JPH05301902A - Microfibrillated highly oriented microbial cellulose and its production - Google Patents

Abstract

PURPOSE:To obtain a microbial cellulose containing highly oriented microfibril fiber and difficult to produce by conventional process by culturing a microbial strain having high capability to produce the microbial cellulose and collecting the objective cellulose from the culture medium. CONSTITUTION:A microbial strain capable of producing microbial cellulose is cultured in a medium under agitation at a rotational speed of 50-250rpm to effect the production and accumulation of the microbial cellulose in the medium and the produced cellulose is separated from the medium. The microbial cellulose produced and accumulated in the medium is heat-treated or treated with a solvent. The microbial strain to be used in the present process is especially preferably Acetobacter pasteurianus ATCC-10245. The medium is preferably incorporated with carbon sources, nitrogen sources and inorganic salts and the cultivation is carried out at 25-35 deg.C and initial pH of 5-6.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to microbial cellulose in which microfibril fibers are highly oriented and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, microbial cellulose produced by microorganisms including bacteria belonging to the genus Acetobacter is structurally β-1,4-glucan and is the same as plant-derived cellulose. The fibers are thin and form strong hydrogen bonds with cellulose hydroxyl groups not only within the microfibrils but also between the microfibrils.

[0003]

[Equation 1] It is used as a high-strength material for speaker diaphragms because it has a high degree of selective plane orientation of the microcrystalline surface. When used as a high-strength material, at present, by treating the microbial cellulose obtained by culturing with alkali or hypochlorous acid, etc., deproteinization is performed and at the same time the crystallinity of microfibrils is increased, The orientation is improved because the above treatment enhances hydrogen bonding within the microfibrils and between the microfibrils.

Microbial cellulose is composed of microfibrils finer than plant cellulose, and if the microbial cellulose can be controlled or utilized in units of microfibrils, its characteristics can be utilized more effectively. It can be expected to be used in a wider range of fields.
However, the microbial cellulose prepared by the conventional method is certainly high in plane orientation, but it is not oriented when viewed at the microfibril level, and has a random arrangement, and microfibril units are not always sufficient. It did not mean that the characteristics of microbial cellulose were utilized.

Until now, attempts have been made to modify the orientation of the microfibrils by adding an optical brightener to the medium or culturing in a high magnetic field. However, the optical brightener after culturing has been tried. Due to the difficulty of removing
It was not a practical method. Further, in the previous studies, the purpose was to modify the orientation of microfibrils, and it was not possible to control the orientation of microfibrils themselves so as to enhance the orientation.

[0006]

DISCLOSURE OF THE INVENTION The present invention provides a microbial cellulose in which microfibril fibers, which have been difficult to produce by the prior art, are highly oriented, and a method for producing the same, which broadens the use of microbial cellulose. Is.

[0007]

[Means for Solving the Problems] The present inventors have diligently studied whether or not the microfibril fibers of microbial cellulose can be oriented by a simple method, and when culturing under a certain specific culture condition, that is, at a low stirring number. It has been found that the microorganism produces microbial cellulose composed of microfibril fibers whose orientation is remarkably enhanced.

Further, it has been found that the orientation of the microfibril fibers can be further enhanced by subjecting the produced microbial cellulose composed of the microfibril fibers having enhanced orientation to a heat treatment or a solvent treatment to complete the present invention.
That is, the present invention, microbial cellulose microfibril fibers are highly oriented, and culturing a microorganism having a microbial cellulose-producing ability in a medium, to produce and accumulate the microbial cellulose in the medium, in a method of collecting it, A method for producing microbial cellulose, which comprises culturing at a stirring number of 50 to 250 rpm, and in the production method, the microbial cellulose produced and accumulated in a medium is subjected to heat treatment and / or solvent treatment, and microfibrils. It is a method for producing microbial cellulose in which fibers are highly oriented.

The microorganisms used in the present invention are not particularly limited as long as they are microorganisms capable of producing microbial cellulose, and for example, microorganisms belonging to the genus Acetobacter, such as Acetobacter pastorianus ATCC 1024.
5, microorganisms belonging to the genus Gluconobacter, microorganisms belonging to the genus Sarsina, for example Sarsina pentriculi ATCC1
9633, microorganisms belonging to the genus Agrobacterium, for example Agrobacterium tumefaciens ATCC 23308 microorganisms belonging to the genus Pseudomonas, microorganisms belonging to the genus Rhizobium, for example Rhizobium leguminosarum ATCC 10004
Is mentioned.

