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Title:
CHITIN-CHITOSAN BLOC CO-POLYMERS
Document Type and Number:
WIPO Patent Application WO/2012/080772
Kind Code:
A1
Abstract:
The invention relates to a chitosan presenting a degree of N-acetylation ranging from 5% to 45%, wherein its X ray powder diffractogram shows at least one characteristic peak of a crystalline form of chitin.

Inventors:
DAVID, Laurent (22 Avenue Cabias, Lyon, F-69004, FR)
SHIRAI MATSUMOTO, Keiko (Auriga NO. 69, CoI. Prado ChurubuscoDel. Coyoaca, CP. MEXICO Distrito Federal, 04230, MX)
TROMBOTTO, Stéphane (7 rue Plein Soleil, Diemoz, Diemoz, F-38790, FR)
ARACELY PACHECO LOPEZ, Neith (CaIz. Ermita Iztapalapa, N°1150. CoI. Barrio san LucasDel. Iztapalap, MEXICO Distrito Federal, MX)
Application Number:
IB2010/003475
Publication Date:
June 21, 2012
Filing Date:
December 17, 2010
Export Citation:
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Assignee:
UNIVERSITE CLAUDE BERNARD LYON I (43 Boulevard du 11 Novembre 1918, Villeurbanne Cedex, F-69622, FR)
UNIVERSIDAD AUTONOMA METROPOLITANA (Prol. Canal de Miramontes3855, Colonia Exhacienda de San Juan de DiosDelegación Tlalpa, México D.F., 14387, MX)
INSTITUT NATIONAL DES SCIENCES APPLIQUEES DE LYON (20 avenue Albert Einstein, Villeurbanne, Villeurbanne, F-69621, FR)
UNIVERSITE JEAN MONNET (34 rue Francis Baulier, Saint-Etienne Cedex 2, Saint-Etienne Cedex 2, F-42023, FR)
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (3 rue Michel-Ange, Paris Cedex 16, Paris Cedex 16, F-75794, FR)
DAVID, Laurent (22 Avenue Cabias, Lyon, F-69004, FR)
SHIRAI MATSUMOTO, Keiko (Auriga NO. 69, CoI. Prado ChurubuscoDel. Coyoaca, CP. MEXICO Distrito Federal, 04230, MX)
TROMBOTTO, Stéphane (7 rue Plein Soleil, Diemoz, Diemoz, F-38790, FR)
ARACELY PACHECO LOPEZ, Neith (CaIz. Ermita Iztapalapa, N°1150. CoI. Barrio san LucasDel. Iztapalap, MEXICO Distrito Federal, MX)
International Classes:
C08B37/00
Domestic Patent References:
WO2001041820A12001-06-14
WO2005019272A12005-03-03
WO2007048974A22007-05-03
WO2005019272A12005-03-03
Foreign References:
FR2892419A12007-04-27
MXPA00011722A2002-05-31
Other References:
BEANEY P ET AL: "Comparison of chitins by chemical and bioprocessing methods", JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, BLACKWELL SCIENTIFIC PUBLICATIONS. OXFORD, GB, vol. 80, no. 2, 12 October 2004 (2004-10-12), pages 145 - 150, XP003010478, ISSN: 0268-2575, DOI: 10.1002/JCTB.1164
ZHANG Y ET AL: "Determination of the degree of deacetylation of chitin and chitosan by X-ray powder diffraction", CARBOHYDRATE RESEARCH, PERGAMON, GB, vol. 340, no. 11, 15 August 2005 (2005-08-15), pages 1914 - 1917, XP004976363, ISSN: 0008-6215, DOI: 10.1016/J.CARRES.2005.05.005
PHUVASATE S ET AL: "Comparison of lactic acid bacteria fermentation with acid treatments for chitosan production from shrimp waste", JOURNAL OF AQUATIC FOOD PRODUCT TECHNOLOGY JULY 2010 TAYLOR AND FRANCIS LTD. USA, vol. 19, no. 3-4, July 2010 (2010-07-01), pages 170 - 179, XP002656950, DOI: DOI:10.1080/10498850.2010.504324
OGAWA KOZO ET AL: "Crystallinity of partially N-acetylated chitosans", BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, vol. 57, no. 9, 1993, pages 1466 - 1469, XP002656951, ISSN: 0916-8451
CIRA LUIS A ET AL: "Pilot scale lactic acid fermentation of shrimp wastes for chitin recovery", PROCESS BIOCHEMISTRY, vol. 37, no. 12, July 2002 (2002-07-01), pages 1359 - 1366, XP002656952, ISSN: 1359-5113
LYDIA ADOUR ET AL: "Combined use of waste materials-recovery of chitin from shrimp shells by lactic acid fermentation supplemented with date juice waste or glucose", JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, vol. 83, no. 12, 1 December 2008 (2008-12-01), pages 1664 - 1669, XP055004982, ISSN: 0268-2575, DOI: 10.1002/jctb.1980
NEITH PACHECO ET AL: "Effect of temperature on chitin and astaxanthin recoveries from shrimp waste using lactic acid bacteria", BIORESOURCE TECHNOLOGY, vol. 100, no. 11, 1 June 2009 (2009-06-01), pages 2849 - 2854, XP055004984, ISSN: 0960-8524, DOI: 10.1016/j.biortech.2009.01.019
RAABE, D., SACHS, C., ROMANO, P, ACTA MATERIALIA, vol. 53, 2005, pages 4281 - 4292
MINKE R., BLACKWELL, J.: "Structure of Alpha-Chitin", JOURNAL OF MOLECULAR BIOLOGY, vol. 120, no. 2, 1978, pages 167 - 181, XP024013722, DOI: doi:10.1016/0022-2836(78)90063-3
ALINE PERCOT, A., VITON, C., DOMARD A.: "Optimization of Chitin Extraction from Shrimp Shells", BIOMACROMOLECULES, vol. 4, no. 1, 2003, pages 12 - 18, XP055221591, DOI: doi:10.1080/14786419.2015.1026341
CAMPANA-FILHO S. P., SIGNINI ROBERTA, BARRETO CARDOSO M., INTERNATIONAL JOURNAL OF POLYMERIC MATERIALS, vol. 51, no. 8, 2002, pages 94,695 - 700
HIRAI, A., ODANI, H., NAKAJIMA, A.: "Determination of degree of deacetylation of chitosan by 1H NMR spectroscopy", POLYMER BULLETIN, vol. 26, 1991, pages 87 - 94, XP026851334
A. MONTEMBAULT, C. VITON, A. DOMARD: "Physico-chemical studies of the gelation of chitosan in a hydroalcoholic medium", BIOMATERIALS, vol. 26, no. 8, 2005, pages 933 - 943, XP027767806
LUIS A. CIRA, SERGIO HUERTA, GEORGE M. HALL, KEIKO SHIRAI: "Pilot scale lactic acid fermentation of shrimp wastes for chitin recovery", PROCESS BIOCHEMISTRY, vol. 37, 2002, pages 1359 - 1366, XP002656952
KEIKO SHIRAI, ISABEL GUERRERO, SERGIO HUERTA, GERARDO SAUCEDO, ALBERTO CASTILLO, R. OBDULIA GONZALEZ, GEORGE M. HALL: "Effect of initial glucose concentration and inoculation level of lactic acid bacteria in shrimp waste ensilation", ENZYME AND MICROBIAL TECHNOLOGY, vol. 28, 2001, pages 446 - 452
ZAINOHA ZAKARIA, GEORGE M. HALL, GILBERT SHAMA: "Lactic acid fermentation of scampi waste in a rotating horizontal bioreactor for chitin recovery", PROCESS BIOCHEMISTRY, vol. 33, 1998, pages 1 - 6
ALIYA ZAFER: "Dissertation", July 2001, UNIVERSITY OF KARACHI, article "Computationnal studies on the structure of beta- chitin and other polysaccharides"
B. FOCHER, P.L. BELTRAME, A. NAGGI, G. TORRI: "Alkaline N-deacetylation of chitin enhanced by flash treatments. Reaction kinetics and structure modifications", CARBOHYDRATE POLYMERS, vol. 12, 1990, pages 405 - 418, XP024146806, DOI: doi:10.1016/0144-8617(90)90090-F
E. L. MOGILEVSKAYA, T. A. AKOPOVA, A. N. ZELENETSKII, A. N. OZERIN: "The Crystal Structure of Chitin and Chitosan", POLYMER SCIENCE, SER. A, vol. 48, no. 2, 2006, pages 116 - 123, XP019317014
OSORIO-MADRAZO A, DAVID L, TROMBOTTO S, LUCAS JM, PENICHE-COVAS C, DOMARD A: "Kinetics study of the solid-state acid hydrolysis of chitosan: evolution of the crystallinity and macromolecular structure", BIOMACROMOLECULES, vol. 11, no. 5, 10 May 2010 (2010-05-10), pages 1376 - 86
BELAMIE, E., DOMARD, A., GIRAUD-GILLE M: "Study of the Solid-State Hydrolysis of Chitosan in Presence of HCI", JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY, vol. 35, 1997, pages 3181 - 3191, XP002469134, DOI: doi:10.1002/(SICI)1099-0518(19971115)35:15<3181::AID-POLA11>3.0.CO;2-7
OSORIO-MADRAZO, LAURENT DAVID, ST6PHANE TROMBOTTO, JEAN-MICHEL LUCAS, CARLOS PENICHE-COVAS, ALAIN DOMARD, BIOMACROMOLECULES, vol. 11, no. 5, 2010, pages 1376 - 1386
Attorney, Agent or Firm:
SARLIN, Laure (Cabinet BEAU DE LOMENIE, 51 Avenue Jean JauresBP 7073, Lyon Cedex 07, F-69301, FR)
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Claims:
CLAIMS

