JPH11181001A - Production of bacteria cellulose - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject substance capable of manifesting an effect of improving yield of filler by culturing a cellulose-producing bacterium in a medium to which water-soluble polysaccharides are added. SOLUTION: This bacteria cellulose(BC) is obtained by culturing a cellulose- producing bacterium (e.g. an acetic acid bacterium such as Acetobacter xylinum subsp. sucrofermentans) in a medium to which water-soluble polysaccharides (e.g. carboxymethyl cellulose or xanthane gum) are added. Preferably, the cellulose-producing bacterium is cultured in an aerated stirring cultivation system. An adding amount of the water-soluble polysaccharide is preferably about 0.01-0.1 wt.% based on the weight of the medium. The BC is used as a filler yield improving agent and a filler-containing paper having excellent opacity and strength is produced by mixing and dispersing the BC with a filler such as talc or clay in water, previously forming aggregates by using a flocculant, adding the resultant aggregates to a paper stock and making the paper stock.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a microorganism capable of producing a cellulosic substance in a medium to which a water-soluble polysaccharide such as carboxymethylcellulose (CMC) is added (hereinafter referred to as "cellulose-producing microorganism"). The bacteria are cultured in a culture system such as aeration and agitation, and the cellulosic substance (hereinafter referred to as
(Hereinafter referred to as "bacterial cellulose" or "BC"), bacterial cellulose obtained by the method, and a filler retention improver comprising the bacterial cellulose.

[0002]

2. Description of the Related Art BC (bacterial cellulose) is edible, is used in the food field, and has excellent water-based dispersibility, so that it retains the viscosity of foods, cosmetics, paints, etc., strengthens food raw material dough, and retains moisture. It has industrial value in improving food stability, as a low calorie additive or as an emulsification stabilizing aid. BC is characterized in that the fibril fragment width is as small as about two digits compared to cellulose produced from wood pulp or the like. Therefore, the dissociated product of BC has various industrial uses as a reinforcing agent for polymers, particularly aqueous polymers, based on such structural and physical characteristics of microfibrils. A material obtained by solidifying such a cellulosic disagglomerated substance into a paper or solid form exhibits a high tensile modulus, so that excellent mechanical properties based on the structural characteristics of microfibrils are expected, and there are applications as various industrial materials. .

[0003] In general, papers used for various purposes such as printing papers and writing papers include opacity improvement, smoothness improvement, whiteness improvement, paper sizing improvement, and reduction of fiber content. However, a fine component such as the filler has a strong tendency to flow out due to dehydration by a wet papermaking method or the like, and there is a problem that it is difficult to remain in the paper. Therefore, a technique using BC as a filler yield improver has been developed on the basis of the excellent properties of BC as described above.

For example, a method of adding a bacterial cellulose disintegration and a chaotic polyelectrolyte to a papermaking raw material suspension for the purpose of increasing the yield efficiency of filler (Japanese Patent Laid-Open No. 1-24)
6495), or a technique using bacterial cellulose obtained by stirring culture as a filler retention improver (JP-A-7-305295).

[0005]

However, generally, when the amount of the filler retention improver is increased, the strength of the paper is reduced. Further, in particular, in the case of BC obtained by aeration and agitation culture under high stirring conditions, there is a problem that the filler yield effect of BC decreases due to the high shearing force applied during the culture. Therefore, there has been a demand for a substance which can be produced even under such culture conditions and has an excellent effect of improving the yield of filler in a smaller amount. As a result of repeated studies to provide such a substance, the present inventor has found that by culturing a cellulose-producing bacterium in a medium supplemented with a water-soluble polysaccharide such as CMC, bacterial cellulose having an excellent filler yield improving effect can be obtained. And completed the present invention.

