JP2010273604A - Method for producing lactic acid by fermentation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing lactic acid having high optical purity by using an inexpensive medium. <P>SOLUTION: The method for producing lactic acid includes a step for culturing microorganisms having lactic acid-producing ability in the medium containing nitrogen sources and a carbon source, and a step for collecting the produced lactic acid. The medium contains a nitrogen source originated from fish and a nitrogen source except the nitrogen source as the nitrogen sources, regulated so that the weight ratio [(the nitrogen source originated from the fish):(other nitrogen source)] of the nitrogen source originated from the fish to other nitrogen source in terms of dried weight may be from 50:1 to 1:1. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for industrially producing high-purity lactic acid useful as a raw material for a lactic acid polymer, which is attracting attention as an alternative plastic, by utilizing microorganisms (fermentation method).

Racemic lactic acid is produced by a chemical method, but optically active lactic acid is mostly produced by a fermentation method.
Lactic acid bacteria such as Lactobacillus delbrueckii have long been known as microorganisms that produce lactic acid (Patent Document 1, Non-Patent Document 1). Lactic acid bacteria can be fermented at a relatively high temperature, and are known to produce lactic acid close to the theoretical value from glucose. However, little research has been conducted on industrial lactic acid production conditions using lactic acid bacteria.
Moreover, since lactic acid is produced as a raw material for a lactic acid polymer that can be used as a substitute for plastics, it is required to produce it at an industrially low cost. However, conventionally, in order to produce lactic acid with high optical purity using lactic acid-producing bacteria, sugars such as glucose, nitrogen sources such as yeast extract and peptone, fatty acids such as Tween 80, and vitamins are used as lactic acid raw materials. It was necessary to use a culture solution containing the above. Since yeast extract, peptone, vitamins and the like are expensive, lactic acid obtained is also expensive. In addition, it is desirable to use starch which is cheaper than glucose.

In addition, since a medium having a relatively high nutritional value is required for the growth of microorganisms that produce lactic acid, the culture medium after fermentation contains medium components at a high concentration. Therefore, a multi-step chemical method is required to purify lactic acid from the culture broth after fermentation. In particular, if the saccharide, which is the main raw material of lactic acid, remains in the culture solution, a side reaction occurs between the carboxyl group of lactic acid and the hydroxyl group of the saccharide when the lactic acid is subjected to esterification. The yield of is reduced. Therefore, it is necessary to remove the remaining saccharide from the culture broth after fermentation, which is a complicated and expensive method.
It has been reported that the hydrolyzate of salmon meat promotes the growth of lactic acid bacteria and reduces the card tension of dairy products, but the lactic acid bacteria that were effective with hydrolysates of dried fish meat were Streptcoccus thermophilus, Streptcoccus lactis. , Streptococcus cremoris, Lactobacillus helveticus (Non-patent Document 2).

JP 62-44188 A

K. Hofvendahl, Enzyme and Microbial Technology, 26, 87-107 (2000) H. Yuguchi, Japanese Journal of Dairy and Food Science, 33,81-91 (1984)

  An object of the present invention is to provide a method for efficiently producing lactic acid with high optical purity using an inexpensive medium.

  The inventors of the present invention have made researches to solve the above problems, and have obtained the following knowledge. That is, in culturing a microorganism having lactic acid-producing ability in a medium containing a nitrogen source and a carbon source, a medium containing a fish-derived nitrogen source and other nitrogen sources is used as the nitrogen source, and the fish-derived nitrogen source and the others are used. If the weight ratio to the nitrogen source is 50 to 1: 1 (fish-derived nitrogen source: other nitrogen source), it is possible to obtain lactic acid with high optical purity while using an inexpensive nitrogen source derived from fish. it can.

This invention is completed based on the said knowledge, and makes it a subject to provide the manufacturing method of the following lactic acids.
Item 1. A method for producing lactic acid comprising a step of culturing a microorganism capable of producing lactic acid in a medium containing a nitrogen source and a carbon source and a step of recovering the produced lactic acid, wherein the medium is a fish-derived nitrogen as a nitrogen source. Source and other nitrogen sources, and the weight ratio of the fish-derived nitrogen source and the other nitrogen source is 50 to 1: 1 in terms of dry weight (fish-derived nitrogen source: other nitrogen source) ) Is the method.
Item 2. Item 2. The method according to Item 1, wherein the concentration of the fish-derived nitrogen source in the medium is 0.5 to 10% by weight, and the concentration of the other nitrogen sources is 0.01 to 5% by weight.
Item 3. Item 3. The method according to Item 1 or 2, wherein the fish-derived nitrogen source is fish meal.
Item 4. Item 3. The method according to Item 1 or 2, wherein the fish-derived nitrogen source is a processed product of proteolytic enzyme.
Item 5. Any one of Items 1 to 4, wherein only the fish-derived nitrogen source is added as a nitrogen source to the culture medium at the start of the culture, and when the lactic acid production rate is reduced or substantially stopped, other nitrogen sources are added. The method described.
Item 6. Item 6. The method according to any one of Items 1 to 5, wherein the microorganism having the ability to produce lactic acid is a lactic acid bacterium belonging to the genus Lactobacillus.
Item 7. Item 7. The method according to Item 6, wherein the microorganism capable of producing lactic acid is Lactobacillus delbrueckii.
Item 8. Item 8. The method according to any one of Items 1 to 7, wherein the carbon source is at least one selected from the group consisting of glucose, sucrose, and sugarcane molasses.
Item 9. Item 9. The method according to any one of Items 1 to 8, wherein the lactic acid is D-lactic acid.

