1.
Chitin/chitosan/chitooligosaccharide
Utilization of Byproducts from Fishery
Industry
(850-517)
Avtar Singh
International Center of Excellence in Seafood Science and Innovation
Faculty of Agro-Industry, Prince of Songkla University, Hat Yai
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2.
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Shrimps Crabs Squid pen
Beta-chitin
Alpha chitin
Different crystalline polymorphic forms
Sources of chitin and chitosan

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Chitin is a polysaccharide composed of 1→4 linked 2-acetamido-2-deoxy-β-D-glucopyranose.
It plays the role of structural element of the outer skeleton of fungi, insects and crustaceans.
Chitin structure

4.
Chitin structure
• Chitin has three different allomorphs, which
differ in the orientation of the respective polymer
chains within the micro-fibril macro structure.
• The most abundant and resilient α-chitin is
formed by antiparallel aligned polysaccharide
chains.
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Source: Arnold, N. D., Brück, W. M., Garbe, D., & Brück, T. B. (2020). Enzymatic modification of native chitin and conversion to specialty chemical
products. Marine drugs, 18(2), 93.
Different types of chitin
• In β-chitin, the sugar chains are ordered in a parallel manner, therefore exhibiting weaker
intramolecular interactions.
• The γ-allomorph of chitin is characterized by a mixture of both antiparallel and parallel
aligned chains, which leads to a polymer with fractions of higher and lower levels of
crystallinity.

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Marine sources of chitin and percentage (dry weight
basis) found in shell discards
Chitin source Chitin in shell wastes(%)
Clam/oyster 3-6
Crab:
Collinectes sapidus 13.5
Chinoecetes opilio 26.6
Shrimp:
Pandalus borealis 17.0
Crangon crangon 17.8
Penaeus monodon 40.4
Prawn: 33.0
Squid pen 20-40
Synowiecki, J., & Al-Khateeb, N. A. (2003). Production, properties, and some new applications of chitin and its
derivatives.

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Extraction of chitin from
crustacean shells by: (A)
biological methods; (B)
lactic acid-mediated
demineralization and (C)
proteases-mediated
deproteinization of shells.
Kaur, S., & Dhillon, G. S. (2015). Recent trends in biological extraction of chitin from marine shell wastes: a review.
Critical reviews in biotechnology, 35(1), 44-61.
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Production of chitin and chitosan
Demineralization and Deproteinization (1 M NaOH, 100 oC for 3 h)
Deacetylation (high concentration of NaOH, temp and time)
Chitin
Chitosan
Generally diluted HCl solutions at room
Temperature
(other acids have also been used; HNO3, H2SO4, CH3COOH)

8.
Demineralization
• Demineralization consists in the removal of minerals, primarily calcium carbonate.
• Demineralization is generally performed by acid treatment using HCl, HNO3, H2SO4,
CH3COOH and HCOOH
• Drastic treatments that may cause modifications, such as depolymerization and
deacetylation of native chitin.
• The use of high temperature accelerates the demineralization reaction by
promoting the penetration of the solvent into the chitin matrix.
2 HCl + CaCO3 → CaCl2 + H2O + CO2 ↑
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9.
Concentration of HCl (M) Time (hr) Ash content (%) Yield (%)
0.5
1 1.27 ± 0.02aA 62.41 ± 0.03aA
2 1.20 ± 0.04aA 61.99 ± 0.10bB
4 1.06 ± 0.11bcB 60.71 ± 0.05cC
1.0
1 1.18 ± 0.02abA 60.06 ± 0.10dA
2 1.04 ± 0.01cdB 59.83 ± 0.03eB
4 0.93 ± 0.12dB 58.89 ± 0.03fC
Ash content and yield of Pacific white shrimp shell demineralized under different HCl
concentrations and treatment times
Mittal, A., Singh, A., Aluko, R. E., & Benjakul, S. (2021). Pacific white shrimp (Litopenaeus vannamei) shell chitosan and the conjugate with epigallocatechin gallate: Antioxidative
and antimicrobial activities. Journal of food biochemistry, 45(1), e13569. 9

