JPH08224093A - Apparatus and method for preparing polysaccharide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an apparatus and a method, capable of realizing good aeration and mixing state even in a high viscosity period at anaphase of cultivation and producing polysaccharides in good productivity even at the high viscosity period.

SOLUTION: This apparatus for producing polysaccharides has an upper part spiral blade 3 and a lower part turbine blade 4, and a stirring shaft of the spiral blade 3 and the turbine blade 4 formed in a fermentation vessel 2. The spiral blade 3 is constituted of at least upper and lower one pair of arms extending in the reverse directions from the stirring shaft 5, positioned at mutually twisted positions and bridged by at least one shear paddle between the upper and lower arms, and the turbine blade is constituted by fixing at least one turbine blade to a rotating disk.

COPYRIGHT: (C)1996,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an apparatus and a method for producing a polysaccharide.

[0002]

2. Description of the Related Art Polysaccharides produced by microorganisms are known as exopolysaccharides and are known to be produced by fermentation in an aerobic environment. Typical examples of such polysaccharides include chitosan,
Examples include dextran, xanthan gum, gellan gum, welan gum, ramzan gum, pullulan, curdlan, schizophyllan, scleroglucan, levan, and sphingan. In the following, the present invention will be described focusing on xanthan gum and pullulan, which are particularly known among these. However, the present invention can be applied not only to xanthan gum and pullulan, but also to other polysaccharides that behave similarly to these during production.

Xanthan gum, for example, is obtained by well known fermentation methods. That is, for example, Xanthomonas campestris (X. campestr ), which is a bacterium belonging to the genus Xanthomonas that produces xanthan gum.
is ) (This compound and its production method are described in US Pat.
No. 59,026 specification, column 4)), by mixing with isopropanol, precipitation, recovery and production are carried out.

In xanthan gum fermentation, the viscosity of the fermentation liquor increases with the production of xanthan gum because the product xanthan gum is soluble in the medium. This increase in viscosity deteriorates the stirring effect and deteriorates the mass transfer in the tank, so that the productivity of xanthan gum decreases in the latter half of fermentation. In order to improve aeration and mixing in high-viscosity xanthan gum fermentation, it is proposed in JP-A-61-173795 and 61-173796 that a gum is formed and that it is precipitated to thereby reduce the viscosity. Has been done. However, the precipitants can cause damage to the microbial cells or they can be removed from the reaction along with the gum. Moreover, the above prior art usually requires removal of the precipitant from the product, which adds significantly to the cost.

Further, Japanese Patent Application Laid-Open No. 58-60997 proposes to reduce the viscosity of the fermentation broth by emulsion fermentation. However, this also requires the removal of oil from the product, increasing cost. In addition, various stirring methods have been investigated for fermentation of xanthan gum and stirring and mixing of a viscous aqueous solution. In particular, turbine blades are frequently used in ordinary fermenters because of their high oxygen solubility, and their effects have been reported in fermentation of xanthan gum and also in xanthan gum aqueous solution (J.Ferment.Te.
chnol.Vol.66, No. 1, pp. 103-109, 1988 / Chemical Engin
eering Science Vol. 35, pp. 2175-2163, 1980). However, the turbine blade only produces a radial fermentation liquid flow in the tank, which is not preferable as a mixed state in the tank.

Further, as other stirring blades, a marine propeller blade, a helical propeller blade, and an inclined blade have been studied. These have been reported to be able to wake up and down due to the slanted wings (Applied Biochemistry and
Biotechnology Vol.28 / 29 667th and later 1991 / Biotechno
logy and Bioengineering Vol.34 pp. 1393-1397 1989 /
Chemical Engineering Progress 1990). However, it has been reported that the use of turbine blades and these stirring blades in combination has a disadvantage in terms of power (Process
Biochemistry Vol. 27 pp. 351-365 1992).

Further, Japanese Patent Laid-Open No. 63-56296 proposes improvement of mass transfer in a tank by a circulating flow using a pump device. However, this technique is not preferable because the device becomes complicated and an important sterilization problem occurs in the fermentation device. In addition, as a fermentation method that does not use a stirring blade, the results of fermentation using a jet stream, a bubbling column fermentation tank, and an air lift fermentation tank have been reported. But,
None of these were sufficiently productive (Biotechno
logy and Bioengeneering Vol.39 pp. 85-94 1992 / App
L. Microbiol. Biotechnol. Vol. 35, pages 330-333, 1991).

