Abstract:
Multiphase biomaterials are provided which are based on bacterially synthesized nanocellulose (BNC) including at least two different bacterial cellulose networks. To this end, a culture median is inoculated with at least two different cellulose-producing bacterial strains.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    The invention relates to multi-phase biomaterials based on bacterially synthesized nanocellulose and a method for producing same. 
         [0002]    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). 
         [0003]    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&#39;s own cells, permanent integration into the body) without requiring disadvantageous additives or composite formations produced in the synthesis with them. 
         [0004]    It is general knowledge that homogeneous or multi-phase biomaterials based on bacterially synthesized nanocellulose (BNC) can be influenced by modifying said material after its synthesis (post-modification) (K.-Y. Lee, J. J. Maker, A. Bismarck: Surface fictionalisation of bacterial cellulose as the route to produce green polylactide nanocomposites with improved properties, Composites Science and Technology (2009), 69(15-16), 2724-2733; D. Klemm, D. Schumann, F. Kramer, N. Heβler, M. Hornung, H.-P. Schmauder, S. Marsch: Nanocelluloses as Innovative Polymers in Research and Application. Advances in Polymer Science (2006), 205 (Polysaccharides II), 49-96). 
         [0005]    However, it is also possible to perform an in situ modification already with the synthesis of the bio-technological cultivation process (H. Wang, F. Guan, X. Ma, S. Ren: Production and performance determination of modified bacterial cellulose, Shipin Keji (2009), (5), 28-31; N. Hessler, D. Klemm: Alteration of bacterial nanocellulose structure by in situ modification using polyethylene glycol and carbohydrate additives, Cellulose (Dordrecht, Netherlands) (2009), 16(5), 899-910; D. Klemm, D. Schumann, F. Kramer, N. Heβler, M. Hornung, H.-P. Schmauder, S. Marsch: Nanocelluloses as Innovative Polymers in Research and Application. Advances in Polymer Science (2006), 205(Polysaccharides II), 49-96). 
         [0006]    In this case, different addition agents are added to the culture medium during the biosynthesis (e.g, M. Seifert: Modifizierung der Struktur von Bakteriencellulose durch die Zusammenstellung des Nährmediums bei der Kultivierung von  Acetobacter xylinum , [Modification of the structure of bacterial cellulose by composing the cultural medium in the cultivation of  Acetobacter xylinum ], doctoral thesis, Friedrich-Schiller-University Jena, Germany, 2004; O. M. Astley, E. Chanliaud, A. M. Donald, M. J. Gidley: Structure of Acetobacter cellulose composites in the hydrated state, International journal of biological macromolecules (2001), 29/3, 193-202; N. Sakairi, H. Asano, M. Ogawa, N. Nishi, S. 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). 
         [0007]    According to this, the addition of carboxymethyl cellulose (CMC) and methyl cellulose (MC) has huge effects on the BNC network. Due to their embedding, both additives have an influence on the pore system and the properties resulting from it, e.g. elasticity, water retention capacity, filter function, and thus novel BNC materials are produced (O. M. Astley, E. Chanliaud, A. M. Donald, M. J. Gidley: Structure of Acetobacter cellulose composites in the hydrated state, International journal of biological macromolecules (2001), 29/3, 193-202; N. Sakairi, H. Asano, M. Ogawa, N. Nishi, S. 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). 
         [0008]    Moreover, the addition of vegetable cell wall accompanying components, such as xyloglucan or pectin, to the culture medium during the BNC biosynthesis was a part of examinations to imitate structural relationships of native cellulose and to analyze its formation in detail (J. Cybulska, E. Vanstreels, Q. T. Ho, C. M. Courtin, V. Van Craeyveld, B. Nicolai, A. Zdunek, K.. Konstankiewicz: Mechanical characteristics of artificial cell walls, Journal of Food Engineering (2009), 96(2), 287-294). 
         [0009]    Unlike water-soluble compounds, solids can also be given as additives to the culture medium during the biosynthesis and are integrated in the produced BNC network. 
         [0010]    Whereas Udhardt (U. Udhardt: Synthese, Eigenschaften und Strukturdesign von Bakteriencellulose mit speziellem Anwendungspotential von BASYC®-Implantaten in der Mikrochirurgie [Synthesis, properties and structural design of bacterial cellulose with a specific application potential of BASYC® implants in microsurgery], doctoral thesis, Friedrich Schiller University Jena, Germany, 2004) described an integration of crystal balls or an integration of silica gel and inorganic salts (calcium carbonate) into the BNC network, Serafica et al. (G. Serafica, R. 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. 
