JPH0833495A - Production of bacterial cellulose - Google Patents

Abstract

PURPOSE:To produce a bacterial cellulose useful e.g. as an additive for foods, cosmetics and coating materials on an industrial scale at a low cost by culturing a cellulose-producing microbial strain while continuously carrying out the extraction and the supply of the culture liquid and keeping the concentration of the cellulosic substance in the culture liquid to a low level. CONSTITUTION:This bacterial cellulose having edibility and excellent dispersibility in aqueous system and useful e.g. as an additive for foods, cosmetics and coating materials is continuously produced by culturing a cellulose-producing microbial strain [e.g. Acetobacter xylinum subsp. sucrofermentans BPR2001 strain (FERM BP-4545)]while continuously extracting the culture liquid from the culture system during the culture period and supplying a fresh culture liquid of a volume nearly equal to that of the extracted culture liquid, thereby keeping the concentration of the cellulosic substance in the culture liquid to a low level (<=10g/L or thereabout) to keep the oxygen consumption rate in the culture liquid to >=15mumol/L/h.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention uses a cell belonging to a microorganism capable of producing a cellulosic substance (hereinafter referred to as "cellulosic bacteria") to produce a cellulosic substance (bacterial cellulose: BC). It relates to a continuous manufacturing method.

[0002]

2. Description of the Related Art BC (bacterial cellulose) is edible and used in the food field and has excellent water-based dispersibility. Therefore, the viscosity of foods, cosmetics, paints, etc. is maintained, the dough for food materials is strengthened, and the water content is retained. It has industrial value as a food stability improvement, low calorie additive or emulsion stabilization aid. BC is characterized in that the width of fibril fragments is as small as about double digits as compared with cellulose produced from wood pulp or the like. Therefore, the disaggregated material of BC has various industrial applications as a reinforcing agent for polymers, particularly aqueous polymers, based on such structural and physical characteristics of microfibrils. A substance obtained by solidifying such a cellulosic dissociated material into a paper-like or solid form exhibits a high tensile elastic modulus, and therefore, excellent mechanical properties based on the structural characteristics of microfibrils are expected, and application as various industrial materials is expected. is there.

Conventionally, stationary culture, shaking, aeration stirring culture or the like has been used as a culture method for culturing microorganisms. Further, as a culture operation method, a so-called batch fermentation method, fed-batch batch fermentation method, repeated batch fermentation method, continuous fermentation method and the like have been used. As the stirring means, for example, an impeller, an air-lift fermenter, a pump-driven circulation of fermentation broth, and a combination of these means are used. B
Regarding the production method of C, JP-A-62-265990
No. 63-202394 and Japanese Patent Publication No. 6-434.
No. 43 and the like have a description on a method for producing BC. As a nutrient medium that is suitable for culturing cellulosic bacteria, a Schramm / Hestrin medium (Schramm et al., J. General Biology, ll , pp, consisting of carbon source, peptone, yeast extract, sodium phosphate and citric acid is used. .123 ~ 129,
l954) is known. Further, in such a nutrient medium, inositol, phytic acid, and pyrroloquinoline quinone (PQQ), which are factors for promoting cellulose production by specific nutrients in the medium, are disclosed in Japanese Examined Patent Publication No. 5-1718; , Vol. 42, No. 3, 237-244
Page) or the like, or further, carboxylic acid or a salt thereof (Japanese Patent Application No. 5-191467), invertase (Japanese Patent Application No. 5-331491) and methionine (Japanese Patent Application No. 5-3).
It has been found that the productivity of cellulosic substances is improved by the addition of No. 35764). By the way, heretofore, no specific report has been made so far regarding the continuous production method of BC using aeration and agitation culture.

[0004]

In the conventional batch and fed-batch fermentation method in aeration and agitation culture, as BC is accumulated in the culture medium by the culture of the cellulose-producing bacteria, the latter half of the culture period is increased. The viscosity of the culture solution increases, and as a result, it becomes extremely difficult to uniformly and sufficiently stir and mix the entire culture system, resulting in insufficient oxygen supply (aeration) and the production rate of BC by bacteria. In addition, a lot of power was required for aeration (supply of bubbles to the culture system) and stirring. An object of the present invention is to eliminate such drawbacks of the prior art and provide an economical and high-yield method for producing BC using a cellulose-producing bacterium.

