JPH09266793A - Production of organic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To perform the culture and fermentation of microbial cells in an extremely loosened state of the cells by proliferating mold capable of producing lactic acid in a culture liquid added with a formite-type clay mineral and adding a coagulant to the liquid before fermentation. SOLUTION: A formite-type clay mineral is added to a liquid medium inoculated with mold cell or its spore capable of producing lactic acid in an amount to get a concentration of 1-50g/L and the cell is cultured. When the proliferation rate of the microbial cell is slowed down after the start of the culture, a coagulant is added to the medium at a concentration of 1-20mg/L and the fermentation is performed while adding an alkaline substance to prevent the lowering of pH.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing lactic acid, and more specifically, it is possible to culture cells in a very well disentangled state, and the rate of mass transfer of oxygen and substrates. The present invention relates to a method for producing lactic acid capable of increasing a substance production rate and improving a substance production rate.

[0002]

BACKGROUND OF THE INVENTION Lactic acid is a substance useful as a food additive, a raw material for brewing, a raw material for medicines and agricultural chemicals, a synthetic raw material for chemicals and plastics, and is produced by a chemical synthesis method and a fermentation method. ing.

In the latter fermentation method, a method is used in which a filamentous fungus having a lactic acid-producing ability is inoculated into a liquid medium containing sugars and the like, and culture and fermentation are carried out while stirring by aeration.
It is often practiced to use filamentous fungi supported on a support or carrier.

Japanese Unexamined Patent Publication (Kokai) No. 57-144985 describes the use of cells immobilized on a gel-like carrier and capable of producing L-lactic acid for fermentation of lactic acid.

JP-A-6-253871 discloses that lactic acid is produced by bringing a lactic acid fermentation liquid into contact with an aggregate of fibers carrying lactic acid-producing microorganisms, in particular, a biocompatible non-woven fabric carrying lactic acid-producing microorganisms. It is described to do.

[0006]

When filamentous fungi are grown in deep culture (liquid culture), they take various forms from pellets in which hyphae are entangled to form lumps, and pulp which is uniformly dispersed in the culture solution. Since the morphology of mycelium in submerged culture affects the production of industrial substances, inducing a highly productive mycelium morphology is a key to high-yield fermentation production.

At present, we are trying to control the morphology of bacterial cells by the production method of lactic acid such as culture conditions, but it is difficult to obtain those conditions, and even if the expected morphology is obtained, it will be affected by subtle environmental changes. Easy to receive. Many filamentous fungi, especially fungi, form large and small spherical pellets during deep culture, but depending on the size, oxygen or substrate diffusion-controlling occurs in the bacterial cells inside the pellet.

When the applicant carries out the culture by supporting the filamentous fungus on a holmite clay mineral such as sepiolite, the mass transfer rate of oxygen, substrate, etc. is increased, and the mass production rate is also improved. It has been found that the above is also effectively performed (Japanese Patent Laid-Open No. 7-115961).

The present inventors have applied this method to a method for producing lactic acid, but in the case of adding sepiolite, the time for keeping the cells in a stable dispersed state is higher than that in the case of not adding sepiolite. Although it was slightly extended, it was unavoidable that the lump of bacterial cells eventually adhered to the vessel wall of the fermenter and the production rate was reduced.

Therefore, the object of the present invention is to cultivate cells in a very well loosened state and to increase the mass transfer rate of oxygen, substrate, etc., thereby increasing the mass production rate and Another object of the present invention is to provide a method for producing lactic acid by a fermentation method capable of significantly increasing the yield. Another object of the present invention is to provide a method capable of fermentatively producing lactic acid with good workability and productivity without inhibiting growth of cells or increasing viscosity.

[0011]

According to the present invention, a holmite clay mineral is added to a liquid medium inoculated with a filamentous fungus capable of producing lactic acid or its spores, and the mixture is cultivated. There is provided a method for producing lactic acid, which comprises adding a flocculant at a time when the speed becomes slow and performing fermentation while adding an alkaline substance so as not to lower the pH.

