Chemical Papers 65 (2) 213–220 (2011) DOI: 10.2478/s11696-011-0001-x

ORIGINAL PAPER

Carnauba wax microparticles produced by melt dispersion technique‡ a

Jelena Milanovic, b Steva Levic, a Verica Manojlovic, b Viktor Nedovic, a Branko Bugarski* a Department

of Chemical Engineering, Faculty of Technology and Metallurgy, University of Belgrade, Karnegijeva 4, 11120 Belgrade, Serbia

b Department

of Food Technology and Biochemistry, Faculty of Agriculture, University in Belgrade, Nemanjina 6, 11080 Belgrade-Zemun, Serbia

Received 18 June 2010; Revised 22 November 2010; Accepted 24 November 2010

Melt dispersion technique was investigated for carnauba wax microparticles production. Microbeads with spherical shape and narrow size distribution were produced. The main objective of this study was to investigate the effect of significant process variables (initial wax concentration, stirring speed, stirring time, and surfactants) on sphericity, size distribution, and morphological properties of wax microparticles. Optimal conditions were evaluated on the basis of particle size distribution and visual analysis. Surface morphology of microparticles was characterized by scanning electron microscopy (SEM). Effects of process conditions on the size distribution of particles were evaluated by sieve analysis. Main purpose of these investigations was to apply optimized parameters to aroma encapsulation for their use in food and feed industry. c 2011 Institute of Chemistry, Slovak Academy of Sciences
Keywords: melt dispersion, carnauba wax, surfactants, microparticles

Introduction In recent years, encapsulation has become a very attractive process for food ingredients, chemicals, drugs, and cosmetics protection (Fuchs et al., 2006; Albertini et al., 2008). The main objective of encapsulation processes is to build a barrier between the components in the particle and the environment. This barrier may protect against oxygen, water, or light. Also, this barrier could be useful in avoiding the contact between ingredients in food products or to ensure a controlled release of the encapsulated materials. The main requests for encapsulation are low price of the process and retention of encapsulated materials inside the barrier (Mellema et al., 2006). The usual materials

used as a barrier in the encapsulation process are proteins (e.g. milk, gelatine), gums (e.g. acacia), carbohydrates (e.g. sucrose, maltodextrins, modified starch, cyclodextrins, and cellulose), lipids, fats, waxes, and fibres (Fuchs et al., 2006). Natural waxes have been extensively used in various fields such as polishes for protection of leather, floors and cars, glazing for paper, coating materials for pharmaceuticals, and foods ingredients (Wang et al., 2001). Waxes are suitable for applications in the food industry and human nutrition due to their water insoluble nature, minimal effects on food properties and on ingredients during digestion (Kamble et al., 2004). Carnauba wax has numerous applications in industry due to the highest melting point among the commercial vegetable waxes.

*Corresponding author, e-mail: [email protected] ‡ Presented at the 37th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 24–28 May 2010.

