JPH09121877A - Production of highly pure l-lactic acid with bacillus group microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To profitably obtain the subject compound useful for producing biodegradable resins, foods, medicines, optical materials, etc., by culturing a Bacillus group microorganism having an ability to produce the highly optically pure L-lactic acid from a carbon source capable of being assimilated and subsequently collecting the product from the culture product. SOLUTION: A microorganism, such as Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus larvae, Bacillus lentimorbus, Bacillus popilliae or Bacillus sphaericus, having an ability to produce L-lactic acid having an optical purity of >=70% from a carbon source capable of being assimilated is cultured, and the product is collected from the culture products to obtain the objective highly pure L-lactic acid at a low cost.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing high-purity L-lactic acid using a microorganism of the genus Bacillus, and more particularly to a method for producing high-purity L-lactic acid at low cost. The present invention also relates to a method for simultaneously producing high-purity L-lactic acid and an insecticidal toxin using a microorganism of the genus Bacillus.

[0002]

L-lactic acid is used as a raw material for polylactic acid which is a biodegradable plastic, food, medicine, brewing, tanning and optical materials. Further, unlike conventional pesticides, insecticidal toxins are attracting attention as pesticides that are harmless to humans and animals.

When lactic acid is used as a raw material for polylactic acid,
A polymer having higher crystallinity can be obtained by using lactic acid having a high optical purity as a raw material. This means, for example, Kulkarni, RK,
Moore, EG, Hegyeli, AF, and Leonard, F. (1971) B
iodegradable poly (lactic acid) polymers. J. Biomed.
Mater. Res. (Journal of Biomedical Materials Research), 5: 169-181. And Ohara, H. (199
4) Poly-L-Lactic acid as biodegradable plastic. Bi
osci. Indust. (Bioscience and Industry), 52: 642-644. Polylactic acid having high crystallinity is suitable for stretched films and spinning.

Further, high-purity L-lactic acid can be used in liquid crystals, for example, Sato, k., Eguchi, T., Toshida, y., Yosh.
inaga, K., and Takasu, Y. (1990) Properties of the f
erroelectric polymer liquid crystals containing a
chiral lactic acid derivative group. Polymer prepr
ints, Japan, (Polymer Chemistry Conference Proceedings) 39: 1962-1964.
And Yoshinaga, K., Eguchi, T., Sato, K., Toshida, Y.,
and Takasu, Y. (1990) Properties of the ferroelectri
c polymer liquid crystals containing a chiral lact
ic acid derivative group (II). Polymer preprings, J
apan, (Polymer Chemistry Conference Proceedings) 39: 1962-1964.

Furthermore, FAO (United Nations Food and Agriculture Organization) and WHO
The (World Health Organization) prefers that lactic acid given to infants is L-lactic acid. This is FAO and
WHO (1974) Toxicological evaluation of certain food
additives with a review of general principles and
of specifications. World Health Organization, Genev
a, p23.

Thus, L-lactic acid is required to be useful and highly pure.

Conventionally, a method of producing L-lactic acid by fermentation has been known. For example, (1) Production of L-lactic acid using Streptococcus faecalis is described in Ohara, H., Hiyama, K., and Yoshida, T. (1).
993) Lactic acid production by a filter-bed-type r
eactor. J. Ferment. Bioeng. (Journal of Fermentation and Bioengineering)
76: 73-75., (2) Production of L-lactic acid using Lactobacillus helvetics, Aeschlimann, A., Di Stasei, L., and von Stoc.
kar, U. Continuous production of lactic acid from w
hey permeate by Lactobacillus helvetics in two che
Mostats in series. Enzyme Microbiol. Technol. (Enzyme Microbiology and Technology) 12: 926-932., (3) L-lactic acid production using Lactobacillus amylovorus is described in Nakamura, LK. and Crowell CD (1979)
Lactobacillus amylovorus, a new starch-hydrolyzing
species from swine waste-com fermentation. Div. I
nd. Microbiol. 20: 531-540., (4) Production of L-lactic acid using Lactobacillus delbruekii is described in Stenroos, SL, Linko, YY,
and Linko, P. (1982) Production of L-lactic acid w
ith immobilized Lactobacillus delbruekii. Bacterio
L. Lett. (Biotechnology Letter) 4: 159-164. (5) Lactococcus lactis.
L-lactic acid production using lactis) is described in Ishizaki, A. and
Kobayashi, G. (1990) Computer simulation of L-lacta
te batch fermentation applying the enzyme inactiva
tion scheme. J. Ferment. Bioeng. 70: 139-140.

