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Extraction, characterization and antimicrobial activity of chitosan

from pen shell, Pinna bicolor

B.P. Sudatta, V. Sugumar, Rahul Varma, P. Nigariga

PII: S0141-8130(20)33743-0
DOI: https://doi.org/10.1016/j.ijbiomac.2020.06.291
Reference: BIOMAC 16048

To appear in: International Journal of Biological Macromolecules

Received date: 14 April 2020

Revised date: 29 June 2020
Accepted date: 30 June 2020

Please cite this article as: B.P. Sudatta, V. Sugumar, R. Varma, et al., Extraction,
characterization and antimicrobial activity of chitosan from pen shell, Pinna bicolor,
International Journal of Biological Macromolecules (2020), https://doi.org/10.1016/
j.ijbiomac.2020.06.291

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EXTRACTION, CHARACTERIZATION AND ANTIMICROBIAL ACTIVITY OF

CHITOSAN FROM PEN SHELL, PINNA BICOLOR

Author Names : Sudatta, B. P., Sugumar, V.,* Rahul Varma, Nigariga, P.

Address : Department of Oceanography and Coastal Area Studies,

Alagappa University, Science Campus, Karaikudi 630 003.

*Corresponding Author: Sugumar, V

Email ID : crustacealab@gmail.com

Contact Number :+919445139906

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Highlights:
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 Extraction of chitosan from the pen shell, P. bicolor by demineralisation and
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deacetylation
 Chitosan characterisation with FTIR, XRD and Micro Raman analysis
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 Extraction of chitosan from chitin of P. bicolor with 59.76% deacetylation

 Elemental analysis revealed higher percentage of carbon than other elements
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 Antibacterial activity of extracted chitosan against both gram-positive and gram-

negative bacteria
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ABSTRACT
Chitosan is a biopolymer discovered abundantly on earth specifically in the
exoskeleton of shrimps, crabs and insects. In the present study, isolation and characterisation
of chitosan from the pen shell Pinna bicolor was carried out. In addition to this, the
chitosan acquired from the pen shell was tested for its antibacterial activity against five
bacterial strains. The FTIR analysis confirmed the presence of NH out of plane bending (872
cm-1) and C-O-C stretching (1016 cm-1) for chitosan with 59.76% degree of deacetylation.
The Micro Raman showed peaks at 1658 cm-1, 1595 cm-1 and at 954 cm-1 corresponding to
chitosan. The XRD was able to establish the crystallinity of the chitosan sample with a
maximum peak at 29.3°. The elemental analysis of chitosan sample confirmed higher level of

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carbon (10.75%) when compared to other elements such as nitrogen, hydrogen and sulphur.

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The antimicrobial activity of extracted chitosan was evident with greater zone of inhibition
against Salmonella typhi (20 mm) and least against Shigella dysenteriae. Thus, the present
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study unravels the properties of chitosan extracted from P. bicolor thereby paving way for its
further use in the field of biomedical science and nanotechnology.
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Keywords: Chitin, Chitosan, Biopolymer, Pen Shell, Characterization, Antimicrobial

Activity
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1. INTRODUCTION
Biopolymers are polymeric structures isolated from the living organisms. They
comprise of discrete components of various covalent bonds that are joined together to form
a longer framework. Chitin is a high molecular weight, non-toxic compostable, naturally
produced polymer. Chitin is the most important part of the fungal cell walls, arthropod
exoskeleton, insects and radula of mollusc cephalopods [1]. Among the ,  and 
crystalline forms of chitin, -chitin is the most stable form of chitin due to the antiparallel
orientation of the polysaccharide chains [2]. Chitin also shares certain similarities with
cellulose, except that a number of -OH groups bonding present in cellulose [3]. The

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formation of chitosan from chitin results from the removal of the acetyl groups (CH3-CO) to