[0005]

[Means for Solving the Problems] The inventors of the present invention have conducted various studies to achieve the above-mentioned object, and have conducted research on B in a culture solution.
The above problem was solved by keeping the concentration of C low. That is, the present invention is a method for culturing a cellulosic substance by culturing a cellulosic bacterium, wherein during the culturing period, a withdrawal of the culture solution from the culture system and a new culture solution with a volume approximately equal to the withdrawal amount are used. The present invention relates to the above-mentioned production method, characterized in that the concentration of the cellulosic substance in the culture broth during culturing is kept low by continuously supplying the broth. In the method of the present invention, B in the culture medium during culturing
The concentration of C can be appropriately selected by those skilled in the art depending on the type of cells, the composition of the medium, the oxygen consumption rate, and other culture conditions, but it is usually preferably about 20 g / L or less, more preferably about 20 g / L or less. It is more preferably 10 g / L or less. Further, when the BC is relatively highly accumulated, it is preferable to maintain the BC concentration so that the oxygen consumption rate in the culture solution is 15 μmol / L / hr or more.

In the production method of the present invention, B in the culture solution is
The concentration of C is maintained in a low range by continuously withdrawing a culture solution containing BC produced from the culture system and continuously supplying an almost equal volume of the culture solution to the culture system as a result, It is intended to prevent an increase in the viscosity of the culture system. In the present invention, “continuous” includes “intermittent” or “intermittent”, and the supply and withdrawal of the culture solution to and from the culture system is performed at a constant flow rate using a peristaltic pump or the like. The concept is not limited to the mode in which the culture solution is continuously supplied at all times, and the mode in which the culture solution is supplied and withdrawn at regular time intervals is also included in the concept. In any case, while monitoring the concentration of BC in the culture medium, the supply amount and the withdrawal amount, and further the amounts of sugar and other medium components in the culture medium supplied are appropriately determined according to the values. As a result, the number of cells and the growth rate in the system can be controlled and the BC concentration can be maintained in a low range. It is preferable that the withdrawal amount and the supply amount of the culture solution are substantially equal to each other in order to keep the liquid volume of the entire culture system constant, but if necessary, these amounts may be independent from each other, especially for a short period. It is also possible to change it.

The cellulose-producing bacterium used in the present invention is, for example, Acetobacter xylinum subsp. Sucroferm .
entans ), Acetobacter xylinum ( Acetobacter
xylinum ) ATCC23768, Acetobacter xylinum ATCC23769, Acetobacter pasteurianus ATCC10245, Acetobacter xylinum ATCC14851, Acetobacter xylinum ATCC11142 and Acetobacter xylinum ATCC10821, and other genus Agrobacterium. , Genus Rhizobium, genus Sarsina, genus Pseudomonas, genus Achromobacter, genus Alcaligenes, genus Aerobacterium, genus Azotobacter and genus Zugrea, and mutating them by a known method using NTG (nitrosoguanidine) etc. Various mutant strains. In addition, BPR2001
The strain was deposited on February 24, 1993, at the Patent Microorganism Depositary Center, Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry (accession number FERM P-13466), and then 199.
Deposit under the Budapest Treaty on the International Recognition of Deposits for Patent Proceedings dated February 7, 4 (deposit number FERM B
P-4545).

Examples of the chemical mutagenesis treatment method using a mutagen such as NTG include Bio Factors, Vol. 1, p. 297-302.
(1988) and J. Gen. Microbiol, Vol. 135, p. 2917-2.
Some are described in 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 such as irradiation. In the composition of the medium used in the production method of the present invention, as the carbon source, 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 Bac
t-Peptone, Bact-Soytone, Ye
Nitrogen-containing natural nutrient sources such as ast-Extract and soybean concentrate may be used. Amino acids, vitamins, fatty acids, nucleic acids, 2,7,9-tricarboxy-as organic micronutrients
1H pyrrolo [2,3,5] -quinoline-4,5-dione, sulfite waste liquor, ligninsulfonic acid and the like may be added.

When using an auxotrophic mutant strain 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, the above-mentioned cellulose production promoting factor can be appropriately added to the medium. For example, when acetic acid bacteria are used as the production bacteria, the pH of the culture is controlled to 3 to 7, preferably around 5. Culture temperature is 10 to 40 ° C,
It is preferably carried out in the range of 25 to 35 ° C. The oxygen concentration supplied to the culture device is 1 to 100%, preferably 21 to 80%
If it is good. Those skilled in the art can appropriately select the composition ratio of each component in these media and the inoculation of bacterial cells into the media according to the culture method. As the stirring means, conventionally known means, for example, impeller, air lift fermenter,
It can be selected from pump-driven circulation of the fermentation broth and any combination of these respective means.

