WO2007063854A1

WO2007063854A1 - Method of producing bacterial cellulose

Info

Publication number: WO2007063854A1
Application number: PCT/JP2006/323726
Authority: WO
Inventors: Yutaka Tamai
Original assignee: National University Corporation Hokkaido University
Priority date: 2005-11-29
Filing date: 2006-11-28
Publication date: 2007-06-07
Also published as: JPWO2007063854A1

Abstract

A method of producing a bacterial cellulose whereby the bacterial cellulose can be produced by using a less expensive medium component. According to this method, an aqueous solution of a water-soluble component originating in a mushroom is obtained by pressing or water-extracting a waste mushroom bed remaining after harvesting mushrooms together with mushroom hard tip, or cooking mushroom fruit body in water and collecting the residue. By diluting and sterilizing the above-described aqueous solution, a medium for culturing a cellulose-producing bacterium is prepared. Subsequently, the cellulose-producing bacterium is cultured in this medium to thereby produce the bacterial cellulose.

Description

Specification

Nocteria cellulose production method

Technical field

[0001] The present invention relates to a method for producing nocteria cellulose.

Background art

[0002] Cellulose is the most abundant natural polymer substance on the earth and has been used in various fields since ancient times. Cellulose is the main component of plant cell walls, and most of the cellulose currently used industrially is derived from higher plants.

[0003] On the other hand, some bacteria are known to produce cellulose! Examples of such butterias include the genus Darconoacetobacter, the genus Agrobacterium, the genus Rhizopium, the genus Sartina, the genus Syudomonas, the genus Achromopacter, the genus Alkagenes, the genus Erotactor and the genus Azotopacter. Among these, one of the genus Darconoacetopactor is known to produce a large amount of cellulose outside the cells.

[0004] A kind of the genus Darconoacetopactor produces a gel-like cellulose film on the surface of a medium when cultured. Cellulose produced by such bacteria (hereinafter referred to as “cellulose-producing bacteria”) is called bacterial cellulose.

[0005] Unlike bacterial cellulose, this bacterial cellulose is pure cellulose containing no hemicellulose or lignin. Bacterial cellulose is also known to have a different fiber thickness from plant cellulose. Plant cellulose forms very fine fibers called microfibrils by concentrating many cellulose molecular chains, and these microfibrils are further bundled to form a higher-order structure. On the other hand, for noceria cellulose, the microfibrils of cellulose secreted from cellulose-producing bacteria form a fine network structure with the same thickness. The thickness of hardwood pulp fibers is about 30 μm in diameter, whereas the thickness of bacterial cellulose microfibrils is about 0.1 μm in diameter.

[0006] As described above, bacterial cellulose has characteristics that are not found in plant cellulose! Therefore, various possibilities as industrial materials are expected. For example, Nocteria cell As a method of using loin, its use in separation membranes and medical pads is being studied by taking advantage of the fine network structure and water retention of bacterial cellulose membranes. In addition, by cutting the bacterial cellulose film by mechanical treatment, it can be used as a papermaking raw material. It is known that when bacterial cellulose fibers obtained by mechanical treatment are mixed with wood pulp, the strength and elastic modulus of paper are improved.

[0007] Conventionally, as a method for producing bacterial cellulose, a method using fruit juice, molasses, waste paper, pulp waste liquid or the like as a raw material has been attempted. In these methods, cellulose-producing bacteria are cultured using the raw material as a medium. However, these methods require a pretreatment such as hydrolysis of the polysaccharides contained in the raw material and conversion of the decomposed monosaccharides into sugar alcohols before culturing, resulting in high production costs. There is a problem of becoming.

[0008] Therefore, in order to improve the production efficiency, many researchers have been studying the components of the culture medium for cellulose-producing bacteria. For example, the methods of Patent Document 1 and Patent Document 2 have been reported. In the method of Patent Document 1, animal serum was added to the standard medium, and in the method of Patent Document 2, hydrolyzed collagen was added to the standard medium, thereby successfully increasing the production efficiency of bacterial cellulose. And

[0009] In addition, Non-Patent Document 1 describes that mannitol is effective in producing bacterial cellulose.

[0010] By the way, in the production and processing of mushrooms, a large amount of wastes such as a waste fungus bed after harvesting mushrooms, stone bumps and boiled residue of mushroom fruit bodies are generated in large quantities. These wastes are reused as compost, fuel, livestock feed or livestock bedding. However, these wastes are thought to contain abundant water-soluble components (especially sugars and sugar alcohols) derived from the fungi of mushrooms. Examples of effective use of these water-soluble components Is almost always an unused resource.

