Preparation, Properties and Biological Applications of Water SolubleChitin Oligosaccharides from Marine Organisms!

A. B. A. Ahmeda, h, Rosna Mat Tahaa, Sadegh Mohajer", M. Elnaiem Elaagiba, and Se Kwon Kimh

a Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, 50603 Malaysiab Marine Bioprocess Research Center, Department of Chemistry, Pukyong National University, Busan, South Korea

E-mail: dr.bakrudeenaliahmed@yahoo.co.in

Abstract-Chitin oligosaccharides (COSs) can be isolated from various natural resources, which have widelybeen used in biological active supplements (BAS) for the benefit of humankind. Several technologicalapproaches for the preparation of COSs such as enzymatic, chemical, acid-catalysts hydrolysis, microwaveradiation, membrane bioreactor methods have been developed and among them, membrane bioreactor, bio-conversion and continuous mass production technologies are reported to be excellent. Compounds isolatedfrom natural products have made a drastic impact on the pharmaceutical industry and especially, water-sol-uble chitin oligosaccharides have shown greater clinical activity, which have been demonstrated in various celllines of disease significance. The activities ofthese COSs were being investigated in different patients, animalsand even plants as a broad phase clinical trial program. In the present article, we have discussed the COSspreparation by different methods through comprehensive diffraction procedures along with the merits anddemerits given in detail. In addition, a summary of recent work describing the synthesis and biological activ-ities of water-soluble COSs has been presented here.

Keywords: Chitin oligosaccharides; enzymatic hydrolysis; membrane bioreactor; cell lines; biological activityDOl: 10. II 34jS 10630740 12040025

Natural products have great economic and ecolog-ical importance, and many of natural products are yetto be discovered. The marine environment is a richsource for production of natural bioactive metabolites,which are used in various clinical trials [49]. Over 60%of natural products can be considered as drugs in thepharmaceutical industry [32]. Many novel compounds(drugs) have been isolated from the sea and screenedfor biological studies includ~g anti-obesity, anti-dia-betes, anti -hypertension, anti -microbial, anti-fertility,anti-tumoral, anti-arthritic, haemolytic and as anantiinflammatory substance. With increasing healthconsciousness among consumers and the rapidprogress of physiologically functional foods, the pro-file of medicinal products containing chitin oligosac-chari des with biological activities seems to be greatlypromising in worldwide [10]. It could be present infood and nutrition. It is emerging as a great potentialto the food industry [53]. Currently, the functionalfood market has significance in the earning ofU.S. $100 billion/year [2].

Nowadays, chitin degradation is of considerableinterest, since the chitin products have potential appli-cations in biomedicine, agriculture, nutrition and bio-techn?logy [50]. Chitin derivatives (monosaccharides,

oligosaccharides and polysaccharides) are water solu-ble and possess lower molecular weight and shows sig-nificant biological activity includ;ng tumouricidal,antimicrobial and antihypertensive [37]. It is reportedthat hexameric chitin oligosaccharides have potent ofbiomedical significance by enhancing the immunesystem [12]. Hence, water-soluble COSs that havebeen applied in biological treatments are needed to beproduced in a large scale for humankind.

COSs are water soluble polymers produced byhydrolysis of chitin (long-chain polymer) that hasbeen refined from crab, prawn, shells, insects, ants,beetles, butterflies, radula of molluscs, cephalopodsand copepods [38]. N-acetyl glucosamine, one of theforms of COSs is a milky white powder, organic innature and sweet to taste. It has low digestibility (slim-ming diets, controlling intestinal function), but isinvolved in the promotion of bifidus proliferation(controlling intestinal function), high hyaluronicacidity (useful for cosmetic applications); immunity(preventing cold and cancer); and shows anti-bacte-rial, anti-fungal, anti-viral, non-toxic, non-allergenicproperties [19]. COS derivatives have strong affinityfor biological systems with distinctive properties with-out any side effects and allergies, and they act on spe-cific organ (or) system, when prepared throughhydrolysis and biodegradable methods [21]. Chitosan,produced by the deacetylation of chitin, is a nontoxic

c::::::::=:J 1998-2001;

_ 2002-04;

_ 2005-10

Fig. 1. Water soluble Chitin Oligosaccharides reputed pub-lications (2010-1998).

biopolymer with versatile chemical and physical prop-erties, but with poor solubility [25].

1. COSS PREPARATION BY DIFFERENTMETHOD

In the last three decades, chitin derivatives haveshown excellent biological activity and are utilized invarious bio-resource technologies and given in theamount of publication (Fig. 1). Researchers are focus-ing more to enhance the efficiency and specificity ofwater soluble COSs by oxidation, ultrasonic treat-ment, mechanical, enzymatic and chemical degrada-tion [56]. Chemical and enzymatic methods have beenalternative conventional processes, but chemicalhydrolysis is a time consuming process and not suit-able for industrial purposes [27]. Hence, enzymaticprocedures are showing many advances in the presentday research and development.

