JP2010029119A - Method for producing d-lactic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provided a method for producing D-lactic acid which uses a fermentation medium (natural biomass) containing cysteine and a low concentration nitrogen, and has a simple operation to produce the D-lactic acid having a high optical purity in high productivity stably and inexpensively, and also reduce the concentrations of byproducts. <P>SOLUTION: This method for producing D-lactic acid by using bacteria belonging to the genus Sporolactobacillus is provided by using a fermentation raw material containing ≥0.6 mg/L of cysteine, and 0.1 to 0.5 g/L of nitrogen source other than cysteine. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for producing D-lactic acid by a fermentation method. More specifically, the present invention relates to a method for producing D-lactic acid by a fermentation method using bacteria belonging to the genus Sporolactocillus.

Polylactic acid, a biodegradable polymer, has attracted strong attention as a sustainability and life cycle assessment (LCA) compatible product with the emergence of CO ₂ and energy problems. There is a demand for an efficient and inexpensive production method for lactic acid.

  Currently produced polylactic acid is an L-lactic acid polymer, but lactic acid has two optical isomers, L-lactic acid and D-lactic acid, and D-lactic acid is also used for polymer raw materials, agricultural chemicals and pharmaceuticals. In recent years, it has attracted attention as an intermediate.

  In nature, there are microorganisms that efficiently produce lactic acid such as lactic acid bacteria, and a method for producing lactic acid using them has already been established. Examples of bacteria that can efficiently produce L-lactic acid include Lactobacillus rhamnosus (see Patent Documents 1 and 2), and examples of bacteria that can efficiently produce D-lactic acid include Bacillus laevolacti. It is known that cas (Bacillus lavolacticus) and the like produce D-lactic acid with high optical purity (see Patent Document 3).

  In addition to lactic acid bacteria that exist in nature, lactic acid production methods using microorganisms that incorporate lactic acid synthase are also known. -Attempts have been made to produce lactic acid. For example, an attempt to produce D-lactic acid using E. coli as a recombinant host has been proposed (see Patent Document 4). There is also an attempt to produce D-lactic acid by yeast by introducing a D-lactic acid dehydrogenase gene derived from a known Lactobacillus plantarum (see Patent Document 5).

However, in the conventional fermentation production of D-lactic acid, the production of a high concentration by-product due to a large amount of nitrogen source contained in the fermentation medium has been a problem. Such a high concentration of by-products causes an increase in costs in the subsequent purification process, and there is a concern that it may increase costs in the entire lactic acid production process. Therefore, any fermentation method such as batch fermentation, fed-batch fermentation (Fed-Batch) method, continuous fermentation, etc. can produce D-lactic acid at a low cost and in a high yield, and D- with high optical purity. There has been a demand for a more efficient production method by suppressing the production of by-products along with the production of lactic acid, and a dramatic improvement in production speed has been demanded.
JP 2007-215427 A JP 2007-215428 A JP 2003-088392 A JP 2005-102625 A JP 2002-136293 A

  Therefore, an object of the present invention is to produce D-lactic acid that can suppress the production of by-products as well as the production of D-lactic acid with a stable high yield and high optical purity in producing D-lactic acid by fermentation using a microorganism. It is to provide a manufacturing method.

As a result of intensive studies, the present inventors have completed the present invention by reducing cysteine and nitrogen sources other than cysteine contained in the fermentation raw material. That is, the present invention is as follows.
(1) A method for producing D-lactic acid by a fermentation method using a bacterium belonging to the genus Sporolactobacillus, comprising 0.6 mg / L or more of cysteine and 0.1 g / L or more of 0.5 g / L The manufacturing method of D-lactic acid using the fermentation raw material containing nitrogen sources other than L or less cysteine.
(2) The method for producing D-lactic acid according to (1), wherein the bacterium is Sporolactobacillus laevolacticus.

  According to the present invention, it is possible to stably produce D-lactic acid, which is a fermentation product, at a lower cost, and to produce D-lactic acid with high optical purity with fewer by-products. D-lactic acid obtained by the method for producing D-lactic acid according to the present invention is useful as a raw material for, for example, acidulants, detergents and pharmaceuticals, and polylactic acid resins.

  The present invention relates to a method for producing D-lactic acid by a fermentation method using a bacterium that belongs to the genus Sporolactobacillus and has the ability to produce D-lactic acid (hereinafter also referred to as D-lactic acid-producing bacterium). It is a method for producing D-lactic acid using a fermentation raw material containing a cysteine of 0.6 mg / L or more and a nitrogen source other than cysteine of 0.1 g / L or more and 0.5 g / L or less.

  Cysteine in the fermentation raw material is important for the growth or growth of the D-lactic acid-producing bacterium, but the present inventors added 0.6 mg / L or more cysteine to the fermentation raw material to produce a by-product. It has been found that when the cysteine concentration in the fermentation raw material is less than 0.6 mg / L, many by-products are produced. Examples of by-products here include organic acids and alcohols, and examples of organic acids include formic acid, acetic acid, pyruvic acid, succinic acid, malic acid, itaconic acid, and citric acid. Examples of the alcohol include ethanol and butanediol. These by-products can be measured by HPLC or GC. The upper limit of the cysteine concentration in the fermentation raw material in the present invention is not particularly limited, but is preferably 2000 mg / L from the viewpoints of good growth of microorganisms, production of lactic acid at a high concentration, and total cost. Moreover, as a preferable cysteine density | concentration in a fermentation raw material, it is 0.6-1500 mg / L, More preferably, it is 0.62-1000 mg / L.

  Moreover, although nitrogen sources other than cysteine are important for good growth of D-lactic acid-producing bacteria, the present inventors set the concentration of nitrogen sources other than cysteine in the fermentation raw material to 0.1 g in addition to the concentration of cysteine. It was found that the production of by-products is suppressed by setting the ratio to / L or more and 0.5 g / L or less. Specific examples of the nitrogen source other than cysteine include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, or organic nitrogen sources (for example, oil cakes, soybean hydrolysate, casein degradation products, and cysteine other than cysteine). Amino acids, vitamins, corn steep liquor, meat extract, peptone, polypeptone S, etc.), carbon sources in fermentation raw materials (eg starch saccharified liquid, sweet potato molasses, sugar beet molasses, high test molasses, cane juice from sugarcane Amino acid or proteinous impurities contained in the extract or concentrated liquid of cane juice, raw sugar or purified sugar purified or crystallized from cane juice, or various fermented cells such as yeast extract or hydrolysates thereof, etc. These may be used alone or in combination. Preferably an organic nitrogen source. A preferable concentration of a nitrogen source other than cysteine in the fermentation raw material is 0.1 g / L or more and 0.45 g / L or less. Note that nitrogen sources other than cysteine may contain cysteine as an impurity, in which case the concentration of nitrogen sources other than cysteine in the fermentation raw material is calculated based on the value obtained by subtracting the cysteine content from the nitrogen source. It shall be.

