar antioxidant activity to that of the ethanolic extract, as
determined by the DPPH method. These authors did not evaluate
the physicochemical properties of the yielded material or its stability
during storage.
The aim of this work was to obtain propolis in an alcohol-free
powder form using the spray-drying technique, as well as to
characterise its moisture, hygroscopicity, solubility, resistance to the
process of spray drying, flavonoid stability during its storage and
antioxidant activity.
Materials and methods Materials A sample of type-12 propolis was collected in the city of Mogi Guaçu
-SP, Brazil. This propolis, which is a Brazilian green propolis,
received the denomination Type-12 according to Park et al. (2000).
The ethanolic extract of the propolis was prepared as described by
Nori et al. (2011). Approximately 30 g of propolis was crushed in a
blender, mixed with 100 mL of ethanol 80% and then placed in a
water bath at 50°C with mechanical stirring (Marconi, model MA
085, Brazil) for 30 minutes.
Spray drying
The extract was atomised in a lab-bench spray dryer, (model MSD
1.0, Labmaq do Brasil Ltda. Ribeirão Preto, Brazil). In the
atomisation process, 3 inlet air temperatures (80ºC, 100ºC and
120ºC) were tested. The other operational parameters of the spray
dryer included an air flow of 0.60 m3/min, a feed flow of 0.60 m3/
min and a nozzle diameter of 1.3 mm.
It was judged that the best drying temperature would be that
which would result in the lowest reduction in total phenolic
compounds from the propolis extract. Thus, the total phenolic
content in both the liquid and dried propolis samples was
determined by spectrophotometry, as described below.
Once the optimum inlet air temperature for the atomisation
process had been determined, it was used to obtain samples that
were analysed according to the methodology described below. All
processes and analyses were carried out in triplicate.
Determination of the amount of total phenolic
compounds in the propolis
Total phenolic compounds in the spray-dried propolis was estimated
according to the Folin-Ciocateau spectrophotometric method, as
described by Woisky and Salatino (1998), employing gallic acid as a
standard. For this assay, a 50 mg sample of spray-dried propolis was
diluted in 80% ethanol in a 50 mL volumetric flask. A 5 mL aliquot
was then transferred to a 25 mL volumetric flask that was filled with
80% ethanol. For the colourimetric reaction, 0.5 mL of the precedent
dilution was transferred to a tube and was mixed with 2.5 mL of a
1:10 water-diluted Folin-Ciocalteau reagent. After resting for 5 min, 2
mL of 4 % sodium carbonate was added to the mixture and was then
allowed to rest for another 2 h protected from light. The absorbance
at 740 nm was measured using a UV/Vis model Libra S22
spectrophotometer (Biochrom, England) and results were expressed
as gallic acid equivalent per 100 g sample.
Determination of the solubility
The method described by Singh and Singh (2003) consisted of
determining the solubility of the spray dried sample in cold water. A
1% (w/v) powder suspension was agitated for 30 min using a shaker
(Tecnal TE-420, Piracicaba, Brazil). The suspension was then
centrifuged at 1200 rpm for 10 min, and a 25 mL aliquot was
obtained from the supernatant, deposited in a porcelain pan and
subjected to a temperature of 110ºC for 4 h in a drying oven.
Solubility was calculated according to the following equation:
grams of supernatant solids x 4
grams of sample x 100
Moisture and water activity
The moisture content of the samples was determined using a
moisture analyser (Ohaus, MB35, USA). The water activity was
measured using an Aqualab 3 analyser (Decagon Devices, USA) at
25ºC after allowing the samples to equalise with this temperature for
1 h (Rocha et al. 2009).
Hygroscopicity
Hygroscopicity was determined according to the method proposed by
Cai and Corke (2000). Samples of powder (approximately 2 g) were
placed at 25°C in a container with a saturated solution of Na2SO4
(81% RH). After one week, samples were weighed, and the
hygroscopicity was expressed as g of adsorbed moisture per 100 g of
dry solids.
Morphology of particles
The morphology of the particles was observed under a scanning
electron microscope (JEOL JSM–T300, Tokyo, Japan) at an
accelerating voltage of 5 kV. Before using the scanning electron
microscope, the samples were coated in an argon atmosphere with
Spray dried propolis 95
gold/palladium using a Balzers evaporator (model SCD 050, Baltec
Lichtenstein, Austria) (Oliveira et al., 2007).
Thermal behaviour
The thermal behaviour was determined by differential scanning
calorimetry according to Ortiz et al. (2009), using a DSC TA 2010
device controlled by a TA 5000 system (TA Instruments, New Castle,
USA).
Evaluation of the antioxidant activity
The measurement of the DPPH sequestrant activity was performed
according to the methodology described by Chen et al. (2003).
DPPH (1,1-diphenyl-2-picryl-hydrazyl) is a stable free radical that
receives an electron or a hydrogen atom to become a stable
diamagnetic molecule that can then be reduced with an antioxidant.
