JP2009142210A - Method for producing lactic acid by continuous fermentation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing lactic acid, which totally reduces a cost throughout a long-term fermentation, prolongs a further fermentation time and effectively improves productivity of lactic acid in a continuous fermentation method for producing lactic acid using a microorganism. <P>SOLUTION: The method for producing lactic acid, which carries out a stable continuous fermentation at a low cost in high productivity, includes separating a fermentation culture liquid into a filtrate and an unfiltered liquid by a separation membrane, recovering lactic acid being a desired fermentation product from the filtrate, and retaining or refluxing the unfiltered liquid in the fermentation culture liquid, reducing a total amino acid concentration in a fermentation raw material and using a natural biomass so as to totally reduce a cost, to prolong a further fermentation time and to effectively improve productivity of lactic acid throughout a stable long-term fermentation. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an improvement in a method for producing lactic acid for producing lactic acid by fermentation for culturing microorganisms. More specifically, the present invention efficiently filters and recovers a liquid containing lactic acid from a microorganism fermentation broth through a porous membrane that is less likely to be clogged while culturing, and removes the unfiltered liquid from the fermentation broth. In continuous fermentation in which the concentration of microorganisms involved in fermentation can be improved and high productivity can be obtained by returning to, the total amino acid concentration in the fermentation raw material is 2 g / L or less, thereby further increasing the productivity of lactic acid. It is related with the manufacturing method of lactic acid which can improve cost and can reduce cost.

  Fermentation, which is a material production method involving the cultivation of microorganisms and cultured cells, is largely divided into (1) batch fermentation (Batch fermentation) and fed-batch fermentation (Fed-Batch fermentation), and (2) continuous fermentation. Can be classified.

  The batch fermentation method and fed-batch fermentation method of the above (1) are simple in terms of equipment, and have an advantage that culture is completed in a short time and damage caused by contamination with bacteria is small. However, the product concentration in the fermentation broth increases with the passage of time, and the productivity and yield decrease due to the influence of osmotic pressure or product inhibition. Therefore, it is difficult to stably maintain a high yield and high productivity over a long period of time.

  On the other hand, the continuous fermentation method of the above (2) is characterized in that high yield and high productivity can be maintained over a long period of time by avoiding accumulation of the target substance at a high concentration in the fermentation reaction tank. . Specifically, such a continuous culture method has been proposed for the production of L-glutamic acid and L-lysine fermentation (see Non-Patent Document 1). However, in these examples, since the raw material is continuously supplied to the fermentation broth and the fermentation broth containing microorganisms and cultured cells is extracted, the microorganisms and cultured cells in the fermentation broth are diluted. Therefore, the improvement in production efficiency was limited.

  Therefore, in a continuous fermentation method, microorganisms and cultured cells are filtered through a separation membrane, and the product is recovered from the filtrate, and at the same time, the filtered microorganisms and cultured cells are retained or refluxed in the fermentation broth. A method for maintaining high concentrations of microorganisms and cultured cells was proposed. Therefore, it can be applied industrially, and a continuous fermentation method and a continuous fermentation apparatus using a porous polymer membrane as a separation membrane so that high material productivity can be stably obtained over a long period of time with a simple operation. Has been developed (see Patent Document 1).

  On the other hand, in substance production using microorganisms, an excessively added YPAD medium (see Non-Patent Document 2) generally known as a medium for culturing microorganisms, yeast extract and peptone contained in YPAD, etc. It is well known that the amount of substance production and the production yield are improved by culturing in the above medium, that is, by adding a large amount of amino acids, vitamins and the like.

However, even if the above-mentioned continuous fermentation method using such a medium is an excellent fermentation method, the production of lactic acid has not been prolonged, and the cost of the medium has become a problem. Therefore, there has been a demand for a method for improving the productivity of lactic acid effectively by reducing the overall cost and further long-term fermentation.
JP 2007-252367 A Toshikiko hirao et. al. (Hirano Toshihiko et al.), Appl. Microbiol. Biotechnol. (Applied Microvials and Microbiology), 32, 269-273 (1989) “Methods in Yeast Genetics”, Cold Spring Harbor Laboratory Volume 194, 1991, pp. 12-18.

  In the continuous fermentation method for lactic acid production using microorganisms, the present invention has an object to effectively improve lactic acid productivity by reducing the overall cost and extending the fermentation time over a long period of fermentation. To do.