  In addition, D-lactic acid from the start of lactic acid fermentation to the end of fermentation can be obtained by using a fermentation raw material containing 0.6 mg / L or more of cysteine and a nitrogen source other than cysteine of 0.1 to 0.5 g / L. The growth rate of the producing bacteria can be delayed to increase the density for a long time, and the production of by-products can be suppressed to improve the lactic acid productivity. In both fermentations, the bacterial death cycle can be delayed and the concentration of by-products generated at the time of death can be reduced. In particular, in continuous fermentation, the clogging of the porous membrane used as a separation membrane for fermentation broth can be suppressed. The lactic acid fermentation can be further prolonged and the production efficiency of the lactic acid fermentation can be increased.

  In the present invention, the fermentation raw material containing 0.6 mg / L or more of cysteine and a nitrogen source other than 0.1 g / L or more and 0.5 g / L or less of cysteine is 0.6 mg / L or more in the lactic acid fermentation medium. And a nitrogen source other than 0.1 g / L to 0.5 g / L of cysteine. Preferable specific examples of the lactic acid fermentation medium include lactic acid fermentation medium (hereinafter sometimes abbreviated as GYC1) containing glucose, 0.1 g / L cysteine and 0.4 g / L yeast extract, glucose, 0. Lactic acid fermentation medium (hereinafter sometimes abbreviated as GYC2) containing 2 g / L cysteine and 0.1 g / L yeast extract, glucose, 0.4 g / L cysteine and 0.1 g / L yeast extract Lactic acid fermentation medium (hereinafter sometimes abbreviated as GYC3), lactic acid fermentation medium (hereinafter referred to as 100 g / L of raw sugar containing 0.3 wt% protein as impurities and 0.15 g / L of yeast extract) RSE may be abbreviated as RSE), lactic acid fermentation medium (hereinafter abbreviated as RSC1) containing the raw sugar and 0.4 g / L of cysteine, and 1.0 g / L of the raw sugar. Lactic acid fermentation medium containing cysteine (hereinafter sometimes abbreviated as RSC2), and lactic acid fermentation medium containing glucose and 0.2 g / L cysteine and 0.4 g / L polypeptone (hereinafter abbreviated as GPC). Etc.).

  The lactic acid fermentation medium can be prepared as follows. For example, among the nutrients, sugars that become carbon sources and nitrogen sources are divided into groups, sterilized by autoclaving for each group, cooled to a temperature below room temperature, and then the concentration of nitrogen contained in each fermentation medium. Prepare as follows.

  The present invention is characterized by using a D-lactic acid-producing bacterium belonging to the genus Sporolactobacillus. The D-lactic acid-producing bacterium may be isolated from the natural environment, or may be partially modified in nature by mutation or genetic recombination. Moreover, it is also possible to purchase as a strain from a biological depository organization such as ATCC.

  The D-lactic acid producing ability of the D-lactic acid producing bacterium used in the present invention is not particularly limited, but is a D-lactic acid producing bacterium having an ability to produce D-lactic acid having an optical purity of 90% or more. Is preferred. A D-lactic acid-producing bacterium having an ability to produce D-lactic acid having an optical purity of 90% or higher is inoculated with bacteria belonging to the genus Sporolactocillus in a culture solution of 100 g / L of sterilized raw material sugar, Incubation was performed, the sugar concentration in the culture solution was measured over time, the optical purity of lactic acid produced in the culture solution after the sugar was completely consumed was measured, and the optical purity of D-lactic acid was 90 If it is% or more, it can be judged that it is a D-lactic acid-producing bacterium having an optical purity of 90% or more.

  Among the D-lactic acid-producing bacteria used in the present invention, Sporolactobacillus lavolacticus, Sporolactobacillus inulinus, Sporolactociloscofuensis (Spololactococcus) Sporolactobacillus sakapie samapi, sama sabai sera, sapora basralus sera, sapora bisra sra sra, sakapi, sama (Sporolactobacillus terrae) etc. are mentioned, Among them, Sporolactobacillus laevolyticus can be preferably used. Moreover, as strains of Sporalactobacillus laevolacticus, ATCC 23492, ATCC 23493, ATCC 23494, ATCC 23495, ATCC 23496, ATCC 223549, IAM 12326, IAM 12327, IAM 12328, IAM 12329, IAM 12330, IAM 12331, IAM 12379, DSM 2315, M14 Specific examples include DSM6763, DSM6764, and DSM6771 strains, and ATCC23492 is preferable.

  In the present invention, when D-lactic acid-producing bacteria are fermented and cultured, the D-lactic acid-producing bacteria may be cultured under aerobic conditions, but preferably under anaerobic conditions. Since bacteria belonging to the genus Sporolactobacillus are aerobic or aeration microorganisms, they are usually cultured under aerobic conditions by aeration, etc., but under aerobic conditions, sugars such as glucose are not Anaerobic conditions are preferably employed from the viewpoint of the yield of D-lactic acid because it is metabolized from pyruvic acid via the Krebs cycle. In order to carry out the culture under anaerobic conditions, the culture can be carried out by standing, but the shaking culture or the stirring culture may be carried out while passing an inert gas. Carbon dioxide, nitrogen, ammonia, argon or the like may be used as the inert gas here, and the amount of ventilation and the ventilation means may be appropriately determined in consideration of D-lactic acid productivity. Here, nitrogen is a gas used for anaerobic culture and does not correspond to a nitrogen source other than cysteine in the present invention.