In the manufacturing method of the present invention, 1. 1. The holmite clay mineral is sepiolite having a fiber diameter of 70 to 400 angstroms and a fiber length of 0.2 to 400 angstroms. Holmite clay mineral in liquid medium at 1 to 50 g /
2. Add it so as to have a concentration of L, particularly 1 to 10 g / L, 3. The aggregating agent is an anionic, nonionic or amphoteric aggregating agent; 4. Add a flocculant to the liquid medium at a concentration of 1 to 20 mg / L, particularly 1 to 10 mg / L. 5. Fermentation while maintaining the pH of the medium at 4-8, especially 5-7. 6. Performing culture and fermentation while stirring with aeration. It is preferred that the alkaline substance is calcium carbonate.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the method for producing lactic acid according to the present invention, a holmite-based clay mineral represented by sepiolite is selected as a carrier for filamentous fungi capable of producing lactic acid, and this holmite-based chain clay mineral is used. When culturing in the added liquid medium, it is a remarkable feature that the flocculant is added at the time when the growth rate of the bacterial cells becomes slow after the start of culturing.

Chain-like clay minerals such as sepiolite are fibrous, have a three-dimensional chain-like structure, and have pores in the gaps between them, so they are more filamentous than ordinary clay minerals and other inorganic carriers. Improves the entanglement with the fungus to firmly and stably fix the filamentous fungus, and the fixed fungus body is made porous to improve the supply of the culture solution and oxygen to the fungus body and improve the filterability. Can be done.

In the present invention, by adding the aggregating agent to the liquid medium at the final stage of the culturing stage of the microbial cells, the production rate of lactic acid is increased as compared with the case where the aggregating agent is not added, as shown in the examples described later. Can be significantly improved and the final yield can also be increased.

It has already been pointed out that, when only the sepiolite is added to the liquid medium, the cells finally aggregate into a large lump and adhere to the vessel wall of the apparatus and the production rate is reduced. Then, by adding an aggregating agent to this medium, the production of large lumps is suppressed, and the production rate and yield can be improved.

In general, the aggregating agent is for aggregating suspended matter in a liquid and the like. Therefore, it is expected that the aggregating of bacterial cells will be promoted by adding the aggregating agent to the medium. Surprisingly, in the medium used in, the strong aggregation of the bacterial cells is prevented, and the loosely dispersed state is maintained until the final stage of fermentation.

According to the observations of the present inventors, the addition of the flocculant brings about a slight floc-like aggregation of the filamentous fungus cells fixed on the holmite clay mineral, but this floc-like aggregation state is extremely stable and It is also believed that it has excellent fluidity and dispersion stability, and that it maintains a high mass transfer rate and mass production rate of oxygen and substrates.

The chain clay mineral used in the present invention is a fibrous magnesium silicate clay mineral typified by sepiolite, attapulgite, palygorskite and the like. These have a three-dimensional chain structure and have a talc structure. Unlike such a two-dimensional crystal structure, the voids formed in the gaps of the chain structure have a BET specific surface area of 100 to 350 m 2 /
It is a porous clay mineral having a large specific surface area such that it falls within the range of g and having an adsorptive action.

Unlike ordinary layered clay minerals represented by montmorillonite, which is also a porous clay mineral, sepiolite and the like are also characterized in that they do not swell in water.

Due to the characteristic of chain-like clay minerals such as sepiolite, namely that they are fibrous and that they are porous, they are more filamentous than ordinary clay minerals and other inorganic carriers. Improves the entanglement with the fungus to firmly and stably fix the filamentous fungus, and the fixed fungus body is made porous to improve the supply of the culture solution and oxygen to the fungus body and improve the filterability. Can be done.

In the present invention, the chain clay mineral can be used alone or in combination with a double-layered fibrous mineral such as halloysite and asbestos, or a volcanic fibrous mineral including Kanuma soil, Akadama soil and the like. Can be used in. If necessary, zeolite or an adsorbent clay mineral represented by acid clay and rocks such as cristobalite, quartz and feldspar may be used in combination with this fibrous clay mineral.

The sepiolite preferably used in the present invention has a chemical structure represented by the formula (1) (OH ₂ ) ₄ (OH) ₄ Mg ₈ Sil ₂ O ₃ 0.6 to 8H ₂ O (1). And a two-dimensional crystal structure such as talc forms a chain-like crystal structure like bricks stacked alternately. Although it is fibrous due to the pores formed in the chain-shaped gaps, it is also characterized by having a large specific surface area and adsorptivity, unlike other fibrous minerals.