The above (1) to (5) are the production of L-lactic acid using lactic acid bacteria. However, these lactic acid bacteria are highly auxotrophic and the cost of the medium is high. High cost of lactic acid production medium using lactic acid bacteria is due to Boer, JPde, Mattos,
MJTde, and Neijssel OM (1990) D (-) Lactic acid
production by suspended and aggregated continuous
cultures of Bacillus laevolacticus. Appl. Microbi
ol. Biotechnol. 34: 149-153. If the cost of the culture medium becomes high, naturally, L-lactic acid as a product becomes expensive.

Therefore, a method for producing L-lactic acid using bacteria other than lactic acid bacteria has also been reported. For example, the production of L-lactic acid by Rhizopus oryzae has
a, M., Bagum, AA, and Sadai, S. (1992) Production of
L (+)-lactic acid by immobilized cells of Rhizopus
oryzae with polymer supports prepared by γ rayin
duced polymerization. J. Ferment. Bioeng. 74: 379-3
83. However, in this method, the fermentation time is as long as 40 to 50 hours, and the production efficiency is not good.

In addition, Bacillus Laevolactis
The production of D-lactic acid by lus laevolactis) is described in Appl.
Microbiol. Biotechnol. 34: 149-153. However, it can only be used with D-lactic acid.

In addition, Bacillus coagulans (Bacillus
Production of L-lactic acid by coagulans) is disclosed in JP-A-58-40.
093, Japanese Patent Publication No. 60-6200, US Patent
It is described in US Pat. No. 5,079,164. However,
Bacillus coagulans has higher auxotrophy than the strains used in the present invention as shown in the examples of the present specification, and the optical purity of L-lactic acid produced is less than 70%. Also, no Bacillus coagulans strain that produces an insecticidal toxin is known.

Further, Japanese Patent Laid-Open No. 3-27291 discloses that
A method for producing lactic acid using Bacillus coagulans is described. However, the publication does not mention that the lactic acid produced is L-lactic acid or its optical purity. Further, Japanese Patent Application Laid-Open No. 2-76592 describes a method for producing lactic acid using Bacillus genus in claim 9 thereof.
-No production of lactic acid is disclosed.

[0013]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing L-lactic acid with high purity and at low cost using a microorganism of a specific species of the genus Bacillus in order to solve the above problems of the prior art. To provide. Further, the object of the present invention is
A method for producing high-purity L-lactic acid in which high-purity L-lactic acid and an insecticidal toxin are simultaneously produced by using a microorganism of a specific species of the genus Bacillus having an ability to produce an insecticidal toxin, and the total cost is further reduced To provide.

[0014]

The method for producing high-purity L-lactic acid according to the present invention has the ability to produce L-lactic acid having an optical purity of 70% or more from an assimilable carbon source, and Bacillus anthracis (Bacillus). anthracis), Bacillus cereus (B
acillus cereus), Bacillus thuringiensis (Bac
illus thuringiensis), Bacillus larvae (Bacillus
larvae), Bacillus lentmorbus (Bacillus lentim)
orbus), Bacillus popilliae and Bacillus sphaericus, and at least one Bacillus genus (Bacillus sphaericus)
illus) microorganism was cultured, and the optical purity of 70% was obtained from this culture.
% Or more of L-lactic acid is collected.

The method for simultaneously producing L-lactic acid and insecticidal toxin of the present invention has the ability to produce L-lactic acid and insecticidal toxin having an optical purity of 70% or more from an assimilable carbon source. Bacillus thuringiensis
uringiensis), Bacillus larvae
, Bacillus lentimorbu
s), at least one Bacillus genus (Bacillus popilliae), and Bacillus sphaericus.
The method is characterized in that the microorganism of (s) is cultured and L-lactic acid and an insecticidal toxin having an optical purity of 70% or more are collected from the culture.

The present invention will be described in detail below.