1 - Chitosan presenting a degree of N-acetylation ranging from 5% to 45%, wherein its X ray powder diffractogram shows at least one characteristic peak of a crystalline form of chitin.

2 - Chitosan according to claim 1 wherein its DA ranges from 10 to 40%, preferentially from 20 to 40%.

3 - Chitosan according to claim 1 or 2 wherein its X ray powder diffractogram shows at least one characteristic peak of the a crystalline form of chitin.

4 - Chitosan according to anyone of the previous claims wherein its X ray powder diffractogram shows the characteristic peak (020) of the a crystalline form of chitin, expressed as interplanar distance at approximately 9.30 A.

5 - Chitosan according to anyone of the previous claims wherein its X ray powder diffractogram shows the characteristic peak (110) of the a crystalline form of chitin, expressed as interplanar distance at approximately 4.60 A.

6 - Chitosan according to anyone of the previous claims wherein its X ray powder diffractogram shows the characteristic peak (013) of the a crystalline form of chitin, expressed as interplanar distance at approximately 3.39 A.

7 - Chitosan according to anyone of the previous claims wherein it is amphiphilic.

8 - Chitosan according to anyone of the previous claims wherein it corresponds to a block copolymer formed of D-glucosamine (GlcN) homopolymeric units and N-acetyl-D-glucosamine (GlcNAc) homopolymeric units, in which each N-acetyl-D-glucosamine (GlcNAc) homopolymeric unit includes at least 3 monomeric units and preferably from 7 to 15 monomeric units.