In the present invention, the specific surface area is in the range of 100 to 350 m ² / g and the oil absorption is 100 to 30.
Sepiolite in the range 0 ml / 100 g is preferably used. If the specific surface area is smaller than this range, the cohesive force of the sludge is small, while if it is larger than this range, no further effect can be obtained.

The fibrous sepiolite preferably used in the present invention generally has a fiber diameter of 70 to 400 Å, a fiber length of 0.2 to 400 μm, and an aspect ratio of 5 to 500. preferable.

Table 1 below shows an example of the general chemical composition of sepiolite (dried product at 110 ° C. for 2 hours). Table 1 General chemical composition of sepiolite SiO ₂ 52.50 (wt%) MgO 22.8 Al ₂ O ₃ 1.7 Fe ₂ O ₃ 0.8 CaO 0.8 H ₂ O + 10.5 H ₂ O- 11.0

The filamentous fungus used in the present invention may be any filamentous fungus capable of producing lactic acid, such as Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Bifidobacterium and the like. Examples include lactic acid bacteria, fungi such as Streptococcus, Enterococcus, Bacillus, Scrotridium, and Sporolactobacillus, and fungi such as Rhizopus. These may be used alone or in combination of two or more kinds. it can. A suitable one is Rhizopus oryzae (Rhizo).
pus Oryzae).

The liquid medium used in the present invention can be used without particular limitation as long as it can grow filamentous fungi having a lactic acid-producing ability. This liquid medium contains a nutrient component that is a raw material of lactic acid and an inorganic component (mineral component) necessary for the growth of bacterial cells. As the nutrient component, sugars such as glucose and polysaccharides such as starch are used. To be done. Examples of the inorganic salt include ammonium nitrate, ammonium sulfate, magnesium sulfate, sodium chloride, zinc sulfate, potassium dihydrogen phosphate and the like. The concentration of the nutritional component is not particularly limited, but is 100 to 250 g / L, preferably 100 to 1
About 50 g / L is suitable.

The holmite-based clay mineral is preferably added to the above liquid medium at a concentration of 1 to 50 g / L, particularly 1 to 10 g / L, more preferably 2 to 7 g / L, and more than the above range. If the amount is small, the effect due to the addition is not recognized, and if the amount is larger than the above range, the production rate decreases.

The method of inoculating the cells or spores thereof into the liquid medium is not particularly limited, and the cells or spores thereof described above are suspended in sterile water, physiological saline or the like, and this suspension is used as a liquid medium. Just inoculate.

Since the cells used in the present invention are aerobic, it is preferable to carry out the culture and fermentation while stirring with aeration. That is, it is preferable that an oxygen-containing gas such as air is supplied to the bacterial cells, and at the same time, the medium is stirred by the rise of air bubbles so that substances such as oxygen and a substrate are uniformly supplied to the bacterial cells.

For this purpose, an air-lift type bioreactor can be used, and an antifoaming agent can be added to the system in order to suppress the generation of bubbles accompanying aeration. As the defoaming agent, a silicone-based surfactant or the like is used.

In FIG. 1 showing an example of an air-lift type bioreactor, a draft tube 5 is provided inside a cylindrical reaction container 16 for containing a culture fermentation liquid, and a gap is formed between both ends of the reaction container. Is provided.
At the center of the bottom of the reaction vessel 16, there is provided a sparger 6 made of, for example, sintered glass or the like, for dispersing air in the liquid medium. On the outer periphery of the reaction vessel 16,
A cooling jacket 4 is provided, and by passing cooling water through it, the liquid medium in the container can be maintained at a constant temperature.

The sparger 6 is connected to an air compressor 8 via a flow meter 7, and a constant flow rate of air is supplied as bubbles from the bottom of the container into the liquid medium. A condenser 12 is provided above the reaction container, and excess air in the reaction container is discharged to the outside after the liquid is removed by the condenser 12. Further, a DO electrode 13 for detecting dissolved oxygen in the medium, an antifoaming agent sensor 14 for detecting a bubble level, and a detection sensor 15 for detecting a liquid level of the liquid medium are provided above the reaction container.

The defoaming agent sensor 14 is connected to the defoaming agent controller 2 so that the defoaming agent can be supplied from the defoaming agent storage tank 1 through the pump 3 according to the foam level.

