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  ISSN 1330-9862  review (FTB-2839) Chitin Extraction from Crustacean Shells Using BiologicalMethods – A Review Wassila Arbia 1 , Leila Arbia 1 , Lydia Adour 1,2 and Abdeltif Amrane 3,4 *  1 Laboratory of Environmental Biotechnology and Process Engineering (BIOGEP),Department of Environmental Engineering, Polytechnic National School, 10 Avenue Hacene Badi,BP 182, El Harrach, DZ-16200 Algiers, Algeria 2 Department of Chemistry, Faculty of Science, Mouloud Mammeri University of Tizi Ouzou,DZ-15000 Tizi Ouzou, Algeria 3 National School of Chemistry of Rennes, CNRS, UMR 6226, Avenue du Général Leclerc, CS 50837,FR-35708 Rennes Cedex 7, France 4 European University of Brittany, 5 Boulevard Laënnec, FR-35000 Rennes, FranceReceived: June 17, 2011Accepted: April 18, 2012 Summary After cellulose, chitin is the most widespread biopolymer in nature. Chitin and its deri-vatives have great economic value because of their biological activities and their industrialand biomedical applications. It can be extracted from three sources, namely crustaceans,insects and microorganisms. However, the main commercial sources of chitin are shells of crustaceans such as shrimps, crabs, lobsters and krill that are supplied in large quantities by the shellfish processing industries. Extraction of chitin involves two steps, deminerali-sation and deproteinisation, which can be conducted by two methods, chemical or biologi-cal. The chemical method requires the use of acids and bases, while the biological methodinvolves microorganisms. Although lactic acid bacteria are mainly applied, other microbialspecies including proteolytic bacteria have also been successfully implemented, as well asmixed cultures involving lactic acid-producing bacteria and proteolytic microorganisms.The produced lactic acid allows shell demineralisation, since lactic acid reacts with cal-cium carbonate, the main mineral component, to form calcium lactate. Key words : chitin, crustacean shells, chitin extraction, biological methods Introduction Enormous amounts of chitin can be found in the bio-sphere; it is the major component of cuticles of insects,fungal cell walls, yeast or green algae ( 1–3 ). Fungi pro-vide the largest amount of chitin in the soil (6–12 % of chitin biomass, which is in the range of 500–5000 kg/ha)( 4 ). Chitin is also widely present in crab and shrimpshells ( 5 ).A working estimate for the annual turnover is in therange of 10 10 –10 11 tonnes ( 6,7 ), making chitin one of themost abundant biopolymers. Chitin can be readily ob-tained by simple extraction ( 8 ). To date, the major sourceof industrial chitin comes from wastes of marine foodproduction, mainly crustacean shells,  e.g.  shrimp, crab orkrill shells ( 9–11 ).In the processing of shrimps for human consump-tion, between 40 and 50 % of the total mass is waste.About 40 % of the waste is chitin, incrusted with cal-cium carbonate and astaxanthin, and containing meatand a small amount of lipid residues. A small part of thewaste is dried and used as chicken feed ( 11 ), while therest is dumped into the sea, which is one of the mainpollutants in coastal areas ( 12,13 ). The utilization of shell-fish waste has been proposed not only to solve environ-mental problems, but as a waste treatment alternative to 12  W. ARBIA  et al. : Chitin Recovery Using Biological Methods,  Food Technol. Biotechnol. 51  (1) 12–25 (2013)*Corresponding author; Phone: ++33 2 2323 8155; Fax: ++33 2 2323 8120; E-mail: abdeltif.amrane @ univ-rennes1.fr  the disposal of shellfish wastes ( 14 ). Crustacean shellwaste consists mainly of 30–40 % protein, 30–50 % cal-cium carbonate, and 20–30 % chitin (Table 1) ( 15–18 ),with species and seasonal variations ( 19 ).Seafood processing and consumption generate eachyear hundreds of tonnes of shellfish waste, like in Tai-wan ( 9 ) or Indonesia ( 20 ), whereas in Germany only22 616 tonnes of shrimp waste is discarded on the sea-shore ( 11 ). By-products from marine food production,mainly shrimp shells, comprise almost 40 % of totalprawn mass and have become a major environmentalconcern due to their slow degradation ( 8 ) .  The majorcomponents (on dry mass basis) of shrimp waste areproteins, chitin, minerals and carotenoids ( 20,21 ) . To extract chitin from shrimp shells using traditionalchemical treatment, 4 % NaOH is used for deproteini-sation and 4 % HCl for demineralization. This processpresents some drawbacks since it is expensive and envi-ronmentally unfriendly ( 22 ), and hence finding alterna-tive processes would be really helpful .  