1.1. Enzymatic Hydrolysis Method

The known enzymes that are involved in the degra-dation of chitins are beta-galactosyl transferases, sialyltransferases, beta-polysaccharide synthases, glycosyl-transferase, chitin synthase, cellulose synthase, hyalu-ronic acid synthase, bacterial NodC protein, endoch-itinases and exochitinases, cellulases, pectinases andlysozymes. Chitinase, ~- N -acetylhexosaminidase,chitin deacetylae and chitin oligosaccharide deacety-lase (COD) significantly increase the water-solublenature ofCOSs [58]. Chitinase catalyze the hydrolyticreaction to form water soluble COSs [14]. When com-bined with ~- N -acetylglucosaminidases, it degradesthe biological molecules, which is very useful for therecycling of COSs (Tanaka, et aI., 2003). Hence,

Bacillus subtilis KHI chitosanases, Streptomyceskurssanovii RCM-Ac-1504 D chitinases and Aeromo-nas hydrophila H-2330 chitinases and other commer-cial enzymes such as papain, cellulase, pectinase andlysozyme have also been used in COSs preparation [7].

Large-scale COSs production requires E. coli andhas to stimulate the chitin oligosaccharide synthaseand NodC [18]. Microorganisms have to be culturedat high densities to yield large scale COSs [41]. How-ever, chitin deacetylase and chitin oligosaccharidedeacetylase with acetamide group significantlyincreased the COSs production than other enzymes[52]. Sometimes, low yield COSs is observed from a-chitin, than P chitin [13]. Under stress conditions,large scale COSs was observed in the presence of opti-mal pH, temperature with endo and NHase in UFLreaction. However, when partially deacetylated 0.-chitin was slightly acidified with media as substrate ofLecanicillium fungicola, chitinases yield 16-foldCOSs [13]. This concludes that, typical enzymatichydrolysis of o.-chitin with crude enzyme significantlyincreased the productibn of COSs [27].

1.2. Chemical Hydrolysis Method

Chitin derivatives are prepared by chemical hydrol-ysis of acetamide groups of chitins are usually isolatedfrom marine crustaceans, mainly because a largeamount of waste is available as a by-product of foodprocessing. In this aspect, chitin derivatives (a, ~, ychitins) are arranged with parallel weaker intermolec-ular forces (~-chitin) and polymorphic form (y-chitin). Chitin could be degrading through chemicalhydrolysis with increasing polarity, electrostatic repul-sion of amino groups, and degree of acetylation forCOSs production [28]. Besides, oligosaccharidederivatives that react with APTS, glacial acetic acidand 1M aqueous sodium cyanoborohydride (10 Ill) aremore suitable in the preparation of COSs [56]. Wehave recently reported that chitin hydrolytic solutionwas neutralized with NaOH and insoluble residueswere removed to leave purified light yellow powderNA-COSs in Micro Acilyzer G3 [33] (Fig. 1).

Chitin was ground, sieved (80 mesh), and thenreacted with 12 N Cone. HCI. The resulted solutionneutralized with NaOH, filtered and then desalted byelectrophoresis. The hydrolysate was freeze-dried andthe production contains chitin oligosaccharides withhigh molecular weight (1-3 kDa) [6], whereas thesame chitin was involved on partial hydrolysis withcone. HCl, phosphoric acid and HF resulted lowmolecular weight chitin oligosaccharides (belowI kDa) [42]. However, mild hydrolyzing of chitin with85% H3P04 yields 43% oligosaccharide [20]. Eisen-beis, et aJ. [15] reported that chitin was suspended with37% HCl and stirted for 2 h at 4°C then cooled to O°Cand adjusted to pH 7 with 50% NaOH. The suspensionsolution was centrifuged for 15 min, and the superna-tant was filtered a glass filter and concentrated to 50 ml

· L.: 1I11thobiJized.·Chitin solution

(Substrate l)

Recycling

IEnzyme

+Substrate II

Partial hydrolysedchitin in

colmn reactor

Substrate II+

Free chitinase

on rotaly evaporator. The sample was fractionated ona Biogel P-2 acrylamide column and analyzed byHPLC. The fractions containing different chitin oli-gosaccharides derivatives such as GlcNAc,(GIcNAc)2, (GlcNAc)3, (GIcNAc)4, (GlcNAc)5,and (GlcNAc)6 were pooled and used as a carbonsource.

In-situ acid hydrolysis must be conducted carefullyto avoid mobilization of heavy metals and wastes,while deriving oligomer products. Sometimes, acidhydrolysis results in many problems of uncontrolledreaction, poor repeatability, concentration variation,which requires extreme desalting during ,he hydrolysisprocess [57]. Acid reacted with N-acetylated deriva-tives are used for COSs preparation [54].