Further, the level sensor 15 is connected to the level controller 10 so that the sterilized water can be supplied from the sterilized water tank 11 by the pump 3 so that the liquid surface becomes constant according to the level of the liquid medium. It has become.

Further, the DO electrode 13 is connected to the DO meter 9 so that the degree of ventilation can be adjusted based on the instruction of the DO meter.

In this air-lift type bioreactor, filamentous fungi or spores thereof are inoculated into a culture fermentation medium of lactic acid, a chain-form clay mineral of formite type is inoculated into this medium, and air is blown into the medium through a sparger 6.

As a result, the oxygen-containing gas such as the blown air becomes bubbles and rises in the draft tube 5,
Due to this upward flow, the draft tube 5 rises,
A circulating flow descending outside the draft tube 5 is formed.
As a result, sufficient mixing and stirring is performed between the liquid medium and the bacterial cells, oxygen and substrates are sufficiently supplied to the bacterial cells, and the bacterial cells are cultured and lactic acid is fermented and produced. Is done.

The production of lactic acid according to the present invention is carried out in two stages, a culturing process of microbial cells in the first stage and a fermentation process of lactic acid in the second stage. During the culture and fermentation process, the temperature of the medium was 20
It is preferable to maintain the temperature in the range of 45 to 45 ° C., particularly 25 to 40 ° C.

The culturing process of the bacterial cells and the fermentation process are not strict, but the end point of the culturing process can be known when the growth of the bacterial cells becomes slow. This time is generally 40 to 80 hours, especially 48 to 72 hours.
It is about time.

In the present invention, the aggregating agent is added to the liquid medium at this point. As the aggregating agent, any aggregating agent known per se is used, but an anionic, nonionic or amphoteric aggregating agent is preferable.

Suitable examples of flocculants include, but are in no way limited to: As an anionic flocculant,
For example, sodium polyacrylate or a copolymer of sodium polyacrylate and polyacrylamide, gelatin, glue, casein, sodium polymethacrylate, sodium alginate, ammonium alginate, carboxymethyl starch, carboxymethyl cellulose, partial hydrolysis of polyacrylamide. , Polyacrylonitrile partial hydrolyzate, acrylamide-acrylic acid copolymer,
Maleic anhydride-vinyl ether copolymer, maleic anhydride-vinyl acetate copolymer, chitosan, sodium styrenesulfonate copolymer and the like are used. Examples of nonionic flocculants include polyacrylamide, polyvinyl alcohol (PVA), starch, cyanated starch, guar gum, tragacanth gum, guaia gum, acacia gum, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, veegum, gelatin, polyethylene glycol, polyoxyethylene, poly Examples thereof include oxypropylene and polyoxyethylene-polyoxypropylene copolymer. Examples of the amphoteric flocculant include a mixture or copolymer of methacrylic acid ester and sodium polyacrylate, a mixture or copolymer of acrylic acid ester and sodium polyacrylate, and the like. Of course, these aggregating agents can be used alone or in combination of two or more kinds.

The amount of the aggregating agent used is such that a slight amount of flocs are formed, so that it may be a very small amount, generally 1 to 20 mg / L, particularly 1 to 10 mg / L in the liquid medium,
More preferably, it is added so as to have a concentration of 2 to 8 mg / L. When the use amount of the aggregating agent is less than the above range, the formation of large lumps of cells cannot be avoided, and when it is more than the above range, dense agglomeration occurs when the aggregating agent is added, in any case. The production rate of lactic acid tends to decrease.

The production of lactic acid is started at the time when the growth of the bacterial cells becomes slow. The temperature of the medium is preferably 20 to 45 ° C., particularly 25 to 40 ° C., as in the culture process. Since the pH of the medium shifts to the acidic side with the production of lactic acid, add an alkaline substance as appropriate to adjust the p
H is in a suitable range for fermentation, generally 4 to 8, especially 5 to 7
To be kept within the range.

As the alkaline substance, calcium carbonate, ammonia, sodium hydroxide, potassium hydroxide and the like can be used, and calcium carbonate is particularly preferable.

The fermentation process is completed when the nutrients in the medium are consumed. This process varies depending on the type of strain, the type or concentration of raw material, but is generally 48 to 80.
About an hour.

