JPH09165403A - Method for freezing bacterial cellulose - Google Patents

Abstract

PROBLEM TO BE SOLVED: To freeze or thaw a bacterial cellulose without detriment to its properties by mixing an aqueous suspension of a bacterial cellulose with a third component except bacterial cellulose and water and freezing the resultant mixture. SOLUTION: An aqueous dispersion of a bacterial cellulose is mixed with a third component except bacterial cellulose and water, and the resultant mixture is frozen. Examples of hydrophilic liquids used as the third component include glycerol, ethylene glycol dimethyl sulfoxide, dimethylformamide, surfactants, lactic acid, gluconic acid, delta-gluconolactone and a mixture thereof. Examples of hydrophilic solids which can be used as the third component include water-soluble substances such as water-soluble low-molecular compounds and water-soluble polymers. Although the freezing temperature can be suitably selected according to the object, etc., it is usually in the range of about 0 deg.C or below.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for freezing a cellulosic substance (hereinafter referred to as “bacterial cellulose” or “BC”) which can be produced by culturing a cellulosic bacterium, and a frozen product obtained by the method. And a method for restoring the frozen product.

[0002]

2. Description of the Related Art BC (bacterial cellulose) is edible and tasteless and odorless, so it is used in the field of foods, and because of its excellent water-based dispersibility, it maintains the viscosity of foods, cosmetics, paints, etc. It has industrial value in strengthening dough, retaining moisture, improving food stability, as a low-calorie additive or as an emulsification stabilizing aid. BC is characterized in that the cross-sectional width of fibrils (or fine fibers) is about two digits smaller than cellulose produced from wood pulp or the like.
Accordingly, the dissociated product of BC has various industrial uses as a reinforcing agent for polymers, especially aqueous polymers, based on such structural and physical characteristics of fibrils. A material obtained by solidifying such a cellulosic disagglomerated product into a paper or solid form exhibits a high tensile modulus, so that excellent mechanical properties based on the structural characteristics of fibrils are expected, and there are applications as various industrial materials. .

[0003]

As described above, when BC is contained in food or the like, it may be subjected to a freezing (freezing) treatment for the purpose of long-term storage or the like. is there. However, BC consists of very fine cellulose fine fibers. This fine fiber has a width of about 100 nm and a number of thicknesses.
It is estimated that the thickness is about nm based on the result of transmission electron microscope observation. Furthermore, it is reported that the width is 20 to 50 nm in scanning electron microscope observation. As is well known, cellulose is a homopolymer formed by β-bonding the carbons at positions 1 and 4 of glucose. Hydroxyl groups are bonded to positions 2, 3, and 6 of the glucose residue, and hydrogen bonds are formed between these hydroxyl groups and oxygen atoms in the pyranose ring of glucose. Water exists between the fine fibers, but large ice crystals grow when freezing is performed for a long time during the freezing process. When water between the fine fibers of BC freezes, if ice crystals larger than the voids between the fine fibers grow, association (hydrogen bond) between the fine fibers of BC occurs. Therefore,
In order to prevent this phenomenon, it is necessary to freeze rapidly. However, a large amount of energy is required for cooling for rapid freezing. Considering such points, a huge amount of energy is required to industrially perform freeze-drying. Ribbon-shaped fine fibers of bacterial cellulose are much finer than ordinary cellulose fibers, for example, plant pulp fibers, and have a large surface area. Therefore, a large portion contributes to hydrogen bonding. The bond is very strong as compared with ordinary pulp fibers and the like.

Therefore, once the composition containing BC is frozen, the bond between the fine fibers is so strong that even if the composition is simply thawed (melted), the original wet state is not restored. For this reason, it was extremely difficult to restore the properties such as solubility, dispersibility, sedimentation degree, and viscosity of BC simply thawed after freezing to the state before freezing. For the reasons described above, there has been no satisfactory method for freezing and thawing while maintaining the various characteristics of BC. The present inventors
After adding BC and a third component other than water to the aqueous suspension of
The present invention has been completed by finding that the conventional problems can be solved by freezing.