In particular, microorganisms belonging to the genus Acetobacter,
For example, the Acetobacter pastorian ATCC 10245
It has a high production capacity of microbial cellulose and is preferably used. The medium for culturing the microorganism preferably includes a medium to which a carbon source, a nitrogen source and an inorganic salt are added.
The carbon source is not particularly limited as long as it can be assimilated by the microorganism, and sucrose, glucose, fructose and the like are usually used alone or in combination. Further, a starch hydrolyzate or a cellulose hydrolyzate containing these carbon sources can also be used.

Any nitrogen source can be used as long as it can be utilized by the microorganism, and inorganic nitrogen sources such as ammonium sulfate, ammonium phosphate and nitrate, and organic nitrogen sources such as yeast extract, polypeptone and corn steep liquor are used. As the inorganic salt, phosphates, magnesium salts, iron salts, calcium salts and the like are used appropriately.

As a micronutrient source of the microorganism, vitamins,
When nucleic acids, amino acids, etc. are required, it is necessary to supplement the required nutritional source. In addition, productivity may be improved by adding inositol, phytic acid, pyrroloquinoline quinone, a cellulase preparation or an inactivated cellulase preparation to the production medium.

Usually, when the microbial cellulose is produced, the medium (B
iochem. J., Vol. 58, p. 345 (1954)) is preferable because of high productivity. The initial pH of the medium is preferably 3 to 7, more preferably 5 to 6. The culture temperature is preferably 10 to 40 ° C, more preferably 25 to 35 ° C. As a culturing method, a method of producing in a culture solution by stirring culture is adopted. The capacity and shape of the culture tank are not particularly limited, and those used for usual culture of microorganisms may be used.

For stirring the culture solution, a magnet or a magnet equipped with a rotary blade is installed at the center of the lower part of the culture tank, and a method of stirring by rotating with a magnetic stirrer or attaching a rotary blade into the liquid from the upper part of the fermentation tank, A general method such as a method in which the rotor is rotated by a motor directly connected to the rotor installed in the upper part of the fermenter and agitated is used. The microorganism is
Since oxygen is required for growth, it is necessary to take care so that oxygen is supplied during the culture period. In some cases, the orientation may be lowered, and natural aeration is normally used without special forced aeration. When forcedly ventilated, the air flow rate is usually 0.05 to 1
vvm is preferred.

The stirring conditions are required to be 50 to 250 rpm, and preferably 100 to 180 rpm. When cultured under the above-mentioned conditions, microbial cellulose is usually produced in the liquid, and microbial cellulose in which the orientation of the microfibril fibers is enhanced is produced. The stirring conditions should be appropriately selected because the optimum production conditions for increasing the orientation of the microfibril fiber are slightly different depending on the shape of the fermentation tank, the stirring method and the strain of the microorganism used.

When culturing at a stirring number lower than 50 rpm, microbial cellulose is produced on the liquid surface of the medium, but the orientation of the produced microfibril fibers is as random as the microbial cellulose obtained by the static culture method. Also, 25
When cultured at a stirring rate of more than 0 rpm, microbial cellulose is produced in the liquid, but the orientation of the produced microfibril fiber is low.

During the culturing, it is preferable to appropriately add an acid or an alkali to control the pH of the culture solution to a pH range preferable for the production of microbial cellulose according to a conventional method. In addition, the yield of microbial cellulose per unit liquid volume can be increased by appropriately supplementing a carbon source such as glucose during culture. Impurities contained in the microbial cellulose collected by the above operation are washed with water, washed with alkali, treated with an organic solvent such as toluene, treated with sodium hypochlorite, treated with a surfactant such as sodium lauryl sulfate (SDS). It is removed either alone or in combination.

The microbial cellulose that has been subjected to deproteinization has a higher orientation of microfibril fibers than the microbial cellulose produced by static culture even in the state as it is, but it should be used in combination with heat treatment, solvent treatment, freeze-drying treatment, etc. Thereby, the orientation is further enhanced. As a heating condition, for example, a method of treating at a high temperature of 105 ° C. for 3 to 12 hours and drying is preferable. The orientation is further enhanced by heating and drying after stretching in advance.