In the present invention, as a microorganism of the genus Bacillus, Bacillus anthasis is used.
racis), Bacillus cereus (Bacillus cereus), Bacillus thuringiensis (Bacillus thuringiensis),
Bacillus larvae, Bacillus lentimorbus, Bacillus popilliae or Bacillus sphaericus are used. By using these microorganisms belonging to the genus Bacillus, L-lactic acid has an optical purity of 70.
It can be obtained with a high purity of at least%. Among these microorganisms, Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are very closely related and their identification is difficult to distinguish. This thing is the Burgies
It is also described on page 1113 of the Manual of Systematic Bacteriology. In addition, these three strains are Egg-yolk lecithinas (Egg-yolk lecithinas).
e) The reaction is positive.

In the present invention, the Bacillus thuringiensis (Bacillus thuringiensis) is used as the microorganism of the genus Bacillus.
uringiensis), Bacillus larvae
, Bacillus lentimorbu
s), Bacillus popilliae or Bacillus sphaericus, it is possible to obtain an insecticidal toxin together with high-purity L-lactic acid.

In addition to glucose, the carbon source used in the present invention is not limited as long as it is assimilable sugar such as sucrose, maltose, fructose, lactose, mannitol and starch. The concentration of these sugars in the medium is usually about 2 to 15% by weight.

As the auxiliary material, inexpensive materials such as polypeptone, cheese whey, corn steep liquor and yeast extract can be used. The concentration of these auxiliary raw materials in the medium is usually about 0.1 to 2% by weight.

The medium may contain inorganic salts such as potassium phosphate and ammonium phosphate, pH adjusting agents such as caustic soda, hydrochloric acid and various buffers, magnesium compounds, manganese compounds and the like.

The culture of the bacterial cells is usually performed by STR (Stirred Tank Re
actor) in a batch manner, but not limited to this, CSTR (Conti
nuous Tank Reactor) can also be performed continuously.
Alternatively, it may be produced by immobilization on calcium alginate, carrageenan, a photocurable resin or the like, or by a membrane reactor or an electrolytic dialysis reactor. Membrane reactors are, for example, those of Dialysis (dialysis type) by Coulman et al.
Applied Environmental Microbiology, 1977, 34, 725
-732 and Biotechnolo by Stieber and Gerhardt
gy and Bioengineering, 1981, 23, 523-534. In addition, the Cross-Flow type membrane reactor was developed by Major and Bull in Biotechnology and Bi
oengineering, Vol. 34, 1989, pages 592-599.

The pH and temperature of the culture depend on the cells used, but are usually pH 6.0-8.0 and temperature 25-40 ° C. Optimal conditions are determined by the bacterial cells used.

In the present invention, the culture can be carried out under aerobic conditions, but it is preferably carried out under anaerobic conditions. The genus Bacillus is an aerobic or facultative aerobic microorganism and is usually aerobically cultured by aeration or the like. Under such aerobic conditions, sugars such as glucose are metabolized from pyruvate through the Krebs cycle. In the present invention, by culturing a bacterium of the genus Bacillus under anaerobic conditions, higher purity L-lactic acid can be obtained from pyruvic acid at a higher conversion rate.

The introduction of the bacterial cells into the medium may be carried out by any conventionally known method, and the produced L-
Separation and purification of lactic acid and the insecticidal toxin obtained in some cases may be performed by any conventionally known method.

According to the present invention, a microorganism of the genus Bacillus having the ability to produce L-lactic acid having an optical purity of 70% or more from an assimilable carbon source, that is, Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis. , Bacillus larvae, Bacillus lenthimbus,
Since Bacillus popirae or Bacillus sphaericus is cultured, L-lactic acid can be produced with a high purity of 70% or more in optical purity. In addition, since these Bacillus microorganisms can be cultured in an inexpensive medium that has lower auxotrophy than lactic acid bacteria, L-lactic acid can be produced at a lower cost.

Further, in the present invention, a microorganism of the genus Bacillus having the ability to produce insecticidal toxins, that is,
Bacillus thuringiensis, Bacillus larvae,
When Bacillus lentilbus, Bacillus popirae or Bacillus sphaericus is cultured, L-lactic acid having an optical purity of 70% or more and an insecticidal toxin can be produced at the same time, and L-lactic acid can be produced at a lower cost as a total cost. It can be manufactured.