Biotechnologicalproduction of chitin has not been commercially availableup to now, but it can offer new perspectives for the pro-duction of highly viscous chitosan, with a promising po-tential for applications in biomedicine and pharmacy( 23,24 ). Fermentation of this biowaste using lactic acid bacteria for the production of chitin has been studiedand reported ( 25 ) .  The use of organic acids such as lacticacid for the demineralisation process is a promising ideasince organic acids can be produced by bacteria at lowcost, are less harmful to the environment, can preservethe characteristics of the purified chitin and the resultingorganic salts from the demineralisation process can beused as an environmentally friendly deicing/anti-icingagents and/or as preservatives ( 22 ) . Properties of Chitin Chitin is one of the most abundant biopolymers innature ( 1 ) and is a major component in the supportingtissues of organisms such as crustaceans, fungi and in-sects ( 26 ) .  It is a linear polysaccharide composed of   a --(1–4)-linked 2-acetamido-2-deoxy- D -glucose units whichmay be de- N  -acetylated to some extent ( 27 ) .  It is a struc-tural polysaccharide similar to cellulose. Chitin mole-cules are known to be ordered into helicoidal, microfi- brillar structures that are embedded into the proteinmaterial of the shells ( 28 ) .  Chitin is closely associatedwith proteins, minerals, lipids and pigments ( 29 ) . Several studies have clearly demonstrated that spe-cific characteristics, namely degree of deacetylation andmolecular mass of chitin and its deacetylated derivative,chitosan, vary with process conditions. The physico-chemical characteristics of chitin and chitosan influencetheir functional properties such as solubility, chemicalreactivity and biological activities ( 30 ), namely biodegrad-ability ( 2,31 ) ,  which differ depending on the crustaceanspecies and preparation methods ( 32 ) .  Recent studieshave revealed notable variability in the dye, water, andfat binding capacities of various chitins, chitosans andtheir derivatives produced from crustacean shell wastesat laboratory scale ( 19 ) ,  as well as notable variability inthe antibacterial activities ( 32 ), biodegradability and im-munological activities ( 33,34 ) . The degree of deacetylation, defined as the molarfraction of deacetylated units in the polymer chain ( 35 ) , is one of the most important factors influencing the prop-erties of chitin and chitosan ( 18 ) ,  such as solubility, flexi- bility, polymer conformation and viscosity ( 16,24,36 ).The degree of acetylation can be employed to differen-tiate between chitin and chitosan; in the case of chitin itis greater than a given value ( e.g.  >50 %) and insoluble,while in the case of chitosan it is smaller than that valueand soluble ( 37 ). A number of methods have been re-ported to determine the degree of deacetylation of chitin( 2 ).Molecular mass determines the viscosity and the rateof degradation, which can be determined by viscometer, 13 W. ARBIA  et al. : Chitin Recovery Using Biological Methods,  Food Technol. Biotechnol. 51  (1) 12–25 (2013)Table 1. Contents of chitin in different organisms ( 15–18 )Organism  w (chitin)/% Organism  w (chitin)/% Crustaceans: Cancer  (crab) Carcinus  (crab) Paralithodes  (king crab) Callinectes  (blue crab) Crangon  and  Pandalus  (shrimp)Alaska shrimp Nephro  (lobster)  Homarus  (lobster) Lepas  (goose barnacle) Insects: Periplaneta  (cockroach) Blatella  (cockroach) Coleoptera  (ladybird) DipteraPieris  (butterfly)72.1 c 64.2  b 35.0  b 14.0 a 17–4028.0 d 69.8 c 60–75 c 58.3 c 2.0 d 18.4 c 27–35 c 54.8 c 64.0 c Bombyx  (silk worm) Galleria  (wax worm) Mollusks: clamshell oysterssquid penkrill, deproteinized shells Fungi:  Aspergillus nigerPenicillium notatumPenicillium chrysogenumSaccharomyces cerevisiae Mucor rouxiiLactarius vellereus 44.2 c 33.7 c 6.13.641.040.242.0 e 18.5 e 20.1 e 2.9 e 44.519.0 a compared to the body fresh mass,  b with respect to the body dry mass,  c  based on the mass of the organic cuticle,  d compared to thetotal mass of the cuticle,  e relative to the dry mass of the cell wall  light scattering and gel permeation chromatography ( 38 ).The chitins differ not only in molecular mass and degreeof deacetylation, but also by their crystalline structure,which controls a number of properties ( 16 ). Chitin oc-curs in three polymorphic forms,  a -,  b -, and  g -chitins,which differ in the arrangement of molecular chains with-in the crystal cell ( 35,39 ).  