J.3. Microwave Radiation Method

Occasionally, electrolytes degrade the chitosan rawmaterials and reduce pollution, saving time therebyplaying a major role in the industrialization and exten-sive marketable potential ofCOSs preparation. Chito-san is composed of NaCl, which can be degraded in3"l2 min at a microwave radiation of 480"800W Inaddition, the cooling temperature, and neutralizingwith NaOH or KOH, and then consequent deposition,

suction filtration and baking dry r~sults in a goodquantity ofCOSs [40].

J.4. Ultra Filtration Membrane Bioreactor Method

COSs can be prepared by biotransformation tech-nologies through a membrane bioreactor, bioconver-sion and continuous mass process technology. Amongthese methods, membrane bioreactor technology withultra filtration has recently been shown to be emergingin the development ofbioactive compounds. This canalso be considered as a potential method [36] (Fig. 2).

Chitin reacts with 12 N HCI while stilTing at 40°Cproducing the different molecular weight COSs. Gen-erally, chitin has been neutralized with NaOH to formCOSs in Micro Acilyzer G 3 (below 1 kDa COSs andMW 1- 3 kDa) by ultra filtration membranes. In addi-tion, COSs production was significantly increased in4-12 N H CI at 30- 70°C in the batch culture process,among which, 12 NHCl and 40°C was suitable forCOSs production [35] (Fig. 3). Bioconversion is a sin-gle reactor system for continuous marine nutraceuti-cals production on 'a large scale. It has a hollow fibercartridge to allow the small particles, whereas it doesnot allow large particles. In this process, enzymes havebeen allowed to catalyze the substrate and the entire

~HITT~

1-4-0o-C-r! -. ---112NHCLI

Stirring for varioustimes (h)*

25% NaOH solution(Neutralize)

IInsoluble residues 1----

IConcentration I

tI Dry I

+Light yellow chitin oligosaccharides

OrN-Acetyl glucosamine powder

~----IDesalting IUFmembrane

system

White chitin oligosaccharidesOr

N-Acetyl glucosamine powder

reaction mixture is pumped into a recycled membranemodel. The proteolytic enzymes cleave the peptidebond specifically to determine the consecutive diges-tion and then, NA-COS products are separated basedon molecular weights. Chemical structure and molec-ular weights (MW) were confirmed by FT - IR, NMR,MALDI-TOFMS [35, 36].

1.5. Maillard Reaction Method (NonenzymaticGlycation)

Chitin was treated with acid according to themethod [43]. Chitin (2 g) was ground to a fine powder,placed into a flask, dissolved in 16 ml of6 M HCl at30°C and stirred for 10 min. This solution was furtherincubated for 110 min at 40°C under continuous stir-ring. Prior to neutralization, the reaction mixture wasplaced in a water bath containing an ice/salt mixturefor a few minutes, and then a 50% aqueous NaOHsolution was carefully added to the continuouslystirred reaction mixture. Insoluble material wasremoved from the chitin hydrolysate by centrifugationat 10000 g for 25 min at 5°C. The supernatant wasapplied to a tandem ultrafIltration system, whichmembranes had cutoffs of 3 and lkDa, respectively.Chitin oligosaccharides obtained were those thatpassed through the 3 kDa membrane but were retainedby the 1 kDa memberane [22]. Hydrolysates were

desalted by ultrafIltration using a membrane of 1 kDacutoff and then subjected to freeze-drying [30].

2. BIOLOGICAL APPLICATIONS OF CHITINOLIGOSACCHARIDES

2.1. COSs Role in Microbes, Plants and AnimalsWater-soluble COSs have attractive and wide vari-

ety of health applications. According to the previousinvestigations, chitin oligomers have shown a varietyof biological activities like controlling plant growth,securing resistance from fungi; and other agriculturaland medical applications [24]. In addition, in themicroorganisms like Vibrio cholera, chitobiose regu-lated by chitin oligosaccharide deacetylase (COD),which can be active in NA-COS media [3, 31]. COSsplay a crucial role in plant biotechnology, secondarymetabolites production and plant resistance (cellwalls) and could assist releasing symbiotic bacteria into the root nodules for nitrogen-fixation [3]. Whenconsidering the innate immune response, COSsresponds as a PAMP (pathogen-associated molecularpattern) and further, lipopolysaccharides (LPS)express the hypersensitive genes, which lead to thehost-specific symbiosis between legumes and rhizo-bium [11]. COSs have been possible precursors in sus-pension culture for the production of diterpenoids and

phytocassanes, which determine chitinase activity inmelon plants [48]. This principle acts as defencemechanism against pathogens in monocots, dicots,mammalian and insect cells [47]. Modified COSsreacts with rhizobial bacteria in roots, which activatephospholipase C (PLC), phospholipase D (PLD) andphytoalexin (PA) syntheses and somatic embryogene-sis [4,60]. The O-acetylated COSs trigger cell divisionin root cortex of Vicia sativa by ballistic micro target-ing 1 [44]. However, the effect of COSs was elucidatedby microinjection of anti-DG42 antiserum in fertil-ized zebrafish eggs, which showed that COSs play animportant role in cell signalling, growth, differentia-tion and development of vertebrates [4, 46].