  In addition, when fermenting and cultivating D-lactic acid producing bacteria, the fermenting material contains a carbon source that can be assimilated, and cultured at an appropriate temperature while neutralizing with calcium carbonate, calcium hydroxide, sodium hydroxide, ammonia, or the like. Is preferred. The pH during the culture is not particularly limited as long as the D-lactic acid-producing bacterium can produce D-lactic acid, but it is preferably within the range of pH 4-8. The culture temperature is not particularly limited as long as the D-lactic acid-producing bacterium can produce D-lactic acid, but is a culture temperature for producing high optical purity D-lactic acid having an optical purity of 90% or more. Specifically, it is preferably from 30 ° C to 45 ° C, more preferably from 31 ° C to 39 ° C, and further preferably from 33 ° C to 38 ° C.

  The carbon source in the fermentation raw material used in the present invention is not particularly limited as long as it is a carbon source that can be assimilated by a D-lactic acid-producing bacterium. Sugars such as raffinose, trehalose, mannitol, sorbitol, sucrose, fructose, galactose, lactose, maltose, starch saccharified solution containing these sugars, sweet potato molasses, sugar beet molasses, high test molasses, cane juice, cane juice extract or concentrate Liquid, raw sugar or purified sugar purified or crystallized from cane juice, saccharified liquid derived from cellulose, or other than sugar, organic acids such as acetic acid and fumaric acid, alcohols such as ethanol, glycerin, etc. Above all, Kanju Purified or crystallized raw sugar from the scan is used preferably as a carbon source.

  The raw sugar is produced by adding lime or lime milk to cane juice or cane koji and removing impurities by heating or removing the impurities by continuous precipitation. Then, it is concentrated and vacuum crystallized to separate molasses and crystals. The raw material sugars are then separated as crystals. Lime or lime milk may be added while heating to remove impurities. You may heat at the time of continuous precipitation and concentration. Crystals separated by vacuum crystallization may be dried. In addition, although there is no limitation in particular about the raw material saccharide | sugar used by this invention, the "super-sugar refinement" by Muso Co., Ltd. is mentioned as a preferable example.

  The concentration of the carbon source used in the fermentation raw material used in the present invention is not particularly limited, and is preferably as high as possible as long as it does not inhibit the production of D-lactic acid. Usually, the concentration of the carbon source is preferably 1.6 (w / v)% or more and 30 (w / v)% or less, more preferably 5 (w / v)% or more and 20 (w / v) in the medium. % Or less.

  In addition, the fermentation raw material may contain inorganic salts, and as the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts and the like can be appropriately added.

  In addition, when the D-lactic acid-producing bacterium used in the present invention requires a specific nutrient for growth, the nutrient is added as a preparation or a natural product containing it. Moreover, an antifoamer can also be used as needed.

  In the present invention, the D-lactic acid-producing bacterium can be fermented and cultured by standing or shaking culture, stirring culture or the like. Furthermore, as a fermentation form, it can be carried out by any conventionally known fermentation method, for example, batch fermentation, fed-batch fermentation, continuous fermentation, or fermentative production. It is preferable that continuous and batch or fed-batch culture operations performed while growing capable fresh cells are usually performed in a single fermentation reaction tank in terms of culture management. However, the number of fermentation reaction tanks is not limited as long as it is a continuous culture method for producing a product while growing cells. A plurality of fermentation reaction tanks may be used because the capacity of the fermentation reaction tank is small. In this case, even if a plurality of fermentation reaction tanks are connected in parallel or in series by piping and continuous or fed-batch culture is performed, high productivity of the fermentation product can be obtained.

  When the present invention is carried out in batch-type or fed-batch culture, the main culture may be started after batch culture or fed-batch culture is performed at an early stage of culture to increase the bacterial concentration. You may seed and you may perform main culture with the start of culture | cultivation.

  When the present invention is carried out in continuous culture, batch culture or fed-batch culture may be performed at the beginning of the culture to increase the bacterial concentration, and then continuous culture (pullout) may be started, or high-concentration cells may be seeded. And you may perform continuous culture with the start of culture | cultivation. In addition, it is possible to continuously and efficiently extract the fermentation broth from the fermentation reaction tank while maintaining the cells in the fermentation reaction tank. It is also possible to efficiently produce the target D-lactic acid by changing the fermentation raw material liquid composition after ensuring sufficient growth. The fermentation raw material supply and the start of extraction of the fermentation broth during continuous culture do not necessarily have to be the same. The supply of the fermentation raw material and the extraction of the fermentation broth may be continuous or intermittent. There may be. Nutrients necessary for cell growth as described above may be added to the fermentation raw material so that the cell growth is continuously performed. As a preferred continuous culture method when the present invention is carried out in continuous culture, a membrane-based continuous fermentation method disclosed in WO2007 / 097260 is preferably employed.

  The concentration of the D-lactic acid-producing bacterium in the fermentation broth of the present invention should be maintained at a high level as long as the environment of the fermentation broth is inappropriate for the growth of bacteria or cultured cells and does not increase the rate of death. Is preferable for obtaining efficient productivity, and as an example, good production efficiency can be obtained by maintaining the dry weight at 5 g / L or more. Moreover, the upper limit of the density | concentration of microorganisms will not be limited if the malfunction on the operation | movement of a continuous fermentation apparatus and the fall of production efficiency are not caused.

  Filtration / separation / purification of D-lactic acid produced according to the present invention can be carried out by a combination of conventionally known methods such as concentration, distillation and crystallization.

  Examples of methods for separating and purifying D-lactic acid include, for example, a method in which the pH of the filtered / separated fermentation broth is 1 or less and extraction with diethyl ether, ethyl acetate, or the like, a method in which elution is performed after adsorption washing on an ion exchange resin, Examples thereof include a method of reacting with an alcohol in the presence of a catalyst to distill as an ester, and a method of crystallizing as a calcium salt or a lithium salt. Preferably, the concentrated D-lactic acid solution obtained by evaporating water from the filtered / separated fermentation broth can be subjected to a distillation operation. Here, when distilling, it is preferable to distill while supplying water so that the water concentration of the undistilled stock solution is constant. After distilling off the D-lactic acid aqueous solution, the water can be concentrated by heating to evaporate to obtain purified D-lactic acid having a target concentration. When a D-lactic acid aqueous solution containing a low-boiling component (ethanol, acetic acid, etc.) is obtained as a distillate, it is a preferred embodiment that the low-boiling component is removed during the D-lactic acid concentration process. After the distillation operation, impurities can be removed from the distillate by ion exchange resin, activated carbon, chromatographic separation, or the like, if necessary, to obtain higher purity D-lactic acid.

