Accepted Manuscript

Title: Citronella essential oil microencapsulation by complex

coacervation with leather waste gelatin and sodium alginate

Authors: Eric F. de Matos, Bianca S. Scopel, Aline Dettmer

PII: S2213-3437(18)30123-4
DOI: https://doi.org/10.1016/j.jece.2018.03.002
Reference: JECE 2243

To appear in:

Received date: 1-12-2017

Revised date: 6-2-2018
Accepted date: 1-3-2018

Please cite this article as: Eric F.de Matos, Bianca S.Scopel, Aline Dettmer,
Citronella essential oil microencapsulation by complex coacervation with
leather waste gelatin and sodium alginate, Journal of Environmental Chemical
Engineering https://doi.org/10.1016/j.jece.2018.03.002

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<AT>Citronella essential oil microencapsulation by complex coacervation with leather
waste gelatin and sodium alginate

<AU>Eric F. de Matosa; Bianca S. Scopelb* bianca.scopel1@gmail.com; Aline Dettmerc*

##Email##alinedettmer@gmail.com##/Email##
<AU>

<AFF>aChemical Engineering - University of Caxias do Sul – 1130, Francisco Getúlio

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Vargas Street, Caxias do Sul, Brazil, ZIP CODE: 95070-560.
<AFF>bPost-Graduation Program in Mining, Metallurgical and Materials Engineering –
Federal University of Rio Grande do Sul – 9500, Bento Gonçalves Avenue, Porto Alegre,

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Brazil, ZIP CODE: 91501-970
<AFF>cChemical Engineering – University of Passo Fundo – BR 285, São José, Passo

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Fundo, Brazil, ZIP CODE: 99052-900
<PA>*
Correspondent authors: Bianca S. Scopel – Aline Dettmer.
ABSTRACT – Thousands of tons of chromium tanned leather wastes (CTLW) are
produced worldwide. Alternatives for their use as raw material in other processes are

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particularly desired. In this study, gelatin extracted from CTLW was used in combination
with sodium alginate as encapsulating agent to prepare microcapsules of citronella essential
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oil (CEO) by complex coacervation. The effect of gelatin and CEO concentration on the
encapsulation efficiency was investigated using commercial gelatin. Best encapsulation
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conditions (mainly determined by morphology parameters) were found when 4% of gelatin
and 10% of CEO were used, resulting in 83.5% of microencapsulation yield.
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Microencapsulation with CTLW gelatin was performed according to the best encapsulation
conditions. It resulted in an efficiency of 73.7%, produced microcapsules with a diameter of
434.06 µm, and with a core completely covered by the encapsulating agent.
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Microencapsulation process with gelatin from CTLW, as presented in this paper, was
shown to be an effective method that will allow controlled release of CEO.
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<KWD>Keywords: – Chromium; Hydrolysis; Microencapsulation efficiency;

Microencapsulation yield; Wet blue
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<H1>1. INTRODUCTION
Chromium tanned leather wastes (CTLW) are generated after hides are tanned with
chromium III, when the leather is shaved to even out its thickness. This process generates,
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more specifically, wet blue leather wastes, which have not being through finishing
processes, as dyeing. Leather waste is made of a fibrous structural protein, called collagen,
which is crosslinked with chromium III. Chromium content in CTLW ranges from 3 to 6%
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[1]. Alternatives targeting CTLW reuse involve from chromium to gelatin recovery.
Chromium can be used in the tanning process and gelatin is applied as raw material to
produce high added value products such as polymeric films or microcapsules [2-5].
Microencapsulation is a technology that applies thin polymeric coatings on solids, liquid
droplets, or gaseous materials in order to form particles called microcapsules. These
particles can release their content under specific conditions at a specific speed [6,7].
Complex coacervation is a process used to produce microcapsules. It forms a coating
capable of protecting the encapsulated active compounds [8,9]. This process consists of a
spontaneous phase separation, which leads to the formation of an insoluble polymer
complex (coacervate – the polymer-rich phase), and of another phase, which is poor in
polymer [10]. Complex coacervation involves at least two substances, typically
biopolymers of opposite ionic charges, in an aqueous system. The basic principle for the
formation of ionic charges on biopolymers, usually a protein and a polysaccharide, is the
pH change [11]. Proteins are positively charged below their isoelectric point and negatively
charged above it. Polysaccharides with anionic carboxyl groups, in their turn, act as
polyanions and, in contact with proteins, produce electrostatic complexes at pH values

