Extraction of Chitosan from Crab Shell and Fungi and Its Antibacterial Activity against Urinary Tract Infection Causing Pathogens

Abstract

In this study, shells of sea crab and fungus Aspergillus niger were subjected for chitosan extraction which has been done following demineralization, deproteination and deacetylation. Chitosan yield from crab shell and fungi was 37.5% and 39.3% respectively and water binding capacity was 58.44% and 60.21% respectively. The extracted chitosan was characterized using Fourier transform infrared spectroscopy (FTIR) and subjected for antibacterial activity against Urinary tract infection (UTI) pathogens – Klebsiella pneumoniae, Proteus mirabilis and E. coli. Chitosan of crab shell showed better antibacterial activity than fungal derived chitosan. Chitosan gel was prepared using the extracted chitosan where it was also showing good antibacterial activity.

Full text

*Correspondence: antonysamrot@gmail.com; abisasi@gmail.com
(Received: August 30, 2020; accepted: May 25, 2021)
 Abirami S, Emilin RR, Antony VS, et al. Extracon of Chitosan from Crab Shell and Fungi and Its Anbacterial Acvity
against Urinary Tract Infecon Causing Pathogens. J Pure Appl Microbiol. 2021;15(2):968-975. doi: 10.22207/JPAM.15.2.55
© The Author(s) 2021. Open Access. This arcle is distributed under the terms of the Creave Commons Aribuon 4.0 Internaonal License which
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Abirami et al. | J Pure Appl Microbiol | 15(2):968-975 | June 2021
Arcle 6620 | hps://doi.org/10.22207/JPAM.15.2.55
Print ISSN: 0973-7510; E-ISSN: 2581-690X
RESEARCH ARTICLE OPEN ACCESS
www.microbiologyjournal.org968Journal of Pure and Applied Microbiology



123*1,
114555
1Department of Microbiology, Kamaraj College, Thoothukudi - 628 003, Tamil Nadu, India.
2Department of Food Processing Technology, School of Agriculture and Biosciences, Karunya Instute of
Science and Technology, Karunya Nagar, Coimbatore - 641 114, India.
3School of Biomedical Sciences, Faculty of Medicine, Nursing, and Bioscience, MAHSA University, Jalan SP 2,
Bandar Saujana Putra, 42610 Jenjarom Selangor, Malaysia.
4Department of Microbiology, Sree Narayana College, Alathur, Palakkad, Kerala - 678 682, Kerala, India.
5Department of Biotechnology, School of Bio and Chemical Engineering, Sathyabama Instute of Science and
Technology, Chennai - 600 119, Tamil Nadu, India.

 Aspergillus niger



Klebsiella
pneumoniae, Proteus mirabilis and E. coli. 
 

Crab shell, fungal cell, chitosan, anbacterial acvity, UTI pathogens

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Journal of Pure and Applied Microbiology

Chitin, an abundant natural amino
polysaccharide which is a constituent of
exoskeleton of shrimp, crab etc and also seen
on fungi cell walls1,2. Deacetylated form of
chin is chitosan which consists of polymer of
N-acetyl D-glucosamine bonded through β-1,4-
glycosidic bond3,4. This chitosan has multiple
functional properties and biological activities
thus used in pharmaceutical, nanotechnology
and agricultural industries 5- 8. Its anmicrobial
acvity made them to be used in food preservaon
as coang agents on wrapper of various foods
8. Its non-toxicity and non-allergenicity made
them to be used in biomedical applications
and also used in treang wounds9- 12,4. Chitosan
coated nanoparcles are used for heavy metal
removal, nanocarrier synthesis, drug delivery
etc13-18. Urinary tract infecons (UTI), commonly
caused by bacteria and their pathogenicity
depends on host biological behavioral factors
and properes of the infecng uropathogens19.
The major bacteria causing UTI are Proteus
mirabilis, Enterococcus faecalis, Escherichia
coli, Pseudomonas aeruginosa, and Klebsiella
pneumoniae20. Emergence of drug resistance
making the treatment harder, and development
of new drugs become inevitable21-23. If derived
from biological sources it might be having less side
eect22, chitosan is one such which can be derived
from crab shell and fungal cell wall. Thus, in this
study, chitosan of crab shells and fungal cell walls
was extracted and used for anbacterial acvity
against UTI pathogens.
MATERIALS AND METHODS

The crab shells required for the
experiments were brought from local fish
processing industries in Tucorin, Tamil Nadu,
India. The crab shells were washed, cleaned,
dried at 70°C in hot air oven, powdered using
blender and stored in air ght container in room
temperature.

Soil fungi Aspergillus niger was isolated
from soil and inoculated into 100 mL of yeast
peptone glucose (YPG) medium (2.0% glucose,
yeast peptone glucose - 0.2% yeast extract, and
1.0% peptone) and incubated at 28°C for 5 days.
The fungal mycelium was transferred into 500 mL
of YPG medium. The asks containing inoculum
were kept for incubation without shaking at
28°C for 5 days. Mycelium was obtained through
filtration process using Whatman filter paper
and harvested mycelia was washed with dislled
water and dried in hot air oven and ground to a
powder and stored in a sterile airght container
at room temperature for extracon for chitosan.
The extracon of chitosan consisted of three steps
such as, deproteinizaon, demineralizaon and
deacetylaon.