[0008]

[Problems to be Solved by the Invention]
In addition to xanthan gum, other polysaccharides such as pullulan have been pointed out in the same manner, and improvements have been desired. Therefore, an object of the present invention is to provide an apparatus and a method capable of achieving good aeration and a mixed state even in the high viscosity stage in the latter stage of culture and capable of producing a polysaccharide with high productivity even in the high viscosity stage. .

[0009]

[Means for Solving the Problems] That is, to achieve the above object, the invention of claim 1 is an apparatus for producing a polysaccharide, which comprises a fermenter containing an aqueous medium containing at least a carbohydrate source and a nitrogen source, An upper spiral blade and a lower turbine blade provided in the fermenter, and a stirring shaft of the spiral blade and the turbine blade, and a pair of arms extending in opposite directions with respect to the stirring shaft are vertically arranged at a twisted position. At least one set and the upper and lower arms are bridged by at least one shear paddle to form the spiral blade, and the turbine blade is fixed to the rotating disk by fixing at least one turbine blade to the rotating disk. It is characterized by comprising.

As one of the features of the present invention, by combining the spiral blade and the turbine blade to stir the inside of the fermentation tank, it is possible to perform fermentation production without lowering the production rate even in the latter stage of fermentation.

The spiral blade used in the present invention preferably has an anti-parallel cylindrical arm perpendicular to the stirring axis, as will be understood from the examples described below. Further, a rod-shaped shear paddle is set between the upper and lower arms, and the upper arm and the lower arm have a twist angle of 20 degrees or more when viewed from above. In addition, this spiral blade can be provided with one or more. In the embodiment described later, the number of stages is two, but the number is not limited to this.

In the present invention, a spiral blade and a turbine blade (generally coaxial and fixed to the same stirring shaft) are arranged in a fermenter containing an aqueous medium containing at least the above-mentioned carbohydrate source and nitrogen source. Set up and use. As the fermenter, various types of fermenters conventionally used and known to those skilled in the art can be used.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION Next, embodiments of the present invention will be described. Conditions and the like when producing xanthan gum In the present invention, conditions and the like when producing xanthan gum will be described. As a xanthan gum-producing bacterium suitable for carrying out the present invention, a bacterium of the genus Xanthomonas can be used. For example, in addition to the above-mentioned Xanthomonas campestris, Xanthomonas carotate (X.carotate ) , Xanthomonas incanae (X.inc )
anae) , Xanthomonas begoniae (X.begoniae) , Xanthomonas papavericola (X.papavericola) , Xanthomonas translucens (X.translucens) , Xanthomonas basculorum (X.vasculorum) , and Xanthomonas hederae (X.hedertan) are used. Can be produced. Among the above, preferred ones are ATCC 55298, ATCC 55258, N
It is Xanthomonas campestris deposited internationally with a number such as RRL B-1459.

As the medium used in the present invention, nitrogen sources and carbon sources usually used for xanthan gum fermentation can be used. As the nitrogen source, a water-soluble inorganic nitrogen component such as ammonium salt, a water-soluble organic nitrogen component such as polypeptone, a water-insoluble organic nitrogen component such as soybean powder, etc. can be used. It is 0.5 g / l. Carbon sources include glucose, sucrose, xylose, molasses, starch, maltose, dextrin, and other sugars, and / or
Alternatively, one or more polyhydric alcohols such as glycerin and sorbitol can be used, and the amount of addition thereof is 5 to 70 g / L. In addition, phosphates, magnesium salts and trace components can be used as the inorganic salts. As the phosphate, 1 potassium phosphate, 2 potassium phosphate,
One or more selected from monosodium phosphate, disodium phosphate and the like can be used, and the addition amount is 1 to 5
g / L. As the magnesium salt, one or more selected from magnesium phosphate, magnesium sulfate, magnesium nitrate and the like can be used, and the addition amount thereof is 0.1 to 1 g / L. Minor components include ferrous chloride, ferric chloride, ferrous nitrate, ferric nitrate, ferrous phosphate, ferric phosphate, zinc sulfate, zinc chloride, zinc nitrate, and zinc phosphate. One or two or more selected from the above can be used, and the addition amount is 0.02 to 0.08.
g / L. The pH during fermentation is 6 ~ with dilute alkaline solution.
It is preferable to adjust to 8. pH below 6 or p
When it is H8 or more, the xanthan gum productivity decreases, which is not preferable. The temperature during fermentation is preferably adjusted to 25 ° C to 35 ° C. Below 25 ℃, the fermentation rate decreases and
A temperature of 5 ° C or higher is not preferable because some of the bacteria die and productivity decreases. Ventilation amount into the fermenter is 0.2-1.0vv
m is preferable, and if it is 0.2 vvm or less, sufficient oxygen for the growth of bacterial cells cannot be supplied. Further, if it is 1.0 vvm or more, there is no further productivity improving effect by ventilation, which is a cost disadvantage.