a -Chitin with its antiparallelchain arrangement is the most abundant chitin in nature(shrimps, crabs),  b -chitin has parallel chains and occursin squid pens ( 11,40,41 ), while  g -chitin presents the mix-ture of   a - and  b -chitins ( 39 ).Similarly to cellulose, chitin is insoluble in water,aqueous solvents and common organic solvents, owingto its strong intra- and inter-molecular hydrogen bonds( 18,42 ). Solubility, which is related to different para-meters, is very difficult to control ( 16,41 ).Chitosan, as a polyelectrolyte, is able to form elec-trostatic complexes under acidic conditions ( 41 ). It is acationic polysaccharide and its cationic nature in acidicmedium is unique among polysaccharides ( 43 ).Owing to its chemical structure, chitosan is a sub-stitute for biological media. Indeed, the glycosidic bondand the  N  -acetylglucosamine residues found in the chi-tosan macromolecules are also present in the structure of the extracellular matrix of most living tissues ( 16 ). Methods of Chitin Extraction Chemical methods In the skeletal tissue, protein and chitin combine toform a protein-chitin matrix, which is then extensivelycalcified to yield hard shells. The waste may also con-tain lipids from the muscle residues and carotenoids,mainly astaxanthin and its esters ( 8 ).A traditional method for the commercial prepara-tion of chitin from crustacean shell (exoskeleton) con-sists of two basic steps (Fig. 1): ( i ) protein separation,  i.e .deproteinisation by alkali treatment, and ( ii ) calciumcarbonate (and calcium phosphate) separation,  i.e . demi-neralisation by acidic treatment under high temperature,followed by a bleaching step with chemical reagents toobtain a colourless product ( 44 – 46 ).Deproteinisation is usually performed by alkalinetreatment ( 29 ). Demineralisation is generally performed byacid treatment including HCl, HNO 3 , H 2 SO 4 , CH 3 COOH,and HCOOH; however, HCl seems to be the preferredreagent ( 29 , 44 ). It was shown that the order of the twosteps may be reversed for shrimp waste containing largeprotein concentrations, which stem primarily from theskeletal tissue and to a lesser extent from the remainingmuscle tissue ( 8 ).The major concern in chitin production is the qual-ity of the final product, which is a function of the mole-cular mass (average and polydispersity) and the degreeof acetylation. Harsh acid treatments may cause hydro-lysis of the polymer, inconsistent physical properties inchitin and are source of pollution ( 47 ). High NaOH con-centrations and high deproteinisation temperatures cancause undesirable deacetylation and depolymerisation of chitin ( 8 ). Percot  et al . ( 29 ) reported that using inorganicacids such as HCl for the demineralisation of chitin re-sults in detrimental effects on the molecular mass andthe degree of acetylation that negatively affect the intrin-sic properties of the purified chitin (Table 2). Similarly,according to Crini  et al.  ( 16 ) this method allows almostcomplete removal of organic salts, but at the same timereactions of deacetylation and depolymerisation may oc-cur (Table 2). Quality improvement can be obtained byimproving the contact of chemicals with the shrimp waste,for instance by using stirred bioreactors. This wouldallow reactions to proceed with the same efficiency atshorter exposure time and at lower temperature ( 24 ).Comparing different chitins (degree of acetylation, mole-cular mass and optical activity), variations of the charac-teristics of the obtained polymer were observed accordingto the acid used for the demineralisation ( 16 ).In addition, chemical chitin purification is energyconsuming and somewhat damaging to the environmentowing to the high mineral acid and base amounts in-volved ( 48 ). These chemical treatments also create a dis-posal problem for the wastes, since neutralisation anddetoxification of the discharged wastewater may be ne-cessary ( 49 ). Another disadvantage of chemical chitinpurification is that the valuable protein components canno longer be used as animal feed ( 22,50 ). Biological methods An alternative way to solve chemical extraction pro- blems is to use biological methods. The use of proteasesfor deproteinisation of crustacean shells would avoid al-kali treatment (Fig. 1). Besides the application of exoen-zymes, proteolytic bacteria were used for deproteinisa-tion of demineralised shells ( 47 ). This approach allowsobtaining a liquid fraction rich in proteins, minerals andastaxanthin and a solid chitin fraction. The liquid frac-tion can be used either as a protein-mineral supplementfor human consumption or as an animal feed ( 25 ).Deproteinisation processes have been reported forchitin production mainly from shrimp waste using me- 14  W. ARBIA  et al. : Chitin Recovery Using Biological Methods,  Food Technol. Biotechnol. 