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In general, natural waxes such as carnauba wax are complex mixtures of different chemical compounds (Wang et al., 2001). They have been used for entrapment of various compounds. Up to now, most research on wax carriers has been done on encapsulation of pharmaceuticals. Thus, a mixture of waxes was used to produce microparticles loaded with verapamil hydrochloride; the obtained microparticles showed promising results in terms of regular shape and high encapsulation efficiency (Passerini et al., 2003). Also, a mixture of carnauba wax and stearic acid was studied for the encapsulation of theophylline and fenbufen by an ultrasonic atomizer (spray congealing technique) (Rodriguez et al., 1999). Cheboyina and Wyandt (2008) reported a freeze pelletization technique for the production of wax pellets loaded with water-soluble drugs. In another study, carnauba wax granules encapsulating diclofenac sodium as a model drug were produced by a twin screw compounding extruder (Miyagawa et al., 1996). Also, carnauba wax has been used for the production of tablets contain¨ ing metronidazole (Ozyazıcı et al., 2006). Recent studies have shown promising potential of the encapsulation process for sunscreens retention into carnauba wax nanoparticles (Villalobos-Hernández & M¨ ullerGoymann, 2005, 2006a, 2006b, 2007). The most widely used techniques for microparticles production are coacervation, solvent evaporation, fluid bed coating, extrusion coating, spray drying, and spray congealing (Albertini et al., 2008). Among these, spray drying is the most extensively used encapsulation technique. Spray dried powders usually have small particle size (10–100 µm), with poor handling and reconstitution properties (Fuchs et al., 2006). Other techniques are less suitable due to many technical problems (Popplewell & Porzio, 2001). There are two most frequently used techniques for wax capsules preparation. The “solid” technique involves deposition of hot wax with the functional ingredient on a plate and provides particles with the diameter in the range of 0.1–1 cm. The “liquid” technique involves an injection of hot wax with a (model) functional ingredient (FI) into cold oil followed by stirring using a high shear mixer. This technique can be used for the preparation of particles with the diameter in the range of 150–500 µm (Mellema et al., 2006). Another promising technique is the melt dispersion technique based on the emulsification of the molten mass in the aqueous phase followed by solidification based on chilling. This technique was successfully used for the incorporation of peptides into glyceryl tripalmitate with high encapsulation efficiency and good release properties (Reithmeier et al., 2001). Solid lipid nanoparticles (SLN) prepared by the hot homogenization technique were used for retinol encapsulation (Jenning & Gohla, 2000). In this study carnauba wax was used for optimization and development of the melt dispersion process

for the production of microparticles of spherical shape with uniform and narrow size distribution. The melt dispersion technique, as a simple, solvent free, and low cost method was applied, with a few modifications compared to the original method described in literature (Gowda & Shivakumar, 2007; Singh et al., 2007). Namely, simultaneous heating of both phases (lipid and water phases) was suggested instead of separate heating of wax and water described in literature (Gowda & Shivakumar, 2007; Singh et al., 2007); this significantly simplified the procedure of emulsification. Recently, this method has been used for ethyl vanillin and certain aromas encapsulation (Milanovic et al., 2010). The effects of internal phase (carnauba wax) concentration, stirring time, and stirring speed on the microparticles properties (shape, size distribution, and surface morphology) were investigated. Also, the influence of surfactants (Tween 20, Span 40, and Span 60) was studied.

Experimental Feed grade carnauba wax was purchased from Carl Roth GmbH (Germany), emulsifiers (Tween 20, Span 40, Span 60) were supplied by Sigma–Aldrich (Germany). Preparation of microparticles was realized by melting carnauba wax in the concentration range of (3.6– 10 %) in purified water (264 cm3 ) at 95 ◦C in a termostated water bath. Carnauba wax was added to the water phase heated at a temperature by 5 ◦C to 10 ◦C higher than the melting point of wax which was completely melted before the mixing started, allowing sufficient time to equalize the temperatures of both phases. Dispersion of melted carnauba wax in water was obtained by different mixing speeds (1000– 1500 min−1 ) and different times of mixing (2–15 min) using a mechanical stirrer with two blade impellers (IKA Werke RW, Germany). Emulsifiers were added in certain ratios (0–1 %) prior to the addition of melted wax. Solidification of microdroplets was performed by cooling with cold water (2–5 ◦C). Carnauba wax microparticles obtained after solidification were collected by filtration under reduced pressure, washed and dried at 50 ◦C to a constant mass. Size distribution of the particles was evaluated by sieve analysis using a set of five standard sieves (U.S. Standard Sieve Series No. 16#, 20#, 40#, 50#, and 120#). Photo images of the produced microparticles were taken by a stereo microscope (Leiter MR6, USA) equipped with a Sony CC camera (model AAVCD5, Japan) and the Image Pro plus v.6.2 program (Media Cybernetics, 2007). Surface morphology of the microcapsules was imaged using scanning electron microscopy (SEM, Jeol JSM 6460LV instrument, Japan). Samples were coated with Au using a Spater coater device Bal-tec SCD 005 (Principality of Leichtenstein). XRD analysis was performed using an APD 2000