The inoculation amount of the pre-culture liquid in the main culture medium of the present invention is 5% by volume or more. When the amount of inoculum is 5% by volume or less, the initial amount of bacterial cells in the main culture solution is small, the bacterial cell growth is delayed, and as a result, the productivity of xanthan gum may be reduced, which is not preferable. During the fermentative production of xanthan gum, the stirring speed of the spiral blade and turbine blade in the fermentation tank (generally, the rotation speed of the stirring shaft) is low at the beginning of the culture, and the viscosity increases due to gum formation. The method is carried out by increasing the stirring rotation speed. It is not preferable to perform high-speed stirring at the initial stage of culture because it is disadvantageous in terms of stirring power. After fermentation, sterilization treatment (heat treatment) is performed, and then a hydrophilic organic solvent that does not dissolve xanthan gum is mixed to crystallize xanthan gum. Such hydrophilic organic solvents include alcohols such as isopropanol, acetone, etc.
Or these aqueous solutions can be mentioned. afterwards,
Dry. Drying with a blast dryer or a vacuum dryer 4
It is performed at 0 ° C. to 100 ° C. for 2 hours or more.

Conditions and the like for producing pullulan In the present invention, conditions and the like for producing pullulan will be described. Pullulan is a type of imperfect bacterium, Aureobasidium pullulans.
ns) is a water-soluble polysaccharide produced in a culture solution when black yeast called ns) is cultured in an aqueous medium in which a carbon source such as a monosaccharide or a decomposed product of starch and an appropriate nitrogen source are combined. Its chemical structure is based on maltotriose, which is a trimer of α-1,4 bond of glucose, as a unit.
It is a linear polymer that is repeatedly linked by 1,6 bonds. Pullulan having a molecular weight of about 80,000 to 300,000 is industrially produced and sold, and excellent properties such as water solubility, adhesiveness and film forming property are widely used in the food industry and the chemical industry. It is also used in the field of chemistry as a standard substance for measuring the molecular weight of water-soluble polymers. In pullulan fermentation production, the viscosity of the fermentation liquor increases with pullulan accumulation because the product pullulan is soluble in the medium. This increase in viscosity deteriorates the stirring effect and deteriorates the mass transfer in the tank, so that the productivity of pullulan decreases in the latter half of fermentation. This tendency is particularly remarkable in the fermentative production of polymer pullulan having a molecular weight of 2,000,000 or more. According to the present invention, the above point is improved.

The high molecular weight pullulan-producing bacterium suitable for carrying out the present invention may be any strain belonging to Aureobasidium pullulans, and may be a similar mutant strain. For example, IFO 6353, IFO commissioned to the Fermentation Research Institute
4464 or ATCC 9348, ATCC74100, ATCC 74101, ATC
C 74102, ATCC 74103, ATCC 74104, ATCC 74105 and the like and mutants thereof can be used. Especially ATCC 74100, ATCC
74101, ATCC 74102, ATCC 74103, ATCC 74104, ATCC 7
4105 is suitable as a strain producing high molecular weight pullulan having a molecular weight of 2,000,000 or more (described in US Pat. No. 5,268,460).