         [0011]    However, these in situ methods have the disadvantage that they require additives to produce novel biomaterials on BNC basis. Thus, the structure and the properties combined with it can only be controlled by using water-soluble organic, inorganic substances or polymers and solid particles. Furthermore, in contrast to pure BNC the integrated additives bear the risk of possible allergic reactions if are used as medical products. 
         [0012]    In the post modification method, a modification of the BNC and the production of homogenous or multiphase materials are achieved by integrating organic or inorganic substances after the cultivation (B. R. Evans, H. O&#39;Neil, M. Hugh, V. P. Malyvanh, I. Lee, J. Woodward: Palladium-bacterial cellulose membranes for fuel cells, Biosensors &amp; Bioelectronics (2003), 18/7, 917-23; B. R. Evans, H. M. O&#39;Neill, E. Greenbaum: Electron Transfer by Enzymes and Photosynthetic Proteins Immobilized in Polysaccharide Composites, Abstracts, 57th Southeast/61st Southwest Joint Regional Meeting of the American Chemical Society, Memphis, Tenn., United States, November 1-4, 2005; W. A. Daoud, J. H. Xin, Y.-H.Zhang; Surface functionalization of cellulose fibers with titanium dioxide nanoparticles and their combined bactericidal activities, Surface Science (2005), 599(1-3), 69-75; D. Zhang, L. Qi: Synthesis of mesoporous titania networks consisting of anatase nanowires by templating of bacterial cellulose membranes, Chem. Commun. (2005), 21, 2735-7). 
         [0013]    By means of this method a multitude of BNC variations have already been realized, e.g. by the use of different types of monomers and synthetic polymers (H. Yano, S. Nakahara: Bio-composites produced from plant microfiber bundles with a nanometer unit web-like network, Journal of Materials Science (2004), 39/5, 1635-8; V. Dubey, L. K. Pandey, C. Saxena: Pervaporative separation of ethanol/water azeotrope using a novel chitosan-impregnated bacterial cellulose membrane and chitosan-poly(vinyl alcohol) blends, Journal of Membrane Science (2005), 251(1-2), 131-136; V. Dubey, C. Saxena, L. Singh, K. V. Ramana, R. S. Chauhan: Pervaporation of binary water-ethanol mixtures through bacterial cellulose membrane, Separation and Purification Technology (2002), 27/2, 163-71; W. A. Daoud, J. H. Xin, Y.-H. Zhang: Surface functionalization of cellulose fibers with titanium dioxide nanoparticles and their combined bactericidal activities, Surface Science (2005), 599(1-3), 69-75), structure-forming polymers, e.g. PVA (T. Wan, Y. Zhu: Preparation of bacterial cellulose/poly(vinyl alcohol) composite gels, Faming Zhuanli Shenqing Gongkai Shuomingshu CN 101570616, 2009), gelatin (K. Yasuda, J. P. Gong, Y. Katsuyama, A. Nakayama, Y. Tanabe, E. Kondo, M. Ueno, Y. Osada; Biomechanical properties of high-toughness double network hydrogels, Biomaterials (2005), 26/2, 4468-75; A. Nakayama, A. Kakugo, J. P. Gong, Y. Osada, M. Takai, T. Erata, S. 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&#39;Neil, M. Hugh, V. P. Malyvanh, I. Lee, J. Woodward: Palladium-bacterial cellulose membranes for fuel cells, Biosensors &amp; Bioelectronics (2003), 18/7, 917-23; B. R. Evans, H. M. O&#39;Neill, E. Greenbaum: Electron Transfer by Enzymes and Photosynthetic Proteins Immobilized in Polysaccharide Composites, Abstracts, 57th Southeast/61st Southwest Joint Regional Meeting of the American Chemical Society, Memphis, Tenn., United States, Nov. 1-4, 2005; Daoud, J. H. Xin, Y.-H.Zhang: Surface functionalization of cellulose fibers with titanium dioxide nanoparticles and their combined bactericidal activities, Surface Science (2005), 599(1-3), 69-75; D. Zhang, L. Qi: Synthesis of mesoporous titania networks consisting of anatase nanowires by templating of bacterial cellulose membranes, Chem. Commun. (2005), 21, 2735-7). 
         [0014]    However, these methods have the disadvantage that they require two production steps (synthesis of BNC and its modification) for developing novel BNC. Moreover, the post modification modifies the BNC partly to such an extent that the unique structure and consequently the excellent properties are lost. In addition to this, these methods require the disadvantageous use of additives, too. 