Patent Document 1: Japanese Unexamined Patent Publication No. 2000-4895

Patent Document 2: JP-A-2005-80571

Non-Patent Document l: Yasumitsu Uraki et al, Holzforschung, Vol.56 (2002), No.4, 341-347. Disclosure of the Invention

Problems to be solved by the invention [0011] The conventional method for producing bacterial cellulose still has the problem that the production cost is still high.

[0012] The conventional manufacturing method and the manufacturing method described in the patent literature require a standard medium such as a Hestrin-Schramm medium (hereinafter abbreviated as "HS medium"). Media must be purchased or prepared. The raw materials (carbon source), serum, hydrolyzed collagen, etc. must also be purchased or prepared. Such medium constituents are used in large quantities and are expensive, and this increases the manufacturing cost of pacteria cellulose. Therefore, if one of these medium constituents can be used at a low price, it can greatly contribute to the reduction of manufacturing costs.

[0013] An object of the present invention is to provide a method for producing bacterial cellulose that can produce bacterial cellulose at low cost and a culture medium for culturing cellulose-producing bacteria used therefor.

Means for solving the problem

The first of the present invention relates to the following method for producing bacterial cellulose.

[1] A method for producing bacterial cellulose, comprising a step of culturing a cellulose-producing bacterium in a medium containing a processed fruit body or mycelium of a mushroom.

[2] The method for producing bacterial cellulose according to [1], wherein the treated product is a pressed or extracted liquid of a fruit body or mycelium of a mushroom.

[3] The production of bacterial cellulose according to [1], wherein the treated product is any one of a mushroom waste fungus bed compressed solution, a mushroom waste fungus bed extract, a mushroom stone squeeze solution, or a mushroom boiled residue solution. Method.

[4] The method for producing bacterial cellulose according to any one of [1] to [3], wherein the mushroom is Pleurotus cornucopiae.

[5] The method for producing bacterial cellulose according to any one of [1] to [3], wherein the cellulose-producing bacterium is Gluconacetobacter xylinus.

[6] The method for producing bacterial cellulose according to any one of [1] to [3], wherein the treated product contains mannitol. [0015] The second of the present invention relates to the following culture medium for culturing cellulose-producing bacteria.

[7] A culture medium for culturing cellulose-producing bacteria, comprising a processed product of mushroom fruit bodies or mycelium. The invention's effect

[0016] According to the present invention, bacterial cellulose can be produced at low cost.

[0017] In the present invention, as a raw material of a medium for producing bacterial cellulose, wastes produced during mushroom production and caking can be used. Since these wastes can be procured almost free of charge from the producer or processor, the cost of raw materials can be kept as low as possible.

[0018] Further, according to the present invention, waste produced during the production and processing of mushrooms such as a waste fungus bed can be effectively used for IJ.

Brief Description of Drawings

[0019] [Fig. 1] Graph showing the results of analysis of water-soluble saccharides in each medium.

[Figure 2] Graph showing the yield of bacterial cellulose in each medium

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail.

[0021] The present inventors paid attention to water-soluble components remaining in wastes during the production and processing of mushrooms as raw materials for the production of nocteria cellulose. Accordingly, the present inventors have completed the present invention as a result of intensive research that it is impossible to produce bacterial cellulose using a water-soluble component in waste as a raw material.

[0022] The method for producing bacterial cellulose according to the present invention is characterized by culturing cellulose-producing bacteria in a medium containing a treated product obtained from mushrooms (here, "treated product" means water-soluble). What was obtained as a result of applying the component to the mushroom was obtained. Specifically, a solution containing a water-soluble component derived from a mushroom (for example, a compressed solution or an extract) or a dried product of a water-soluble component derived from a mushroom (for example, a product obtained by drying a compressed solution or an extract) Take as an example.

[0023] In this specification, the term "mushroom" does not mean "a body of a macroscopic size of ascomycetes or basidiomycetes" or "ascomycetes and basidiomycetes forming a large fruiting body" Used to mean “fungi”. That is, “mushroom” in the present specification includes not only fruit bodies but also mycelia. [0024] The method for obtaining a processed product of mushroom is not particularly limited as long as water-soluble components such as mannitol and glucose can be obtained from mushrooms. The treated product may be a dry product or an aqueous solution, but an aqueous solution is preferable from the viewpoint of work efficiency. That is, the pressing liquid obtained by the pressing method, the extracting liquid obtained by the water extraction method, or the like can be used as a processed product. By using these simple methods, human costs can be suppressed. When the processed product is obtained as a dry product, it can be treated in the same manner as the above-mentioned pressing solution or extract by making it into an aqueous solution.