According to the method of the present invention, lactic acid with high optical purity can be produced using an inexpensive medium containing a fish-derived nitrogen source. Moreover, since the residual rate of carbohydrates becomes extremely low by using the above-mentioned medium, it is possible to save time and effort for removing carbohydrates from the culture solution after culture. Furthermore, by culturing using the above medium, a culture solution having a high lactic acid concentration can be obtained. That is, the concentration of the raw sugar in the medium can be increased, and as a result, a culture solution having a high lactic acid concentration can be obtained after culturing.
The method of the present invention is particularly suitable for the production of D-lactic acid.

It is a figure which shows transition of D-lactic acid density | concentration at the time of adding another nitrogen source to a fish origin nitrogen source after that, and residual glucose concentration. It is a figure which shows transition of D-lactic acid density | concentration, L-lactic acid density | concentration, and a residual glucose density | concentration at the time of adding calcium carbonate at the time of culture | cultivation start, and adjusting pH using sodium hydroxide after that.

Hereinafter, the present invention will be described in detail.
Nitrogen sources in the medium
<Fish-derived nitrogen source>
Examples of the fish-derived nitrogen source include fish meat; “ra” (parts other than fish meat) such as fish bones, skins, eyeballs and internal organs; and whole fish; and dried products thereof. Among them, fish meal (fish meal) is preferable. Fish meal is a whole fish dried and crushed. The type of fish is not particularly limited, but sardines, bonito, sharks, cod, herring, menhaden, etc. that can be obtained in large quantities may be used.
For example, the fish-derived nitrogen source may be pulverized and added to the medium as it is. However, it is preferable to use a fish-treated nitrogen source that has been treated with a proteolytic enzyme in view of improving the lactic acid fermentation performance of the lactic acid-producing bacteria. The proteolytic enzyme-treated product can be obtained by treating fish meat, arabe, whole fish, and dried products thereof with a protease, for example, at about 30 to 80 ° C. for several hours.

As the fish-derived nitrogen source, a fish meal proteolytic enzyme-treated product is preferable in that the fermentation performance of lactic acid-producing bacteria is high and the price is low. As the proteolytic enzyme, a known enzyme can be used without limitation. Moreover, any of alkaline protease, acidic protease, and neutral protease may be used, and the species of origin is not limited. In the case of producing D-lactic acid, an alkaline protease is preferable. By treating fish meat, arabe, whole fish, and their dried products with an alkaline protease, it is possible to sterilize miscellaneous bacteria such as L-lactic acid producing bacteria, and the optical purity of the obtained D-lactic acid. Can be prevented.
One fish-derived nitrogen source can be used alone, or two or more fish-derived nitrogen sources can be used in combination.

<Other nitrogen sources>
As the nitrogen source other than the fish-derived one, that is, a known nitrogen source used for culturing microorganisms can be used without limitation. Non-fish-derived nitrogen sources include non-fish animal-derived nitrogen sources, microbial-derived, and plant-derived nitrogen sources. Examples of such nitrogen sources include yeast extract, peptone (excluding fish-derived peptone, non-fish-derived peptone, including plant-derived peptone), and polypeptone (excluding fish-derived polypepton. Non-peptidone such as milk casein-derived polypepton. Including fish-derived polypeptone, plant-derived polypeptone such as soybean and pea-derived polypeptone.), Beer yeast, meat extract, soybean hydrolysate, pea hydrolyzate, wheat hydrolyzate, casein hydrolyzate, casamino acid And peptides or amino acids such as oil cake; inorganic nitrogens such as ammonia and nitrate; urea and the like. Among them, peptides or amino acids are preferred in that the fermentation performance of lactic acid-producing bacteria is high, yeast extract, non-fish animal or plant-derived polypeptone is more preferred, Torula yeast extract (especially Nippon Paper Chemicals, SK yeast extract) S2, SK yeast extract HUAP), soybean-derived polypeptone (especially, Maruhanichiro, bacterion SH, bacterion SS), and pea-derived polypeptone (especially, Maruhanichiro, bacterion PF) are particularly preferred.
Other nitrogen sources can be used alone or in combination of two or more.