The fermented liquor thus obtained is separated from the cells by filtration or the like, calcium lactate is neutralized with sulfuric acid or the like, and gypsum by-product is separated to obtain crude lactic acid, which is concentrated by a means known per se, Purify into product.

[0049]

The embodiments of the present invention will be described below with reference to examples, but these are merely examples and do not limit the present invention in any way. In addition, the quantification of glucose and lactic acid in the examples was performed by the following method.

(1) Glucose quantification method The culture solution was diluted 2000-fold. Take 1 ml and add 0.5 ml of 80% phenol aqueous solution. Add 5 ml concentrated sulfuric acid there. Leave for 30-40 minutes. 500 nm
Colorimetric determination with. (2) Lactic acid quantification method Trichloroacetic acid was added to 1 ml of the sample to obtain 3000 r
After centrifugation at pm for 10 minutes, the supernatant was collected and diluted. 1 ml of the solution was added to a 3% copper sulfate solution, calcium hydroxide was added, and the mixture was allowed to stand at room temperature. Then, it is centrifuged and the supernatant is collected and copper sulfate and sulfuric acid are added. Then, heat in a boiling bath for 5 minutes, then cool, add p-hydroxyphenyl, and add 3 in a 30 ° C water bath.
Leave it for 0 minutes, put it in a boiling bath for 90 seconds, then cool it to 56
Colorimetric determination was performed at a wavelength of 0 nm.

<Fermentation production of lactic acid in a stirred culture tank>
L (+)-lactic acid producing strain Rhizopus oryzae NRRL395
Was suspended in physiological saline so as to have a concentration of 10'spores / ml, and the medium was inoculated in a medium having a medium volume. Glucose 120g /
L, ammonium sulfate 3.02 g / L, potassium dihydrogen phosphate 0.25 g / L, zinc sulfate heptahydrate 0.04 g /
L, magnesium sulfate heptahydrate 0.15 g / L, Aed Plus 3 g / L. Cultivation is at 35 ℃, stirring speed is 300 rp
m. After the start of the culture, polyethylene oxide was added as a flocculant at 5 mg /
L was added so that the concentration would be L, and then calcium carbonate was added so that the pH was not lowered by the lactic acid produced.
The progress of culture is shown in FIG. The production rate of lactic acid was very fast as compared with the case of no addition (Comparative Example 1), and the final production amount was 69.6 g / L in the case of no addition, whereas AdePlus and the coagulant were added. The product weighed 115.1 g / L. Therefore, the lactic acid concentration increased about 1.67 times that of the case of no addition. When sepiolite and a flocculant were added, the cells kept their loose morphology stable until the end of the culture.

(Example 2) The same procedure as in Example 1 was performed except that the ratios of sepiolite and coagulant (polyethylene oxide) were changed. The result is shown in FIG.

(Comparative Example 1) The same procedure as in Example 1 was performed except that the sepiolite and the aggregating agent (polyethylene oxide) were not added. The result is shown in FIG. as a result,
The microbial cells became a large mass, the amount of lactic acid produced was low, and the microbial cells adhered to the baffle plate and the stirring shaft in the reactor, which placed a burden on the device.

(Comparative Example 2) The same procedure as in Example 1 was carried out without adding the aggregating agent (polyethylene oxide). The result is shown in FIG. As a result, the bacterial cells became a large mass, and the production amount of lactic acid was between Example 1 and Comparative Example 1 as is clear from FIG. In addition, the amount of the bacterial cells attached to the baffle plate and the stirring shaft in the reactor is the same as that in Comparative Example 1
It is less than that, but it put a burden on the device.

Example 3 Lactic Acid Fermentation Production in Air Lift Bioreactor> By adding Aed Plus, the viscosity of the culture solution during the culture can be kept almost the same as that of water. Attempts were made to culture in an advantageous airlift bioreactor (Fig. 1). The method was the same as in Example 1. The result is shown in FIG. When sepiolite and polyethylene oxide as a coagulant were added so as to have a concentration of 5 mg / L, 116.6 after 56 hours
The amount of lactic acid produced was g / L.