9 - Chitosan according to anyone of the previous claims wherein its weight average macromolecular weight (/%) ranges from 450,000 g/mol to 200,000 g/mol.

10 - Chitosan according to anyone of the previous claims wherein it forms a colloidal solution in an acidic aqueous solution. 11 - Chitosan according to anyone of the previous claims wherein it is in the form of nanoparticules, colloidal solutions, physical hydrogels or solid forms like films or fibers.

12 - Preparation process of a chitosan according to anyone of claims 1 to 11 comprising the following steps:

a) obtaining of a Biochitin with a crystalline phase by treatment of a naturally occurring chitin source, in a medium containing lactic acid bacteria and a fermentable carbon source,

b) deacetylation of the recovered Biochitin in the solid state to remove the acetyl group from N-acetyl-D-glucosamine (GIcNAc) units and to form an amine group, yielding D-glucosamine (GIcN) units.

13 * Preparation process according to claim 12 wherein the naturally occurring chitin source is the exoskeletons of crustaceans.

14 - Preparation process according to claim 12 or 13 wherein the treatment of step a) is performed with sucrose.

15 - Preparation process according to anyone of claims 12 to 14 wherein at the end of the step a), a mild acid treatment is performed for elimination of the remaining minerals.

16 - Preparation process according to claim 15 wherein the mild acid treatment is performed with HCI, H-COOH, CH3COOH, lactic acid, citric acid, H3PO4, HNO3 and H2SO4, preferably at a concentration ranging from 0.2 to 0.6 N, for instance at a temperature between 10 and 40°C, and preferentially at room temperature.

17 - Preparation process according to anyone of claims 12 to 16 wherein at the end of step a), the obtained Biochitin includes a highly crystalline chitin, in comparison to available commercial chitin or chitin obtainable by chemical route, and an amorphous phase.

18 - Preparation process according to anyone of claims 12 to 17 wherein at the end of step a), the obtained Biochitin contains crystals of chitin with a largest dimension higher than 6 nm, and preferably higher than 10 nm. 19 - Preparation process according to anyone of claims to 18 wherein the deacetylation of the recovered chitin is performed with a concentrated aqueous solution of NaOH.

20 - Chitosan according to anyone of claims 1 to 11 obtainable by the process defined in anyone of claims 12 to 19.

Description:
CHITIN-CHITOSAN BLOC CO-POLYMERS

The invention relates to the field of biopolymers, more particularly to alternated 'bloc'-copolymers of chitin and chitosan family.

Chitin is the most widespread natural polymer with cellulose. Chitin is a linear co-polysaccharide constituted by D-glucosamine and N-acetyl D- glucosamine repeat units, linked by a β-(1->4) glycosidic bond. The degree of N-acetylation or DA, is the molar fraction of the D-glucosamine residues. The DA in chitin is frequently higher than 80%, resulting in an insoluble polymer in aqueous media.

Industrially, chitin is mainly extracted from the exoskeleton (shell) of arthropods (lobster, crab and shrimp). Chitin can also be extracted from endoskeleton of cephalopods such as squids. Chitin is a fibrillar semicrystalline polymer, organized at several length scales [The crustacean exoesqueleton as an example of a structurally and mechanically graded biological nanocomposite material. Raabe, D., Sachs, C. and Romano, P, 2005, Acta Materialia, 53, pp 4281-4292.]. It is thus composed of crystalline and amorphous phases. Crystalline chitin can be found in two allomorphs, and β-chitin (specifically found in the endosqueleton of cephalopods), but a- chitin is the main (crystalline) form found in biomass. Such a crystalline form is well described at atomic level [Structure of Alpha-Chitin Minke R. and Blackwell, J., 1978, Journal of Molecular Biology, 120 (2) pp 167-181 ] with a dense network of hydrogen bonds. This induces a rigidity and low molecular mobility of the chains and a weak accessibility to the deacetylation reactants. a-chitin is not soluble in aqueous acidic media, and swells only weakly in neutral aqueous media. The extraction of chitin from biomass (e.g. shrimp shells) can be operated from a chemical process involving (i) a demineralization process at ambient temperature in HCI 1M or 0.25M for about 30 min and (ii) a deproteinization treatment in aqueous solution of sodium hydroxide NaOH 1M at moderate temperatures (room temperature to 70°C during 24 to 48 hours). [Optimization of Chitin [Extraction from Shrimp Shells Aline Percot, A. Viton, C. and Domard A. Biomacromolecules, 2003, 4 (1), pp 12-18 DOI: 10.1021/bm025602k]. Chitosan is a de-acetylated derivative of chitin, and is therefore also a co-polysaccharide with D-glucosamine and N-acetyl D-glucosamine repeat units linked by a β-(1->4) glycosidic bond. Chitosan usually contains a proportion of N-acetyl-D-glucosamine monomer units lower than 60%. Chitosan is notably obtained by total or partial deacetylation of chitin. Chitosan is, by definition, soluble in acidic media (except sulfuric and phosphoric a ds). This feature is mainly resulting from the lower value of the DA, but is also impacted by the intra-macromolecular distribution of acetylated and deacetylated residues. As an example, chitosans with pure statistical Bernoulli distribution of the residues stay soluble up to a critical DA close to 60%. Such statistical structure is obtained by reacetylation of a low- DA chitosan in solution. After chemical deacetylation of chitin in the solid state, the DA range of soluble chitosan in acidic aqueous solution is typically 0-40%. Chitosan has a higher number of reactive free amine groups than chitin, and also exists in two allomorphs: hydrated (tendon) allomorph and anhydrous allomorph.