[0005]

That is, the present invention is characterized in that an aqueous suspension containing bacterial cellulose is added with bacterial cellulose and a third component other than water and then frozen, and then frozen. Involved in the method. The present invention also relates to a frozen bacterial cellulose product obtained by such a freezing method. Examples of the hydrophilic liquid used as the third component in the method of the present invention include glycerin, ethylene glycol, dimethyl sulfoxide, dimethylformamide, a surfactant, lactic acid, gluconic acid and delta-gluconolactone, and a mixture of at least one of them. Can be mentioned. Further, the hydrophilic solid that can be used as the third component includes a water-soluble substance such as a water-soluble low molecule and a water-soluble polymer.

Among these, water-soluble low-molecular weight compounds include, for example, sugars (glucose, fructose, galactose, xylose, mannose, arabinose, sucrose, lactose, cellobiose, palatinose, maltose, gentiobiose, trehalose, rhamnose, oligosaccharides,
Isomaltooligosaccharide, soybean oligosaccharide, fructooligosaccharide, galactooligosaccharide, lactosucrose, coupling sugar, liquid sugar, cyclodextrin, sugar alcohol, sorbitol, erythritol, lactitol,
Maltitol, xylitol, mannitol, dursit), salts (sodium sulfate, ammonium sulfate, salt, calcium chloride, sodium bicarbonate, sodium carbonate, rossel salt), amino acids, amino acid salts, organic acids, organic acid salts, nucleic acids, nucleic acid salts , Alkyl ketene dimer, styrene-acrylic sizing agent, olefin-maleic anhydride sizing agent, higher fatty acid sizing agent, water resistant material composed of epoxy compound, fluorescent brightener, defoamer, antistatic agent, pigment, dye , Sulfuric acid band, aluminum chloride, sodium aluminate, basic aluminum chloride, basic polyaluminum hydroxide, alumina sol, water-soluble aluminum compound, ferrous sulfate, ferric chloride, and alkenyl succinic anhydride-based sizing agent and the like Refers to one or more mixtures.

The water-soluble polymer includes, for example, cellulose derivatives (carboxymethyl cellulose, methyl cellulose, hydroxypropyl cellulose, ethyl cellulose), fibron, xanthan gum, xyloglucan, dextrin, dextran, carrageenan, echo gum, locust bean gum, alginic acid, Alginate, Pullulan, Starch, Potato starch, Kudzu flour, Positive starch, Phosphorylated starch, Corn starch, Gum arabic, Locust bean gum, Gua gum, Gellan gum, Polydextrose, Pectin, Chitin, Water-soluble chitin, Chitosan, Casein, Albumin, Soybean Protein lysate, peptone, polyvinyl alcohol, polyacrylamide, sodium polyacrylate, polyvinylpyrrolidone, sodium polyphosphate, polyvinyl acetate , Polyamino acids, polylactic acid, polymalic acid, polyglycerol, latex, rosin sizing agents, petroleum resin sizing agents, urea resins, melamine resins, epoxy resins, polyamide resins, polyamide
Polyamine resin, polyethyleneimine, polyamine, vegetable gum, polyethylene oxide, hydrophilic cross-linked polymer, polyacrylate, starch polyacrylic acid copolymer, tamarind gum, gellan gum, pectin, guar gum and colloidal silica, and mixtures of one or more of them. Say. Among these, xanthan gum and carboxymethyl cellulose are preferable.

The third component described above is selected by a person skilled in the art depending on the purpose of use of the frozen product of the present invention. A solid component, a low-molecular soluble component, a high-molecular component and the like may be used in combination. For example, when used as a thickener in the food field, xyloglucan, salt, sucrose, etc. may be used in combination at a ratio of 100: 5: 20, for example. In particular, when tasteless, odorless, colorless, and harmless substances are required for use in foods, the third component is preferably an edible soluble polymer. Those skilled in the art can appropriately select the addition amount of these third components depending on the type of substance and the like, and are usually 2 to 1,000% by weight based on the weight of BC. Furthermore, as an example of the bacterial cellulose aqueous suspension containing the third component, the culture solution of the cellulose-producing bacterium itself or the one containing the third component may be used.