<Ratio of nitrogen source>
The ratio of fish-derived nitrogen source and other nitrogen source in the medium (fish-derived nitrogen source: other nitrogen source) may be, for example, about 50 to 1: 1 in terms of dry weight, 45 to 1: 1 is preferred, about 41 to 1: 1 is more preferred, about 40 to 1: 1 is even more preferred, about 20 to 1: 1 is even more preferred, and about 10 to 1: 1 is particularly preferred. If it is the said range, high optical purity lactic acid (especially D-lactic acid) will be obtained, using an inexpensive fish-derived nitrogen source.
The concentration of the fish-derived nitrogen source in the medium is preferably about 0.5 to 10 w / v%, more preferably about 1 to 8 w / v%, and more preferably about 2 to 6 w / v in terms of dry weight. % Is more preferable, and about 3 to 5 w / v% is particularly preferable. Within the above range, lactic acid with high optical purity can be obtained. Moreover, it does not become difficult to stir because there are too many fish-derived nitrogen sources.

The concentration of other nitrogen sources in the medium is preferably about 0.01 to 5 w / v%, more preferably about 0.05 to 4.5 w / v%, in terms of dry weight, 0.05-4 w / v% is even more preferable. If it is the said range, culture medium cost can be restrained to such an extent that it can be industrially used, manufacturing lactic acid of high optical purity.
The inoculum may be cultured in a medium using any nitrogen source. As the inoculum, a medium containing a fish-derived nitrogen source may be used.
The nitrogen source may be added to the medium at once, or the fish-derived nitrogen source and other nitrogen sources may be added to the medium at different timings. Specifically, the culture may be performed in a medium to which a fish-derived nitrogen source and other nitrogen sources are added at the start of the culture. At the start of the culture, only the fish-derived nitrogen source is used. However, another nitrogen source may be added at the time when the operation is stopped. In the present invention, the time point when the production of lactic acid is reduced or substantially stopped is, for example, the time point when the production amount of lactic acid is 0.3 mg / ml / hr or less, preferably 0.1 mg / ml / hr or less. means.

If only the fish-derived nitrogen source is used, not all of the carbohydrates in the medium are converted to lactic acid, and although saccharides remain in the medium, lactic acid production tends to be delayed or stopped. By adding the source, lactic acid fermentation proceeds and all or almost all of the remaining carbohydrate can be converted to lactic acid.
In addition, the method of post-adding another nitrogen source includes a method of post-adding only the other nitrogen source, a method of post-adding the other nitrogen source and a carbon source, and other necessary components other than the nitrogen source and the nitrogen source. Examples include a method of post-adding a medium containing, a method of post-adding the nitrogen source, a fish-derived nitrogen source, and a medium containing necessary components other than the nitrogen source.

As the carbon source carbon source in the medium, a saccharide that can be lactic acid fermented by the lactic acid-producing bacteria to be used may be used. Examples of the saccharide include monosaccharides such as glucose and fructose; disaccharides such as sucrose, maltose and trehalose; polysaccharides such as starch, cellulose, hemicellulose and xylan. When using polysaccharides such as starch, cellulose, hemicellulose, and xylan as saccharides, saccharides such as amylase, cellulase, hemicellulase, endoxylanase, and xylosidase, which have been previously treated with an enzyme that degrades the polysaccharide, Alternatively, it is preferable to carry out lactic acid fermentation in parallel with the degradation of the polysaccharide by adding a polysaccharide-degrading enzyme together with the polysaccharide, so that lactic acid can be produced effectively. In addition, sugar cane molasses containing these saccharides, waste molasses such as sugarcane waste molasses, and the like can also be used.

Among these, monosaccharides and disaccharides are more preferable, and glucose and sucrose are even more preferable in that the fermentation performance of lactic acid-producing bacteria is high. Moreover, sugarcane molasses is also preferable from the viewpoint of increasing the productivity of lactic acid.
In addition to carbohydrates, organic acids such as acetic acid and fumaric acid; monohydric alcohols such as ethanol; polyhydric alcohols such as glycerin and the like can also be used as the carbon source.
The carbon source containing a saccharide can be used alone or in combination of two or more.
The sugar concentration in the medium at the start of the culture is preferably about 5 to 15 w / v%, more preferably about 8 to 14 w / v%, and even more preferably about 10 to 13 w / v%. If it is the said range, while being able to produce lactic acid efficiently, the residual sugar mass is suppressed.
The carbon source and the nitrogen source may be sterilized by a general sterilization method such as steam sterilization, filter sterilization, or instantaneous sterilization, respectively, and added to the medium.

