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Archives of Razi Institute, Vol. 77, No. 4 (2022) 1349-1356 Copyright © 2022 by
Razi Vaccine & Serum Research Institute
DOI: 10.22092/ARI.2022.358342.2204
1. Introduction
Chitosan is a derivative of chitin. The ratio of
Glucosamine and N-acetyl-D-glucosamine indicates the
degree of deacetylation (DD) in chitosan. Two types of
straight-chain copolymers of chitosan and chitin exist
(1). Chitosan has high biodegradability and
biocompatibility as well as some functional moieties
such as N-H and O-H. Therefore, specific
physicochemical properties of chitosan that are crucial
for drug targeting can be chemically modified and
customized (2). Chitin is the second most prevalent
polysaccharide in nature after cellulose and is made from
(-(1-4)-poly-N-acetyl-D-glucosamine). Fungal spore
germination, hyphal elongation, and radial growth are
well documented to be inhibited by chitosan (3).
Most studies have examined the effectiveness of
Chitosan against food-related yeasts and molds, plant
damage, and entomopathogenic fungi (4). Chitosan has
antimicrobial properties which prevent germs from
growing such as its significant effect on the growth
inhibition of the Enterobacteriaceae family as it
contains multiple biochemically and genetically related
species. Escherichia coli, Shigella sonnei, Salmonella
sonnei, Enterobacter sonnei, Proteus sonnei, and
Original Article
Evaluation of the Biological Activity of Laboratory-Prepared
Chitosan from Shrimp Shells against Pathogenic Bacterial
Isolates
Jawad, S. M 1 *
1. Department of Soil Science and Water Resources, Faculty of Agriculture, University of Kufa, Najaf, Iraq
Received 12 April 2022; Accepted 21 May 2022
Corresponding Author: saharm.alkarawy@uokufa.edu.iq
Abstract
Chitin is the most substantial natural polysaccharide after cellulose, found in the shells of crabs, shrimps, and
other crustaceans. Several medical and environmental applications have been recognized for chitosan.
Therefore, the present study aimed to evaluate the biological activity of laboratory-prepared chitosan from
shrimp shells against pathogenic bacteria isolates. In the present study, chitosan was extracted from chitin
acetate of shrimp shells at different temperatures (room temperature, 65 and 100 ° C) for equal amounts of
shells at specified time intervals. The degree of acetylation of different treatments of RT1, RT2, and RT3
reached 71%, 70%, and 65%, respectively. The laboratory-prepared chitosan was examined and antibacterial
properties were observed against clinical isolates of bacterial causative agents of urinary tract infections (E. coli,
Klebsiella Pneumonia, Pseudomonas spp., Citrobacter freundii, and Enterobacter spp.). The inhibitory activity
of all types of treatments ranged between 12 to 25 mm for all isolates with the highest for Enterobacter spp. and
the lowest for Pseudomonas isolates. The results also indicated a large relative discrepancy between the
inhibitory activity of laboratory-prepared chitosan and antibiotics. These results were in the S-R range of the
isolates. The similarity of laboratory production conditions and treatments is due to the different proportions of
chitin formed in shrimp, environmental conditions, nutrition factors, pH, the extent of heavy metals in the water,
and the age of the organism.
Keywords: Antimicrobial Activity, Chitosan, Enterobacteriaceae
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Yersinia sonnei are all members of the
Enterobacteriaceae family which may be found in the
soil, water, plants, animals, insects, and even humans.
Enterobacteria are opportunistic pathogens and as a
result, this is a clinically significant family (5).
Thousands of people die each year due to antibiotic
resistance, which h is predicted to become a significant
health issue worldwide (6, 7). As many as ten million
people could die annually as a result of antibiotic
resistance by 2050 (8, 9). The treatment of infectious
diseases has significantly changed since the discovery,
manufacture, and use of biological materials (10).
Therefore, the present study aimed to evaluate the
biological activity of laboratory-prepared chitosan from
shrimp shells against pathogenic bacterial isolates.
2. Materials and Methods
2.1. Experimental Procedures
2.1.1. Preparation of Chitosan Solutio
Chitosan solution was prepared by dissolving
different weights of chitosan in 100 ml of a solution
containing acetic acid: distilled water in a ratio of 99:1
with constant stirring until dissolution, with weights of
2, 3, 4, 5, and 10 mg (AOAC, 2006). The scales were
obtained from carp after washing and drying with
deionized water .
2.1.2. Chitin Preparation
Chitin was extracted from shrimp shells using the
method described by Toan, Ng (11) with some
modifications.