Classical chemical treatments are industrially used to derive chitin and chitosan from food industry wastes. They usually produce high amount of pollutants, without any possibility to recover and valorize the protein and pigments initially present in the waste. Alternative biotechnological routes based on the effect of lactic acid bacteria on arthropods shells [see Mexican patent No. MXPA0001 1722, 28/1 1/2000. Instituto Mexicano de Propiedad industrial (IMPI) 247295. Gasification lnt.CI.8:A23J1/04; C08B37/08; C22B26/20, Autors: Keiko Shirai, Maribel Plascencia, Luis, Cira y Sergio Huerta.] now offer the possibility to recover valorizable products from these wastes and of course extract chitin after an eco-friendly process.

Chitosan is used or offers potential in a variety of applications from water treatment, cosmetics, nutraceutics, food conservation, agriculture, tissue engineering, biotechnologies... This polymer is biodegradable, bioresorbable, biocompatible and non toxic, it is widely used in pharmacy as an excipient, drug-delivery applications, and also as an active principle (fungistatic, bacteriostatic, wound healing formulation and devices). The interest of chitosan for such applications is a function of the molecular mass and the DA or the amount of reactive amine groups. Such macromolecular features will in turn impact the solubility in acidic media, the reactivity for chemical modification, but also the biological properties such as the biodegradation rate, the immune activity, the processing ability (possibility to form physical hydrogels, high performance fibers, and highly viscous solutions) and the mechanical and rheological properties of the chitosan materials in various physical forms.

In a majority of applications, low DA chitosan (with DA < 5%) with high weight average macromolecular weight (/ ) are needed. This explains why the strategies to obtain chitosan from chitin by a purely chemical treatment [for instance, see WO2005/019272; and also CAMPANA-FILHO S. P.; SIGNINI Roberta; BARRETO CARDOSO M.; International journal of polymeric materials ISSN 0091-4037; 2002, vol. 51, no 8 (94 p.) pp. 695-700] were optimized to (i) preserve the structure of macromolecular chains by avoiding depolymerization, in particular in presence of oxygen and (ii) favor the diffusion and access of deacetylation reagents within the crystalline phase to obtain a fully deacetylated chitosan with high molecular weight. In this context, the interest of β-chitin was clearly shown since this crystalline form is not stabilized by a dense hydrogen bond network and offers an easier access to the deacetylation reagents. In the optimized method presented in WO2005/019272, the aim of the treatment is to disrupt the crystalline domains of a or β-chitin in order to favor the accessibility to NaOH and water for the deacetylation hydrolysis.

The patent application WO2007/048974 describes a method for preparing novel D-glucosamine and N-acetyl-D-glucosamine hetero- oligomers, enabling the size, the degree of acetylation and the architecture of the resulting oligomers to be controlled. Nevertheless, this method requires the synthesis of specific monomers, their coupling in order to form dimers and so on ... So, this method is complicated in order to obtain hetero- oligomers of DP (degree of polymerisation) higher than 6 and polymers of higher molecular weight. In this context, the invention proposes a new chitosan presenting a degree of N-acetylation (DA) ranging from 5% to 45%, for instance from 10 to 45%, preferably from 10 to 40%, and preferentially from 20 to 40%, wherein its X ray powder diffractogram shows at least one characteristic peak of a crystalline form of chitin.

The chitosan according to the invention presents a bloc structure defined by the alternation of highly acetylated blocs (with mainly N-acetyl-D- glucosamine rich blocs) and more deacetylated blocs (sequences with mainly D-glucosamine residues) and, as a result, is an amphiphilic polymer. Due to its amphiphilic property, the chitosan according to the invention forms a colloidal solution in an acidic aqueous solution.

An other aspect of the invention is related to the different materials and coatings obtained from such a chitosan, namely the nanoparticules the colloidal solutions, the physical hydrogels and the solid forms (films and fibers).

The invention also concerns a preparation process of such a chitosan comprising the following steps:

a) obtaining of a Biochitin with a crystalline phase by treatment of a naturally occurring chitin source, in a medium containing lactic acid bacteria and a fermentable carbon source,

b) deacetylation of the recovered Biochitin in the solid state to remove the acetyl group from N-acetyl-D-glucosamine (GlcNAc) units and to form an amine group, yielding D-glucosamine (GlcN) units.

The chitosan obtainable by such a process detailed hereafter is an integral part of the invention.

The following specification in reference to the enclosed Figures permits to understand the invention.

Figure 1 displays the diffraction diagram (Cu Ka X-ray radiation at room temperature) of the shrimp shell before and after different treatments at different times. Figure 2 displays the evolution of the diffraction diagrams for initial Biochitin and samples obtained after an FTP deacetylation step in the solid state for various reaction times.

Figure 3 studies the second Freeze-Pump-Thaw deacetylation cycle on a sample deacetylated 20 minutes.

Figure 4 displays the diffraction diagram of a commercial chitosan sample (from Mahtani Chitosan).

In preliminary, certain definitions of terms used in the specification will be given.

Degree of N-acetylation (DA) defined above is the mean DA measured by H NMR spectroscopy in liquid media. A specific method for DA determination is that reported by Hirai et al., 1991, (Hirai, A., Odani, H. and Nakajima, A. (1991). Determination of degree of deacetylation of chitosan by 1H NMR spectroscopy. Polymer Bulletin. 26: 87-94).

The weight average macromolecular weight {! ) can be determined by the technique described in «Physico-chemical studies of the gelation of chitosan in a hydroalcoholic medium » A. MONTEMBAULT, C. VITON, A. DOMARD Biomaterials, 26(8), 933-943, 2005.

Within the scope of the invention, a chitin obtained by a lactic fermentation bacteria (LFB) method is named Biochitin.