The freezing method in the method of the present invention may be any conventionally known method, for example, leaving it in an atmosphere of about 0 ° C. or lower. Generally, the higher the freezing rate is, the more preferable it is, but those skilled in the art can appropriately set it in consideration of various conditions. The freezing temperature can be appropriately selected according to the purpose, etc., but is generally in the range of about 0 ° C or lower. An example of a freezing device specifically used in the method of the present invention is a freezer.

In the method of the present invention, the cellulosic material is preferably one that has been subjected to disaggregation treatment. The disintegration phenomenon of bacterial cellulose 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. Accordingly, the disaggregation treatment of bacterial cellulose can be performed by applying a mechanical external force to bacterial cellulose. Further, the disaggregation treatment can be carried out by acid hydrolysis, enzymatic hydrolysis and a bleaching agent. 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. The actual application of these mechanical external forces to bacterial cellulose can be achieved by using, for example, a mixer, a polytron, an ultrasonic oscillator or the like.

In the disaggregation treatment by the mixer, the mechanical external force is mainly composed of the impact force caused by the collision between the stirring blade and the bacterial cellulose and the shear force generated by the displacement phenomenon due to the speed difference of the medium. In the disaggregation process using Polytron, the mechanical external force is the compressive force of bacterial cellulose sandwiched between the external teeth and the internal teeth, the impact force of the collision of high-speed rotating teeth with bacterial cellulose, the static external teeth and the high speed of stationary external teeth. The shear stress generated in the medium existing in the gap between the rotating internal teeth is mainly involved. In the disintegration by the ultrasonic pulverizer, the mechanical external force is mainly cavitation (cavity phenomenon) continuously generated in the medium due to the oscillation of the ultrasonic oscillating unit, and significant shear stress locally generated. The defibration treatment of the present invention can be performed by any method other than the above-mentioned specific examples as long as a certain load (mechanical external force) can be applied to the bacterial cellulose. Other disaggregation treatment conditions can be appropriately selected by those skilled in the art.

The disaggregation treatment has been described above. The disaggregation treatment according to the present invention is carried out after stirring culture of the cellulose-producing bacterium.
It is obvious to those skilled in the art that the present invention is not limited to an independent secondary operation performed on bacterial cellulose separated and purified from a culture solution. That is, the stirring operation has a function of disaggregating the bacterial cellulose, and in the stirring culture, the dissociation treatment of the bacterial cellulose can be sufficiently performed even by the stirring function for the purpose of culturing. Furthermore, the same can be said for the operations of separating, washing, purifying and transporting the bacterial cellulose obtained by stirring culture, and additional disaggregation treatment in these operations is also included in the disaggregation treatment of the present invention. Please note.

The agitation culture as referred to in the present invention is a culture method carried out while agitating a culture solution, and the agitation effect received during the agitation culture causes the structure of bacterial cellulose to decrease, for example, due to a decrease in crystallization index. The crystal parts change so as to increase. As the stirring means, for example, an impeller, an airlift fermenter, a pump-driven circulation of fermentation broth, a combination of these means, and the like can be used. The culture operation method includes a so-called batch fermentation method, a fed-batch batch fermentation method, a repeated batch fermentation method and a continuous fermentation method. Furthermore, a method for producing a cellulosic substance by circulating a culture solution containing bacterial cells between a culture apparatus and a separation apparatus described in Japanese Patent Application No. 6-192287 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 Japanese Patent Application No. 6-192288 in the name of the present applicant. A method for producing a cellulosic material by continuously extracting a culture solution from a culture system and supplying a new culture solution having substantially the same volume as the amount withdrawn during the culture period. The production method is characterized in that the concentration of the cellulosic substance in the culture solution is kept low.

