International Journal of Advanced Research and Publications

ISSN: 2456-9992

Effect Of Degrees Of Deacetylation On The

Antimicrobial Activities Of Chitosan
ENYERIBE C.C, KOGO A.A, YAKUBU M.K, OBADAHUN J, AGHO B.O , KADANGA, B
NIGERIAN INSTITUTE OF LEATHER AND SCIENCE TECHNOLOGY (NILEST), ZARIA Department of Science Technology, P.M.B
1034 Samaru, Zaria. Kaduna State, 08069434300
enyeribecollins88@gmail.com.

AHMADU BELLO UNIVERSITY, Department of Polymer and Textile Science, 08037400148

aakogo@abu.edu.ng

AHMADU BELLO UNIVERSITY, Department of Polymer and Textile Science, 08030964967

mkyakubu@gmail.com

NIGERIAN INSTITUTE OF LEATHER AND SCIENCE TECHNOLOGY (NILEST), ZARIA Department of Science Technology, P.M.B
1034 Samaru, Zaria. Kaduna state, 08067280257
joshuaobadahun@gmail.com.

NIGERIAN INSTITUTE OF LEATHER AND SCIENCE TECHNOLOGY (NILEST), ZARIA Department of Science Technology, P.M.B
1034 Samaru, Zaria. Kaduna state, 08063216117
aghoosazebright@gmail.com.

NIGERIAN INSTITUTE OF LEATHER AND SCIENCE TECHNOLOGY (NILEST), ZARIA Department of Science Technology, P.M.B
1034 Samaru, Zaria. Kaduna state, 08036936391
ellidelovely@yahoo.com.

ABSTRACT: Chitosan, a natural polysaccharide polymer derived from chitin and was investigated for the effect of degree of
deacetylation and its antimicrobial activity. The chitin was subjected to modification using 30%, 40% and 50% NaOH to obtain chitosan of
various degree of Deacetylation and were subjected to physiochemical analysis such as: solubility, moisture content, ash content, viscosity
and degree of deacetylation, Ultra-violet visible spectroscopy (UV-Visible), Fourier-Transform Infrared Spectroscopy Analysis (FT-IR)
spectroscopy, Differential Scanning Calorimetry (DSC) and the antimicrobial study (zone of inhibition, minimum inhibitory concentration
and minimum bacteriocidal/ fungicidal concentration) The degree of deacetylation was 80% when 50%w/v NaOH was used and 67% 30%
w/v NaOH, which shows that as the concentration of NaOH increases the more acetyl groups are been removed.

Keywords. Chitin, Chitosan, Deacetylation and antimicrobial study

1.0 INTRODUCTION functional groups such amine groups and hydroxyl

Chitin, poly [β-(1-4)-N-acetyl-D- glucosamine) is a
natural polysaccharide of major importance, first
identified in 1884. It consist of 2-acetamide-2-deoxy-β-D-
glucose through a β – (1,4) linkage (poly-N-acetyl-D-
glucosamine). This kind of linear muco-polysaccharide is
abundant in nature mainly present in animal support or
tissue of lower evolutionary organisms such as
crustaceans, insects and mushrooms, only less abundant in
nature than cellulose[1]. Chitin, the second-most abundant
biopolymer, and its deacetylated product, chitosan are
high molecular- weight biopolymers and are recognized as
versatile environmentally friendly raw materials in many
applications [2]. Chitosan, poly [(1,4)-β-linked 2-amino-2-
deoxy-D-glucose] [polyglucosamine], is the N-
deacetylated form of chitin with amino groups along with
the chitosan structure (polyglucosamine) also known as 2-
amino-2-deoxy-(1,4)-β-D-glucopyranan[3][4]. This N-
deacetylation is almost never complete. Chitosan is
considered to be the most widely distributed biopolymer,
it is a cationic, non-toxic, biodegradable and
biocompatible polyelectrolyte with a pKa of
approximately 6.5[5][6]. The solubility of chitosan in
acidic solutions is the generally accepted criterion for
identifying chitin and chitosan. Chitosan polymer is
hydrophobic in nature although consisting of hydrophilic
groups. The polyamine chitosan is normally insoluble in
water at near neutral pH and in most common organic
solvents such as dimethylsulphoxide (DMSO) and organic
alcohols, mainly due to its crystalline structure with
extensive intra-molecular and intermolecular hydrogen
bonding [7]. Chitosan is a semi-crystalline polymer in its
solid state. The crystallinity of chitosan is lower than that
of chitin. the free amino groups at the C2 position of the
polymer backbone (D-glucoamine unit) are protonated
and the polymer becomes fully soluble below pH 5[8].
Therefore, chitosan salt particles or flakes or other form-
like polymers can be prepared from these aqueous
solutions.
2.0 MATERIALS AND METHODS
2.1 Materials
Chitin, Hydrochloric acid, sodium hydroxide, acetic acid,
Mueller Hinton agar, Mueller Hinton broth, potato
dextrose agar and nutrient agar ( oxido),Thermometer,
beaker, crucible, desiccator, conical flask, cork borer,
burette, Heating mantle, magnetic stirrer,
brookfiedsynchro-lectic viscometer, Agilent pH meter,
Agilent FT-IR spectrophotometer, Agilent UV-Visible