According to the invention, the new alternated 'bloc' chitosans are prepared from the deacetylation of a 'Biochitin' extracted from biomass with a biotechnological process, namely lactic acid fermentation (LAF) process. For more details about such a process, it is possible to refer to Mexican patent No. MXPA000 722 previously cited. Advantageously, such a process includes the treatment of a naturally occurring chitin source, in a medium containing lactic acid bacteria (LAB) and a fermentable carbon source to produce lactic acid for mineral solubilization and the activation of endogenous and produced enzymes for chitin deproteinization.

In a particular embodiment, the naturally occurring chitin source is exoskeletons of crustaceans that leads to the a crystalline form of chitin. According to a particular embodiment that can be combined with the previous one, the treatment of step a) is performed with sucrose (i.e. saccharose), but glucose or lactose could be employed, as well as spray dried cheese whey ["Pilot scale lactic acid fermentation of shrimp wastes for chitin recovery" Luis A. Cira, Sergio Huerta, George M. Hall, Keiko Shirai, Process Biochemistry 37 (2002) 1359-1366]. As examples of lactic acid bacteria, Lactobacillus plantarum Lactobacillus casei, Lactobacillus pentousus, Pediococcus acidolactici ["Effect of initial glucose concentration and inoculation level of lactic acid bacteria in shrimp waste ensilation" Keiko Shirai, Isabel Guerrero, Sergio Huerta, Gerardo Saucedo, Alberto Castillo, R. Obdulia Gonzalez, George M. Hall, Enzyme and Microbial technology 28 (2001): 446-452] L. paracasei ["Lactic acid fermentation of scampi waste in a rotating horizontal bioreactor for chitin recovery" Zainoha Zakaria, George M. Hall, Gilbert Shama, Process Biochemistry 33 (1998): 1-6] can be cited. For instance, exoesqueletons of crustaceans are mixed with sucrose (10 to 20 wt wt %) and Lactobacillus plantarum (5-10 vol/wt%). Then, the mixture can be incubated in a column reactor at temperatures, for instance, ranging from 25 to 40 °C, and preferably during a time superior to 90h, for instance ranging from 96 to 144h.

The step a) can be followed by a mild acid treatment in order to remove the residual minerals. The necessity of such a treatment is function of the duration of the LAF step. This acid treatment is qualified as mild as it does not destroy the crystals of the chitin obtained by the LAF process, but improves its chitin crystallinity fraction and increases the size and degree of perfection of the chitin crystals as can be deduced of the characteristic diffraction peaks of the chitin (see equation 4.1 and 4.2 below). This mild treatment can be performed with an acid, for instance chosen among HCI, H- COOH, CH3COOH, lactic acid, citric acid, H 3 P0 4 , HN0 3 and H 2 S0 4 , preferably at concentrations from 0.2 to 0.6 N and for instance at a temperature ranging from 10 to 40°C, and preferentially at room temperature (20°C). Subsequently, to this mild acid treatment, a mild treatment with alkali (for instance NaOH or KOH) preferentially at a concentration range of 0.2 to 0.6 N can be applied to pursue the removal of the residual proteins. This alkali treatment is optional as the residual proteins can be removed, during the deacetylation step b).

In this invention, the Biochitin extracted by lactic fermentation bacteria (LAB) treatment was found to exhibit a particular well defined crystalline structure. As a result, it was possible to obtain alternated 'bloc' chitosans by deacetylation (for instance, but not preferably according to WO2005/019272) of the 'Biochitin' extracted from biomass with such a biotechnological process.

Figure 1 displays the diffraction diagram (Cu Ka X-ray radiation at room temperature) of the shrimp shell before and after different treatments at different times. Figure 1 presents the X-ray diffraction patterns of the samples obtained after different fermentation times (0; 24; 48; 72; 96; 120 and 144h) of a Biochitin (BIO-C) obtained at the end of the fermentation process after 144 h, with an additional mild HCI treatment (0.04M HCI), and of chemical (CH-C) and commercial (CO-C) chitins. The diffraction diagram exhibits the 6 characteristic reflections, identified as (020), (021), (110), (120), (130) and (013), corresponding to the crystalline structure of a-chitin according to the model proposed by Minke R. and Blackwell, J., 1978, Structure of Alpha-Chitin. Journal of Molecular Biology. 120(2), pp 167-181.). Unexpectedly, after removing of the mineral phase by dissolution with lactic acid, and deproteinization by the proteases produced by lactic fermentation bacteria, it can be deduced that the crystallinity index (area contribution of the crystalline peaks in the diffractogram), the degree of perfection and the size of the crystallites (as deduced from a decrease of the width of the diffraction peaks) is increasing with fermentation time, as shown in Table 1 hereafter. In the cases where the chitin phase corresponds to the β-chitin, for the characteristic reflection picks of the X-ray diffractogram, it is possible to refer to Aliya Zafer, "Computationnal studies on the structure of beta- chitin and other polysaccharides", Dissertation presented for the degree of Doctor of Philosophy in the University of Karachi, Dep. Biochemistry, 75270 Pakistan, July 2001; available at http://eprints.hec.gov.pk/1340/l/1045.html.htm.

Table 1. Crystalline index (ICR) and apparent crystallite size (D ap ) in - chitin obtained from shrimp waste {Litopenaeus vanameii) at different fermentation times and BIO, chemical and commercial chitins. ICR was calculated from normalized diffractograms according to the method reported by Focher et al., (1990).[ Focher, B., Beltrame, P. L, Naggi, A. and Torn, G. (1990). Alkaline N-Deacetylation of Chitin Enhanced by Flash Treatments. Reaction Kinetics and Structure Modifications. Carbohydrate Polymers. 12: 405-418.] The apparent crystallite size is deduced from the Scherrer equation (D=0.9A/(cos(e) Δ2Θ)) where λ . is the incident wavelength (~1.54A); Δ2Θ is the FWHM of the diffraction peak and 2Θ is the diffraction.