The other components of the medium include inorganic salts such as phosphate, magnesium sulfate, calcium salts, iron salts, manganese salts, vitamins, polysorbate Such fatty acids may be included.
Lactic acid producing bacteria Lactobacillus delbrueckii, Lactobacillus plantarum, Leuconostoc mesenteroides, Spololactobacillus genus, etc. are known as lactic acid producing bacteria, in particular D-lactic acid producing bacteria. delbrueckii subsp. lactis IAM 12476, Lactobacillus delbrueckii subsp. Bulgaricus IAM 12472, Lactobacillus delbrueckii subsp. delbrueckii IAM 12474, Lactobacillus delbrueckii IFO 3534, Lactobacillus delbrueckii subsp. Among them, Lactobacillus delbrueckii subsp. Delbrueckii NRIC0761 is preferable because it has good fermentation performance and is suitable for industrial production of lactic acid, particularly D-lactic acid. In addition, Leuconostoc pseudomesenteroides JCM 9696 etc. can be used as a lactic acid strain for heterofermentation.
Microorganisms with IFO numbers can be obtained from the National Institute of Product Evaluation Technology Biotechnology Headquarters Biogenetic Resources Division (NBRC). Microorganisms with IAM numbers and JCM numbers can be obtained from the RIKEN BRC-JCM, RIKEN BioResource Center. Microorganisms with NRIC numbers can be obtained from the Tokyo University of Agriculture strain storage room.

Culture conditions The temperature of lactic acid fermentation may be any temperature at which the lactic acid-producing bacteria to be used grow, and is preferably about 0 to 60 ° C, more preferably about 30 to 50 ° C, and still more preferably about 35 to 45 ° C.
During fermentation, the medium pH decreases with the production of lactic acid. If the pH of the medium is too low, the growth of the bacteria is inhibited. Therefore, the pH may be adjusted with an alkali such as calcium carbonate, sodium hydroxide, or ammonia. Of these, sodium hydroxide and ammonia are preferable. This is because sodium lactate and ammonium lactate are in a liquid state at room temperature, so that they can be easily separated from insoluble components such as cells after culturing. In addition, if a liquid neutralizer such as sodium hydroxide is used, the amount of lactic acid produced is correlated with the required amount of neutralizer, so the degree of fermentation can be easily known from the amount of neutralizer added. it can. Furthermore, sodium hydroxide is inexpensive and advantageous for industrial production. The pH of the fermentation broth varies depending on the strain used, but is preferably adjusted to about 4 to 7, and more preferably about 5 to 6.5.
It is also preferable to add about 0.1 to 1 w / v% of calcium carbonate in the medium in advance or at the start of the culture and adjust the pH to the above range using a different kind of alkali as the pH decreases. Thereby, lactic acid production rate becomes still higher and lactic acid can be manufactured efficiently.
The culture may be any of batch culture, semi-batch culture, and continuous culture. Among these, batch culture is preferable in that the residual sugar mass can be reduced. Further, semi-batch culture or continuous culture in which only carbohydrates are added during culture may be used.
The culture time varies depending on the strain used, medium components, particularly the amount of carbohydrates, etc., but in the case of batch culture, it is preferably about 1 to 8 days, more preferably about 2 to 7 days. However, the present invention is not limited to this when continuous culture or semi-batch culture is performed.
By the method of the present invention, lactic acid with high optical purity can be produced using an inexpensive medium containing a fish-derived nitrogen source, and a culture solution having a high lactic acid concentration can be obtained. In addition, by using the above medium, the residual sugar concentration in the medium can be extremely lowered finally.

Lactic acid can be recovered in the form of lactic acid or lactic acid alkali salt by removing the cells from the culture solution after the culturing of lactic acid. In addition, when calcium carbonate is used as an alkali for pH adjustment, calcium lactate is produced. However, since calcium lactate is insoluble in water, the culture solution is acidified with, for example, sulfuric acid (pH 3.5 or lower), and calcium is sulfated. What is necessary is just to make it precipitate as calcium and to remove with a microbial cell. Furthermore, after removing insoluble components such as bacterial cells, lactate can be produced with ethanol or methanol and recovered in an organic solvent.