[0006]

That is, the present invention provides a method for producing bacterial cellulose, which comprises culturing a cellulose-producing bacterium in a medium to which a water-soluble polysaccharide has been added, and a method for producing bacterial cellulose (so obtained). Hereinafter, it is simply referred to as “the present bacterial cellulose (BC)”. Examples of the water-soluble polysaccharide include carboxymethyl cellulose, xanthan gum, methyl cellulose and the like. The molecular weight of these water-soluble polysaccharides is not particularly limited. These substances are commercially available and can be easily obtained by those skilled in the art. The production method of the present invention
In particular, it is suitable to be performed in an aeration stirring culture system. The amount of the water-soluble polysaccharide to be added to the medium can be appropriately selected by those skilled in the art according to the type and the like, but it is not necessary to add too much from an economical point of view. In the production method of the present invention, the water-soluble polysaccharide is preferably used in an amount of about 0.
01-0.1% by weight, particularly preferably about 0.01-0.1%
Excellent effects can be achieved with an addition amount in the range of 0.05% by weight. The water-soluble polysaccharide is preferably added to the medium from the start of the culture, but may be added at any stage during the culture.

[0007] Cellulose producing bacteria used for producing bacterial cellulose in the present invention include, for example, BPR2
Acetobacter xylinum subspecies schlofermentans ( Acetobac
ter xylinum subsp. sucrofermentans ), Acetobacter xylinum ATCC23
768, Acetobacter xylinum ATCC 2376
9. Acetobacter pasteurianus ( A. pasteurianu)
s ) ATCC 10245, Acetobacter xylinum ATCC 14851, Acetobacter xylinum AT
CC11142 and Acetobacter xylinum ATC
C10821 and other acetic acid bacteria (genus Acetobacter), in addition to Agrobacterium, Rhizobium, Sarsina, Pseudomonas, Achromobacter, Alcaligenes, Aerobacterium, Azotobacter and Zugrea and NTG ( And various mutant strains created by performing a mutation treatment by a known method using, for example, nitrosoguanidine). In addition, BPR2001
The strain was deposited on February 24, 1993 at the Patented Microorganisms Depositary Center, National Institute of Bioscience and Human-Technology, Ministry of International Trade and Industry (Accession No. FERM P-13466), and then 199
Deposit under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms in Patent Procedures on February 7, 2004 (Accession No. FE
RM BP-4545). Chemical mutation treatment methods using a mutation agent such as NTG include, for example, Bi
o Factors, Vol. l, p. 297-302 (1988) and J. Gen. Mi
crobiol, Vol. 135, p. 2917-2929 (1989). Therefore, those skilled in the art can obtain the mutant strain used in the present invention based on these known methods. The mutant strain used in the present invention can also be obtained by other mutation methods, for example, irradiation.

[0008] In the composition of the medium used for the culture, sucrose, glucose, fructose, mannitol, sorbitol, galactose, maltose, erythrit, glycerin, ethylene glycol, ethanol, etc. may be used alone or in combination as a carbon source. Can be. Furthermore, starch hydrolyzate containing these, citrus molasses, beet molasses, beet juice, sugarcane juice,
Juices such as citrus fruits can also be used in addition to the carbon source. As the nitrogen source, an organic or inorganic nitrogen source such as ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, nitrate, and urea can be used, or Bacto-Pepton
e, Bacto-Soytone, Yeast-Ext
Nitrogen-containing natural nutrients such as rac and soybean may be used. As organic trace nutrients, amino acids, vitamins, fatty acids, nucleic acids, 2,7,9-tricarboxy-1H pyrrolo [2,3,5] -quinoline-4,5-dione, sulphite pulp waste liquor, ligninsulfonic acid, etc. are added. You may.

