US20130004784A1 - Multi-phase bacterially-synthesized-nanocellulose biomaterials and method for producing the same - Google Patents

Multi-phase bacterially-synthesized-nanocellulose biomaterials and method for producing the same Download PDF

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US20130004784A1
US20130004784A1 US13/634,788 US201113634788A US2013004784A1 US 20130004784 A1 US20130004784 A1 US 20130004784A1 US 201113634788 A US201113634788 A US 201113634788A US 2013004784 A1 US2013004784 A1 US 2013004784A1
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phase
bnc
bacterial cellulose
different bacterial
biomaterials
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Nadine Hessler
Barno Sultanova
Dieter Klemm
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Jenacell GmbH
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Assigned to FRIEDRICH-SCHILLER-UNIVERSITAET JENA reassignment FRIEDRICH-SCHILLER-UNIVERSITAET JENA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HESSLER, NADINE, SULTANOVA, BARNO, KLEMM, DIETER
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Assigned to JeNaCell GmbH reassignment JeNaCell GmbH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FRIEDRICH-SCHILLER-UNIVERSITAET JENA
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B15/00—Preparation of other cellulose derivatives or modified cellulose, e.g. complexes
    • C08B15/10—Crosslinking of cellulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/31504—Composite [nonstructural laminate]
    • Y10T428/31971—Of carbohydrate
    • Y10T428/31975—Of cellulosic next to another carbohydrate
    • Y10T428/31978—Cellulosic next to another cellulosic

