JP2006034235A - Culture apparatus of polymer compound-producing microorganism and culture method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culture apparatus of microorganisms, capable of solving such a problem that deterioration of oxygen supply capacity is caused by viscosity rise of a culture solution accompanied by accumulation of a polymer compound, when the polymer compound is produced by aeration-agitation culture, and to provide a culture method. <P>SOLUTION: This culture apparatus 1 cultures the microorganisms, by supplying oxygen to the culture solution containing the microorganisms which produce the polymer compound, such as γ-PGA, wherein the culture apparatus 1 is equipped with an aeration means 6 which relieves the deterioration of the oxygen supply capacity caused by the viscosity rise of the culture solution. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention reduces a decrease in oxygen supply capacity accompanying the increase in viscosity of a culture solution during culture, and a microorganism having the ability to produce a polymer such as a polymer such as poly-γ-glutamic acid (hereinafter referred to as “γ-”). The present invention relates to a method and an apparatus for producing high molecular weight poly-γ-glutamic acid (hereinafter referred to as “γ-PGA”) using “PGA-producing bacteria”.

  A high molecular compound such as γ-PGA is a protein produced by bacteria belonging to the genus Bacillus, and has recently been used as a functional material in many fields such as foods, pharmaceuticals, cosmetics, and water purification.

  However, when γ-PGA is produced by liquid culture suitable for industrial production, especially when high molecular weight γ-PGA having a molecular weight of 3 million or more is to be produced, the high molecular weight of γ-PGA produced in the culture solution. The problem is that the viscosity of the culture broth increases due to the increase in concentration and / or concentration, the contact between the culture broth and air becomes non-uniform, and production stops or becomes unstable. It was an obstacle to industrial production of PGA.

  As a method for solving the problem due to the increase in the viscosity of the culture solution, there is an attempt to increase the oxygen supply efficiency by maintaining the culture solution homogeneously by first increasing the stirring speed of a stirring blade such as a disk turbine blade in the culture apparatus. . However, the stirring of the culture solution by this method is subject to damage such as the cleavage of the molecular chain of γ-PGA by the shearing force, so the stirring speed must be suppressed. That is, the enhancement of the stirring speed is not a means that can cope with the increase in the viscosity of the culture solution during the culture. Therefore, production of high molecular weight γ-PGA has been difficult.

  Next, there is an attempt to use an oxygen supply device such as a stainless mesh sparger that forms fine bubbles and has a high oxygen supply efficiency compared to a commonly used ring sparger. This method forms microbubbles under low viscosity but cannot form microbubbles under high viscosity, so the reduction in oxygen supply capacity cannot be reduced and the culture solution cannot be made highly viscous.

  Furthermore, there is an attempt to increase the oxygen partial pressure of the air that flows to the culture apparatus. In this case, the purpose cannot be sufficiently achieved in an environment where the stirring speed is suppressed under the high viscosity described above and fine bubbles cannot be formed.

  On the other hand, manufacturing γ-PGA by liquid culture using microorganisms is disclosed in Patent Documents 1 to 4 and the like, but the molecular weight of γ-PGA to be manufactured is about several thousand to 2 million. This was an effective means for avoiding the above-mentioned problem of increasing the viscosity of the culture solution and producing a significant amount of γ-PGA, but as a result, a high molecular weight γ-PGA cannot be produced.

  By the way, among γ-PGA-producing bacteria, γ-PGA of Bacillus subtilis natto, which has food experience and is used for safe and secure natto production, has a molecular weight of several hundred to about 5 million and other γ-PGAs. It has a higher molecular weight than the producing bacteria, and its usefulness can be expected. However, due to the high molecular weight, the technical difficulties brought about by the high viscosity of the culture solution described above are significant. For example, in the case of a culture volume of 300 liters or more compared to a relatively small amount of culture volume of 3 liters or less, the productivity is remarkably unstable, and Bacillus natto used for natto production with a culture volume exceeding 100 liters. There has been no report on the production of γ-PGA, and as a result, consumers such as γ-PGA are more familiar with safety and missed the opportunity to obtain high molecular weight γ-PGA.

