JP2003159091A - Method for producing lactic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a lactic acid production technique by fermentation method so as to effect high production efficiency. <P>SOLUTION: This method for producing lactic acid comprises the steps of culturing lactic acid-producing bacteria in the presence of an externally added pyruvic acid to produce the lactic acid in the medium and then collecting the thus produced lactic acid. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing lactic acid with a high production efficiency, which uses a specific fermentation (lactic acid production reaction) accelerator.

[0002]

2. Description of the Related Art Lactic acid is used as a food additive in sake, beer,
It is used for the production of soft drinks, pickles, soy sauce, bread and the like, and is also used as a chemical raw material for pharmaceuticals, agricultural chemicals, cosmetics and the like. Esters such as ethyl lactate and butyl lactate, which are derivatives of lactic acid, are also used as highly safe solvents and cleaning agents. Polylactic acid, which is a polymer of lactic acid, is expected to grow in the future for biodegradable polymer applications and biocompatible medical polymer materials. Lactic acid is useful as a raw material for these chemicals.

Conventionally, lactic acid production by a fermentation method has been carried out by utilizing fermentation of various microorganisms capable of producing lactic acid, using saccharides from sucrose, glucose, starch and cellulosic materials as raw materials. However, fermentation production efficiency such as production rate is still insufficient, and improvement in this respect is desired.

Therefore, for the purpose of improving the production efficiency of lactic acid, a method of adding yeast extract, peptone, meat extract, malt extract or the like to the fermentation medium during lactic acid fermentation is well known. The addition of these substances is considered to provide essential nutrients for the growth of microorganisms, which mainly include vitamins, amino acids, nucleic acids and minerals. However, these compounds enable the efficiency of lactic acid fermentation production in microorganisms to be increased. The reason for the improvement is not clear.

[0005] In addition, soybean protein hydrolyzate (soy bean) containing mevalonic acid and various amino acids in the fermentation medium for the purpose of increasing the production rate of lactic acid.
It is also known to add hydrolyzate (Biosci
ence Biotechnology & Biochemistry, 61 (4): 604-608,1
997). However, again it is not clear why these additives also increase the rate of lactic acid fermentation.

[0006]

DISCLOSURE OF THE INVENTION The object of the present invention is to produce lactic acid by a fermentation method capable of achieving high production efficiency with a compound which has not been known to have an effect of improving the production efficiency of lactic acid fermentation. To provide the technology.

[0007]

Means for Solving the Problems The present inventor is keenly aware of a substance that promotes lactic acid fermentation (the term "fermentation" used in the present invention also means a lactic acid production reaction without the growth of microorganisms used). As a result of research, it was found that the efficiency of lactic acid production was remarkably improved by adding a specific compound whose lactic acid production promoting action was not known to the fermentation medium. A particular compound of the present invention is pyruvic acid. It has been clarified that lactic acid fermentation from saccharides by lactic acid-producing bacteria produces lactic acid from pyruvic acid produced in the glycolytic pathway in the microorganism. Although pyruvic acid added to the fermentation medium of the present invention is the same compound as pyruvic acid produced in the glycolytic pathway, it was found that the added pyruvic acid had an unexpected effect.
Usually, in a general chemical reaction system, the increased amount of the reactant in the raw material system results in the increase in the substance in the production system. However, the increase in the production system does not exceed the increase in the raw material system.

[0008] However, in the present invention, by adding pyruvic acid to the fermentation medium, the increase in the amount of lactic acid produced is significantly higher than the amount of lactic acid derived from pyruvic acid added to the fermentation medium, and the effect is increased. It continues continuously, and the rate of lactic acid production is significantly increased as compared with the case of no addition. And the glucose consumption rate also increases. In other words, the pyruvic acid added to the fermentation medium has the effect of priming water when pumping well water. It is not clear why pyruvic acid added to the fermentation medium has such an effect. However, the following reasons can be considered as the reason for the estimated effect.