The reaction mixture was composed of 0.5 mL of a 50 ppm solution
of the yielded powder and ethanol (80%), 3 mL of ethanol PA and
0.3 mL of DPPH solution (0.5 mM in ethanol). After 40 minutes, the
absorbance was recorded at 517 nm at room temperature. The
antiradical activity was determined as an inhibitory percentage (IP),
estimated as the decline rate of the DPPH-extract solution
absorbance after the reaction took place (stable phase), compared
with the reference solution (DPPH-ethanol).
Determination of total flavonoids
The analysis of total flavonoids in the spray-dried propolis was
performed according to the method described by Park et al. (1995).
A colourimetric reaction was obtained by mixing 0.5 mL of the initial
powder dilution in ethanol, 4.3 mL of 80% ethanol solution, 0.1 mL
of 10% aluminum nitrate and 0.1 mL of 1M potassium acetate. After
40 minutes, the absorbance of each sample was recorded at 415 nm
using a spectrophotometer. A standard curve was prepared using
quercetin, and results were expressed as quercetin equivalent / 100
g sample.
Stability of total flavonoids during storage
After the atomisation process, the yielded material was placed in
vacuum-sealed rubber-capped flasks (Fig. 1) and was stored at
25ºC, away from light. The amount of total flavonoids was assayed
at 0, 30, 60 and 120 days after the drying process, when
approximately 50 mg of the powder was diluted in an 80% ethanol
solution in a 50 mL volumetric flask and the colourimetric reaction
described above was performed.
Statistical analysis
A complete randomised design (CRD) was employed, taking into
account the temperature effect (80ºC, 100ºC and 120ºC) and the
storage period (0, 30, 60 and 120 days) while studying the influence
of the drying temperature on the loss of total phenolic compounds
and the stability of total flavonoids, respectively. The experimental
data was statistically analysed by SAS (Statistic Analysis System)
(2001), version 8.02, using the PROCANOVA procedure. Tukey’s
Honestly Significant Difference (HSD) was adopted as the multiple
comparison procedure. P-value below 1% (p<0.01) was considered
to be significant.
Results The spray dried propolis powder tended to agglomerate, as
shown in Figure 1 and by scanning electron microscopy as shown in
Figure 2. These observations suggest that dispersing such material in
food and drugs would be difficult because their flow might be
impaired.
The total phenolic content of the ethanolic extract of propolis
(feed) was 30.6 ± 0.8 g of gallic acid/100 g of solids, on a dry weight
basis.
The total level of total phenolic content did not vary with inlet
air temperature (Table 1). The spray dried propolis samples obtained
at air inlet temperature of 120oC demonstrated low water solubility
(Table 2) and agglomerated material was observed on the water
surface. Hyroscopicity was relatively low (13.1 g absorbed water /
100 g sample) and a sticky mess resulted (Fig. 3). Ethanolic extract
of spray dried propolis (50 ppm) retained antioxidant activity (Table
2) and was found to inhibit 35% of the DPPH free radical activity.
On the basis of the thermogram (Fig. 4), it was not possible to
identify the glass transition temperature (Tg) of spray-dried propolis.
Storage of spray dies propolis for up to 120 days had no effect on
total falvonoid content (Fig. 5).
96 da Silva, Favaro-Trindade, de Alencar, Thomazini,
Fig. 1. Sample of spray-dried propolis, stored in vacuum-sealed
flask.
Spray dried propolis 97
Fig. 2. Scanning electron of particles of spray-dried propolis
(5000x).
Fig. 3. Spray-dried propolis agglomeration after water uptake in the
hygroscopicity test.
Drying temperature
(ºC)
Total phenolic compounds
(mean ± SD) (g of gallic acid/100 g of sample)
80 21.2 ± 0.4
100 20.9 ± 0.2
120 20.6 ± 0.6
Table. 1. The influence of drying temperature on the total phenolic
content.
Parameter Mean ± SD (n = 3)
Moisture (%) 11 ± 1
Water activity 0.43 ± 0.05
Hygroscopicity (g of absorbed
water/100 g propolis) 13.1 ± 0.7
Solubility (%) 9 ± 1
Antioxidant activity [50 ppm] (%) 35 ± 2
Table. 2. Characterisation of the spray-dried propolis.
Fig. 4. Thermogram of the spray-dried propolis.
Discussion
Spray drying of the propolis extract
Alcohol-free propolis powder has benefits in terms of storage,
handling and use, and it facilitates applications where alcoholic
residues would not be convenient, such as, in drugs or food.
However, in the present study, it was noted that a large portion of
the powder remained adhered to the spray dryer surface inside the
chamber and cyclone, which resulted in low efficiency and
production. An alternative to improving the efficiency of this system
might be to add a carrier agent, such as maltodextrin or gum arabic.
