WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Jaganathan et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 5.210

Volume 5, Issue 01, 1290-1301 Research Article ISSN 2278 – 4357

EXTRACTION AND CHARACTERIZATION OF CHITIN FROM

MARINE BYCATCH CRUSTACEANS EMPLOYING FERMENTATION
METHOD

Kamalesan Jaganathan1, Sirajudeen Mohammed Raffi2 and Peyil Soundarapandian1*

1
Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai
University, Parangipettai- 608 502, Tamil Nadu, India.
2
Kerala University of Fisheries and Ocean Studies, Panangad, Kochi- 682 506, Kerala, India.

Article Received on ABSTRACT

16 Nov 2015, Chitin is an important biopolymer next to cellulose, extracted in the
Revised on 06 Dec 2015, present study. The exoskeleton of marine bycatch brachyuran crabs,
Accepted on 28 Dec 2015
namely Calappa lophos, Dromia dehaani, Dorippe facchino and also
from stomatopod Squilla spp. were used to extract chitin through
*Correspondence for
fermentation methods by employing two bacterial strains such as
Author
Peyil Soundarapandian Pseudomonas aeruginosa, Serratia marcescens. The yield of chitin
Centre of Advanced Study was 44.24%, 37.45%, 11.56% and 27.24% in C. lophos, D. dehaani, D.
in Marine Biology, facchino and Squilla spp. respectively. FT-IR spectra of the produced
Faculty of Marine
chitin exhibit peaks which is more or less coherent to that of standard
Sciences, Annamalai
chitin which is further analysed by Scanning Electron Microscope. The
University, Parangipettai-
608 502, Tamil Nadu, quality of produced chitin was assessed through moisture, protein, ash
India. and lipid content analysis ensured that chitin obtained from trash
crustaceans are on par with that of standard chitin.

KEYWORDS: chitin, fermentation, bycatch, Calappa lophos, Pseudomonas aeruginosa,

FT-IR.

INTRODUCTION
Demersal trawling is the most destructive forms of fishing that reduces the structural
heterogeneity of benthic habitats of a range of fish and invertebrates.[1,2] Along with the target
fishes, this type of fishing will indiscriminately catch quite a large number and biomass of
non-target species (bycatch) which is estimated to be between 6.8 and 20 million tons per

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annum globally.[3,4] Production of byproducts from the non-edible crabs and stomatopods will
amplify its cost-effectiveness. Marine trash fish are considered as a potential economic
resource and is no more considered as waste as the production of valuable substances and
compounds from bycatch resources gained momentum in recent past.[5] Trash crustacean
exoskeleton shells are rich chitin content.[6] Based on this evidence the present study focused
on the production of chitin from trash crustaceans.

Chitin is mainly used as a raw material to produce chitin-derived products, such as chitosan,
chitin/chitosan derivatives, oligosaccharides and glucosamine. An increasing number of
useful products derived from chitin have tremendous industrial and pharmaceutical
applications.[7] An estimated 75% of chitin produced is used to manufacture products of
nutraceutical importance. Currently, the major driving force in the market is the increasing
sales of glucosamine as a dietary supplement.[8] Commercially the chitin occupies high
economic value. Practical grade chitin rated at a price of 220 USD per kilogram in the
international market.

Production of chitin employing chemical method is widely used worldwide. The common
procedure for isolating chitin from crustacean shells involves demineralisation,
deproteinisation and decoloration. Demineralisation is generally performed by acids and
deproteinisation by alkaline treatments. Moreover, these chemical treatment methods bring
about quite a large question of waste water and its disposal pave way to several
environmental problems. Above all, the cost of the chemicals involved in these process is
another drawback[9], makes it less lucrative.

An interesting alternative method involves in the production of chitin is the biological

method, through the process of fermentation by employing microbes such as bacteria and
fungi. Application of bacterial proteolytic enzymes for protein removal from chitin rich
fractions gains momentum in recent past as it ensures effective conversion of crustacean shell
waste into useful chitin fractions. In the present study two bacterial strains were used to
produce chitin. P. aeruginosa, is used for the production of organic acids by fermenting the
D-Glucose. The S. marcescens is used to produce protease enzyme for the deproteinization
step.