As the medium used in the present invention, nitrogen sources and carbon sources usually used for polymer pullulan fermentation can be used. As the nitrogen source, a water-soluble inorganic nitrogen component such as ammonium salt, a water-soluble organic nitrogen component such as polypeptone, a water-insoluble organic nitrogen component such as soybean powder, etc. can be used. It is 0.5 g / l. Sugars such as glucose, sucrose, molasses, decomposed products of starch, decomposed products of cellulose, and maltose can be used as the carbon source, and the addition amount thereof is 5 to 150 g / L. In addition, inorganic salts can be used, and examples thereof include salts of metal ions such as magnesium, iron, calcium, sodium and potassium. Specifically, for example, magnesium sulfate, ferrous sulfate, calcium carbonate,
Examples thereof include sodium chloride, potassium phosphate, manganese sulfate, potassium chloride, zinc sulfate, cobalt chloride and ammonium molybdate.

It is suitable that the initial pH is about 6.0 to 8.0, but it is not always necessary to adjust to this range during the culturing, and it is general that the pH lowers as the culturing progresses. . The temperature is appropriately 25 to 35 ° C, and the culturing time is 2 to 7
It is about a day. Furthermore, the conditions under which the cells grow in a yeast-like form are more preferable. The amount of aeration is preferably 0.1 to 1.0 vvm, and if it is 0.1 vvm or less, sufficient oxygen for the growth of bacterial cells cannot be supplied. Further, if it is 1.0 vvm or more, there is no further productivity improving effect by ventilation, which is a cost disadvantage. The inoculation amount of the pre-culture liquid in the main culture medium of the present invention is 2 volume% or more. When the amount of inoculum is 2% by volume or less, the initial amount of bacterial cells in the main culture solution is small, the bacterial cell growth is delayed, and as a result, the productivity of polymer pullulan may be reduced, which is not preferable.

During the fermentation production of polymer pullulan, the stirring speed of the spiral blade and the turbine blade in the fermenter (generally, the rotation speed of the stirring shaft) is low at the initial stage of the culture to produce pullulan. It is carried out by a method of increasing the stirring rotation speed as the viscosity increases due to. It is not preferable to perform high-speed stirring at the initial stage of culture because it is disadvantageous in terms of stirring power. After fermentation, sterilization treatment (heating treatment) (method described in JP-A-5-328988) is performed, bacteria are sterilized by filtration or centrifugation, and pullulan is mixed with a hydrophilic organic solvent that does not dissolve to give a polymer pullulan. To crystallize. As such a hydrophilic organic solvent,
Examples thereof include alcohols such as isopropanol, acetone and the like, or aqueous solutions thereof. Then, it is dried. Drying with a blast dryer or vacuum dryer at 40 ° C
It is performed at -100 ° C for 2 hours or more. It should be noted that pullulan can be recovered by direct drying without crystallization.

Conditions for Producing Other Polysaccharides In the present invention, conditions for producing other polysaccharides are substantially the same as those for xanthan gum and pullulan. However, an appropriate strain is used according to the polysaccharide produced, and a medium with high production efficiency is selected according to the strain.

Next, an embodiment of the polysaccharide production apparatus according to the present invention will be described with reference to the accompanying drawings. FIG.
FIG. 1 shows an outline of a polysaccharide production apparatus according to an embodiment of the present invention. As shown in the figure, the polysaccharide manufacturing apparatus 1 is
The fermentation tank 2 is provided with an upper and lower two-stage spiral blade 3 and a lower turbine blade 4 provided in the fermentation tank 2, and a stirring shaft 5 for the spiral blade 3 and the turbine blade 4. The fermentation tank 2 contains an aqueous medium 6 containing at least a carbohydrate source and a nitrogen source. In the present embodiment, the upper spiral blade 3 and the lower turbine blade 4 are fixed to a common stirring shaft 5. In FIG. 1, 7 is a motor that is a driving means of the stirring shaft 5, and 8 is a mechanical seal. Also, what is shown as D in the figure is the distance between the blades.

As shown in FIGS. 2 and 3, in each stage of the spiral blade 3, a pair of columnar arms 9 and 10 extending in opposite directions with respect to the stirring shaft 5 are provided in a pair at upper and lower positions at twisted positions. At the same time, the upper and lower arms 9 and 10 are bridged by eight shear paddles 11. Reference numeral 12 denotes a mounting portion of the arms 9 and 10, and the spiral blade 3 is fixed to the stirring shaft 5 by inserting the stirring shaft 5 into the through hole 13 at the center thereof. The ratio of blade diameter / body diameter (of the fermenter) of this spiral blade is 0.77. The blade length / blade diameter ratio (L / W) is 0.40. The twist angle θ of the upper and lower arms is 65.5 degrees. Figures 4 and 5
Indicates the turbine blade 4. Turbine blade 4 is rotating disk 1
It is constituted by fixing turbine blades 15 of 4 to 6. Reference numeral 16 denotes a mounting portion of the rotating disk 14, and the turbine blade 4 is fixed to the stirring shaft 5 by inserting the stirring shaft 5 into the through hole 17 at the center thereof. The turbine blade has a blade diameter / body diameter (of the fermenter) ratio of 0.50.

