JP3803443B2 - Method for producing bacterial cellulose filament and culture apparatus used therefor - Google Patents

Description

【００４５】
本発明におけるバクテリアセルロース繊維の引張強度は、レーヨン繊維及びアセテート繊維の同様の試験方法における引張強度が、それぞれ1.7 〜5.2g/ デニール及び1.2 〜1.4g/ デニール程度であることから、アセテート繊維の引張強度よりは優れ、レーヨン繊維の引張強度と同等であった。
【００４６】
なお、上記表１における引張強度測定における引張試験は、インストロン試験機（東洋測容器株式会社製ＵＴＭ−１）を用い、初期荷重： 2.5ｇ、引張速度： 2mm/min、試験長：25mmの条件下で、含水率３％に調整した試験片を用いて行った。
【００４７】
【発明の効果】
本発明の方法では、従来、フイルム状又は懸濁液状としてのみ分離回収されていた微生物の生産するバクテリアセルースを、バクテリアセルロースフイラメントとして分離回収すること初めて可能となった。
【００４８】
しかも、本発明の方法により得られたバクテリアセルロースフイラメントは、適度の緊張下で培養されるため、優れた分子配向性及び引張強度を有するとことから、さらに新たな機能の付与が可能できることも大きな特徴である。
【００４９】
また、本発明はセルロース源を含む各種の食品廃棄物から機能性セルロースフィラメントをバイオ的手法で直接に培養育成に合わせて連続的に再構築するために応用可能と考えられることから、その工業的意義は極めて大である。
【図面の簡単な説明】
【図１】バクテリアセルロースフイラメントの製造に用いる培養装置を説明する模式図である。
【符号の説明】
１．巻き取りロール
２．ＳＤＳ水溶液通過ロール
３．ＳＤＳ水溶液槽
４．無菌チャンバー
５．ピックアップロール
６．培養液供給槽
７．培養液
８．平板状培養容器
９．ピックアップされているバクテリアセルロース
10．培養液仕切板
11．培養液供給チューブ[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for separating and recovering bacterial cellulose produced by a microorganism from a culture solution. In particular, the present invention separates a gelatinous bacterial cellulose thin layer formed in an upper layer of the culture solution directly from the culture layer, thereby separating it as a bacterial cellulose filament. The present invention relates to a method for producing bacterial cellulose filaments to be recovered and a culture apparatus used therefor.
[0002]
[Prior art]
Cellulose is the main component of plant cell walls, and as a raw material for paper and pulp, cellulose of wood, which is a higher plant, is widely used as natural cellulose.
[0003]
However, in addition to cellulose produced by higher plants, bacteria, seaweed, and sea squirt are known as sources of cellulose, but in particular, certain types of Acetobacter have a cellulose membrane in a medium containing carbohydrates. Since reporting its formation, this system has been noted and studied as a biosynthetic model.
[0004]
By the way, bacterial cellulose produced by acetic acid bacteria is discharged out of the cells as pure cellulose, but has a fine structure, high purity as cellulose, high Young's modulus, and high biodegradability. In addition to medical applications such as artificial skin, etc., it is known to be used for electrical applications such as acoustic diaphragms (speaker cones). Only use was seen.
[0005]
On the other hand, as a method for separating and recovering bacterial cellulose produced by microorganisms, a method for collecting gel-like cellulosic substances produced by microorganisms as a disaggregated product having excellent dispersibility (Japanese Patent Laid-Open No. 61-113601), cellulose produced by microorganisms A cellulosic material obtained by drying a filtrate of a suspension of a soluble material (Japanese Patent Laid-Open No. Sho 62-175190), microbial treatment of cellulose-producing bacteria and a cellulose-containing mixture after ultrasonic treatment There is known a method for separating and recovering by a method (Japanese Patent Laid-Open No. 63-68093), but a method for separating and recovering bacterial cellulose produced from a culture vessel directly as a bacterial cellulose filament has never been known.
[0006]
Therefore, if bacterial cellulose produced by microorganisms can be separated and recovered in a form other than the filtrate or sheet of suspension, for example, in the form of fibers, a new application is expected.