Hereinafter, the present invention will be described more specifically with reference to examples.
Analysis method
(1) Determination of Lactic Acid and Measurement of Optical Purity In consideration of the solubility of calcium lactate, the sample for lactic acid quantification was obtained by adding 0.8 ml of ethanol to 0.2 ml of the sample diluent and centrifuging the resulting precipitate (15,000 rpm, 5 min) and removed, and the supernatant was appropriately diluted with water. This sample was analyzed by HPLC (chiral column). The HPLC conditions are as follows.
Column: Sumichiral OA Chiral Column (Column, Sumichiral OA-5000 (4.6mm ID x 15cm)
Temperature: Room temperature Mobile phase: 2 mM Cooper (II) sulfate-5H ₂ O (249.69) in water-isopropanol (98: 2) solution elution rate: 1.0 ml / min
Detection: UV at 254nm
The enantiomeric excess ee (Enantiomeric excess) was calculated by the following formula.
ee (%) = ([D-form]-[L-form]) / ([D-form] + [L-form]) × 100

(2) Determination of sugar The sugar was determined by the phenol-sulfuric acid method. Glucose was quantified using a glucose quantification kit (Glucose CII Test Wako, Wako Pure Chemical Industries).
Example 1 (comparison of production rate of D-lactic acid by each lactic acid bacterium)
Reagent medium (fish extract 1 w / v (Maruha Nichiro Foods, Inc.), 1 w / v% yeast extract (Nippon Paper Chemical Co., Ltd.), 3 w / v% CaCO ₃ , 5 w / v% glucose, pH 6.8, 121 ° C., 15 minutes Each bacterium was inoculated into 10 ml of autoclaved) from a cryopreservation vial, and allowed to stand for 24 hours in a 37 ° C. incubator to obtain a seed culture solution. Reagent medium (1 w / v% fish extract (Maruha Nichiro Foods, Inc.), 1 w / v% yeast extract (Nippon Paper Chemical Co., Ltd.), 3 w / v% CaCO ₃ , 5 w / v% glucose, pH 6.8, 121 ° C. 15 1 ml of each fungus was inoculated from 100 ml of seed culture solution into 100 ml of sterilized autoclave, and cultured at 37 ° C. for 72 hours on a rotary shaker. Table 1 shows the tested bacterial species, culture time, D-lactic acid production concentration, residual sugar concentration, and optical purity of the produced D-lactic acid.

菌種１：Lactobacillus delbrueckii subsp. delbrueckii NRIC0760
菌種２：Lactobacillus delbrueckii subsp. delbrueckii NRIC0761
表１から、窒素源として、魚エキスと酵母エキスとを１：１で用いることにより、何れの菌株を用いた場合も、高濃度の乳酸が得られたことが分かる。

Species 1: Lactobacillus delbrueckii subsp. Delbrueckii NRIC0760
Species 2: Lactobacillus delbrueckii subsp. Delbrueckii NRIC0761
From Table 1, it can be seen that by using a fish extract and a yeast extract at a ratio of 1: 1 as a nitrogen source, a high concentration of lactic acid was obtained when any strain was used.

Example 2 (Comparison of growth of lactic acid bacteria by medium type)
The growth of lactic acid bacteria according to the type of medium was compared. Lactobacillus delbrueckii NRIC0761 from a cryopreservation vial to 100 ml of test media 1 to 4 having different nitrogen sources (each nitrogen source, 3 w / v% CaCO ₃ , 5 w / v% glucose, pH 6.8, autoclaved at 121 ° C. for 15 minutes) ) Was inoculated in an amount of 1%, and statically cultured in a 37 ° C. incubator for 24 hours.
Each nitrogen source in the medium is as follows.
Test medium 1: 0.5 w / v% polypeptone N (soybean origin, Nippon Pharmaceutical Co., Ltd.), 1 w / v% yeast extract (HY-yest, KERRY)
Test medium 2: MRS (non-fish-derived animal-derived meat extract and yeast-derived, Kanto Chemical Co., Inc.)
Test medium 3: 0.5 w / v% polypeptone NF (fish origin, Nippon Pharmaceutical Co., Ltd.), 1 w / v% SK yeast extract S-2 (yeast origin, Nippon Paper Chemicals Co., Ltd.)
Test medium 4: 1 w / v% fish extract (fish origin, bacterion KN, Maruha Nichiro Foods Co., Ltd.), 1 w / v% SK yeast extract HUAP (yeast origin, Nippon Paper Chemicals Co., Ltd.)
The results are shown in Table 2.

表２から、魚由来窒素源と酵母由来窒素源とを併用した試験培地３、４では、魚由来以外の窒素源だけ使用した試験培地１、２に比べて、Ｄ-乳酸の生産性が高いことが分かる。

From Table 2, test mediums 3 and 4 using both a fish-derived nitrogen source and a yeast-derived nitrogen source have higher productivity of D-lactic acid than test media 1 and 2 using only nitrogen sources other than fish-derived nitrogen sources. I understand that.

