JP2007159569A - Method for producing polysaccharide-containing product containing chitin/chitosan using geotrichum species - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for safely, easily and efficiently producing a large quantity of a chitin/chitosan-containing polysaccharide compared to production methods using conventional microorganisms. <P>SOLUTION: The method for safely, easily and efficiently producing a large quantity of a chitin/chitosan-containing polysaccharide is provided, comprising culturing and proliferating Geotrichum bacteria in a liquid medium. A fibrous product containing a large amount of the chitin/chitosan-containing polysaccharide is provided, being in the form of a multi-laminated structure. The fibrous product thus obtained can be used as a food material, a cosmetic material, a medical material, a cell-culturing sheet serving as a foothold in culturing totipotent stem cells or multipotent stem cells, or a drug carrier in DDS(drug delivery system). This fibrous polysaccharide-containing product, because of being in the form of a multi-laminated structure, can be put to a simple process to be formed into nonwoven fabrics or sheets. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing a polysaccharide-containing material containing chitin / chitosan using a genus Geotrichum, and its use. According to the present invention, fibrous polysaccharide-containing materials composed of chitin / chitosan as a main component and other trace amounts of insoluble polysaccharides, water-soluble polysaccharides and the like can be safely and easily formed in a multi-layered structure in a large amount. Can be produced efficiently. The obtained polysaccharide-containing material is used to culture food materials, cosmetic materials, medical materials (wound protection coating materials), and stem cells (including totipotent and pluripotent stem cells) and cells used in the field of regenerative medicine. It can be used as a cell culture sheet and a drug carrier used for DDS, and is provided as a biomaterial usable in various fields.

  Chitin / chitosan is widely known to be a component present in cell walls of insects and fungi in addition to crustacean epidermis such as shrimp and coral in nature. Therefore, methods have been devised for producing chitin / chitosan from mycelium of fungi, which are fungi, and polysaccharides produced by some bacteria outside the fungus. Representative examples of those conventional methods are described in the following Patent Documents 1 to 7 and the like, but in the production method using these techniques and cells, fibrous chitin / chitosan having a multilayer structure is used. It was not possible to produce large quantities efficiently.

  For example, by culturing a filamentous fungus containing chitin / chitosan on a solid medium, the mycelium is subjected to heat treatment and alkali treatment to produce a chitin nonwoven fabric, and the loss of the medium is extremely large. Chitin / chitosan-containing polysaccharides are inefficient with low yields, and it is not possible to obtain non-woven fabric-like polysaccharides, but only to obtain polysaccharides in the form of thin threads.

In addition, other conventional methods, such as those in which mycelia are cultured in a liquid medium using filamentous fungi to produce a polysaccharide containing chitin / chitosan, are low in production, or are not specified The cost is expected to increase by at least a dozen times compared to the production method of chitin and chitosan derived from shrimp and sea bream. Furthermore, the mycelium produced is not like a multi-layered pulp slurry, but is solidified in a ball shape with a diameter of about 2 mm to 10 mm. As it is, it is processed as a cosmetic material, and processed into a nonwoven fabric and a sheet. It was a structure that was not suitable for this. Similarly, the amount of chitin / chitosan produced outside the bacterial body is not large, and the safety is unknown. From this background, there have been almost no attempts to put it into practical use in the industry, and in order to truly industrialize the production of microbial-derived chitin and chitosan, it is necessary to either solve the high production cost factor or create new functions. There was no way other than finding gender and increasing added value.
JP-A-4-343762 JP-A-5-184378 JP-A-5-199892 Japanese Patent Laid-Open No. 7-97721 JP-A-8-9922 JP 10-316702 A JP-A-11-276160

  Chitin and chitosan are used in products and materials in various fields such as food, cosmetics, and medical products, but the physical properties required for these products include high bioactivity and biocompatibility. The structure is suitable for processing. An object and problem of the present invention is to provide a method for safely and efficiently producing a polysaccharide-containing material containing chitin / chitosan as a novel biomaterial that meets the requirements of products (or materials) in these various fields. That is.

As a result of accumulating research to achieve the above object, the present inventor has obtained a mycelium containing chitin / chitosan and its cell wall as compared with the methods for producing chitin / chitosan by filamentous fungi and bacteria previously published. As a result, the present inventors have completed the present invention by establishing preferable production conditions for producing polysaccharide-containing substances by efficiently producing fibrous mycelium containing chitin / chitosan. .
That is, the present invention is a method for producing a polysaccharide-containing product containing chitin / chitosan by culturing and growing Geotrichum, and preferably includes a step of culturing and growing Geotricam in a liquid medium.
The “polysaccharide-containing substance” of the present invention includes (1) a fibrous mycelium containing chitin / chitosan, (2) a polysaccharide-containing substance comprising a cell wall of mycelium, and (3) acetylation in the subsequent step. The polysaccharide-containing material in which the chitin component is increased by the treatment is included (hereinafter, the polysaccharide-containing material may be simply referred to as “polysaccharide”). The fibrous mycelium of (1) can be produced by a step of culturing and growing Geotrichum in a liquid medium and a step of sterilizing the obtained mycelium after separating it from the culture solution. The polysaccharide-containing material comprising the cell walls of the mycelium of (2) can be produced by detaching the cytoplasm component from the fibrous mycelium of (1). The polysaccharide-containing material having an increased chitin component in (3) can be produced by subjecting the polysaccharide-containing material comprising the cell walls of the mycelium in (2) to acetylation treatment (see FIG. 13).
In the production method of the present invention, in order to obtain a desired mycelium, preferably Geotrichum armillariae, Geotrichum capitatum, Geotrichum linkii, Geotrichum sericeum, Geotrichum citrium-auranti (Geotrichum citri-aurantii), Geotrichum candidum, Geotrichum pulmoneum, Geotrichum clavatum, Geotrichum eriense, Geotrichum famentans Polysaccharides containing chitin and chitosan using one or more Geotricum species selected from Geotrichum klebahnii species It may be produced inclusions.
As will be described later, the mycelium preferably becomes a fiber having a diameter of around 5 μm and a length of 1,000 μm or more as segmental spores (segmented conidia) in the process of growth and proliferation of the genus Geotricum, It is in the form of an assembly that is stacked in a complex manner. The preferred composition of the mycelium cell wall mainly contains a trace amount of insoluble polysaccharides and water-soluble polysaccharides in addition to chitin / chitosan.
As an example, the polysaccharide-containing product containing chitin / chitosan of the present invention can be produced using Geotrichum fragrans strain CBS127.76 and Geotrichum fragrans strain CBS164.32. CBS is an abbreviation for a microorganism preservation institution in the Netherlands, and contributes to microorganism research by, for example, distributing cells when requested by researchers. The CBS127.76 strain and the CBS164.32 strain are endemic species registered with this institution, and no other identical cells exist.
The polysaccharide-containing material containing chitin / chitosan of the present invention can be used as a raw material for foods and cosmetics, as well as a medical wound protective covering material, a cell culture sheet material, a drug carrier used for DDS, etc. Can also be used. This point will be described in detail later. In particular, wound protectors made of polysaccharide-containing materials manufactured using Geotricum Fragrance species are superior in biocompatibility (compatibility) and have new blood vessel inducing ability, which is not found in conventional products. The feature was shown. Therefore, it is preferable to use a polysaccharide-containing material produced using Geotricham fragrance species for the use of a wound protection material. In addition, since the polysaccharide-containing material produced using Geotricham Fragrance species was extremely excellent in biocompatibility (compatibility) as described above, it is used for cell culture sheets and drug carriers. It is preferable. The present invention includes such a wound protection material, a cell culture sheet and a drug carrier. In the present specification, the terms “wound protective material” and “wound protective covering material” both mean medical wound protective material, and are exemplified by sheet-like materials that cover and protect wound sites. However, it may be a wound protective material of other shapes and usage methods.