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Table 1. Mean particle diameter of wax microparticles as a function of process variables Wax phase mass fraction

Stirring time

Stirring speed

Concentration of the emulsifier

Mean particle diameter

%

min

min−1

mass %

µm

1.0 2.0 3.6 5.0 6.0 8.0 10 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6

4 4 4 4 4 4 4 2 4 7 10 15 4 4 4 4 4 4 4 4 4 4 4 4

1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1100 1200 1500 1100 1100 1100 1100 1100 1100 1100 1100

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.3a 0.5a 0.7a 1.0a 0.3b 0.5b 0.7b 1.0b

231.9 244.3 241.5 273.2 284.8 480.8 400.7 366.7 257.6 294.3 322.0 294.5 313.1 257.6 255.9 238.3 243.3 212.7 189.5 176.6 295.9 220.8 210.3 169.4

a) Tween 20/Span 40; b) Tween 20/Span 60.

80 60 F /%

Multifunctional X-ray diffractometer (Ital Structure, Italy) with a step of 0.4◦ s−1 and the 2θ range 10–40◦. Surface tension was measured by a Sigma Tenziometer 703D, KSV Instruments Ltd. (Finland) using the Du Nouy ring method (Katona et al., 2010) at 80 ◦C and the drop height on the carnauba wax surface was determined using a Contact anglometer, model 1501, (Micromeritics, USA) at 23 ◦C.

40 20 0

Results and discussion

0

250

750

1000

1250

Particle size/µm

Effect of oil to water phase mass ratio Microparticles were produced in the size range suitable to for food and feed applications. Namely, powders of wax microparticles entrapping aromas can be used as ingredients of food premixes. Constituents of food premixes have particle size in the range of 150– 300 µm (Card et all., 1960); therefore, this size range was the target fraction in our experiments. One of the parameters which can affect the size of the microspheres is the volume ratio of both the oil and the aqueous phases (McClements, 1999). For these experiments, the speed of the stirrer was set to 1000 min−1 . The carnauba wax bulk lipid phase was added to 264 cm3 of the aqueous phase in amounts ranging from 3.6 % to 10 % of the mass ratio. The influence of the wax phase ratio on the particle size distribution is shown in Fig. 1. The results have shown that with low mass ratio

500

Fig. 1. Particles size frequency distribution curves obtained at different wax phase mass ratios: (
) 3.6 %, ( ) 5 %, (∗) 6 %, ( ) 8 %, and ( ) 10 %. Process parameters: stirring time 4 min; stirring speed 1000 min−1 .

◦

of the wax phase, the obtained microparticles have relatively narrow size distribution while with the increase in the internal phase concentration, the obtained microparticles become larger (Table 1) and non-uniform (Fig. 1). When producing waxes/fat microspheres loaded with lithium carbonate for a controlled release (Gowda & Shivakumar, 2007), it was observed that irregularly shaped larger particles were obtained when the volume of the external phase decreased. In the turbulent regime, stable drops are determined by the balance between the fluctuation in the hydrodynamic pressure of the continuous phase