10.
Deproteinization
• It involves the disruption of chemical bonds between chitin and proteins using alkali such as
NaOH.
• Many proteases such as alcalase, pepsin, papain, pancreatine, trypsin, etc. has been used
to remove proteins
• Enzymes minimize the deacetylation and depolymerization during chitin isolation
• Enzymatic methods is inferior to chemical methods with approximately 5%–10% residual
protein typically still associated with the isolated chitin.
• The final isolated chitin could be then treated with an additional NaOH treatment after
enzymatic hydrolysis.
• In addition, fermentation process, or auto-fermentation) or by adding selected strains of
microorganisms have been used for the deproteinization.
-Lactic Acid Fermentation
-Non-lactic-Acid Fermentation (Bacillus sp., Pseudomonas sp., and Aspergillus sp.)
• Besides chitin fraction, liquid fraction rich in proteins, minerals and astaxanthin could be
obtained.
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Temperature (ºC) Time (min) Remaining protein (mg/g) Yield (%)
60
60 140.77 ± 0.07aA 37.72 ± 0.20aA
80 122.44 ± 0.02bB 34.26 ± 0.23bB
70
60 83.24 ± 0.04cA 31.74 ± 0.21cA
80 67.06 ± 0.07dB 29.96 ± 0.49dB
Residual protein content and yield of chitin from Pacific white shrimp deproteinized
under different temperatures and times
Mittal, A., Singh, A., Aluko, R. E., & Benjakul, S. (2021). Pacific white shrimp (Litopenaeus vannamei) shell chitosan and the conjugate with epigallocatechin gallate: Antioxidative
and antimicrobial activities. Journal of food biochemistry, 45(1), e13569. 11

12.
Chitosan from chitin
• Chitin is differentiated from chitosan by the degree of deacetylation, which is the
balance between two types of residues (amine rich and N-acetyl rich).
• The degree of deacetylation of higher than 50% makes chitosan highly soluble in
acidic aqueous solutions of pH less than 6.0 because of the protonation of NH2
groups.
• Factors affecting chitosan extraction and properties:
-Source of chitin
-degree of deacetylation
-reaction conditions (reactants concentration, alkali/chitin ratio, and
temperature)
• Chitosan produced by the removal of acetyl groups from chitin using strong alkali
treatment at high temperature:
1. Homogenous deacetylation
2. Heterogenous deacetylation
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13.
Yield, degree of deacetylation (DDA), intrinsic viscosity, and viscosity-average molecular weight
of chitosan from squid pen chitin deacetylated under different temperatures and times
Temperature
(°C)
Extraction time
(hr)
Yield (%) DDA (%) [η] (dL/g) MW (Da)
110
2 64.99 ± 1.83aA 78.21 ± 1.28eC 6.52 ± 0.02aA 3.2 × 105
4 54.12 ± 1.67bB 84.65 ± 0.42dB 4.19 ± 0.04bB 1.7 × 105
8 50.54 ± 0.80cC 86.84 ± 0.4cA 3.47 ± 0.02dC 1.3 × 105
130
2 53.51 ± 1.22dA 86.55 ± 0.73cC 3.79 ± 0.02cA 1.5 × 105
4 51.91 ± 1.21bcB 87.74 ± 0.49bA 3.39 ± 0.08eB 1.3 × 105
8 50.07 ± 1.02cB 89.72 ± 0.37aB 3.24 ± 0.02fC 1.2 × 105
Singh, A., Benjakul, S., & Prodpran, T. (2019). Ultrasound‐assisted extraction of chitosan from squid pen: Molecular characterization and fat binding capacity. Journal of food
science, 84(2), 224-234. 13