When lactic acid is produced by fermentation from pyruvic acid, consumption of NADH (nicotinamide adenine dinucleotide reduced form) is involved in this reaction. on the other hand,
NADH is considered to be an inhibitor of an enzyme that catalyzes the reaction of pyruvic acid, which is a precursor of pyruvic acid in the glycolytic system, to pyruvic acid. Therefore, by adding pyruvic acid to the lactic acid fermentation medium from outside the glycolysis system, NADH is consumed and the catalytic inhibition is eliminated. As a result, the reaction pathway from glyceraldehyde 3-phosphate to pyruvic acid and lactic acid is increased. It is presumed that lactic acid production is promoted by progressing particularly smoothly as compared with before the addition of pyruvic acid. Of course, the present invention is not limited to such presumed reasons.

That is, the present invention provides (1) culturing a lactic acid-producing bacterium in the presence of pyruvic acid externally added,
A method for producing lactic acid, which comprises producing lactic acid in a medium and collecting the produced lactic acid, (2) The lactic acid-producing bacterium according to (1) above, wherein the lactic acid-producing bacterium belongs to the genus Corynebacterium. Production method, (3) The method for producing lactic acid according to (2) above, wherein the lactic acid-producing bacterium is Corynebacterium glutamicum, and (4) the lactic acid-producing bacterium belongs to the genus Brevibacterium. (1) The method for producing lactic acid according to (1) above, (5) The method for producing lactic acid according to (4) above, wherein the lactic acid-producing bacterium is Brevibacterium lactofermentum, (6)
Lactic acid-producing bacteria are cultivated in the presence of pyruvic acid that is added from the outside (the term "culturing" in the present invention includes the meaning of performing substance production without the growth of microorganisms), and lactic acid is added to the medium. The present invention relates to lactic acid obtained by a method of producing and collecting from a medium, and (7) a lactic acid production promoter characterized by containing pyruvic acid.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a known lactic acid fermentation technique can be used except that a specific compound is added to a fermentation medium, and there is no particular limitation. The present invention also provides a method for producing lactic acid with high production efficiency by adding a predetermined amount of pyruvic acid to a fermentation medium.

The fermentation of the present invention may be carried out under anaerobic or aerobic conditions as long as production of lactic acid is recognized. Whether fermentation is performed under anaerobic conditions or under aerobic conditions is appropriately selected depending on the microorganism species used. The fermentation medium of the present invention means a "field" in which lactic acid is produced under anaerobic or aerobic conditions.
In the above, depending on the microorganism species used, lactic acid is usually produced with the growth of microorganisms, or lactic acid is produced without the growth of microorganisms, as shown in Examples and the like described later.

The anaerobic conditions can be carried out, for example, by aeration of carbon dioxide gas, inert gas (nitrogen gas, argon gas, etc.), or by a non-aeration method. The aerobic condition can be achieved by aeration of a gas containing oxygen gas.

The fermenting microorganism used in the present invention is one obtained from nature capable of producing L-lactic acid or D-lactic acid in the presence of saccharides, or transformed so that the lactate-producing gene functions. Any microorganism can be used. Specifically, for example, Lactobacillus (La
genus ctobacillus, Streptococcu
s) genus, Pediococcus genus, Aerococcus genus, Carbobacterium
Acterium genus, Enterococcus genus,
Erysipclothorix genus, Gemella genus, Globicatella genus, Lactococcus genus, Leuconostoc (L)
genus euconostoc, Tetragenococcu
s) genus, Vagococcus genus, Corynebacterium, Brevibacterium, Mycobacterium genus, Arthrobacter (Art
hrobacter genus, Escherichia genus, Serratia genus, Anaerobiospirum (Anaero)
The microorganisms belonging to the genera bioapirillum and Rhizopus can be mentioned, and in addition, microalgae can also be used.

Examples of the transformed microorganisms include recombinant yeast (Appl. Environ. Microbiol., 1999, Sep; 65 (9); 421.
1-4215) and recombinant E. coli (Appl.Environ.Microbiol.,
1999, Apr; 65 (4); 1384-1389). These microorganisms capable of producing lactic acid may be used alone or in combination of two or more. In addition, as a matter of course, when the lactic acid produced by the method of the present invention is used for applications such as food additives, the use of pathogenic microorganisms among the above-mentioned microorganisms to be used should be avoided.