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make the material be soluble in most diluted acids. The deacetylation process releases
amine groups (NH) and provides the chitosan a cationic characteristic. As the degree of N-
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acetylation decreases to an amount less than 50%, chitin becomes soluble in aqueous acidic
solutions (pH < 6.0) and is then converted to chitosan. Chitosan is a linear polysaccharide
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comprising of deacetylated and acetylated units of D-glucosamine linked by (1,4)
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glycosidic bonds. Chitosan contains nitrogen in comparison to cellulose, which makes it a

potential chelator of metal ions such as iron, magnesium and cadmium.
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Chitin and its derivative, mainly chitosan, have anticoagulant, antimicrobial and
antioxidant properties and it may be used for various pharmaceutical and medical
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applications thanks to its biodegradability, biocompatibility and nontoxicity [4]. Chitosan

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has been a potential drug carrier owing to its biocompatible properties. The use of fabricated
chitosan nanoparticles in anticancer drug delivery has been documented by Saravanabhavan
et al., [5] and Wang et al. [6] have studied the use of BSA and chitosan in the preparation of
nanoparticles of polyphenols from the pine cones of Pinus koraiensis. The blue crab shells
have been utilised for the synthesis of chitosan nanoparticles using sodium tripolyphosphate
[7]. Alternative methods to chemical processes in the production of chitin/chitosan
nanofibers has been documented. Kadokawa [8] has reported an environment – friendly
approach to produce chitin-chitosan nanofibers by self-assembly mechanism using
amphiphilic chitosan derivatives.

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The pen shell, Pinna bicolor is the species of bivalve mollusc that lives in the soft
bottoms of seagrass regions, muddy area, sandy area as well as in the coral regions. They
are the largest bivalve family found on earth which are capable of attaining lengths up to
120 cm [9]. It is normally found at depths between 0.5 and 60 m. The structure of the fan
present in the shell allows the animal to live in soft sediments, suppressed into the substrate
up to at least one third of its sharp anterior apex, attached by fine byssal thread [9,10].
Different methods of extraction of chitosan and characterization using facilities such
as FTIR, Micro Raman and XRD are briefly mentioned in the studies carried out by
Abdulwadud et al. [10], Majekodunmi et al. [11] and Furuhashi et al. [12]. Weiss and
Schönitzer [13], Winkler [14] and Zentz et al. [15] have extracted chitin from mollusc’s

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shells Atrina rigida, Aplysia californica, Bulla gouldiana, Haliotis tuberculata and

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the oyster Pinctada maxima.
Kumirska et al. [16] have described the application of spectroscopic methods for the
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structural analysis of chitosan. A simplest and faster technique for the determination of
allomorphic forms of chitosan is by utilizing the Fourier Transform Infrared spectroscopy
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[17]. It is reported in many studies that the FTIR spectra of α- and β-chitin gives two sharp
bands at 1660 and 1620 cm-1. The FTIR spectra of chitosan shows different
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bands resembling -NH2 group which might be allocated to the symmetrical COO- gather
extending vibration [18,19]. The results of FTIR, in the case of chitosan, shows major bands
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of C=O secondary amide stretch (1660 cm-1) and N-H bend (1550 cm-1) [20]. FTIR studies
are based on the vibrations of particles and they ordinarily records vitality of the
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electromagnetic radiations which are conducted through a sample as a work of wavenumber

[21].
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The Micro Raman may be a way used for the determination of vibrational modes of
molecules. The Micro Raman analysis is utilized for obtaining the structural fingerprint of a
material. This technique profoundly depends on the inelastic diffusing of photons [22]. The
examination of the chemical structure of chitosan reveals a six-membered ring containing
oxygen. The foremost peaks are found to be present at 471 cm-1 (bending vibration of C-C-
O bonds) and 895 cm-1, 1146 cm-1 (stretching vibration of C-C-O bonds) [23]. Zhu et al.
[24] have studied the adsorption kinetics and thermodynamics of novel magnetic chitosan
enwrapping nanosized -Fe2O3. Being crystalline is one among the most prominent
characteristics of chitin and chitosan as identified using the X-ray diffraction (XRD). The
XRD gives data with relevance the physical and chemical form of a molecule [25].