(Example 4) <Changing coagulant (air lift)> Rhizopus oryzae NRRL395, which is an L (+)-lactic acid producing strain, was added at 10 ⁷ spores / m 2.
The suspension was suspended in physiological saline so that the concentration became l, and 1% of the medium solution volume was inoculated on the medium. The ingredients of the medium were the same as in Example 1, and the amount of sepiolite added was 5 g / L. The soil temperature was 35 ° C., and the ventilation was 0.5 to 1.0 vvm.
After the start of culturing, add a flocculant (sodium polyphosphate) to the soil at a concentration of 7 mg / L around the time when the cell growth rate has become moderate, and make sure that the pH is not lowered by the lactic acid produced thereafter. Calcium carbonate was added. The final amount of lactic acid produced was 98.3 g / L, which was slightly inferior to that when polyethylene oxide was added, but the cells remained loose.

(Comparative Example 3) The same procedure as in Example 3 was carried out without adding sepiolite and a flocculant (polyethylene oxide). FIG. 4 shows the results. In the case of no addition, since it adhered to the vent and proliferated in a lump, the supply of substrates and oxygen to the bacterial cells was hindered, and the production rate also decreased.

[0058]

INDUSTRIAL APPLICABILITY According to the present invention, by adding a holmite clay mineral to a culture solution to grow a filamentous fungus capable of producing lactic acid and adding a flocculant to the filamentous fungus prior to fermentation, the mycelia It became possible to culture and ferment in a well-disassembled state.

Since the amount of holmite clay mineral and flocculant to be added is very small, there is no adverse effect such as growth inhibition or increase in viscosity on bacterial cells. By disintegrating the cells, the mass transfer rate of oxygen, substrate, etc. is increased compared to when the cells on the surface are in a lumpy form, which is involved in production. . When culturing is performed by this method, the microbial cells contain the holmite chain-like clay mineral inside to form loosely aggregated flocs, so that the microbial cells and the culture solution can be easily separated. Therefore, it can be applied to all culture methods such as batch, semi-batch, and continuous. If continuous culture becomes possible, it will be possible to reuse the cells that had been discarded until now. Not only the productivity is improved, but also the microbial cell separation property is improved in the recovery and purification of the fermentation product, so that the cost required for the recovery and purification can be minimized. Since the viscosity of the culture solution itself is suppressed, it is also effective in improving the physical properties of the culture solution in the culture of filamentous fungi in which the hyphae are intertwined intricately and the viscosity of the culture solution is inevitably high. It becomes possible to control the morphology of the microbial cells, and it becomes possible to use an air-lift type bioreactor with less damage due to shearing of the microbial cells, and further improve the productivity.

[Brief description of drawings]

FIG. 1 is a layout view showing an example of an air-lift type bioreactor used in Examples.

FIG. 2 is a diagram showing the time course of lactic acid production by a stirred bioreactor.

FIG. 3 is a diagram showing the relationship between the ratio of sepiolite and a flocculant and the amount of lactic acid produced.

FIG. 4 is a diagram showing a time course of lactic acid production in an airlift bioreactor.

[Explanation of symbols]

 1 Defoaming agent storage tank 2 Defoaming agent controller 3 Pump 4 Cooling jacket 5 Draft tube 6 Sparger 7 Flow meter 8 Air compressor 9 DO meter 10 Level controller 11 Sterilized water tank 12 Condenser 13 DO electrode 14 Defoaming agent sensor 15 Level Sensor 16 reaction vessel

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[Procedure amendment]

[Submission date] May 10, 1996

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Name of invention

[Correction method] Change

[Correction contents]

Title: Method for producing organic acid

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0012

[Correction method] Change

[Correction contents]

In the manufacturing method of the present invention, 1. 1. The holmite clay mineral is sepiolite having a fiber diameter of 70 to 400 angstrom and a fiber length of 0.2 to 400 μm . Holmite clay mineral in liquid medium at 1 to 50 g /
2. Add it so as to have a concentration of L, particularly 1 to 10 g / L, 3. The aggregating agent is an anionic, nonionic or amphoteric aggregating agent; 4. Add a flocculant to the liquid medium at a concentration of 1 to 20 mg / L, particularly 1 to 10 mg / L. 5. Fermentation while maintaining the pH of the medium at 4-8, especially 5-7. 6. Performing culture and fermentation while stirring with aeration. It is preferred that the alkaline substance is calcium carbonate.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0013

[Correction method] Change

[Correction contents]