  The main substance that composes the cell wall of the mycelium of the genus Geotricham is chitin / chitosan, a linear high-molecular polysaccharide with β-1,4-linked glucosamine, and a trace amount of insoluble polysaccharides. And water-soluble saccharides. The mycelium is characterized by a multi-layered structure in which fibrous mycelia are intertwined and laminated in a complicated manner. These structures have excellent features.

  Until now, chitin derived from shrimp and shark has already been commercialized as a surgical suture and a wound protective covering material because of its excellent biocompatibility. Also, it is used as a cosmetic material because it can be moisturized when applied to the skin surface, and it can be expected to prevent rough skin and soften the skin due to the peeling effect. Furthermore, chitosan obtained by deacetylating chitin is used as a food additive because it exhibits broad antibacterial properties against bacteria. In addition, since the basic skeleton has a hydroxyl group and an amino group rich in chemical modification properties, the range of use is widespread such as application as a carrier for minerals, metal components, drugs and the like.

  In addition to these known functions, the polysaccharide containing chitin / chitosan obtained from the mycelial cell wall of the genus Geotrichum of the present invention can be easily dispersed in water with a multi-layered structure in which fibers are intertwined and laminated. Because of its features, it can be used in various ways without the need to dissolve it in special solvents like shrimp and sea bream chitin / chitosan. In addition, it has very good biocompatibility and many physiological activities. Therefore, it can be said that it has extremely superior functionality compared to conventional chitin / chitosan cosmetic materials.

  Eating an appropriate amount can be used as a functional dietary fiber that works on the immune system of the intestinal tract and enhances the activity of immune cells.

  Furthermore, since it has a multi-layered structure in shape as described above, it can be processed into a nonwoven fabric and a sheet by a simple method, and can be commercialized as a wound protection covering material for medical use. Further, it can be used as a cell culture sheet as a scaffold when culturing stem cells used in the field of regenerative medicine, and further as a drug carrier used in DDS, and can be widely used as a completely new functional biomaterial.

Hereinafter, an embodiment of the present invention will be described with reference to FIG.
In this embodiment, Geotrichum is shaken under aerobic conditions in a liquid medium containing carbon sources such as glucose and molasses, and nitrogen / phosphoric acid and mineral components such as polypeptone, yeast extract and malto extract. Alternatively, a fibrous polysaccharide is produced in a pulp slurry-like, gel-like, or sheet-like manner by stirring culture or stationary culture.

  The fungus belonging to the genus Geotricum is used as a starter fungus for cheese (camembert) production in Europe and the United States, and is included in the microorganism group that works during the mash fermentation of white liquor, which is distilled liquor. In Africa and the Middle East, palm wine ( It is traditionally used for food processing around the world, such as being involved in ethanol fermentation of palm wine). However, there has been no example in which a fibrous polysaccharide is taken out from the genus Geotricum and processed and prepared as a biomaterial.

As shown in FIG. 13, the culture of the genus Geotricum can be performed by aeration stirring liquid culture or the like (S1). For example, in a jar fermenter 5 L volume, the culture solution is 3 L, pH 4.0 to 7.5, the culture conditions are temperature 27 ° C. to 30 ° C., rotation speed 150 to 400 rpm, aeration rate 0.3 to 1.5 vvm, culture time 72 ~ 168 hours.
The liquid mycelium of the genus Geotricum cultivated in liquid culture has a multi-layered structure in which intricately intertwined layers are stacked. The mycelium can be separated from the culture solution by a centrifuge or the like, then washed with water and further filtered by suction filtration with a microfilter or the like (S2). Moreover, fibrous mycelium can be efficiently collected in large quantities in a short time by pumping with a pressure pump to a cylindrical large filter (pore diameter 1 to 3 μm) without performing centrifugation.

  Further, the mycelium is heat sterilized in demineralized water at a temperature of 100 to 121 ° C. and then dehydrated (S3) to obtain a fibrous mycelium having a multi-layer structure (S4). The mycelium of the genus Geotricham is roughly divided into a cell periphery including a cell wall and a cytoplasm. As the composition, the cytoplasm has many nutritious ingredients such as proteins, nucleic acids, lipids, etc., so it can be used as it is as a food or cosmetic material, but when using only polysaccharides on the cell wall from the mycelium, A step (S5) of effectively removing cytoplasmic contents is required.

  As this process, for example, there is a method in which mycelium is added to 1 to 3 M sodium hydroxide or sodium carbonate aqueous solution so as to be 1 to 20 w / v% and heated at 100 to 121 ° C. for 60 minutes or more. By this heating operation, sterilization is performed and most of the cytoplasm components are separated and eluted from the mycelium. Moreover, you may perform the protease process for decomposing | disassembling the protein which remains slightly as needed. Filamentous chitin / chitosan-containing polysaccharide (S6) composed of mycelium cell walls is obtained by filtering and washing the mycelium after deproteinization in this manner.

In addition, the mycelium is added to 1 to 3M sodium hydroxide or sodium carbonate aqueous solution so as to be 5 to 20 w / v%, and in the treatment at a heating temperature of 120 ° C. and a heating time of 6 hours or less, Although the fibrous polysaccharide, which is a cell wall component, was thinned by about 10% due to elution or the like, the multi-layered structure did not change greatly. This fibrous polysaccharide has a white or light skin color unlike the shrimp and persimmon-derived chitin / chitosan-derived crimson or mild yellow color, and therefore does not require bleaching. The taste was tasteless and odorless, with no peculiar peculiar smell or smell like chitin and chitosan derived from shrimp and sea bream. These features are important and advantageous when added to food and cosmetics.
Furthermore, by performing acetylation treatment on the polysaccharide, a fibrous polysaccharide having a multi-layered structure with an increased chitin component can be obtained (S7). By processing this into a non-woven fabric or a sheet (S8), it can be used as a medical wound protective covering or cell culture sheet. In addition, there is no inconvenience even if the fibrous polysaccharide before acetylation treatment is used as it is. The fibrous polysaccharide that has been subjected to acetylation treatment or before acetylation treatment can be used as a drug carrier for DDS by crushing to the nano level with an ultrasonic crusher or a homogenizer.

According to the method for producing a fibrous polysaccharide using Geotrichum of the present invention, a fibrous polysaccharide mainly composed of chitin / chitosan, which is expected to have various physiologically active effects in recent years, can be safely and easily obtained. It can be produced efficiently in large quantities at low cost. For example, as shown in the examples described later, the production method of the present invention using a liquid medium provides a yield several hundred times higher than the production method using a solid medium, and is a fibrous material containing chitin / chitosan. Polysaccharides could be produced very efficiently. Furthermore, if it is refined and processed according to the purpose of the product to be used, it will have sufficient functionality required for next-generation biomaterials such as foods, cosmetics, medical products, cell culture sheets for regenerative medicine, and drug carriers used for DDS. Can respond to this.
Below, the specific example of the utilization method of the polysaccharide containing chitin * chitosan of this invention is demonstrated.

