Encapsulation of Anthocyanins Extract by the

Supercritical Anti-Solvent Process

Ana Paula F. Machado a,*, Camila A. Rezendeb, Rodney A. Rodriguesc, Paulo T. V. Rosab and
Julian Martíneza
a
Department of Food Engineering, College of Food Engineering, University of Campinas, 80,
Monteiro Lobato Street, 13083-862 Campinas, SP, Brazil,
b
Institute of Chemistry, University of Campinas, 126, Josué de Castro Street, 13083-861 Campinas,
SP, Brazil,
c
Biological and Agricultural Pluridisciplinary Research Center (CPQBA), University of Campinas,
999, Alexandre Cazelatto Street, 13148-218 Paulínia, SP, Brazil.
* email: anapfmachado@hotmail.com

ABSTRACT
The present work carried out the encapsulation of anthocyanin extracts obtained from
blackberry (Rubus fruticosus) residues in polyvinylpyrrolidone (PVP) using the supercritical
anti-solvent process (SAS). For comparison, the conventional methods spray-drying (SD) and
freeze-drying (FD) were also applied for the production of encapsulated particles. The
encapsulation carried out by these three techniques was made from an ethanolic extract rich in
anthocyanins obtained from the residue of blackberry lyophilized and crushed. The produced
particles were evaluated in terms of precipitation efficiency, residual ethanol content,
monomeric anthocyanin content, antioxidant capacity (by DPPH and FRAP methods) and
morphology. The SAS, SD and FD processes produced particles with good precipitation
efficiency (> 76%), low residual ethanol content (< 0.26%), high antioxidant capacity,
without involving large degradations of these compounds. They were efficient to concentrate
the anthocyanin pigments under the polymeric material (PVP), highlighting the SAS
technique, which produced particles with anthocyanin content and antioxidant capacity five
and two times higher than those obtained by SD and FD, respectively. Scanning electron
microscopy showed that the particles produced by SAS and SD had spherical shape and were
agglomerated, whereas those produced by FD presented irregular shapes. The SAS process
was quite interesting for drying the ethanol solution, showing a preferential precipitation of
anthocyanins compared to the other techniques under investigation, since supercritical CO2
has no affinity for these compounds.

INTRODUCTION
Anthocyanins are phenolic compounds belonging to the group of flavonoids and are pigments
responsible for a wide variety of colors (most of the colors blue, purple, violet and almost all
shades of red) present in vegetables, flowers, fruits and derivatives. From to the secondary
metabolism of plants, anthocyanins are of great importance for their survival, and when
ingested they are responsible for several health benefits due to their biological properties,
such as high antioxidant power. Among these benefits, we can highlught the reduction in the
incidence of many oxidative, inflammatory and cancerous diseases [1].
However, several external factors such as heat, light, acids, enzymatic and non-enzymatic
oxidation, pH, the presence of oxygen and enzymes, metals and the use of high temperatures
trigger the oxidative degradation of anthocyanins, limiting not only their final application, but
also restricting the entire process chain, from the choice of the extraction method until the
conditions that the product will face after its formulation. Therefore, encapsulation techniques
in polymer matrices can be applied with great benefits to anthocyanin extracts, aiming greater
dissolution, bioavailability, protection and stabilization of the pigment [2].
Encapsulation can be performed by several techniques, which may involve conventional
processes (coacervation, spray-dryer, lyophilization, interfacial polymerization,
emulsion/evaporation) that are well known and studied, beyond of the new technologies such
as the use of supercritical fluids [3]. The conventional techniques to produce materials in the
nanometric and/or micrometric range, besides not allowing the size control of the formed
particles, use organic solvents that lead to high residual indexes, requiring post-processing
steps, in addition to being suspected of conferring toxicity to the final product. Additionally,
these techniques result in significant loss of biological activity due to the high temperatures
applied, making them unattractive for the precipitation of thermosensitive compounds. These
facts limit their use, since the functionality and the application properties of a micro or
nanoparticulate material are highly dependent on the size, morphology and crystalline
structure of the particle [4].
The use of supercritical fluids is an alternative to conventional encapsulation processes,
motivated by the exploration of their peculiar characteristics, especially carbon dioxide (CO2),
which is the most used supercritical fluid for precipitation. CO2 is versatile, non-toxic, non-
flammable, chemically inert, environmentally acceptable, low cost, besides not leaving toxic
residues in the final product, and its critical properties are relatively mild (critical temperature
(Tc) = 31.1 ° C, critical pressure (Pc) = 7.38 MPa - relevant factors to reduce operating costs
and thermal degradation of bioactive compounds) [3, 5]. Encapsulation/precipitation
processes using supercritical fluids are classified according to their function in the process:
solvent (Rapid Expansion of Supercritical Solutions (RESS), Supercritical Solvent
Impregnation (SSI)); solute (Particles from Gas Saturated Solutions (PGSS)) ou anti-solvent
(Supercritical Anti-Solvent (SAS); Supercritical Fluid Extraction of Emulsions (SFEE)) [3,
5]. For the processes employing pressurized fluids as antisolvent, particles can be obtained on
the nanometric or micrometer scale with narrow size distribution, in addition to high
percentages of encapsulation [3]. Literature reports studies that indicate the possibility of
using this technique for precipitation and encapsulation of several materials [3, 5-7].
In this context, the present work aimed to carry out the encapsulation of anthocyanin
extracts obtained from blackberry residues in polyvinylpyrrolidone (PVP) using the
supercritical anti-solvent process (SAS), with supercritical CO2 acting as antisolvent and
ethanol as solvent for the organic solution (extract + PVP). For comparison, the conventional
methods spray-drying (SD) and freeze-drying (FD) were also applied for the production of
encapsulated particles.