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Characterization of chitin and chitosan
• Degree of acetylation and deacetylation (DDA) using FTIR, NMR
FTIR- (1−A1320/A1420×0.3192)×100
HNMR- {1−(1/3 ICH3/1/6 IH2−H6
)}×100
ICH3and IH2−H6
are the integral intensities of CH3of N-acetyl and H2, H3, H4,
H5, H6, H6′protons
• The crystallinity index (Icr) using X-ray diffraction (XRD) analysis
• Intrinsic viscosity using an Ubbelohde capillary type viscometer
• Degree of depolymerization (DDP) using reducing sugar using
dinitrosalicylic acid (DNS) method
• Degree of polymerization
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15.
FTIR spectra of chitosan prepared from Pacific white shrimp shell chitin
deacetylated using different temperatures and times. NaOH (50% w/v) and
chitin/ alkaline solution ratio (1:40 w/v) were used. CS-110-2, CS-110-4,
and CS-110-6: Chitosan prepared by deacetylation using 50% NaOH at 110
ºC for 2, 4, and 6 h, respectively. CS-130-2, CS-130-4, and CS-130-6:
Chitosan prepared by deacetylation using 50% NaOH at 130 ºC for 2, 4, and
6 h, respectively.
Bands Wavenumber (cm-1)
O-H stretching and N-H
stretching (amide A)
3350 and 3280
CH2 stretching 2921-2875
C=O stretching (amide I) 1650
N-H bending (amide II) and C-N
stretching
1560 and 1320
CH2 bending 1420-1425
C-O-C stretching 1150 and 1030
C-O stretching 897
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FTIR analysis of chitosan samples

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A B
XRD patterns of chitin (A) and chitosan (CS-130-4) (B) prepared from Pacific white shrimp shell.
Crystallinity index: 40.75% Crystallinity index: 19.75%
04-02-2023 16
XRD patterns
20.22º (110)
19.28º (110)
9.09º (020)
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17.
1H-NMR spectra of chitosan (CS-130-4) (A) and CE-8 conjugate (B) prepared from Pacific white shrimp shell. CE-8 conjugate was prepared by free
radical grafting method in the presence of 8% EGCG (w/w of chitosan). For caption: See Fig. 1. CE-8: Conjugate prepared with chitosan and 8%
EGCG (w/w of chitosan) using free radical grafting method.
A
1
2
3
4
5
6
1
2
3
4 6
5
04-02-2023 17
B
NMR spectra
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Chitosan solubility
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Chitosan as natural flocculant for
beer clarification
Wastewater
treatment

20.
Anti-obesity properties of chitosan
Fat Blocker
Reduced
weight
Obesity
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21.
Chitosan
Binding of chitosan to oil
Oil droplet surrounded by chitosan
Oil
droplet
Oil
Soluble in acidic
condition
pKa of amino group is 6.5
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22.
Esophagus
pH: 5-7
Stomach
pH: 1.5-3.5
Duodenum
pH: 7-8.5
Chitosan
Fat/Lipids
Soluble chitosan under
acidic conditions
Chitosan/fat emulsion
Stable fat binding/
Micelles formation
Reduced fat
absorption into
blood Resistance to
hydrolysis by
lipase or
phospholipase
How Chitosan function as fat blocker
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23.
A B C
D E F
110
C
130
C
2 h 4 h 8 h
Microstructure of emulsion stabilized by FITC-labelled chitosan from squid pen with
different deacetylation conditions (A-F) under the simulated gastrointestinal tract.
Chitosan was deacetylated under different temperature (110 and 130 °C) at different times
(2 to 8 h)
FITC: Fluorescein
Singh, A., Benjakul, S., & Prodpran, T. (2019). Ultrasound‐assisted extraction of chitosan from squid pen: Molecular
characterization and fat binding capacity. Journal of food science, 84(2), 224-234. 23