Example 3 (Influence of proteolytic enzyme treatment on skipjack fish powder)
Neutral protease (Orientase 90N, HIBI Co., Ltd.) or 400 mg of alkaline protease (Orientase 22BF, HIBI Co., Ltd.) is added to 8 g of bonito fish powder (Maruishi Co., Ltd.) and enzyme-treated at 60 ° C. The treatment solution before and after the enzyme treatment was applied to a 100 μl PYG medium plate, and the number of germs contained in the treatment solution was measured. The results are shown in Table 3 below.
As shown in Table 3, those treated with the neutral protease contained more bacteria than before treatment, and those treated with the alkaline protease had fewer germs than before treatment. In Table 3, + indicates 10 to 100 miscellaneous bacteria in 1 ml of the enzyme treatment solution, ++ indicates 100 to 1000, and +++ indicates 1000 or more.

Example 4 (Examination of fish powder types)
Various fish meals were used as a nitrogen source derived from fish to prepare a medium. Using these, D-lactic acid was produced by lactic acid bacteria fermentation, and the lactic acid concentration and optical purity were compared. That is, 4 g of various fish powders, 50 ml of 0.4% K ₂ HPO ₄ (pH 7.2), 80 mg of orienter 22BF (alkaline protease, HI Corporation) were added to a 200 ml Erlenmeyer flask, and the mixture was kept at 60 ° C. for 3 hours. Reacted. After the reaction, 5 g of CaCO ₃ was added and made up to 90 ml with water. This was autoclaved at 121 ° C. for 15 minutes, and 10 ml of 100% (w / v) glucose solution autoclaved separately (the glucose concentration in the medium was about 10 w / v%) was added to obtain a fish powder medium. 1 ml of a seed culture solution of Lactobacillus delbrueckii NRIC0761 prepared in the same manner as in Example 1 was added to these media, followed by culturing at 37 ° C. for 72 hours. The concentration of fish-derived nitrogen source in the medium is about 4 w / v%. The results are shown in Table 4 below. From Table 4, it can be seen that each fish powder was able to produce an equivalent concentration of D-lactic acid.

Example 5 (Production of D-lactic acid in fish powder medium by each lactic acid bacterium)
Lactic acid was produced by lactic acid bacteria fermentation. Into a 200 ml Erlenmeyer flask, 4 g of Menhaden fish powder (USA), 50 ml of 0.4% K ₂ HPO ₄ (pH 7.2), 80 mg of orientase 22BF (alkaline protease, HBI Co., Ltd.) are added, and 3 at 60 ° C. Reacted for hours. After the reaction, 5 g of CaCO ₃ was added, and the volume was adjusted to 90 ml with water. The mixture was autoclaved at 121 ° C. for 15 minutes, and 12 ml of a separately autoclaved 100% (w / v) glucose solution (the glucose concentration in the medium was about 12 w / v%) was added to obtain a fish powder medium. The concentration of fish-derived nitrogen source in the medium is about 4 w / v%. 1 ml of Lactobacillus delbrueckii NRIC0760 strain and NRIC0761 strain seed culture solution prepared in the same manner as in Example 1 was added to these media and cultured at 37 ° C. for 96 hours. The results are shown in Table 5 below.

Example 6 (concentration of fish-derived nitrogen source)
Lactic acid was produced by lactic acid bacteria fermentation. To a 200 ml Erlenmeyer flask, 4 g or 2 g of Menhaden fish powder (USA), 50 ml of 0.4% K ₂ HPO ₄ (pH 7.2), 80 mg or 40 mg of orientase 22BF (alkaline protease, HTV Corporation), The reaction was carried out at 60 ° C. for 3 hours. After the reaction, 12 g of CaCO ₃ was added, and the volume was adjusted to 90 ml with water. The mixture was autoclaved at 121 ° C. for 15 minutes, and 12 ml of 100% (w / v) glucose solution autoclaved separately (the glucose concentration in the medium was 12 w / v%) was added to obtain a fish powder medium. The fish-derived nitrogen source concentration in the medium is 4 w / v% or 2 w / v%. 1 ml of Lactobacillus delbrueckii NRIC0760 strain and NRIC0761 strain seed culture solution prepared in the same manner as in Example 1 was added to these media and cultured at 37 ° C. for 96 hours. The results are shown in Table 6 below.

表６から、魚由来の窒素源が２ｗ／ｖ％あるいは４ｗ／ｖ％の濃度で調製された培地で高効率な乳酸発酵が可能であることが分かる。また、魚由来の窒素源を２倍にすることにより、得られる乳酸濃度がさらに向上したことが分かる。

From Table 6, it can be seen that highly efficient lactic acid fermentation is possible with a medium prepared with a nitrogen source derived from fish at a concentration of 2 w / v% or 4 w / v%. It can also be seen that the concentration of lactic acid obtained was further improved by doubling the fish-derived nitrogen source.