[0025] As a specific method for obtaining such a treated product, for example, a method for obtaining a compressed liquid by pressing a waste fungus bed after harvesting mushrooms, extraction from the waste fungus bed with water (cold water or hot water). And a method of obtaining an extract, a method of obtaining a squeezed solution by squeezing a stone tip remaining on a fungus bed after harvesting mushrooms, and a method of obtaining a simmered juice (boiled residue) by boiling a mushroom fruit body. . In this case, the pressed solution, the extract solution, and the boiled residue solution correspond to the processed product. The waste fungus bed contains not only mushroom mycelia but also oga powder and bran, etc., but it may be a processed product of the entire waste fungus bed. Of course, it does not matter if it is a bed after inoculation with the inoculum and before harvesting the mushrooms.

[0026] The treated product obtained by the above method has different power depending on the type of mushroom to be used and the method of obtaining the treated product. Water-soluble components such as mannitol, glycerol, glucose, fructose, trehalose, arabitol and inositol (Such as sugar and sugar alcohol). These water-soluble components are contained in many mushrooms, although their amounts and proportions vary depending on the species (Journal of Japanese Food Industry Association, Vol.31, No.12, 765-771). Among them, it is preferable that the ratio of mannitol, glycerol and glucose is large, and particularly the ratio of mannitol is large. As shown in Non-Patent Document 1, a medium containing mantol is considered suitable for production of bacterial cellulose. In fact, the method for producing bacterial cellulose according to the present invention is generally used, and it has been shown that the yield of bacterial cellulose is increased as compared with the method using HS medium (see Examples).

[0027] The treated product obtained by the above method can be used as a medium as it is simply by adjusting the concentration or sterilizing. The concentration of the medium is the conditions under which the cultivating cellulose-producing bacteria can grow May be selected as appropriate. For example, when culturing dalconoacetopacter xylinus, the Brix concentration should be 3.4%, which is equal to HS medium. As long as no growth failure occurs, the Brix concentration of the medium is not particularly limited.

[0028] The medium according to the present invention includes a mushroom processed product, or a concentration-adjusted or sterilized product thereof, but may further include an optional component. Examples of optional components include a carbon source (eg, glucose), a nitrogen source (eg, amino acid), a metal ion source (eg, calcium salt hydrate), vitamins (eg, riboflavin, etc.) ) And the like, which may be added to the culture medium for microbial culture. The medium according to the present invention may be mixed with a medium such as an HS medium.

[0029] In the present invention, the type of mushrooms to be treated is not particularly limited as long as the processed product can be obtained, but it is an edible mushroom because a boiled mushroom residue can be used. It is preferable. Moreover, since a waste mushroom bed and a stone thrust can also obtain a processed material, it is more preferable that it is a cultivation mushroom. Examples of edible mushrooms include Tamogitake (Pleurot us cornucopiae: uolden Oyster), Enokitake (Flammulina velutipes: Velvet Foot), Gifutake (Pleurotus ostreatus: Oyster Mushroom), and Shirotamogitake (Hypsizygus Elumyus). .

[0030] The type of cellulose-producing bacterium used in the present invention is not particularly limited as long as it can produce cellulose in the medium. Examples include bacteria belonging to the genera Darconoacetobacter, Agrobacterium, Rhizopium, Sartina, Syudomonas, Achromopacter, Alkaligenes, Elopacter, Azotopacter, and the like. Of these, Gluconacetobacter xylinus (former name: AcetoDacter xyiinum).

[0031] Next, the culture method will be described.

[0032] As a culture method, a normal culture method for bacteria can be applied. For example, static culture, shaking culture or aeration and agitation culture can be mentioned. As the culture operation method, for example, a batch culture method, a fed-batch culture method, a repeated batch culture method or a continuous culture method can be used. As the stirring means, for example, an impeller (stirring blade), an air lift type culture device, a pump-driven circulation of culture broth, etc. may be used alone or in combination. Can do.

[0033] As the culture apparatus, a culture apparatus for normal bacteria can be applied. For example, a jar armor with a stirrer that can keep the inside of the tank at a constant temperature can be used.

[0034] The culture conditions may be appropriately selected so that the cultivating cellulose-producing bacteria can grow. For example, when culturing darconoacetopacter xylinus, the culture temperature may be room temperature, for example, 10 to 40 ° C., preferably 25 to 35 ° C. The pH of the medium should be in the range of 3-7, preferably 4-6. However, the processed product derived from mushrooms in the aqueous solution is weakly acidic (pH 5.0-5.5), so it remains in the dull state as it is. It is suitable for culturing Conoacetobacter-1 'xylinus. Of course, adjust the pH with any alkali and acid.