MATERIALS AND METHODS

Raw Material
The raw material used as source of anthocyanins in this work was the residue generated from
the processing of blackberry (Rubus fruticosus).
Production of Antocianic Ethanol Extract
The ethanolic extract rich in anthocyanins was produced by manual maceration and
mechanical agitation using a shaker (Eberbach, E6010). A volume of 350 mL of acidified
ethanol (pH 3.0 - adjusted by the direct addition of citric acid) was added in 10 erlenmeyers
containing 15 grams of lyophilized and triturated blackberry residue in each one. The samples
were then macerated, sealed and taken to the shaker. They were simultaneously subjected to
90 min stirring and then filtered through a vacuum filtration system. The extract produced had
1.50% total solids.

Production of the Polymer Solution

The organic solutions (extract + polymer) to be used directly in the particle formation tests by
SAS, SD and FD were prepared in the proportions of total solids of extract and
polyvinylpyrrolidone (PVP - average molecular weight 10000 g/mol) polymer of 1:0.4 and
1:2.5 w/w, respectively.

Particle Formation Processes

Supercritical Anti-solvent
The precipitation of the anthocyanin extract by the SAS technique was performed in the
equipment shown schematically in Figure 1. The experiments were performed in triplicate, at
the following conditions: supercritical antisolvent = carbon dioxide (CO2); temperature (T) =
40 °C; pressure (P) = 12.5 MPa; flow rate of feed organic solution (Qsolution) = 0.5 mL/min;
flow rate supercritical anti-solvent (QCO2) = 20.4 g/min; volume of injected organic solution
(Vinjection) = 35 mL; and time for removal of the residual organic solvent (ethanol) from the
precipitated particles (tdrying) = 60 min.
Spray-Drying
For the formation of particles by spray-drying (SD), the organic solutions were dried using the
Mini Spray-Dryer BÜCHI B-290 (Switzerland). The experiments were performed in triplicate
at the following process conditions: Drying fluid = compressed nitrogen (N2); input
temperature (Tin) = 105 ºC; outlet temperature (Tout) = 80 ºC; Qsolution = 3.0 mL/min; QN2 =
536 L/h e Vinjeçtion = 200 mL.
Freeze-Drying
In order to obtain a material dried by the freeze-drying process (FD) from the ethanol
solutions, they had first undergone a rotavaporation process (Marconi, MA-120, SP, Brazil) to
completely remove the ethanol present in the mixture. After being evaporated, the samples
were resuspended with water and then frozen in glass flasks at -18 °C for one day. Then, they
were submitted to the freeze-drying process using a benchtop freeze-drier Liotop L101 (São
Carlos, Brazil).
 Figure 1. Schematic diagram of the precipitation unit using the supercritical anti-solvent configuration. C1 –
CO2 cylinder; F1 – CO2 filter; M1 and M2 – pressure gauges; CB – cooling bath; C – compressor; V1, V2, V3,
V4, V5 e V6 – blocking valves; F2 – compressed air filter; Pump – pneumatic pump; HB – heating bath; HPLC
Pump; SV – safety valve; CP = cell of precipitation; V7 – micrometric valve; I1 – temperature indicator; CV –
collection vase; FL – flow meter; TOT – gas totalizer.