(1) Specific examples of methods used as food materials Chitosan, a mucopolysaccharide, and chitosan, a deacetylated derivative of chitin, are low toxic and resistant to digestion in the human intestine. The utility value of is recognized. Already, shrimp and persimmon-derived chitin and chitosan are processed into dry powder and distributed in the market. As an example of use, by adding an appropriate amount to confectionery, noodles, ice cream, cheese, etc., effects such as thickening action, water retention / moisturization action, emulsification action, etc. are obtained, and these are used in their production.

  In addition, chitosan is also recognized in specific nutritional foods because it has the effect of reducing cholesterol by being adsorbed and excreted with bile acids in the human intestine. In addition, it exhibits wide antibacterial properties against various bacteria and filamentous fungi and is used for the purpose of preserving food. However, in order to exert such an effect, it must be once water-soluble and kneaded throughout the food without any unevenness, but shrimp and persimmon-derived chitin and chitosan do not dissolve in water as they are.

  Chitin is generally dissolved in a combination of dimethylformamide, dimethylacetamide, N-methylpyrrolidone, etc. and lithium chloride, and chitosan is dissolved in an organic acid such as malic acid or acetic acid, or in an acidic solvent such as inorganic acid. I have to let it. Therefore, shrimp and persimmon-derived chitin dissolved in these special solvents is extremely difficult to use in foods. Since chitosan is also an acidic solvent, its taste and aroma are altered, so that it can be used only in a very small amount.

  From these facts, the fibrous polysaccharide containing chitin / chitosan obtained from the genus Geotricum of the present invention can be expected to have the same effect as described above, and can be easily dispersed in water to make a pulp slurry. Therefore, it can be kneaded uniformly in food. In addition, there is an advantage that the degree of freedom of the blending ratio is high and the setting can be made arbitrarily. Therefore, it can greatly contribute to the food industry field.

(2) Specific Examples of Methods Used as Cosmetic Materials As described above, shrimp and persimmon-derived chitin and chitosan cannot be dissolved or dispersed in water as they are. To use it as a cosmetic, complicated treatments such as dissolving in a special solvent and chemically modifying it into a water-soluble derivative are required. Furthermore, the process which does not have toxicity with respect to skin is also required.

  On the other hand, the fibrous polysaccharide containing chitin / chitosan obtained from the genus Geotricum of the present invention is characterized by having a hydrophilic structure in addition to being fibrous, so that it can be easily dispersed in water. It is possible and the pH value is also neutral. The dispersion concentration can be adjusted as appropriate according to these characteristics (characteristics), and it can be easily processed into an emulsion-like, cream-like or gel-like. If applied to the skin, it has sufficient moisture retention and film-forming ability, so that a barrier can be formed between the epidermis and the outside air. Therefore, it becomes possible to protect skin and hair from ultraviolet rays and outside air. In addition, ultra-fine crushing and nano-particle formation using an ultrasonic crusher or homogenizer improves the permeability from the skin to the dermis layer, and promotes skin metabolism due to its extremely high physiological activity. You can expect

  Furthermore, since this chitin / chitosan has a hydroxyl group and an amino group that are rich in chemical modification properties in the basic skeleton, by adding a compound showing a positive or negative charge, these substances react and are adsorbed and retained. There are features. In addition, it is considered that hydrophobic polymer compounds are easily retained between the cross-links of the constituent sugars. Therefore, by utilizing this property and adding a compound having physiologically active ability or high functionality, the application range is greatly expanded as a drug carrier for DDS and as a material for various cosmetics.

(3) Specific Examples of Methods Used as Medical Wound Protection Dressing Products As medical wound protection coating materials, products derived from shrimp and sea bream have already been used in clinical settings. The therapeutic effect described in 4 is obtained. That is, by covering the surface of burns, deep wounds, wounds with a large amount of exudate, etc., the exudate is absorbed to create an appropriate moist environment in the wound, and the effect of promoting the formation of epidermis and granulation tissue is achieved.
1. New drugs and Linyi 41 (8): 192-202, 1992
2. Western sun skin 54 (6): 1182-1189, 1992
3. Western skin 55 (5): 941-946, 1992
4). Chitin, Chitosan Handbook: 324-354, Chitin, Chitosan Study Group

  However, the chitin / chitosan-containing polysaccharide obtained from the mycelium of the genus Geotricum of the present invention can be used as a material for a wound protection dressing as it is after deproteinization, and acetylation can be achieved by chemical treatment. By promoting and further increasing the chitin component, a chitin component composition derived from shrimp and persimmon can be obtained. This can be used as a wound protective covering material by refining it into a nonwoven fabric or sheet. In the production of this wound protective dressing material, the nonwoven fabric has a simpler process (method) compared to the conventional production process (method) by having a multi-layered structure which is a characteristic (characteristic) of the mycelium of the genus Geotricum. And can be processed into a sheet shape. Details are described in Example 7. The excellent effect will be described in Example 9.

(4) Specific examples of methods used as cell culture sheets Currently, cell culture sheets include polyglycolic acid or synthetic products made of polyglycolic acid and polylactic acid copolymers, and human amniotic membrane as a biological material. In addition to collagen sheets derived from collagen, there are collagen sheets derived from pigs and fish (skin). It is essential that the biological properties required for them are not toxic, pathogenic, or antigenic. For example, since human amniotic membrane contains epithelial cells, a process to remove it is necessary, but in order to remove pathogenicity and antigenicity as close to zero as possible and obtain a purified collagen sheet, it is an extremely complicated chemistry. A processing step is required. Therefore, the manufacturing cost is increased. Collagen sheets derived from pigs and fish (crust) have similar problems. Currently, research is progressing in the direction of using synthetic materials as the main raw material for cell culture sheets used in regenerative medicine, but for synthetic materials, physiological activities such as inducing new blood vessels in vivo and acting on immunity There are almost no reports of studies showing prominent performance. As for biological materials, it is currently suggested that collagen sheets derived from human amniotic membrane have an overall advantage, and this cell culture sheet has already been used clinically as a scaffold for culturing corneal cells in medical colleges and the like. Although advanced, manufacturing costs are very high.

  Based on these facts, it is recognized that the excellent bioactivity, which is a feature of the fibrous chitin / chitosan-containing polysaccharide obtained from the mycelium of the genus Geotricum of the present invention, can be greatly utilized, and a method for using it as a cell culture sheet has been newly developed. I found it. In other words, fibrous chitin / chitosan-containing polysaccharides having a multi-layer structure are eluted and removed from proteins and nucleic acids, which are the main causes of pathogenicity and antigenicity, by alkaline hydrothermal treatment, protease treatment, etc. Furthermore, it is a method of obtaining a cell culture sheet having more excellent biocompatibility and in-vivo digestibility by increasing the chitin component by a chemical treatment that promotes acetylation. Details are described in Example 8.