  D-lactic acid obtained by the method for producing D-lactic acid according to the present invention is used as a raw material for a polyester resin, and the polyester resin is used for injection molding, extrusion molding, blow molding, vacuum molding, melt spinning, film molding, and the like. It can be molded into a desired shape by any molding method, and can be used as a resin molded article such as a machine part, a fiber such as clothing or industrial material, and a film such as packaging or magnetic recording. By using the method for producing D-lactic acid according to the present invention, it is possible to efficiently produce D-lactic acid having a wide range of uses, and thus it is possible to provide lactic acid at a lower cost.

  Hereinafter, the fermentation production of D-lactic acid by batch fermentation will be specifically described based on Examples in order to more specifically describe the method for producing D-lactic acid of the present invention. The present invention is not limited to these examples.

  In the examples of the present invention, among the D-lactic acid-producing bacteria belonging to the genus Sporolactobacillus, the lactic acid bacterium, Sporolactobacillus laevolacticus, is used as a bacterium that produces D-lactic acid. ) ATCC23492 strain was selected. The lactic acid fermentation medium (GYC1, GYC2, GYC3, RSE, RSC1, RSC2, or GPC medium) described above was used as a fermentation raw material. The method for analyzing the D-lactic acid concentration by HPLC is as follows.

[Analysis method of D-lactic acid concentration by HPLC]
The culture solution was centrifuged, and the resulting supernatant was subjected to membrane filtration, and then the amount of lactic acid was measured by the HPLC method under the conditions shown below.

[Analysis conditions]
Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation)
-Mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min)
-Detection method: electrical conductivity-Temperature: 45 ° C.

The optical purity of D-lactic acid was measured by the HPLC method under the following conditions.
Column: TSK-gel Enantio L1 (manufactured by Tosoh Corporation)
-Mobile phase: 1 mM aqueous copper sulfate-Flow rate: 1.0 ml / min
・ Detection method: UV254nm
-Temperature: 30 degreeC.

Moreover, the optical purity of D-lactic acid is calculated by the following formula.
Optical purity (%) = 100 × (DL) / (L + D)
Here, L represents the concentration of L-lactic acid, and D represents the concentration of D-lactic acid.

  By-products produced simultaneously with the production of D-lactic acid include organic acids and alcohols. Formic acid, acetic acid, pyruvic acid, and succinic acid as organic acids were analyzed in the same manner by the HPLC used for the analysis of the D-lactic acid concentration described above, and the concentration was further measured by F-kit (Roche). went. Ethanol as an alcohol was measured by the following GC method.

[Analysis method by GC]
The culture solution was centrifuged, and the resulting supernatant was subjected to membrane filtration, and then measured under the following conditions.
・ Device: GC-2010 (manufactured by Shimadzu Corporation)
Column: TC-1; 15 m * 0.53 mm * 1.5 μm (manufactured by GL Sciences Inc.)
・ Injection method: Split (30: 1)
Carrier gas: Helium Makeup gas: Helium Vaporization chamber temperature: 200 ° C
-Column temperature: 100 ° C
・ Detector: FID
-Detector temperature: 200 ° C
-Linear velocity: 59.2 cm / sec.

(Reference Example 1) Cysteine content as an impurity contained in a nitrogen source other than cysteine Hereinafter, yeast extract (Bacto ^™ Yeast Extract (BD)) used as a nitrogen source other than cysteine in Examples of the present invention, Polypeptone S ( Japan Pharmaceutical Co., Ltd.), and the cysteine content in the raw sugar (Musau Corporation “Yuyakusei”), which mainly contains sucrose and contains 0.3% protein by weight, is determined by the following amino acid analyzer under the following conditions: And measured.

[Method of analyzing cysteine by amino acid analyzer of yeast extract and polypeptone S]
For pretreatment of the yeast extract and polypeptone S, 0.2201 g and 0.1229 g of each were collected and dissolved in purified water to make yeast extract 12.12 ml and polypeptone S 12.29 ml. Thereafter, 450 μl of the sample solution was collected in another tube, and 50 μl of 20% sulfosalicylic acid was added to the solution and stirred. Subsequently, 50 μl was collected by filtration through a 0.22 μm filter. Samples with 10 times the concentration of each sample were also prepared, which were used as analysis samples, and the cysteine content was measured under the following conditions using an L-8500 type amino acid analyzer.

[Analysis conditions]
・ Amino acid analyzer: Model L-8500 (manufactured by Hitachi, Ltd.)
Column: Strongly acidic ion exchange resin # 2620M SC (Hitachi, Ltd.) 4.6 mm D. × 8cm
Mobile phase: Buffer PS-1, PS-3, PS-4 and PS-RG (for L-8500 Hitachi High-Speed Amino Acid Analyzer, manufactured by Wako Pure Chemical Industries, Ltd.)
-Mobile phase flow rate: 0.19 mL / min
Reaction solution: ninhydrin test solution-L8500 set (ninhydrin solution, buffer solution) (for L-8500 type Hitachi high-speed amino acid analyzer, manufactured by Wako Pure Chemical Industries, Ltd.)
-Reaction solution flow rate: 0.2 mL / min
・ Chemical reactor temperature: 130 ℃
-Reaction time: 50 seconds-Detection wavelength: 570 nm, 440 nm
Sample injection volume: 50 μl.

[Method of analyzing cysteine with amino acid analyzer of raw sugar]
For pretreatment of raw sugar, 100 g of raw sugar was dissolved in 1 L of water, 180 μl was collected, and 20 μl of 20% sulfosalicylic acid was added and stirred. Subsequently, 25 μl was collected by filtration through a 0.22 μm filter, and this was used as an analysis sample. Using an L-8800A type amino acid analyzer, the cysteine content was measured under the following conditions.