Sample ICR (no) Dap (020) Dap (110)

Raw 42.6 62.3 ND t24 60.8 62.31 49.4

t48 59.0 63.54 51.23

t72 65.1 63.86 58.39

t96 77.3 64.71 62.56

tl20 77.1 64.93 63.6 tl44 78.4 65.02 62.82

BIO-C 86.4 67.99 69.61

CH-C 78.5 64.21 63.19

CO-C 80.2 66.19 67.23

ND. Not determined

The crystalline microstructure of the obtained Biochitin can be compared with chitin obtained by a full chemical treatments (i.e. according to WO2005/019272 procedure see CH-C in Figure 1) and commercial chitin (alfa-chitin Mahtani P.V.T) see CO-C in Figure 1). The crystalline ratio, crystalline perfection and crystallite size are higher for Biochitin. As a result, Biochitin can be defined as a highly crystalline substrate with large nanocrystalline blocs, in comparison with chitins obtained after a full chemical extraction treatment. According to the invention, the inventors show that if such a Biochitin obtained at the end of step a), which includes a highly crystalline chitin in comparison to available commercial chitin or chitin obtainable by chemical route and an amorphous phase, is deacetylated in the solid state, then the N-acetyl D-glucosamine residues located inside the large crystals exhibit low accessibility to deacetylation reagents and are not deacetylated or undergo a limited deacetylation process, while the amorphous phase undergoes a pronounced deacetylation process. For instance, the Biochitin which will be deacetylated presents a Crystalline Index (ICR) higher than 80%, preferentially higher than 85%.

Moreover, at the end of step a), the obtained Biochitin corresponding to the Biochitin which will be deacetylated contains preferably crystals of chitin with a largest dimension higher than 6 nm, and preferentially rather than 10 nm. According to the invention, the largest dimension of the crystals corresponds to the large value os D ap deduced from the breadth of the diffraction peaks (see equation 4.2 below) the X-ray diffractogram.

The Crystallinity index is determined according to the method reported by B. Focher, P.L. Beltrame, A. Naggi and G. Torri , Alkaline N-deacetylation of chitin enhanced by flash treatments. Reaction kinetics and structure modifications. Carbohydrate Polymers 12 (1990), pp. 405-418, as detailed in the examples.

The step b) of deacetylation can be performed with any method of deacetylation of chitin known by the man skilled in the art. Most of the time, a concentrated aqueous solution of NaOH (30 to 60 % w/w, preferably 40-55 %w/w) is employed. The temperature usually ranges from 50°C to 110°C and the time of treatment from 1.5 to 5 hours. These parameters are dependent on the desired degree of deacetylation. Even the process described in WO 2005/019272 can be used whereas it could have been unfavourable as it generally leads to the fragmentation of the crystals. Nevertheless, the examples hereafter show that, even with this process, a chitosan bloc with at least one characterised peak of a crystalline form of the chitin is obtained. This process of heterogeneous deacetylation includes one or several steps of curing a chitin suspension in an aqueous concentrated solution of sodium hydroxide at a temperature lower or equal to 100°C, for instance during 20 to 30 minutes, this curing being performed in a reactor under reduced pressure and without O2. Each step of curing is preceded by a succession of at least 6 cycles of freezing/defreezing as described in WO2005/019272 that can be consulted for more details. Advantageously, this process comprises only one step of curing. Additionally, the curing can be optionally followed by one or several of these optional steps :- a neutralisation of the hydroxide sodium in the reactive medium until a pH equal to 8.5 ; - a washing step with demineralised water consisting of several washings separated by centrifugation ; - a step of lyophilisation after freezing. Preferably, a deacetylation process in standard conditions, without steps of freezing/defreezing and lyophilisation, will be performed.

Of course, the conditions of the deacetylation process will be chosen in order to obtain the required DA, from 5 to 45%, preferably from 10 to 40%, and preferentially from 20 to 40%.

The difference of accessibility in the crystalline and in the amorphous phase of the chitin induces a difference in the acetylation degree, whatever the deacetylation process used. The resulting chains are constituted by highly deacetylated segments (preferentially located in the amorphous phase and at the surface of crystals during the deacetylation treatment) and weakly deacetylated segments (preferentially located in the bulk of the nanocrystals during the deacetylation treatment), even if the mean DA is lower than 45%, even lower than 40%.

The secondary structure of the chitosan issued from Biochitin is then constituted by:

-highly deacetylated blocs (with mainly D glucosamine residues) corresponding to the chain portions confined in the amorphous phase, and separated by -weakly deacetylated sequences (with mainly N-Acetyl D-glucosamine residues) corresponding to the chain portions confined in the chitin crystalline domains.

So, according to a particular embodiment, its X ray powder diffractogram shows at least one characteristic peak of the a crystalline form of chitin, and for instance the characteristic peak (020) of the a crystalline form of chitin, that can be expressed as interplanar distance at approximately 9.30 A, and, in particular, 9.30 A. ±0.40 A.

According to a particular embodiment which can be combined to the previous one, the X ray powder diffractogram of the chitosan of bloc structure according to the invention shows the characteristic peak (110) of the a crystalline form of chitin, that can be expressed as interplanar distance at approximately 4.60 A, and, in particular, 4.60 A. ±0.40 A.

According to a particular embodiment which can be combined to the previous ones, the X ray powder diffractogram of the chitosan of bloc structure according to the invention shows the characteristic peak (013) of the a crystalline form of chitin, that can be expressed as interplanar distance at approximately 3.39 A, and, in particular, 3.39 A. ±0.40 A.