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According to the XRD studies conducted by Jang et al. [18] and Kaya et al. [19], there have
been four weak peaks (13⁰ , 21⁰ , 23⁰ , 26⁰ ) in α-chitin. Elemental analysis may be a
tool utilized for the recognizable proof of components shown during a material. The
elemental analysis in some cases are used for the detection of isotopic compositions in the
material. Elemental analysis gives only a baseline data of the particles present within the
sample. Therefore, the differentiation of the specific modification within the material is a
difficult process.
Chitosan which incorporates a higher positive charge, is expected to possess a
stronger antimicrobial activity. It is discovered that the degree of deacetylation of the
chitosan may additionally affect its antimicrobial activity [26, 27, 28]. Studies about the

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antimicrobial activities and properties of chitosan is a major component in scientific

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exploration and technological development. Based on the previous studies, the current work
is aimed to extract, characterize and estimate the antimicrobial activity of chitosan from the
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pen shell Pinna bicolor.
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2. MATERIAL AND METHODS
2.1 Collection of pen shells
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The pen shells, Pinna bicolor were obtained from the local fishermen at Thondi Coast
(9⁰ 45’ N, 79⁰ 04’ E). The collected shells were dried for seven days. The dried shells
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were weighed and grounded for further use (Fig. 1).

2.2 Extraction of chitin
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The extraction of chitin was done as per the methodology of Kaya et al. [19]. The
extraction method proposed here involved four chemical treatment steps, with each step
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followed by rinsing with distilled water until a neutral pH was reached. The samples (4 g of
powder sample) were refluxed in 40 mL sodium hypochlorite (NaOCl) solution (3%, v: v)
at 100 °C for 10 min. After washing with distilled water, the procedure was repeated.
Demineralization was done by refluxing the samples in 20 mL of 1 M HCl at 75 °C for 15
min. Then, the samples were refluxed in 20 mL of 1 M NaOH (sodium hydroxide) solution
at 100 °C for 20 minutes to get rid of any protein residues. Finally, the extracts were filtered
off and placed in an oven at 60 °C for five days. The dry mass of the chitin sample was
calculated and then the share of chitin content was determined.
2.3 Extraction of chitosan
The extraction of chitosan was followed according to the methodology of Rasti [29].
This process of extraction of chitosan was administered by dissolving the deproteinized

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product in 45% sodium hydroxide solution (15 ml/g) at 110 ºC for 24 h. The product
acquired was found to be soluble in 2% acetic acid which indicates a high degree of
deacetylation. Then, the resulting product was continuously washed with distilled water till
the pH was neutral and filtered so as to separate the solid matter, which was the
ultimate product.
2.4 Characterization of chitin and chitosan
2.4.1 Fourier Transform Infrared Spectroscopy (FTIR)
KBr supported sample chitosan was taken for the FTIR analysis over the frequency
range between 4000 - 400 cm-1 at a resolution of 4 cm-1 employing a model of Perkins-Elmer
spectrometer (Spectrum RX I, MA, USA). The degree of acetylation (DA) was determined in

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triplicate from FTIR [30].

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DA (%) = (A1792/A3448) * 115
where the A1792 represents the degree of absorption at 1792 cm-1 and A3448 represents the
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degree of absorption at 3448 cm-1.
2.4.2 Micro Raman Spectroscopy
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In the present investigation, Micro Raman spectra were used to study the vibrational
modes of films using of Princeton instrument Acton SP 2500 (Japan) under an excitation
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wavelength of 632 nm by using argon laser.

2.4.3 X-ray Powder Diffractometry (XRD)
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X-ray diffraction analysis (XRD) was carried out for the detection of the crystallinity
of the extracted samples. X’ Pert PRO PAN analytical (Netherlands) instrument was
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operated at 40 kV and 30 mA with Cu kα= 1.5406A0.