Example 7 (Combination of fish-derived nitrogen source and other nitrogen sources)
In the same manner as in Example 6, 2 ml of Menhaden fish powder (USA) was used to prepare 100 ml of a fish powder proteolytic enzyme-containing product medium (fishmeal medium) in a 200 ml Erlenmeyer flask. Further, a nitrogen source shown in Table 7 below was added to the medium so as to be 1 w / v%. Nitrogen source Barlex 60 (Barley Fermentation Laboratory Co., Ltd.) is derived from wheat, Bacterion-N-PF (Maruha Nichiro Foods Co., Ltd.) is derived from peas, Bacterion-N-SH (Maruha Nichiro Foods Co., Ltd.), And Bacterion-N-SS (Maruha Nichiro Foods Co., Ltd.) is derived from soybeans.
Using these media, the Lactobacillus delbrueckii NRIC0761 strain was cultured at 37 ° C. for 96 hours.
The results are shown in Table 7. The glucose concentration in Table 7 is the residual glucose concentration in the medium after completion of fermentation.

表７から、魚由来窒素源とその他の窒素源とを併用することにより、魚由来窒素源のみ用いる場合に比べて、高濃度の乳酸が得られたことが分かる。

From Table 7, it can be seen that a high concentration of lactic acid was obtained by using a fish-derived nitrogen source and another nitrogen source in combination, compared with the case where only the fish-derived nitrogen source was used.

Example 8 (Combination of fish-derived nitrogen source and other nitrogen sources)
For D-lactic acid production method using fish-derived nitrogen source and non-fish-derived nitrogen source, effective range of addition amount of nitrogen source other than fish-derived (concentration range that brings merit in D-lactic acid production efficiency by addition) ) Was investigated. In addition, SK yeast extract (HUAP) was used as a nitrogen source other than fish-derived.
0.1 ml of frozen cells of Lactobacillus delbrueckii NRIC0761 strain were added to a seed culture medium (SK yeast extract HUAP 1 w / v%, Polypeptone (bacterion KN) 1 w / v%, glucose 5 w / v%, CaCO _{3 3} w / v%, pH 6.8) 9.9 ml was inoculated, allowed to stand at 37 ° C. for 24 hours, and seed-cultured.
This medium was prepared as follows. Herring fish meal (Ecuador) 8 w / v% was suspended in 0.4% K ₂ HPO ₄ and adjusted to pH 10. Orientase 22BF (alkaline protease, HBI Corporation) was added in an amount of 1/20 (0.4 w / v%) to fish meal and treated at 60 ° C. for 3 hours. The supernatant obtained by centrifugation was adjusted to pH 6.3 and used as a fish meal supernatant medium.

In test groups 1 to 9 shown in Table 8 below, 5 ml of the fishmeal supernatant medium, 3.9 ml of ion-exchanged water, CaCO ₃ 6 w / v%, and HUAP were added in the amounts shown in Table 8 at 121 ° C. Heat treatment was performed for 15 minutes. In test groups 10 to 18, 2.5 ml of fish meal supernatant medium, 6.4 ml of ion-exchanged water, 6% w / v CaCO ₃ and HUAP were added to the test tube and heat-treated at 121 ° C. for 15 minutes. 1 ml of a 1 g / ml glucose solution sterilized in a separate container (glucose preparation concentration of 10 w / v% in the medium) was added to the test tube of each test group. 9.9 ml of the main culture medium was inoculated with 0.1 ml of the seed culture solution, allowed to stand at 37 ° C., and cultured for 96 hours. The results are shown in Table 8.

表８から、魚由来窒素源とその他の窒素源とを約４０〜１：１で使用することにより、高乳酸濃度が得られたことが分かる。

From Table 8, it can be seen that a high lactic acid concentration was obtained by using a fish-derived nitrogen source and other nitrogen sources at about 40 to 1: 1.

Example 9 (combination of fish-derived nitrogen source and other nitrogen sources)
60 g of herring fish meal (Ecuador) 1.2 kg, 0.4% K ₂ HPO ₄ (pH 7.2) 15 L, orientase 22BF (alkaline protease, HTV Corporation) was added to Mitsuwa 50 L jar fermenter, The reaction was performed at 60 ° C. for 3 hours (rotation speed: 100 rpm). After the reaction, make up to 27 L with water, sterilize with steam at 121 ° C. for 30 minutes, add 3 L of a 100 w / v% glucose solution that has been autoclaved separately (the concentration of glucose in the medium is 10 w / v%) and add fish meal. The medium was used.
To this fish meal medium, 300 ml of a seed culture solution of Lactobacillus delbrueckii NRIC0761 prepared in the same manner as in Example 1 was added and cultured. The culture conditions were a stirring speed of 100 rpm, a culture temperature of 37 ° C., aeration was conducted in the gas phase only with 5% vvm, and the pH was controlled to 6.0 using a 25% NaOH aqueous solution. At the time when the production of lactic acid was stopped 47 hours after the start of the culture, 30 g (0.1 w / v%) of fish extract bacterion KN and yeast extract HUAP were added. The concentration of the fish powder proteolytic enzyme degradation product in the medium is 4 w / v% (1.2 kg / 3 L), the concentration of the fish extract added after is 0.1 w / v%, and the concentration of the yeast extract added after is Since it is 0.1 w / v%, the weight ratio of the final fish-derived nitrogen source and yeast-derived nitrogen source is 41: 1 (fish-derived nitrogen source: yeast-derived nitrogen source).
The results of measuring the D-lactic acid concentration in the culture solution over time are shown in Table 9 below and FIG. From Table 9 and FIG. 1, it can be seen that the fermentation of D-lactic acid once stopped was resumed by the addition of fish extract bacterion KN and yeast extract HUAP.