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION In the method for producing lactic acid according to the present invention, a holmite-based clay mineral represented by sepiolite is selected as a carrier for filamentous fungi having a lactic acid-producing ability, and the holmite-based clay mineral is added. When culturing in a liquid medium, a remarkable feature is to add an aggregating agent at the time when the growth rate of the bacterial cells becomes slow after the start of culturing.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0019

[Correction method] Change

[Correction contents]

The chain clay mineral used in the present invention is a fibrous magnesium silicate clay mineral typified by sepiolite, attapulgite, palygorskite and the like. These have a three-dimensional chain structure and have a talc structure. Unlike such a two-dimensional crystal structure, the voids formed in the chain structure have a BET specific surface area of 100 to 350 m ² /
It is a porous clay mineral having a large specific surface area such that it falls within the range of g and having an adsorptive action.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0023

[Correction method] Change

[Correction contents]

The sepiolite preferably used in the present invention has a chemical structure represented by the formula (1) (OH ₂ ) ₄ (OH) ₄ Mg ₈ Si ₁₂ O ₃₀ · 6-8H ₂ O (1). It has a two-dimensional crystal structure such as talc, which forms a chain crystal structure in which bricks are alternately stacked. Although it is fibrous due to the pores formed in the chain-shaped gaps, it is also characterized by having a large specific surface area and adsorptivity, unlike other fibrous minerals.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0027

[Correction method] Change

[Correction contents]

The filamentous fungus used in the present invention may be any filamentous fungus capable of producing lactic acid, such as Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Bifidobacterium and the like. Examples include lactic acid bacteria, fungi such as Streptococcus, Enterococcus, Bacillus, Scrotridium, and Sporolactobacillus, and fungi such as Rhizopus. These may be used alone or in combination of two or more kinds. it can. A suitable one is Rhizopus oryzae ( Rhizo
pus Oryzae ).

[Procedure amendment 7]

[Document name to be amended] Statement

[Correction target item name] 0028

[Correction method] Change

[Correction contents]

The liquid medium used in the present invention can be used without particular limitation as long as it can grow filamentous fungi having a lactic acid-producing ability. This liquid medium contains a nutrient component that is a raw material of lactic acid and an inorganic component (mineral component) necessary for the growth of bacterial cells. As the nutrient component, sugars such as glucose and polysaccharides such as starch are used. To be done. Examples of the inorganic salt include ammonium nitrate, ammonium sulfate, magnesium sulfate, sodium chloride, zinc sulfate, potassium dihydrogen phosphate and the like. The carbon source concentration is not particularly limited,
100 to 250 g / L, preferably 100 to 150
About g / L is suitable.

[Procedure amendment 8]

[Document name to be amended] Statement

[Correction target item name] 0038

[Correction method] Change

[Correction contents]

In this air-lift type bioreactor, filamentous fungi or spores thereof are inoculated into a culture fermentation medium of lactic acid, formite clay mineral is inoculated into this medium, and air is blown through the sparger 6 into this medium.

[Procedure amendment 9]

[Document name to be amended] Statement

[Correction target item name] 0042

[Correction method] Change

[Correction contents]

In the present invention, at the time when the bacterial growth becomes slow
Add the flocculant to the liquid medium with. As the aggregating agent, any aggregating agent known per se may be used.
It is preferably a nonionic or amphoteric aggregating agent.

[Procedure amendment 10]

[Document name to be amended] Statement

[Correction target item name] 0051

[Correction method] Change

[Correction contents]

(Example 1) <Fermentation production of lactic acid in a stirring culture tank> Rhizopus oryzae NRRL 395 , which is an L (+)-lactic acid producing strain, was added to 10 ⁷ spores.
/ ml of were suspended in physiological saline to a concentration were inoculated with 1% of the culture liquid amount in the following culture land. Glucose 120g /
L, ammonium sulfate 3.02 g / L, potassium dihydrogen phosphate 0.25 g / L, zinc sulfate heptahydrate 0.04 g /
L, magnesium sulfate heptahydrate 0.15 g / L, sepio
Light 3g / L. Cultivation is at 35 ℃, stirring speed is 300 rp
m. After the start of the culture, polyethylene oxide was added as a flocculant at 5 mg /
L was added so that the concentration would be L, and then calcium carbonate was added so that the pH was not lowered by the lactic acid produced.
The progress of culture is shown in FIG. Compared to the case of no addition (Comparative Example 1), the production rate of lactic acid was very fast, and the final production amount was 69.6 g / L in the case of no addition, whereas Sepio
It was 115.1 g / L when the light and the aggregating agent were added. Therefore, the lactic acid concentration increased about 1.67 times that of the case of no addition. When sepiolite and a flocculant were added, the cells kept their loose morphology stable until the end of the culture.