(5) Specific example of method used as drug carrier of DDS (drug delivery system) In order for a drug to be effective in vivo, the drug must be allowed to act appropriately on the site where it is required. However, most drugs have no site selectivity and are administered more than necessary to develop their pharmacological action, which causes various side effects. Therefore, it is required to work the drug at the required concentration at the required concentration only at the required time. This is the idea of DDS. Currently, as a drug carrier used for DDS, in addition to a synthetic product using the above-mentioned polyglycolic acid, polylactic acid, or a copolymer of polyglycolic acid and polylactic acid, soy lecithin derived from soybean is used as a biological material. Products that have been processed to the particle level and made into ribosomes have been used in animal studies for clinical application to humans. By deproteinizing the chitin / chitosan-containing polysaccharide of the present invention and finely crushing it to a nano level, it can be easily introduced into a living body. Since the basic structure of chitin / chitosan has an amino group and a hydroxyl group that are rich in chemical modification properties, by incorporating the target drug in the chitin / chitosan-containing polysaccharide, the drug is released gradually in vivo at a predesigned time. By doing so, it can be expected to obtain the effect required at the target site. This application to a drug carrier used for DDS makes use of the excellent biocompatibility and in-vivo digestibility that are the characteristics of the chitin / chitosan-containing polysaccharide of the present invention.

  Next, a method for producing a fibrous chitin / chitosan-containing polysaccharide having a multi-layered structure in a liquid medium will be described in more detail with reference to Examples, but the present invention is not limited thereto. Moreover, the culture | cultivation for confirming the difference with productivity by a solid culture medium was also performed together.

[Example 1]
After preparing a medium consisting of 3,000 ml of demineralized water, 60 g (2%) of D-glucose, 15 g (0.5%) of polypeptone, 9 g (0.3%) of yeast extract and 5 g (0.0167%) of malto extract to pH 5.6, The mixture was placed in a 5 L capacity jar fermenter, autoclaved at 121 ° C. for 15 minutes, and then cooled to 28 ° C. This is used as the main culture medium (medium). In addition, you may change and use suitably the mixture ratio of each nutrient component.

  Dispense 7 ml each of sterilized medium with the same composition as above into 5 test tubes, cool and inoculate 1 platinum ear of Geotrichum fragrans strain CBS127.76 on a clean bench and shake incubator And culturing for 24 hours under aerobic conditions at a temperature of 30 ° C. and a shaking speed of 140 rpm. From the pre-cultured bacterial solution, 10 ml of the best-growing bacterial cell solution was inoculated into the main culture solution (medium) of the jarfamenter and the culture was started. In addition, the inoculation amount of the precultured bacterial solution may be approximately 1 ml or more. The culture conditions were as follows, and each set value was arbitrarily combined and carried out seven times in total. The aeration amount was 0.3 vvm to 1.5 vvm, the culture solution temperature was in the range of 24 ° C to 32 ° C, and the rotor blades were stirred at 100 rpm to 400 rpm for cultivation.

  The cultivation time was 40 hours to 160 hours, preferably around 90 hours, and the yield was maximum. In addition, what is necessary is just to increase the amount of inoculation suitably when shortening culture | cultivation time. The pH of the bacterial cell liquid after the culture was in the range of 7.0 to 7.3 on average.

  Further, as a result of taking out a sample after 30 hours from the start of culture and observing under a microscope (× 1,000 times), it was easily observed that fibrous mycelia were intertwined and laminated in multiple layers (see FIG. 1). The mycelia had an average diameter of about 5 μm and a length of 1,000 μm or more.

  This mycelium culture solution was separated into a culture solution and mycelium by a centrifuge by a known method, and suction filtered with an aspirator using a filter paper to obtain a mycelium having a wet weight of 102.34 g. Subsequently, it dried at 100 degreeC with the constant temperature dryer for 3 hours. The dry weight of the obtained mycelium was 14.44 g. The sugar yield (D-glucose ratio) was 24.06%. In addition, when the amount of D-glucose in the culture supernatant from which the mycelium was separated was confirmed by the Somoginelson method, it was confirmed that about 96% was used for assimilation and polymerization of polysaccharides. These values vary slightly depending on the culture conditions.

  Next, when the raw fibrous mycelium was heated in demineralized water at 100 ° C. for 6 hours, it was fractionated into a water-soluble substance and an insoluble substance. Among these, a water-soluble substance was dried at 70 ° C., precipitated with 99% ethanol, and dried again at 90 ° C. This was designated as Soluble Part (water-soluble substance: not including chitin / chitosan). The insoluble substance was Unsoluble Part (insoluble substance: containing chitin / chitosan). In addition, the raw fibrous mycelium was dried at 90 ° C. without being subjected to hot water treatment. This was designated as Original Sample (containing chitin / chitosan). When these were analyzed by infrared absorption spectroscopy (FT-IR), it was determined that most of the unsoluble part was chitin with an acetylation degree of about 63%, and the original sample was chitin with an acetylation degree of about 61%. It was. In addition, it was determined that the water-soluble substance Soluble Part contains a polysaccharide having a carboxyl group (see FIG. 2).

  The fibrous mycelium obtained by the above method can be used as it is as a food or cosmetic material without chemical treatment, and if it is chemically treated and purified and processed, it can be used as a wound for medical products. As a protective coating material, a cell culture sheet for regenerative medicine, and a drug carrier for DDS, it becomes possible to produce a product suitable for the purpose.

[Example 2]
A medium consisting of 200 ml of demineralized water, D-glucose 2%, polypeptone 0.5%, yeast extract 0.3%, malto extract 0.016%, powder agar 2% was prepared at pH 5.6, and then autoclaved at 121 ° C for 15 minutes. Then, 15 ml each was dispensed into 10 previously sterilized petri dishes on a clean bench and cooled to 28 ° C. This is used as the main medium.

  Dispense 7 ml of sterilized liquid medium with the same composition as above, except for powdered agar, into 5 test tubes, and after cooling, use a platinum loop of Geotrichum fragrans CBS127.76 strain on a clean bench. The cells were inoculated and cultured with shaking in a shaker at a temperature of 30 ° C., a shaking speed of 140 rpm, and aerobic conditions for 24 hours. From this pre-cultured bacterial solution, 50 μl of the most viable bacterial solution was dispensed with a micropipette to one medium in a Petri dish, inoculated, spread over the entire surface with a spreader, and culture was started. In addition, the inoculation amount of the precultured bacterial solution may be in the range of approximately 30 μl to 100 μl. The culture conditions were set in a temperature range of 27 ° C. to 32 ° C. with a thermostat and cultured under aerobic conditions. This was performed a total of 5 times.

  The growth time of the mycelium (colony) was maximum when the culture time was 40 to 160 hours, preferably around 70 hours (see FIG. 3).

  In addition, as a result of taking out a sample after 30 hours from the start of culture and observing under a microscope (× 1,000 magnification), there are few places where mycelium is intertwined in a complicated manner as observed in liquid culture, and growth of yeast and Bacillus It was very similar to the situation.

  These facts are thought to be due to the characteristics similar to the dimorphic fungus, which is a major feature of Geotrichum fragrans. In other words, the cells of the genus Geotricum are very similar to filamentous fungi, but they are included in the Ascomycetes yeast family group in molecular phylogeny, so they are different from filamentous fungi such as Aspergius (Koji) and Rhizopus (Tempe). On the solid medium, the growth and proliferation of mycelium does not progress greatly and almost no aerial hyphae are seen. However, the hyphae (segmental conidia) begin to grow at an overwhelming speed in the liquid medium. The ratio is probably hundreds to thousands. Many polysaccharides containing chitin / chitosan are contained in this mycelium. Therefore, in the case of industrial use of the genus Geotricum, a liquid medium is preferable.