2.1.3. Deproteinization
Shrimp shells were treated with a solution of 1.2N
sodium hydroxide (NaOH) for 24 h at a ratio of 10:1
wt:v at 70-75°C. The sample was then filtered using a
Buechner funnel and rinsed several times with distilled
water at a high discharge rate to obtain PH7.
2.1.4. Demineralization
The sample was treated with 0.7 N hydrochloric acid
(HCl) at room temperature for 15 min at a ratio of 1:10
wt:v. Then the sample was washed well with water to
remove acid and calcium carbonate to obtain pH7 and
dried in an air oven at 60 °C for 24 h.
2.1.5. Discoloration
The sample was treated with acetone at a ratio of 1:10
wt:v for 10 min. The sample was filtered and dried for
2 h, and then shortened with sodium hypochlorite 32%
solution at a ratio of 5:1 wt:v for 15 min at room
temperature with constant stirring. Then it was washed
with distilled water and dried in an air oven at 60 °C for
24 h.
2.2. Methods
Three different temperatures were used for extracting
chitosan from shrimp shells:
2.2.1. Preparation of Chitosan
According to Toan, Ng (11), chitosan was made in
four distinct methods by treating laboratory-prepared
chitin with a 50% NaOH solution at a ratio of 1:13
wt:v:
- First treatment (RT1): Primary treatment for 48 h at
room temperature.
The sample was immediately filtered and washed
with distilled water under vacuum after treatment for
48 h at room temperature, then dried in a hot air oven at
60° C for 24 h.
- Second treatment (RT2): This is considered the
starting point.
After the first treatment, the sample was quickly
filtered and washed with distilled water under vacuum
to obtain pH7, then dried in an oven at 61°C for 24 h.
Then the precipitate was dried at 55°C in a vacuum
oven.
-Third treatment (RT3): A 20-hour base treatment at
100 °C.
The sample was treated for 20 h at 100 °C and then
filtered and washed several times with distilled water to
obtain pH7, and then dried in a hot air oven at 60 °C for
24 h.
2.2.2. Detection of Chitosan by Fourier Transform
Infrared Spectroscopy (FTIR)
Chitosan prepared from carp scales was detected at
the Faculty of Pharmacy, University of Kufa, Najaf,
Iraq. The dried chitosan was mixed with dry potassium
bromide at a ratio of 1:5 wt:v with a ceramic mortar for
10 min and compressed by a hydraulic press at a
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1351
pressure of 8 bar for 60 s before being analyzed by
FTIR (Biotech. Engineering Co.Ltd).
2.2.2.1. Degree of Deacetylation (DD%)
The degree of removal of acetyl groups (DD) was
estimated based on the FTIR results. The absorbance at
wavelength A 1655 (1655) represents the amine group
compared to that at wavelength A 3450 (3450),
representing the hydroxyl group and serving as an
internal standard. It does not decompose and is
unaffected by the transactions that occur during the
extraction of Chitosan. The absorbance was calculated
based on Beer-Lambert law according to the equation:
(A: absorbance, T: permeability)
A%= 2-log T%
The degree of removal of acetylcholine groups was
calculated as mentioned by Maghsoudi, Razavi (12).
2.2.2.2. Specimens Collection
In this study, 33 samples were collected from patients
with urinary tract infections at Al-Sadr Teaching
Hospital, Najaf, Iraq, and cultured on agar plates. The
plate was incubated for 18-24 h at 37 ° C.
Amount of absorbed water (ml/g) = amount of added
water (10ml) - the amount of water after separation
2.2.2.3. Preparation of the Bacterial Suspension
Each bacterial suspension was produced to a turbidity
of 0.5 McFarland standard (1.5x108 CFU / ml).
Turbidity was determined using the Kirby-Bauer
method by a spectrophotometer at 625 nm in turbid
suspension (13).
2.2.2.4. Determination of Antimicrobial Activity
The Vitek 2 system isolated and identified E. coli,
Klebsiella pneumonia, Pseudomonas spp, Citrobacter
freundii, and Enterobacter spp. Then, 0.1 ml of culture
was spread on Mueller Hinton Agar using a sterile
brush and dried at room temperature for 10-15 min.
The agar well diffusion technique (13) was employed.
Then, three wells with a diameter of 10 mm were
created on the surface of the culture medium after
sterilizing with the cork borer, and 50 L was added to
each well of prepared chitosan. The plate was
incubated at 37 °C for 18-24 h. The diameter of the
zone of inhibition was measured.