Definitions

  • the invention relates to multi-phase biomaterials based on bacterially synthesized nanocellulose and a method for producing same.
  • the proposed BNC materials are suitable for a broad range of applications, for example in medicine (wound dressings, great variety of implants), in engineering (membranes, foils, barrier layers) and in food industry (zero-calorie nutrition, packaging) due to highly versatile determinable structures and material properties).
  • This application-designed method for obtaining defined structures and properties that are even new for BNC materials in particular refers to mechanical strengths, elasticity, transparency and water balance, particularly the capability to re-expand appropriately and completely after drying, as well as so-called filter/membrane functions (permeability), scaffold-properties (pore system, surface characteristics, colonization by cells) and bio-compatibility (body compatibility, endothelialization, immigration of body's own cells, permanent integration into the body) without requiring disadvantageous additives or composite formations produced in the synthesis with them.
  • filter/membrane functions permeability
  • scaffold-properties pore system, surface characteristics, colonization by cells
  • bio-compatibility body compatibility, endothelialization, immigration of body's own cells, permanent integration into the body
  • CMC carboxymethyl cellulose
  • MC methyl cellulose
  • Tokura A method for direct harvest of bacterial cellulose filaments during continuous cultivation of acetobacter xylinum . Carbohydrate Polymers (1998), 35/3-4, 233-7; C. H. Haigler, A. R. White, R. M. Brown Jr., K. M. Cooper: Alteration of In Vivo Cellulose Ribbon Assembly by Carboxymethylcellulose and Other Cellulose Derivative, J Cell Biology (1982), 94, 64-9).
  • solids can also be given as additives to the culture medium during the biosynthesis and are integrated in the produced BNC network.
  • Mormino Bungay: Inclusion of solid particles in bacterial cellulose, Applied Microbiology and Biotechnology (2002), 58/6, 756-60) mainly reported about the integration of metals (aluminum) or metal oxide (ferric oxide) particles.
  • Kawano High mechanical strength double-network hydrogel with bacterial cellulose, Advanced Functional Materials (2004), 14/11, 1124-8 and by inorganic substances e.g. calium salts, metals, metal oxides (B. R. Evans, H. O'Neil, M. Hugh, V. P. Malyvanh, I. Lee, J. Woodward: Palladium-bacterial cellulose membranes for fuel cells, Biosensors & Bioelectronics (2003), 18/7, 917-23; B. R. Evans, H. M. O'Neill, E.
  • inorganic substances e.g. calium salts, metals, metal oxides (B. R. Evans, H. O'Neil, M. Hugh, V. P. Malyvanh, I. Lee, J. Woodward: Palladium-bacterial cellulose membranes for fuel cells, Biosensors & Bioelectronics (2003), 18/7, 917-23; B. R. Evans, H. M. O'Neill, E.
  • A. Seto et al. (A. Seto, Y. Saito, M. Matsushige, H. Kobayashi, Y. Sasaki, N. Tonouchi, I. Tsuchida, F. Yoshinaga, K. Ueda, T. Beppu: Effective cellulose production by a coculture of Gluconacetobacter xylinus and Lactobacillus mali , Applied Microbiology and Biotechnology (2006), 73(4), 915-921), C. Choi et al. (KR 2002/067226) and H. Seto et al.
  • JP 10201495 demonstrated that the yield of synthesized cellulose could be optimized by co-cultivating a cellulose-forming bacterial strain ( Acetobacter xylinum (st-60-12)) with a lactobacillus strain ( Lactobacillus mali (st-20)).
  • This effect is mainly due to the metabolites of the lactobacillus strain, such as acetic acid, that support the biosynthesis of cellulose (A. Seto, Y. Saito, M. Matsushige, H. Kobayashi, Y. Sasaki, N. Tonouchi, T. Tsuchida, F. Yoshinaga, K. Ueda, T.
  • Hayashi Enhancement of bacterial cellulose productivity and preparation of branched polysaccharide-bacterial cellulose composite by co-cultivation of Acetobacter species, Sen'i Gakkaishi (1995), 51(7), 323-32; K. Tajima, M. Fujiwara, M. Takai: Biological control of cellulose. Macromolecular Symposia (1995), 99 (Functional Polysaccharides), 149-55).
  • a cultivation of several different bacterial strains in order to influence the structure and properties of BNC has not been disclosed. Modifications of the BNC properties are exclusively caused by additives that are added during the cultivation process or after it and settle in the BNC structure. Moreover, the accessibility of multi-phase biomaterial systems is strongly restricted because only homogeneous structures can be achieved due to a resulting composite formation.
  • the aim of the invention is to create multi-phase biomaterials based on bacterially synthesized nanocellulose without required additives and composite formations, whereby the bacterial cellulose properties of said biomaterials can be specifically influenced in very wide limits in the synthesis process.
  • the biomaterials based on bacterially-synthesized nanocellulose are synthesized from at least two different cellulose-producing bacterial strains to a plurality, i.e. at least two, different bacterial cellulose networks in a common culture medium.
  • the properties of the bacterial cellulose are not achieved by deliberately added additives or composite formations developed in the synthesis with them but by the controlled generation of the synthesized phase system consisting of a plurality of different bacterial cellulose networks.
  • Said bacterial cellulose networks which differ from each other in their molecular and/or supra-molecular structure in particular, can be synthesized, for example, as a combined homogeneous phase system and thus generate a common homogeneous phase of the biomaterial.
  • a linked formation of the aforementioned phase systems can also be generated if the at least two different bacterial cellulose networks are formed as a layered phase system consisting of at least one combined homogeneous phase and of at least one single phase.
  • new biomaterials are generated only by the synthesized bacterial cellulose networks and thus without the disadvantageous absolutely required additives as starting components of the synthesis, and the bacterial cellulose properties of said biomaterials can be influenced in very wide limits and consequently clearly controlled in the production.
  • the structure and properties of the BNC materials can be specifically defined in very wide limits by the volumetric relation of the aqueous cell dispersions of the bacterial strains used and can be controlled in the synthesis in a “tailored” manner. Said “tailoring” can be applied to all structures and properties that are relevant for the application of BNC materials in a wet or dried (hot-pressed, air- or freeze-dried) form, for example, in medicine (wound dressings, implants), in technology (membranes, foils, barrier layers) and in food industry (zero-calorie nutrition, packaging).
  • the structure and properties of the BNC materials can be influenced particularly by the variation of the cultivation (combination of the bacterial strains before or after the inoculation) of the corresponding cellulose-producing bacterial strains, by the use of different culture media or by the use of different cultivation parameters (temperature, duration, volume, cultivation vessels).
  • the invention is not restricted to so called “pure” BNC materials but also includes the use of bacterial strains that produce cellulose-like structures on the basis of modified C-sources, e.g. the use of N-acetyl glucosamine or glucosamine as C-source.
  • FIG. 1 Bacterially synthesized nanocellulose (BNC) consisting of a plurality of different bacterial cellulose networks that form a common homogeneous phase system