As the solvent used for the solvent treatment, for example, methanol, acetone, acetonitrile and the like are preferably used. Solvent treatment, for example, immersing the water-containing microbial cellulose after deproteinization treatment in a solvent several times,
It is carried out by replacing the solvent with water. In addition, heat treatment or solvent treatment may be used in combination.

The microbial cellulose prepared as described above has not only higher selective crystal plane orientation of microcrystalline planes than microbial cellulose produced by the conventional method, but also remarkably high orientation in the microfibril fiber unit. Therefore, it can be used as it is as a material having higher strength than conventional microbial cellulose. Furthermore, by using the microbial cellulose of the present invention, by a simple treatment such as freezing treatment, it becomes possible to disaggregate microfibril units that could not be carried out with microbial cellulose prepared by a conventional method, and plant origin such as viscose It is possible to easily fiberize the disaggregated microfibrils by twisting them as they are, without performing the regenerating treatment required when producing fibers made of cellulose as a raw material. Etc. can be used as a high-performance fiber raw material.

[0021]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to these examples.

[0022]

Example 1 Hestrin-Schlum medium (HS medium) (D-glucose 2.0 g, Bactopeptone (Difco) 0.5 g)
, Yeast extract (manufactured by Difco) 0.5 g, citric acid 0.115
g, disodium hydrogen phosphate 0.27g, distilled water 100ml, pH
6.0) 450 ml to a fermenter with a capacity of 1 L (CORNING model 2650
4-1L), and culture medium of Acetobacter pastorianus ATCC 10245 that had been statically cultured in HS medium for 3 days in advance.
15 ml was inoculated and cultured at 28 ° C. for 7 days under the condition of stirring speed of 120 rpm. During the culture, aeration was not forcibly performed, but natural aeration was performed from the air intake.

After completion of the culture, the microbial cellulose produced in the culture solution was taken out by filtration, boiled in a 2% SDS aqueous solution, immersed in a 1% NaOH aqueous solution, and washed with ultrasonic waves. After further neutralization with a 1% acetic acid aqueous solution, the product was washed with water to obtain microbial cellulose. When the obtained microbial cellulose was dried and the weight of the dried product was weighed, the amount of microbial cellulose produced was 100 mg per 450 ml of the culture solution. The obtained water-containing microbial cellulose membrane was examined with a polarizing microscope (Olympus BH-2) at a magnification of 100 and photographed (FIG. 1).

The microbial cellulose dried at room temperature for 3 hours and the microbial cellulose dried at 105 ° C. for 3 hours were examined under a polarizing microscope and photographed (FIG. 2 was dried at room temperature, and FIG. 3 was dried at 105 ° C.). .. As a control, microbial cellulose prepared by the same method as above except that it was cultured by static culture without stirring, was dried at 105 ° C. for 3 hours and examined under a microscope (FIG. 4).

As shown in FIG. 1, when stirring culture was carried out even in the presence of water, the microfibrils were oriented in a considerable part. When dried at room temperature, the orientation of the microfibrils increased as shown in FIG. When dried at 105 ° C., the orientation of microfibrils was significantly enhanced as shown in FIG. Microbial cellulose produced by static culture as a control showed no orientation of microfibrils even when dried at 105 ° C. as shown in FIG.

The microbial cellulose produced by stirring culture produced by drying at 105 ° C for 3 hours and the microbial cellulose produced by static culture produced at 105 ° C for 3 hours were subjected to X-ray diffraction using a goniometer. When it went, it became like FIG. 5 and FIG. 6, respectively.

[0027]

[Equation 2] The area ratio was 1.38 for the suspension culture and 0.995 for the static culture, indicating that the microbial cellulose produced in the suspension culture had a higher plane orientation. I had.

[0028]

[Example 2] Using the same culture apparatus as in Example 1, 450 ml of the same HS medium as in Example 1 was dispensed, and precultured in the same manner as in Example 1 for Acetobacter pastorianus ATCC.
Inoculate 15 ml of 10245 culture solution and let it aerate naturally with stirring speed of 12
Culturing was carried out at 28 ° C. for 7 days under the condition of 0 rpm.

