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![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](https://image.slidesharecdn.com/chitinchitosanandcos-230204084221-2c16d48b/85/Chitin-chitosan-and-COS-pptx-13-320.jpg)
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Chitin, chitosan and COS.pptx
- 1. Chitin/chitosan/chitooligosaccharide Utilization of Byproductsfrom 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 1
- 2. 2 Shrimps Crabs Squidpen Beta-chitin Alpha chitin Different crystalline polymorphic forms Sources of chitin and chitosan
- 3. 3 Chitin is apolysaccharide 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 • Chitinhas 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. 4 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.
- 5. 5 Marine sources ofchitin 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.
- 6. Extraction of chitinfrom 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. 6
- 7. 7 Production of chitinand 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 consistsin 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 ↑ 8
- 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 involvesthe 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. 10
- 11. 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 12
- 13. Yield, degree ofdeacetylation (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
- 14. Characterization of chitinand 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 14
- 15. FTIR spectra ofchitosan 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 15 FTIR analysis of chitosan samples
- 16. A B XRD patternsof 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) 16
- 17. 1H-NMR spectra ofchitosan (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 17
- 18.
- 19. 19 Chitosan as naturalflocculant for beer clarification Wastewater treatment
- 20. Anti-obesity properties ofchitosan Fat Blocker Reduced weight Obesity 20
- 21. Chitosan Binding of chitosanto oil Oil droplet surrounded by chitosan Oil droplet Oil Soluble in acidic condition pKa of amino group is 6.5 21
- 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 22
- 23. A B C DE 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
- 24. Chitosan as packagingmaterial 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) 24
- 25. Chitosan conjugate Mechanism offree 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) 25
- 26. Chitosan as coating material/nanoliposome/emulsion Protectbioactive compounds from harsh environment (high-low pH) Act as delivery agent and targeted release of the bioactive compound Solubilizers for various ingredients 26
- 27. Chitosan as awall material 27
- 28.
- 29. Preparation of chitooligosaccharidesfrom chitosan via different methods 29
- 30. Leakage of proteinaceouscomponents 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 30
- 31. Degree of depolymerizationof 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 31
- 32. Staphylococcus aureus 20 mg/mL 20mg/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 32
- 33. Preparation of COSusing 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
- 34. Effect of chitooligosaccharidefrom squid pen on gel properties of sardine surimi gel and its stability during refrigerated storage (Singh et al., 2019) 34 COS in surimi gel
- 35. Chemistry of MeatDiscoloration 35
- 36. Photographs of tunaslices 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
- 37.
Editor's Notes
- #9 Most of the other minerals present in the shellfish cuticle react similarly and give soluble salts in presence of acid. Then, salts can be easily separated by filtration of the chitin solid phase followed by washing using deionized water.
- #10 solute/solvent ratio. The latter depends on the acid concentration, since it needs two molecules of HCl to convert one molecule of calcium carbonate into calcium chloride.
- #11 Lactic acid reacts with the calcium carbonate, leading to the formation of a precipitate of calcium lactate separated from lighter shells which are recovered and rinsed with water. This process may be realized either on purified crustaceous shells, or on complete shrimp waste (including heads and viscera).
- #16 Bands between 3350 and 3280 belongs to OH and NH stretching. CH stretching both symmetric and asymmetric was observed at 2921 and 2875. Amide 1 band appeared at 1650. Amide 2 band shifted to higher wavenumber with increase in temperature and treatment time due break down of hydrogen bond of the amide group. The bands near 1,559–1,589 cm−1 correspond to NH2 bending (Kumirska et al., 2010). The shift of band to higher wavenumber was attained when deacetylation temperature and time were increased, related to the removal of the acetyl group from C‐2 of chitosan. The amide‐III indicating CN stretching was observed around 1,320 cm−1 for all the samples. The intensity of the amide‐III band was decreased with increasing deacetylation temperature and times, which is consistent with the removal of the N‐acetyl group (Singh et al., 2019a). The difference in the wavenumber of the peak for CH2 bending (1,420–1,425 cm−1) of all samples was mainly determined by the reordering of hydrogen bonds at primary O‐H groups (Kasaai, 2008). The wavenumber of 1,375 cm−1 was mostly involved with the symmetrical deformation of CH3 (Trung & Bao, 2015). Antisymmetric stretching of the C–O–C bridge representing glycosidic linkage was seen around 1,150 cm−1. The prominent peaks around 1,030 cm−1 represented skeletal vibrations with C–O–C stretching (Trung & Bao, 2015). The characteristic bands of chitosan at low intensity were observed near 897 cm−1 (C–O stretching of glycosidic linkage) (Kasaai, 2008; Kumirska et al., 2010).
- #17 Chitin showed two crystalline planes (020 and 110) observed a reflection of 9.10 and 19.17º, respectively. Additionally, minor reflections at 26.25º and higher ones were also noticed. For the CS‐130‐4 sample, only one crystalline peak was detected at 19.73º. This was more likely associated with a reduction in crystallinity or the formation of the amorphous structure due to chitin deacetylation (Hajji et al., 2014). Heating of chitin at high temperature and alkaline concentration distorted crystalline structure. The results are supported by the reduction in the CrI of chitin from 40.75% to 19.75% after the deacetylation.
- #18 For CS‐130–4, the resonance for H‐1 (D), H‐2/6, H‐2 (D), and H‐Ac were assigned at a chemical shift of 5.32, 4.12, 3.05, and 1.97 ppm, respectively. H‐1 (D) and H‐2 (D) are assigned for protons of H‐1 and H‐2 deacetylated monomer, respectively. H‐Ac denotes signal for the proton of acetyl group and H‐2/6 shows resonance for the protons of pyranose ring and other between 3.55 and 4.12 ppm. The absence of peak for H‐1 (A) around 4.9 ppm, which denotes proton for H1 of acetylated monomers, indicate chitin deacetylation For CE‐8, a similar spectrum was obtained when compared to that of CS‐130–4, indicating that the conjugate was derivative of chitosan (Figure 3b). However, a new peak was observed at 6.9 ppm, which corresponds to aromatic protons, mainly from EGCG.
- #21 Due to the higher molecular weight, viscosity, gelling ability, chitosan has been known entrap oil, which can pass though body without lipase hydrolysis. This result into no absorption of the fats into blood and can help to reduce the body weight.
- #25 Chitosan is also used for the packaging material in the form of edible film. Edible films are used in the preservation of various perishable food. Generally, chitosan film are prepared by casting method, in which chitosan solution was poured on the casting tray and allowed for solvent evaporation. However, chitosan film are low in activity therefore, chitosan based composite film was prepared.
- #26 Free radical grafting method was opted for preparation on Chitosan-EGCG conjugate. The reason behind selection of this method that, it is cheap, non-toxic, reaction carried out room temperature and oxidation of phenolics into semiquinones were not occurred. In this method ascorbic acid is used with hydrogen peroxide to generate hydroxyl free radicals. The free radicals further abstracted hydrogen from C 2, 3 and 6 from chitosan to generate chitosan macroradical. These chitosan macroradical interacted with polyphenol via covalent interaction, resulted into formation of chitosan-polyphenol conjugate.
- #27 Similar to the emulsion formation, chitosan can be used to prepare nanoliposome, which can be used as delivery agent. Chitosan can protect the bioactive components or drugs from the harsh conditions of the digestive tract by forming coating around them.