Gene expression mechanjsms of cellular recogni-tion, embryomc development, tumorigenesis or infec-tious diseases after COSs treatment are still not under-stood. Hence, chemo attractants are extracellularchjtinases and the chemotaxis systems for specificCOSs act as "nutrient sensor" cells, which supportprotein, monosaccharide (GluNAc) and disaccharide(GlcNAc)2 syntheses [39]. Bottomley and Myrold[5], reported that lipochitin oligosaccharides containN -acetyl glucosamine residues and different enzymessuch as acyl transferase (nodA), chitin oligosaccharidedeacetylase (nodB) and chitin oligosaccharide syn-thase (nodC) regulate plant and animal cells throughlipochitin oligosaccharides synthesis. In V furnissii,mutagenesis processes through certain sensor compo-nents are able to induce the COSs, which are encodedby the Chis homologous gene. This Chjs gene releasesCBP (periplasmic chitin oligosaccharide binding pro-tein), which activates cellular regulation [16]. Chitinoligosaccharides and chitinase have been activated incarrot cells by incubation with mycelia walls offungus(Chaetomium globosim) [29]. S. oneidensis contllinseleven adjacent genes S03514 thru S03503, encodestwo permeases specific to COSs and-chemotactic pro-tein, and also encodes certain enzymes that areinvolved in converting N -acetylglucosamine into fruc-tose [61]. Chitin is a component of fugal cell walls, andits fragments act as elicitors in many plants. Theplasma membrane glycoprotein (CEBiP), wruch pos-sesses LysM domains, is a receptor for the chitin oli-gosaccharides elicitor (CE) in rice. Kisrumoto et al.[26] constructed chimeric genes composed of CEBiPand Xa21, which mediate resistance to rice bacterialleafblight. During investigation, rice plants expressionof the chimeric receptor exhibited necrotic lesions inresponse to CE and became more resistant to Mag-naporthe oryzae. These results suggest that chitin oli-gosaccharide elicitors are produced and recognizedthrough the LysM domain ofCEBiP during the inter-action between rice and M. oryzae and imply that anengineering pattern recognition receptors represents anew strategy for crop protection against fungal dis-eases [26].

2.2. COSs Role in Human DiseaseHuman pathogens produces immune-stimulants

that modulate the innate immune responses in host,and specifically, T -cells, cytokines, interleukin 2, 6 arestimulated [3, 45]. COSs has enhanced the humanimmune system with the protein agglutination, anti-coagulant processes [55]. COSs are strongly inhibitingthe agglutination of blood, which contains lectin [9].COSs reduce the blood pressure in animal andhumans, which also can prevent the formation of theangiotensin converting enzyme I, II and reduce serumcholesterol [3, 8]. But COSs decrease the intestinaltoxicants such as ammonia, amines, rutrosamines,phenols, skatoles, and in addition, they reduce carci-nogenic and mutagenic incidences along with hepa-toxicity inmbition. On the other hand, bifidobacteriabind up cholesterol and heavy fatty acids in the gut bythe help of COSs and produce B1, B2, B6, B12 andfolic acid vitamins [3]. Malignant growth progressionof the cancer and the invasive properties like anchor-ing of the extracellular, pericellular matrix polysac-charide, hyaluronan are reduced by the COSs treat-ment [3]. COSs are involved in a peculiar mechanismof biocherillcal recognjtion processes on the cell sur-face, and decreases the excretion of body substancesthere by activating the absorption of fats in the colonas well as growth of bacteria. Xenopus, zebrafish andcarp embryos synthesize COSs during late gastrulastages, which inmbit the antibodies against DG42,further entering into fertilized eggs for the develop-ment of trunk and tail [17]. COSs have detoxificationproperties and reduces the enzymes responsible forfatty liver, hepatitis and cirrhosis [3]. In Minamata'sdisease, mercury released from body and decreasebradykinin (pain hormone). It was noticed that thegrowth rate in K562 cells was increased at 24, 48, 72 htreatment ofCOSs [59]. These results could be recom-mended forTh1 disease (caused by Psoriasis vulgaris),rheumatic and cachet disease [23].