2.4.4 Elemental Analysis
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The Vario micro cube CHNS (Elementar, Germany) was used for the analysis. The
samples were coated with a tin boat and then introduced to the elemental analyser and was
combusted within the presence of helium and oxygen gases.
2.4.5 Antimicrobial Activity
The antibacterial activity of the chitosan on the gram positive and gram-negative
bacteria was studied by well diffusion method. Samples were dissolved in water and
constantly stirred until an even colloidal solution was formed [31]. Different concentrations
of sample (50µg/mL,100 µg/mL 150µg/mL, and 200µg/mL) were prepared from the stock
solution (1mg/ml) mixed in appropriate amount of 0.1% acetic acid. Muller Hinton agar
(5.3 g) was dissolved in 100 ml of distilled water and kept at 121oC about 15-20 minutes for
nutrient medium sterilization. Sterilized medium was poured on the sterilized plates while

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the temperature of medium was bearable. To assess the toxicity range of the sample against
Escherichia-coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa,
Klebsiella pneumonia, Salmonella typhi, and Shigella dysenteriae an appropriate quantity of
these bacteria was swabbed gently on the nutrient broth medium and equitized wells were
created. Samples of different concentrations were injected into the respective wells together
with positive and negative control. 0.1% acetic acid served as negative control and for
positive control, (30µg/mL) tetracycline was used for E. coli, S. aureus, cefuroxime was
used for B. subtilis and ceftriaxone (30µg/mL) was used for K. pneumonia, S. typhi, S.
dysenteriae. The plates were kept overnight for incubation at 32°C. After the incubation
time, the zone of inhibition was measured in mm and the best range was proposed from 50-

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200 µg/mL of samples.

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3. RESULTS -p
3.1 Extraction of chitin and chitosan
Chitin and chitosan were extracted from Pinna bicolor with an average weight of 50
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g. The amount of chitin and chitosan extracted from 100 g of grounded powder was 80.15%
and 0.021% respectively.
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3.2 Characterization of chitin and chitosan

3.2.1 Fourier Transform Infrared Spectroscopy (FTIR)
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FTIR studies of chitosan revealed major bands between 700 to 3000 cm-1. The
samples showed absorbance bands at peaks of 711, 872, 968, 1016, 1215, 1395, 1741, 1792,
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2137, 2511, 2855, 2926, 3448 and 3941 cm-1 for chitosan. Among them, 711, 871 and 872
cm-1 indicate -NH out of plane bending for chitosan and ring stretching (Fig. 2). The C-O-C
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stretching was observed at 1016 cm-1 and amide I and II bands were observed at 1741 cm-1
and 1395 cm-1 respectively. Aliphatic CH stretching was observed at 2855 cm-1 in both
chitin and chitosan. The peak at 2926 cm-1 showed the presence of an asymmetric CH2
stretching in chitosan. Assigned –OH stretching was observed at 3448 cm-1 in chitosan
(Table 1). Peaks for commercial standard chitosan were found at 1604 cm-1, 1598 cm-1 and
1592 cm-1. The degree of deacetylation of chitosan extracted from Pinna bicolor was
estimated from the FTIR spectrum. The result showed 59.76% deacetylation for the
extracted chitosan.
3.2.2 Micro Raman Spectroscopy

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The vibrational shift of chitosan was observed at 474 cm-1 and 2433 cm-1. The
extracted chitosan gave the Raman peaks at 1658 cm-1, 1595 cm-1 and at 954 cm-1 at the shift
distribution ranging from 4000 – 500 cm-1 (Fig. 3).
3.2.3 X-ray powder diffractometry (XRD)
A total of 13 strong peaks were obtained from chitosan, among which sharp peaks
were observed between 23-50° (1200 - 1300 counts/s) with the strongest peak at 2θ 29.3°
(2144 counts /s), 19.63° and at 20° (Fig. 4).
3.2.4 Elemental analysis
The elemental analysis was carried out to find out the percentage of elemental and
isotopic compounds in the samples. The percentage of nitrogen, carbon, hydrogen and

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sulphur in the extracted chitosan was 2.90%, 10.75%, 0.64% and 0.91% (Table 2).