In addition to the production of γ-PGA, for example, in the cultivation of microorganisms in the production of biocellulose, xanthan gum, pullulan, dextran, fructan, levan, etc., when the viscosity of the culture solution increases, it relates to the supply of oxygen to the culture solution. Similar problems occur and the production of useful products is limited.
JP 43-24472 A Japanese Patent Laid-Open No. 01-174597 Japanese Patent Laid-Open No. 03-47087 Patent No.3081901

  The object of the present invention is to solve the problem of a decrease in oxygen supply capacity caused by an increase in the viscosity of a culture solution accompanying the accumulation of a polymer compound in the production of the polymer compound by aeration and agitation culture. In the production of γ-PGA by culture, the problem of decrease in oxygen supply capacity due to increase in viscosity of the culture solution accompanying accumulation of γ-PGA was solved, and high molecular weight γ-PGA having an average molecular weight of 3 million or more was obtained in high yield. It is another object of the present invention to provide a culture apparatus and culture method for microorganisms that produce high molecular weight γ-PGA that can be stably produced.

  The present invention reduces the decrease in oxygen supply capacity under high-viscosity conditions by suspending or destabilizing γ-PGA production that accompanies an increase in the viscosity of the culture solution observed in liquid culture of γ-PGA-producing bacteria. It is based on the new knowledge that it can prevent by doing.

  Based on the above knowledge, the present invention provides a culture apparatus for culturing by supplying oxygen to a culture solution containing a microorganism that produces a polymer compound, and reducing the oxygen supply capacity caused by an increase in the viscosity of the culture solution in the culture device. It is characterized by having a ventilation means for reducing the above.

  The microorganism culturing apparatus for producing high molecular weight γ-PGA of the present invention is equipped with aeration means for reducing a decrease in oxygen supply capacity caused by an increase in the viscosity of a culture solution, and produces high molecular weight γ-PGA having a molecular weight of 3 million or more. It is characterized by doing.

  The method for culturing microorganisms producing high molecular weight γ-PGA according to the present invention is a method of cultivating microorganisms producing high molecular weight γ-PGA having a molecular weight of 3 million or more by supplying oxygen to the culture medium of microorganisms producing high molecular weight γ-PGA. The culture method is characterized in that aeration is performed to reduce a decrease in oxygen supply capacity caused by an increase in the viscosity of the culture solution.

  According to the present invention, there is provided an oxygen supply device or a high-viscosity fluid aeration device and a stirring blade that form fine bubbles of a gas containing oxygen even under conditions where the viscosity of the culture solution increases with the progress of culture. By combining the stirrer, damage of the high molecular weight γ-PGA due to the shearing force of stirring can be reduced and oxygen can be supplied efficiently.

  In the culture of natto for use in the production of natto using the present invention, by reducing the decrease in oxygen supply capacity under high-viscosity conditions, culture medium conditions exhibiting a viscosity of 1000 millipascal seconds (mPa · s) or more Can produce high-molecular-weight γ-PGA with high safety and average molecular weight of 3 million or more at a recovery rate of 2% or more per culture medium. It becomes possible to do. This is about 2.3 times the production amount compared to the case of culturing using a ring sparger generally used in fermenters. In addition, most of conventional γ-PGA has a molecular weight of about 1 million or less, and it is expected that new functions or enhancement of known functions will be expected due to the high molecular weight. Therefore, the present invention expands the application of γ-PGA to cosmetics, foods, medical-related products and the like, and is extremely useful industrially.

  The γ-PGA-producing bacterium targeted by the present invention is not particularly limited, but natto bacterium used for commercial natto production is particularly preferable. Representative natto bacteria used in the production of natto are Miyagino, Takahashi, Naruse, Asahikawa, and Matsumura. Any of these strains may be used for the production of γ-PGA. Cultures (eg, IFO 3009, IFO 3013, IFO 3336, IFO 3936, IFO 13169, ATCC 7058, ATCC 7069, ATCC 15245) may be used. Alternatively, a high-viscosity expression strain selected from commercially available natto by a known method (Natto Test Method Research Group, Natto Test Method, pp. 65-73, Kohan (1990)) may be used.

  In addition, any bacteria other than natto bacteria may be used as long as they produce γ-PGA, such as Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, and the like. Bacillus genus bacteria, Xanthobacter, Mycobacterium tuberculosis, Corynebacterium glutamicum, and the like may be used.

  Moreover, the mutant strain obtained from the said microbe using various well-known mutation processing methods can be used. Examples of the mutation treatment method include an ultraviolet irradiation method, an X-ray irradiation method, and a chemical contact.