In the polysaccharide manufacturing apparatus according to the present embodiment having the above-mentioned structure, the spiral blade 3 is rotated by rotating the stirring shaft 5.
And the turbine blade 4 is rotated to stir the inside of the fermenter 2. At this time, the broth is sheared by the shear paddle of the spiral blade 3, which lowers the apparent viscosity of the high-viscosity broth, and the inclined shear paddle allows the fermenter to efficiently flow up and down. It is possible to maintain the mass transfer in the tank in a good state.

The spiral blade 3 can be modified in various ways by changing the specifications, and not only two stages but also
The one-stage type can also be a multi-stage type.
6 to 13 show modified examples thereof. These are used in the examples described below. By the way, Figures 6 and 7
Is a type in which the mounting portion 12 is vertically divided.
The stirring blades shown in FIG. 14 to FIG.
Used. The one shown in FIGS. 20 and 21 uses an inclined blade, and the inclination angle θ ₂ of the stirring blade 18 in FIG.
5 degrees. As a structure for joining the upper and lower spiral blades 3, various known structures can be considered. 22 and 23 show an example thereof. The spiral wing 3'includes lower opposing arms 3'a and 3'b, and the lower spiral wing 3 "includes upper opposing arms 3" c and 3 "d. And the upper portion of the spiral wing 3 "are configured to fit into each other as illustrated, and the arms 3'a and 3'b and the arms 3" c and 3 "d are located at right angles to each other. To be joined. This state is shown in Figure 2.
3 (θ ₃ is a right angle). 22 and 23, the relative positional relationship between the arms 3'c, 3'd, 3 "a, and 3" b is also understood.

The polysaccharide production apparatus according to the present invention can be modified in various manners other than the above-mentioned embodiment, and all changes and modifications within the scope of the technical idea of the present invention can be made. All are included in the present invention. For example, the rotational speeds of the spiral blade and the turbine blade can be made different by known means. However, it is preferable to comply with the following conditions. The ratio of blade diameter / body diameter (of the fermenter) of the spiral blade is preferably 0.5 or more. When it is less than 0.5, a non-fluid region is generated in the fermentor, which lowers the fermentation productivity and is not preferable. The ratio of blade length / blade diameter of the spiral blade is preferably 0.2 or more. If it is less than 0.2, it is necessary to increase the number of blade stages in order to obtain equivalent agitation, which increases power and is not preferable. The twist angle of the upper and lower arms is preferably 20 degrees or more. If it is less than 20 degrees, sufficient up-and-down flow cannot be obtained and the productivity is lowered. Also, 180
Degrees or less. Above this, the shape of the paddle cannot be maintained during stirring. It is preferable that four or more shear paddles are set. If the number is less than 4, sufficient shear force cannot be obtained. The width of the shear paddle is not particularly limited.
The cross-sectional shape of the shear paddle is circular in the above embodiment, but various shapes such as an ellipse, a square, a rectangle, an equilateral triangle, and an isosceles triangle are conceivable.

The turbine blade used in the present invention has a blade diameter /
The ratio of the body diameter (of the fermenter) is preferably 0.3 or more. When it is less than 0.3, the solubility of oxygen released from the sparger is low and the productivity is lowered, which is not preferable. The turbine blades preferably have a ratio between blades (D in FIG. 1) / blade diameter of 1 or less. When it exceeds 1, the blade interval is too wide, and a non-fluid region is generated, which lowers the fermentation productivity, which is not preferable. The set angle with respect to the tangential direction of the circumference of the rotating disk 14 of the turbine blade 15 is 30 to 9 as shown in FIG.
It is preferably 0 degree. When it is 30 degrees or less, the shearing force of the turbine blade is weak and the bubble diffusion effect is low, which is not preferable. Further, the set angle of the turbine blade 15 with respect to the disk surface of the rotating disk 14 is preferably 30 to 150 degrees as shown in FIG. When it is 30 degrees or less, the shearing force of the turbine blade is weak and the bubble diffusion effect is low, which is not preferable. The shape of the turbine blade 15 may be a triangle or a pentagon, as well as a quadrangle as in the present embodiment.