[Example 3]
Prepare a medium as in Example 1, dispense 7 ml of each sterilized liquid medium into five test tubes, and after cooling, inoculate one platinum ear inoculation with Geotrichum fragrans CBS164.32 strain on a clean bench. Then, the cells were cultured with shaking in a shaking culture machine at a temperature of 30 ° C., a shaking speed of 140 rpm, and aerobic conditions for 24 hours. From the pre-cultured bacterial solution, 10 ml of the best-growing bacterial cell solution was inoculated into the main culture solution (medium) of a jar fermenter prepared as in Example 1, and the culture was started. In addition, the inoculation amount of the preculture bacterial solution should just be 1 ml or more. The culture conditions were as follows, and each set value was arbitrarily set and combined for a total of 3 times. The aeration amount was 0.3 vvm to 1.5 vvm, the culture solution temperature was in the range of 24 ° C to 32 ° C, and the rotor blades were stirred at 100 rpm to 400 rpm for cultivation.

  The cultivation time was 40 hours to 160 hours, preferably around 90 hours, and the yield was maximum. In addition, what is necessary is just to increase the amount of inoculation suitably when shortening culture | cultivation time. The pH of the bacterial cell liquid after the culture was in the range of 7.0 to 7.3 on average.

  Further, as a result of taking out a sample after 30 hours from the start of culture and observing under a microscope (× 1,000 times), it was easily observed that fibrous mycelia were intertwined and laminated in multiple layers (FIG. 4). The mycelia had an average diameter of about 5 μm and a length of 1,000 μm or more.

  This mycelium culture solution was separated into a culture solution and mycelium using a centrifuge, and further filtered with suction using a filter paper with an aspirator to obtain a mycelium having a wet weight of 86.98 g. Subsequently, it dried at 100 degreeC with the constant temperature dryer for 3 hours. The dry weight of the obtained mycelium was 12.27 g. These values vary slightly depending on the culture conditions. This dried product is added to 1 to 3 M sodium hydroxide or sodium carbonate aqueous solution so as to be 1 to 10 w / v%, heated at 100 to 121 ° C. for 60 to 300 minutes, cooled, and then thoroughly washed with demineralized water. And neutralized. The insoluble material was dried and analyzed by infrared absorption spectroscopy (FT-IR method). As a result, it was confirmed that chitin / chitosan was contained (see FIG. 5).

[Example 4]
In a flask with a capacity of 5,000 ml, 1,000 ml of demineralized water and 5 g of polypeptone were placed, sterilized in an autoclave, cooled to 50 ° C., and then added with 200 g of rice bran fermented with Aspergillus oryzae for 40 hours. It hydrolyzed with the thermostat for 20 hours. After cooling to 28 ° C. as a medium, 1 ml of Geotrichum fragrans CBS127.76 strain pre-cultured in a test tube for 24 hours is inoculated, and the shaking speed is 100 rpm under aerobic conditions in a shaking incubator. The culture was performed with shaking at ˜140 rpm and a temperature of 27 ° C. to 31 ° C. for 30 hours.

  Then, static culture was performed at 28 ° C. with a thermostat. From the time when 30 hours had passed since the start of stationary culture, milky white fungal membranes began to cover the surface of the culture medium. The fungus membrane was gradually laminated thicker as time passed, and grew to a sheet-like substance having a maximum thickness of about 3.5 mm and a diameter of about 150 mm when 240 hours passed. During this time, almost no growth of aerial hyphae was observed (see FIG. 6). This was washed with demineralized water, and medium components other than the mycelium sheet were removed. The physical properties of this sheet also included tensile strength and did not break easily.

From this experiment, it was found that Geotrichum spp. Easily formed into a sheet shape depending on the medium composition and culture conditions. By utilizing this feature, it is possible to easily create a cell culture sheet used in the field of regenerative medicine.
In order to confirm the components, the culture sheet was washed with water, added to 1 to 3 M sodium hydroxide or sodium carbonate aqueous solution so as to be 1 to 10 w / v%, and heated at 100 to 121 ° C. for 60 to 300 minutes. After cooling, it was thoroughly washed with demineralized water and neutralized. As a result of drying and analyzing by infrared absorption spectroscopy (FT-IR method), it was confirmed that chitin / chitosan having an acetylation degree of about 45% was contained (see FIG. 7).

[Example 5]
Five test tubes each containing 7 ml of a sterilized and cooled liquid medium prepared as described in Example 1, with the Geotricam Candidum strain under the trade name SIGMA-54 ordered from the French bacterial cell sales company ETS Andre COQUARD on a clean bench One platinum loop was inoculated, and cultured with shaking in a shaking culture machine at a temperature of 30 ° C., a shaking speed of 140 rpm, and aerobic conditions for 24 hours. From the pre-cultured bacterial solution, 10 ml of the best-growing bacterial cell solution was inoculated into the main culture solution of a jar fermenter prepared as in Example 1, and the culture was started. In addition, the inoculation amount of the preculture bacterial solution should just be 1 ml or more. The culture conditions were as follows, and each set value was arbitrarily set and combined for a total of 3 times. The aeration amount was 0.3 vvm to 1.5 vvm, the culture solution temperature was in the range of 24 ° C to 32 ° C, and the rotor blades were stirred at 100 rpm to 400 rpm for cultivation.

  The cultivation time was 40 hours to 160 hours, preferably around 90 hours, and the yield was maximum. In addition, what is necessary is just to increase the amount of inoculation of a pre-culture | cultivation microbe liquid suitably, when shortening culture | cultivation time. The pH of the bacterial cell solution after the culture was in the range of 6.9 to 7.3 on average.

  In addition, as a result of taking out a sample after 30 hours from the start of culture and observing under a microscope (× 1,000 times), it was easily observed that fibrous mycelia were intertwined and laminated in multiple layers (see FIG. 8). The mycelium had an average diameter of about 5 μm and a length of about 100 μm.

  This mycelium culture solution was separated into a culture solution and mycelium using a centrifuge, and suction filtered with an aspirator using filter paper to obtain a mycelium with a wet weight of 46.95 g. Subsequently, it dried at 100 degreeC with the constant temperature dryer for 3 hours. The dry weight of the obtained mycelium was 6.35 g. These values vary slightly depending on the culture conditions. This dried product was added to 1 to 3 M sodium hydroxide or sodium carbonate aqueous solution so as to be 1 to 10 w / v%, and heated at 100 to 121 ° C. for 60 to 300 minutes. After cooling, it was thoroughly washed with demineralized water and neutralized. As a result of drying and analyzing by infrared absorption spectroscopy (FT-IR method), it was confirmed that chitin / chitosan was contained (see FIG. 9).

[Example 6]
Chitin and chitosan derived from shrimp and sea bream cannot be dissolved or dispersed in water as they are. In order to use it as a cosmetic, a complicated treatment such as dissolving in a special solvent is required, and a treatment not to be toxic to the skin is also required. Chitin is generally dissolved in a combination of dimethylformamide, dimethylacetamide, N-methylpyrrolidone, etc. and lithium chloride, and chitosan is dissolved in an organic acid such as malic acid or acetic acid, or in an acidic solvent such as inorganic acid. I have to let it. Shrimp and persimmon-derived chitin and chitosan dissolved in such a solvent are difficult to use as cosmetics as they are. To be used as a cosmetic, it must be dissolved in a milder solvent. Therefore, there are examples in which chitin is used as an aqueous solution of carboxymethyl chitin, and chitosan is used as an aqueous solution of hydroxypropyl ether synthesized by propylene oxide. However, they cannot be used as cosmetics unless they are prepared to have a very low concentration.