The Crystalline structures of α-chitin and hydrated "tendon" chitosan are reviewed in 'The Crystal Structure of Chitin and Chitosan", E. L. Mogilevskaya, T. A. Akopova, A. N. Zelenetskii, and A. N. Ozerin, Polymer Science, Ser. A, 2006, Vol. 48, No. 2, pp. 116-123. , ISSN 0965-545X

When the size of the crystals is about 70-80nm (as shown in previous Table 1), then about n=15 to 20 repeat units of a chain are trapped within a crystal, the weakly acetylated blocs will contain about M=(N+l)xn units length where N is the number of re-entrant folds in the crystals. The typical number of re-entrant folds N can be deduced from acid hydrolysis of chitosan in the solid state (see Osorio-Madrazo A, David L, Trombotto S, Lucas JM, Peniche-Covas C, Domard A. Kinetics study of the solid-state acid hydrolysis of chitosan: evolution of the crystallinity and macromolecular structure BiomacromoJecules. 2010 May 10;ll(5):1376-86 and, also Belamie, E.; Domard, A.; Giraud-Gille M; Study of the Solid-State Hydrolysis of Chitosan in Presence of HCI, Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 35, 3181-3191 1997) in the physic-chemical conditions where the acid hydrolysis occurs preferentially in the amorphous phase.

In agreement with such studies, the most probable values for N in the case of chitin/chitosan extracted from shrimp shells are close to 0, 2 and 5 (resulting in segments with about 16, 50 and 90 repeat units in the paper by Cartier 1997 and Osorio-Madrazo 2010, values which are consistent with the value obtained with the formula M=(N+l)xn).

In short, the degree of polymerization of the chitin blocs is controlled by crystallite sizes according to a multimodal distribution with modes at 16, 50 and 90; whereas the chitosan blocs result from chain portions from the amorphous phase and are thus following a distribution of sizes with larger breadth.

According to one embodiment of the invention that can be combined with the previous ones, the obtained chitosan corresponds to a block copolymer formed of D-glucosamine (GIcN) homopolymeric units and N- acetyl-D-glucosamine (GIcNAc) homopolymeric units, in which each N-acetyl- D-glucosamine (GIcNAc) homopolymeric unit includes at least 3 monomeric units and preferably from 7 to 25 monomeric units.

According to one embodiment of the invention that can be combined with the previous ones, the obtained chitosan presents a weight average macromolecular weight (/ ,) ranging from 450,000 g/mol to 200,000 g/mol.

The chitosan according to the invention with alterned bloc GIcN / GIcNAc structures is an amphiphilic polymer. This property can be qualitatively evaluated by the solubilization speed in a diluted acid solution, since chitin (GIcNAc blocs) is more hydrophobic than chitosan (GIcN blocs): chitin is not soluble in acidic aqueous solutions, whereas chitosan is soluble in acidic aqueous solutions thanks to the protonation of amine groups. In particular, spontaneous self-organization of alterned bloc chitosan/chitin macromolecules for example in micelles will result in biocompatible, bioresorbable, bacteriostatic and fungistatic nano-objects, thus with a major advantage in comparison with all synthetic polymers that do not exhibit such combined properties. In an aqueous solution, the core of such micelles will be composed of chitin chain portions, whereas the shell will be composed of chitosan chain portion. This core-shell structure is useful to encapsulate, protect and enhance the biological effect hydrophobic drugs, proteins or active principle in general. Chitosan is a polyamine, and thus the -NH 2 moieties can be used as reactive moieties for the grafting of functional molecules with biological or physico-chemical interest (ex: implied in a specific interaction in order to induce a targeted drug delivery or a specific biological response-adjuvant for vaccines...). The use of alternated bloc chitosan/chitin macromolecular structures can also be envisioned in the field of functional surfaces for lab-on chip and in vitro diagnostics applications. The alterned bloc chitosan/chitin structures can also be used for their interaction with colloids (for steric and electrostatic stabilization purposes) and vesicle. At last, the amphiphilic structure and resulting self-organization into hydrophilic and hydrophobic domains is to be exploited for bulk materials or coatings such as solid dry materials or gels, hydrogels and in particular physical hydrogels, in particular physical hydrogels with hydrophobic nanodomains, containing active principles and exhibiting a controlled release of active species, in particular hydrophobic active principles.

The following examples will illustrate the invention. EXAMPLES

The X-ray diffraction measurements were carried out in a diffractometer (Bruker D8 Advance) with an Incident radiation CuKa and a wave length of λ = 1.5418 A, in the range of 2Θ = 4.5 - 50° with steps of 0.02°. D ap was determined according to the method reported by Focher et al., cited supra (1990) after the mathematical treatment of the peaks corresponding to its deconvolution and application of the Lorentzian function. The ICR of the samples was determined using the intensities of the peaks at (110) lattice around 2Θ = 20° (corresponding to the maximal intensity) and at 2Θ≥ 16° (corresponding to the amorphous diffraction) according to the equation 4.1 while the values of D ap were determined by the equation 4.2.

I x 100(4.1)

A

¾ 11Ο ] = ^0 (4 · 2)

Where K is a constant; λ (Α) is the wave length of the incident radiation; βθ (rad) is the width of the crystalline peak at half height and Θ (rad) is half the Bragg angle corresponding to the crystalline peak.

Preparation of Biochitin

Biotechnologically prepared chitin (Biochitin) is needed for the preparation of chitosans with block sequence structure with chemical deacetylation process. This Biochitin was obtained by the application of Lactic acid fermentation (LAF), see Mexican patent MXPA00011722 previously cited. The exoesqueletons of crustacean were minced to a particle size from 0.5 - 6, mixed with sucrose (10 wt wt %) and Lactobacillus plantarum (5 vol/wt%). The mixture was placed into a packed bed column reactor and incubated at operational temperatures between 25 to 40 °C, during 144h. The product obtained after fermentation was treated with HCI (0.5M) ratio 1:15 (w/v) during lh at room temperature (25°C) then treated with NaOH (0.4M) at ratio 1:15 (w/v), during lh at room temperature (25°C), to eliminate the remaining minerals and proteins.