[Procedure amendment 11]

[Document name to be amended] Statement

[Correction target item name] 0055

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Example 3 Lactic Acid Fermentation Production in Air Lift Bioreactor> By adding Aed Plus, the viscosity of the culture solution during the culture can be kept almost the same as that of water. the culture in chromatic advantage is the airlift bioreactor (Fig. 1) was attempted. The method was the same as in Example 1. The result is shown in FIG. When sepiolite and polyethylene oxide as a coagulant were added to a concentration of 5 mg / L, 116.6 g after 56 hours
/ L of lactic acid production was shown.

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(Example 4) <Changing coagulant (air lift)> L (+)-lactic acid producing strain Rhizopus oryzae NRRL 395 was added at 10 ⁷ spores / m 2.
were suspended in physiological saline to a concentration of l, it was inoculated with 1% of the culture liquid amount in the culture ground. Component of the culture ground is the same as in Example 1, the amount of sepiolite was 5 g / L. Culture ground temperature, 35 ℃, ventilation was carried out at 0.5~1.0vvm.
To culture locations flocculant (sodium polyphosphate) in the vicinity of the start of culture after the cell growth rate became gentle, it was added to a concentration of 7 mg / L, so that pH does not drop by then produced lactic acid Calcium carbonate was added. The final amount of lactic acid produced was 98.3 g / L, which was slightly inferior to that when polyethylene oxide was added, but the cells remained loose.

[Procedure amendment 13]

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Since the amount of holmite clay mineral and flocculant to be added is very small, there is no adverse effect such as growth inhibition or increase in viscosity on bacterial cells. By disintegrating the cells, the mass transfer rate of oxygen, substrate, etc. is increased compared to when the cells on the surface are in a lumpy form, which is involved in production. . If the culture was performed in this way, since the bacterial cells form a loosely aggregated floc contained therein the hormite type clay mineral,
It is easy to separate the cells from the culture solution. Therefore, it can be applied to all culture methods such as batch, semi-batch, and continuous.
If continuous culture becomes possible, it will be possible to reuse the cells that had been discarded until now. Not only the productivity is improved, but also the microbial cell separation property is improved in the recovery and purification of the fermentation product, so that the cost required for the recovery and purification can be minimized.
Since the viscosity of the culture solution itself is suppressed, it is also effective in improving the physical properties of the culture solution in the culture of filamentous fungi in which the hyphae are intertwined intricately and the viscosity of the culture solution is inevitably high. It becomes possible to control the morphology of the microbial cells, and it becomes possible to use an air-lift type bioreactor with less damage due to shearing of the microbial cells, and further improve the productivity.

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[Fig. 2]

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FIG. 4

Claims (8)

[Claims] 1. A flocculant is added at the time when the growth rate of microbial cells becomes slow after the start of culturing, by adding holmite clay mineral to a liquid medium inoculated with a filamentous fungus having lactic acid-producing ability or its spores and culturing. And a method for producing lactic acid, wherein the fermentation is carried out while adding an alkaline substance so that the pH does not decrease. 2. A holmite clay mineral having a fiber diameter of 70 to 400 angstrom and a fiber length of 0.2 to 400.
The production method according to claim 1, which is sepiolite having a size of μm. 3. The method according to claim 1, wherein the holmite-based clay mineral is added to the liquid medium at a concentration of 1 to 50 g / L. 4. The production method according to claim 1, wherein the aggregating agent is an anionic or nonionic aggregating agent. 5. A liquid medium containing a flocculant in an amount of 1 to 20 mg / L.
5. The manufacturing method according to claim 1, wherein the addition is performed so as to have a concentration of. 6. The production method according to claim 1, wherein the fermentation is performed while maintaining the pH of the medium at 4 to 8. 7. The production method according to claim 1, wherein the culture and the fermentation are performed while stirring with aeration. 8. The production method according to claim 1, wherein the alkaline substance is calcium carbonate.