Deproteinizaon of both the fungi and crab
shell was done as prescribed24. Demineralizaon
and deacetylaon of chin were done following
the earlier reports25- 26. Determinaon of yield of
crab shell and fungal biomass chitosan was done27.
The fat binding capacity and water binding capacity
of chitosan were measured using the protocol of
No et al.28.

FTIR characterization of sample was
performed with a Perkin Elmer spectrum RX1
instrument within a frequency range of 400 – 4000
cm-1. Degree of deacetylaon of chitosan was
determined29.


Urine sample isolates were collected from
AVM hospital, Tucorin, Tamil Nadu, India. Sterile
nutrient agar plates were prepared and inoculated
with urine sample. After 24 h of incubation,
observed colonies were idened through the
morphological and biochemical characteriscs.
All the organisms were checked for their anbioc
resistance paern against commercial anbioc
discs. Anbioc suscepbility was determined
using commercially available disc diusion assay.

Chitosan soluon was done by dissolving
chitosan in 1% of acec acid. This soluon was used
for tesng anbacterial acvity30. For anbacterial
acvity, Mueller Hinton agar (MHA) medium was
prepared and sterilized. The culture of pathogenic
microorganisms was swabbed onto the surface
of sterile MHA plates. The anbacterial acvity
was performed by agar well diusion method31.
Chitosan soluon of dierent concentraon (10 µg,

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20 µg, 30 µg) was added using a micropipee into
the wells (6 mm) punched over the MHA plates
using a sterile cork borer. These agar plates were
incubated for 24 h at 37°C. Zone of inhibion was
observed and measured around the wells aer the
period of incubaon.
      

In order to form the gel, method
described by Samano-Valencia et al.32 was followed
and anbacterial acvity was performed by agar
well diusion method.

The yield of extracted chitosan of crab
shell and fungi was found to be 38% and 39.3%
respecvely (Table 1). The moisture content of
the chitosan extracted from crab and fungi was
found to be 64.34% and 66% respecvely. The
water binding capacity and fat binding capacity of
crab and fungal chitosan was found to be 58.44%,
60.21% and 40%, 41% respecvely (Table 2).
 Percentage of extracted crab and fungal chitosan
S.No Source Inial weight Final weight % of extracted
(g) (g) Chitosan
1. Crab shell 20 7.5 37.5
2. Fungal biomass 20 7.86 39.3
 Physicochemical characterizaon of extracted chitosan
S.No Physicochemical Characteriscs Values
Crab Shell Fungal Biomass
chitosan Chitosan
1 % Water binding capacity of 58.44 60.21
Extracted Chitosan
2 % Fat binding capacity of 40 41
extracted Chitosan
3 Moisture content 64.34 66
4 Degree of deacetylaon (%) 90 60
Extracted chitosan a) Crab shell derived b) fungal derived
The appearance of extracted chitosan
from crab shell was yellowish white in color
(Fig.1a) and from fungi it was black in color due
to its conidial nature (Fig.1b). FTIR spectrum of
crab chitosan recorded major peaks lying between
500.05 cm-1 and 3880.60 cm-1 (Fig. 2). FT-IR
spectrum of the fungal chitosan recorded major
peaks lying between 564.67cm-1 and 3451.92 cm-
1. In this way, our study of FT-IR analysis showed
spectral peaks of extracted chitosan from crab

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Journal of Pure and Applied Microbiology
and fungi were conrmed with their funconal
groups and crab and fungal chitosan with degree
of deacetylaon (DD) of 90%, 60% respecvely.
The idened organisms of urine samples
were Escherichia coli, Proteus mirabilis and
Klebsiella pneumoniae through its biochemical
characteriscs. All the organisms were showing
resistance to one or two antibiotics used in
this study (Table 3). 20 µg of crab shell derived
chitosan showed 6 mm zone of inhibion against
Escherichia coli whereas 30 µg showed 8 mm
against K.pneumoniae (Table 4 and Fig. 3). The
highest zone of inhibion was observed at 30 µg
of both crab shell chitosan and fungal chitosan
against all bacteria (Table 4, Fig. 3 and 4).
 Anbioc suscepbility of UTI pathogens
S.No Pathogens Tetracycline Streptomycin Noroxacin Methicillin
(30mcg) (10mcg) (10mcg) (10mcg)
1 E. coli R I R R
2 P. mirabilis I I R R
3 K. pneumoniae R R I R
R- resistant, I - intermediate
 Anbacterial acvity of crab chitosan against the urinary pathogens
Organism Zone of inhibion (mm)
used Crab Fungal Crab Fungal Crab Fungal
chitosan chitosan chitosan chitosan chitosan chitosan
(10 µg) (10 µg) (20 µg) (20 µg) (30 µg) (30 µg)
E. coli - 2 6 5 8 6
P. mirabilis - - - 2 5 2
K. pneumoniae - - 1 - 2 3
 Fourier transform infrared spectroscopy (FTIIR) of chitosan a) Crab shell derived b) fungal derived