When using an auxotrophic mutant which requires an amino acid or the like for growth, it is necessary to supplement the required nutrient. As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, cobalt salts, molybdates, red blood salts, chelate metals and the like are used. Regarding the method for producing BC, see JP-A-62-26.
No. 5990, JP-A-63-202394 and JP-B-6-202.
No. 43443 describes a method for producing BC. The nutrient medium suitable for culturing cellulose-producing bacteria includes a carbon source, peptone, yeast extract, Schramm / He containing sodium phosphate and citric acid.
strin medium (Schramm et al., J. General Biology, 11 , p.
pp. 123-129, 1954) are known. In addition, in such a nutrient medium, inositol, phytic acid and pyrroloquinoline quinone (PQQ) which are factors for promoting the production of cellulose by specific nutrients in the medium (Japanese Patent Publication No. 5-1718; , Vol. 42, No. 3, 23rd
7 to 244), and carboxylic acid or a salt thereof (Japanese Patent Application No. 5-191467), invertase (Japanese Patent Application No. 5-331149) and methion (Japanese Patent Application No. 5-335768). It has been found that the productivity of cellulosic substances is improved by adding.
In the present invention, conventionally known methods including the above-mentioned methods can be arbitrarily selected. For example, when acetic acid bacteria are used as production bacteria, the culture pH is 3 to 7
, Preferably at around 5. Culture temperature is 10-4
The reaction is performed at 0 ° C, preferably at 25 to 35 ° C. The concentration of oxygen supplied to the culture device is 1 to 100%, preferably 21 to 100%.
80% is sufficient. Those skilled in the art can appropriately select the composition ratio of each component in the medium, the inoculation of the cells into the medium, and the like, depending on the culture method. Bacterial cellulose is conventionally known as a stationary, shaking or aeration-stirring culture method, and the culture operation method is a so-called batch fermentation method, a fed-batch batch fermentation method, a repeated batch fermentation method, or a known method such as a continuous fermentation method. Can be manufactured.

The stirring culture is a culture method in which the culture solution is stirred by an impeller, an air-lift fermenter, a pump-driven circulation of a fermentation broth, or a combination of these means. Thereby, the structure of the bacterial cellulose changes, for example, such that the crystallization index decreases and the amorphous part increases. Further, a method for producing a cellulosic substance in which a culture solution containing cells is circulated between a culture apparatus and a separation apparatus described in JP-A-8-33494 in the name of the present applicant. And separating the cellulosic substance, which is a product, from the cells and the culture solution, and culturing the cellulosic bacteria described in JP-A-8-33495 in the name of the present applicant. A method for producing a cellulosic material by culturing, by continuously withdrawing the culture solution from the culture system and supplying a new medium having a volume substantially equal to the amount of the culture medium during the culture period, The above production method is characterized in that the concentration of the cellulosic substance in the culture solution is kept low. In the stirring culture referred to in the present invention, aeration is performed simultaneously with stirring, if necessary.

Bacterial cellulose obtained by stirring culture is separated from the culture solution by a centrifugation method, a filtration method, or the like.
Bacterial cellulose may be collected together with the cells,
Further, a treatment for removing impurities other than the cellulosic substance including bacterial cells contained in the substance can be performed. To remove impurities, washing with water, pressure dehydration, washing with diluted acid, washing with alkali, treatment with bleach such as sodium hypochlorite and hydrogen peroxide, treatment with cell lysing enzymes such as lysozyme, sodium lauryl sulfate, Impurities can be almost completely removed from the cellulosic material by performing a treatment with a surfactant such as deoxycholic acid, washing with heat in the range of room temperature to 200 ° C., alone or in combination. The bacterial cellulose thus obtained contains cellulose, a heteropolysaccharide having cellulose as a main chain, and a glucan such as β-1,3, β-1,2 or the like. Components other than cellulose in the case of heteropolysaccharide are mannose, fructose, galactose, xylose,
Hexoses such as arabinose, rhamnose, glucuronic acid,
Pentose and organic acids. These polysaccharides may be a single substance, or two or more polysaccharides may be mixed by hydrogen bonding or the like.

In the present invention, the BC used for the filler yield improver can be further defibrated. BC
The disintegration phenomenon is considered to be a phenomenon in which a stress generated inside the cellulose due to a mechanical external force or the like deforms or breaks it. Therefore, the BC disaggregation process can be performed by applying a mechanical external force to the BC. Further acid hydrolysis,
Disintegration treatment can also be performed by enzymatic hydrolysis and bleach. The mechanical external force referred to herein includes, for example, stresses such as tension, bending, compression, torsion, impact, and shearing, but generally includes compression, impact, and shearing stress. Actually applying these mechanical external forces to the BC can be achieved by using, for example, a mixer, a polytron, or an ultrasonic oscillator such as a self-excited ultrasonic crusher.