  On the other hand, the fibrous polysaccharide containing chitin / chitosan obtained from the genus Geotricum of the present invention can be easily dispersed in water by taking advantage of its hydrophilic property and can be made into a pulp slurry. (See FIG. 10). By ultra-fine crushing using an ultrasonic crusher or a homogenizer and making it into nanoparticles, it can be easily processed into an emulsion-like, cream-like, or gel-like without using a special solvent. Moreover, since pH value is neutral, it can use as it is. In addition to high skin permeability and extremely high bioactivity, it not only promotes skin metabolism, but also forms a barrier between the epidermis and outside air with sufficient moisture retention and film-forming ability. can do. Therefore, it becomes possible to protect skin and hair from ultraviolet rays and outside air.

[Example 7]
Currently, products made from shrimp and persimmon-derived chitin (poly-N-acetylglucosamine) and processed into non-woven fabrics are used as medical wound protective covering materials in clinical settings. However, the manufacturing process of this nonwoven fabric is extremely complicated, and the fiber of the previous stage must be processed with a spindle using a special solvent. Therefore, the manufacturing cost is high. Further, a special adhesive is required to make the fiber processed with the spindle into a non-woven fabric, and this has been revealed as a problem to be solved, such as a slight loss of biocompatibility.

  On the other hand, the fibrous polysaccharide containing chitin / chitosan obtained from the genus Geotricham of the present invention is eluted in an alkaline solution to elute cytoplasmic components and remove pathogenicity and antigenic components by a known method. And hot water treatment. Further, it can be used as a material for a wound protective covering material by protease treatment, acetylation treatment, or the like, if necessary. In the production of this wound protective dressing, a non-woven fabric manufacturing process using chitin material derived from shrimp and persimmons as a raw material by utilizing the multi-layered structure that is a characteristic of the mycelium of the genus Geotricum and the features rich in hydrophilicity Can be processed into a nonwoven fabric by a simpler process. The production method using CBS127.76 strain is described in detail below.

  First, the cultured mycelium is collected by a known method, and then added to 1 to 3 M sodium hydroxide or sodium carbonate aqueous solution so as to be 1 to 10 w / v%, and 100 to 121 ° C. and 60 to 300. Heat for minutes. By this heating operation, sterilization is performed, and cytoplasm components are separated and eluted from the mycelium, thereby increasing the purity of chitin / chitosan. The pulp slurry-like mycelium cell wall obtained by this alkaline hot water treatment can be obtained as a fibrous multi-layered fibrous chitin / chitosan-containing polysaccharide with a neutral pH value by performing filtration and washing with water several times. It is done. If necessary, a trace amount of protein component is removed by protease treatment or the like.

  Next, acetylation treatment is performed to increase the chitin component. The fibrous chitin / chitosan obtained by the protein removal treatment described above has an average degree of acetylation as high as 60% or more, and can be used as a biomaterial as it is. Is possible. In the acetylation operation, first, the mixture is mixed with demineralized water so as to be 90 w / v% or more. Then, the composition having the required acetylation rate is obtained by chemical treatment similar to a known method (references: chitin chitosan study group, chitin, chitosan handbook: 228-354). Thereafter, it is washed and neutralized with demineralized water to a pH value of 6.8 to 7.1. The segmented spores (segmented conidia) constituting the multilaminate structure that is a characteristic of the mytricium of the genus Geotricum are fibrous having an average diameter of 5 μm and a length of 1,000 μm or more. This multi-layered structure hardly changes even though the fiber diameter is reduced by about 10% even after a long acetylation treatment.

  Next, a fibrous chitin / chitosan-containing polysaccharide having a multi-layered structure obtained by the method as described above is added to demineralized water so as to be in the range of 0.02 w / v% to 1 w / v%, and a mixer is added. Stir well and store in a flat bottom / rectangular stainless steel container open at the top so that the dispersion concentration does not become uneven. Then, using the traditional method of making Japanese paper, the method of scooping out fibrous chitin / chitosan-containing polysaccharides is made in the same way that Japanese paper is used. The scooping device and the scissors of the girder preferably have a structure that has been used since ancient times. If this instrument is rolled several times, it will become a nonwoven fabric, so when the nonwoven fabric has a thickness of 200 μm to 2,000 μm, scooping is stopped and a Teflon (registered trademark) processed stainless steel flat plate is used. Only the non-woven fabric is taken out, spread and naturally dried in a temperature range of 30 ° C. to 80 ° C. or air-dried. A nonwoven fabric is obtained by these operations (see FIG. 11).

It should be noted here that when the nonwoven fabric is completely dehumidified, it loses its elasticity, plasticity and flexibility and its strength is insufficient, so that the moisture content is in the range of 90% to 5%. It is important to prepare. The size of the nonwoven fabric to be created can be adjusted to a maximum of about 1 m ² by appropriately changing the size of the stainless steel container that stores the aqueous solution of fibrous chitin / chitosan-containing polysaccharide and the size of the scooping device. Is possible. Since there are other excellent techniques for manufacturing this nonwoven fabric, the most suitable one may be used. It is important that all these operations be performed under aseptically controlled conditions (clean room, etc.).

[Example 8]
A method for producing a cell culture sheet used in the field of regenerative medicine will be described. The most important biological property required for the cell culture sheet is that there is no pathogenicity or antigenicity. The present inventors have found a method for producing a cell culture sheet that takes advantage of the fibrous chitin / chitosan-containing polysaccharide having a multi-layered structure obtained from the genus Geotricum of the present invention. That is, substances such as proteins and nucleic acids that are the main cause of pathogenicity and antigenicity by treating the fibrous chitin / chitosan-containing polysaccharides having a multi-layered structure with alkaline hot water treatment and further protease treatment as necessary This is a method for obtaining a cell culture sheet excellent in biocompatibility and in-vivo digestibility by increasing the chitin component by chemical treatment that elutes and removes acetylation and further promotes acetylation. The production method using CBS127.76 strain is described in detail below.

  Similar to the method for producing the nonwoven fabric described in Example 7, first, the cultured mycelium was collected by a known method and then 1 to 10 w / v% in 1 to 3 M sodium hydroxide or sodium carbonate aqueous solution. And heated at 100 to 121 ° C. for 60 to 360 minutes. By this heating operation, sterilization is performed and most cytoplasmic components are separated and eluted from the mycelium, and the purity of chitin / chitosan is increased. The pulp slurry-like mycelium cell wall obtained by this alkaline hot water treatment is obtained as a fibrous chitin / chitosan-containing polysaccharide having a multi-layered structure by performing water washing by filtration several times and neutralizing. Moreover, a trace amount protein is also removed by protease treatment as needed.

  Next, acetylation treatment is performed to increase the chitin component. The fibrous chitin / chitosan obtained by the protein removal treatment described above has an average degree of acetylation as high as 60% or more, and can be used as a biomaterial as it is. Is possible. For the acetylation operation, a composition having a required acetylation rate is obtained by chemical treatment similar to a known method (references: chitin chitosan study group, chitin, chitosan handbook: 228-354). The segmented spores (segmented conidia) constituting the multilaminate structure that is a characteristic of the mytricium of the genus Geotricum are fibrous having an average diameter of 5 μm and a length of 1,000 μm or more. This multi-layered structure hardly changes even though the fiber diameter is reduced by about 10% even after a prolonged acetylation treatment.