As a tank for carrying out the stirring culture,
For example, stirring tanks such as jar fermenters and tanks,
In addition, baffled flasks, Sakaguchi flasks, and air-lift type stirring tanks can be used, but are not limited thereto. In the stirring culture according to the present invention, at the same time as stirring,
Ventilation may be performed if necessary. The aeration referred to here may be any of an oxygen-containing gas such as air and an oxygen-free gas such as argon and nitrogen. These gases may be used by those skilled in the art according to the conditions of the culture system. Will be selected as appropriate. For example, in the case of anaerobic microorganisms, if an inert gas is ventilated, the culture solution can be stirred by the bubbles. In the case of aerobic microorganisms, the culture solution can be agitated while supplying oxygen necessary for the growth of the microorganisms by aerating an oxygen-containing gas.

The cellulose-producing bacterium producing BC used in the present invention is, for example, Acetobacter xylinum subsp. S, which is represented by the BPR2001 strain.
ucrofermentans ), Acetobacter xylinum ( Acet
bacterium xylinum ) ATCC 23768, Acetobacter xylinum ATCC 23969, Acetobacter
Pasteurianus ( A. pasteurianus ) ATCC1024
5, Acetobacter xylinum ATCC 14851,
Acetic acid bacteria (genus Acetobacter) such as Acetobacter xylinum ATCC11142 and Acetobacter xylinum ATCC10821, and others, Agrobacterium, Rhizobium, Sarsina, Pseudomonas,
The genera are Achromobacter, Alcaligenes, Aerobacterium, Azotobacter and Zooglare, and various mutants created by subjecting them to mutation treatment by a known method using NTG (nitrosoguanidine) or the like. The BPR2001 strain was deposited on February 24, 1993 at the Patent Microorganisms Depositary Center of the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry (accession number FERM P-
13466) and subsequently transferred to a deposit under the Budapest Treaty on the International Recognition of Patent Deposits on February 7, 1994 (accession number FERM BP-4545).

Examples of the chemical mutation treatment method using a mutagen such as NTG include, for example, Bio Factors, Vol. 1, p. 297-302.
(1988) and J. Gen. Microbiol, Vol. 135, p. 2917-2.
929 (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. Among the cellulose-producing bacteria created by the above-mentioned method, a high polymerization degree having a weight average polymerization degree of not less than 1.6 × 10 ⁴ , preferably 1.7 × 10 ⁴ or more in terms of polystyrene is obtained by aeration and stirring culture. Or a strain that produces bacterial cellulose having a high degree of polymerization having a weight-average degree of polymerization in terms of polystyrene of 2.0 × 10 ⁴ or more by producing the bacterial cellulose or by culturing it in a static state. Among the bacterial cells producing a high degree of polymerization that can be used in the present invention, BPR
3001A was deposited on June 12, 1995, with the Patented Microorganisms Depositary Center, Biotechnology and Industrial Technology Research Institute, Ministry of International Trade and Industry, under the accession number FERM P-14982. In general, it is known that the higher the degree of polymerization of a polymer, the higher the strength and elastic modulus of the polymer material. Similarly, in the case of bacterial cellulose, various products obtained using high polymerization degree bacterial cellulose as a raw material have higher strength and elasticity than those obtained using relatively low polymerization degree bacterial cellulose as a raw material. High rate. Therefore, when it is desired to produce a product having a high strength and an elastic modulus, the use of bacterial cellulose having a high degree of polymerization as described above provides a higher effect.