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 International Journal of Advanced Research and Publications
ISSN: 2456-9992

spectrophotometer, TA instrument differential scanning The crucible was dried by holding in the Bunsen flame for
calorimeter, autoclave and incubator. about two minutes, then transferred into a desiccator to
cool before weighing (W1)g. 2g of the sample was
2.2 Methodology weighed inside the crucible (W2)g, the crucible with the
sample was heated gently in a Bunsen burner in a fume
2.2.1 Conversion of Chitin to Chitosan cupboard till the smoke ceased, which was then
(Deacetylation) transferred to the muffle furnace, preheated at 550°C. The
The chitin was deacetylated in 30% (w/v) NaOH for 5 heating was continued until all the carbon had been burnt
hours at 100°C using a heating mantle. After away; the crucible was taken away with a pair of tong and
deacetylation, the chitosan was washed thoroughly with immediately covered and placed in a desiccator to cool
water followed by distilled water. The resulting chitosan before weighing (W3)g. The ash content was calculated
was dried to constant weight at 65°C. Further using the formula:
deacetylation were carried out using 40% and 50% (w/v)
NaOH, to obtain other samples with different degrees of
deacetylation as shown in the reaction scheme below. ……………………..Equation 3
CH3 CH3
O O
NH NH 2.2.6 Viscosity
OH O OH O Viscosity of chitosan was determined with a Brookfield.
O O
O
O
O Chitosan solution was prepared in 1% acetic acid at a 1%
O
O O concentration on a dry basis at 25°C.
OH OH
NH NaOH HO NH2
HO
o 2.2.7 Fourier-Transform Infrared Spectroscopy
O CH3 n 100 C n Analysis (FT-IR)
5Hrs
The sample was thoroughly mixed with KBr, the dried
Scheme 1: Extraction of chitosan from chitin mixture was pressed which resulted into a homogeneous
sample disk. The measurement was carried out over the
2.2.2 Determination of Degree of Deacetylation frequency range of 400-4,000 cm- 1 using the Agilent FTIR
Dried chitosan (0.2 g) was dissolved in 20 cm30.1 M spectrophotometer [9].
hydrochloric acid and 25 cm3deionized water. After 30
minutes continuous stirring, next portion of deionized 2.2.8 UV-Visible Spectroscopy (UV-VIS)
water (25 cm3) was added and stirring continued for 30 The UV-Visible absorption spectra were measured using
minutes. When the chitosan completely dissolved, the Agilent UV-visible spectrophotometer. 0.1M acetic acid
solution was titrated with a 0.1 M sodium hydroxide solution of the sample was used while for the cationated
solution using a pH meter to monitor the pH. Degree of samples distilled water was used and the spectra was
deacetylation (DA) of chitosan will be calculated using recorded from 200-650nm wavelength frequency range
formula: [10].