  As a result of intensive studies by the present inventors in order to solve the above problems, in lactic acid fermentation using yeast having lactic acid synthesis ability, the fermentation broth is filtered through a porous polymer membrane, and the product is recovered from the filtrate. In addition, when producing lactic acid by continuous fermentation in which the unfiltered liquid is retained or refluxed in the fermentation broth and the fermentation raw material is added to the fermentation broth, the concentration of all amino acids in the fermentation raw material is lowered and natural. By using biomass, the present inventors have found that it is possible to effectively improve the productivity of lactic acid by effectively reducing the cost and further extending the fermentation time over stable long-term fermentation, thereby completing the present invention.

  In the method for producing lactic acid by continuous fermentation according to the present invention, a yeast fermentation broth having the ability to produce lactic acid is filtered through a separation membrane, the product is recovered from the filtrate, and an unfiltered liquid is used as the fermentation broth. A method for producing lactic acid by continuous fermentation by holding or refluxing and adding a fermentation raw material to the fermentation broth, wherein a porous membrane having an average pore size of 0.01 μm or more and less than 1 μm is used as the separation membrane. A method for producing lactic acid by continuous fermentation, wherein the transmembrane pressure difference is 0.1 to less than 20 kPa and filtration is performed, and the total amino acid concentration in the fermentation raw material is 2 g / L or less. .

According to a preferred aspect of the method for producing lactic acid by continuous fermentation of the present invention, the porous water has a pure water permeability coefficient of 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 6 × 10 ⁻⁷ m ^3. / M ² / s / pa or less.

  According to a preferred embodiment of the method for producing lactic acid by continuous fermentation of the present invention, the average pore diameter of the porous membrane is 0.01 μm or more and less than 0.2 μm, and the standard deviation of the average pore diameter is 0.8. 1 μm or less.

  According to a preferred embodiment of the method for producing lactic acid by continuous fermentation of the present invention, the membrane surface roughness of the porous membrane is 0.1 μm or less, and the porous membrane is a porous membrane including a porous resin layer. .

  According to a preferred aspect of the method for producing lactic acid by continuous fermentation of the present invention, a polyvinylidene fluoride resin can be used as the membrane material of the porous membrane.

  According to a preferred aspect of the method for producing lactic acid by continuous fermentation of the present invention, the fermentation raw material is a medium containing at least one kind obtained from the group consisting of sucrose, fructose, glucose, galactose, lactose, maltose and the like. .

  Further, according to a preferred aspect of the method for producing lactic acid by continuous fermentation of the present invention, the fermentation raw material is starch, starch hydrolyzate, sweet potato molasses, sugar beet molasses, cane juice, sugar beet molasses or cane juice extract. Or concentrate, sugar beet molasses or cane juice filtrate, syrup (high test molasses), purified or crystallized raw sugar from sugar beet molasses or cane juice and purified or crystallized refined from sugar beet molasses or cane juice It is a medium containing at least one kind obtained from the group consisting of sugar and the like.

  According to a preferred embodiment of the method for producing lactic acid by continuous fermentation of the present invention, the lactic acid is L-lactic acid, and the yeast is a yeast belonging to the genus Saccharomyces or a yeast into which a lactic acid synthase gene has been introduced. .

  According to a preferred embodiment of the method for producing lactic acid by continuous fermentation of the present invention, the lactic acid synthase gene is a gene encoding L-lactate dehydrogenase derived from Xenopus laevis, and the L -The gene encoding lactate dehydrogenase is a gene having the nucleic acid sequence shown in SEQ ID NO: 1.

  According to the present invention, by reducing the total amino acid concentration using an amino acid-reduced medium or the like, the fermentation time and lactic acid production can be improved significantly more stably than the conventional continuous lactic acid fermentation proposed in Patent Document 1. Production efficiency can be increased. Moreover, cheaper lactic acid fermentation is possible by using natural biomass as the sugar source.

  As described above, according to the present invention, continuous fermentation that stably maintains high productivity over a long period of time can be performed, and in the fermentation industry, lactic acid that is a fermentation product can be stably produced at low cost. It becomes possible.

  In the method for producing lactic acid by continuous fermentation of the present invention, the fermentation culture solution of microorganisms is filtered through a separation membrane, the product is recovered from the filtrate, and the unfiltered solution is retained or refluxed in the fermentation culture solution, and the fermentation is performed. In continuous fermentation in which the raw material is added to the fermentation broth, a porous membrane having pores with an average pore diameter of 0.01 μm or more and less than 1 μm is used as a separation membrane, and the transmembrane pressure difference is 0.1 to 20 kPa. The range is filtered and the fermentation raw material having a total amino acid concentration of 2 g / L or less in the fermentation raw material is used.