In the fermentation medium in the method for producing lactic acid of the present invention, pyruvic acid, which is an essential constituent of the present invention, is used in an amount of about 1/2000 to 1/2 of the assimilated carbon source saccharide as a raw material for lactic acid production. It is added at a molar ratio of about (a molar ratio converted to a monosaccharide when a polysaccharide is used), preferably about 1/500 to 1/5. By adding this pyruvic acid, the production efficiency of lactic acid is remarkably increased.

The pyruvic acid used in the present invention may be in a free acid form, for example, in a salt form such as sodium salt and potassium salt.

Other assimilating carbon sources for the fermentation medium include:
Any conventionally known assimilating carbon source for a microorganism having a lactic acid-producing ability can be used, and is appropriately selected depending on the microorganism used as long as the fermenting microorganism grows and produces lactic acid. As the assimilated carbon source, for example, glucose, maltose, corn starch, spent sugar syrup, corn steep liquor, and sugars derived from cellulosic materials are used.

As the nitrogen source, for example, urea, yeast extract, heptone and whey are used. Also,
As an inorganic nutrient source, for example, potassium phosphate, magnesium sulfate, Fe (iron), Mn (manganese) compounds, etc. are also used. Furthermore, a pH adjusting agent for the medium can also be used.

The production of lactic acid can be performed by either a batch method or a continuous method. Further, a production method in which fermenting microorganisms are supported on an immobilization carrier is also possible. In order to carry out lactic acid fermentation, the above-mentioned medium is inoculated with a microorganism capable of producing lactic acid, and the pH of the medium is, for example, about 2-9, preferably about 5-8. The lactic acid production temperature is not particularly limited, but is usually about 20 to 40 ° C. However, higher temperatures are possible depending on the fermenting microorganism used. The reaction time required for the production of lactic acid is not particularly limited, and the reaction may be performed at any production reaction time as long as the effects of the present invention are observed. Optimization of these conditions can be easily determined by those skilled in the art.

The present invention comprises collecting the lactic acid thus produced, and as a means for separating and concentrating lactic acid, a known method used in a conventional production process by lactic acid fermentation can be used. Such known methods are
For example, 1) a calcium lactate recrystallization method comprising adding lime milk to neutralize, 2) an organic solvent extraction method using a solvent such as ether, 3) an esterification separation method in which the produced lactic acid is esterified with alcohol, 4 ) Chromatographic separation method using an ion exchange resin, 5) Electrodialysis method using an ion exchange membrane, and the like. Therefore, the lactic acid obtained by the method of the present invention may be not only free lactic acid, but also a salt with, for example, sodium or potassium, or an ester such as methyl ester or ethyl ester.

[0022]

The present invention will be described below with reference to examples, but the present invention is not limited to such examples.

[Example 1] Corynebacterium glutamicum (Corynebacterium 
glutamicum) Production of lactic acid by ATCC13032 and
Sampling Urea 4g, (NH_Four)_TwoSO_Four14g, KH_TwoPO_Four 
0.5g, K_TwoHPO _Four 0.5 g, MgSO_Four・ 7H
_TwoO 0.5g, FeSO_Four・ 7H_TwoO 20mg, Mn
SO_Four・ NH_TwoO 20mg, D-biotin 200μ
g, thiamine hydrochloride 100 μg, yeast extract 1 g, thistle
A medium containing 1 g of noic acid and 500 ml of distilled water (pH 6.6)
Dispense 100 ml each into a 500 ml Erlenmeyer flask.
And sterilized at 120 ° C for 15 minutes 5
Add 4 ml of 0% glucose solution, and add Corynebacterium
Mu Glutamicum (Corynebacterium glutamicum) AT
Inoculate CC13032 and shake at 14 ° C for 14 hours.
Fed (aerobic culture). After culturing, centrifuge (800
The cells were collected at 0 g for 20 minutes. All the obtained bacterial cells
The amount was tested in the following reaction.