The fibrous chitin / chitosan-containing polysaccharide obtained by the method as described above is neutralized with water washing to a pH value of 6.8 to 7.1, further centrifuged and dehydrated, and then 1 w / v in demineralized water. Add to a range of 5% to 5% w / v and stir well with a mixer. Subsequently, suction filtration is performed with an aspirator using filter paper by a known method. The suction filtration operation is stopped when the thickness of the sheet is in the range of 100 μm to 5,000 μm, and the sheet is removed from the filter paper. Since the moisture content of the obtained sheet is about 90%, dehumidification operation (drying treatment) is performed with a thermostat until the required moisture content, but the sheet is dried in a temperature range of 30 ° C to 80 ° C. It should be noted here that when the sheet is completely dehumidified, it loses its elasticity, plasticity and flexibility, and its strength is insufficient. Therefore, the sheet is dried and prepared so that the moisture content is in the range of 20% to 8%. It is important to. The surface area of the cell culture sheet can be adjusted to a maximum of about 0.5 m ² by appropriately changing the diameter of the filter (see FIG. 12). It is important that all these operations be performed under aseptically controlled conditions (clean room, etc.).

[Example 9]
Next, verification of whether or not the chitin / chitosan-containing polysaccharide sheet of the present invention has characteristics and functions suitable as a medical wound protective covering material is performed with the cooperation of the Foundation for Advanced Medical Promotion. It was. The mice used in the experiment were 3 inbred C57BL / 6 Cr Slc males (21 g to 23 g body weight) 7 weeks old and reared and managed under SPF conditions. As a material, a chitin / chitosan-containing polysaccharide sheet (hereinafter referred to as “the sheet of the present invention”) prepared in the same manner as described in Examples 7 and 8 was formed into a piece of 8 mm square and weight of 18 mg. Cut and used. As a control, a commercially available shrimp / crab-derived medical material and chitin wound protective material (hereinafter referred to as “commercial sheet”) were cut into the same size and weight and used.

  In order to embed both biomaterials under the skin of the mouse, anesthesia was first performed as a pretreatment, and the entire back of the back was subjected to hair removal treatment. Then, the skin on the left and right sides of the back is incised over 12 mm in width, and the skin is spread with an insulator, and the sheet of the present invention is individually provided on the left side of the back and the commercial sheet on the right side of the back. After being inserted into a flat and spread out flat so as not to be rounded, the incision site was closed and sutured. Thereafter, all three animals were reared and managed under the same conditions in the same breeding cage, followed by follow-up, and sacrificed on the 35th, 63rd, and 91st days, and the surgical site was dissected in vivo. It was verified and confirmed what kind of physiological activity is seen.

  After the operation, all three animals showed no particular changes in food and behavior, and there was no abnormal behavior until the sacrifice. However, from about 5 days after the treatment, swellings, which are inflammatory reactions, occurred as external changes visible in both of the three biomaterials. The degree of swelling gradually progressed and reached a peak at the second week, and it was confirmed that all of them were somewhat smaller on the commercial sheet side. This swelling gradually subsided with the passage of days, and only the scar at the sutured part was 4 weeks after the operation. From these circumstances, the inflammatory reaction is a mild one that does not have a fatal effect on the living body, and it is considered that none of the sheets is toxic to the living body or very little.

  Individuals necropsied 35 days after treatment showed significant differences from controls. About 50% of the sheet was absorbed in the living body at the buried portion of the sheet of the present invention. Above all, a large number of new blood vessels were observed (about 10 times that of a commercially available sheet) toward the buried portion of the sheet (see FIGS. 14 and 20). On the other hand, in the buried part of the control commercial sheet, most of the commercial sheet was not absorbed in the living body and remained almost intact and adhered. In addition, only one new blood vessel was observed toward the buried site (see FIG. 15).

  As a factor causing such a large difference between the two biomaterials, it is considered that a factor that induces the expression of new blood vessels is more significantly contained in the sheet of the present invention than in the commercially available sheet. In addition, the expression of new blood vessels is a self-healing mechanism induced by treatment, and works preferentially in repairing wounds at an early stage. It can be determined that the chitin / chitosan-containing polysaccharide sheet of the present invention has a bioactive ability to promote such a mechanism.

  FIGS. 16 and 17 show the results obtained from an individual who was necropsied on day 63 after the operation. In the buried part of the sheet of the present invention, nearly 100% of the sheet was absorbed into the living body and no adhesion occurred. However, the number of new blood vessels was clearly reduced as compared to the individual autopsied on day 35 (see FIGS. 16 and 20). On the other hand, most of the commercially available sheet remained in the living body without being absorbed in the living body at the buried part of the control commercial sheet, and adhered to the dermis part. In addition, no new blood vessels were observed (see FIG. 17).

  18 and 19 show the results of an individual who was necropsied 91 days after the operation. In the buried part of the sheet of the present invention, the sheet was already absorbed into the living body and no adhesion occurred. New blood vessels were observed to decrease (see FIGS. 18 and 20). On the other hand, about 30% of the commercial sheet remained in the living body without being absorbed in the living body at the buried part of the control commercial sheet, and adhered to the dermis part. In addition, no new blood vessels were observed (see FIG. 19).

  Judging from the results so far, the chitin / chitosan-containing polysaccharide sheet of the present invention has a rate of absorption of 100% or more faster in the living body than the control commercially available sheet, and the toxicity in the living body. Is also not allowed. That is, it is recognized that the biocompatibility (compatibility) is excellent. Furthermore, it was confirmed that about 10 times as much neovascularization ability as a commercially available sheet was provided around 35 days after the sheet was buried (see FIG. 20). The mechanism of these physiological responses will be studied in detail in the future, but in light of the findings so far (the chitin, chitosan handbook: description on pages 324-354, etc.), the immune-inflammatory response of the biological defense mechanism Is considered to be involved.

  First, the absorption mechanism into the living body is poor as a foreign substance due to dendritic cells and macrophages (or lysozyme positive cells) present around cells (tissue) in contact with the chitin / chitosan-containing polysaccharide sheet of the present invention. It is eaten and presented to other immunocompetent cells as an antigen. It is considered that the chitin / chitosan-containing polysaccharide sheet of the present invention is gradually eaten, decomposed and absorbed by these cells.

  Next, regarding the ability to induce neovascularization, the transplantation of the chitin / chitosan-containing polysaccharide sheet of the present invention may cause an inflammatory reaction caused by a foreign substance and activate blood vessel growth factors widely distributed in the living body. Conceivable. The main causative substance of the activation may be chitin / chitosan contained in the sheet of the present invention, other polysaccharides or sugar chains contained in the sheet of the present invention, or may be a complex substance thereof. There is. In addition, when the commercially available sheet | seat used this time was analyzed by the infrared absorption spectrum method (FT-IR), it was chitin chitosan with an acetylation degree of about 56%, and this invention sheet with an acetylation degree of about 63% is significant. There was a big difference. This may be a factor affecting the difference in bioactivity. Next, regarding the mechanism by which the new blood vessels decrease, the sheet of the present invention is absorbed into the living body, granulations at the buried site are newly formed, and wound healing progresses, so that the wound at the buried site is advanced. The existence and role of new blood vessels themselves are no longer necessary, and it is thought that they decreased. In other words, it is thought that the homeostasis of the living body is maintained.