  In the method for producing lactic acid by continuous fermentation of the present invention, the total amino acid concentration in the fermentation raw material is more preferably 1 mg / L or more and 1.5 g / L or less, and further preferably 2 mg / L or more and 1 g / L or less. The lower the total amino acid concentration, the better.

  The porous membrane used as a separation membrane in the present invention is less likely to be clogged by microorganisms and cultured cells used for fermentation, and has a performance in which filtration performance continues stably for a long period of time. Therefore, it is important that the porous membrane used in the present invention has an average pore diameter of 0.01 μm or more and less than 1 μm.

  The configuration of the porous membrane used as the separation membrane in the present invention will be specifically described. The porous membrane used in the present invention has separation performance and water permeability according to the quality of water to be treated and the application. The porous membrane is preferably a porous membrane including a porous resin layer from the viewpoint of separation performance such as blocking performance, water permeability performance, and dirt resistance. Such a porous membrane has a porous resin layer that acts as a separation functional layer on the surface of the porous substrate. The porous substrate supports the porous resin layer and gives strength to the separation membrane.

  The material of the porous substrate is made of an organic material and / or an inorganic material, and among them, an organic fiber is desirably used. Preferred porous substrates are woven fabrics and nonwoven fabrics made of organic fibers such as cellulose fibers, cellulose triacetate fibers, polyester fibers, polypropylene fibers and polyethylene fibers. Among these, non-woven fabrics that are relatively easy to control density, easy to manufacture, and inexpensive are preferably used.

  Further, as described above, the porous resin layer functions as a separation functional layer, and an organic polymer membrane can be suitably used. Examples of the material of the organic polymer film include polyethylene resin, polypropylene resin, polyvinyl chloride resin, polyvinylidene fluoride resin, polysulfone resin, polyethersulfone resin, polyacrylonitrile resin, cellulose resin, and the like. Examples thereof include cellulose triacetate resins. The organic polymer film may be made of a mixture of resins mainly composed of these resins. Here, the main component means that the component is contained in an amount of 50% by weight or more, preferably 60% by weight or more. Among them, as a membrane material constituting the porous resin layer, a polyvinyl chloride resin, a polyvinylidene fluoride resin, a polysulfone resin, which is easy to form by a solution and has excellent physical durability and chemical resistance. Polyether sulfone resins and polyacrylonitrile resins are preferably used. For the film material, a polyvinylidene fluoride resin or a resin containing it as the main component is most preferably used.

  Here, as the polyvinylidene fluoride resin, a homopolymer of vinylidene fluoride is preferable, but a copolymer of a vinyl monomer copolymerizable with vinylidene fluoride is also preferably used. Examples of vinyl monomers copolymerizable with vinylidene fluoride include tetrafluoroethylene, hexafluoropropylene, and ethylene trichloride fluoride.

  The porous membrane used in the present invention may be a flat membrane or a hollow fiber membrane. When the porous membrane is a flat membrane, the average thickness is selected according to the use, but is preferably 20 μm or more and 5000 μm or less, more preferably 50 μm or more and 2000 μm or less.

  As described above, the separation membrane used in the present invention is desirably a porous membrane formed of a porous substrate and a porous resin layer. At that time, either the porous resin layer permeates the porous base material or the porous resin layer does not permeate the porous base material, and it is selected according to the application. The average thickness of the porous substrate is preferably selected in the range of 50 μm to 3000 μm. When the porous membrane is a hollow fiber membrane, the inner diameter of the hollow fiber is preferably selected in the range of 200 μm to 5000 μm, and the film thickness is preferably selected in the range of 20 μm to 2000 μm. Further, a woven fabric or a knitted fabric in which organic fibers or inorganic fibers are formed in a cylindrical shape may be included in the hollow fiber.

  First, an example of an outline of a method for producing a flat film among the porous films will be described.

  A film of a stock solution containing a resin and a solvent is formed on the surface of the porous base material, and the porous base material is impregnated with the stock solution. Thereafter, only the coating-side surface of the porous substrate having a coating is brought into contact with a coagulation bath containing a non-solvent to solidify the resin, and a porous resin layer is formed on the surface of the porous substrate.