[0028]

[Examples] Results of xanthan gum production using the stirring blade described in the above embodiment (Examples 1 to 1)
6) is shown below. Further, the results of Comparative Examples 1 to 6 for clarifying the difference from the conventional technique are also shown.

Examples 1-6 Xanthomonas camp in a fermentor containing the following medium components of I
After culturing estris for 24 hours, it contains the following medium components of II 30
Inoculated into L fermentor. As the stirring blade, fermentation was performed using the stirring blade having the shape described in the above example.
Table 1 shows the parameters of the stirring blade and the mounting method. I. Preculture medium component composition Glucose 5.8 g / L Polypeptone 5.2 g / L Yeast extract 2.6 g / L NaCl 9.0 g / L water 1.8 L II. Main culture medium component composition Glucose 58 g / L Soybean powder 3. 3 g / L (nitrogen content: 0.3 g / L) KH ₂ PO ₄ 2.0 g / L MgSO ₄ / 7H ₂ O 0.5 g / L water 16.2 L

[0030]

[Table 1]

After fermentation for 2 days under the above conditions, sterilization was performed at 70 ° C. for 1 hour. After that, it was crystallized with an 85% IPA aqueous solution of 1.5 times by weight using a mixer, and then dried with a blast dryer at 60 ° C.
It was dried for 3 hours. After drying, the weight of xanthan gum was measured. Table 2 shows the final fermentation broth viscosity, residual glucose concentration, xanthan gum concentration, and final motive power under each fermentation condition. The viscosity was measured at 20 ° C. using a BL type viscometer (Tokimec Co., Ltd.) rotor No. 4, 30 rpm. The residual glucose concentration was measured by Enzyme Electrode Analyzer M-100 (Asahi Kasei Corporation).

[0032]

[Table 2]

It was confirmed that good xanthan gum production was achieved even at a low speed stirring rotation speed equivalent to that of Comparative Example 5 described later, and it was found that the spiral blade had good mass transfer.

Example 7 Fermentation was carried out under the same conditions as in Example 1, and after 2 days of fermentation, a further 4
500 ml of an aqueous solution containing 14 g of glucose was sterilized and added to the fermentor to continue fermentation. Table 3 shows the results after 3 days
Shown in It was confirmed that the spiral blade was well produced even at 30,000 cP.

[Table 3]

Comparative Examples 1 to 6 Fermentation was carried out using the same medium as in Examples and using the stirring blades shown in Table 4. The conditions are shown in Table 4. The results are shown in Table 5.

[0036]

[Table 4]

[0037]

[Table 5]

By contrasting the above examples with the comparative examples, it is understood that the present invention has significantly higher productivity.

Results of polymer pullulan production using the stirring blade described in the above examples (Examples 8 to 1)
3) is shown below. In addition, the results of a comparative example for clarifying the difference from the prior art are also shown.

Examples 8-13 Aureobasidium pu in a fermentor containing the following medium components of I
After culturing llulans (ATCC 74105) for 24 hours, it was inoculated into a 30 L fermenter containing the following medium component II. Fermentation was performed using a stirring blade having the shape shown in the figure as the stirring blade.
Table 6 shows the parameters of the stirring blade and the mounting method. I. Preculture medium component composition Sucrose 10.0 g / L Yeast extract F 2.0 g / L (NH ₄ ) ₂ SO ₄ 0.5 g / L K ₂ HPO ₄ 3.0 g / L MgSO ₄ / 7H ₂ O 0.2 g / L FeSO ₄ .7H ₂ O 0.01 / L MnSO ₄ .7H ₂ O 0.01 g / L ZnSO ₄ .7H ₂ O 0.01 g / L Water 2.5 L pH 7.0 II. Main culture medium composition Sucrose 100 g / L (NH ₄ ) ₂ SO ₄ 1.0 g / L K ₂ HPO ₄ 2.0 g / L FeSO ₄ · 7H ₂ O 0.01 g / L MnSO ₄ · 7H ₂ O 0.01 g / L ZnSO ₄・ 7H ₂ O 0.01 g / L water 16.2 L pH 7.0