  In conclusion, the chitin / chitosan-containing polysaccharide sheet of the present invention has a feature that surpasses commercially available sheets (medical materials derived from shrimp and crab, chitin wound protective materials) that are currently used in many medical settings. Therefore, it is a biocompatible material suitable for use as a medical device for promoting healing of burns and wounds, and further as a cell culture sheet.

  As described above, the present invention relates to a method for producing a polysaccharide-containing product containing chitin / chitosan using Geotrichum, and as described above, cell culture used in foods, cosmetics, medical products, and regenerative medicine. It can be used as a biomaterial in various fields such as sheets and drug carriers for DDS.

It is a microscope picture (* 1,000 times) of the filamentous mycelium in the liquid culture of Geotrichum fragrans (Geotrichum fragrans) CBS127.76. It is an analysis figure in the infrared absorption spectrum method (FT-IR) of the filamentous mycelium of Geotrichum fragrans (Geotrichum fragrans) CBS127.76. It is the photograph of the mycelium on the solid culture medium of Geotrichum fragrans (Geotrichum fragrans) CBS127.76. It is the photograph (* 1,000 times) of the filamentous mycelium in the liquid culture of Geotrichum fragrans (Geotrichum fragrans) CBS164.32. It is an analysis figure by the infrared absorption spectrum method (FT-IR) of the filamentous mycelium of Geotrichum fragrans (Geotrichum fragrans) CBS164.32. It is a photograph of the sheet-like substance of Geotrichum fragrans CBS127.76. It is an analysis figure of the infrared absorption spectrum method (FT-IR) of the sheet-like substance of Geotrichum fragrans (Geotrichum fragrans) CBS127.76. It is a microscope picture (* 1,000 times) of the filamentous mycelium in the liquid culture of Geotricam candidum SIGMA-54. It is an analysis figure by the infrared absorption spectrum method (FT-IR) of the filamentous mycelium of Geotricam candidum SIGMA-54. It is a photograph of a state where a fibrous mycelium of Geotrichum fragrans CBS127.76 is dispersed in water to form a pulp slurry. It is the photograph of what made the fibrous polysaccharide obtained from Geotrichum fragrans (Geotrichum fragrans) CBS127.76 into a nonwoven fabric. It is a photograph of a fibrous polysaccharide obtained from Geotrichum fragrans CBS127.76 in a sheet form. It is a flowchart which shows the manufacturing process of the polysaccharide containing material containing chitin and chitosan based on one Embodiment of this invention. (A) and (b) are photographs showing the result of observing the state of the treatment site on the 35th day after the operation of embedding the sheet of the present invention in the mouse dorsal subcutaneous tissue. (A) and (b) are photographs showing the results of observing the state of the treatment site on the 35th day after the operation of burying a commercial sheet in the mouse dorsal subcutaneous tissue. It is a photograph which shows the result of having observed the mode of the treatment site | part 63 days after the operation which embeds this invention sheet | seat in a mouse | mouth dorsal subcutaneous tissue. (A) and (b) are photographs showing the result of observing the state of the treatment site on day 63 after the treatment of burying a commercial sheet in the mouse dorsal subcutaneous tissue. It is a photograph which shows the result of having observed the mode of the treatment site on the 91st day after the operation of embedding the sheet of the present invention in the mouse dorsal subcutaneous tissue. It is a photograph which shows the result of having observed the mode of the treatment site on the 91st day after the operation of burying a commercially available sheet in the mouse dorsal subcutaneous tissue. A graph showing the comparison between the results of examining the bioabsorption rate of each sheet and the amount of neovascularization at the treatment site over time after treatment of embedding the sheet of the present invention and the commercially available sheet (control) in the mouse dorsal subcutaneous tissue, respectively. It is.

Claims (23)

  A method for producing a polysaccharide-containing product containing chitin / chitosan by culturing and growing Geotrichum spp.   The production method according to claim 1, comprising a step of culturing and growing Geotrichum in a liquid medium.   The production method according to claim 1, wherein a fibrous mycelium is produced as the polysaccharide-containing material.   The production method according to claim 3, comprising a step of culturing and growing Geotrichum spp. In a liquid medium, and a step of sterilizing the obtained mycelium after separating it from the culture solution.   The manufacturing method of Claim 1 which manufactures the polysaccharide containing material which consists of a cell wall of a mycelium as a polysaccharide containing material.   The production method according to claim 5, wherein a cytoplasmic component is detached from the fibrous mycelium obtained by the method according to claim 4 to produce a polysaccharide-containing material comprising a cell wall of the mycelium.   The manufacturing method of Claim 1 which manufactures the polysaccharide containing material which raised the chitin component by the acetylation process as a polysaccharide containing material.   The manufacturing method of Claim 7 which manufactures the polysaccharide containing material which increased the chitin component by acetylating the polysaccharide containing material which consists of the cell wall of the mycelium obtained by the method of Claim 6.   Geotrichum armillariae, Geotrichum capitatum, Geotrichum linkii, Geotrichum sericeum, Geotrichum citri-aurantii, Geotrichum citri-aurantii, Geotrichum candihum pulmoneum, Geotrichum clavatum, Geotrichum eriense, Geotrichum fermentans, Geotrichum fragrans, and Geotrichum klebahnii species from several species of Geotrichum klebahnii A method for producing a polysaccharide-containing product containing chitin and chitosan.   A wound protection material comprising a polysaccharide-containing material produced using Geotricam Fragrance.   A cell culture sheet comprising a polysaccharide-containing material produced using Geotricam Fragrance.   A method for producing a polysaccharide-containing product containing chitin and chitosan using Geotrichum fragrans CBS127.76 strain.   A method for producing a polysaccharide-containing product containing chitin and chitosan using Geotrichum fragrans strain CBS164.32.   The manufacturing method of the foodstuff which uses the polysaccharide containing material containing chitin and chitosan manufactured using the genus Geotricum as a raw material.   A method for producing a cosmetic using, as a raw material, a polysaccharide-containing material containing chitin / chitosan produced using a genus Geotricum.   A method for producing a wound protection dressing for medical use, using as a material a polysaccharide-containing material containing chitin / chitosan produced using a genus Geotricum.   A method for producing a cell culture sheet using a polysaccharide-containing material containing chitin / chitosan produced using a genus Geotricum as a material.   An edible composition comprising a polysaccharide-containing material produced by the method according to any one of claims 1 to 9, 12, and 13.   A cosmetic composition comprising a polysaccharide-containing material produced by the method according to any one of claims 1 to 9, 12, and 13.   A medical wound protective dressing produced using the polysaccharide-containing material produced by the method according to any one of claims 1 to 9, 12 and 13 as a material.   The cell culture sheet manufactured using the polysaccharide containing material manufactured by the method of any one of Claims 1-9, 12, and 13 as a material.   The drug carrier used for DDS (drug delivery system) manufactured using the polysaccharide containing material manufactured by the method of any one of Claims 1-9, 12 and 13 as a material. A method for producing a drug carrier for use in a drug delivery system (DDS) using a polysaccharide-containing material containing chitin / chitosan produced using a genus Geotricham as a material.