         [0015]    Another solution for producing new BNC material is based on the common cultivation of germs of different strains. Thus, 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. Beppu: Effective cellulose production by a coculture of  Gluconacetobacter xylinus  and  Lactobacillus mali , Applied Microbiology and Biotechnology (2006), 73(4), 915-921; KR 2002/067226; JP 10201495). 
         [0016]    In contrast to the aforementioned method, the co-cultivation of  acetobacter aceti  subsp.  xylinum  (NCI 1005) with the strains ATCC 10245 or NCI 1051 led to the increase of the respective polymer synthesis. Thus, the additional cellulose production and its subsequent decomposition cause, on the one hand, the increase of the nutrients in the culture solution and consequently an increased yield of the polymers. On the other hand, the presence of cellulose in the culture solution made the formation of water-soluble branched polysaccharides possible (K. Tajima, H. Ito, M. Fujiwara, M. Takai, J. Hayashi: Enhancement of bacterial cellulose productivity and preparation of branched polysaccharide-bacterial cellulose composite by co-cultivation of  Acetobacter  species, Sen&#39;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). 
         [0017]    However, experts exclusively know co-cultivation methods that refer to the increased productivity of the yield of cellulose or to a composite formation and always cultivate one cellulose-producing bacterial strain known for the cellulose synthesis. 
         [0018]    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. 
         [0019]    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. 
         [0020]    According to the invention, 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. Thus, 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. 
         [0021]    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. 
         [0022]    It is also possible that as a result of the synthesis the at least two different bacterial cellulose networks lead to the formation of a layered phase system comprising firmly connected BNC-network-specific separate single phases. 
         [0023]    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. 
         [0024]    Depending on the selection and number of the different cellulose-producing bacterial strains used for the synthesis and depending on the selected synthesis conditions, particularly the composition of the culture medium, 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. 
         [0025]    Surprisingly, even the achievement of the properties contradicting in themselves for the synthesis of BNC materials, such as high water content with gelatinous, soft consistency and dense material structure of high strength, in one and the same material of bacterially synthesized nanocellulose can be realized and could open up new fields of application. 
         [0026]    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). This refers to the control of the mechanical strength, elasticity, permeability, transparency and water balance as well as of scaffold-properties (pore system, surface characteristics, colonization by cells) and bio-compatibility (body compatibility, endothelialization, immigration of body&#39;s own cells, permanent integration into the body). 
         [0027]    In the synthesis process, 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). 
         [0028]    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. 
     
    
     
       DETAILED DESCRIPTION OF THE INVENTION 
         [0029]    The invention will be explained in more detail by virtue of the following embodiments illustrated in the figures. 
           [0030]    They show: 
           [0031]      FIG. 1 : Bacterially synthesized nanocellulose (BNC) consisting of a plurality of different bacterial cellulose networks that form a common homogeneous phase system 
           [0032]      FIG. 2 : BNC consisting of two different bacterial cellulose networks each of them forming a separate layered single phase 
           [0033]      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 
       
    
    
       [0034]      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). 
         [0035]    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 . However, said area can be freely selected for the special phase formation in this embodiment. After separate preparation the two bacterial strains are added together into the cultivation vessel and thus they are inoculated for the common synthesis. 
         [0036]    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). 
         [0037]    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. 
         [0038]    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 
         [0039]    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. With 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. 
         [0040]    Furthermore, the addition of glacial acetic acid up to 2% can improve the homogeneity of the generated BNC material. 
         [0041]      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. 
         [0042]    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. In this embodiment, the two bacterial strains are separately prepared, too, and are added together into the cultivation vessel for the common synthesis. 
         [0043]    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). 
         [0044]    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. 
         [0045]    In this synthesis, a stable layered system is obtained from the two separated but firmly combined single phases 2, 3 with a relationship of 20:1 between the cultivation medium and the mentioned bacterial strains as well as by the use of  Gluconacetobacter  strains different from the ones used in the first embodiment, although these single phases 2, 3 are—at least for the bacterial strains used here—externally almost not visible (a two-phase system of the BNC networks almost not visible). Thus, the synthesized BNC biomaterial gives the external impression of a homogenous carded web but structurally consists of said two different bacterial cellulose networks. 
         [0046]    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. 
         [0047]      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). 
         [0048]    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 (inoculation relationship of 50:50) 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). 
         [0049]    The 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 . 
         [0050]    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). 
         [0051]    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. 
         [0052]    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. 
         [0053]    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.). 
       LIST OF REFERENCE NUMERALS 
       [0054]      1 —combined homogeneous phase 
         [0055]      2 , 3 —separate single phase