The weight average degree of polymerization of various types of cellulose such as BC in the present invention is determined by GPC having RI as a detector.
It measures as follows using a system (Tosoh HLC-8020). Various cellulose samples were treated with fuming nitric acid-phosphorus pentoxide solution by WJ Alexander, RL Mitchell, Anal.
It is nitrated by the method of ytical chemistry 21, 12, 1497-1500 (1949). A nitrated cotton linter is used as a control. The cellulose nitrate is dissolved in THF (Wako Pure Chemicals first grade) at a concentration of 0.05%, and then filtered through a 1.0 μm pore size filter. THF is also used as the eluent for GPC. Flow rate is 0.5
ml / min, the pressure is 10-13 kgf / cm ² , and the sample injection volume is 100 μl. Column is TSKgel GMH
-Using HR (S) (7.5 ID x 300 mm x 2) and guard column (HHR (S)) (Tosoh Co., Ltd.) 35
Measure in ° C. The standard polystyrene (Tosoh) is used to calculate the molecular weight, and the relative molecular weight in terms of polystyrene is calculated. Elution time (t) and logarithm of molecular weight (log M) using polystyrene having a molecular weight of 2 × 10 ⁷ to 2630
For the cubic equation: (log M = At ³ + Bt ² + Ct
+ D) is approximated to create a standard curve.
Molecular weight: Tosoh's data processing machine (SC-8020)
Calculate the weight average molecular weight by the program installed in. From these molecular weight values, the weight average polymerization degree is calculated in consideration of the degree of substitution after nitration.

In the composition of the medium used for the stirring culture of the present invention, carbon sources include sucrose, glucose, fructose, mannitol, sorbitol, galactose,
Maltose, erythritol, glycerin, ethylene glycol, ethanol and the like can be used alone or in combination. Furthermore, starch hydrolyzate containing these, citrus molasses, beet molasses, beet juice,
It is also possible to use sugar cane juice, fruit juice such as citrus fruits, etc. in addition to sucrose. As the nitrogen source, ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, nitrates, organic or inorganic nitrogen sources such as urea can be used, or Bacto
-Natural nitrogen-containing nutrient sources such as Peptone, Bacto-Soytone, Yeast-Extract, and soybean concentrate may be used. Amino acids, vitamins, fatty acids, nucleic acids, 2,7,9 as organic micronutrients
-Tricarboxy-1H pyrrolo [2,3,5] -quinoline-4,5-dione, sulfite waste liquor, ligninsulfonic acid, etc. may be added.

When using an auxotrophic mutant that requires amino acids and the like for growth, it is necessary to supplement the required nutrients. 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. Furthermore, inositol, phytic acid, pyrroloquinoline quinone (PQQ) (Japanese Patent Publication No. 5-1718; Mitsuo Takai, Paper and Paper Cooperative Journal, Vol. 42, No. 3, 237-2).
44), carboxylic acid or a salt thereof (Japanese Patent Application No. 5-1914).
No. 67), invertase (Japanese Patent Application No. 5-331491)
) And methionine (Japanese Patent Application No. 5-335564) can be added to the medium as appropriate. For example, when acetic acid bacterium is used as a production bacterium, the pH of the culture is controlled at 3 to 7, preferably around 5. The culture temperature is 10 to 40 ° C, preferably 25 to 3
Perform at 5 ° C. The oxygen concentration supplied to the culture device is 1 to
100%, preferably 21 to 80%. 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.

The bacterial cellulose of the present invention can be produced, for example, by the following method. Bacterial cellulose obtained by aeration and agitation culture is separated from the culture solution by a centrifugation method, a filtration method or the like. The bacterial cells produced by the method of the present invention may be recovered as the bacterial cells as they are, and may be further treated to remove impurities other than the cellulosic material including the bacterial cells contained in the substance. 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 cellulosic material thus obtained in the present invention includes cellulose, a substance containing a heteropolysaccharide having cellulose as a main chain, and a substance containing glucan such as β-1,3, β-1,2. In the case of the heteropolysaccharide, components other than cellulose include hexoses such as mannose, fructose, galactose, xylose, arabinose, rhamnose, and glucuronic acid, pentoses, and organic acids. These polysaccharides may be a single substance, or two or more polysaccharides may be mixed by hydrogen bonding or the like.