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3.2.5 Antimicrobial Activity
The antimicrobial activity of chitosan was test against gram positive and gram-
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negative bacteria. The zone of inhibition against E. coli was observed only at 200 µg/ml
concentration of chitosan (15 mm). Zones of inhibition of chitosan against P. aeruginosa
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were prominent in all the concentrations studied, with greater zone of inhibition at 200
g/ml (19 mm) followed by 150 g/ml (13 mm). Eminent antimicrobial activity of chitosan
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was observed at 150 µg/ml and 200 µg/ml against K. pneumonia (7 and 13 mm), S. typhi
(18 and 20 mm), S. aureus (7 and 15 mm) and B. subtilis (9 and 19 mm). S. dysenteriae
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failed to show any activity in the chitosan sample (Table 3: Fig 5).
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4. DISCUSSION
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Extensive studies have been carried out highlighting the application of chitin and
chitosan from marine waste resources and its transformation into valuable commercial
products as a solution of waste management. The present study deals with the
characterisation of chitosan from the pen shell, Pinna bicolor. The percent of chitin
extracted in the present study from P. bicolor is 80.15%, which is much higher than those
reported for tiger prawn (16.75%), jinga shrimp (19.13%), blue crab (20.8% for males, and
20.14% for females), scyllarid lobster (21.26%), and also the cuttlefish (7.4%) within
the Persian Gulf [32]. In previous studies, the amount of chitin extracted from the organisms
such as crab, crayfish and shrimp shells were observed to be in the range of 10% to 20%
[33] which is a smaller amount than our yield product.

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. Majekodunmi et al. [11] reported that the yield of chitosan from Mytilus edulis was
51.8% and from Laevicardium attenuatum was 43.8% which is beyond that of P. bicolor.
The studies conducted by Paul et al. [34] reported that the chitosan yield from
Fenneropenaeus indicus was 67%.
The present study showed different absorption peaks with FTIR studies and among
them 711 cm-1 indicated the potentiality of –NH out of plane bending in chitosan. The ring
stretching in chitosan was observed at 871 cm-1 and also at 872 cm-1. The results of the
present study were similar to those observed by Lavall et al. [35] in Loligo sp. and
Shanmugam et al. [36] in donacid clam. FTIR spectra for chitosan gave a characteristic –
NH2 band of 3447 cm-1 and a carbonyl group band of 1447 cm-1 from mussel shell [10].

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Dahmane et al. [37] attributed characteristic peaks for chitosan at 3400 cm-1 to the

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stretching vibration and intermolecular hydrogen bonding of -NH2 and -OH groups. The
FTIR analysis by Thillai Natarajan et al. [38] reported a characteristic-NH2 band at 2921
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cm-1 and a carbonyl group band at 2523 cm-1 for chitosan extracted from Achatinodes.
The degree of deacetylation of chitosan extracted from the pen shell, Pinna bicolor
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was calculated from FTIR studies and was observed to be 59.76%. The ratio between the
absorbance of amide group to that of the hydroxyl group determines the degree of
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deacetylation of chitosan. A higher degree of deacetylation has been obtained for Mytilus
edulis chitosan (69.60 ± 0.12%) and a lower degree of deacetylation has been obtained for
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Laevicardium attenuatum (37.30 ± 0.31) [19] when compared to the present study. Al-
Hassan [39] reported 54.65% in shrimp shell while Muñoz et al. [40] observed a
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deacetylation degree of 73.6% from Aspergillus niger mycelium.