  The object can be achieved by culturing γ-PGA-producing bacteria in the culture apparatus of the present invention or holding a culture solution of γ-PGA-producing bacteria in the apparatus for a certain period. The aeration means for reducing the decrease in oxygen supply capacity under high viscosity conditions is an oxygen supply apparatus (for example, Japanese Patent Application Laid-Open No. 2001-000890) having the ability to form fine bubbles of gas containing oxygen under the conditions. JP, 2002-045667, A) or a high-viscosity fluid diffuser.

  Furthermore, the culture apparatus provided with the aeration means can be used as long as it is a culture apparatus capable of purely culturing microorganisms, or a container capable of holding a purely cultured culture solution aseptically. The oxygen partial pressure of the air ventilated to the culture apparatus can be arbitrarily changed according to the viscosity of the culture solution.

  In addition, γ-PGA having a molecular weight of 3 million or more is produced by culturing a culture solution of γ-PGA producing bacteria, or γ-PGA having a molecular weight of 3 million or less is accumulated at a high concentration and the viscosity of the culture solution is 1000 millipascal second (mPa · s) If it becomes more than this, this invention can be used. In addition, the production of γ-PGA according to the present invention is preferably used for the production of high molecular weight γ-PGA having a molecular weight of 3 million or more by culturing a culture solution of Bacillus natto used for natto production.

  In addition to the production of γ-PGA, the present invention can be used for the production of a polymer compound that increases the viscosity of a culture solution, for example, (1) production of biocellulose (producing bacteria: Acetobacter xylinum subspecies Schcrofermentans ( Acetobacter xylinum subsp. Sucrofermentans), Acetobacter xylinum ATCC 23768, ATCC 23769, ATCC 14851, ATCC 11142, ATCC 10821, Acetobacter pasteurianus ATCC 10245, etc. , Achromobacter genus, Alkaligenes genus, Aerobacter genus, Azotobacter genus, Zoogrea genus, etc.), (2) Production of xanthan gum (producing bacteria: Zanthomonas campe) (3) Pullulan production (producer: Aureobasidium pullulans, etc.), (4) Dextran production (producer: Leuconostoc mesenteroides, etc.) (5) Production of fructans and levans (Producing bacteria: Bacillus subtilis, Bacillus subtilis natto, and other genus Bacillus, Rahnella aquatilis, Zymomonas mobilis) , Pseudomonas aurantiaca, Gluconobacter oxydans, Aerobacter levanicum, Corynebacterium laevaniformans, L It can be applied to Winwina amylobora (Erwinia amylovora), Streptococcus salivarius, Acetobacter genus, Aspergillus genus and the like.

  FIG. 1 is a schematic view showing an example of the culture apparatus of the present invention.

  At the top of the container (fermenter) 1 for storing the culture solution, there is a culture solution supply port 2 with a lid 2a for supplying the culture solution into the container 1, an exhaust port 3 for discharging the air supplied into the container, and the culture A stirring blade 4 that is rotated by a motor M to stir the liquid, and a jacket 5 through which a heat exchange medium flows are provided to keep the temperature of the container 1 constant. A circulating flow generator 6 is disposed in the container as a ventilation means for supplying air below the stirring blade 4. The circulating flow generator 6 is supplied with high-pressure air from the compressor 8 through the pipe 7 (see FIG. 2).

  FIG. 2 shows a circulating flow generator as an example of aeration means used in the present invention, (a) is a longitudinal sectional view of a pipe, (b) is a transverse sectional view of a gas blowing hole portion, and (c) is a culture solution. The longitudinal cross-sectional view of a discharge port part, (d) is a figure which shows the path | route of the high pressure air supply of a circulating flow generator.

  In FIG. 2A, a flow path 12 is formed in the pipe 11 of the circulating flow generator 6, and a culture medium discharge port 13 is formed at one end of the flow path 12 so as to expand in the shape of a frustum. Is formed with a culture solution suction port 14.

    A plurality of gas blowing holes 15 are formed in the lower part of the pipe 11. In the vertical cross section of the pipe 11, the gas blowing hole 15 is provided so as to be inclined at an acute angle (at an angle θ <b> 1) with respect to the traveling direction (arrow a direction) of the culture solution in the flow path 12. Further, as shown in FIG. 2B, the gas blowing hole 15 is formed in the tangential direction of the wall surface of the flow path 12 in the horizontal cross section of the pipe 11.