[0028]

Next, the present invention will be described more specifically with reference to examples.

[Example 1] Bacillus cereus
S. cereus) JCM 2152 was cultured in Brain Heart Infusion Medium (Becton Dickinson) at 34 ° C. for 10 hours, and this was used as an inoculum. Each 0.1 ml was inoculated into 10 ml of a liquid medium having the following composition in two test tubes. Polypeptone S (Nippon Pharmaceutical Co., Ltd.) 10 g / l, glucose 20 g / l, and dipotassium phosphate 35 g / l
1: The medium had a pH of 7.
Adjusted to zero. The mouth of each test tube has a porous silicon stopper that can be vented, and one test tube is used for anaerobic culture.
Aerobic culture was performed in the other test tube as follows. <Anaerobic culture> Use test tubes with BBK GasPak (Becton
Dickinson) and statically cultured at 34 ° C. for 10 hours. Since it was performed in a gas pack, it is more anaerobic than the next shake culture. <Aerobic culture> Test tubes with BBK GasPak (Becton
The culture was carried out at 34 ° C. for 10 hours with shaking at 120 rpm without being placed in Dickinson.

After culturing, the amount of lactic acid produced (g / l), the amount of glucose consumed (g / l), the conversion rate (%) and the optical purity (%) were determined as follows.

<Amount of lactic acid produced and amount of glucose consumed> The amount of lactic acid produced and the amount of glucose consumed are the lactic acid concentration (g / l) and the amount of glucose consumed (g / l) in the culture solution, respectively.
l) as high performance liquid chromatography (HPLC)
Was quantified under the following conditions. The amount of lactic acid produced was L-
The total amount of body and D-body. HPLC; (manufactured by Shimadzu Corporation, LC-6A), detector; differential refractometer (RID-6)
A, Shimadzu), column: Shim-pack SCR-101H (Shimadzu), column temperature: 60 ° C, eluent: 2.5 m
Molar perchloric acid aqueous solution, flow rate; 0.9 ml / min.

<Conversion rate> The conversion rate is the conversion rate (%) = (lactic acid production amount (g / l) / glucose consumption amount)
(g / l)) × 100. Here, the production amount of lactic acid is L-form and D-form.
The total amount of body.

<Optical Purity> The optical purity of L-lactic acid is calculated by the following formula: Optical purity (%) = 100 × (LD) / (L + D) where L is the concentration of L-lactic acid and D Represents the concentration of D-lactic acid. The culture solution sample was filtered through a UF membrane (UFPI, Millipore) to cut off molecules having a molecular weight of 5000 or more. This was measured by high performance liquid chromatography to quantify the concentrations of L-lactic acid and D-lactic acid in the culture solution. HPLC;
(Shimadzu Corporation, LC-6A), Detector; Spectrometer (Shimadzu Corporation, SPD-6AV), Column; CRS10W
(Manufactured by Mitsubishi Kasei), column temperature; 30 ° C, detection wavelength; 25
4 nm, eluent; 2 mM CuSO ₄ , flow rate; 0.5 m
l / min.

Example 2 In place of Bacillus cereus JCM 2152 of Example 1, Bacillus thuringiensis was used as a strain.
Culture was performed in the same manner as in Example 1 except that subsp. kurustaki ATCC 33679 was used to produce lactic acid (g / g).
1), glucose consumption (g / l), conversion rate (%) and optical purity (%) were determined.

[Comparative Example 1] In place of Bacillus cereus JCM 2152 of Example 1, Bacillus coagulans JCM 2257 was used as a strain.
Culturing was performed in the same manner as in Example 1 except that the amount of lactic acid produced (g / l) and the amount of glucose consumed (g / g) were used.
l), conversion rate (%) and optical purity (%) were determined.

[Comparative Example 2] The same procedure as in Example 1 was repeated except that Bacillus subtilis JCM 1465 was used as the strain instead of Bacillus cereus JCM 2152 of Example 1. After culturing, the amount of lactic acid produced (g / l), the amount of glucose consumed (g / l), the conversion rate (%) and the optical purity (%) were determined.