J. Milanovic et al./Chemical Papers 65 (2) 213–220 (2011)

80 60

F /%

(acting on the drop surface and inducing drop deformation) and drop capillary pressure which opposes drop deformation (Kolmogorov, 1949; Hinze, 1955). Characteristic time is needed for the deformation of droplets (Walstra & Smudlers, 1997). Fusion process of two small droplets into one bigger requires an encounter between the droplets. The encounter time is based on the diameter of the droplets and on the volume fraction of the oil in the system as well as on the degree of turbulence. The encounter time decreases with the increasing volume fraction of the oil. This might explain larger particles production as the internal phase concentration increases in our investigations. The smallest microparticles with a satisfactory size distribution were produced starting at 3.6 % of the internal phase, where the prevalent fraction was in the range of 125 µm to 297 µm (69.6 %). In relation to the ratio of the lipid phase (molten wax), it can be assumed that the emulsion will become more viscous with increasing amounts of the dispersed phase added. This may suppress the formation of eddies that can reduce the particle size. The size of the smallest eddies in the turbulent flow (Kolmogorov eddies) (Kolmogorov, 1949) depends on the viscosity and energy dissipation rate per unit mass. The size of the smallest eddies increases with the increasing viscosity value determining thus the maximal droplets size in the turbulent flow (Curle & Davies, 1968). While the microsphere size is the smallest at the lowest phase mass ratio of 3.6 %, the amount of microspheres generated at such a low ratio is also reduced. The optimum phase mass ratio should be chosen between low values giving small and uniform microspheres and high values providing large amounts of micropaticles.

40 20 0 0

250

500

750

1000

1250

Partcle size/µm Fig. 2. Particles size frequency distribution curves obtained at different stirring times: (
) 2 min, ( ) 4 min, ( ) 7 min, (∗) 10 min, ( ) 15 min. Process parameters: stirring speed 1000 min−1 ; wax phase fraction 3.6 %.

◦

80 60

F /%

216

40 20 0 0

250

500

750

1000

1250

Particle size/µm Fig. 3. Particles size frequency distribution curves obtained at different stirring speed: ( ) 1000 min−1 , (
) 1100 min−1 , ( ) 1200 min−1 , and (∗) 1500 min−1 . Process parameters: stirring time 4 min; wax phase fraction 3.6 %.

Effect of stirring time The effect of stirring time (2 min, 4 min, 7 min, 10 min, and 15 min) on the particle size was also investigated. The particle size distribution as a function of stirring time is presented in Fig. 2. Obtained results suggest that it is necessary to apply mixing time up to 4 min as microparticles with narrow distribution are needed. The sample produced by employing stirring time of 4 min is considered as the most suitable since it has the highest contribution of the fraction in the size range of 125 µm to 297 µm (70.7 %). Mixing time of 2 min was insufficient for the formation of droplets with the appropriate size distribution. Effect of stirring speed The size distribution of microparticles as a function of stirring speed is shown in Fig. 3. Applied speeds were: 1000 min−1 , 1100 min−1 , 1200 min−1 , and 1500 min−1 . The values of Reynolds number (Re) and impeller power were calculated on the basis of

the used vessel and impeller geometries. The calculated values for Re and for the impeller power were 1.06 × 105 and 165 W, respectively. Results obtained for different mixing speeds show that with increasing speed, smaller particles with the mean diameter of less than ≈ 240 µm are obtained (Table 1). At the lowest speed (1000 min−1 ), the distribution is wide, encompassing a wide mass range of particles (≈ 45 %) larger than 300 µm. Producing carbazepine loaded cetyl palmitata liposphere, Barakat and Yassin (2006) found that by increasing the stirring speed from 400 min−1 to 800 min−1 , the average size of microspheres decreased gradually from 441 µm to 220 µm. The relationship between the mean size of the microspheres and the speed of agitation is not linear. Similar impact of the speed increase on the production of microparticles was observed by Singh et al. (2007) and Gowda and Shivakumar (2007). As the droplet or microsphere size is determined by the balance between the interfacial forces existing between the phases, it can be assumed that disruptive

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80

F/%

60 40 20 0 0

250

500

750

1000

1250

Particle size/µm Fig. 4. Particles size frequency distribution curves obtained at emulsifier Tween 20/Span 40 concentration of ( ) 0.3 %, ( ) 0.5 %, (
) 0.7 %, and ( ) 1.0 %. Process parameters: stirring time 4 min; stirring speed 1000 min−1 ; wax phase fraction 3.6 %.