  In the circulating flow generating device 6, when a high-pressure gas containing oxygen is blown into the flow path 12 through the gas blowing holes 15 as indicated by an arrow b, bubbles are formed along the wall surface of the flow path 12. While rising. The culture solution is entrained in the swirling flow of such bubbles, and is sucked from the culture solution suction port 14 by the ejector effect, mixed with the swirling flow, flows through the flow path 12, and the gas blown from the gas blowing hole 15. After being mixed, it flows through the flow path 12 and is discharged from the culture solution discharge port 13. As a result, a gas-liquid mixed circulation flow as shown by an arrow c is generated.

  The flow path 12 from the culture solution suction port 14 to the culture solution discharge port 13 has a cylindrical shape with a constant radius, but the culture solution discharge port 13 continuous to the flow channel 12 has a divergent shape. is doing. For this reason, the pressure of the culture solution flowing in the flow path 12 in a high energy state is rapidly reduced and a strong shearing force is generated, so that bubbles contained in the culture solution are separated by the shearing force, and the culture is performed. The contact area between the liquid and the bubbles is expanded, and the dissolved gas (oxygen) is accelerated.

  The angle θ1 of the gas blowing hole 15 is set in the range of 10 degrees to 80 degrees. In this range, the swirl flow becomes the most intense, and the swirl flow is insufficient at angles less than 10 degrees or greater than 80 degrees. Note that θ2 = 90−θ1. Further, the gas blowing hole 15 is formed in the tangential direction of the wall surface in the horizontal cross section of the pipe 11. Thus, when it forms in the tangent direction of the wall surface of the flow path 12, the strongest swirl | vortex flow can be generated. In addition, although the number of the gas blowing holes 15 shall be about 3-8, you may increase / decrease suitably as needed.

  The high-pressure gas blown from the gas blow hole 15 formed as described above becomes bubbles, rises while turning along the wall surface of the flow path 12, and sucks the culture solution from the culture solution suction port 14. The sucked culture medium is mixed with bubbles while swirling, rises, reaches a culture solution discharge port 13 provided so as to spread, and a strong shear stress occurs, so that the bubbles become finer. It will be divided into bubbles. Here, the pressure of the high-pressure gas blown from the gas blow hole 15 into the flow path 12 is appropriately in the range of about 0.1 to 10 kgf / square meter. The high-pressure gas is air and oxygen necessary for culture.

  In FIG. 2C, an angle λ formed by a tangent line d in contact with the curved surface of the culture medium discharge port 13 at a connection point A between the flow path 12 and the culture solution discharge port 13 is defined as an opening angle of the culture solution discharge port 13. To do. The opening angle λ of the culture solution outlet 13 is most preferably not less than 50 degrees and not more than 70 degrees. Even if this is not the case, the opening angle λ is preferably 30 degrees or more. If the angle is less than 30 degrees, the bubbles generated by the blown high-pressure gas cannot be refined, and if the opening angle λ is greater than 90 degrees, the bubbles cannot be refined.

  The lower end of the pipe 1 is connected to a holder 16, and the holder 16 has a rectangular shape in a vertical cross section, and a cavity 17 is formed inside thereof. Further, a hole conforming to the outer shape of the pipe 11 is formed in the lower inner surface of the holder 16. Furthermore, the culture solution suction port 14 is formed so as to penetrate from the hole to the outside of the holder 16. Further, high-pressure air is blown into the side portion of the holder 16 through the pipe 7 from the compressor 8 to the cavity 17.

  In this example, Bacillus natto used for natto production was cultured in a 500 liter culture volume culture apparatus equipped with the circulating flow generator shown in FIG. The comparative example shows an example of culturing in a conventional 500 liter culture volume culture apparatus equipped with the conventional ring sparger shown in FIG. The culture apparatus shown in FIG. 4 is shown in FIG. A ring-shaped ring sparger 18 having a large number of air ejection holes for air supply is arranged in the container 1 instead of the circulating flow generator 6, and the same members as those in FIG. Omitted.

The culture medium composition used for the culture was weight / volume: sodium glutamate 5.0%, sodium chloride 0.05%, disodium hydrogen phosphate 12 water 0.42%, potassium dihydrogen phosphate 0.27% Glucose 2.0%, magnesium sulfate 7 water 0.05%, biotin 1.0ppm. 400 L of the culture solution was put into a fermenter equipped with a circulating flow generator and sterilized at 121 ° C. for 30 minutes. This was inoculated with 10 ⁴ cfu / ml of commercially available edible natto bacteria, and cultured for 80 hours at an aeration rate of 0.6 vvm, a stirring speed of 80 rpm, and a culture temperature of 40 ° C.