The frozen bacterial cellulose obtained by the method of the present invention can be thawed with running water or the like and then mixed with stirring to restore the original wet state of the disaggregated product or the like. When the frozen product of bacterial cellulose obtained by the method of the present invention is thawed and restored to its original disaggregated state, the cellulose fine fibers constituting BC are in a state where they are not firmly bound to each other. . As mentioned earlier, BC
Consists of very fine cellulose fine fibers. As described in the present invention, when restored to the original disaggregated state, a large amount (cellulosic fine fibers) is present on the surface of such fine fibers or in the voids of the network structure composed of fine fibers. (A few times to several hundred times the weight of the liquid component). As described above, since a large amount of liquid component is contained, good dispersibility, sedimentation property, and liquid viscosity can be obtained.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail by way of examples, but the examples do not limit the present invention. In addition, in each Example, various characteristic values were measured as follows. Sedimentation degree of suspension Sedimentation degree is measured by adding 10 ml of 0.2% bacterial cellulose (BC) suspension to a 15 ml tube made of Falcon, centrifuging at 3000 rpm for 15 minutes, and then sedimenting. It was expressed as a ratio of the volume of a part to the whole. The larger the value of the degree of sedimentation, the harder the sedimentation, and the more dispersed the particles. The value of the sedimentation degree recovery ratio (the sedimentation degree of the disaggregated matter after drying and condensate / the sedimentation degree of the disaggregated matter before drying) was used. Viscosity The viscosity in the present invention means a dynamic viscosity at an angular velocity of 10 rad / sec at 30 ° C. when an aqueous suspension having a BC content of 0.1% is measured by a dynamic liquid viscoelasticity measuring method (hereinafter, Simply called viscosity). More specifically, a dynamic liquid viscometer (“FLUIDS SPECTROMETER RF” manufactured by Rheometrics) is used.
S II "was used to measure 2 ml of an aqueous suspension of BC disaggregate having a concentration of 0.1% between parallel rotating disks having a diameter of 5 cm, and the temperature was 30 ° C and the angular velocity was 10 rad / sec and the strain was 10%. It has a high viscosity.

[0023]

Example 1 Production of bacterial cellulose and disaggregation treatment (1) Preparation of seed bacterial solution (proliferation of bacterial cells) Cellulose-producing bacteria were grown by a flask culture method. Basic composition of 40 g / L fructose, 1.0 g / L potassium phosphate, 0.3 g / L magnesium sulfate, 3 g / L ammonium sulfate, 5 g / L bacto-peptone, 1.4 ml / L lactic acid, initial pH 5.0 Medium 100
BPR into a 750 ml Roux flask into which the
1 ml of a cryopreserved bacterial solution of the 2001 strain (FERM BP-4545) was inoculated, and statically cultured at 28 ° C. for 3 days in a constant temperature incubator. After the seed culturing, the Roux flask was shaken well, and the contents were subjected to gauze filtration under aseptic conditions to obtain a seed bacterial solution.

(2) Production of Bacterial Cellulose by Stirring Culture Aseptically inoculating 60 ml of the above seed bacterial solution into a small jar fermenter (total volume 1000 ml) in which 540 ml of sterilized medium for stirring culture to be described later is put. 20 at 30 ° C
PH for 1 hour or 30 hours with 1N NaOH or 1N H
While controlling to 5.0 with ₂ SO ₄ , the stirring speed was initially 400 rpm, and the dissolved oxygen (DO) was 3.0.
Stirring culture was performed with a jar fermenter while automatically controlling the number of revolutions so as to fall within 0 to 21.0%. For the stirring culture, a medium having the following composition was used. Fructose 40 g / L, KH ₂ PO ₄ 1.0 g / L, MgSO ₄ 0.3 g / L, (NH ₄ ) ₂ SO ₄ 3 g / L, Bacto-Soytone (manufactured by Difco) 5 g / L and sono (soybean) 5 g / L Initial pH 5.0 After cultivation, the solids in the jar fermenter were collected, washed with water to remove the medium components, and then placed in a 1% NaOH aqueous solution at 110 ° C. The cells were removed by treatment for 20 minutes. Further, the produced cellulose was washed with water until the washing liquid became nearly neutral to obtain bacterial cellulose.

