The Effect of Polysorbate 80 Addition on the Size of

Chitosan Nanoparticles from Shrimp Shells Vannamei

(Litopenaeus vannamei)
Firdaus, Rossa A.a), Iksan, M.M, Jannah, Lutfiatul and Suyadhi, Ardian A.

Universitas Jember
a)
220820101025@mail.unej.ac.id

Abstract. Nanoparticle study is growing rapidly because it has very specific character and new characters that have many
positive opportunities for the development of science and industry. In case of chitosan material, nanoparticles have a big
role in the pharmaceutical study such as good agent of parenteral drugs, oral, ocular. However, smaller nanoparticles
have a greater risk of aggregation of particles during storage and transportation dispersion thus blocking its function as a
good medicine agent. The addition of the surfactant in the production of nanoparticles with an ionic gelation method is a
solution that can be used to stabilize the emulsion particles in solution by preventing agglomeration and aggregation, as
well as to reduce the particle size. One type of surfactant that is widely used is Polysorbate 80 which has the trade name
as Tween 80. This study aims to determine the effect of Tween 80 and the optimum concentration of Tween 80 addition
to be able to produce the smallest size of nanoparticles produced by ionic gelation methods. The method were used in this
research are an experimental method with a statistical analysis of the data. The results showed that Tween 80 have the
effect to reducing the size of chitosan nanoparticles. The addition of 0.2% Tween 80 concentrations capable to forming
nanoparticles in the average size 444,6nm with polydispersity index value 0.520. An addition of Tween 80 were too little
and too much of it will cause agglomeration and increased the size of the chitosan nanoparticles.

Keywords: Chitosan nanoparticles, Particle Size Analyzer, ionic gelation, shrimp shells

PENDAHULUAN

Shrimp is one of the potential commodities, especially in the markets of Japan, China, Vietnam, United
States, Canada, and United Kingdom (Badan Pusat Statistik, 2012). Shrimp production for the export market
produces waste including the heads and shells of shrimp (Suptijah and Zahiruddin, 2005). In developed countries
such as Japan and the United States, shrimp shells have been widely used as raw material for making chitosan
(Anggraini, 2011). To support the wider use of chitosan, physical modifications have been developed, one of which
is by reducing chitosan particles to nanoparticle sizes in the range of 1-1000 nm (Tiyaboonchai, 2003). One of the
functions of chitosan nanoparticles is as a drug delivery agent either parenterally, orally, or ocularly. The ability of
chitosan nanoparticles as drug delivery and release is influenced by particle size. Smaller particles have a larger
surface area, therefore most of the drug that is on the surface of the particles will be released quickly. Meanwhile,
larger particles have larger nuclei that allow more drug to be carried and have a relatively slower release/diffusion
time. However, smaller particles have a greater risk of particle aggregation during storage and transportation of
nanoparticle dispersions. (Mohanraj and Chen, 2004). The addition of surfactants to the manufacture of
nanoparticles by ionic gelation is a solution that can be used to stabilize particle emulsions in solution by preventing
clumping and reducing particle size (Silva et al., 2006). One type of surfactant that is widely used is Polysorbate 80
or many are sold in the market with Tween 80. Based on this background, it is necessary to conduct research on the
effect of the addition of Polysorbate 80 on the size of chitosan nanoparticles of white shrimp carapace (L.vannamei).

RESEARCH MATERIALS
Research Place and Time
Research on the manufacture of chitosan nanoparticles was carried out at the Educational Laboratory of the
Faculty of Fisheries and Maritime Affairs, Airlangga University. Testing the Degree of Deacetylation (DD) using
the Fourier Transform Infra-Red (FT-IR) Spectrophotometer instrument was carried out at the Material
Characterization Division of the Sepuluh Nopember Institute of Technology, Surabaya. Testing the particle size of
chitosan nanoparticles using a Particle Size Analyzer (PSA) was carried out at the Solid Substance Physics
Laboratory, Sepuluh Nopember Institute of Technology. All these activities were carried out in June 2016.