  The stock solution can be prepared by dissolving the resin in a solvent. The temperature of the stock solution is usually preferably selected within the range of 5 to 120 ° C. from the viewpoint of film forming properties. The solvent dissolves the resin and acts on the resin to encourage them to form a porous resin layer. As the solvent, N-methylpyrrolidinone (NMP), N, N-dimethylacetamide (DMAc), N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone, methyl ethyl ketone, tetrahydrofuran, Tetramethylurea, trimethyl phosphate, cyclohexanone, isophorone, γ-butyrolactone, methyl isoamyl ketone, dimethyl phthalate, propylene glycol methyl ether, propylene carbonate, diacetone alcohol, glycerol triacetate, acetone and methyl ethyl ketone can be used. Of these, N-methylpyrrolidinone (NMP), N, N-dimethylacetamide (DMAc), N, N-dimethylformamide (DMF) and dimethylsulfoxide (DMSO), which have high resin solubility, are preferably used. These solvents may be used alone or in combination of two or more. The stock solution can be prepared by dissolving the above-mentioned resin in the above-mentioned solvent at a concentration of preferably 5 wt% or more and 60 wt% or less.

  Further, for example, components other than the solvent such as polyethylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone and glycerin may be added to the solvent. Non-solvents can also be added to the solvent. The non-solvent is a liquid that does not dissolve the resin. The non-solvent acts to control the pore size by controlling the rate of solidification of the resin. As the non-solvent, water and alcohols such as methanol and ethanol can be used. Of these, water or methanol is preferably used as the non-solvent from the viewpoint of cost. Components other than the solvent and the non-solvent may be a mixture.

  A pore opening agent may be added to the stock solution. The pore-opening agent is extracted when immersed in the coagulation bath, and has a function of making the resin layer porous. By adding a pore opening agent, the average pore size can be controlled. The pore-opening agent is preferably one having high solubility in the coagulation bath. As the pore opening agent, for example, an inorganic salt such as calcium chloride or calcium carbonate can be used. As the pore opening agent, polyoxyalkylenes such as polyethylene glycol and polypropylene glycol, water-soluble polymer compounds such as polyvinyl alcohol, polyvinyl butyral, and polyacrylic acid, glycerin, and the like can be used.

  Next, an example of an outline of a method for producing a hollow fiber membrane among the porous membranes will be described.

  The hollow fiber membrane discharges a stock solution composed of a resin and a solvent from the outer tube of the double-tube base, and discharges a hollow portion forming fluid from the inner tube of the double-tube base in a cooling bath. It can be produced by cooling and solidifying.

  The stock solution can be prepared by dissolving the resin described in the above-described method for producing a flat film, preferably in a concentration of 20% by weight or more and 60% by weight or less, in the solvent described in the above-described method for forming a flat film. . Moreover, a gas or a liquid can be normally used for the hollow portion forming fluid. In addition, a new porous resin layer can be coated (laminated) on the outer surface of the obtained hollow fiber membrane. Lamination can be performed in order to change the properties of the hollow fiber membrane, such as hydrophilicity / hydrophobicity or pore diameter, to desired properties. A new porous resin layer to be laminated can be produced by bringing a stock solution obtained by dissolving a resin in a solvent into contact with a coagulation bath containing a non-solvent to coagulate the resin. As the material of the resin, for example, the same material as that of the organic polymer film is preferably used. As a lamination method, the hollow fiber membrane may be immersed in the stock solution, or the stock solution may be applied to the surface of the hollow fiber membrane. After the lamination, a part of the attached stock solution is scraped off or an air knife is used. It is also possible to adjust the stacking amount by blowing it away.

  The porous membrane used in the present invention can be made into a separation membrane element by combining with a support. A separation membrane element in which a support plate is used as a support and the porous membrane used in the present invention is disposed on at least one side of the support plate is a preferred embodiment of the separation membrane element having a porous membrane used in the present invention. One. In this form, when it is difficult to increase the membrane area, it is a preferable aspect to dispose a porous membrane on both surfaces of the support plate in order to increase the water permeability.

  When the average pore diameter of the separation membrane is within the range of 0.01 μm or more and less than 1 μm as described above, it is possible to achieve both a high exclusion rate without leaking bacterial cells or sludge and high water permeability, Further, clogging is less likely to occur and the water permeability can be maintained for a long time with higher accuracy and reproducibility. When bacteria are used as the microorganism, the average pore diameter of the porous membrane is preferably 0.4 μm or less, and the average pore diameter is preferably less than 0.2 μm. If the average pore diameter is too small, the water permeation rate may decrease. Therefore, in the present invention, the average pore diameter is 0.01 μm or more, preferably 0.02 μm or more, more preferably 0.04 μm or more. is there.