[0041]

[Table 6]

After fermentation for 5 days under the above conditions, 6 with stirring
Sterilized at 0 ° C. for 30 minutes. Then, the cells were removed by centrifugation. This pullulan solution is 1.5% by weight 86%
It was mixed with IPA and pullulan was recovered. Table 7 shows the final fermentation broth viscosity, residual sucrose concentration, pullulan concentration, pullulan molecular weight, and final power under each fermentation condition. Viscosity is 20
℃, BL type viscometer (Tokimec Co., Ltd.) rotor N
o. It was measured using 4,30 rpm. The residual sucrose concentration was measured by Enzyme Electrode Analyzer M-100 (Asahi Kasei Co., Ltd.). Pullulan molecular weight was measured by measuring the intrinsic viscosity, and the relationship between intrinsic viscosity and molecular weight of Buliga et al. (Int. J. Biol. Macromol., Vol 9, 71-7
6 (1987)) [η] = 0.000258 × Mw− ⁰⁶⁴⁶ .

[0043]

[Table 7]

It was confirmed that good polymer pullulan production was achieved even at a low stirring speed equal to that of Comparative Example 11 described later, and it was found that the spiral blade had good mass transfer.

Comparative Examples 7 to 12 Fermentation was carried out using the same medium as in Examples and using stirring blades other than the spiral blade. The conditions are shown in Table 8 and the results are shown in Table 9.

[0046]

[Table 8]

[0047]

[Table 9]

By contrasting the above examples with the comparative examples, it is understood that the present invention has remarkably higher productivity.

[0049]

As is apparent from the above, according to the present invention, good aeration and a mixed state are realized even in the high viscosity stage in the latter stage of culture, and the polysaccharide is produced with good productivity even in the high viscosity stage. An apparatus and method are provided that are capable of doing so.

That is, in the present invention, by using the spiral stirring blade and the turbine blade in combination, the dissolved oxygen level necessary for the growth of the bacterial cells is maintained at the early stage of fermentation by the high shearing force of the turbine blade, and the polysaccharide is used. It is possible to effectively mix the high-viscosity fermented liquid with the spiral blade even after the viscosity is increased due to the production, and it is possible to perform the fermentation production in the latter stage of the fermentation without lowering the polysaccharide production rate. Also,
It is possible to achieve good productivity with lower power consumption as compared with the case of using the conventional turbine blade only in combination with the turbine blade and the inclined blade.

[Brief description of drawings]

FIG. 1 is a conceptual diagram outlining an embodiment of a polysaccharide production apparatus according to the present invention.

FIG. 2 is a side view of the spiral blade used in Examples 1 and 8 of the spiral blade used in the polysaccharide manufacturing apparatus according to the present invention.

FIG. 3 is a bottom view of the spiral blade used in Examples 1 and 8 of the spiral blade used in the polysaccharide manufacturing apparatus according to the present invention.

FIG. 4 is a side view of a turbine blade used in each of Examples and Comparative Examples of the turbine blade used in the polysaccharide manufacturing apparatus according to the present invention.

FIG. 5 is a bottom view of a turbine blade used in each of the examples and comparative examples of the turbine blade used in the polysaccharide manufacturing apparatus according to the present invention.

FIG. 6 is a side view of a spiral blade used in Examples 2, 4, 9 and 11 of the spiral blade used in the polysaccharide manufacturing apparatus according to the present invention.

FIG. 7 is a bottom view of a spiral blade used in Examples 2, 4, 9 and 11 of the spiral blade used in the polysaccharide production apparatus according to the present invention.

FIG. 8 is a side view of the spiral blade used in Examples 3 and 10 of the spiral blade used in the polysaccharide production apparatus according to the present invention.

FIG. 9 is a bottom view of the spiral blade used in Examples 3 and 10 of the spiral blade used in the polysaccharide manufacturing apparatus according to the present invention.

FIG. 10 is a side view of the spiral blade used in Examples 5 and 12 of the spiral blade used in the polysaccharide production apparatus according to the present invention.

FIG. 11 is a bottom view of a spiral blade used in Examples 5 and 12 of the spiral blade used in the polysaccharide production apparatus according to the present invention.