[Analysis conditions]
Amino acid analyzer: L-8800A type (manufactured by Hitachi, Ltd.)
Column: Hitachi custom ion exchange resin # 2622 (manufactured by Hitachi, Ltd.) 6 mm × 25 mm × 2 Ammonia filter column: Hitachi custom ion exchange resin # 2650 (manufactured by Hitachi, Ltd.) 4.6 mm × 60 mm
Mobile phase: L-8500 buffer solution PF-1, PF-2, PF-3, PF-4, L-8500 column regeneration solution PF (for L-8500 type Hitachi high-speed amino acid analyzer, Wako Pure Chemical Industries, Ltd. Made)
-Reaction solution: ninhydrin test solution Wakonin hydrin test solution-L8500 set (ninhydrin solution, buffer) (for L-8500 type Hitachi high-speed amino acid analyzer, manufactured by Wako Pure Chemical Industries, Ltd.)
・ Chemical reactor temperature: 135 ℃
・ Detection wavelength: 570 nm, 440 nm
Sample injection volume: 25 μl.

  According to the above analysis, the cysteine contained in the yeast extract was 1.11 mg per gram of yeast extract, the cysteine contained in Polypeptone S was 0.48 mg per gram of Polypeptone S, and the cysteine contained in 100 g of raw sugar was 0.48 mg.

(Example 1) Production of D-lactic acid by batch fermentation (part 1)
Sporactobacillus laevolacticus ATCC23492 strain was statically cultured at a temperature of 30 ° C. for 24 hours in a GYC1 medium purged with 5 ml of nitrogen gas in a test tube (preculture). The preculture was inoculated with the same medium and cultured with shaking at 37 ° C. and 120 rpm until the end of the culture.

  As a result of conducting a fermentation test for 150 hours, the optical purity was improved, and it was 99% or more, and the production yield was 90%. In addition, pyruvic acid, acetic acid, formic acid and ethanol were produced as by-products, but the concentration of by-products was very low at 0.4 g / L, which was more than 1/3 of that of the comparative example. We were able to reduce production (Table 1).

  The above GYC1 medium is prepared by dissolving 100 g of glucose and 0.1 g of cysteine in 1 L of water, 0.4 g of yeast extract as a nitrogen source other than cysteine, and 0.2 g of potassium dihydrogen phosphate as a phosphorus source. And GYC1.

(Example 2) Production of D-lactic acid by batch fermentation (part 2)
Sporalactobacillus laevolacticus ATCC23492 strain was statically cultured at a temperature of 30 ° C. for 24 hours in a GYC2 medium purged with 5 ml of nitrogen gas in a test tube (preculture). The preculture was inoculated with the same medium and cultured with shaking at 37 ° C. and 120 rpm until the end of the culture.

  As a result of a 160-hour fermentation test, the optical purity was improved and 99% or more and the production yield was 93%. In addition, pyruvic acid, acetic acid, formic acid and ethanol were produced as by-products, but the concentration of by-products was very low at 0.1 g / L, which was 1/15 or more compared to the comparative example. We were able to reduce production (Table 1).

  The above GYC2 medium is prepared by dissolving 100 g of glucose, 0.2 g of cysteine, 0.1 g of yeast extract as a nitrogen source other than cysteine, and 0.2 g of potassium dihydrogen phosphate in 1 L of water. did.

(Example 3) Production of D-lactic acid by batch fermentation (part 3)
Sporactobacillus laevolacticus ATCC23492 strain was statically cultured at a temperature of 30 ° C. for 24 hours in GYC3 medium purged with 5 ml of nitrogen gas in a test tube (preculture). The preculture was inoculated with the same medium and cultured with shaking at 37 ° C. and 120 rpm until the end of the culture.

  As a result of performing the fermentation test for 150 hours, the optical purity was improved and 99% or more and the production yield was 95%. In addition, pyruvic acid, acetic acid, formic acid and ethanol were produced as by-products, but the concentration of by-products was very low at 0.1 g / L, which was 1/15 or more compared to the comparative example. We were able to reduce production (Table 1).

  The above GYC3 medium is prepared by dissolving 100 g of glucose, 0.4 g of cysteine, 0.1 g of yeast extract as a nitrogen source other than cysteine, and 0.2 g of potassium dihydrogen phosphate in 1 L of water. did.

(Example 4) Production of D-lactic acid by batch fermentation (part 4)
Sporactobacillus laevolacticus ATCC23492 strain was statically cultured at a temperature of 30 ° C. for 24 hours in an RSE medium purged with 5 ml of nitrogen gas in a test tube (preculture). The preculture was inoculated with the same medium and cultured with shaking at 37 ° C. and 120 rpm until the end of the culture.

  As a result of the 180-hour fermentation test, the optical purity was improved, and it was 99% or more, and the production yield was 90%. In addition, pyruvic acid, acetic acid, formic acid and ethanol were produced as by-products, but the concentration of by-products was very low at 0.5 g / L, which was more than 1/3 of that of the comparative example. We were able to reduce production (Table 1).

  The above-mentioned RSE medium was prepared by dissolving 100 g of raw sugar (Musou Co., Ltd., “Yakusakusei”), 0.15 g of yeast extract and 0.2 g of potassium dihydrogen phosphate in 1 L of water, and RSE was used. Since 100 g of raw sugar contains 0.48 mg of cysteine, the RSE medium contains 0.65 mg / L of cysteine. Moreover, 0.45 g / L of nitrogen sources other than cysteine is contained.

(Example 5) Production of D-lactic acid by batch fermentation (part 5)
Sporactobacillus laevolacticus ATCC23492 strain was statically cultured at a temperature of 30 ° C. for 24 hours in a GPC medium purged with 5 ml of nitrogen gas in a test tube (preculture). The preculture was inoculated with the same medium and cultured with shaking at 37 ° C. and 120 rpm until the end of the culture.

  As a result of a 160-hour fermentation test, the optical purity was improved and 99% or more and the production yield was 90%. In addition, pyruvic acid, acetic acid, formic acid and ethanol were produced as by-products, but the concentration of by-products was very low at 0.5 g / L, which was more than 1/3 of that of the comparative example. We were able to reduce production (Table 1).

  The GPC medium is prepared by dissolving 100 g of glucose and 0.2 g of cysteine in 1 L of water, 0.4 g of polypeptone S as a nitrogen source other than cysteine, and 0.2 g of potassium dihydrogen phosphate as a phosphorus source. GPC.