(NH ₄ ) ₂ SO ₄ 23 g, KH ₂ PO
_{4 0.5g, K 2 HPO 4 0.5g} , MgSO 4 · 7
_{H 2 O 0.5g, FeSO 4 ·} 7H 2 O 20mg, M
nSO ₄ · nH ₂ O 20 mg, D-biotin 200 μ
g, 100 μg of thiamine hydrochloride, and 500 ml of distilled water were added to a 1 L jar fermenter, and glucose 1
80 mM was added as an assimilating carbon source, and 10 mM sodium pyruvate was added. Then, the whole amount of the above-mentioned bacterial cells was added and the mixture was allowed to react in a sealed state at 33 ° C. with gentle stirring (200 rpm). After 4 hours, the culture solution was centrifuged (8000 rp
m, 15 minutes, 4 ° C.), and the resulting supernatant was analyzed to find that the amount of lactic acid produced was 77.4 mM. The glucose consumption was 64.1 mM.

[Example 2] to [Example 5] Except that the reaction time of lactic acid production was changed to the reaction time shown in Table 1, the same method and conditions as in Example 1 were used, and the results of these reactions are shown. Shown in 1.

[Comparative Example 1] to [Comparative Example 5] The same reaction as in Example 1 was carried out in the reaction time shown in Table 1 without adding sodium pyruvate to the reaction system. The results of these reactions are shown in Table 1.

[0027]

[Table 1]

As is clear from Table 1, the effect of significantly increasing the amount of lactic acid produced was found to be greater than the amount of sodium pyruvate added, and the rate of lactic acid production was significantly improved.
Then, the addition of pyruvic acid greatly enhances glucose consumption and consumption rate. It is also clear that such a pyruvic acid addition effect continues for a long time.

Example 6 Corynebacterium glutamicum ( Corynebacterium) used in Example 1 except that the amount of sodium pyruvate added was 30 mM.
glutamicum ) ATCC13032 was used to carry out the lactic acid production reaction under the same reaction method and conditions. Reaction time is 2
The results are shown in Table 2.

[Example 7] Corynebacterium glutamicum ( Corynebacterium) used in Example 1 except that the amount of sodium pyruvate added was 30 mM.
glutamicum ) ATCC13032 was used to carry out the lactic acid production reaction under the same reaction method and conditions. Reaction time is 4
The results are shown in Table 2.

[0031]

[Table 2]

As is clear from Table 2, significant increases in the amount of lactic acid produced and the amount of glucose consumed are recognized, and their rates are also greatly increased.

Example 8 Brevibacter
ium lactofermentum) ATCC13869 same method as Production Example 1 of lactic acid by, according to the conditions, Burebibakuteriu-lactofermentum (Brevibacterium lactofermen
tum ) ATCC13869 after aerobically culturing, Example 1
The lactic acid production reaction was anaerobically carried out under the same method and conditions.
The reaction time was 2 hours. Table 3 shows the result of analysis of the reaction solution after completion of the reaction.

Example 9 According to the same method and conditions as in Example 1, Brevibacterium lactofermentum ( Br
evibacterium lactofermentum) ATCC13869 after aerobically cultured, the same method as that in Example 1 was carried out anaerobically lactic acid producing reaction under the conditions. The reaction time was 4 hours.
Table 3 shows the result of analysis of the reaction solution after completion of the reaction.

The method similar to Example 10 Example 1 in accordance with the conditions, Burebibakuteriu-lactofermentum (Brevibacterium lactofermentum) ATCC1386
After culturing 9 aerobically, the lactic acid production reaction was anaerobically performed under the same method and conditions as in Example 1. The reaction time was 6 hours. Table 3 shows the result of analysis of the reaction solution after completion of the reaction.