2.3. COSs Role in Animal Cell Lines

Very few reports on biological activities of COSswere reported because of the toxic nature of the prod-ucts, when prepared by acid hydrolysis [59]. However,successful non cytotoxic effects are observed in celllines such as MRC-5, RAW 264.7, HL-60, Changliver, U937, HT-29, SW 480, HT 1080 and Bl6F10[33]. Low molecular weight (below 1 kDa) and 1-3 kDa COSs reduce the antioxidant activity, reactiveoxygen species (ROS), myeloperoxidase (MPO), oxi-dation of DNA and intracellular H202 level in2',7' dichlorofluorescein (DCF), however, the intrac-ellular glutathione (GSH) level was significantlyincreased. This result shows that COSs and its deriva-tives can be used as food supplements, marine nutra-ceutical and cosmeceuticals products [33]. COSsinhibit mtric acid (NO) production, and inflamma-tory studies of the epithelial cells indicate that they

stimulate mitochondria activity in HaCaT cell lines.Wound healing, chronic bowel diseases [1], free radi-cal scavenging effects in live cells are all well affectedwith the COSs treatment [33, 35]. Sometimes, below1 kDa and 1-3 kDa NA-COSs inhibit NO productionand stimulates lipopolysaccharides (LPS) in mousemacrophage. However, NO inhibitory action washigher in COS 1-3 kDa than COS £lkDa at 1000Ilg/ml and it regulates the iNOS, COX-2, TNF-a, IL-1~ gene, matrix metalloproteinases (MMP-2, 9)expression by western blot analysis, and RT - PCR andthese results could be supported by the anti-inflamma-tory agent [33, 35]. In addition, chitin oligosaccha-rides (NA-COS) with low molecular weight (229-21-593-12 Da) was produced from crab chitin by acidhydrolysis. They showed reducing power and a scav-enging effect on 1,1 diphenyl-2-picrylhydrazyl(DPPH), hydroxyl and alkyl r~dicals. The radicalscavenging action of NA-COS increased in a dose-dependent manner. Their ICso values for DPPH,hydroxyl and alkyl radicals were 0.8, 1.75 and 1.14mg/ml respectively. It was observed that NA-COSexhibited the inhibitory effect on the oxidative damageof DNA from human lymphoma U937 cell lines andthe direct radical-scavenging effect in human fibrosa-rcoma cells (HT 1080) in 2,7 dichlorofluorescin diac-etate (DCFH-DA). The results suggest that NA-COScan exert antioxidant effects in live cells and have thepotential to be applied to food supplements or nutra-ceuticals [34].

This review has fully addressed water soluble COSsproperties when prepared by enzymatic, chemical,acid-hydrolysis, microwave radiation, membranebioreactor and their merits and demerits in biologicalstudies has also been discussed. Different colouredCOSs products have desirable properties be are oftenunsuitable for large scale reduction. Neverthless,COSs showed minimum number of biological activitywith different cell lines and the mechanism of actionneeds to be further studied in deaph. This review willlend support to the major changes in industry for com-merical production of COSs.

1. Alexandra, D., Frank, P, and Jorg S., Andreas, H., N-Acetyl-D-glucosamine Oligosaccharides Induce MucinSecretion from Colonic Tissue and Induce Differentia-tion of Human Keratinocyte, J. Pharm. Pharmacol.,2008, vol. 60, pp. 197-204.

2. Aluko, R., Functional Foods and Nutraceuticals, IFISPublishing, 2006, http://www.ifis.orgjfscjixidI4335.

3. Asaoka, K., Chitin-chitosan: The choice food supple-ment for over 10000 physicians in Japan, A CompleteAnalysis of its Clinical Applications to VariousDiseases,vol. XXII, Vantage Press, New York, 1996, p. 143.

4. Bakkers, J., Semino, C.E., Stroband, H., Kijine, J.W,Robbins, PW, Spaink, H.P, An Important Role in Oli-gosaccharides During Early Embryo Development ofCarp and Zebrafish, PNAS, 1997, vol. 94, pp. 7982-7986

5. Bottomley, PJ. and Myrold, D.D., Biological N Inputs,Soil Microbiology, Ecology and Biochemistry, 3rd Edi-tion, Ch.14, Alvin Paul, Ed., Elsevier, 2007, pp. 365-386.

6. Chen, H.C., Chang, c.c., Mau, WJ., and Yen, L.S.,Evaluation of N-acetyl Chitooligosaccharides as theMain Carbon Sources for the Growth oflntestinal Bac-teria, FEMS Microbiol. Left., 2002, vol. 209, pp. 53-56.

7. Chen, S.H., Yen, YH., Wang, c.L., and Wang, S.L.,Reversible Immobilization of Lysozyme via CouplingReversibly Soluble Polymer, En:(JImeMicrob. Technol.,2003, vol. 33, pp. 643-649.

8. Chuosuisanken, S., Effect of Polysaccharides and itsMetabolites from Marine Organisms on Homeostasisand Cell Functions, Kagaku Gijutsu Shinko Choseihi niyoru Juten Kiso Kenkyu Seikashu (Japan), 2002, vol. 12,pp.336-337.