Variations in the degree of deacetylation (54.6%, 60.5% and 84.7%) was observed by
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Hussain et al. [41] for untreated, 4 h treated and 8 h alkali treated samples. Pursetyo et al.
[42] have framed a multistage deacetylation process of chitin to obtain the deacetylation
degree of 75% and solubility of 82.91%. The alkali concentration, pressure and non-
pulverisation of chitin used for the deacetylation could have attributed for the low degree of
deacetylation in the present study. Hossain and Iqbal [43] have extended heating time and
high alkali concentration to drastically improve the degree of deacetylation.
The Micro Raman Spectra obtained in the present study, showed a Raman shift for
chitosan at 2433 cm-1 and 474 cm-1 which is similar to those observed in Sepia pharaonis
[44]. Further, C-O stretching (1085 cm-1) and the vibrational shift of the extracted samples
at 2178 cm-1 which exhibited linear variation were also concomitant with previous studies.
The weak spectral peaks at 446, 479 and 507 cm-1 are the major indicators of the

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disintegration of vibrations of O=S=O groups [18, 19]. Sagheer et al. [32] have reported
five sharp peaks which were similar to those obtained in the present study. Rasti et al. [29]
reported stronger sharp reflections for chitosan from mollusc chiton around 30-35° which
are similar to those observed for chitosan in the present study.
Molluscs play a predominant role in the accumulation and circulation of nitrogen,
carbon and calcium in their habitats. The elemental analysis was administered to seek out the
percentage composition of carbon, nitrogen, hydrogen and sulphur within the extracted
chitosan. The result showed that the contribution of carbon is high in chitosan (10.75%) when
compared to that of nitrogen, hydrogen and sulphur. These results are concomitant with those
obtained by Karnkowska [45] in snails (Viviparus viviparus and Lymnaea stagnalis) as well

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as in bivalves (Dreissena polymorpha and Anodonta anatina from the Zegrzyński reservoir

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ecosystem. The mean content of nitrogen ranged from 0.19 to 0.56%, whereas mean carbon
content was on an average of 13%. A higher percentage of elements has been reported in the
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chitosan extracted from the shell of Sepia pharaonis (C- 42.9%, H - 6.78%, N - 5.7%) and
from Persian Gulf Chiton (N - 6.45%, C - 40.86%) [44]. The percentage of organic matter
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increases with the length of individuals due to the increasing ratio between the shell weight
and tissue weight and moreover the organic matter contained in shells will cycle at a much
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slower rate than body fraction [46].

The antibacterial activity of chitosan extracted from P. bicolor is evident against the
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bacterial strains except S. dysenteriae. The present study demonstrated the highest zone of
inhibition for chitosan against S. typhi at the concentration of 200 µg/ml while on the
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other hand the antimicrobial activity of chitosan against S. dysenteriae displayed minimum
inhibition. Greater inhibitory activity was witnessed at 200 µg/ml concentration of chitosan
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against the bacteria studied these results were found to be similar with the other studies
[47,48]. Packirisamy et al. [49] have synthesized novel chitosan nanocomposites and
investigated its antibacterial properties.
The antibacterial effect of chitosan has been reported due to increase its hydrophobic
character which in turn is dependent on the number of N-acetyl groups [44, 47, 49]. Earlier
studies indicated higher antibacterial activity of chitosan against gram-positive bacteria (S.
aureus, Bacillus subtilis, Sarcinia lutea) than gram-negative bacteria (E. coli, Serratia
marcescens) [50]. Chitosan with a lower degree of acetylation or a higher number of free
amino groups has been witnessed to have increased antimicrobial activity against various
strains of fungi, gram-positive and gram-negative bacteria (S. aureus and E. coli) [25].

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5. CONCLUSIONS
The quality and chemical properties of both chitin and chitosan are highly economical
and commercially valuable. Chitosan extracted from the pen shell, Pinna bicolor was
characterized using various methods like FTIR, Micro Raman, XRD and elemental analysis.
The FTIR and also the Micro Raman analysis confirmed the functional groups present within
the chitosan sample. The degree of deacetylation of 59.76% was obtained from the FTIR
studies of chitosan. The results of XRD showed the crystalline nature of the sample and also
the elemental analyses proved the presence of carbon, nitrogen hydrogen and sulphur within
the sample. The study also reveals the antibacterial activity of chitosan against both gram-
positive and gram-negative bacteria at a concentration of 200 µg/ml. Further antimicrobial

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studies to elucidate the effect of chitosan over a wide range of microbes and extraction of

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antimicrobial proteins from the pen shell, Pinna bicolor are underway and the properties of
chitin and chitosan vouch them suitable for nanodrug delivery.
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ACKNOWLEDGEMENT
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We gratefully acknowledge the funding received from the RUSA-Phase 2.0 grant
sanctioned vide letter. No F. 24-51/2014-U, Policy (TN Multi-Gen), Department of
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Education, Govt. of India. Dt. 09.10.2018.