  Here, the average pore diameter can be obtained by measuring and averaging the diameters of all pores that can be observed within a range of 9.2 μm × 10.4 μm in a scanning electron microscope observation at a magnification of 10,000 times. it can.

  The standard deviation σ of the average pore diameter is preferably 0.1 μm or less. Further, a uniform permeate can be obtained when the standard deviation of the average pore diameter is smaller, that is, when the pore diameters are uniform. Since fermentation operation management becomes easy, the smaller the standard deviation of the average pore diameter, the better.

  The standard deviation σ of the average pore diameter is N (the number of pores that can be observed within the above-mentioned range of 9.2 μm × 10.4 μm), each measured diameter is Xk, and the average pore diameter is X (ave) It is calculated by the following (Formula 1).

In the porous membrane used in the present invention, the permeability of the fermentation broth is one of the important points, and the pure water permeability coefficient of the porous membrane before use can be used as the permeability index. In the present invention, the pure water permeation coefficient of the porous membrane is 2 × 10 ⁻⁹ m ³ / when the water permeability is measured at a head height of 1 m using purified water at a temperature of 25 ° C. by a reverse osmosis membrane. It is preferably m ² / s / pa or more. If the pure water permeability coefficient is 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 6 × 10 ⁻⁷ m ³ / m ² / s / pa or less, a practically sufficient amount of permeated water can be obtained. A more preferable pure water permeability coefficient is 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 1 × 10 ⁻⁷ m ³ / m ² / s / pa or less.

  The membrane surface roughness of the porous membrane used in the present invention is a factor that affects the clogging of the separation membrane. When the membrane surface roughness is preferably 0.1 μm or less, the separation coefficient and membrane resistance of the separation membrane can be suitably reduced, and continuous fermentation can be carried out with a lower transmembrane pressure difference. Therefore, since stable continuous fermentation becomes possible by suppressing clogging, the smaller the surface roughness, the better.

  In addition, by reducing the membrane surface roughness of the porous membrane, it can be expected that the shearing force generated on the membrane surface will be reduced during filtration of microorganisms and cultured cells. It is considered that stable filtration is possible for a long time by suppressing clogging of the conductive film.

Here, film | membrane surface roughness can be measured on the following apparatus and conditions using the following atomic force microscope apparatus (AFM).
・ Device: Atomic force microscope device (Nanoscope IIIa manufactured by Digital Instruments)
・ Conditions: Probe SiN cantilever (manufactured by Digital Instruments)
: Scanning mode Contact mode (in-air measurement)
Underwater tapping mode (underwater measurement)
: Scanning range 10μm, 25μm square (measurement in air)
5μm, 10μm square (underwater measurement)
: Scanning resolution 512 × 512
-Sample preparation The membrane sample was immersed in ethanol at room temperature for 15 minutes, then immersed in RO water for 24 hours, washed, and then air-dried. The RO water refers to water that has been filtered using a reverse osmosis membrane (RO membrane), which is a type of filtration membrane, to exclude impurities such as ions and salts. The pore size of the RO membrane is approximately 2 nm or less.

Membrane surface roughness _{d rough} from the Z-axis direction of the height of each point by the AFM, is calculated by the following equation (2).

  The fermentation raw material for microorganisms used in the present invention can promote the growth of microorganisms for fermentation and culture, and can satisfactorily produce lactic acid, which is a target fermentation product. As a fermentation raw material, for example, a normal liquid medium that appropriately contains a carbon source, a nitrogen source, and, if necessary, organic micronutrients such as amino acids, inorganic salts, and vitamins is preferably used.

  Examples of the carbon source include sugars such as glucose, sucrose, fructose, galactose and lactose, starch containing these sugars, starch hydrolysates, sugarcane molasses, sugar beet molasses, cane juice, sugar beet molasses or cane juice. Extract or concentrate, sugar beet molasses or cane juice filtrate, syrup (high test molasses), refined or crystallized raw sugar from sugar beet molasses or cane juice, refined or crystallized from sugar beet molasses or cane juice Purified sugars, organic acids such as acetic acid and fumaric acid, alcohols such as ethanol, glycerin and the like are used. Sugars are the first oxidation products of polyhydric alcohols, and are carbohydrates that have one aldehyde group or ketone group, sugars with aldehyde groups are classified as aldoses, and sugars with ketone groups are classified as ketoses. Point to.

  In addition, as raw material sugar, lime or lime milk or the like is added to cane juice or cane koji, and the impurities are removed by heating or are continuously precipitated to remove impurities. 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.