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MATERIALS AND METHODS

Collection and preparation of raw material
The non-edible crabs viz C. lophos, D. dehaani and D. facchino and also stomatopod Squilla
spp. were procured from Mudasalodai fish landing centre (Lat. 11°29’N; Long. 79°46’E),
Parangipettai, Cuddalore district, Tamilnadu. The specimens were thorough and repeatedly
washed in seawater to remove all the dirt and sand. They were then taken to the laboratory
and the viscera and tissues were removed. The exoskeletons of the crustaceans were
thoroughly washed with running tap water with ample care so as to remove sand adhered to
it, and placed in hot air oven at 60°C for 24 hours.

Fermentation method
The demineralization[10] and deproteinization[11] steps were carried out for the production of
chitin. The bacterial strains P. aeruginosa and S. marcescens were isolated from sediment
samples procured from Vellar estuary, Parangipettai (11°29′N; 79°46′E). Thirty percentage of
powdered exoskeleton was mixed with 10.0% inoculum of P. aeruginosa in a 500 ml flask
containing 200 ml of 10.0% D-glucose. The fermentation was carried out at 30°C in an
orbital shaker at 180 RPM for 7 days. After 7 days the shell powder was washed with
distilled water and dried at 60°C for 4 hours; subsequently 2-3 drops of 1% H2SO4 was added
with 1 mg of fermented crab shell powder to confirm the demineralization. The foam
appearance indicates the presence of minerals. The demineralization step was extended until
complete removal of minerals. All the experiments were done in triplicate to get concurrent
values.

The S. marcescens inoculum was prepared for demineralization process. The washed
powdered exoskeleton was mixed with 10.0% of inoculum in a flask (500 ml) containing
200ml of 10.0% D-glucose. The fermentation was carried out at 30°C an orbital shaker at 180
RPM for 7 days. After that the sample was washed with distilled water and dried at 60°C for
4hours, subsequently 2-3 drops of I2/KI-solution (0.2 g I2 in 100 ml of 5% KI solution) was
added with a pinch of fermented exoskeleton powder to confirm the chitin production. The
appearance of brownish-yellow colour indicates the removal of protein and non appearance
of colour indicates protein presence. The deproteinization step was extended until complete
protein removal. All the experiments were done in triplicate.

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FTIR spectral analysis

IR characterization of chitin was performed with PERKIN ELMER SPECTRUM RX1 type
FT-IR instrument in the Central Instrumentation Laboratory, Annamalai University. The
standard grade chitin (Marine Chemicals, Cochin, India) was compared with that of the chitin
obtained from all the species. The chitin sample was dried in hot air oven at 60°C for 6 hours
for dehydration and was subjected for FTIR analysis.

Scanning Electron Microscope Analysis

The physical structure and nature of chitin was obtained with the help of Scanning Electron
Microscope (SEM) (Model: JEOL.JSM 5160 with INCAEDS, version 1.1, Japan) at the
Central Instrumentation Laboratory, Annamalai University. Powdered chitin was well dried
in hot air oven at 60°C for 6 hours. Prior to analysis the chitin samples were sprinkled onto
Carbon tapes which are adhesive and supported on metallic disks and coated with Au. Images
of the sample surfaces were recorded at different areas and magnifications. Chitin produced
from C. lophos alone were subjected to SEM analyse; since it fetched higher yield.

Proximate analysis
The extracted chitin from various species was subjected to moisture and ash content
analysis.[12] Protein[13] and Lipid[14] estimations were carried out by following the standard
methodologies.