Tools and Materials

The equipment used in the manufacture of chitosan nanoparticles is a glass beaker, blender/grinder, basin,
fume hood, gloves/gloves, stirrer, hot plate, magnetic stirrer, spin bar, thermometer, glass spatula, pH meter, stative,
oven, micropipe, pipette, analytical balance, Fourier Transform Infra-Red (FT-IR) Spectrophotometer from the
Therma Scientific Smart Orbit Nicolet iS10 brand, Particle Size Analyzer (PSA) from Malvern Zetasizer brand.
The research material used in this study was the carapace of vannamei shrimp (L. vannamei) obtained from
PT. Manage Mina Laut Gresik. The materials needed during the chitosan nanoparticle production process are HCl,
NaOH, acetic acid, Tween 80, Sodium Trypolyphosphate (STPP) and sterile distilled water.
Research Procedure
a. Manufacturing of Chitosan
The process of making chitosan begins with washing the shrimp carapace from the remaining meat and
swimming legs that are still attached using running water. Furthermore, the shrimp carapace was dried under the sun
and maximized its drying by drying the oven to get samples in dry form without water. The dried carapace was then
reduced in size using a grinder.
The initial stage of making chitosan was demineralization with 1.5 N HCl at 90°C for 60 minutes. After that
it is neutralized using distilled water until the pH reaches neutral. Next is deproteination treatment with 3.5 N NaOH
at 90°C for 60 minutes and neutralization again using distilled water until the pH reaches neutral to get chitin. After
that, the deacetylation step was carried out with 55% NaOH at 130°C for 60 minutes and chitosan was produced
(Suptijah et al., 2004). The results of chitosan were then tested for characteristics including water content, ash
content, nitrogen content, amount of yield and degree of deacetylation.
 b. Manufacturing of Chitosan Nanoparticles
The main research includes the manufacture of nanoparticles from the results of preliminary research
chitosan. The initial stage of making chitosan nanoparticles was the formation of a chitosan gel by dissolving 0.2
gram of chitosan in 100 ml of acetic acid with a concentration of 1% (Nadia et al, 2014). Chitosan was then sized
using a magnetic stirrer at 2500 rpm (based on the ability of IKA's Big Squid stirrer) at room temperature for one
hour. Furthermore, the formation of nanoparticles was carried out by adding a chitosan solution to a 0.1%
tripolyphosphate solution in a beaker glass (Nadia et al, 2014) with a ratio of 5:1 (Mardliyati et al, 2012). The final
stage of forming chitosan nanoparticles using the ionic gelation method is the addition of Tween 80 by mixing the
Tween 80 solution into the chitosan-TPP solution and then homogenizing at 2500 rpm for one hour (Wijaya, 2014).

Research Parameters
The main test parameters in this study used quantitative parameters, namely data obtained from calculating the size
of chitosan nanoparticles using the Particle Size Analyzer MALVERN Nano Zetasizer instrument. The supporting
parameters in this study were the polydispersity index value, moisture content, ash content, nitrogen content, and the
degree of deacetylation.

Data Analysis
This study used an experimental method using a completely randomized design (CRD) experiment (Sasmita,
2012) with five treatments, namely without the addition of Tween 80 surfactant, with the addition of Tween 80 at
each concentration of 0.1%, 0.2%, 0.3% and 0.4% (Suptijah et al, 2011); (Rachmania, 2011) with four repetitions.
The effect of treatment was analysed using Analysis of Variance (ANOVA). Based on the Analysis of Variance
(ANOVA), the treatment that showed a significant effect (P, 0.05) was further tested with the Duncan Multiple
Range Test (DMRT) using SPSS 16.0.