9. Ciopraga, J., Angstrom, J., Bergstrom, J., Larsson, T.,Karlsson, N., Motas C. et aI., Tsolectinsfrom Solanumtuberosum with Different Detailed Carbohydrate-bind-ing Specificities: Unexpected Recognition ofLactosyl-ceramide by N-acetyllactosamine-binding Lectins,J. Biochem., 2008, vol. 128, pp. 855-867.

10. Clydesdale, FM., Functional Foods: Opportunitiesand Challenges, Food Technol., 2004, vol. 58, p. 34.

11. Cohn, J.R., Day, R.B., and Stacey, G., Legume NoduleOrganogenesis, TrendsPlantSci., 1998,vol. 3, pp. 105-110.

12. Collin, M. and Fischetti, V.A, A Novel SecretedEndoglycosidase from Enterococcusfaecalis with Activ-ity on Human Immunoglobulin G and Ribonuclease B,J. BioI. Chem., 2004, vol. 279, pp. 22558-22570.

13. Coutino, L.R., Marin-Carmen, M.D., Huerta, S.,Revah, S., and Shirai, K., Enzymatic Hydrolysis ofChitin in the Production of Oligosaccharides UsingLecanicillium fungicola Chitinases, Process Biochem.,2006, vol. 41, pp. ] ]06-1110.

]4. Dahiya, N., Tewari, R., and Hoondal, G.S., Biotech-nological Aspects of Chitinolytic Enzymes: a Review,Appl. Microbiol. Biotechnol., 2006, vol. 7], pp. 773-782.

15. Eisenbeis, S., Lohmiller, S., Valdebenito, M., Leicht, S.,and Braun v., NagA-dependent Uptake of N-acetylGlucosamine and N-acetyl Chitin OligosaccharidesAcross the Outer Membrane of Caulobacter crescentus,J. Bacteriol., 2008, vol. 190, pp. 5230-5238.

16. Flintoft, L., Bacterial Physiology - Unlocking ChitinDegradation, Nat. Rev. Microbiol., 2004, vol. 2, pp. 90.

17. Gagneux, P and Varki, A, EvolutionaIy Consider-ations in Relating Oligosaccharide Diversity to Biolog-ical Function, Glycobiology, 1999, vol. 9, pp. 747-755.

18. Geremia, R.A, Mergaert, P, Geelen, D., Van Mon-tagu, M., and Holsters M., The NodC Protein ofAzorhizobium caulinodans is a N-acetylglucosaminylTransferase, PNAS, 1994, vol. 91, pp. 2669-2673.

19. Gordon, D.R. and Besch-Williford, c., Modulation ofNutrient Absorption: Gut Dynamics of Chitin, Chito-

san and Oligosaccharides, Chitin Workshop, ICNHP,Raleigh, NC; North Carolina State University, 1983.

20. Hasegawa, M., Isogai, A, Onabe, F, and Usuda M.,Preparation of Low-molecular Weight Chitosan UsingPhosphoric Acid, Carbohydr. Polym., 1993, vol. 20,pp. 279-283.

21. Hsiao, Yc., Lin, YW, Su, c.K., and Chiang, B.H.,High Degree Polymerized Chitooligosaccharides Syn-thesis by Chitosanase in the Bulk Aqueous System andReversed Micelle Micro Reactors, Process Biochem.,2008, vol. 43, pp. 76-82.

22. Jeon, YJ., and Kim, S.K., Production ofOligosaccha-rides Using an Ultrafiltration Membrane Reactor andtheir Antibacterial Activity, Carbohydr. Polym., 2000,vol. 41, pp. 133-141.

23. Kan, K., Clinical Efficacy of Chitin Oligosaccharidefor Th I Diseases, Kichin Kito~anKenkyu (Korea), 2006,vol. 12, pp. 134-135. .

24. Karasuda, S., Tanaka, S., Kajihara, H., Yamamoto, Y,and Koga, D., Plant Chitinase as a Possible BiocontrolAgent for Use Instead of Chemical Fungicides, Biosci.Biotechnol. Biochem., 2003, vol. 67, pp . 221-224.

25. Kim, S.K. and Ye, J.Y, Continuous Production of Chi-tooligosaccharides by Enzymatic Hydrolysis, Chitin,Chitosan, Oligosaccharides and Their Derivatives,Kim, S.K, Ed., CRC Press, FL, 2011. pp. 47-51.

26. Kishimoto, K., Kouzai, Y, Kaku, H., Shibuya, N.,Minami, E., and Nishizawa, Y, Perception of theChitin Oligosaccharides Contributes to Disease Resis-tance to Blast Fungus Magnaporthe oryzae in rice, PlantJ., 2010, vol. 64, pp. 343-354.