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ranging generally from 2 to 5, depending on the type of protein [12,13]. Due to these
characteristics, gelatin and sodium alginate are appropriate coating materials for
microencapsulation processes [14].
Essential oils are volatile substances made of a complex mixture of components found in

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herbs. These components are terpenes, sesquiterpenes, and, in some plants,
phenylpropanoids, with small molecules such as alcohols, aldehydes and short-chain

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ketones. Oils have limited use due to their volatile characteristics, sensitivity to heat, and
easy oxidation. Thus, they require some external protection for appropriate use [15].
Citronella essential oil (CEO) has been widely used due to its medicinal and aromatic
properties. Its main components are citronellal, geraniol, and citronellol. Citronellol is

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considered an excellent insect repellent, and presents bactericide and antifungal action [16].
The microencapsulation of volatile essential oils can be an effective solution to increase
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their stability and, consequently, to improve their efficiency, giving them a longer lifetime.
The aim of this study was, therefore, to produce microcapsules containing CEO using
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CTLW gelatin and sodium alginate by complex coacervation.
<H1>2. MATERIAL AND METHODS
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<H2>2.1. Material
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CTLW used in this work (wet blue shavings) were provided by a local tannery (Ritter –
Brazil). Gelatin was extracted from it with magnesium oxide (Neon, Brazil). Sodium
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alginate (Dinâmica, Brazil), glutaraldehyde (Dinâmica, Brazil), and acetic acid (Vetec,
Brazil) were used in the microencapsulation process of CEO (Cimbopogon winterianus).
For microencapsulation, it was also used a commercial gelatin (Dr. Oetker, Brazil) and the
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gelatin extracted from CTLW.

<H2>2.2. Methods
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<H3>2.2.1. CTLW gelatin extraction

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For alkaline hydrolysis, 50 g of CTLW were mixed with 2 g of MgO and 250 mL of
deionized water in a 500 mL Erlenmeyer flask. Hydrolysis was conducted in an orbital
shaker (MA 832, Marconi, Brazil) for 6 h at 70°C and 180 rpm. Gelatin in aqueous medium
was separated from chrome cake through vacuum filtration [2,5].
 CTLW gelatin in aqueous medium was concentrated by water evaporation at 85°C

for as long as it was needed in order to achieve the proper concentration used for complex

coacervation.

Before being used for encapsulation process, possible traces of Cr(VI) present in the gelatin
were reduced to Cr(III) by reaction with ferrous sulfate. The reduction of Cr(VI) – a toxic

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oxidation state of chromium - to Cr(III) was then proved by UV-Vis Spectrophotometry
with 1-5-diphenylcarbazide, according to the method of ASTM D 1687 – 92. When
converted to Cr(III), chromium no longer produces a red-violet complex with 1-5-
diphenylcarbazide.

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<H3>2.2.2. Gelatin characterization

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Both CTLW and commercial gelatin were characterized according to their total Kjeldahl
nitrogen content (TKN), as described by SMEWW Method 4500-Norg-B. Chromium
content was determined according to SMEWW Method 3111-B through atomic absorption

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using a spectrophotometer (Model Analyst 200, Perkin Helmer, USA) in samples
previously prepared according to SMEWW Method 3030-E.
<H3>2.2.3. Microencapsulation process N
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The best complex coacervation condition was obtained from experiments conducted with
commercial gelatin and CEO in the concentrations shown in Table 1. The production of
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microcapslules with gelatin from CTLW was performed only under the conditions
determined as the optimum ones for the microencapsulation with commercial gelatin.
Aqueous solutions of gelatin and sodium alginate (2%) were prepared separately by heating
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under constant stirring at 50-55 °C for 5 minutes. Then, 200 ml of an aqueous gelatin
solution and the specific amount of were heated at 50-55°C under constant stirring for CEO
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(values according to the ones presented in Table 1) 30 minutes. Finally, 50 ml of sodium

alginate solution (2% m/v) were added to the oil-gelatin solution and stirred for another 30
minutes.
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The fundamental principle for the formation of ionic charges in biopolymers is pH

modification. According to McClements [20], protein molecules are positively charged
below their isoelectric point and negatively charged above it. On the other hand,
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polysaccharides form electrostatic complexes when in contact with proteins at pH ranging