Effect of the drying temperature on the total
phenolic content in the propolis extract
The process of spray drying led to approximately 30% loss of
content. However, the observed loss found in this study was lower
than those observed by Marquele et al. (2006), who dehydrated the
ethanolic extract of propolis by atomising it under different
temperatures, which resulted in losses varying from 45.1% to
54.9%. Contrary to our findings, González et al. (2009), studied the
thermal stability of propolis from Tucumán, Argentina and reported
that components in propolis were stable between room temperature
and 120ºC. Such disagreement may be related to the different
sources of propolis used in each study, where the phenolic
composition profile may render different resistance characteristics to
the process. Furthermore, the operational conditions of the spray
dryer, such as the outflow, may also influence the final result.
Because the inlet temperature did not affect the content of
total phenolic compounds, we decided to carry out the remaining
experiments using the samples dried at 120ºC, because this
treatment yielded more material after the cyclone, allowing us to
recover a larger volume of sample.
In addition, as the temperature increased, there was a lower
amount of moisture in the powder, which helped to maintain its
stability.
Moisture, water activity, hygroscopicity
and solubility
In general, the moisture content and water activity values obtained
herein coincide with those for powders and reasonably guarantee the
microbiological stability of the material (Favaro-Trindade et al. 2010) The low solubility of spray-dried propolis was expected because
propolis is composed of both hydrophilic and hydrophobic
compounds, which impair its solubility in solvents with strong
polarity, such as water. Due to its constituents, the material
solubilises in solvents with intermediate polarity such as alcohol,
ether, acetone and dichloromethane, which can better interact with
those classes of substances. Such properties indicate that it would be
difficult to utilise propolis powder, especially in food applications,
where water solubility is almost always required. This problem may
be overcome by using a hydrophilic carrier during the drying process,
such as maltodextrin.
A phenomenon observed in spray-dried propolis powder after
storage is named ‘caking’ and is characterised by a loose, low
moisture powder that absorbs water forming rocks, thus becoming a
sticky material. According to Aguilera et al. (1995), the caking phase
begins with bridge formation, which results in a deformed surface
and adhesion between particles. In this phase, the small bridges
among the particles may disintegrate with any light agitation. In the
next phase, agglomeration is observed, which implies a reversible
98 da Silva, Favaro-Trindade, de Alencar, Thomazini, Balieiro
Fig. 5. Flavonoid stability during storage of spray-dried propolis.
consolidation of the bridges. This phase results in groups of particles
with structural integrity. Compacting, the next phase of caking, is
associated with the loss of integrity as a result of the enlargement of
bridges among the particles, leading to a decrease of the space
between particles and group deformation. In the final phase of
caking, the sample is liquefied. This liquidation is observed with the
dried propolis extract. Thus, the product cannot be stored or
handled in conditions where relative humidity is high.
Thermal behaviour
The glass transition temperature (Tg) for the spray-dried propolis
would be useful in determining an optimal storage temperature that
could prevent events such as caking. However, based on the
thermogram, it was possible to infer that many compounds in the
spray-dried propolis were in the crystal form, because several
melting points of the crystals were identified in the temperature
range from 53.75ºC to 153.22ºC.
Evaluation of the antioxidant activity
The spray-dried propolis showed high antioxidant properties that
were retained in the process of spray-drying. The results obtained
by the antioxidant property were higher than those obtained by
Souza et al. (2007), who observed 50% inhibition for the
concentration of 500 ppm of spray-dried propolis. Compared with
the present work, a 10-fold lower concentration led to a 35%
inhibition. The higher sequestrant activity of the DPPH radical may
be due to the different sources of propolis, which could result in
differences in their chemical profile, antioxidant capability and thus,
the resistance of the phenolic compounds to the operational
conditions in the spray dryer.
Some authors state that the biological activities of Brazilian
green propolis are mostly due to its high levels of prenylated p-
coumaric acids, mainly artepillin C and baccharin (Banskota et al.
1998; Park et al. 2002; Simões-Ambrosio et al. 2010), as well as
flavonoids such as aromadendrine-4’-methyl-ether and 5,6,7-
trihydroxy-3’-4-dimethoxiflavone (Marcucci and Bankova, 1999).
Total flavonoids stability during storage
Dehydrating the propolis using a spray-dryer did not lead to
flavonoid loss during the storage period studied here at 25ºC. On
the basis of these findings, it can be concluded that the process did
not affect the conservation of the material for at least 120 days.
Conclusions
It is possible to dry the propolis by spray drying, and such processes
pose little harm to the phenolic compounds contained in the sample.
However, the spray drying process presented low productivity, and
the yielded material tended to agglomerate during storage; that is,
it was not physically stable, which prevents it being handled or stored
in environments with high relative humidity. The yielded powder
displayed low water solubility. The propolis powder kept its
antioxidant activity and flavonoid content after storage at room
temperature (25ºC).
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