[0035] The culture period may be a normal culture period. For example, when using darconoacetopactor xylinus, a bacterial cellulose film on a gel can be obtained in about 7 days.

[0036] The bacterial cell mouth produced by the method for producing bacterial cellulose according to the present invention may be recovered as it is, or may be subjected to a treatment for removing impurities contained in the recovered material. Examples of the method for removing impurities include washing with water, pressure dehydration, dilute acid washing, alkali washing, treatment with a bleaching agent such as sodium hypochlorite or hydrogen peroxide, and a lytic enzyme such as lysozyme. Treatment with a surfactant, treatment with a surfactant such as sodium lauryl sulfate or deoxycholate, or heat washing may be performed alone or in combination.

[0037] The method for producing bacterial cellulose according to the present invention can use mushroom production and waste from the cultivation as a raw material of the medium. Since these wastes can be procured almost free of charge from the producer or processor, the raw material costs can be kept as low as possible. In addition, mushrooms are cultivated all year round, so each waste can be procured stably. Furthermore, since medium preparation from waste can be performed by simple operations such as pressing and water extraction, human costs can be kept low. The residue after the medium is prepared can be used as it is for reuse of conventional waste such as compost, fuel, livestock feed and livestock bedding. [0038] In addition, the method for producing bacterial cellulose according to the present invention includes the reduction of the waste transportation cost when the mushroom production and the waste at the time of Karoe are used for the above-mentioned conventional waste reuse, In addition, it is possible to improve the storage stability of the waste. The water content of the waste fungus bed is about 70%, and the water content of stone heads and fruit bodies is about 90%. However, in order to use these wastes for the above-mentioned reuse applications, the transportation cost and the preservability are also important. Considering that it is desirable to remove moisture from these wastes. In the method for producing bacterial cellulose according to the present invention, by obtaining a treated product by a pressing method, it is possible to remove moisture from the waste without hindering reuse of the waste.

Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example

[0040] (Preparation of medium)

The raw material used for the treatment was waste bed after harvest of Tamogitake (Kalamatsuga powder + bran), Tamogitake crest and fruit body of Tamogitake. The treated product was obtained as an aqueous solution from the waste microbial bed by pressing, cold water extraction and hot water extraction, pressing from the stone tip, and boiling from the fruit body. The resulting treated product was adjusted to a Brix concentration of 3.4% so that the Brix concentration was equal to the HS medium used as a control. A medium obtained by further sterilizing the processed product obtained by the above method was used.

[0041] Hereinafter, a method for preparing a medium having each raw material strength will be described more specifically.

[0042] First, the culture medium is prepared by squeezing the waste bacteria bed. The waste microbial bed after harvesting Tamogitake was squeezed at about 500 kN to obtain a squeezed solution. The obtained compressed solution was filtered, the brick concentration was adjusted to 3.4%, and sterilized with an autoclave to obtain a medium.

[0043] Next, it is a case where the culture medium is prepared by extracting the waste fungus bed with cold water. Tamogitake Deionized water was added to the harvested fungus bed and stirred for 1 hour at room temperature. The obtained extract was filtered, the Brix concentration was adjusted to 3.4%, and sterilized by autoclave to obtain a medium.

[0044] Next, it is a case where the culture medium is prepared by hot water extraction of the waste fungus bed. Tamogitake Deionized water was added to the harvested fungus bed and boiled for 1 hour. The resulting extract is filtered and filtered. The medium was obtained by adjusting the Lix concentration to 3.4% and sterilizing with autoclave.

[0045] Next, a culture medium is prepared by squeezing a stone tip. After harvesting the bamboo shoots, the stone bumps remaining on the fungus bed were crushed with a mixer. The crushed stone bump was squeezed to obtain a squeezed liquid. The obtained compressed solution was filtered, the Brix concentration was adjusted to 3.4%, and sterilized with an autoclave to obtain a medium.

[0046] Finally, a case where the medium is prepared by boiling the fruit body in water. Boiled soup produced when boiled tamamotake was obtained. The resulting broth was filtered, the Brix concentration was adjusted to 3.4%, and sterilized with an autoclave to obtain a medium.