[0007]
[Problems to be solved by the invention]
Therefore, as a result of various studies on the method for separating and recovering bacterial cellulose, the present inventors have found that the culture medium upper layer in a plate-shaped culture medium container having a plurality of culture medium partition plates provided parallel to the filament convergence direction. A plurality of gelled bacterial cellulose thin layers accumulated on the substrate are treated with an aqueous solution of sodium dodecyl sulfate (hereinafter abbreviated as “SDS”) via a pick-up roll, and then continuously converged into a filament shape under tension by a take-up roll. The present inventors have found that it is possible to produce a bacterial cellulose filament characterized by the above.
[0008]
Accordingly, a first object of the present invention is to provide a method for producing bacterial cellulose filaments, in which bacterial cellulose obtained by stationary culture such as acetic acid bacteria is separated and recovered directly from bacterial cell culture medium as bacterial cellulose filaments. . Furthermore, the second object of the present invention is to provide a plate-like culture container that is directly used in the method for producing bacterial cellulose filaments.
[0009]
[Means for Solving the Problems]
The first problem of the present invention is that a plurality of gelled bacterial cellulose thin layers accumulated in a culture medium upper layer in a flat culture vessel having a plurality of culture solution partition plates provided parallel to the filament convergence direction. This was solved by a method for producing a bacterial cellulose filament, characterized in that after being treated with an aqueous SDS solution via a pick-up roll, it was continuously converged into a filament shape under tension by a take-up roll.
[0010]
Further, the second problem described above has been solved by a bacterial cellulose culture apparatus characterized by using a plate-like culture vessel having a plurality of culture liquid partition plates provided in parallel to the filament convergence direction.
[0011]
Hereinafter, the present invention will be described in detail.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
As the cellulose-producing bacterium used in the present invention, conventionally known Acetobacter xylinum or the like can be used.
[0013]
Similarly, a Hestrin-schrum medium using glucose as a carbon source can be used as the medium. For example, glucose 2.0%, peptone 0.5%, yeast extract 0.5%, phosphate A medium comprising 0.15% disodium salt, 0.27% citric acid, and 0.2 to 2.0% by weight of carboxymethyl cellulose can be exemplified.
[0014]
As the culture temperature, static culture is performed at a temperature of about 28 ° C. at which the cellulose production rate is maximum. After culturing for a predetermined period, the bacterial cellulose filament of the present invention can be obtained by treating the bacterial cellulose filament obtained by a conventional method with an aqueous SDS solution under tension and then converging as a cellulose filament.
[0015]
Next, a specific method for producing the bacterial cellulose filament of the present invention will be described together with an outline of the culture apparatus.
[0016]
The stationary culture apparatus used in the present invention is as shown in FIG. 1, and its main parts are a culture solution supply tank (6), a stationary culture container (8), an SDS aqueous solution tank (3), and a winding. Although it is composed of a take-up roll (1), these devices are housed in a sterile chamber (4) having a filter to prevent contamination by various bacteria.
[0017]
As a stationary culture vessel used for cultivation of bacterial cellulose by acetic acid bacteria, etc., a rectangular plate-shaped stationary culture vessel (8) made of stainless steel or plastic having a width of 10 to 20 cm, a length of 40 cm, and a depth of about 7 mm is used. Can be used.
[0018]
The flat stationary culture vessel (8) has a plurality of culture liquid partition plates (10) provided in parallel to the filament convergence direction.
[0019]
The interval between the culture medium partition plates (10) is appropriately determined according to the thickness of the target bacterial cellulose filament.
[0020]
In addition, a culture solution supply tank (6) is provided on the upper end of one end of the culture vessel via a culture solution supply tube (11), and a pickup roll (5) and an SDS aqueous solution passing roll (2) are provided at the other end. And a winding roll (1) attached with a winder capable of converging at a speed of 1 to 15 mm per hour.
[0021]
The culture solution (7) is continuously added from the culture solution supply tank (6) provided at the top of the stationary culture vessel so that the amount of the culture solution in the culture vessel (8) is always about 3 to 4 mm deep. Then, static culture is performed, and bacterial cellulose is accumulated on the upper layer of each culture solution divided into a plurality of culture solution partition plates.
[0022]
Here, the culture medium partition plate (10) provided in parallel to the filament convergence direction of the flat culture vessel (8) converges the gel-like bacterial cellulose thin layer formed on the upper layer of the culture medium into a filament shape. Thus, the thickness of the bacterial cellulose filament for separation and recovery is adjusted.
[0023]
Next, the plurality of gelled bacterial cellulose thin layers (9) accumulated in the upper layer of the culture solution are converged as filaments by the take-up roll (1) through the pick-up roll (5) and the SDS aqueous solution passing roll (2).