from 2 to 5. In order to reduce reagent consumption and still induce coacervation, in this
work the pH was adjusted to 4.5-5.0 by the addition of acetic acid (10% v/v). The solution
was then stirred for 1 h (total coacervation time).
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The system temperature was gradually reduced to 10 °C in an ice bath during coacervation.
For the microcapsules crosslinking, glutaraldehyde solution (50% w/v) was slowly added,
until the gelatin coating appeared to be stiff.
Finally, the reticulated microcapsules were separated by centrifugation (Centrifuge Model
MA832, Marconi, Brazil) for 5 minutes at 1500 rpm and 25°C, and oven dried at 30°C
(oven model A35ED, DeLeo, Brazil). The microcapsules were stored in a desiccator.
<H3>2.2.4. Microcapsules characterization
The microcapsules were morphologically characterized by optical microscopy (OM) using
a trinocular microscope with infinity optics (TNB-40T-PL, Opton). An image capture
system coupled to a software (ISCapture, Tucsen) was employed to measure and edit the
images. Magnifications from 40 to 100X were used.
Equation 1 was used to determine average particle diameter of the microcapsules.

𝑑𝑝 ∗𝑓𝜇𝑚

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𝐷𝑎 = 𝑖𝑛 (1)
𝑅𝑖 ∗𝑀

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Where:

Da: avarage particle diameter [μm];

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dp: diameter [linear pixels];

Ri: image resolution [pixels/in];

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𝑀: magnifying power [40X or 100X];

𝑓𝜇𝑚 : conversion factor [μm/in];

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𝑖𝑛
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Encapsulation yield, which relates the microcapsules mass obtained to the mass of
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non-solvents that are fed in the system, was calculated using Equation 2 [21].
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𝑊𝑀
𝑌𝑖𝑒𝑙𝑑 = 𝑥 100 (2)
𝑊𝑁𝑆
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Where:

Yield = microencapsulation yield [%];

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WM: microcapsules mass [g];

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WNS: non-solvent mass fed in the system (gelatin + sodium alginate) [g];

Encapsulation efficiency, which indicates the amount of active agent encapsulated in the
process, is defined by the Equation 3 [13]. For the determination of encapsulation
efficiency, 3 g of microcapsules were dispersed in 10 mL of hexane. This dispersion was
then stirred in an orbital shaker (MA 832, Marconi, Brazil) for 6 h at 130 rpm and 30°C to
extract the core material of the microcapsules. The solution was allowed to stand for two
hours and then filtered. The quantitative analysis of the supernatant was performed by gas
chromatography coupled to mass spectrometry (GC-MS) (6890-GC / MS-5973, HP, USA).
𝑚1
𝐸𝐸 (%) = 𝑥100 (3)
𝑚2

Where:

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EE: encapsulation efficiency [%];

m1: mass of encapsulated CEO [g];

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m2: mass of CEO fed in the process [g];

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Ash content was determined according to ASTM D2617-2012 in order to compare the
amount of non-volatile inorganic content in microcapsules produced with commercial

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gelatin to the same content in microcapsules produced with CTLW gelatin.
<H1>1. RESULTS AND DISCUSSION
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<H2>1.1. Commercial gelatin and CTLW gelatin characterization
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Commercial and CTLW gelatin should present similar protein concentration in aqueous
media, so that microencapsulation with both can be compared. Table 2 presents their
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characterization. CTLW gelatin was concentrated until reaching TKN value similar to the
one of the diluted commercial gelatin.
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Even though hydrolysis was performed in this study under the same conditions used by
Scopel et al. [2], the authors obtained gelatin with 2200 mg NH3-N/L, and with chromium
content lower than 0.04 mg/L. CTLW used by Scopel et al. [2] and the one used in this
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study were obtained from different tanneries. This probably explains the different protein
and chromium concentrations in both gelatin samples produced by hydrolysis. Despite
these differences, hydrolysis process was still able to increase protein:chromium proportion
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in gelatin when compared to CTLW: from 13:1 to 1296:1. It proves that the process was
able to extract gelatin with low amounts of chromium solubilized in it.
Commercial and CTLW gelatin extraction processes are also different. Gelatin extraction
from CTLW involves a more aggressive process, since collagen is bonded to chromium,
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and this chemical bond has to be broken so that collagen can be extracted as gelatin. More
aggressive processes tend to result in partial hydrolysis of gelatin, reducing its molar mass.
In addition, commercial gelatin undergoes a purification step after its extraction, removing
inorganic substances, which was not performed in the gelatin here produced, resulting in
higher ash content for CTLW gelatin, as it is seen in Table 2. Therefore, CTLW gelatin
quality is different from commercial gelatin’s quality.
<H2>1.2. Determination of gelatin and CEO concentration for better quality
microcapsules production
Figures 1 to 3 show the commercial gelatin microcapsules produced by complex
coacervation. When low gelatin concentrations were used (4ComG/4CEO and
4ComG/10CEO samples), microcapsules with similar morphologies were produced. In
these samples, the only change was the CEO concentration: from 4% v/v to 10% v/v. At
100X magnification, it is possible to observe the active agent (CEO) without the cover of
the encapsulating agent.