51  (1) 12–25 (2013) Shell Biological method Chemical method Washed and crushedAlkali treatment (1 M NaOH for Deproteinisation byProteinsDeproteinised shellDemineralisation byorganic acid-producing bacteria protease-producing bacteriaAcidic treatment (0.275-2.0 M HClfor 1–48 h at 0–100 °C) ( ) 31 Minerals (calcium carbonateand calcium phosphate)Discolouration and bleaching (organic mixture of chloroform, methanol and water (1:2:4) at 25 °C) ( ) 8 Raw chitin1–72 h at 65–100 °C) ( ) 31 Fig. 1.  Chitin recovery by chemical and biological methods ( 8 , 31 )  chanical ( 47 ), enzymatic ( 51,52 ) and microbial processesinvolving species like  Lactobacillus  ( 25 ),  Pseudomonas ae-ruginosa  K-187 ( 53 ) and  Bacillus subtilis  ( 54 ). Biologicaldemineralisation has also been reported for chitin pro-duction from crustacean shells; enzymatically, using forinstance alcalase ( 52 ), or by microbial process involvingspecies like  L. pentosus  4023 ( 45 ) or by a natural probiotic(milk curd) ( 55 ). In these biological processes, deminera-lisation and deproteinisation occur mainly simultane-ously but incompletely ( 47 ). An overview of the various biological methods available in the literature is given inTable 3 ( 11,25,45,46,48,49,53,56–70 ).Fermentation has been applied to fish for manyyears and represents a low-level (artisanal) and afford-able (neither capital nor energy intensive) technology( 71 ). It consists in the ensilation of crustacean shells anda low-cost  in situ  production of lactic acid from by--products such as whey, lignocellulose and starch. Lacticacid production by lactic acid bacteria induced a lique-faction of the semi-solid waste and led to a low pH andactivation of proteases ( 50 ). The protein-rich liquid could be separated from the chitin, which remained in the sedi-ment ( 11 ). This method might offer a commercial routefor the recovery of chitin ( 45 ).Lactic acid is formed from the breakdown of glu-cose, creating the low pH, which improves the ensilationthat suppresses the growth of spoilage microorganisms.Lactic acid reacts with the calcium carbonate componentin the chitin fraction, leading to the formation of calciumlactate, which precipitates and can be removed by wash-ing. The resulting organic salts from the demineralisa-tion process could be used as de- and anti-icing agentsand/or preservatives ( 56 ). Deproteinisation of the bio-waste and simultaneous liquefaction of the shrimp pro-teins occurs mainly by proteolytic enzymes produced bythe added  Lactobacillus,  by gut bacteria present in the in-testinal system of the shrimp, or by proteases present inthe biowaste ( 25 ). It results in a fairly clean liquid frac-tion with a high content of soluble peptides and freeamino acids ( 71 ).Khanafari  et al . ( 22 ) extracted chitin and chitosanfrom shrimp waste by chemical and microbial methods.Their results showed that the microbial method is moreeffective especially for the recovery of chitin, comparedto chemical method.Lactic acid fermentation of shrimp wasteRecently, biological processes for chitin productionhave been reported using bacteria that produce organicacids and enzymes for the demineralisation and depro-teinisation of crustacean shells. Fermentation of shrimp( Penaeus monodon ) waste with lactic acid bacteria for chi-tin recovery was studied with added carbohydrates suchas lactose or cassava extract as a natural energy source( 56 ) and date juice for extraction of chitin from head andshell of shrimp  Parapenaeus longirostris  using  Lactobacillushelveticus  ( 57 ). Raw heads of African river prawn (  Macro-brachium vollenhovenii ) were fermented with  Lactobacillus 15 W. ARBIA  et al. : Chitin Recovery Using Biological Methods,  Food Technol. Biotechnol. 