(Example 6) Production of D-lactic acid by Fed-Batch fermentation (Part 6)
Sporalactobacillus laevolacticus ATCC23492 strain was statically cultured at a temperature of 30 ° C. for 24 hours in an RSE medium purged with 5 ml of nitrogen gas in a test tube (pre-culture). The preculture was inoculated with the same medium and cultured with shaking at 37 ° C. and 120 rpm until the end of the culture. The residual total sugar concentration was measured, it was confirmed that the residual sugar concentration was 0.5 g / L, 200 ml of the culture solution was inoculated into 800 ml of RSC1 medium prepared in another fermentor, and 37 ° C., 120 rpm. And shaking culture until the end of the culture.

  As a result of performing the fermentation test for 150 hours, the optical purity was improved and 99% or more and the production yield was 95%. In addition, pyruvic acid, acetic acid, formic acid and ethanol were produced as by-products, but the concentration of by-products was as low as 0.3 g / L, which was 1/5 or more compared to the comparative example. We were able to reduce production (Table 1).

  The above RSC1 medium was prepared by dissolving 100 g of raw material sugar (“Musou Co., Ltd.,“ Yakuseisei ”), 0.4 g of cysteine, and 0.2 g of potassium dihydrogen phosphate in 1 L of water, and designated RSC1. Since the raw material sugar contains 0.3% by weight of protein as an impurity, the RSC1 medium contains 0.3 g / L of a nitrogen source other than cysteine.

(Example 7) Production of D-lactic acid by Fed-Batch fermentation (part 7)
Sporactobacillus laevolacticus ATCC 23492 strain was statically cultured at a temperature of 30 ° C. for 24 hours in a RSE medium purged with 5 ml of nitrogen gas in a test tube (pre-culture). The preculture was inoculated with the same medium and cultured with shaking at 37 ° C. and 120 rpm until the end of the culture. The residual total sugar concentration was measured, it was confirmed that the residual sugar concentration was 0.5 g / L, 200 ml of the culture solution was inoculated into 800 ml of RSC2 medium prepared in another fermentor, and 37 ° C., 120 rpm. And shaking culture until the end of the culture.

  As a result of performing the fermentation test for 150 hours, the optical purity was improved and 99% or more and the production yield was 95%. In addition, pyruvic acid, acetic acid, formic acid and ethanol were produced as by-products, but the concentration of by-products was very low at 0.4 g / L, which was more than 1/3 of that of the comparative example. We were able to reduce production (Table 1).

  The above RSC2 medium was prepared by dissolving 100 g of raw material sugar (Musau Co., Ltd., “Sugar Seika”), 0.4 g of cysteine, and 0.2 g of potassium dihydrogen phosphate in 1 L of water, and designated as RSC2. Since the raw material sugar contains 0.3% by weight of protein as an impurity, the RSC2 medium contains 0.3 g / L of a nitrogen source other than cysteine.

(Comparative Example 1) Production of D-lactic acid by batch fermentation (Part 8)
A batch fermentation test was conducted in the same manner as in Example 1 except that GY1 medium prepared by dissolving 100 g of glucose and 0.5 g of yeast extract in 1 L of water was used. Since 1.11 mg of cysteine is contained per 1 g of added yeast extract, the GY1 medium contains 0.56 mg / L of cysteine.

  As a result of performing the fermentation test for 220 hours, the optical purity was 97% or less, and pyruvic acid, acetic acid, formic acid and ethanol were produced as by-products, and the by-product concentration in the fermentation broth was 1.5 g / L or more. Many were produced (Table 1).

Comparative Example 2 Production of D-lactic acid by batch fermentation (part 9)
A batch fermentation test was conducted in the same manner as in Example 1 except that a GY2 medium prepared by dissolving 100 g of glucose and 1 g of yeast extract in 1 L of water was used. Since 1.11 mg of cysteine is contained per 1 g of added yeast extract, the GY2 medium contains 1.11 mg / L of cysteine.

  As a result of performing the fermentation test for 180 hours, the optical purity was 97.5%, but pyruvic acid, acetic acid, formic acid and ethanol were produced as by-products, and the by-product concentration in the fermentation broth was 2 g / L or more. (Table 1).

Comparative Example 3 Production of D-lactic acid by batch fermentation (part 10)
A batch fermentation test was conducted in the same manner as in Example 1 except that a GY3 medium prepared by dissolving 100 g of glucose and 5 g of yeast extract in 1 L of water was used. Since 1.11 mg of cysteine is contained per 1 g of yeast extract added, the GY3 medium contains 5.55 mg / L of cysteine.

  As a result of a 160-hour fermentation test, the optical purity was 97.9%, but by-products such as pyruvic acid, acetic acid, formic acid and ethanol were produced, and the by-product concentration in the fermentation broth was 5 g / L or more. (Table 1).

  Hereinafter, in order to describe the method for producing D-lactic acid of the present invention more specifically, continuous fermentation production of D-lactic acid by using a continuous fermentation apparatus disclosed in WO2007 / 097260 is specifically described based on Examples. However, the present invention is not limited to these examples.

(Example 8) Production of D-lactic acid by continuous fermentation (part 1)
In order to investigate whether D-lactic acid is obtained by continuous fermentation by operating the continuous fermentation apparatus shown in FIG. 1, a continuous fermentation test using the apparatus was performed. GYC1 was used as the medium, and used after high-pressure steam sterilization at 121 ° C. for 15 minutes. As the separation membrane element, the form shown in FIG. 3 is adopted. As the separation membrane, a porous membrane mainly composed of polyvinylidene fluoride (PVDF) produced according to Reference Example 2 of WO2007 / 097260 is used as a separation membrane element member. Used a molded product of stainless steel and polysulfone resin. When the characteristics of the porous membrane were examined, the average pore diameter was 0.1 μm, and the pure water permeability coefficient was 50 × 10 ⁻⁹ m ³ / m ² / s / pa. The operating conditions in Example 8 are as follows unless otherwise specified.

[Operating conditions]
・ Fermentation reactor capacity: 1.5 (L)
-Separation membrane used: PVDF filtration membrane-Membrane separation element Effective filtration area: 120 square cm
・ Temperature adjustment: 35 (℃)
-Aeration volume of fermentation reaction tank: 100 (mL / min)
・ Fermentation reactor stirring speed: 800 (rpm)
・ Sterilization: The culture tank containing the separation membrane element and the used medium are all autoclaved at 121 ° C. for 20 minutes. ・ PH adjustment: pH adjusted to 6.0 with 8N calcium hydroxide ・ Membrane permeate flow control: The flow rate was controlled by the membrane separation tank water head difference (water head difference was controlled within 2 m).