The method similar to Example 11 Example 1 in accordance with the conditions, Burebibakuteriu-lactofermentum (Brevibacterium lactofermentum) ATCC1386
After culturing 9 aerobically, the lactic acid production reaction was anaerobically performed under the same method and conditions as in Example 1. The reaction time was 8 hours. Table 3 shows the result of analysis of the reaction solution after completion of the reaction.

The method similar to Example 12 Example 1 in accordance with the conditions, Burebibakuteriu-lactofermentum (Brevibacterium lactofermentum) ATCC1386
After culturing 9 aerobically, the lactic acid production reaction was anaerobically performed under the same method and conditions as in Example 1. The reaction time was 10 hours.

[0038] Comparative Example 6 except that no addition of sodium pyruvate was used in Example 8 Burebibakuteriu-lactofermentum (Brevibacterium lacto
fermentum ) ATCC 13869 was used to carry out a lactic acid production reaction according to the method and conditions of Example 8 with a reaction time of 2 hours. Table 3 shows the result of analysis of the reaction solution after completion of the reaction.

[0039] Comparative Example 7, except that no addition of sodium pyruvate was used in Example 8 Burebibakuteriu-lactofermentum (Brevibacterium lacto
fermentum ) ATCC 13869 was used to carry out a lactic acid production reaction according to the method and conditions of Example 8 with a reaction time of 4 hours. Table 3 shows the result of analysis of the reaction solution after completion of the reaction.

[0040] Comparative Example 8, except that no addition of sodium pyruvate was used in Example 8 Burebibakuteriu-lactofermentum (Brevibacterium lacto
fermentum ) ATCC 13869 was used to carry out a lactic acid production reaction according to the method and conditions of Example 8 and a reaction time of 6 hours. Table 3 shows the result of analysis of the reaction solution after completion of the reaction.

[0041] [Comparative Example 9 except that no addition of sodium pyruvate was used in Example 8 Burebibakuteriu-lactofermentum (Brevibacterium lacto
fermentum ) ATCC 13869 was used to carry out a lactic acid production reaction in a reaction time of 8 hours according to the method and conditions of Example 8. Table 3 shows the result of analysis of the reaction solution after completion of the reaction.

[Comparative Example 10] Brevibacterium lactofermentum ( Brevibacterium lac) used in Example 8 except that sodium pyruvate was not added.
tofermentum ) Example 8 using ATCC 13869
A lactic acid production reaction was carried out for a reaction time of 10 hours according to the method and conditions described in 1. Table 3 shows the result of analysis of the reaction solution after completion of the reaction.

[0043]

[Table 3]

From Table 3, it was revealed that the effect of adding pyruvic acid was exhibited regardless of the microorganism species.

[0045]

The method for producing lactic acid according to the present invention can remarkably improve the production efficiency of lactic acid during lactic acid fermentation,
Lactic acid having a good flavor can be obtained. The lactic acid is used as a food additive for sake, beer, soft drinks, pickles,
It can be used as a chemical raw material for the production of soy sauce, bread, etc., and pharmaceuticals, agricultural chemicals, cosmetics, etc.

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Claims (7)

[Claims] 1. A method for producing lactic acid, which comprises culturing a lactic acid-producing bacterium in the presence of pyruvic acid externally added to produce lactic acid in a medium and collecting the produced lactic acid. 2. The method for producing lactic acid according to claim 1, wherein the lactic acid-producing bacterium belongs to the genus Corynebacterium. 3. The method for producing lactic acid according to claim 2, wherein the lactic acid-producing bacterium is Corynebacterium glutamicum. 4. The method for producing lactic acid according to claim 1, wherein the lactic acid-producing bacterium belongs to the genus Brevibacterium. 5. The method for producing lactic acid according to claim 4, wherein the lactic acid-producing bacterium is Brevibacterium lactofermentum. 6. A lactic acid obtained by a method of culturing a lactic acid-producing bacterium in the presence of externally added pyruvic acid to produce lactic acid in the medium, and collecting the lactic acid from the medium. 7. A lactic acid production accelerator, which comprises pyruvic acid.