Pe-acetylation of Biochitin

The chemical deacetylation of the Biochitin was carry out by heterogeneous deacetylation according to the Freeze-Pump-Thaw (FPT) method described by Lamarque et al. (2005), Patent Application WO2005/019272. Biochitins were deacetylated by one cycle of FPT process during different times of reaction at 100°C in a solution of NaOH at 50%. In each case only the water-insoluble fractions (pH 8) of each time were considered for characterization. Samples with values below 50% of deacetylation degree (DD) were re-acetylated according to the method reported by Sorlier et al. (2001). Samples with DD greater than 50% were purified by solubilization in the presence of the amount of acetic acid necessary to achieve the stoichiometric protonation of the -NH 2 sites. The obtained solution was then filtered through a 0.45 micron filter (Millipore) before addition of aqueous ammonia to fully precipitate the polymer. After repeated washings with deionized water followed by centrifugations, until a neutral pH was achieved, the product was dispersed in distilled water and then dried by lyophilization (see Patent Application No. WO2005/19272).

Evidence that the structure of the obtained co-polymer is related from the initial crystallinity of chitin, is given by X-ray diffraction of samples at different steps of the deacetylation process. Figure 2 displays the evolution of the diffraction diagrams for initial Biochitin and samples obtained after an FTP deacetylation step in the solid state for various reaction times (i.e. the conditions were chosen as in WO2005/19272, but other deacetylation conditions would lead to similar results): 20, 40, 60 and 80 min for 1D20B, 1D40B, 1D60B and 1D80B respectively), ID represents one deacetylation step. Their caracteristics are given in Table 2 hereafter.

Table 2. Acetylation degree (DA), Crystalline Index (ICR) and apparent crystallite size (Dap) corresponding to the 2 principal reflexions of a-chitin (020 and 110) after a first FTP heterogeneous deacetylation process.

DA Dap(020) Dap(llO) ICR(IIO)

Biochitin 94 66.2 67.6 85.4

1D10BC* 85 64.9 39.1 80.0

1D20BC 68 60.2 37.9 74.8

1D30BC 63 22.5 23.4 70.1

1D40BC 50 12.3 19.6 43.8

1D60BC 45 11.7 15.1 40.5

1D80BC 42 9.8 13.6 39.9

ND = Not determined.* 1D10B ID first FPT deacetylation, 10, 20, 30, 40, 60, 80 min are reaction times, BC represents biochitin.

The diffraction diagrams are characteristic of α-chitin all through the first deacetylation step, although the value of the DA decreased down to about 40%. This means that the crystals of the obtained semicrystalline materials are made of chitin, and in view of the combined values of the DA and crystalline fraction, the amorphous phase should be completely deacetylated. The copolysaccharide macromolecules thus obtained are thus constituted by the amorphous deacetylated (chitosan) chain portions separated by chitin chain portions inherited from the crystallites.

The second Freeze-Pump-Thaw deacetylation cycle on a sample deacetylated 20 minutes was studied in Figure 3. Figure 3 is the X-Ray diffraction patterns after the second heterogeneous FPT deacetylation at several reaction times, 2D two steps, 10, 20, 30 reaction time in min, from Biochitin deacetylated 20 after a first FTP cycle (1D20BC). The deacetylation procedure led to a decrease in the DA (see Table 3) and to the preservation of the crystalline structure of chitine.

Table 3. Deacetylation degree (DA), Crystalline index (ICR) and apparent crystallite size (Dap) corresponding to the 2 principal reflexions of a-chitin (020) and (110) after the second FTP deacetylation process.

DA D ap (020) Dap (110) IC (IIO)

2D10BC* 52 35.0 35.9 62.8

2D20BC 32 38.6 25.9 55.6

2D30BC 22 19.7 16.1 46.2

* 2D10, 2D second FPT deacetylation, 10, 20, 30 are reaction time in minutes, BC represents biochitin.

At these different deacetylation times, the crystalline structure is still a- chitin like and the chitosan is still organized in blocs of highly deacetylated chain portions separated with chitin chain portions. This still shows an alterned bloc structure with DA as low as 20%.

As a comparison, Figure 4 displays the diffraction diagram of a commercial chitosan sample (from Mahtani Chitosan). This commercial sample was obtained from classical chemical deacetylation procedures of shrimp shell wastes. The degree of acetylation is DA=22% (see table 1 in Osorio-Madrazo et al 2010). Figure 4 presents the diffraction diagram of sample CHI in "Kinetics Study of the Solid-State Acid Hydrolysis of Chitosan: Evolution of the Crystallinity and Macromolecular Structure" Anayancy Osorio-Madrazo, Laurent David, Stephane Trombotto, Jean-Michel Lucas, Carlos Peniche-Covas , Alain Domard, Biomacromolecules, 2010, 11 (5), pp 1376-1386 DOI: 10.1021/bml001685. The diffraction pattern was obtained on B2AM beamline at the ESRF (Grenoble France) in transmission mode at an incident photon energy close to 16 keV.

The diffraction peaks are characteristic of the hydrated tendon chitosan crystal structure with (202) (at q~0.73k l corresponding to an inter-reticular distance d=2n/q~ 8.6A) and (200) (at ^1.42A "1 ) diffraction peaks. In comparison, at this value of DA, the deacetylated sample obtained from Biological chitin (see Figure 3-2D30BC) is exhibiting the crystalline structure of a-chitin.




 
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