Example 10 (Improved sodium hydroxide neutralization method in which calcium carbonate is added)
Calcium carbonate was added as an additive to the culture solution in an amount of 1 w / v%, and the pH stat was subjected to a fermentability test by a neutralization method using sodium hydroxide.
<Method>
40 g (4 w / v%) of herring fish meal (Ecuador) and 2 g of orientase 22BF (Hichiai Co., Ltd.) are put into a 2 L jar fermenter, 500 ml of water is added, and the mixture is stirred and mixed at 80 ° C. for 30 minutes at 60 ° C. Enzyme treatment. Here, 1.0 g of yeast extract (HUAP) (final concentration 0.1 w / v%), 10 g of calcium carbonate (final concentration 1 w / v%), and 360 ml of water were added, and autoclaved at 121 ° C. for 20 minutes. Was done. Separately sterilized 70 w / v% aqueous glucose solution was added to a 140 ml culture tank (final glucose concentration of about 10 w / v%) to obtain a main culture medium. The fish-derived nitrogen source concentration in this medium is 4.0 w / v%, the yeast-derived nitrogen source concentration is 0.1 w / v%, and the glucose concentration is 10 w / v%.

  As the inoculum, the first seed culture was performed in a medium containing 1 w / v% each of peptone (bacterion KN) and yeast extract (HUAP), which was inoculated into a medium having the same composition and cultured for 2nd seed. Specifically, 1% of the inoculum was inoculated from a Lactobacillus delbrueckii NRIC0761 frozen strain vial, and statically cultured at 37 ° C. (1st seed). A 1% amount of 22.5 hr (OD; 10.0) was transferred to the 2nd seed medium. The turbidity of the 2nd seed cultured in the same manner and transplanted into the main culture was 17.2. 10 ml of this was transplanted into the main culture medium, and cultured for 120 hours at 37 ° C. with gentle stirring at 75 rpm.

<Result>
The transition of lactic acid concentration and residual glucose concentration is shown in FIG. As is apparent from FIG. 2, by adding other nitrogen sources and calcium carbonate to the medium of fish-derived nitrogen source, the fermentation rate becomes very high, and extremely efficient D-lactic acid fermentation can be performed. It was.

  According to the method of the present invention, lactic acid as a polylactic acid raw material can be produced efficiently at low cost, and lactic acid, particularly D-lactic acid, can be industrially produced at low cost.

Claims (9)

  A method for producing lactic acid comprising a step of culturing a microorganism capable of producing lactic acid in a medium containing a nitrogen source and a carbon source and a step of recovering the produced lactic acid, wherein the medium is a fish-derived nitrogen as a nitrogen source. Source and other nitrogen sources, and the weight ratio of the fish-derived nitrogen source and the other nitrogen source is 50 to 1: 1 in terms of dry weight (fish-derived nitrogen source: other nitrogen source) ) Is the method.   The method according to claim 1, wherein the concentration of the fish-derived nitrogen source in the medium is 0.5 to 10% by weight, and the concentration of the other nitrogen sources is 0.01 to 5% by weight.   The method according to claim 1 or 2, wherein the fish-derived nitrogen source is fish meal.   The method according to claim 1 or 2, wherein the fish-derived nitrogen source is a proteolytic enzyme-treated product.   Either the fish-derived nitrogen source is added as a nitrogen source to the culture medium at the start of culture, and other nitrogen sources are added when the lactic acid production rate is reduced or substantially stopped. The method described in 1.   The method according to any one of claims 1 to 5, wherein the microorganism capable of producing lactic acid is a lactic acid bacterium belonging to the genus Lactobacillus.   The method according to claim 6, wherein the microorganism capable of producing lactic acid is Lactobacillus delbrueckii.   The method according to any one of claims 1 to 7, wherein the carbon source contains a saccharide, and the saccharide is at least one selected from the group consisting of glucose, sucrose, and sugarcane molasses.   The method according to any one of claims 1 to 8, wherein the lactic acid is D-lactic acid.

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