RESULTS AND DISCUSSION

Production of Chitin
The results of chitin extracted from the exoskeleton of trash crustaceans by fermentation
method are presented in table 1. The average yield of chitin from 30 g of raw exoskeleton of
C. lophos, D. dehaani, D. facchino and Squilla spp. were 13.27 g, 11.233 g, 3.47 g and 8.17 g
respectively. The percentage yield of chitin also followed similar trend as average yield of
chitin. Among the species used in the present study, chitin obtained from C. lophos, showed
higher yields of 44.24% and minimum yield fetched from D. facchino as 11.57%.

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Table 1: Table showing the average yield of chitin produced from marine trash
crustaceans.
Yield of chitin in
Raw material Average yield Percentage
Source three replicates
(g) of the chitin Yield of Chitin
A B C
C. lophos 30 13 13.6 13.2 13.27 44.24
D. dehaani 30 11 11.1 11.6 11.233 37.45
D. facchino 30 3.4 3.5 3.5 3.47 11.56
Squilla spp. 30 7 9 8.5 8.17 27.24

The strains of P. aeruginosa have been applied for demineralization process. This strain
exhibits high protease activity (~50 U/ml) in the culture medium and produced organic acids,
mainly of lactic, succinic and citric acids (roughly in the ratio of 1:1:1, w/w/w, about 0.3
mg/ml each). It is unraveled that the strains also secreted substantial amounts of chitinase (0.5
U/ml) but little chitosanase.[15,16]

The demineralization rate was gradually increased for the samples during culture. During 7th
day, the demineralization rate was low for the exoskeleton of C. lophos. To rectify this,
secondary incubation with fresh strains of P. aeruginosa was conducted for another 7 days at
same parameters to achieve complete demineralization. The pH (pH7) was gradually
decreased from the second day of incubation and finally reduced to pH3. Double
fermentation with P. aeruginosa alone achieved higher level of demineralization. It is
interesting to note that, during fermentation, P. aeruginosa produce sufficient amount of
organic acids to solubilize CaCO3; and it produce less quantum of protease, which indicates
that the partial deproteinization occurs in the first step itself.

The parameters such as initial glucose amount, inoculation level and culture time have a
significant effect on organic acid production.[16, 26] Since, crab shell waste is a poor source of
fermentable carbon, so an additional energy source like glucose, lactose, malt, or whey
[18]
powder must be added to the medium for better growth of bacterial strains. The
concentration of glucose as carbon source were standardised for the tune of 10.0%.
Supplementation of 10.0% of glucose resulted in better efficiency for demineralization and
deproteinization .[10] In the present study, nearly 91% of demineralization rate attained on the
8th day which was more or less similar with previously reported that the highest
demineralisation value of 90% in lactic acid fermentation (with 7.2% of TTA) of crayfish
waste.[19]

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In order to improve the extraction efficiency of chitin from crab shell waste, deproteinisation
was conducted using protease producing bacterium S. marcescens secretes mainly proteases
but not sufficient amount of organic acids.[11] In this step, the pH reached to 5 at the 4th day.
Deproteinisation reached its peak at 14th day of incubation for C. lophos; for Squilla spp. it
achieved on 10th day, whereas for D. facchino it was achieved on the 7th day. Earlier works
stated that 23% of chitin extracted from crab shells using P. aeruginosa[10] and 33% of chitin
from red crab shells by fermentation method using Lactobacillus paracasei KCTC-3074 and
S. marcescens FS-3.[11] In the present study, the yield obtained for C. lophos, D. dehaani, D.
facchino, and Squilla spp. were to the tune of 44.24%, 37.45%, 11.56 and 27.24%
respectively by employing fermentation method. From the results it is suggested that
fermentation method produced higher yield in all the species.

FTIR Analysis
The results of FT-IR C. lophos, D. dehaani, D. facchino and Squilla spp. were represented in
Fig. (1-5). FT-IR investigation proved the existence of the helical arrangement of chitin. The
amide-A band of standard chitin was recorded at 3449 cm-1 showed that there were OH
groups involved in free hydroxyl bonds. The amide-B band of chitin was found at 2960 cm-1
which is related to asymmetric and symmetric stretching H-C-H, were as amide-I, amide-II
and amide-III bands were observed at 1425 cm-1, 1418 cm-1 and 1261 cm-1 respectively.