  D-lactic acid contained in the fermentation broth was confirmed by measuring the amount of lactic acid by the HPLC method.

  First, Sporalactobacillus laevolacticus ATCC23492 strain was statically cultured at a temperature of 30 ° C. for 24 hours in the GY2 medium of the above Comparative Example 2 purged with 5 ml of nitrogen gas in a test tube (previously) culture). The obtained culture solution was inoculated into 150 ml of a fresh lactic acid fermentation medium GYC purged with nitrogen gas, and statically cultured at a temperature of 30 ° C. for 48 hours (pre-culture). The culture solution was inoculated in 1 L of lactic acid fermentation medium GYC1 purged with nitrogen gas from the continuous fermentation apparatus shown in FIG. 1, and the fermentation reaction tank 1 was stirred at 800 rpm by the attached stirrer 5. The aeration was adjusted and the temperature was adjusted to a temperature of 35 ° C., followed by culturing for 24 hours (preculture). Immediately after completion of the pre-culture, continuously supply GYC 1 medium, continuously culture while controlling the amount of permeated water so that the fermentation culture volume of the continuous fermentation apparatus becomes 1.5 L, and by continuous fermentation at 37 ° C. D-lactic acid was produced. At this time, nitrogen gas was supplied from the gas supply device into the fermentation reaction tank 1, and the discharged gas was recovered and supplied to the fermentation reaction tank 1 again. That is, recycling supply of gas containing nitrogen gas was performed. Control of the amount of permeated water through the continuous fermentation test is performed by appropriately changing the water head difference so that the head of the fermentation reaction tank is within 2 m at maximum, that is, the transmembrane pressure difference is within 20 kPa, by the water head difference control device 3. went. The produced D-lactic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately.

  As a result of conducting a 500-hour fermentation test, it is confirmed that by using this continuous fermentation apparatus, it is possible to produce inexpensively stable 99% or higher optical purity stable D-lactic acid by continuous fermentation. I was able to. As the by-products, pyruvic acid, acetic acid, formic acid and ethanol were produced, but the concentration of by-products was as low as 0.3 g / L, which could be reduced to 1/5 or more compared to the comparative example ( Table 2).

(Example 9) Production of D-lactic acid by continuous fermentation (part 2)
In order to investigate whether D-lactic acid is obtained by operating the continuous fermentation apparatus shown in FIG. 2, a continuous fermentation test using the same apparatus was performed. GYC3 was used as the medium, and used after high-pressure steam sterilization at 121 ° C. for 15 minutes. The separation membrane and separation membrane element used in Example 8 were used. The operating conditions in Example 9 are as follows unless otherwise specified.

[Operating conditions]
・ Fermentation reactor capacity: 1.5 (L)
-Separation membrane used: PVDF filtration membrane-Membrane separation element Effective filtration area: 120 square cm
・ Temperature adjustment: 37 (℃)
-Aeration volume of fermentation reaction tank: 100 (L / min)
・ Fermentation reactor stirring speed: 800 (rpm)
・ Sterilization: The culture tank containing the separation membrane element and the medium to be used are all autoclaved at 121 ° C. for 20 minutes. ・ PH adjustment: pH adjusted to 6.0 with 8N calcium hydroxide ・ Membrane permeate flow control: The flow rate was controlled by the membrane separation tank water head difference (water head difference was controlled within 2 m).

  The products D-lactic acid and glucose were measured by the method shown in Example 8.

  First, Sporalactobacillus laevolacticus ATCC23492 strain was statically cultured at a temperature of 30 ° C. for 24 hours in a GY2 medium purged with 5 ml of nitrogen gas in a test tube (pre-culture). The obtained culture solution was inoculated into 50 ml of GYC medium as a fresh lactic acid fermentation medium purged with nitrogen gas, and statically cultured at a temperature of 30 ° C. for 48 hours (pre-culture). The culture broth was inoculated in 1 L of lactic acid fermentation medium GYC3 purged with nitrogen gas from the membrane separation type continuous fermentation apparatus shown in FIG. 2, and the fermentation reaction tank 1 was stirred at 800 rpm by the attached stirrer 5 for fermentation reaction. The aeration rate of the tank 1 was adjusted and the temperature was adjusted to a temperature of 37 ° C., followed by culturing for 24 hours (pre-culture). Immediately after completion of the pre-culture, continuously supply GYC3 as a continuous / batch fermentation medium and continuously culture while controlling the amount of permeated water so that the fermentation culture volume of the continuous fermentation apparatus is 1.5 L. D-lactic acid was produced. At this time, nitrogen gas was supplied into the fermentation reaction tank 1 from the gas supply device, and the discharged gas was recovered and supplied to the fermentation reaction tank 1 again. That is, recycling supply of gas containing nitrogen gas was performed. Control of the amount of permeated water through the continuous fermentation test is performed by appropriately changing the water head difference so that the head of the fermentation reaction tank is within 2 m at maximum, that is, the transmembrane pressure difference is within 20 kPa, by the water head difference control device 3. went. The produced D-lactic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately.

  As a result of conducting a 500-hour fermentation test, it is confirmed that by using this continuous fermentation apparatus, it is possible to produce inexpensively stable 99% or higher optical purity stable D-lactic acid by continuous fermentation. I was able to. As the by-products, pyruvic acid, acetic acid, formic acid and ethanol were produced, but the concentration of by-products was as low as 0.1 g / L, and could be reduced to 1/15 or more compared to the comparative example ( Table 2).

(Example 10) Production of D-lactic acid by continuous fermentation (part 3)
Instead of the porous membrane used in Example 8, a hollow fiber membrane mainly composed of polyvinylidene fluoride (PVDF) produced according to Reference Example 13 of WO2007 / 097260 was used as a separation membrane. The form shown in FIG. 4 was adopted as the separation membrane element. Moreover, the continuous fermentation test was done like Example 8 except having used GPC culture medium.

  As a result of conducting a 500-hour fermentation test, it is confirmed that by using this continuous fermentation apparatus, it is possible to produce inexpensively stable 99% or higher optical purity stable D-lactic acid by continuous fermentation. I was able to. As by-products, pyruvic acid, acetic acid, formic acid and ethanol were produced, but the concentration of by-products was as low as 0.45 g / L, which could be reduced to 1/3 or more compared to the comparative example ( Table 2).