In the disaggregation process by the mixer, the mechanical external force is mainly composed of an impact force caused by collision of the stirring blade with the BC and a shear force generated by a displacement phenomenon caused by a difference in speed of the medium. In the disintegration process using Polytron, the mechanical external force is the compression force due to the BC being pinched between the external teeth and the internal teeth, the impact force due to the collision between the high-speed rotating teeth and the BC, and the high-speed rotation with the stationary external teeth. The shear stress generated in the medium existing in the gap between the internal teeth is mainly caused. In the disintegration by the ultrasonic crusher, the mechanical external force is mainly cavitation (cavitation phenomenon) continuously generated in the medium due to the oscillation of the ultrasonic oscillating section, and the local shear stress is mainly generated. The disaggregation process of the present invention can be performed by any method other than the above-described specific examples as long as a constant load (mechanical external force) can be applied to the BC. Other disaggregation treatment conditions can be appropriately selected by those skilled in the art. Furthermore, it can be sieved with a screen having a predetermined opening.

The BC of the present invention thus produced has an excellent yield improving effect. Accordingly, the present invention also relates to a filler retention improver comprising such bacterial cellulose. Furthermore, by mixing and dispersing the filler and the BC of the present invention in water and forming an agglomerate in advance using an aggregating agent, the opacity can be improved more effectively without reducing the strength of the paper. . Examples of the filler used in the present invention include talc, clay, titanium dioxide, precipitated calcium carbonate, heavy calcium carbonate, calcium sulfate, barium sulfate, aluminum hydroxide, activated clay, synthetic silicate, kaolin, calcined kaolin, Fillers which can be usually used for paper such as plastic pigments can be used alone or in combination. In order to obtain sufficient opacity and strength, the average particle diameter of the filler is preferably 2.0 μm or less. The average particle size in the present invention is determined by using a particle size distribution curve obtained by dispersing an aqueous dispersion of a filler with an ultrasonic disperser for 5 minutes and then applying a light transmission type particle size distribution analyzer (SKN type, manufactured by Seishin Enterprise Co., Ltd.). , The diameter of the particles whose cumulative weight percent determined corresponds to 50%.

As the coagulant in the present invention, among the cationic polymer electrolytes, cationic polyacrylamide having a molecular weight of 100,000 or more, cationic starch, cationic guar gum and the like can be used. The amount of addition depends on the type of filler and BC used.
It is appropriate that the content is 0.005% by weight or more and 10.0% by weight or less. Further, an anionic polyelectrolyte such as forming an complex with these cationic polyelectrolytes to enhance aggregation, such as anionic polyacrylamide, or anionic inorganic fine particles, such as colloidal silica or bentonite aqueous dispersion, Further, or by using an amphoteric polymer electrolyte or an aqueous dispersion of amphoteric inorganic fine particles in combination, it is also possible to form an aggregate of the filler and the bacterial cellulose disintegrated product of the present invention.

Filler (A) and BC of the present invention (in terms of dry matter)
The content ratio (A / B) with (B) is preferably 0.5 or more and 50.0 or less, more preferably 20.0 or less by weight.
If (A / B) is less than 0.5, sufficient opacity improving effect cannot be obtained for the required paper strength, and (A / B) is 5
If it exceeds 0.0, the filler yield efficiency will be poor, and if the opacity is to be satisfied, the paper strength will decrease. In addition to the filler retention improver of the present invention, the filler-incorporated paper may also contain additives usually used in papermaking, such as sizing agents, defoamers, slime control agents, dyes, coloring pigments, fluorescent agents, and dry paper strength. An enhancer, a wet paper strength enhancer, a water repellency enhancer, a retention agent, and the like can be included as necessary. Also,
Starch, polyvinyl alcohol,
It is also possible to smear various surface sizing agents and the like.

