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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