[0024]
At that time, it is important to synchronize the growth rate with the pick-up roll (5) of the gel-like bacterial cellulose thin layer at a speed of about 16 mm per hour and then 3 to 5 mm per hour. .
[0025]
The SDS aqueous solution tank (3) is for denaturing proteins in the separated and collected bacterial cellulose, sterilizing the bacteria, and removing the added culture, and a 2% SDS aqueous solution is usually used.
[0026]
Usually, bacterial cellulose is two-dimensionally formed into a net-like surface, and orientation cannot be expected. However, by culturing under moderate tension, bacterial cellulose filaments with a certain degree of orientation and tensile strength can be obtained. be able to.
[0027]
In the present invention, the reason why a bacterial cellulose filament having an oriented and excellent tensile strength is obtained is clear from the observation result by a scanning electron micrograph, but the upper layer of the culture solution under moderate tension while culturing from the culture solution. It is considered that a filament excellent in molecular orientation and tensile strength can be obtained by continuously winding up the cellulose layer formed in (1).
[0028]
In the flat plate culture vessel used in the present invention, the average thickness of the bacterial cellulose filaments separated and recovered was about 100 denier, so that the bacterial cellulose is converged and separated and recovered as a filament having a thickness of 10 denier. In other words, it is necessary to divide the culture solution into at least about 10 mm width as the interval between the culture solution partition plates (10) in the flat plate culture vessel.
[0029]
As described above, in the present invention, the culture solution is divided by a plurality of culture solution partition plates provided in parallel to the filament convergence direction, and the gelatinous bacterial cellulose thin layer generated on each divided culture solution upper layer is formed. By collecting and converging continuously with a winding roll in a wet state, a culture product such as acetic acid bacteria can be directly separated and recovered as a filament.
[0030]
In addition, the obtained bacterial cellulose filament can be made into a bacterial cellulose fiber by performing a purification treatment, a distraction treatment, and a tension drying by a conventional method.
[0031]
Hereinafter, the present invention will be described in more detail with reference to examples.
[0032]
【Example】
1. Culture medium composition of bacterial cells and culture medium of hestrin-schram (glucose 2.0%, peptone 0.5%, yeast extract 0.5%, disodium phosphate 0.15%, citric acid 0.27%, carboxymethylcellulose 0.2-2.0% by weight Acetobacter xylinum (bacterial cell number: ATCC10245) was inoculated at a temperature of 28 ° C. into the above-described flat plate static culture container of the present invention.
[0033]
As a static culture container, use a rectangular culture apparatus (a large culture container with a length of 38 cm and a width of 20 cm, and a small culture container with a length of 38 cm and a width of 10 cm) with a culture volume ranging from 150 ml to 300 ml. The culture solution is supplied from the culture solution supply tank so that the depth of the culture solution is always 3 to 4 mm, and air for continuous culture is aseptically supplied during the period (2 weeks). went.
[0034]
The amount of the medium used was 300 ml at the beginning for the large culture container, the final 375 ml by dropping halfway, the first 150 ml for the small culture container, and the final 200 ml by dropping halfway.
[0035]
2. Separation and recovery of the produced bacterial cellulose As the winding speed of the thin gelatinous bacterial cellulose layer formed on the upper layer of each culture broth, convergence is started by the winding roll at a slow speed (16 mm / 1 hour) at first, After that, it converged by a winding roll at a stable and high speed (4 mm / 1 hour) according to the growth speed.
[0036]
Subsequently, in order to denature proteins, sterilize bacteria, and remove added culture, 2% (weight / volume) aqueous SDS solution was passed through a thin gel-like bacterial cellulose filament and converged by a winding roll.
[0037]
3. Purification of bacterial cellulose filament separated and recovered Bacterial cellulose filaments converged by a take-up roll as described above are stretched by human power and boiled for 2 hours, then boiled again in 4% sodium hydroxide aqueous solution for 1.5 hours, followed by alkali removal. For this purpose, the sample was sufficiently washed with distilled water (samples 1 to 5) or sufficiently washed with a 10 (v / v)% ethylene glycol aqueous solution (samples 6 to 8). In addition, the sample sufficiently washed with distilled water was further treated with a 10% ethylene glycol aqueous solution at room temperature (samples 9 to 12).