(Example 11) Production of D-lactic acid by continuous fermentation (part 4)
A continuous fermentation test was performed in the same manner as in Example 9 except that the separation membrane element used in Example 10 was used and RSC1 medium was used.

  As a result of conducting a 500-hour fermentation test, it is confirmed that by using this continuous fermentation apparatus, it is possible to produce inexpensively stable 99% or higher optical purity stable D-lactic acid by continuous fermentation. I was able to. As the by-products, pyruvic acid, acetic acid, formic acid and ethanol were produced, but the concentration of by-products was as low as 0.3 g / L, which could be reduced to 1/5 or more compared to the comparative example ( Table 2).

(Example 12) Production of D-lactic acid by continuous fermentation (part 5)
A continuous fermentation test was conducted in the same manner as in Example 11 except that the RSE medium was used. As a result of conducting a 500-hour fermentation test, it is confirmed that by using this continuous fermentation apparatus, it is possible to produce inexpensively stable 99% or higher optical purity stable D-lactic acid by continuous fermentation. I was able to. As the by-products, pyruvic acid, acetic acid, formic acid and ethanol were produced, but the concentration of by-products was as low as 0.45 g / L, which could be reduced to 1/3 or more compared to the comparative example.

(Comparative Example 4) Production of D-lactic acid by continuous fermentation (part 6)
A continuous fermentation test was conducted in the same manner as in Example 8 except that the GY1 medium of Comparative Example 1 was used. As a result of performing the fermentation test for 200 hours, the optical purity was 88% or less, and the sugar in the fermentation broth was not consumed, and many sugars remained. As by-products, pyruvic acid, acetic acid, formic acid and ethanol were produced, and the concentration of by-products was as high as 1.5 g / L (Table 2).

(Comparative Example 5) Production of D-lactic acid by continuous fermentation (part 7)
A continuous fermentation test was performed in the same manner as in Example 8 except that the GY2 medium of Comparative Example 2 was used. As a result of performing the fermentation test for 200 hours, the optical purity was 99%, and the sugar in the fermentation broth was not consumed, and many sugars remained. As by-products, pyruvic acid, acetic acid, formic acid and ethanol were produced, and the concentration of by-products was as high as 1.7 g / L (Table 2).

(Comparative Example 6) Production of D-lactic acid by continuous fermentation (part 8)
A continuous fermentation test was conducted in the same manner as in Example 8 except that RSE1 medium (containing 1.59 mg / L of cysteine) prepared by dissolving 100 g of raw sugar and 1 g of yeast extract in 1 L of water was used. . As a result of performing the fermentation test for 200 hours, the optical purity was 99.2%, and the sugar in the fermentation broth was not consumed, and many sugars remained. As by-products, pyruvic acid, acetic acid, formic acid, and ethanol were produced, and the concentration of by-products was as high as 1.7 g / L.

  From the examples and comparative examples, it became clear that batch fermentation can produce lactic acid while significantly reducing the production of by-products. It has also been clarified that F-Batch fermentation can reduce by-product production and produce D-lactic acid with high lactic acid production yield and high optical purity. Furthermore, it has been clarified that the continuous fermentation using the membrane separation type continuous fermentation apparatus of FIGS. 1 and 2 significantly improves the production rate and optical purity of lactic acid while greatly reducing the production of by-products. . That is, using a membrane separation type continuous fermentation apparatus incorporating a porous membrane disclosed by the present invention, by controlling the transmembrane pressure difference, the fermentation broth is separated into a filtrate and an unfiltered liquid by the separation membrane, A desired fermentation product is recovered from the filtrate, and a continuous fermentation method in which the unfiltered solution is returned to the fermentation broth is enabled, and it is possible to produce D-lactic acid by continuous fermentation while maintaining a high amount of microorganisms. It became clear. Furthermore, by using a lactic acid fermentation medium containing a low concentration of nitrogen, the production of D-lactic acid is lower than conventional D-lactic acid production, reducing the production of by-products and improving the optical purity of D-lactic acid. It was revealed that long-term production of D-lactic acid is possible while maintaining the production of high optical purity D-lactic acid.

  According to the method for producing D-lactic acid according to the present invention, high productivity of D-lactic acid and optical purity of D-lactic acid are improved by using a fermentation medium containing cysteine and low concentration of nitrogen and simple operation conditions. At the same time, production of high optical purity D-lactic acid became possible, and the concentration of by-products could be reduced. Widely in the fermentation industry, it becomes possible to stably produce D-lactic acid, which is a fermentation product, at a low cost by producing high optical purity, reducing the concentration of by-products, and using natural biomass. D-lactic acid obtained by the method for producing D-lactic acid according to the present invention is useful, for example, as a raw material for polylactic acid resin.

FIG. 1 is a schematic side view for explaining one embodiment of a membrane separation type continuous fermentation apparatus used in the present invention. FIG. 2 is a schematic side view for explaining one embodiment of another membrane separation type continuous fermentation apparatus used in the present invention. FIG. 3 is a schematic perspective view for explaining one embodiment of a separation membrane element using a porous membrane used in the present invention. FIG. 4 is a schematic perspective view for explaining one embodiment of a separation membrane element using a hollow fiber membrane used in the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fermentation reaction tank 2 Separation membrane element 3 Water head difference control device 4 Gas supply device 5 Stirrer 6 Level sensor 7 Medium supply pump 8 pH adjustment solution supply pump 9 pH sensor / control device 10 Temperature controller 11 Fermentation liquid circulation pump 12 Membrane separation Tank 13 Support plate 14 Channel material 15 Separation membrane 16 Concave portion 17 Water collecting pipe 18 Separation membrane bundle 19 Upper resin sealing layer 20 Lower resin sealing layer 21 Support frame 22 Water collection pipe

Claims (2)

  A method for producing D-lactic acid by fermentation using a bacterium belonging to the genus Sporolactobacillus, comprising 0.6 mg / L or more of cysteine and 0.1 g / L or more and 0.5 g / L or less. A method for producing D-lactic acid using a fermentation raw material containing a nitrogen source other than cysteine.   The method for producing D-lactic acid according to claim 1, wherein the bacterium is Sporolactobacillus laevolacticus.

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