(3) Stirring culture with addition of carboxymethyl cellulose In the stirring culture described in (2), as the medium component,
Furthermore, carboxymethylcellulose was added at 5 g / l for culturing. The resulting culture was more viscous than the culture obtained in (2). (4) Production of Bacterial Cellulose (Comparative Example) by Static Culture 600 ml of a medium having the composition described in (2) was dispensed into a sterile petri dish by 30 ml each, and allowed to stand at 30 ° C. for 7 days. did. After completion of the culture, the bacterial cellulose formed on the surface of the Petri dish was washed with water to remove the medium components.

(5) Disaggregation treatment of bacterial cellulose Water was added to the washed bacterial cellulose obtained by the stirring culture method of (2), and suspended at a disaggregation treatment concentration (bacterial cellulose dry weight / volume) of about 0.2% by weight. A suspension was prepared. Similarly, water was added to the bacterial cellulose obtained by the stationary culture in (4) to prepare a suspension having a disintegration treatment concentration of about 0.2% by weight (dry weight / volume of bacterial cellulose). Next, these suspensions were disintegrated for 3 minutes at 25 ° C. using a stirrer (Blender manufactured by Auster). The rotation speed of the stirrer was set to the highest level. By this disaggregation treatment, a BC disintegration product (A) was obtained from the stirring culture, and a BC disintegration product (B) was obtained from the stationary culture. Water was added to the culture solution in the jar fermenter containing bacterial cellulose after completion of the stirring culture in (2) and (3) to adjust the bacterial cellulose content to 0.2%. Then, the mixture was stirred at 25 ° C. for 3
Disaggregated for a minute. The rotation speed of the stirrer was set to the highest level. By this disaggregation treatment, BC disaggregates (C) and (D) were prepared from the dilutions of the respective culture solutions. In addition,
The concentration of BC in the above was determined by centrifuging to remove a wet solid from the culture solution, and then in a 0.2 N sodium hydroxide solution (20 times the solid content) at 100 ° C.
After immersion for a time to remove bacterial cells and medium components other than bacterial cellulose, the cells were sufficiently washed and dried and calculated from the measured dry weight.

Example 2 The disaggregated material (A) prepared in Example 1 was used as the third component of the present invention.
As a component, carboxymethyl cellulose (CMC),
Echo gum, fibron, dextrin, and sodium polyphosphate at 50% and 100% by weight of BC
%, And frozen to -20 ° C over about 60 minutes.
After being kept in a frozen state for about 1 hour, it was thawed by leaving it in an atmosphere at room temperature to restore the original suspension state. The degree of sedimentation was measured according to the method described above at each time point before freezing and after thawing.

[0028]

[Table 1]

When the third component is added (No. 1 to No. 1)
It was shown in 0) that the recovery rate of sedimentation degree was higher than that in the case of no addition (No. 11). Using the disaggregated materials (C) and (D) prepared in Example 1 instead of the disaggregated material (A), the same test was conducted. As a result, disaggregated material (A)
The same effect as described above was obtained.

Example 3 Static of Cellulose Producing High Polymerization Cellulose
Incubation and Preparation of Cellulose (BC) BPR3001A was added to the medium 10 from glycerol stock.
Inoculation was carried out at a concentration of 1% in a 750 ml volume flask equipped with 0 ml, and the cells were 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 3 ml of the bacterial solution was inoculated into a Petri dish (diameter: 90 mm) containing 27 ml of CSL-Fru medium and cultured at 28 ° C for 10 days. After the cultivation, the cellulose membrane of each bacterium was washed with running water and heated in about 500 ml of water at 80 ° C. for 20 minutes. After heating, each cellulose membrane was further washed with running water, and thereafter, about 500
Lysis was achieved by heating at 80 ° C. for 20 minutes in 0.1 ml NaOH. After lysis, each cellulose membrane is
Washing was carried out by heating in 00 ml of distilled water at 80 ° C. for 20 minutes. The same washing was performed 3 to 5 times while exchanging distilled water to obtain purified BC.