FIG. 1 is a graph showing the results of sugar analysis for each medium. As a result of sugar analysis by high performance liquid chromatography (HPLC), it was found that each medium contained mannitol, glycerol, dalcose, fructose, trehalose, arabitol, inositol and the like. Depending on the type of waste and the treatment method, the concentration of each sugar and sugar alcohol was slightly different, but the composition was almost the same. In all the media, mannitol had the largest content. The total content of the above seven sugars in each medium adjusted to the same Brix concentration is 1.2% (bacteria bed press), 1.0% (cold water extraction), 1.1% (hot water extraction), 0.9% (stone bump press) and 1.1% (boiled residue liquid). As a control, the strength of the sugar analysis was also obtained for the extract of only kala maggot powder.

[0048] (culture)

As a cellulose-producing bacterium, darconoacetobacter xylinus ATCC-10245 strain was used. Pre-culture was performed using HS agar medium, and then one platinum loop of the pre-culture liquid was inoculated into 40 mL of each medium in a deep bottom petri dish (diameter 9 cm). As a control experiment, 40 mL of HS medium (glucose 2.0%, peptone 0.5%, yeast extract 0.5%, disodium phosphate 0.15%, and ken Acid 0.27% dissolved in sterile water: pH = 6.0) was also inoculated. The culture temperature was 29 ° C, the culture period was 1 week, and stationary culture was performed.

[0049] (Quantification of bacterial cellulose)

Bacterial cellulose membrane formed on the surface of the culture medium is separated, and 1M sodium hydroxide The solution was neutralized after boiling, dried in an oven at 105 ° C for 15 hours, and weighed. Figure 2 is a graph showing the results. The yield of bacterial cellulose in petri dishes cultured in the above media was 1.2 times (bacterial bed press), 1.2 times (cold water extraction), 1.1 times (hot water extraction) of the HS medium petri dishes in the control experiments. ), 2.8 times (stone squeezed) and 1.7 times (boiled residue). From these data, it was estimated that bacterial cellulose having a residue 1 ton force of 0.2 to 2.0 kg could be produced.

[0050] Further, after culturing, when the total content of the seven sugars in each medium derived from the fungus bed was prayed, 0.28% (bacteria bed pressing), 0.21% (cold water extraction), 0.19% (hot water) Extraction), and various sugars were significantly reduced compared to the medium before the culture. Furthermore, although data are not shown here, man-tol, glucose and inositol were consumed almost entirely (over 95%) in 7 days of culture. These results suggest that the water-soluble components remaining in the waste generated during mushroom production and processing are effective for the production of bacterial cellulose.

[0051] Nov. 29 29, Japanese Patent Application No. 2005-344633 The contents of the description, drawings and abstract contained in this application are all incorporated herein by reference. This application claims priority based on that application.

Industrial applicability

[0052] According to the present invention, the production cost of bacterial cellulose can be reduced, so that it can be used in various applications such as separation membranes, medical's cosmetic pads, speaker cones, cellulose film-forming paints, thickeners, paper, and composite materials. Therefore, bacterial cellulose can be provided at a low cost.

Claims

The scope of the claims

[I] A method for producing bacterial cellulose comprising a step of culturing a cellulose-producing bacterium in a medium containing a processed product of mushroom fruit bodies or mycelium.

[2] The method for producing bacterial cellulose according to claim 1, wherein the treated product is a pressed or extracted liquid of a fruit body or mycelium of a mushroom.

[3] The bacterial cellulose according to claim 1, wherein the treated product is any one of a mushroom waste fungus bed compressed solution, a mushroom waste fungus bed extract, a mushroom stone squeeze solution, or a mushroom boiled residue solution. The manufacturing method.

[4] The method for producing bacterial cellulose according to claim 1, wherein the mushroom is Pleurotus cornucopiae.

5. The method for producing bacterial cellulose according to claim 2, wherein the mushroom is Pleurotus cornucopiae.

6. The method for producing bacterial cellulose according to claim 3, wherein the mushroom is Pleurotus cornucopiae.

[7] The method for producing bacterial cellulose according to [1], wherein the cellulose-producing bacterium is Gluconacetobacter xylinus.

[8] The method for producing bacterial cellulose according to [2], wherein the cellulose-producing bacterium is Gluconacetobacter xylinus.

[9] The method for producing bacterial cellulose according to claim 3, wherein the cellulose-producing bacterium is Gluconacetobacter xylinus.

10. The method for producing bacterial cellulose according to claim 1, wherein the treated product contains mannitol.

[I I] The method for producing bacterial cellulose according to claim 2, wherein the treated product contains mannitol.

12. The method for producing bacterial cellulose according to claim 3, wherein the treated product contains mannitol.

[13] A culture medium for culturing cellulose-producing bacteria, comprising a processed product of mushroom fruit bodies or mycelium.