[0038]
Each sample was dried under room temperature tension after the above treatment to obtain cellulose filament fibers.
[0039]
The fiber yield of the bacterial cellulose produced by the above examples (yield after 6 days after the induction period of 2 days) was 7.5 g in the large culture container (length 38 cm, width 20 cm), and the small culture container (long It was 3.2 g at a length of 38 cm and a width of 10 cm).
[0040]
In addition, when 14 days of culturing was performed with an induction period of 2 days, 9.0 g (the medium volume starts at 300 ml and finally 500 ml) in the large culture container, and 5.1 g (similarly in the small culture container) The amount of the medium was 150 ml, and the cultivation was started, and the final yield was 300 ml).
[0041]
In the conventional sown culture method, even if the medium volume is 500 ml, the fiber yield of bacterial cellulose is only about 0.8 to 1.0 g. Therefore, according to the method of the present invention, it can be said that the fiber yield of bacterial cellulose is quite high. .
[0042]
4). Properties of Bacterial Cellulose Filament Properties of bacterial cellulose filaments obtained by the method of the present invention are shown in Table 1. As is apparent from Table 1, the fibers obtained from the bacterial cellulose filaments of the present invention have an average tensile strength of 2.55 g / denier in a normal washed sample (samples 1 to 5), and a washed sample with 10% ethylene glycol aqueous solution (sample). In 6-8), it was 1.50 g / denier, and after washing with 10% ethylene glycol aqueous solution, it was 1.48 g / denier. In addition, in the ethylene glycol-treated sample, although the denier value of the obtained filament increased about twice as much as that of the water-washed sample, the molecular arrangement in the filament was slightly due to the residual ethylene glycol in the cellulose matrix. It was speculated that it was disturbed.
[0043]
Further, the Young's modulus of the filament showed the same tendency as the average tensile strength.
[0044]
[Table 1]

[0045]
The tensile strength of bacterial cellulose fiber in the present invention is that the tensile strength in the same test method for rayon fiber and acetate fiber is about 1.7 to 5.2 g / denier and 1.2 to 1.4 g / denier, respectively. It was superior to the strength and equivalent to the tensile strength of rayon fiber.
[0046]
In addition, the tensile test in the above-mentioned tensile strength measurement in Table 1 uses an Instron testing machine (UTM-1 manufactured by Toyo Sokki Co., Ltd.), initial load: 2.5 g, tensile speed: 2 mm / min, test length: 25 mm Under the conditions, a test piece adjusted to a moisture content of 3% was used.
[0047]
【The invention's effect】
According to the method of the present invention, it has become possible for the first time to separate and recover bacterial cellulose produced by microorganisms, which has been conventionally separated and recovered only as a film or suspension, as a bacterial cellulose filament.
[0048]
Moreover, since the bacterial cellulose filament obtained by the method of the present invention is cultured under moderate tension, it has excellent molecular orientation and tensile strength, so it is also possible to add new functions. It is a feature.
[0049]
In addition, since the present invention is considered to be applicable for continuously reconstructing functional cellulose filaments from various food wastes containing a cellulose source by a biotechnological method according to culture cultivation, Significance is extremely great.
[Brief description of the drawings]
FIG. 1 is a schematic diagram for explaining a culture apparatus used for production of bacterial cellulose filaments.
[Explanation of symbols]
1. 1. Winding roll 2. SDS aqueous solution passing roll 3. SDS aqueous solution tank 4. Aseptic chamber 5. Pickup roll 6. Culture solution supply tank Culture solution 8 8. Plate culture vessel Bacterial cellulose picked up
Ten. Culture medium divider
11． Culture solution supply tube

Claims (2)

  Bacterial cellulose-producing bacteria are cultured in a plate-like culture vessel having a plurality of culture solution partition plates provided parallel to the filament convergence direction, and a plurality of gelled bacterial cellulose thin layers accumulated in the upper layer of the culture solution are picked up. A method for producing bacterial cellulose filaments, characterized in that after treatment with a sodium dodecyl sulfate aqueous solution through a roll, the filament is continuously converged into a filament shape under tension by a winding roll. A flat plate culture vessel having a plurality of culture medium partition plates provided in parallel to the direction in which the filaments converge, a pick-up roll for converging a plurality of gelled bacterial cellulose accumulated in the culture medium upper layer into a filament shape, and a take-up roll An apparatus for producing bacterial cellulose filaments , comprising:

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