2.2.9 Differential Scanning Calorimetry (DSC)

The differential scanning calorimeter (TA Instrument,
…………………Equation 1 Q200, USA) was employed to study the thermal property
⁄
of the sample. 2.5mg of the sample was placed in an
2.2.3 Solubility Analysis aluminum pan and sealed. The lid was perforated before
Solubility analysis of the sample was performed in sealing. Empty closed aluminum pan was used as the
different selected solvents. For this purpose, the sample reference. Samples was scanned from a temperature range
was added to the solvent at the concentration of 5 mg/mL of 50°C to 450°C at a heating rate of 10°C/min under
at 25°C and their solubility was evaluated. nitrogen atmosphere.

2.2.4 Determination of Percentage Moisture 2.3 Antimicrobial Profile (Sensitivity Test) of the
Content polymer using agar well diffusion method.
The crucible was dried in an oven at 80°C for 20 minutes, The standardized inocula of both the bacterial and fungus
cooled in a dessicator and weighed (W1)g, 2g of the isolates were streaked on sterilized Mueller Hinton and
sample was then placed into the crucible and reweighed potato dextrose agar plates respectively with the aid of a
(W2)g, the crucible with the sample was dried in the oven sterile swab sticks. Wells were pounched on each
at 105°C until a constant weight was obtained after inoculated plates with a sterile cork borer with a diameter
successive cooling in desiccator and weighing. It was of 6mm. The wells were properly labeled according to
finally transferred from the oven to the desiccator to cool different concentrations of the polymer which were 50,
and then quickly weighed (W3)g. The percentage moisture 25, 12.5 and 6.25mg/ml. each well was filled up with
content was calculated using formula: approximately 0.2ml of the polymer solution. The
inoculated plates with the polymers were allowed to stay
on the bench for about one hour, this was to enable the
..Equation 2
polymer solution to diffuse into the agar. The plates were
then incubated at 37°C for 24 hours (plates Mueller
2.2.5 Determination of Ash Content Hinton agar) while the plates of potato dextrose agar were