RESULTS AND DISCUSSION

Size of Chitosan Nanoparticles
Based on the results of ANOVA analysis it was known that the results of chitosan nanoparticle sizes were
significantly different between treatments (P<0.05). The results of the analysis were then followed by Duncan's test
to determine the significance between treatments. The average size value of chitosan nanoparticles in each treatment
is presented in Table 1.
Table 1. The average results of testing chitosan nanoparticles’ size with the addition of Tween 80 (Average ±SD)
Treatment Average particle size ±SD (nm)
P0 (Without Tween 80 addition) 485,225d ± 6,69
P1 (Tween 80 0,1%) 482,025cd ± 2,26
P2 (Tween 80 0,2%) 444,600a ± 2,47
P3 (Tween 80 0,3%) 464,175b ± 7,26
P4 (Tween 80 0,4%) 475,775c ± 6,87
Information :
Different superscript letter notations in the same column indicate that there is a significant difference in the
comparison between treatments (F count > F table 0.05).
 The results of the treated chitosan nanoparticle solution were then characterized including particle size and
polydispersity index (PdI) values. From the results, it can be seen that chitosan nanoparticles in the control treatment
had a size of 485.22nm, then decreased to 482.02nm with the addition of 0.1% concentration of Tween 80 and
reached the smallest size with the addition of 0.2% concentration of Tween 80, namely 444.6nm. Chitosan
nanoparticles had a larger size than the previous concentration at Tween 80 concentration of 0.3% at 464.17nm and
0.4% at 475.77nm respectively. From the results of Duncan's further test, it is known that the addition of Tween 80
with a concentration of 0.2% has the highest notation results so that it can be interpreted as having the best effect.
The occurrence of the optimum point for adding Tween 80 surfactant can be influenced by the nature of the
surfactant itself on the formation of droplet size and the stability of an emulsion. According to Ben et al (2013)
medium chain length surfactants allow these surfactants to produce smaller droplets, conversely the longer the
surfactant chain used the larger the size of the droplets formed.
In the research that has been done, the size of chitosan nanoparticles with the addition of 0.1% Tween 80
with a rotational speed of 2500rpm is 482.02nm. The same study was reported by Suptijah et al (2011), the
formation of chitosan nanoparticles with the addition of 0.1% Tween 80 and a stirrer rotation speed of 3000rpm
resulted in a chitosan nanoparticle size of 450nm. The difference in the speed of rotation of the stirrer used during
the process of forming chitosan nanoparticles is thought to have caused the results of different particle sizes. The
kinetic molecular theory of gases states that gas molecules often collide with each other and reacting molecules. The
rate of reaction will be directly proportional to the number of molecular collisions per second, or directly
proportional to the frequency of molecular collisions. The faster the rotation, the greater the intensity of the solvent
molecules to come into contact with chitosan, so that the greater the intensity of the rotational speed of the magnetic
stirrer the resulting particles will be smaller (Chang, 2005).

Polydispersity Index (PdI) Value of Chitosan Nanoparticles

Based on the results of ANOVA analysis, it was found that the polydispersity index value of chitosan nanoparticles
between treatments was not significantly different (P>0.05). The average value of the polydispersity index of
chitosan nanoparticles in each treatment can be seen in Table 2.
Table 2. The average test results for the polydispersity index value of chitosan nanoparticles with the addition of
Tween 80 (± SD)
Treatment Polydispersity Index Value ±SD (PdI)
P0 (Without Tween 80 addition) 0,537 ± 0,107
P1 (Tween 80 0,1%) 0,613 ± 0,056
P2 (Tween 80 0,2%) 0,520 ± 0,097
P3 (Tween 80 0,3%) 0,559 ± 0,066
P4 (Tween 80 0,4%) 0,518 ± 0,103
The uniformity of particle size distribution/Polydispersity Index can be determined based on measurements
using a Zetasizer. From the measurement results, the PdI values were obtained successively starting from the
concentration control treatment with the addition of Tween 80 0.1%, 0.2%, 0.3% and 0.4% respectively, namely
0.537, 0.613, 0.52, 0.559 and 0.518. The addition of 0.4% Tween 80 showed the lowest polydispersity index value
compared to the control and other treatments. Nevertheless, all treatments and controls are still classified as samples
with a good level of monodispersed because they have a polydispersity index value of less than 0.7 (Malvern, 2011).
Good sample distribution uniformity can be due to stable and even stirrer rotation in all parts of the beaker glass so
that all parts experience the same ionic gelation process.

Chemical Characterization of Vaname Shrimp Carapace and Chitosan (L. vannamei)