2.2.2.5. Statistical Analysis
The data were obtained and transferred to a Microsoft
Excel spreadsheet and descriptive statistics were
calculated. SAS software (version 9.1) was used to
analyze the data. A two-way ANOVA was used to
investigate whether an interaction was observed between
the effect of extract concentration and the pathogenic
bacteria. In both tests, a P-value less than 0.05 is
considered to be statistically significant (Tukey’s test). In
addition, an analysis was performed to determine the
difference between the means. One-way ANOVA was
performed to reveal statistical differences using various
zones of inhibition when chitosan extracts were used
against the isolates in this study.
3. Results and Discussion
3.1. Diagnosis of Chitosan
Chitosan was prepared in the laboratory using shrimp
shells purchased from local markets in Najaf and Basra,
Iraq, and then exposed to three different temperature
and time treatments. Figure 1 illustrates the chitosan
yield obtained from the three treatments (RT3, RT2,
RT1) of shrimp depending on the extraction procedure.
Statistically significant differences (P= 0.05) were
observed between the treatments (17.5, 13.5, and
11.25%, respectively, based on the dry weight of
shells). The yield variation might be due to differences
in base treatments, extraction temperature, and duration
since higher extraction temperatures and periods result
in lower yields than lower extraction times (14).
According to Hosseinnejad and Jafari (15), chitosan
from shrimp residues varied from 17.36 to 13.12%.
Also, they discovered that the temperature and time
required to eliminate acetylcholine aggregates
significantly affected yield, and the yield decreases by
increasing temperature.
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Many variables contribute to the variance in shrimp
yield percentages, including the age of the shrimp, the
pH of the environment, and the presence of heavy
metal ions in the water, which inhibit the development
of chitin in the shells.
3.1.1. Detection of Chitosan by Fourier Transform
Infrared Spectroscopy (FTIR)
FTIR is one of the most important and fastest techniques
used to qualitatively detect chitin and chitosan due to
sensitivity and no need for high purification or dissolution
in specific solvents. However, one of its disadvantages is
the difference in the percentage of DD according to the
equation used to calculate it . Determining the active groups
in organic compounds and calculating the optical
transmittance are important in the FTIR technique (16). The
results indicated that the studied chitosan samples had
different characteristics than the chitin produced from it and
amino groups are among the essential active groups with an
absorption peak appearing at a frequency of 1658 cm-1 of
this spectrum as the presence of this group indicates the
existence of chitin. Additionally, chitosan represents the
absorbance for limited wave numbers between 1157-1024,
1155-1020, and 1155-1024 cm-1 for the RT1, RT3, and
RT2 chitosan models, respectively. The primary group in
chitosan is one of its stable properties and a guide to the
formation of acetylation as presented in figure 2. The
results of the present study are consistent with previously
published work by Sini, Santhosh (17).
3.1.2. Degree of Deacetylation (DD %)
Chitosan treatments RT1 to RT3 were evaluated
using the FTIR technique to determine how much of
the acetyl groups had been removed and the results are
shown in table 1. The amide group was used to
measure the content of N-acetyl groups, while the
frequency 3455 cm-1 was used as a scale for hydroxyl
groups.
Figure 1. Percentage of chitosan prepared from shrimps
Figure 2. Detection of chitosan extracted from shrimp shells
(FTIR)
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The results revealed a difference in the percentage of
DD for chitosan treatments, as the removal values for the
treatments were 71, 70, and 65%, respectively. Increasing
the temperature leads to a decrease in the percentage of
acetylcholine groups for chitosan treatments. RT3
treatment performed weakly in removing and distributing
acetyl groups in chitosan compared to other treatments.
Alves, Furman (18) report that when performing the
adaptation for this simplified method and stirring at room
temperature, the DD values in the range of 77 to 80%
demonstrate the viability of this method for the intended
purpose of the produced chitosan.
3.1.3. Analysis of the Biological Activity of Chitosan
According to the results, chitosan prepared from
shrimp shells showed biological activity with high and
various rates in different concentrations (2,3,4,5,10
mg/ml) and treatments against the growth of gram-
negative bacteria isolated from patients with urinary tract
infection. Increased concentration of chitosan has a
significant inhibitory effect on the growth of pathogenic
bacteria, as the zone of inhibition ranged from 18-24, 22-
12, 15-25, and 14-21 mm in E. coli, Proteus,
Enterobacter, and Klebsiella, respectively in different
treatments of shrimps (Table 2). The laboratory-prepared
chitosan from shrimp shells had a great inhibitory
activity against gram-negative pathogenic bacteria.
The antibacterial ingredient is chitosan (AB). Activity
increases with concentration until a critical
concentration (CC ( is reached, which then decreases
(19). The discovery of the main peaks of chitosan
properties at 1345, 1420, 1560, 1655, and 3290 cm1,
respectively, validated the solubility of chitosan
suspensions (18). The temperature should be noted to
have an essential effect on the activity of chitosan as an
antibacterial. As presented in figure 3, chitosan
prepared at room temperature showed significant
biological activity against pathogenic isolates compared
to those of other temperatures.