  The apparent viscosity of the culture broth was measured using a B-type viscometer. 2 rotor or no. Three rotors were used, and measurement was performed at a measurement rotation speed of 30 rpm and a measurement temperature of 20 ° C. In addition, when the apparent viscosity is less than 1000 millipascal seconds (mPa · s), no. The measured value with 2 rotors is adopted as it is, and when it is 1000 millipascal second (mPa · s) or more, No. No. 3 rotor was measured in advance. 2 rotor measured value and No. No. 3 from the regression equation of the measured values of the three rotors. It was converted into a value with 2 rotors.

  As a result, in the fermenter equipped with the conventional ring sparger, as shown by Δ in FIG. 3, the apparent viscosity of the culture solution reached 1000 millipascal second (mPa · s) at 45 hours of culture. Thereafter, the increase in viscosity was stopped and production of γ-PGA was stopped. In contrast, in the fermenter according to the present invention equipped with a circulating flow generator, as shown by the circles in FIG. 3, the apparent viscosity subsequently increased and reached 3500 millipascal seconds (mPa · s) at 76 hours of culture. Reached.

  An example is shown in which γ-PGA is recovered from the culture solution produced in this example by alcohol precipitation, which is a known method. The alcohol-precipitated γ-PGA was vacuum-dried to obtain 9.1 kg of white pellets. The purity of this white pellet was 90% or more. In addition, as a result of measuring the molecular weight at a UV region of 214 nm using a 50 mM sodium phosphate buffer as a mobile phase in a high performance liquid chromatograph equipped with TSK-GEL GMPW (7.8 × 300 mm) as a separation column, the molecular weight was 300 It was ten thousand.

Table 1 shows a comparison of the viscosity of the culture solution and the amount of γ-PGA produced and the average molecular weight of the obtained γ-PGA when edible natto bacteria are cultured in the present invention and when cultured with a conventional ring sparger. .

  From Table 1, it can be seen that the production amount of γ-PGA according to the present invention is superior to the case of culturing with a conventional ring sparger.

  The present invention expands the use of γ-PGA for cosmetics, foods, medical-related products, and the like, and can also be applied to culture production of high-molecular substances produced by microorganisms.

  In addition, the present invention can be expected to be effective in the production of a polymer substance produced from microorganisms when the production of the polymer substance is suppressed by reducing the oxygen supply capacity accompanying an increase in the viscosity of the culture solution. .

It is the schematic which shows an example of the culture apparatus of this invention. It is a figure which shows the circulating flow generator which is a ventilation means used by this invention. It is a figure which shows the time-dependent change of the culture solution viscosity of this invention and a prior art example. It is the schematic which shows the conventional culture apparatus.

Explanation of symbols

1: container 2: culture solution supply port 3: exhaust port 4: stirring blade 5: jacket 6: circulating flow generator 7: piping 8: compressor 11: pipe 12: channel 13: culture solution discharge port 14: culture solution suction Mouth 15: Gas blowing hole 16: Holder 17: Cavity 18: Ring sparger

Claims (5)

  A culture apparatus for culturing by supplying oxygen to a culture solution containing a microorganism that produces a polymer compound, and comprising a ventilation means for reducing a decrease in oxygen supply capacity caused by an increase in the viscosity of the culture solution. A microorganism culturing apparatus for producing a polymer compound.   A culture apparatus for culturing by supplying oxygen to a culture solution containing microorganisms that produce poly-γ-glutamic acid, and comprising aeration means for reducing a decrease in oxygen supply capacity caused by an increase in the viscosity of the culture solution in the culture device A culture apparatus for microorganisms producing high molecular weight poly-γ-glutamic acid, characterized by producing high molecular weight poly-γ-glutamic acid having a molecular weight of 3 million or more.   3. The culture apparatus for microorganisms producing high molecular weight poly- [gamma] -glutamic acid according to claim 2, wherein the aeration means is an apparatus for forming fine gas bubbles containing oxygen in the culture solution.   Oxygen supply ability produced by an increase in the viscosity of a culture solution in a microorganism culture method for producing high molecular weight γ-PGA having a molecular weight of 3 million or more by supplying oxygen to a culture solution of a microorganism producing high molecular weight poly-γ-glutamic acid A method for culturing a microorganism that produces high-molecular-weight poly-γ-glutamic acid, wherein aeration is performed to reduce the decrease in the amount. The method for culturing microorganisms producing high molecular weight γ-PGA poly-γ-glutamic acid according to claim 4, wherein fine bubbles of gas containing oxygen are formed in the culture solution.

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