Fig.1: FT-IR spectral analysis of standard grade chitin.

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Fig.2: FT-IR spectral analysis of chitin produced from C. lophos.

Fig.3: FT-IR spectral analysis of chitin produced from D. dehaani.

Fig.4: FT-IR spectral analysis of chitin produced from D. facchino.

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Fig.5: FT-IR spectral analysis of chitin produced from Squilla spp.

In the case of C. lophos, D. dehaani, D. facchino and Squilla spp. chitin, amide-I were
showed at 1638 cm-1, 1638 cm-1 and 1639 cm-1respectively in fermentation method against
those of standard chitin which is 1653 cm-1. Amide-II was represented at 1425 cm-1- 1425
cm-1, 1413 cm-1 – 1426 cm-1, 1379 cm-1 – 1328 cm-1 and 1411 cm-1 for C. lophos, D. dehaani,
D. facchino and Squilla spp. respectively against 1418 cm-1 for standard chitin representing
CH2 bend and CH3 deformation. The amide-III band position of standard chitin was in the
range of 1262 cm-1, 1261 cm-1 – 1262 cm-1, 1262 cm-1 – 1262 cm-1 and 1258 cm-1 – 1262 cm-
1
respectively.

The stretching bands of the OH groups involved in hydrogen bonds O-3-H…O-5 occurs at
3440cm-1.[20, 21] The C=O stretching region of the amide-moiety between 1600 and 1500cm-1
for chitin, the Amide-I band is split at 1656 and 1621cm-1 for β–chitin.[21] From the results of
the FT-IR spectral analysis it is evident that, the chitin produced from the C. lophos, D.
dehaani, D. facchino and Squilla spp. showed helical arrangement and their functional
properties more or less matching with that of standard grade chitin.

SEM Analysis
The SEM images of the chitin produced from C. lophos with different magnifications and
different surface areas of chitin are shown in Fig.6. It was observed that chitin biopolymer
produced in the present study exhibits porous and fibril structures. The structure of chitin as
crystalline and dense those were extracted from the exoskeleton of shrimps, Penaeus
semisulcatus, (de Haan), Metapenaeus affinis (Milne-Edwards); from brachyuran crabs

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Portunus pelagicus (Linne), from lobster Thenus orientalis, (Lund) and from cephalopod
Sepia spp.[22]

Fig.6: SEM images of chitin produced from C. lophos by fermentation method (a. 500X
and b.750X).

Proximate Composition and Quality Analysis of Chitin

The extracted chitin contains very little amount of moisture, lipid, protein, and ash content
and is satisfactory in line with commercial international standards (Fig.7). The proximate
composition of chitin used to vary in its chitin and non – chitinous fractions which might be
due to the variation in parent source (raw material), difference and modifications in the
methodologies, hygiene, less sand content, etc. The quality of chitin is assessed based on its
proximate composition, moisture content, ash content in such a fashion that lower the levels
of protein, lipid, moisture and ash contents were low indicates a better quality of chitin. Non
– chitinous materials negatively affects the quality and property of chitin by interacting with
it.[23] The proportion of chitin and the non– chitinous fractions varies with species.[17]

Fig.7. Moisture content, Protein content, Ash content and Lipid content of chitin
produced from C. lophos, D. dehaani, D. facchino and Squilla spp.

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CONCLUSION
The chitin extracted, contains very little amount of moisture content, lipid, protein, and ash
content and is satisfactory in line with commercial international standards. Moreover, this
method involves little application of chemicals, thus ensures an economically feasible and an
ecofriendly method. In general, it is evident that the chitin produced from the trash
crustaceans showed better yield and the quality assessment proved that the chitin produced
from trash crustaceans are in line with that of the standard grade chitin available at
international markets.

ACKNOWLEDGEMENT
The author Kamalesan Jaganathan is thankful to the University Grants Commission, New
Delhi for providing Rajiv Gandhi National Fellowship to done this work.

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