  • FIG. 2 BNC consisting of two different bacterial cellulose networks each of them forming a separate layered single phase
  • FIG. 3 BNC with two different bacterial cellulose networks that form a layered phase system consisting of two layered single phases and one combined, homogeneous phase
  • FIG. 1 shows bacterially synthesized nanocellulose (BNC biomaterial) that, according to the invention, consists of a plurality (two in the example) of different bacterial cellulose networks forming a common phase system of one combined homogeneous phase (1).
  • This phase system is synthesized from two kinds of Gluconacetobacter strains, in the example ATCC 23769 and DSM 11804, in a not shown cultivation vessel with a synthesis area of 7 cm 2 .
  • said area can be freely selected for the special phase formation in this embodiment.
  • the two bacterial strains are added together into the cultivation vessel and thus they are inoculated for the common synthesis.
  • An added cultivation medium consists of a carbon source (preferentially different sugars and their derivatives), a nitrogen source (preferentially peptone) and, if required, a buffer system (preferentially disodium hydrogen phosphate and citric acid).
  • the biosynthesis was carried out at a temperature ranging from 28 to 30° C. during a period from 3 to 21 days and it was tested for both a discontinuous and a continuous synthesis procedure.
  • a common, very stable and transparent combined homogenous BNC phase system (see FIG. 1 ) of the two synthesized BNC networks is achieved with a relationship of 5:1 or 2:1 of the culture medium and the bacterial strains
  • the so called inoculation relationship (relationship of the inoculated bacterial strains to each other) is 50:50 (ATCC 23769 DSM 11804), i.e. the quantities of the bacterial strains that take part in the synthesis are identical.
  • a change of this inoculation relationship would additionally allow the control of the pore system and thus of the stability as well as of the transparency of the homogenous BNC hiomaterial.
  • an inoculation relationship of 10:90 for example, a solid/stable, transparent and simultaneously elastic BNC carded web was generated. If the inoculation relationship is reverse (e.g. 90:10), both the strength and the elasticity can be reduced without changing the transparency.
  • glacial acetic acid up to 2% can improve the homogeneity of the generated BNC material.
  • FIG. 2 shows a BNC material that, as proposed, also consists of two different bacterial cellulose networks which, however, have been synthesized to a layered phase system comprising separate single phases 2, 3.
  • Each of the separate single phases 2, 3 corresponds to one BNC carded web and its properties known per se and are firmly combined with each other.
  • This phase system is synthesized from two kinds of Gluconacetobacter strains, ATCC 10245 and DSM 14666 in this example, in the cultivation vessel that was mentioned in the first example and has a synthesis area that can be freely selected for this special phase formation.
  • the two bacterial strains are separately prepared, too, and are added together into the cultivation vessel for the common synthesis.
  • the added cultivation medium consists again of a carbon source (preferentially different sugars and their derivatives), a nitrogen source (preferentially peptone), a vitamin source (preferentially yeast extract) and, if required, a buffer system (preferentially disodium hydrogen phosphate and citric acid).
  • a carbon source preferentially different sugars and their derivatives
  • a nitrogen source preferentially peptone
  • a vitamin source preferentially yeast extract
  • a buffer system preferentially disodium hydrogen phosphate and citric acid
  • the biosynthesis was performed at a temperature ranging from 28 to 30° C. during a period from 3 to 21 days and was tested both for a discontinuous and continuous synthesis procedure.
  • the selected inoculation relationship between the bacterial strains used is 50:50 (ATCC 10245 : DSM 14666). If this relationship is changed in favor of one bacterium, the thickness of the single phases 2 or 3 and the resulting properties (water absorption and water retention, etc.) can be specifically controlled. Furthermore, an inoculation relationship of 70:30 (the relationship of 20:1 between the cultivation medium and the bacterial strains was maintained) results in an improved transparency without a change of the thickness of the BNC carded web.
  • FIG. 3 shows a BNC that also consists—as proposed—of two different bacterial cellulose networks which, however, have been synthesized to a special layered phase system and always two separate single phases (2, 3) correspond to a respective BNC carded web of the corresponding bacterial strain and its properties known per se, and both single phases (2, 3) are firmly combined via a combined homogenous phase (1).
  • This special phase system is synthesized from the two Gluconacetobacter strains ATCC 23769 and DSM 14666 again in tile mentioned and not shown cultivation vessel with a. synthesis area of 7 cm 2 , If this synthesis area is changed, the formation of the single phases 2, 3 can be deliberately influenced.
  • the increase of the area supports the formation of the single phase 2 (corresponding to the bacterial strain DSM 14666) more than the formation of the single phase 3 (corresponding to bacterial strain ATCC 23769).
  • phase system of the BNC biomaterial shown in FIG. 3 is achieved by the use of the bacterial strains mentioned before and by their separate preparation and subsequent common inoculation. However, a common cultivation of these bacterial strains, common preparation included, would generate a combined homogeneous phase system (see FIG. 1 .
  • the cultivation medium used here is also a mixture of a carbon source (preferentially different sugars and their derivatives), a nitrogen source (preferentially peptone), a vitamin source (preferentially yeast extract) and, if required, a buffer system (preferentially disodium hydrogen phosphate and citric acid).
  • a carbon source preferentially different sugars and their derivatives
  • a nitrogen source preferentially peptone
  • a vitamin source preferentially yeast extract
  • a buffer system preferentially disodium hydrogen phosphate and citric acid
  • the biosynthesis was carried out at a temperature ranging from 28 to 30° C. during a period from 3 to 21 days with a relationship of 20:1 between the cultivation medium and the bacterial strains and was tested both for a discontinuous and continuous synthesis procedure.
  • the inoculation relationship of 50:50 between the bacterial strain leads to the externally visible layered BNC phase system ( FIG. 3 ) comprising the aforementioned two single phases 2, 3 and the homogenous phase 1 located between them. Moreover, with this inoculation relationship the proportions of the single phases are identical.
  • the change of the inoculation relationship in favor of one bacterial strain allows the deliberate control of the thickness of the single phases 2, 3 and of the resulting properties (water absorption and water retention, etc.).

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CN102791300B (zh) 2015-07-29
JP5975221B2 (ja) 2016-08-23
US10829567B2 (en) 2020-11-10
DE102010012437A1 (de) 2011-09-22
JP2013522399A (ja) 2013-06-13
EP2547372A1 (de) 2013-01-23
PL2547372T3 (pl) 2017-10-31
CA2792381A1 (en) 2011-09-22
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