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 International Journal of Advanced Research and Publications
ISSN: 2456-9992
incubated at room temperature for about 3-5 days. At the
end of the incubation period, the plates were observed for
any evidence of inhibition which appeared as a clear zone
that was completely devoid of growth around the wells
(zone of inhibition) the diameter of the zones were
measured using a transparent ruler calibrated in
millimeter.
2.3.1 Determination of Minimum Inhibitory
Concentration (MIC)
The minimum inhibitory concentration of the sample was
determined using the broth dilution method[11][12].
Mueller Hinton broth was used as the diluent. The lowest
concentration of the polymer solution showing inhibition
for each organism when the polymer solution was tested
during sensitivity test was serially diluted in the test tubes
containing Mueller Hinton broth. The organisms were
inoculated into each tube containing the broth and the
polymer solution. The inoculated tubes were then
incubated at 37°C for 24 hours. At the end of the
incubation period the tubes were examined for the
presence or absence of growth using turbidity as a
criterion, the lowest concentration in the series without
visible sign of growth (turbidity) was considered to be the
minimum inhibitory concentration (MIC).
2.3.2 Determination of Minimum Bactericidal/
fungicidal Concentration (MBC/MFC)
The result from the minimum inhibitory concentration
(MIC) was used to determine the minimum bactericidal
concentration (MBC) of the polymers.A sterilized wire
loop was dipped into the test tubes that did not show
turbidity (clear) in the MIC test and a loopful was taken
and streaked on a sterile nutrient agar plates. The plates
were incubated at 37°C for 18-24 hours.At the end of
incubation period, the plates were examined for the
presence or absence of growth. This is to determine
whether the effects of the polymers are bacteriostatic or
bacteriocidal[11][12].
3.0 RESULTS AND DISCUSSION
Synthesis of chitosan was carried out by heating chitin
from shrimp with various percent Concentrations of
sodium hydroxide 30%, 40% and 50% so as to obtain
chitosan of various degrees of deacetylation, the chitosan
obtained was dissolved in 1% acetic acid for solubility
which is an indication that chitosan have been
synthesized,[13] stated that the solubility of chitosan is
also controlled by the distribution of the acetyl groups
remaining along the chain. The deacetylated chitin
(chitosan) obtained was tested with acetic acid to see its
solubility in organic acid, they were all soluble in 1%
acetic acid but with a variation in solubility, 50%w/v
NaOH chitosan had the highest solubility, followed by
that of 40%w/v NaOH chitosan and 30%w/v NaOH
chitosan had the least solubility this can be attributed to
the various degree of deacetylation of the chitin. Thus, the
more deacetylated the chitin is, the better the solubility.
This is a necessary condition to ascertain that chitosan
have been obtained from chitin, since chitin is not soluble
in acetic acid. The moisture content of the chitosan ranges
from 8.07%-11.6%, that produced using 30%w/v NaOH
has 8.07% moisture content, 40%w/v NaOH had 9.86%
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moisture content and 50%w/v NaOH had 11.60%
moisture content this is because as the degree of
Deacetylation increases the polymer becomes less
crystalline and absorbs more water, the permitted level is
below 20%. The moisture content may vary depending on
season, relative humidity and intensity of sunlight [14].
The ash content of the 50%w/v NaOH chitosan is 6.02%,
while that of 40%w/v NaOH is 5.97% and 30% w/v
NaOH is 5.88%. The ash content is an indication of the
effective removal of inorganic minerals from shrimp. The
ash content is due to the presence of calcium carbonate
which is found in large amount in shrimp shells[15].
Viscosity is an important factor in the conventional
determination of molecular weight of chitosan[16]. Higher
molecular weight chitosan often provides highly viscose
solutions, which may not be desirable for industrial
application. The chitosan extracted have a viscosity of
144.9 cp, 131.8cp and 108.3cp for 30%w/v NaOH, 40%
w/v NaOH and 50% w/v NaOH respectively. From
literature the viscosity of chitosan general ranges from 60
to 780cp[17]. The decrease in viscosity of the extracted
chitosan as the concentration of NaOH increases may be
attributed to the increase in the degree of deacetylation.
The viscosity of chitosan is affected by some factors such
as temperature, pH, ionic strength, concentration,
molecular weight and degree of deacetylation [18].
Table 1: physiochemical characterization of chitosan
physicochemical characterization of chitosan
30% 40% 50%
Parameters
NaOH NaOH NaOH
DD% 67.02 74.64 80.39
Solubility (1% acetic
soluble soluble
acid) soluble
Moisture content (%) 8.07 9.86 11.6
Ash content (%) 5.88 5.97 6.02
Viscocity (cp) 144.9 131.8 108.3
The degree of deacetylation of the chitosan extracted was
determined using potentiometric titration with a degree of
deacetylation of 67%, 73% and 80% for 30%w/vNaOH,
40%w/v NaOH and 50%w/vNaOH respectively, which
are higher than that reported by Isa et al., (2012), of 50%
for shrimp shell lower than the reported degree of
deacetylation of 98.38-98.79% achieved by [19] using
shrimp shell. This may be attributed to the nature of the
raw material used, its immediate environment and also the
methods applied during the processes. There is an
indication that chitosan has been extracted in the work,
since the necessary condition as stated by some literature
is that the degree of deacetylation should be above 50%
and it should in acidic media [2]. The chitosan was found
to dissolve completely in 1% acetic acid.
57
 International Journal of Advanced Research and Publications
ISSN: 2456-9992

14
FT-IR spectra of chitosan synthesized using different
12
DEGREE OF DEACETYLATION concentrations of sodium hydroxide 30%w/v, 40% w/v
and 50%w/v NaOH respectively. They all showed a broad
pH of solution