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Chitosan as packaging material
Application and preparation method of chitosan film
Source: Wang et al., 2018
Proposed mechanism of chitosan film formation
Low bioactivities Composite film (used
two different
polymers)
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25.
Chitosan conjugate
Mechanism of free radical grafting
 Cheap
 Non-toxic
 Reaction occurred at room temperature to
avoid degradation and oxidation of phenolics
Conjugation methods:
-Activated ester-mediated (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC))
-Enzyme-mediated strategy (polyphenol oxidases, such as tyrosinase and laccase)
-Free radical induced grafting reaction (Hydrogen peroxide and ascorbic acid)
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Chitosan as coating
material/nanoliposome/emulsion
Protect bioactive compounds from harsh environment (high-low pH)
Act as delivery agent and targeted release of the bioactive
compound
Solubilizers for various ingredients
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Chitosan as a wall material
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Chitooligosaccharide preparation
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Preparation of chitooligosaccharides from chitosan via different methods 29

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Leakage of proteinaceous components
and other intracellular constituents from
the cell
caused by interaction between the
negatively charged microbial cells
membranes and positively charged
chitooligosaccharides
Inhibition of mRNA and protein
translation as a result of the interaction
between the microbial DNA with the
diffused hydrolysis product
Chelate the ion required for microbial
growth
Antimicrobial
mechanisms of
chitooligosaccharides
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31.
Degree of depolymerization of chitooligosaccharides (COSs) from squid pen prepared using pepsin, amylase or
lipase for different hydrolysis times.
Bars represent the standard deviation (n=3). Enzymes at 8% (w/w) were used.
Depolymerization (DDP): reducing sugar using dinitrosalicylic acid (DNS) method in comparison with total sugar content.
Pepsin: 37 C, amylase and lipase: 50 C
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Staphylococcus aureus
20 mg/mL
20 mg/mL
10 mg/mL
5 mg/mL
2.50 mg/mL
10 mg/mL
5 mg/mL
2.50 mg/mL
Escherichia coli
20 mg/mL
10 mg/mL 5 mg/mL
2.50 mg/mL
20 mg/mL
10 mg/mL 5 mg/mL
2.50 mg/mL
Pseudomonas aeruginosa
20 mg/mL
10 mg/mL
5 mg/mL
2.50 mg/mL 20 mg/mL
10 mg/mL
5 mg/mL
2.50 mg/mL
Vibrio parahaemolyticus
20 mg/mL
10 mg/mL
5 mg/mL
2.50 mg/mL
20 mg/mL
10 mg/mL
5 mg/mL
2.50 mg/mL
20 mg/mL
10 mg/mL
5 mg/mL
2.50 mg/mL
200 mg/mL
10 mg/mL
5 mg/mL
2.50 mg/mL
Listeria monocytogens
Antimicrobial activity of COS against various bacteria
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Preparation of COS using H2O2 and AsA/H2O2 redox pair reaction
H2O2 AsA/H2O2
CS (1%, w/v) was dissolved in acetic acid (2%, v/v)
Hydrolysis
• H2O2 was added to CS solution to obtain final
concentrations of 0.5 and 1 M.
• The mixtures were then shaken for 2 h at
60 C with the aid of a shaker water bath.
• Several molar ratios of AsA/ H2O2 (0.05/0.05, 0.05/0.1
and 0.1/0.05, M/M) was used to prepare stock
solutions.
• The radical generated solutions (2 mL) were mixed in
100 mL of CS (1%, w/ v) solution. To start hydrolysis,
mixtures were incubated at 60 C for 2 h.
 The undissolved matter was removed from mixtures using a centrifuge
 Supernatant was dialyzed and thereafter subjected to lyophilization
COS powder 33

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Effect of chitooligosaccharide from squid pen on gel properties of sardine surimi gel and its stability during
refrigerated storage (Singh et al., 2019)
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COS in surimi gel

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Chemistry of Meat Discoloration
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Photographs of tuna slices treated without and with COS or EGCG or their
mixture at different concentrations during storage of 12 days at 4 °C.
CON: samples without any treatment, C2: sample added with 200 ppm COS, C4: sample added with 400 ppm COS,
E2: sample added with 200 ppm EGCG, E4: sample added with 400 ppm EGCG, CE2: sample added with 100 ppm
COS and 100 ppm EGCG, CE4: sample added with 200 ppm COS and 200 ppm EGCG. 36

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Thank you
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