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Anbacterial acvity of crab shell chitosan
gel showed maximum zone of inhibion i.e. 6
mm against Escherichia coli (Fig. 5) where fungal
chitosan showed maximum zone of inhibion at
100 µg against Proteus mirabilis (4 mm).
 Anbacterial acvity of crab shell chitosan a) E.coli b) Proteus mirabilis c) K.pneumoniae
 Anbacterial acvity of fungal chitosan a) E. coli b) Proteus mirabilis c) K. pneumoniae
 Anbacterial acvity of chitosan gel against UTI pathogens

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
The wider applicaons of chitosan are
because of its biodegradability, biocompability
and low toxicity33. The maximum yield of chin and
chitosan in fungi was obtained in late logarithmic
phase34. Yield% of chitosan from Scylla serrata
was 38.23%35,36 where in this study, it has also
shown crab chitosan yield% of 37.5%. Chitosan
extracon using NaOH helps in binding with acetyl
group of chins, forms sodium acetate aids in the
extracon of chitosan37. It was found that the fat
binding capacity was more with fungal derived
chitosan than the crab derived chitosan. No et al.28
reported this as a physical property of chitosan to
hold water or fat held and geng trapped in the
structure and swells, thus can be used for drug
delivery. Chemical methods for deproteinaon
and demineralizaon are eecve38, the colour of
chitosan obtained was dierent which might the
inuence of conidia of fungal strain. Kobayashi et
al.39 pointed that increased advantage of chitosan
of fungi made them to receive great aenon
compared to chitosan extracted from crustacean
shell. The major advantage of using chitosan
extracted from fungi mycelium is, it is obtained
easily by fermentaon process and available at all
seasons and geographical locaon but collecon
of crustacean waste supplies is limited by shing
industry locations and seasons40. chitin from
fungal species can be derived from Phycomycetes,
Basidiomycetes, and Ascomycetes41.
Peaks at 564.67cm-1 and 3451.92 cm-1 of
fungal chitosan and 500.05 cm-1 and 3880.60 cm-1 of
crab shell derived chitosan where there are on par
with the earlier reports of chitosan extracted from
various sources42-44. The degree of deacetylaon
(DD) is calculated by idenfying the funconal
group present in the extracted chitosan45. The DD
of crab shell derived chitosan and fungal chitosan
were 90% and 60% respecvely, Thus, DD was
more with crab shell chitosan than fungal, so crab
shell chitosan was purer than fungal chitosan.
Chitosan behaves like a polyelectrolyte by geng
dissolved in the acid aqueous soluon when the
degree of deacetylaon reaches above 50%46. In
our study, both extracted chitosan was insoluble
in water and highly soluble in acidic aqueous
soluons. Chitosan molecules do have a pH below
6 with a strong posive charge when the DD is
high (> 85%)47. Chitosan extracted from crab shell
showed excellent anbacterial acvity compared
to fungal chitosan.
Escherichia coli, Proteus mirabilis and
Klebsiella pneumoniae were idened by their
biochemical characteriscs48. The highest zone
of inhibion was observed for crab shell chitosan
as well as the chitosan gel than fungal chitosan
/ chitosan gel against all bacteria. Kong et al.49
reported that crab shells are rich source of chitosan
and this chemical constituent greatly effects
anbacterial acvies. And also, Klaykruayat et
al.50 reported that smaller oligomeric chitosan
can penetrate into the cell membrane and inhibits
RNA transcripon and prevents the cell growth
therefore it is observed that molecular weight
plays a major role in determining the anbacterial
acvies of chitosan. Moreover, chitosan absorbs
the electronegave substrate of proteins and may
disrupt the physiological acvies of microbes
and leads to cell death51. Lim and Hudson52
reported that the interacon between the anionic
components and intracellular components are due
to the leakage caused by changing the cellular
permeability by the polycaonic nature of chitosan
and leading to cell death.

In this study, crab shell and fungi were
used for deriving chitosan. The derived chitosan
were characterized using FTIR and used for making
chitosan gel. Both the chitosan and chitosan gel
were found to have anbacterial acvity against
UTI pathogens, where crab shell derived chitosan
and chitosan gel were showing more acvity than
the fungal chitosan.

S. Abirami would like to thank the
Management, Kamaraj College for their support
to do this work.

The authors declare that there is no
conict of interest.

All authors listed have made a substanal,
direct and intellectual contribuon to the work,
and approved it for publicaon.

www.microbiologyjournal.org974Journal of Pure and Applied Microbiology
Abirami et al. | J Pure Appl Microbiol | 15(2):968-975 | June 2021 | hps://doi.org/10.22207/JPAM.15.2.55

None.

All datasets generated or analyzed during
this study are included in the manuscript.
ETHICS STATEMENT
Not applicable.
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