The reason why a filler-filled paper having excellent opacity and strength can be produced by preliminarily aggregating the filler and BC and then adding it to a paper material to form a paper is that when only the filler is agglomerated, The effective interface for scattering is significantly reduced,
Since the refractive index is different from that of the filler and fine BC is agglomerated together with the filler, it is conceivable that the number of interfaces effective for scattering light is not reduced. In addition, since the filler is held between the fibers as agglomerates, it is considered that the amount of fine filler that inhibits the bonding between the fibers is small, and the decrease in strength is reduced.

[0018]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to examples, but the examples do not limit the scope of the present invention.

【Example】

Example 1 strain 757-3-5-11 (deposited on April 12, 1996, with the Patent Microorganisms Depositary Center, National Institute of Biotechnology, Ministry of International Trade and Industry; Accession number: FERM P-1556)
4, then transferred to the International Deposit under the Budapest Treaty on February 10, 1997; Accession No. FERM BP-5
815) and 67-2 strains (deposited on May 29, 1997 with the Patent Microorganisms Depositary Center, Biotechnology and Industrial Technology Research Institute, Ministry of International Trade and Industry; accession number FERM P-1624).
9) was converted to CSL-S from glycerol stock.
1% was inoculated into a 750 ml roux flask charged with 100 ml of uc medium, and cultured at 28 ° C. for 3 days. After the culture, the roux flask was shaken well to remove the cells from the cellulose membrane, and 12.5 ml of the bacterial solution was added to 500 ml containing 112.5 ml of the medium.
The cells were inoculated into a ml flask and cultured at 28 ° C. and 180 rpm for 3 days. Aseptically loosened away the resulting culture by the blender was inoculated the 60ml to 1l jar charged with CSL-Suc medium 540 ml, 4.9 to pH with NH ₃ gas and 1 N H ₂ SO ₄ Main culture was performed under aeration and agitation while controlling the rotation speed automatically so that the dissolved oxygen amount (DO) became 3.0% or more while controlling to 5.1. After completion, the obtained culture solution was diluted about 5 times with an acetate buffer, and then centrifuged to collect a precipitate. The precipitate was diluted with distilled water to about 8 times the initial culture volume, heated at 80 ° C. for 20 minutes, and collected by centrifugation after heating. The precipitate was suspended in 8 volumes of 0.1N NaOH at 80 ° C.,
The cells were lysed by heating for 0 minutes, and the precipitate was recovered by centrifugation after lysis. Thereafter, the precipitate was suspended in an eight-fold amount of distilled water, heated at 80 ° C. for 20 minutes, centrifuged after heating, and the precipitate was collected to wash the cellulose. By performing the same washing three times, the purified BC of the present invention is obtained.
I got The composition of CSL-Suc used in the above examples is shown below. In addition, at the time of aeration and agitation culture, CMC (sodium carboxymethylcellulose: Code 073-26 manufactured by Nacalai Tesque) having predetermined concentrations shown in Table 4 in the medium was used.
) Was added.

[0020]

TABLE 1 Medium Composition CSL-Suc medium Sucrose _{4.0 (%) KH 2 PO 4} 0.1 MgSO 4 · 7H 2 O 0.025 (NH 4) 2 SO 4 0.33 Vitamin mixture 1.0 Salt mixture 1.0 CSL (corn staple liquor) 4.0 pH 5.0

[0021]

Table 2 Vitamin mixture liquid compound mg / L Inositol 200 Niacin 40 Pyridoxine HCl 40 Thiamine HCl 40 Calcium pantothenate 20 Riboflavin 20 p-Aminobenzoic acid 20 Folic acid 0.2 Biotin 0.2

[0022]

TABLE 3 saline mixture _{FeSO 4 · 7H 2 O 360mg /} L CaCl 2 · 2H 2 O 1470mg / L Na 2 MoO 2 · 2H 2 O 242mg / L ZnSO 4 · 7H 2 O 173mg / L MnSO 4 · 5H 2 _{O 139mg / L CuSO 4 · 5H} 2 O 5mg / L