The results of Examples 1 and 2 and Comparative Examples 1 and 2 are shown in Table 1.
Shown in

[Table 1]

From Table 1, when Bacillus cereus and Bacillus thuringiensis were used as the strains, L-lactic acid was produced with a high conversion rate and a high optical purity.
In addition, the conversion rate and the optical purity were higher in the anaerobic culture than in the aerobic culture. On the other hand, when Bacillus coagulans was used as the strain, the amount of lactic acid produced was small even under aerobic culture, and the optical purity was less than 70%. No lactic acid production was observed in the case of anaerobic culture. When Bacillus subtilis was used as the strain, lactic acid was produced under both anaerobic and aerobic conditions, but the concentration and conversion rate were 0.1 g / l and 2%, respectively.
The results were 5.0%, 3.7 g / l, and 49.3%, which were not practical.

Also noteworthy in these results is that polypeptone S is good as an auxiliary material other than glucose, which is unlikely in lactic acid bacteria.
That is, these Bacillus cereus and Bacillus thuringiensis strains have lower auxotrophy than lactic acid bacteria and Bacillus coagulans and do not select a medium, and can produce high-purity L-lactic acid in a cheaper medium. Became clear.

[Example 3] Bacillus thuringiensis subsp. Kurustaki ATCC 33
679, the medium is polypeptone S; 10 g / l, ammonium phosphate; 5 g / l, and glucose 100 g / l
It was cultured in a medium consisting of The culture was performed using a 500 ml incubator (500 ml of culture solution), stirring at 30 ° C. and 60 rpm, and the pH was maintained at 7.0 with 6M caustic soda. Further, in order to maintain the anaerobic condition, nitrogen gas was aerated at 30 ml / min, and the cells were cultured at 30 ° C. for 15 hours. The lactic acid concentration of the culture broth was 98 g / l, and the optical purity was 99.5%. Centrifugation (20,000 G, 1
After collecting the bacterial cells for 5 minutes, the wet weight was 30
g of cells were obtained and observed with a phase contrast microscope,
0.6 × 2μ spindle-shaped crystals were observed. 150 cells
It was crushed for 10 minutes using an ultrasonic crusher of w, and this was fed to 20 larvae of the American white-hit, as a feed. 7 animals within 1 hour, 10 animals within 1 to 2 hours The animal died within 2 to 5 hours. Thus, when Bacillus thuringiensis was used as the strain, L-lactic acid was obtained with a high conversion rate and a high optical purity, and an insecticidal toxin was obtained at the same time. Therefore, it became clear that L-lactic acid can be produced at a lower cost as a total cost.

[0041]

According to the method of the present invention, as described above,
L-lactic acid with very high optical purity can be produced at low cost. According to the more preferred method of the present invention, an insecticidal toxin can be simultaneously produced together with L-lactic acid, and L-lactic acid with high optical purity as a total cost can be produced at a lower cost. Thus, the method of the present invention is
It is very useful as compared with conventional L-lactic acid production methods using microorganisms.

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Claims (3)

[Claims] 1. A Bacillus anthracis or Bacillus cereus (B) having an ability to produce L-lactic acid having an optical purity of 70% or more from an assimilable carbon source.
acillus cereus), Bacillus thuringiensis (Bac
illus thuringiensis), Bacillus larvae (Bacillus
larvae), Bacillus lentmorbus (Bacillus lentim)
orbus), Bacillus popilliae and Bacillus sphaericus, and at least one Bacillus genus (Bacillus sphaericus)
illus) microorganism was cultured, and the optical purity of 70% was obtained from this culture.
% Or more L-lactic acid is collected, The manufacturing method of L-lactic acid characterized by the above-mentioned. 2. Bacillus thuringiensis having the ability to produce L-lactic acid and an insecticidal toxin with an optical purity of 70% or more from an assimilable carbon source.
nsis), Bacillus larvae, Bacillus lentimorbus, Bacillus popilliae, and Bacillus sphaericus, at least one microorganism belonging to the genus Bacillus. A method for producing L-lactic acid and an insecticidal toxin at the same time, which comprises culturing L. lactic acid and an insecticidal toxin having an optical purity of 70% or more from the culture. 3. The method according to claim 1, wherein the microorganism of the genus Bacillus is cultured under anaerobic conditions.
The method described in the section.