Characterization results of raw materials for Vaname shrimp carapace and chitosan can be seen in Table 3.
Table 3. Chemical characteristics of dried Vaname shrimp carapace and chitosan
Percentage (%)
Characteristics
Shrimp Carapace Chitosan
Water content 6,71 14,39
Ash Content 31,11 0,28
Nitrogen Levels - 5,46
Degree of Deacetylation - 83
Yield 100 23,9
From the results of the analysis it is known that the percentage of water content increased from the value of the
water content in the shrimp carapace of 6.71% to 14.39% in the chitosan sample. The ash content in the sample has
the opposite value, which decreases from the carapace sample of 31.11% to 0.28% in chitosan.
The results of the proximate test showed that the Vaname shrimp carapace had a moisture content of 6.71%
and an ash content of 31.11%. Based on Rachmania's research (2011) the water and ash content of Vaname shrimp
carapace was 15.04% and 18.02%. The ash content of the samples and the comparison in the literature after being
converted in 100% DM were 33.34% and 21.20%, respectively. The ash content of the carapace samples used in the
study has a higher value compared to the literature. The diversity of chemical composition is thought to be caused
by habitat, food, season, species, and age of the shrimp on the carapace used.
Nitrogen content in chitosan shows a value of 5.46%. The low nitrogen content is due to the deproteination
stage in the manufacture of chitosan. At the deproteination stage, the protein and chitin bonds will be broken by the
addition of sodium hydroxide (Rochima, 2007). When compared with the standards set by BSN (2013) the nitrogen
content contained in chitosan is still above the limit, this can happen because the deproteination process is not
running perfectly. According to Silvia et. al (2005) the factors that influence the deproteination process are the
concentration of NaOH used and the length of time used in the deproteination process. The high concentration of
NaOH and the length of time for the deproteination process will cause the reaction between the protein and the ester
forming solution (Na-proteinate) to be more perfect, so that more protein is removed.
Based on the calculation of the sample weight after going through each treatment stage, the yield results
were 23.9%. The results of the overall yield analysis calculated from dry carapace to chitosan products amounted to
24.83%. Suptijah et al research (2011) on the manufacture of chitosan produced a yield of 13.50% and Nadia et al
research (2014) of 19.08%. The resulting yield of chitosan samples has a greater value than the literature. According
to Nadia et al (2014) the high yield is due to chitin/chitosan which is not lost much with solvents in the
demineralization, deproteination and deacetylation processes. In addition, washing and neutralizing techniques using
distilled water which are carried out carefully also minimize the amount of chitin/chitosan weight loss.
Determination of the degree of deacetylation of chitosan was carried out by analysis using the Fourier
Transform Infrared Spectroscopy (FTIR) instrument. The spectrum that appears in chitosan is at wave number
 1636.96, namely the functional group C=O stretching and 3292.94 in the NH (-NH2) group stretching (Gyliene et
al., 2003). Based on the results of the FTIR spectrophotometric analysis, calculations were carried out using the
Domszy and Robert (1985) formula and the deacetylation degree was 83%. The results of the analysis using the FT-
IR instrument can be seen in Picture 1.

Picture 1. FTIR chromatogram of chitosan samples

The deacetylation process is the process of forming chitosan from chitin using NaOH to replace acetamide
groups with amino groups (Hargono et al, 2008). The deacetylation process will produce chitosan with a certain
degree of deacetylation. The degree of deacetylation indicates the content of free amino groups in the polysaccharide
(Purwanti, 2014). The degree of deacetylation is an important quality parameter of chitosan. The higher the degree
of deacetylation, the higher the purity, meaning that chitosan has been separated from impurities such as proteins,
minerals, pigments and acetyl groups (Suptijah, 2004) and the more amine groups (NH2) in the chitosan molecular
chain so that chitosan is more reactive (Suptijah, 2006 ). The value of the degree of deacetylation of chitosan
samples is comparable to research conducted by Purwanti (2014) that the results of chitosan production from shrimp
carapace have a degree of deacetylation in the range of 80%. The results of the degree of deacetylation of the sample
chitosan also show that chitosan has met the standard regulations set by the National Standardization Agency (2013)
in SNI 7949:2013 of ≥ 75%.

CONCLUSION
The addition of Tween 80 influences the resulting particle size in the manufacture of chitosan nanoparticles by the
ionic gelation method. The optimum concentration for adding Tween 80, which is 0.2%, is known from the average
size of the chitosan nanoparticles produced which is 444.6nm with a polydispersity index value of 0.520. Based on
the research results, it is suggested to optimize the ionic gelation method and use Tween 80 surfactant in the
manufacture of chitosan nanoparticles which can prevent aggregation between particles, to improve the size of the
nanoparticles which are still high and maintain the stability of chitosan nanoparticles so that they can be applied in
various fields.
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