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Table 1: Wavenumber and possible assignment of absorption band of chitosan

from Pinna bicolor

Wavenumber (cm-1) Vibration modes or possible

assignment
711 NH out of plane bending
872 Ring stretching
1016 CO stretching
1395 Amide II
1741 Amide I
2855 Aliphatic CH stretching

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2926 Symmetric CH stretching

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3448 -OH stretching
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Table 2: Elemental Composition in the extracted chitin and chitosan of Pinna bicolor

Nitrogen Carbon Hydrogen Sulphur

S. No. Name
(%) (%) (%) (%)
1. Chitin 6.07 12.32 0.31 0.21
2. Chitosan 2.90 10.75 0.64 0.91

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Table 3: Antimicrobial activity of chitosan extracted from Pinna bicolor

Zone of Inhibition (mm)

Species 50 (g/ml) 100 (g/ml) 150 (g/ml) 200 (g/ml)
Gram Positive
Escherichia coli - - - 15
Pseudomonas aeruginosa 8.5 8 13 19
Klebsiella pneumonia - - 7 13
Salmonella typhi 12 15 18 20
Shigella dysenteriae - - - -
Gram Negative

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Staphylococcus aureus - - 7 15
Bacillus subtilis - - 9 19

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Fig. 1 Dorsal view of the pen shell Pinna bicolor

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Fig. 2 FTIR spectra of chitosan extracted from P. bicolor

100

90

3941.99cm-1
80
473.54cm-1

601.96cm-1
70 2137.88cm-1 417.78cm-1
2511.27cm-1
3448.76cm-1 1792.62cm-1
2855.90cm-1 448.67cm-1
60 572.72cm-1
2926.29cm-1
534.30cm-1
516.05cm-1
%T

50
1741.29cm-1 1215.32cm-1

711.77cm-1
40 1016.33cm-1

968.75cm-1

30

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872.34cm-1

1395.60cm-1
10

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0
4000 3500 3000 2500 2000 1500 1000 500 400
cm-1
Name Description
ALU-IR-Sud 4-
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Fig. 3 Raman shift of the extracted chitosan from P. bicolor

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Fig. 4 XRD of extracted chitosan from P. bicolor

Counts
SuKi_1

800

600
Counts

400

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200

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20 30 40 50 60 70 80
Position [°2Theta] (Copper (Cu))
2
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Fig 5 Antimicrobial activity of chitosan extracted from P. bicolor

A B C

G) S. aureus

D E F

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A) B. subtilis B) E. coli C) K. pneumonia D) P. aeruginosa E) S. typhi F) S. dysenteriae

G) S. aureus

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31.05.2020

From

Dr. Sugumar Vasudevan

Assistant Professor

Department of Oceanography and Coastal Area Studies

Alagappa University

Karaikudi – 630003

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Tamilnadu, India.

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Dr. Ian Sims
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Editor
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International Journal of Biological Macromolecules

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Sub: Authors Statement - Manuscript ID: IJBIOMAC-D-20-00916

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With subject to the above, this is to certify that all authors have seen and approved the final
version of the manuscript being submitted (“Extraction, characterization and antimicrobial activity of
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chitosan from the pen shell, Pinna bicolor” (Manuscript ID: IJBIOMAC-D-20-00916)). We warrant that
the article is our original work, hasn't received prior publication and isn't under consideration for
publication elsewhere.

Sincerely,

Sugumar Vasudevan

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