51  (1) 12–25 (2013)Table 2. Comparison of chemical and biological methods for chitin recovery ( 31,38 )Chemical method Biological methodChitinrecovery in situ Demineralisation Mineral solubilisation by acidic treatmentincluding HCl, HNO 3 , H 2 SO 4 , CH 3 COOHand HCOOH.Carried out by lactic acid produced by bacteriathrough the conversion of an added carbonsource.Deproteinisation Protein solubilisation by alkaline treatment. Carried out by proteases secreted into thefermentation medium. In addition,deproteinisation can be achieved by addingexo-proteases and/or proteolytic bacteria.Effluent treatment after acid and alkalineextraction of chitin may cause an increase inthe cost of chitin.Extraction cost of chitin by biological methodcan be optimised by reducing the cost of thecarbon source.Solubilised proteins and minerals may be usedas human and animal nutrients.ChitinqualityThe major concernin chitin productionis the quality of thefinal product, whichis a function of themolecular mass(average andpolydispersity) andthe degree of acetylation.A wide range of quality properties of thefinal product.Using inorganic acids such as HCl for chitindemineralisation results in detrimental effectson the molecular mass and the degree of acetylation that negatively affect the intrinsicproperties of the purified chitin ( 31 ) . This method allows almost complete removalof organic salts, but at the same time thereactions of deacetylation and depolymeri-sation may occur ( 38 ).The comparison of different chitins (degree of acetylation, molecular mass, optical activity)obtained with four different acids showed thatthe polymer characteristics varied according tothe extraction method used ( 38 ).Homogeneousness and high quality of the finalproduct.  16  W. ARBIA  et al. : Chitin Recovery Using Biological Methods,  Food Technol. Biotechnol. 51  (1) 12–25 (2013)Table 3. Overview of the biological methods for chitin recoveryWaste source Strains and/orproteolytic enzymesCarbonsourceDurationdayEfficiency/%Refs.deprotein-isationdemineral-isationLactic acid fermentation Penaeus  sp.  Lactobacillus  spp. B2 sucrose whey 6 85 87.6 ( 59 )demineralised  Nephrops norvegicus  Stabisil:  Streptococcus faecium  M74, L. plantarum ,  Pediococcus acidilactici lactose 7 40 n.d. ( 60 ) Nephrops norvegicus  Sil-All 4×4 :  L. plantarum, L. salivarius,S. faecium, P. acidilactici glucose 7 n.d. 90.99 ( 48 ) Nephrops norvegicus L. paracasei  A3 glucose 5 77.5 61 ( 58 )one-step shrimp fermentation  L. plantarum  541 glucose – 75 86 ( 25 )pretreated  Procambarus clarkii (crayfish) L. paracasei  A3 dextrose 3 94 97.2 ( 61 ) Procambarus clarkii  immobilized  Lactobacillus pentosus  4023whey 2.1 81.5 90.1 ( 45 ) Chionoecetes japonicus L. paracasei  ssp.  tolerans  KCTC-3074 glucose 1 54.7 55.2 ( 56 ) Parapenaeus longirostris L. helveticus  date juice 14 91 44 ( 57 )Non-lactic acid fermentation  Metapenaeus dobson Bacillus subtilis  jaggery – 84 72 ( 62 )shrimp and crab shell  Pseudomonas aeruginosa  K-187 – 5 82 – ( 49 )shrimp and crab shell powdernatural shrimp shellsacid treated natural shrimp shellshrimp and shell crab powderproteases of   P. aeruginosa  K-187immobilized proteases of  P. aeruginosa – 7 72784567– ( 53 )( 53 )crab shell powder  P. aeruginosa  F722 – 7 63 92 ( 63 ) Chionoecetes opilio (natural crab shell waste) Serratia marcescens  FS-3Delvolase ® Combination of Delvolase ® and Serratia marcescens  FS-3 S. marcescens  FS-3 supernatantculture7 4790858184 ( 64 )shrimp shell waste  Bacillus cereusExiguobacterium acetylicum – 97.192.89592( 65 )squid pen  Bacillus  sp. TKU 004 73 n.d. ( 66 ) Penaeus monodon Pediococcus acidilactici  CFR2182 97.9 ± 0.3 72.5 ± 1.5 ( 70 )shrimp shells  Pediococcus  sp. L1/2 sucrose 1.5 n.d. 83 ( 46 )Cofermentationtwo-step fermentation of   Penaeusmonodon  and  Crangon crangon first step: anaerobicdeproteinisation by autochthonousflora of Indonesian shrimp shellsand/or proteolytic bacteriasecond step:  L. casei  MRS197.490.899.699.7( 11 )prawn waste  Lactobacillus lactisTeredinibacter turnerae co-fermentation of both species66.577.89578.823.395( 67 )red crab shell waste one-step fermentation:  L. paracasei ssp.  tolerans  KCTC-3074 and S. marcescens  FS-3successive two-step fermentation7 52.694.397.268.9( 68 )two  Bacillus licheniformis  strains withtreatment of the final fermentationproduct with 0.9 % lactic acid2 99 98.8 ( 69 )n.d.=not determined
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