10
peak at 3500-3300cm-1 for OH and NH2 stretching
8 vibrations [21][22][23]. The amide I band or C=O and
30% NaOH
6 amide II band or NH deformation appeared at 1651cm-1
4 40% NaOH and 1580cm-1 respectively[21][24] They all showed
2 50% NaOH absorption peaks at 1423-1416cm-1which are due to CH2
0 bending[24][25]. They also showed peaks at the range of
0 20 40 60 1155-1148cm-1 and 1077-1069cm1ascribed to C-O and C-
O-C stretching frequencies respectively [21][26][27]. The
Volume of NaOH used (cm3)
various chitosan synthesized using 30%w/v, 40%w/v and
50%w/vNaOH all showed the chitosan saccharide ring
stretching observed at 895cm-1 .
Figure 1: degree of deacetylation
The UV spectra of the chitosan, was recorded in the range
of 180mm-600mm as shown in Figure 2-4 the absorption
band of chitosan were noticeably shown in the UV
spectrum at 237nm, 227nm and 226nm for 30%, 40% and
50%NaOH respectively, these values are in agreement
with the report of [20].

Figure 5: FT-IR spectra of 30% NaOH chitosan

Figure 2: Uv-Visible spectra of 30% w/v NaOH chitosan

Figure 6: FT-IR spectra of 40% NaOH chitosan

Figure 3: Uv-Visible spectra of 40% NaOH chitosan

Figure 7: FT-IR spectra of 50% NaOH chitosan

The DSC curves as shown in figure 8-10 the extracted

Figure 4: Uv-Visible spectra of 50% NaOH Chitosan
chitosans showed endothermic peaks at a range of
79.33°C- 84.74°C. The endothermic peaks, often called
dehydration temperature are due to the evaporation of
water molecules. This values are in close agreement with
the report of of 79°C. The presence of the dehydration
temperature suggests that some bound water was still not
removed from the samples after drying. While it gives an
exothermic transition at 345°C which likely corresponds

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 International Journal of Advanced Research and Publications
ISSN: 2456-9992

to chitosan decomposition since polysaccharides do not Table 3: Minimum Inhibitory Concentration of chitosan
melt.
minimum inhibitory concentration of chitosan in
mg/ml

40% 50%
Test organisms 30%NaOH
NaOH NaOH
Staphylococus aureus 25 12.5 12.5
Bacillus subtilis 25 12.5 12.5
Escherichia coli 25 6.25 6.25
Salmonella typhi 25 25 6.25
Pseudomonas
25 12.5 12.5
aeruginosa
Klebsiella pneumonia 25 25 12.5
Figure 8: DSC spectra of 30% NaOH chitosan Candida albicans 0 0 0
20 Aspergillus niger 25 25 25

15 The antimicrobial studies, showed that the polymers have

a strong activity against Bacillus subtilis, Escherichia coli,
10 Staphylococus aureus, Salmonella typhi, Pseudomonas
aeruginosa, Klebsiella pneumonia, Candida albicans and
Aspergillus niger. The zone of inhibition of the organisms
5 that showed sensitivity ranges from 16mm-26mm with
30%w/v NaOH having the least activity while and the
0 synthetic antimicrobial polymer having the highest
0 100 200 300 400 500 activity as shown in Table 2-3 The MBC/MBF values of
-5 the polymers are 50mg/ml for 30% NaOH for all the
organisms that showed sensitivity except it was unable to
Figure 9: DSC spectra of 40% NaOH chitosan kill it at that concentration, 40%w/v NaOH, with MBC of
12.5mg/ml for Escherichia coli, 25mg/ml for
Staphylococus aureus, Bacillus subtilis and Pseudomonas
aeruginosa. Staphylococus aureusis known to play an
important role in skin diseases including superficial

Conclusion
The concentration of sodium hydroxide used in the
deacetylation, as the concentration increases the degree of
deacetylation of the chitosan increases, which further
enhance the antimicrobial activity of the selected
microbial organisms, while the thermal stability decreases
as the degree of deacetylation of the chitosan decreases,
because the chitosan became less crystalline.
Figure 10: DSC spectra of 50% NaOH chitosan
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