FIG. 12 is a side view of a spiral blade used in Examples 6 and 13 of the spiral blade used in the polysaccharide manufacturing apparatus according to the present invention.

FIG. 13 is a bottom view of the spiral blade used in Examples 6 and 13 of the spiral blade used in the polysaccharide manufacturing apparatus according to the present invention.

FIG. 14 is a side view of a spiral blade used in Comparative Examples 1 and 7.

FIG. 15 is a bottom view of the spiral blade used in Comparative Examples 1 and 7.

FIG. 16 is a side view of the spiral blade used in Comparative Examples 2 and 8.

FIG. 17 is a bottom view of a spiral blade used in Comparative Examples 2 and 8.

FIG. 18 is a side view of a spiral blade used in Comparative Examples 3 and 9.

FIG. 19 is a bottom view of a spiral blade used in Comparative Examples 3 and 9.

FIG. 20 is a side view of one of the inclined blades used in Comparative Examples 4, 5, 10, and 11.

FIG. 21 is a bottom view of one of the inclined blades used in Comparative Examples 4, 5, 10, and 11.

FIG. 22 is a side view for explaining a manner of joining upper and lower spiral blades.

FIG. 23 is a plan view from above illustrating a state in which the spiral blades of FIG. 22 are joined. This figure corresponds to a state in which the upper and lower spiral blades in FIG. 1 are viewed from above.

FIG. 24 is a plan view from above illustrating an inclination angle with respect to the circumference of a turbine blade of a turbine blade that can be used in the present invention.

FIG. 25 is a perspective view from above for explaining the inclination angle of the turbine blade of the turbine blade that can be used in the present invention with respect to the rotating disk.

[Description of sign]

 1 Polysaccharide Manufacturing Equipment 2 Fermenter 3 Spiral Blade 4 Turbine Blade 5 Stirring Shaft 6 Aqueous Medium 7 Motor 8 Mechanical Seal 9 Arm 10 Arm 11 Shear Paddle 12 Mounting Part 13 Through Hole 14 Rotating Disk 15 Turbine Blade 16 Mounting Part 17 Through hole 18 Stirrer

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:64) (72) Inventor Kanji Murobushi Niigata Prefecture Nakakubiki-gun Kubiki-mura 28 Nishi-Fukushima 1 Shin-Etsu Chemical Co., Ltd., Synthetic Technology Laboratory (72) Inventor Shigehiro Nagura 28, Nishi-Fukushima, Kubiki-mura, Nakakubiki-gun, Niigata Prefecture 1 Shin-Etsu Chemical Co., Ltd. Synthetic Technology Laboratory (72) Inventor Taira Honma Niigata 28, Nishi-Fukushima, Kubiki-son, Nakakubiki-gun, Shizuoka 1 Shin-Etsu Chemical Co., Ltd.

Claims (5)

[Claims] 1. A fermenter containing an aqueous medium containing at least a carbohydrate source and a nitrogen source, an upper spiral blade and a lower turbine blade provided in the fermenter, and a stirring shaft for the spiral blade and the turbine blade. And a pair of arms extending in mutually opposite directions with respect to the stirring shaft are provided at least one set up and down at mutually twisted positions, and the upper and lower arms are bridged with at least one shear paddle to form the spiral blade. An apparatus for producing a polysaccharide, characterized in that the turbine blade is configured by fixing at least one turbine blade to the rotating disk. 2. The polysaccharide production apparatus according to claim 1,
The polysaccharide manufacturing apparatus, wherein the twist angle of the pair of arms at the upper and lower positions is 20 degrees or more. 3. The polysaccharide production apparatus according to claim 1 or 2, wherein the ratio of the blade diameter of the spiral blade / the barrel diameter of the fermentation tank is 0.5 or more. 4. The polysaccharide production apparatus according to claim 1, wherein the ratio of blade diameter of turbine blade / body diameter of fermentation tank is 0.3 or more. Manufacturing equipment. 5. A polysaccharide producing microorganism is inoculated into an aqueous medium containing a carbohydrate source and a nitrogen source using the polysaccharide production apparatus according to claim 1, and the medium is fermented. A method for producing a polysaccharide, which comprises fermenting by mechanically stirring and aerating under conditions.