◦

80 60

F /%

forces produced by the mixer at a speed lower than 1100 min−1 are not strong enough to break interfacial forces, which would result in smaller microparticles. In addition, further optical microscopy examination showed irregular shape of larger particles produced. When speed was increased, the distribution narrowed significantly and the microsphere size reduced since there were less than 20 % of fractions with large particles (in the range of 297 µm to 420 µm). Generally, at the speed of 1100 min−1 and 1200 min−1 , very little difference in the polydispersity of the suspensions can be seen. The highest speed, 1500 min−1 , resulted in undesirable small particles with the diameter below 125 µm (15 %). On base of the results presented here, the following process variables were selected as the optimal: wax to water mass ratio in dispersion: 3.6 %; stirring time: 4 min, stirring speed: 1100 min−1 . Based on these parameters, the effect of different ratios of surfactants was investigated. In an oil-in-water system, such as the one used in this study, the role of the emulsifier is to reduce the surface tension of the oil phase allowing easier spreading and also to lower the interfacial tension between phases. This enables the formation of a stable microemulsion. A surfactant or a surfactant pair from a number of available chemical types with correct solubility for a unique application has to be selected. Singh et al. (2007) used carnauba wax matrix and produced wax microparticles by the emulsification method with an addition of Tween 80 as the emulsifier; this was the first step of the production of pellets by the thermal sintering technique. Two different mixtures (Tween 20/Span 40 and Tween 20/Span 60) were used as surfactant agents in this study. Tween surfactants are ethoxylated sorbitan esters while Span surfactants are fatty acid esters of sorbitol; they were selected based on their chemical similarity with carnauba wax. Namely, carnauba wax also consists of esters of hydroxylated unsaturated long-chain fatty acids with long-chain alcohols. The particular blends (mass ratio of Tween 20 to Span 40 was 0.53 : 0.47, and mass ratio of Tween 20 to Span 60 was 0.60 : 0.40) have hydrophilic–lipophilic balance (HLB) values matching the required hydrophilic– lipophilic balance value (HLBreq 12 of carnauba wax) (Holmberg et al., 2003). Singh et al. (2007) used the Tween 80 surfactant to produce carnauba wax microparticles with the help of a high-shear homogenizer. Wax particles of 45 µm to 75 µm were obtained. When the concentration of the surfactant was kept below 1 %, significant changes in the particle size and shape were observed. Increasing the concentration to 1.5 % did not make much difference; thus, the concentration of the surfactant was optimized at 1 %. In our study, both surfactant mixtures were added in the concentration range from 0.3 % to 1 % and their effects on the particle size distribution were compared. The results of particle size analysis for Tween 20/Span

40 20 0

0

250

500

750

1000

1250

Particle size/µm Fig. 5. Particles size frequency distribution curves obtained at different concentration of emulsifiers Tween 20/Span 60: 0.3 % ( ), 0.5 % ( ), 0.7 % (
), and 1.0 % ( ). Process parameters: stirring time 4 min; stirring speed 1000 min−1 ; wax phase fraction 3.6 %.

◦

40 and Tween 20/Span 60 are shown in Figs. 4 and 5, respectively. As it can be seen, high ratio (≈ 80 %) of the target fraction (125–297 µm) was reached in all experiments. Worse results were obtained at the concentration of 0.3 % of the surfactant mixture Tween 20/Span 40. Herein, the fraction with larger particles (in the region of 420–833 µm) was higher than 10 % and thus considered as unacceptable. When high concentration of the surfactant is present (0.7 % and 1 %), small particles (< 125 µm) are formed in high portions (19 % and 27 %, respectively). The most suitable concentration of the surfactant mixture Tween 20/Span 40 appeared to be 0.5 % providing microparticles with the mean diameter of 212.7 µm, high percentage of the target fraction (79 %) and a small amount of large particles (≈ 10 %).