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When concentration of gelatin is increased to 10% w/v (10ComG/4CEO and
10ComG/10CEO samples) it is seen the formation of a gelatinous layer among the
spherical particles, which is seen in details at 100X magnification. According to Schmitt et
al. [23], high concentrations of encapsulating agents in solution can lead to an excess of

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charges, negatively influencing the formation of the complexes. Another important factor
observed by Saravanan, Rao [24] is the increase in viscosity caused by higher

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concentrations of gelatin. It changes the stirring pattern, since the mobility of the magnetic
stirring bar oscillates according to the viscosity of the medium and affects the
homogeneous formation of microparticles.
When 7% w/v of active agent and gelatin is used (sample 7ComG/7CEO), it is not

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observed such a prominent formation of the gelatinous layer surrounding the

microcapsules.
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The images indicate that the sample 4ComG/10CEO presented better morphological results
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in microencapsulation.
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Particle diameter results confirmed the differences found in the micrographs for
microcapsule sizes. Average diameter for each sample, as well as their encapsulation yield,
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is presented in Table 3. As determined by Oxley (2014) [7], due to their diameter, the
samples can be classified as microcapsules.
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Even though the sample 10ComG/4CEO presented the highest encapsulation yield, its

morphology was not as good as expected. CEO and gelatin were dispersed around the
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microcapsules.

Taking into account morphology, the microcapsules produced with the concentration of 4%
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of commercial gelatin and 4% or 10% of CEO (4ComG/4CEO and 4ComG/10CEO,

respectively) presented the most appropriate morphology. When comparing average
diameter of both, due to its lower diameter, sample 4ComG/10CEO was chosen as the most
suitable one. Therefore, in order to produce CEO microcapsules with CTLW gelatin, it was
used gelatin and CEO concentrations of 4 and 10%, respectively.
<H2>1.3. Characterization of the microcapsules obtained with CTLW gelatin
Figure 4 shows the microcapsules produced with CTLW gelatin (4% of CTLW
gelatin and 10% of CEO). The microcapsules had a predominantly spherical shape with the

core completely covered by the CTLW gelatin and sodium alginate. Their average diameter

was of 434.06 µm ± 104.68 and the encapsulation efficiency was equal to 73.7%. Xing et

al. [25] considered efficiencies from 70 % to 84 % relatively high, and so can be considered

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the efficiency found for the process here described.

Table 4 shows the characterization of microcapsules produced with commercial gelatin and

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gelatin extracted from CTLW. The analysis confirmed what had already been observed by
other authors: CTLW gelatin presents higher ash content than commercial gelatin [2,26].

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Figure 5 shows the results of thermogravimetry (TG / DTG) of the microcapsules

produced using CTLW gelatin. A moisture loss stage (at around 100°C) and two other main

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mass loss stages were observed. From 230°C to 270°C it is observed the thermal event

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related to the volatilization of the essential oil of citronella present in the sample. Asbahani
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et al. [27] indicate that citronella essential oil typically decomposes at 201-207°C. This
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temperature is increased when the oil is encapsulated since degradation of shell material

(gelatin and sodium alginate) needs to start so that the oil may be released. Bezerra et al.
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[28] have also pointed to citronella oil release at the temperature interval of 233°C to 272°C
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from microcapsules produced with gelatin and gum Arabic. A mass loss of about 14% is
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noticed in this temperature interval, which is an estimated percentage of citronella essential

oil in the microcapsules.

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The decomposition of the gelatin-sodium alginate complex is seen from 280°C to 420°C.
As stated by both Bezerra et al. [28] and Devi, Kakati [29], gelatin’s degradation event has
its peak temperature at ≈ 300°C. Sodium alginate degrades at ≈ 245°C, gelatin-sodium
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alginate complex, however, presents a thermogravimetric behavior more similar to the one
of gelatin, degrading from about 270°C to 430°C [29].
Toxicity of microcapsules produced with CTLW gelatin should not be a matter of concern
due to reduced chromium concentration. Possible direct contact with the skin should not
cause toxic effects, since leather itself has a certain amount of soluble chromium, which is
released to human skin when leather goods are used [30]. Disposition of these
microcapsules are not a problem as well, since CTLW hydrolysate – a product similar to
CTLW gelatin, but with smaller molecular chain size and higher concentration of
chromium – is widely used as fertilizer and is in accordance to The European Commission,
Fertilizer Working Group Meeting [31].
<H1>2. CONCLUSION
The main objective of this study was to determine whether it was possible to produce
microcapsules using CTLW gelatin, which is known to present properties different from the
ones presented by commercial gelatin. Further studies are needed in order to determine
CTLW gelatin microcapsules physical and thermal stability, shelf life, biological and