27. Kuk, J.H., Jung, WJ., Jo, G.H., Kim, K.Y, andPark, R.D., Production of N, N'-Deacetylchitobiosefrom Chitin Using Temperature-sensitive ChitinlyticEnzyme Preparations of Aeromonas sp. GJ-18, WorldJ. Microbiol. Biotechnol., 2006, vol. 22, pp. 135-139.

28. Kurita, K., Controlled Functionalization of thePolysaccharide Chitin, Prog. Polym. Sci., 200 I, vol. 26,pp.I92I-1971.

29. Kurosaki, F, Nobuhiko, T, and Nishi, A, Role ofChitinase and Chitin Oligosaccharides in Lignifica-tions Responses of Cultured Carrot Cells Treated withMycelial Walls, Plant Cell Physiol., 1988, vol. 29,pp.527-531.

30. Ledesma-Osuna, AI., Ramos-Clamont, G., Guzman-Partida, AM., and Vazquez- Moreno, L., Conjugates ofBovine Serum Albumin with Chitin OligosaccharidesPrepared Through the Maillard Reaction, J. Agric.Food. Chem., 2010, vol. 58, pp. 12000-12005.

31. Li, X., Wang, L.X., Wang, X, and Saul, R., The ChitinCatabolic Cascade in the Marine Bacterium Vibriocholerae: Characterization of a Unique Chitin Oli-gosaccharide Deacetylase, Glycobiology, 2007, vol. 17,pp.1377-1387.

32. Newman, D.J., Cragg, G.M., and Snader, K.M., Nat-ural Products as Sources of New Drugs Over the Period1981-2002, J. Nat. Prod., 2003, vol. 66, pp. 1022.

33. Ngo, D.N., Kim, M.M., and Kim, S.K., Chitin Oli-gosaccharides Inhibit Oxidative Stress in Live Cells,Carbohydr. Polym., 2008, vol. 74, pp. 228-234.

34. Ngo, D.N., Kim, M.M., Qian, Z.J., Jung, WK.,Lee, S.H., and Kim, S.K., Free Radical-scavenging

Activities of Low Molecular Weight Chitin Oligosac-charides Lead to Antioxidant Effect in Live Cells,J. FoodBiochem., 2010, vol. 34,pp. 161-177.

35. Ngo, D.N., Lee, S.H., Kim, M.M., and Kim, S.K.,Production of Chitin Oligosaccharides with DifferentMolecular Weights and their Antioxidant Effect inRAW 264.7 cells, J. Funct. Foods, 2009, vol. I, pp. 188-198.

36. Ngo, D.N., Biological Activities of Chitin Oligosac-charides and their Derivatives, Ph.D thesis, Depart-ment of Chemistry, Pukyong National University,Busan, South Korea, 2008.

37. Osborn, H. and Khan, T, Oligosaccharides: Their Syn-thesis and Biological Roles, Oxford: Oxford Univ. Press,2000.

38. Park, J.K., Keyhani, N.O., and Roseman, S., ChitinCatabolic Cascade in the Marine Bacterium Vibriofur-nissii: Identification, Molecular Cloning and Charac-terization of a N, N' Diacetylchitobiose PhosphOlY-lase, J. BioI. Chem., 2000, vol. 275, pp. 33077-33083.

39. Park, J.K., Wang, L.X, and Roseman, S., Isolation ofaGlucosamine-specific Kinase, a Unique Enzyme ofVibrio cholera, J. Bioi. Chem., 2002, vol. 277,pp.15573-15578.

40. Pengcheng, L.I., Ronge, X, Song, L., andHuahua, YD., A Chitin Oligosaccharide Compoundand its Preparation Process, Shenyang Patent andTrademark Agency Academia Sinica, 2005.

41. Samain E., Chazalet V, and Geremia, KA., Produc-tion of O-acetylated and Sulphated Chitooligosaccha-rides by Recombinant Escherichia coli Strains Har-bouring Different Combinations of nod Genes, J. Bio-technol., 1999, vol. 72, pp. 33-47.

42. Sashiwa, H., Fujishima, S., Yamano, N., Kawasaki, N.,Nakayama, A., Einosuke, M., et aI., Enzymatic Pro-duction ofN-acetyl-D-glucosamine from Chitin.

Degradation Study ofN -acetylchitooligosaccharide andthe Effect of Mixing of Crude Enzymes, Carbohydr.Polym., 2003, vol. 51, pp. 391-395.

43. Scheel, O. and Thiem, J., Cleavage of Chitin by Meansof Aqueous Hydrochloric Acid and Isolation of Chito-Oligosaccharides, Chitin Handbook; Musszarelli,KA.A., Peter, M.G., Eds.; European Chitin Society,Atec Edizioni: Grottammare, Italy, 1997, pp. 167-168.