Particle Characterization
Precipitation Efficiency
The precipitation efficiency (PE) was evaluated by the ratio between the mass of the
encapsulated active principle, which in this case were the anthocyanins, and the theoretical
mass of the encapsulated particles, which is the total mass of active principle inserted in the
precipitation vessel.
Residual Ethanol Content
The residual ethanol content (REC) present in the particles was determined using a gas
chromatograph (Agilent, model HP-6890) coupled to a mass selective detector (Agilent,
model HP-5975). The REC was expressed in percentage.
Total Monomeric Anthocyanins Content
The quantification of the monomeric anthocyanins content (MAC) was performed by the
differential pH method of GIUSTI and WROLSTAD [8]. The results were expressed in mg of
cyanidin-3-O-glycoside equivalent (Cy3GlE)/g of particle.
Antioxidant Capacity
The antioxidant capacity (AC) of the particles was evaluated using the free radical capture
method DPPH (1,1-diphenyl-2-picrylhydrazyl) and the iron reduction method (FRAP)
according to the adaptations of BRAND-WILLIAMS, CUVELIER and BERSET [9] and
SILVA et al. [10], respectively. The CA, by both methods, was expressed as µmol trolox
equivalent (TE)/g of particle.
Morphology
The analysis particle morphology was performed using a scanning electron microscope
(SEM) equipped with a field emission cannon (FESEM - FEI ®, Inspect F50). A large
number of images at different magnifications (150 to 50000x) was obtained in different areas
of the samples to ensure the reproducibility of the results.
RESULTS
Table 1 shows the precipitation efficiency, residual ethanol content, anthocyanin content and
antioxidant capacity (DPPH and FRAP methods) of the particles obtained in each drying
process. It is noted that the SAS and SD processes did not differ significantly in terms of
efficiency in precipitating anthocyanins. However, a significant loss of the pigments initially
contained in the feed solution can be verified. This is because anthocyanins are highly
sensitive to adverse environmental conditions and also certain processing conditions, thus
affecting their stability. It can be seen that the processes SAS and FD produced particles
without presence of residual ethanol, whereas those obtained by SD resulted in average REC
of 0.26%, which can be considered low. The SAS technique was far more efficient in
concentrating compounds with antioxidant character, resulting in approximately five and two
times more anthocyanins and antioxidant capacity, respectively, than those obtained by SD
and FD. It was clear, then, that supercritical CO2, a predominantly non-polar substance,
besides efficiently removing ethanol from the organic solution under the SAS studied
conditions, also extracted substances with lipophilic character contained in the extracts, thus
generating a precipitate more concentrated in anthocyanins when compared with the other
investigated methods.
Table 1. Precipitation efficiency, residual ethanol content, monomer anthocyanin content and antioxidant
capacity of the dry samples produced by SAS, SD and FD.

Antioxidant Capacity
PE REC MAC DPPH FRAP
Samples
(%) (%) (mg Cy3GlE/g) (μmol TE/g) (μmol TE/g)
A,B A A A
SAS 85,23 ± 2,53 0,00 ± 0,00 6,61 ± 0,05 253,81 ± 7,82 321,88 ± 9,22A
SD 76,26 ± 2,82A 0,26 ± 0,01B 1,36 ± 0,08B 98,05 ± 3,69B 183,12 ± 1,28B
FD 92,76 ± 1,20B 0,00 ± 0,00A 1,10 ± 0,04B 113,60 ± 2,88B 188,48 ± 2,75B
- Results expressed by their mean ± standard deviation (SD).
- Equal capital letters in the same column indicate that there is no significant difference at the 5% level of
significance by the Tukey’s test.

Figure 2 shows the micrographs obtained by MEV of the precipitates produced by each
process. It can be noted that different morphologies were obtained from each process, but
each with the typical characteristics described in literature [5-7, 11]. It is observed that
particles obtained by SAS (A) and SD (B) presented spherical forms. However, those
obtained by SAS are quite agglomerated, which led to the deformation of their shapes due to
their coalescence, whereas those obtained by SD are slightly agglomerated, presenting a better
defined shape and almost completely smooth surface. The particles obtained by FD (C)
exhibited irregular shape, having a continuous porous surface.

CONCLUSION
In conclusion, the SAS process was interesting for both encapsulation and concentration of
the extract, and allowed obtaining a preferential precipitation of the anthocyanins, since
supercritical CO2 has no affinity with the anthocyanins and the process conditions are
relatively mild.
 (A) (B) (C)

Figure 2. Micrographs by scanning electron microscopy of polymer precipitates containing anthocyanin extract.
(A), (B) and (C) refer to the images of the samples obtained by SAS, SD and FD.

ACKNOWLEDGEMENTS
The authors wish to thank CNPq (140268/2014-6) for the PhD scholarship, FAPESP
(2013/02203-6 and 2015/11932-7) and CAPES (2952/2011) for the financial support.

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