Example 4 High-Polymerization Cellulose-Producing Bacteria
Bacterial agitation culture and preparation of cellulose (BC) BPR3001A was prepared from glycerol stock by CSL-
1% of the cells were inoculated into a 750 ml roux flask charged with 100 ml of Fru medium and statically 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. The culture was aseptically disaggregated with a blender, 60 ml of which was charged with 540 ml of CSL-Fru medium.
Inoculate the jar and adjust the pH to NH ₃ gas and 1N H ₂ S
While controlling the O ₄ at 4.9 to 5.1, the amount of dissolved oxygen (D
Main culture was performed while automatically controlling the rotation speed so that (O) was 3.0% or more. After the completion, the obtained culture solution was diluted about 5 times with acetate buffer and then centrifuged to collect the 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 is also 8 times the amount of 0.1N
Lysis was carried out by suspending in NaOH and heating at 80 ° C. for 20 minutes, and then the precipitate was recovered by centrifugation after lysis. Thereafter, the precipitate was suspended in 8 volumes of distilled water,
The cellulose was washed by heating for 1 minute and centrifuging after heating to collect the precipitate. Purified BC was obtained by performing the same washing three times.

The CSL-Fru used in the above-mentioned embodiments is used.
Is as shown below.

[0033]

[Table 2]

[0034]

[Table 3] 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

[0035]

[Table 4] Salt mixture ammonium iron citrate 1.5 g / l calcium chloride 1.5 g / l ammonium molybdate 0.1 g / l zinc sulfate heptahydrate 0.2 g / l manganese sulfate tetrahydrate 0.1 g / l Copper sulfate pentahydrate 2 mg / l

Example 5 The purified BC obtained in Examples 3 and 4 was disaggregated in the same manner as in the disaggregated product (A) described in Example 1 to obtain a disaggregated product. These are called disintegration products (E) and (F), respectively.
Using the disaggregates (E) and (F), Table 1 of Example 2
As a result of performing the same test as described above, the same results as in Table 1 were obtained as in the disaggregated material (A). Example 6 A sample for molecular weight analysis was prepared by drying the disaggregated material (A), the disaggregated material (B), the disaggregated material (E) and the disaggregated material (F) under reduced pressure at 80 ° C. for 12 hours. After nitration, the weight-average molecular weight was measured and the weight-average degree of polymerization was calculated according to the method already described for these samples. As a result, the weight-average degree of polymerization was 10,60 in terms of polystyrene, respectively.
0, 14,900, 22,500 and 17,400.

[0037]

[Effect] The bacterial cellulose frozen product obtained by the freezing method of the present invention is about 60 to 60% after thawed.
The recovery rate of sedimentation degree was 100%, and it was found that the physical form of bacterial cellulose fiber was not substantially impaired by the freezing process of the method of the present invention.

Claims (7)

[Claims] 1. A method for freezing bacterial cellulose, which comprises adding bacterial cellulose and a third component other than water to an aqueous suspension containing bacterial cellulose, and then freezing. 2. The method according to claim 1, wherein the bacterial cellulose has been subjected to disaggregation treatment. 3. The method according to claim 1, wherein the bacterial cellulose is bacterial cellulose obtained by stirring culture. 4. The method according to claim 2, wherein the disaggregation treatment method is mechanical shearing force, ultrasonic wave, high pressure treatment, acid hydrolysis, hydrolysis using an enzyme or a method using a bleaching agent, or a combination thereof. 5. The method according to claim 1, wherein the third component is a hydrophilic liquid. 6. The method according to claim 1, wherein the third component is a hydrophilic solid. 7. After freezing the bacterial cellulose by the method according to any one of claims 1 to 6, water is added to form a dispersion liquid, and the dispersion liquid is stirred and mixed, and if necessary, A method for reconstituting a frozen bacterial cellulose product, which comprises heat-treating before or after stirring and mixing.