44. Schlaman, H.KM., Gise,1 AA, Quaedvlieg, E.M.,Bloemberg, G.V, Lugtenberg, B.J.J., Kijne J.W, et aI.,Chitin Oligosaccharides Can Induce Cortical CellDivision in Roots of Vicia sativa when Delivered byBallistic Microtargeting, Development, 1997, vol. 124,pp.4887-4895.

45. Seferian, PG. and Martinez, M.L., Immune Stimulat-ing Activity of Two New Chitosan Containing AdjuvantFormulations, Vaccine, 2000, vol. 19, pp. 661-668.

46. Semino, C.E. and Allende, M.L., Chitin Oligosaccha-rides as Candidate Patterning Agents in ZebrafishEmbryogenesis, Tnternational Journal of DevelopmentalBiology, 2000, vol. 44, pp. 183-193.

47. Shibuya, N. and Minami, E., Oligosaccharide Signal-ling for Defence Responses in Plant, Physiol. Mol. PlantPathol., 2001, vol. 59, pp. 223-233.

48. Shimura, K., Okada, A, Okada, K., Jikumaru, Y,Ko, K.W, Toyomasu, T., et aI., Identification of Bio-

synthetic Gene Cluster in Rice for Momilactones,J. BioI. Chem., 2007, vol. 282, pp. 34013-34018.

49. Skropeta, D. Deep-sea Natural Products, Nat. Prod.Rep., 2008, vol. 25, pp. 989-1216.

50. Songsiriritthigul, c., Pantoom, S., Aguda, AH., Rob-inson, R.C., and Suginta, W, Crystal Structures ofVibrio harveyi Chitinase a CompJexed with Chitooli-gosaccharides: Implication for the Catalytic Mecha-nism, J. Struct. BioI., 2008, vol. 162, pp. 491-499.

51. Tanaka, T., Fukui, T., Atomi, H., and Imanaka, T.,Characterization of an exo- ~- D-glucosaminidaseInvolved in a Novel Chitinolytic Pathway from theHyperthermophilic Archaeon Thermococcuskodakaraensis KODl, J. Bacteriol., 2003, vol. 185,pp.5175-5181.

52. Tsigos, I., Martinou, A, Kafetzopoulos, D., and Bou-riotis V, Chitin Deacetylases: ~ew, Versatile Tools inBiotechnology, Trends Biotechnol., 2000, vol. 18.pp.305-312.

53. Tucker, M., Waley S.R., and Sharp J.S., Consumer Per-ception of Food Related Risks, Int. J. FoodSci. Tech-nol., 2006, vol. 41, pp. 135.

54. Varum, K.M., Ottoy, M.H., and Smidsrod, 0., AcidHydrolysis of Chitosans, Carbohydr. Polym., 2001,vol. 46, pp. 89-98.

55. Vongchan, P, Sajomasang, W, Kasinrerk, W,Subyen, D., and Kongta-WeJert, P. AnticoagulantActivities of the Chitosan Polysulfate Synthesized fromMarine Crab Shell by Semi-Heterogeneous Condi-tions, 2003, vol. 29, pp. 115-120.

56. Wang, c.Y and Hsieh, YZ., Analysis of Chitin Oli-gosaccharides by Capillary Electrophoresis with Laser-induced Fluorescence, 1. Ch rom atogr. A., 2002,vol. 979. pp. 431-438.

57. Wang, S.L. and Chang, WT., Purification and Charac-terization of Two Bifunctional Chitinases/LysozymesExtracellulariy Produced by Pseudomonas aeruginosaK-187 in a Shrimp and Crab Shell Powder Medium,Appl. Environ. Microbiol., 1997, vol. 63, pp. 380-386.

58. Wang, S.L., Lin, H.T., Liang, T.W, Chen, YJ.,Yen, YH., and Guo S.P, Reclamation of ChitinousMaterials by Bromelain for the Preparation of Antitu-mor and Antifungal Materials, Bioresour. Technol.,2008, vol. 99, pp. 4386-4393.

59. Wang, S.L., Lin, T.Y, Yen, YH., Liao, H.E, andChen, YJ., Bioconversion of Shellfish Chitin Wastesfor the Production of Bacillus subtilis W-1l8 Chitinase,Carbohydr. Res., 2006, vol. 341, pp. 2507-2515.

60. Yamaguchi, T., Minami, E., Ueki, J., and Shibuya, N.,Elicitor-induced Activation ofPhospholipases Plays anImportant Role for the Induction of DefenseResponses in Suspension-cultured Rice Cells, PlantCell Physiol., 2005, vol. 46, pp. 579-587.

61. Yang, c., Rodionov, D.A, Li, x., Laikova, O.N., Gel-fand, M.S., Zagnitko, O.P, et aI., ComparativeGenomics and Experimental Characterization of N-acetylglucosamine Utilization Pathway of Shewanellaoneidensis, J. Bioi. Chem., 2006, vol. 281, pp. 29872-29885.