Furthermore, AB activity of chitosan is highly
dependent on incubation temperature which is
significantly boosted at 37 °C (20). As a result, AB
activity of chitosan is nevertheless promising in
refrigerated conditions despite the limiting effect of
temperature, and 100% bacterial suppression was not
achieved. Similar findings have been recently
published, suggesting that when bacteria are exposed to
high temperatures, they become vulnerable to the
action of chitosan nanoparticles (21). Low temperatures
might alter the structure of bacteria by reducing the
number of surface binding sites (or electronegativity).
As a result, fewer protonated chitosan amino groups
Table 1. The degree of acetylation in chitosan treatments
Deacetylation
DD%
Treatment Details
Chitosan
Treatments
71%
Base treatment at room
temperature for 48 h
RT1
70%
Base treatment at 65°C
for 20 h
RT2
65%
Base treatment at 100°C
for 20 h
RT3
Table 2. The zone of inhibition for chitosan extracted from shrimp shells
Type of Bacteria
Zone of Inhibition of RT
E. coli
18 – 24 mm
Proteus
12 – 22 mm
pseudomonas
12 – 22 mm
Enterobacter
15 – 25 mm
Klebsiella
14 – 21 mm
Figure 3. Biological activity of pathogenic isolates of
different chitosan treatments prepared from shrimp shells
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may interact with negatively charged sites on the
bacterial surface and reduce the AB activity of
chitosan. The data of the present study demonstrated
that the AB activity of both chitosan significantly but
not intensely decreased as the temperature decreased.
3.1.4. Effect of Bacterial Species
Figure 4 presents the percentage survival and
reduction of five species of bacteria when in contact
with chitosan. Chitosan is more effective against E. coli
than other germs. The chitosan solution is more
sensitive to gram-negative bacteria (22, 23). Gram type,
hydrophilicity, negative charge density, and adsorptive
capacity are different. A higher electrostatic interaction
between positively charged chitosan amino groups and
negatively charged bacterial surfaces may occur in
gram-negative bacteria (24). Gram-negative bacteria
have higher hydrophilicity and chitosan adsorption on
their cell walls than those of gram-positive bacteria,
which may contribute to the A.B. effect (3, 21). Also,
the structural arrangement of envelope/membrane
components in gram-positive and gram-negative
organisms. Gram-negative bacteria have a bilayered
phospholipid membrane with a single phospholipid
layer and a thin layer of peptidoglycan on the outside.
This difference in peptidoglycan layer thickness may
render gram-negative bacteria more vulnerable to
chitosan action (25, 26). This might explain why
different authors came to conflicting conclusions when
comparing the effect of chitosan. Data in the present
study indicate that partial solubilization is required for
chitosan to have an A.B. action. Consequently, reduced
M.W. (including low-MW species or
chitooligosaccharides, even in trace levels) and
increased DDA are preferred (27-29). Also, reducing
the size of chitosan particles was found to increase its
antibacterial properties. Additionally, chitosan has
antibacterial, wound healing, and mucoadhesive
properties which makes it an ideal drug carrier (30).
4. Conclusion
The present study demonstrates that chitin extracted
from shrimp shells may be employed in various
applications, particularly when transformed into the
more beneficial component of chitosan. Chitosan is
made by mixing several sources and treating them with
diluted HCl and NaOH. Chitosan has significant
antibacterial activity against Enterobacteriaceae when
produced at room temperature and this action is
influenced by pH, temperature, chitosan content, purity,
and bacterial type. Further study is required on chitosan
activity to fully understand the methods and variables
involved in extracting Chitosan and improving its
effectiveness in suppressing and eliminating the growth
Figure 4. Zones of inhibition formed by the laboratory
prepared chitosan
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of harmful bacteria and promoting its usage as an
alternative to the antibiotic. Furthermore, chitosan
producers might collect and treat these wastes before
donating or selling them to research organizations,
especially those focused on nanotechnology. Chitosan
production provides businesses with potential for future
investments on a national and global scale and
generates new sources of profit that may help build the
economy.
Authors' Contribution
Study concept and design: S. M. J.
Acquisition of data: S. M. J.
Analysis and interpretation of data: S. M. J.
Drafting of the manuscript: S. M. J.
Critical revision of the manuscript for important
intellectual content: S. M. J.
Statistical analysis: S. M. J.
Administrative, technical, and material support: S. M.
J.
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