After completion of the culture, the microbial cellulose produced in the culture solution was taken out by filtration, boiled in a 2% SDS aqueous solution, immersed in a 1% NaOH aqueous solution, and washed with ultrasonic waves. After further neutralization with a 1% acetic acid aqueous solution, the product was washed with water to obtain microbial cellulose. When the obtained microbial cellulose was dried and the weight of the dried product was weighed, the amount of microbial cellulose produced was 100 mg per 450 ml of the culture solution. The obtained gel-like microbial cellulose film containing water was examined under a polarizing microscope at a magnification of 100 and photographed (FIG. 7).

Further, microbial cellulose containing water is sandwiched between a cover glass and a slide glass, acetone is poured from one end of the cover glass, and the water contained in the microbial cellulose is absorbed by filter paper from the other end. The water content was replaced with acetone. This operation was repeated 6 times, and the microbial cellulose obtained by substituting acetone for water contained in the gel was examined under a polarizing microscope and photographed (FIG. 8).

Further, the operation of immersing the microbial cellulose containing water in methanol and taking it out, and then immersing it in fresh methanol and taking out it 6 times was repeated, and the microbial cellulose obtained by substituting the water contained in the microbial cellulose gel with methanol was polarized. It was examined under a microscope and photographed (FIG. 9). The microbial cellulose in which the water content has been replaced with methanol is sandwiched between the cover glass and the slide glass, acetonitrile is poured from one end of the cover glass, and the methanol contained in the microbial cellulose is extracted from the other end with a filter paper and contained. The methanol was replaced with acetonitrile.

The operation of immersing in acetonitrile was repeated 6 times, and the microbial cellulose in which methanol was replaced with acetonitrile was observed under a polarizing microscope and photographed (Fig. 1).
0). As shown in FIG. 7, the microfibrils were partially oriented even in the presence of water, but when treated with acetone, the orientation was enhanced as shown in FIG. In addition, when replacing with methanol or acetonitrile,
The microfibrils were generally oriented as shown in FIG.

[0033]

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide microbial cellulose in which microfibrils, which have been difficult to produce in the past, are highly oriented, and the obtained microfibrils are
Not only can it be used as a high-strength material or highly functional material, but it can also be used as an ultrafine fiber material.

[Brief description of drawings]

FIG. 1 is a photograph showing the fiber shape of a membrane of microbial cellulose containing water produced by agitation culture.

FIG. 2 is a photograph showing the fiber shape of microbial cellulose produced by stirring culture after being dried at room temperature for 3 hours.

FIG. 3 shows the microbial cellulose produced by the agitation culture as 105
It is a photograph which shows the shape of the fiber about what was dried at ° C for 3 hours.

FIG. 4 shows microbial cellulose produced by static culture as 105
It is a photograph which shows the shape of the fiber about what was dried at ° C for 3 hours.

FIG. 5 shows microbial cellulose produced by stirring culture as 105
It is an X-ray diffraction diagram of what was dried at 3 degreeC.

FIG. 6 shows microbial cellulose produced by static culture as 105
It is an X-ray diffraction diagram of what was dried at 3 degreeC.

FIG. 7 is a photograph showing the fiber shape of a water-containing gelled microbial cellulose obtained by stirring culture.

FIG. 8 is a photograph showing the fiber shape of microbial cellulose produced by stirring culture in which the water content is replaced with acetone.

FIG. 9 is a photograph showing the fiber shape of microbial cellulose produced by stirring culture in which the water content is replaced with methanol.

FIG. 10 is a photograph showing the fiber shape of microbial cellulose obtained by substituting methanol for acetonitrile of microbial cellulose substituted with methanol.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Masahiro Fukaya 28, No. 1 Hakuikeike, Morioka, Higashiura-cho, Chita-gun, Aichi Prefecture (72) Inventor Yoshiya Kawamura 132, Furuwato, Kochino-cho, Konan-shi, Aichi Prefecture

Claims (3)

[Claims] 1. Microbial cellulose in which microfibril fibers are highly oriented. 2. A method of culturing a microorganism capable of producing microbial cellulose in a medium, producing and accumulating the microbial cellulose in the medium, and collecting the microorganism, wherein the stirring number is 50 to 250 r.
A method for producing microbial cellulose, which comprises culturing at pm. 3. The method according to claim 2, wherein the microbial cellulose produced and accumulated in the medium is heat-treated and / or solvent-treated.