[0010] BC produced by the method of the present invention may be obtained by recovering the bacterial cells as they are, and further, a treatment for removing impurities other than the cellulosic material containing the bacterial cells contained in this substance can be performed. To remove impurities,
Washing with water, pressure dehydration, dilute acid washing, alkaline washing, treatment with bleaching agents such as sodium hypochlorite and hydrogen peroxide, treatment with microbial enzyme such as lysozyme, surface activity such as sodium lauryl sulfate and deoxycholic acid Impurities can be almost completely removed from the cellulosic substance by performing treatment with the agent, washing with heating at room temperature to 200 ° C., etc., alone or in combination. The thus-obtained cellulosic material in the present invention includes those containing cellulose and a heteropolysaccharide having cellulose as a main chain, and β-1,3, β.
It includes glucans such as -1, 2 and the like. In the case of the heteropolysaccharide, the constituent components other than cellulose are hexoses such as mannose, fructose, galactose, xylose, arabinose, rhamnose and glucuronic acid, pentose sugars and organic acids. Note that these polysaccharides may be a single substance, or two or more types of polysaccharides may be mixed due to hydrogen bonds or the like.

[0011]

The present invention will be described in more detail by the following examples. Example 1 The manufacturing method of the present invention was carried out under the following conditions. As preculture, BPR2001 strain was inoculated into a 500 ml baffled flask containing 125 ml CSL-Fru medium, and 2
It was shake-cultured at 8 ° C. for 3 days. Next, the total amount of this bacterial solution was added to a 3 liter jar containing 2 liters of CSL-Fru medium. Then, each culture was performed as shown below. -The present invention (continuous culture): 40 g / L of sucrose, continuously adding the culture solution from 48 hours, and withdrawing the same amount of the culture solution. Culture time 120 hours, control the concentration of added sugar,
The BC concentration in the culture solution is maintained below about 8 g / L. -Control (fed-batch culture method): 40 g / L of sucrose, continuously added concentrated medium (10 times) after 48 hours. Culture time 12
0 hours. The aeration rate was 660 ml / min, and the stirring was performed at 300 to 1000 rpm so that the dissolved oxygen was satisfied. The results obtained are shown below.

[0012]

[Table 1] 1. Increased production rate, increased production Fed-batch culture Continuous culture Average BC production rate (g / day) 5.2 8.6 Total BC production (g) 26.0 43.0 Total consumed sugar (g) 160 160 Culture BC concentration (g / L) in the system at the end 13.0 4.9 ─────────────────────────────────

[0013]

[Table 2] 2. Reduction of required power Fed -batch culture Continuous culture At the end of culture BC concentration (g / L) 13.0 4.9 Maximum required power (kg.cm/L/sec) 80 45 ────────── ───────────────────────

[0014]

[Table 3] 3. Oxygen consumption rate during culturing Cultivation time Fed-batch culturing Continuous culturing 48 hours 19 1996 96 hours 14 22 120 hours 12 20 (μmol / L / hr)

Method of Measuring Oxygen Consumption Rate In the examples, the oxygen consumption rate is defined as the oxygen consumption amount per 1 L of the culture broth, and the oxygen amount of the supplied sterile air (including oxygen-enriched air) It was calculated from the difference between the product of oxygen concentration and the oxygen content in the exhaust gas.

[0016]

[Table 4]

[0017]

Table 5 Vitamin mixture 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

In Tables 1 and 2 above, the amount of BC (g /
L) is a mixture of solids in the culture broth, washed with water to remove the medium components, and then in a 0.1N NaOH aqueous solution at 80 ° C. for 2
The cells were removed by treatment for 0 minutes. Further, it was determined by washing the produced cellulose with water until the washing liquid became nearly neutral, followed by vacuum drying at 80 ° C. for 12 hours and measuring the dry weight.

[0019]

From the above results, in the production method of the present invention, since the concentration of BC in the culture system was kept low, the system was sufficiently mixed and oxygen was sufficiently supplied to the entire system. Therefore, compared with the conventional method, the total sugar consumption is the same, but the production rate of BC (productivity)
And, since the total amount of production increases and the BC concentration in the culture solution is low, the required power is small and the equipment is reduced.
It has become possible to reduce power consumption. As described above, according to the present invention, the production of BC can be performed very efficiently,
BC productivity is further improved.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yasushi Morinaga 3-2-1, Sakado, Takatsu-ku, Kawasaki-shi, Kanagawa Biopolymer Research Co., Ltd.

Claims (3)

[Claims] 1. A method for culturing a cellulosic substance by culturing a cellulosic bacterium, which comprises withdrawing a culture solution from a culture system during the culture period and supplying a new culture solution having a volume substantially equal to the amount of withdrawal. The production method is characterized in that the concentration of the cellulosic substance in the culture medium during culturing is kept low by continuously performing the above step. 2. The method according to claim 1, wherein the concentration of the cellulosic substance in the culture medium is maintained at about 10 g / L or less. 3. The method according to claim 1, wherein the concentration of the cellulosic substance in the culture solution is maintained so that the oxygen consumption rate in the culture solution can be maintained at 15 μmol / L / hr or more. .