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functional activities.
Complex coacervation of CEO with commercial gelatin and sodium alginate produced
better morphological qualities in microcapsules when lower gelatin concentrations (4%)

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and higher CEO concentrations (10%) were used. These concentrations, when employed to
the same process, but with CTLW gelatin instead of the commercial one, allowed a high
encapsulation efficiency (73.7%) and the production of microcapsules with 434.06 µm of

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diameter. Microcapsules produced with CTLW gelatin presents higher ash content due to
the absence of any purification step of the CTLW gelatin. CTLW gelatin also changed the
morphology of the microcapsules, which still were successfully produced.

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Even though toxicity of microcapsules produced from CTLW gelatin should not be an
issue, until it is properly tested, uses that do not lead to direct contact with human skin –
such as in mosquito nets placed in windows – are suggested.
AKNOWLEGMENTS N
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This work was supported by University of Caxias do Sul and by the Brazilian National
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Council for Scientific and Technological Development (CNPq) for the financial support
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provided to this study.

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importance of environmental parameters. Contact Dermatitis 72(5), 275-285 (2015).

<DOI>10.1111/cod.12334</DOI>
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[31] ENTR/F2/EL: Minutes of the Fertiliser Working Group Meeting. In: Chemicals, m.,
mechanical, electrical and construction industries; Raw materials; Chemicals -
Classification & Labelling, Specific Products, Competitiveness (ed.). European Comission
EP

Brussels, (2013)
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</BIBL>
<Figure>Figure 1 – Micrographs of CEO microcapsules produced by complex coacervation using

commercial gelatin and sodium alginate: (a) 4ComG/4CEO / 40X magnification, (b) 4ComG/4CEO / 100X
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magnification; (c) 4ComG/10CEO / 40 X magnification, and (d) 4ComG/10CEO / 100X magnification.

<Figure>Figure 2 – Micrographs of CEO microcapsules produced by complex coacervation using

commercial gelatin and sodium alginate: (a) 10ComG/4CEO / 40X magnification, (b) 10ComG/4CEO /
100X magnification; (c) sample 10ComG/10CEO / 40 X magnification, and (d) sample 10ComG/10CEO /

100X magnification.

<Figure>Figure 3 - Micrographs of CEO microcapsules produced by complex coacervation using

commercial gelatin and sodium alginate: (a) 7ComG/7CEO / 40X magnification, (b) 7ComG/7CEO / 100X

magnification

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<Figure>Figure 4 - Microcapsules produced by complex coacervation using CTLW

gelatin and sodium alginate. 100X magnification

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<Figure>Figure 5 - Thermogravimetry of microcapsules produced with CTLW (a): TG

curve; (b): DTG curve

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<Table>Table 1 – Gelatin and CEO concentrations tested for the microencapsulation
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process.
Gelatin concentration CEO concentration (%
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Sample
(%m/v) v/v1)
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4ComG/4CEO 4 4
4ComG/10CEO 4 10
10ComG/4CEO 10 4
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10ComG/10CEO 10 10
7ComG/7CEO 7 7
1
As a function of the gelatin volume used
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<Table>Table 2 – CTLW and commercial gelatin characterization

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Gelatin Chromium TKN

(mg/L) (mg NH3-N/L)
Commercial (4% m/v) Not Determined 5047.23 ± 879.15
CTLW 0.57 ± 0.035 739.73 ± 19.71
CTLW concentrated 1.50 ± 0.040 4385.02 ± 185.14
<Table>Table 3 - Characterization of microcapsules produced by complex coacervation
using commercial gelatin and sodium alginate
Sample Encapsulation Yield (%) Average diameter (μm)
4ComG/4CEO 83.41 608.42 ± 153.68
4ComG/10CEO 83.52 561.06 ± 115.68
10ComG/4CEO 91.83 453.21 ± 266.04
10ComG/10CEO 88.61 557.62 ± 204.94

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7ComG/7CEO 79.36 485.68 ± 211.82

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<Table>Table 4- Ash content in the microcapslules produced by complex coacervation
using commercial gelatin or CTLW gelatin and sodium alginate

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Ashes
Sample
(% m/m. dry basis)

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4ComG/10CEO <1.0
4% of CTLW/10% of CEO 5.86
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