Example 2 Filler yield test The BC of the present invention prepared in Example 1 was used in accordance with JIS-P-8209.
LBKP disintegrated in accordance with JIS and weight ratio 2.5: 97.5
Alternatively, 100 parts of light calcium carbonate and 1 part of positive starch are added to 100 parts of pulp prepared by mixing at 5:95, and fillers are passed through a screen using this papermaking material in accordance with TAPPI standard method T261. I asked for the yield. The filler was quantified at 400 ° C. for 8 hours in accordance with TAPPI standard method T269. Table 4 shows the results
It was shown to.

[0024]

[Table 4]

For comparison, the purified BC (C
(Obtained by adding 0.05% by weight of MC to the medium and culturing)), the above-described purification obtained by culturing without adding CMC to the medium so as to contain CMC at substantially the same ratio. A similar filler yield test was performed using a material obtained by adding CMC to BC later (0.5 g of CMC was added to 11 g of BC). Table 5 shows the results.
Shown in

[0026]

[Table 5]

As is evident from the results shown in Tables 4 and 5, the bacterial cellulose obtained by the production method of the present invention has a superior filler yield improving effect as compared with the bacterial cellulose alone. It is. It is also found that such an effect cannot be obtained by simply mixing purified BC and the water-soluble polysaccharide after culturing.

Example 3 A suspension of BC of the present invention obtained by culturing strain 67-2 in the medium supplemented with CMC in Example 1 and a suspension obtained by culturing 67-2 strain without adding CMC. Each of the BC suspensions was placed on a copper mesh for transmission electron microscopy observation and dried. Next, each sample was negatively stained and dried with a 1% aqueous potassium phosphotungstate solution, and then observed with a transmission electron microscope (JEM1200-EX II, manufactured by JEOL Ltd.). The obtained results are shown in FIGS. In BC (FIG. 1) obtained by culturing strain 67-2 without adding CMC, a number of native bands (cellulose II) were observed. In the BC of the present invention obtained by culturing (FIG. 2), this native band was hardly observed. For the native band, see the report by Kumagura (Polymer, 34, 3293 (1993)).

[0029]

According to the present invention, a small amount of BC obtained by the production method of the present invention has an excellent filler yield improving effect, and can be used as a filler yield improver.

[Brief description of the drawings]

FIG. 1 is a transmission electron micrograph of a BC obtained by culturing strain 67-2 without adding CMC (at a magnification of 10,000).
0 times).

FIG. 2 is a transmission electron micrograph (magnification: 1) of BC of the present invention obtained by culturing strain 67-2 in a medium supplemented with CMC.
0000 times).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:02) (72) Inventor Hiroshi Ogiya 3-2-1 Sakado, Takatsu-ku, Kawasaki-shi, Kanagawa Prefecture Biopolymer Research Inc. (72) Inventor Takayasu Tsuchida 3-2-1 Sakado, Takatsu-ku, Kawasaki-shi, Kanagawa Prefecture Inside Biopolymer Research Inc. (72) Inventor Fumihiro Yoshinaga 3-2-1 Sakado, Takatsu-ku, Kawasaki-shi, Kanagawa Bio Inc. Within Polymer Research

Claims (6)

[Claims] 1. A method for producing bacterial cellulose, comprising culturing a cellulose-producing bacterium in a medium to which a water-soluble polysaccharide has been added. 2. The method according to claim 1, wherein the water-soluble polysaccharide is carboxymethyl cellulose. 3. The method according to claim 1, wherein the cellulosic bacteria are cultured by aeration and agitation culture. 4. The method according to claim 1, wherein the water-soluble polysaccharide is contained in a medium in an amount of 0.01 to 0.1.
The production method according to any one of claims 1 to 3, wherein the addition is performed by weight%. 5. A bacterial cellulose obtained by the production method according to claim 1. 6. A filler retention improver comprising the bacterial cellulose according to claim 5.