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A

B Fig. 6. SEM image of wax microparticles produced by the melt dispersion technique with an addition of emulsifier Tween 20/Span 40 (0.5 %).

Fig. 8. SEM image of a carnauba wax microparticle (A) and a cracked carnauba wax microcapsule (B).

Fig. 7. Microscope image of wax microparticles produced by the melt dispersion technique.

Curves of the particle size distribution presented in Fig. 5 show that the Tween 20/Span 60 surfactant mixture gives microparticles of non-uniform size in all concentrations. According to the mixed surfactant theory, the most favourable surfactant combination is that in which the hydrophilic surfactant is capable of forming a complex with the hydrophobic one. Due to the molecular complex of the surfactant molecules formed, the interfacial film withstands higher pressures than either of the components alone (Dunker, 1960). Mixtures produce a stronger film with higher resistance to rupture forming a more stable emulsion, i.e. an

emulsion whose droplets are less liable to coalescence. Span 40 has a shorter hydrocarbon chain compared to Span 60 (for two CH2 groups). Better packing of Span 40 with Tween 20 and the formation of an interfacial layer which is more resistant to breakage might explain better emulsifying properties and finally better size distribution of the produced particles. Surfactant mixture concentrations which resulted in satisfying particle size distribution were used for surface tension and drop height measurements: 0.5 % for Tween20/Span40 and 1 % for Tween20/Span60. The following values of the surface tension and drop height were obtained: 25.03 mN m−1 , 2.016 mm for Tween20/Span40, and 28.81 mN m−1 , 2.285 mm for Tween20/Span60. Higher values obtained with the Tween 20/Span 60 mixture indicate lower affinity to carnauba wax, which consequently resulted in worse emulsifying properties. The SEM and microscope images of wax microparticles produced by the melt dispersion technique with

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5000

of some thermostable food compounds or pharmaceuticals were optimized.

Intensity/a.u.

4000

Acknowledgements. This work was supported by the Ministry of Science and Technological Development, Republic of Serbia (grant III46010) and by the COST action FA0907 Bioflavour.

3000 2000

References 1000 0 10

20

30

40

50

2θ /° Fig. 9. XRD pattern of carnauba wax microparticles.

and without an addition of emulsifiers are shown in Figs. 6 and 7, respectively. According to these pictures, both microparticle samples have mainly spherical shape. Fig. 8 indicates smooth surface of the beads. In Fig. 8B, a crushed particle is shown indicating that the particles do not show matrix type appearance with homogenous structure. Instead, the fine core/shell microstructure can be seen. The rupture of surface opened the empty interior of beads and showed their wall thickness of the order of several tenths of micrometers. This finding can be significant for the aroma encapsulation and aroma release and is to be an object of further investigations in this field. Powder X-ray diffraction analysis (XRD) was carried out to investigate crystal structure of the carnauba wax particles obtained using the melt dispersion technique. The X-ray diffraction pattern is shown in Fig. 9. No new peaks were detected in the XRD pattern as compared to carnauba wax. Donhowe and Fennema (1993) reported two significant peaks of carnauba wax at (21.5◦ and 23.8◦) 2θ, which can also be seen in Fig. 9.

Conclusions In this study, microparticles of carnauba wax with a narrow size distribution were successfully produced by the melt dispersion technique. From the practical point of view, the goal was to produce particles in the size range of 150 µm to 300 µm. Spherical and uniform microbeads were obtained in the presence of a Tween 20/Span 40 mixture as the emulsifier. The optimum process variables were selected as the wax to water phase ratio in dispersion of 3.6 %; stirring time of 4 min, stirring speed of 1100 min−1 , Tween 20/Span 40 mass fraction of 0.5 %. Under these conditions, microparticles with the mean diameter of 212.7 µm, with high percentage of the target fraction (79 %) and a small amount of large particles (≈ 10 %) were produced. SEM images showed smooth microparticle surface. Experimental conditions of the melt dispersion technique which can be applied for encapsulation

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