  Examples of the nitrogen source include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other auxiliary organic nitrogen sources such as oil cakes, soybean hydrolysates, casein decomposition products, Other amino acids, vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and hydrolysates thereof are used.

  Examples of the amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or These hydrates are used.

  Further, if the above amino acid is a free amino acid and promotes the growth of microorganisms to be fermented and cultured, and can successfully produce lactic acid, which is the target fermentation product, a combination of amino acids containing at least one or more types Appropriate additions can be used.

  Amino acids may be added to the medium as protein or peptide containing as an auxiliary nitrogen source. Examples of protein or peptide-containing materials include oil cakes, soybean hydrolysates, casein degradation products, other amino acid polymers, corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, and various biological proteins And peptide-containing materials. The concentration of the amino acid derived from the auxiliary nitrogen source is preferably in the concentration range of 1 mg / L to 2 g / L.

  Moreover, as said inorganic salt, a phosphate, magnesium salt, calcium salt, iron salt, manganese salt etc. can be added and used suitably, for example.

  When the microorganism used in the present invention requires a specific nutrient for growth, the nutrient can be added as a preparation or a natural product containing it. An antifoaming agent can be added and used as necessary.

  In the present invention, the fermentation culture liquid refers to a liquid obtained as a result of the growth of microorganisms on the fermentation raw material. The composition of the fermented raw material to be added can be appropriately changed from the fermented raw material composition at the start of the culture so that the target lactic acid productivity is increased.

  In the present invention, the sugar concentration in the fermentation broth is preferably maintained at 5 g / L or less. The reason is to minimize the loss of sugar due to withdrawal of the fermentation broth. Therefore, it is desirable that the saccharide concentration be as small as possible.

  In many cases, the fermentation culture of microorganisms is usually performed at a pH of 4 to 8 and a temperature of 20 to 40 ° C. The pH of the fermentation broth is adjusted to a predetermined value within the above range with an inorganic acid or an organic acid, an alkaline substance, urea, calcium carbonate, calcium hydroxide, ammonia gas, or the like.

  In the fermentation culture, if it is necessary to increase the oxygen supply rate, oxygen is added to the air to keep the oxygen concentration preferably at 21% (v / v%) or higher, the fermentation culture solution is pressurized, and the stirring rate is increased. Alternatively, a means such as increasing the air flow rate can be used. Conversely, if it is necessary to reduce the oxygen supply rate, it is also possible to supply a gas containing no oxygen such as carbon dioxide, nitrogen and argon mixed with air.

  In the present invention, batch culture or fed-batch culture may be performed in the initial stage of culture to increase the microorganism concentration, and then continuous culture may be started, or high-concentration cells are seeded, and continuous culture is performed at the start of culture. May be. It is possible to supply and filter the fermentation raw material from an appropriate time. The fermentation raw material supply and the start time of filtration do not necessarily need to be the same. Moreover, supply and filtration of fermentation raw material may be continuous, and may be intermittent. Nutrients necessary for cell growth as described above may be added to the fermentation raw material so that the cell growth is continuously performed.

  In the present invention, the fermentation broth is filtered using a porous membrane having pores having an average pore size of 0.01 μm or more and less than 1 μm.

  The continuous culture operation performed while growing fresh cells having fermentation production ability is usually preferably 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 fermentation culture method that produces products while growing cells. A plurality of fermentation reaction tanks may be used because the capacity of the fermentation reaction tank is small. In that case, high productivity of the fermentation product can be obtained even if continuous fermentation is performed by connecting a plurality of fermentation reaction tanks in parallel or in series by piping.

  In the present invention, continuous and efficient filtration of the fermentation broth from the fermentation reaction tank is possible while maintaining the microorganisms in the fermentation reaction tank. Therefore, it is also possible to efficiently produce the target lactic acid by continuously fermenting and culturing microorganisms and changing the composition of the fermentation raw material solution after ensuring sufficient growth.

  Lactic acid obtained by the method for producing lactic acid by continuous fermentation of the present invention is a substance produced by the above-mentioned microorganism in the fermentation broth.

  In the present invention, the transmembrane pressure difference during filtration of the fermentation broth of microorganisms with a separation membrane may be any condition as long as the microorganisms and medium components are not easily clogged, but the transmembrane pressure difference is 0.1 kPa or more. It is important to perform the filtration treatment in the range of 20 kPa or less. The transmembrane pressure difference is preferably in the range of 0.1 kPa to 10 kPa, and more preferably in the range of 0.1 kPa to 5 kPa. If the range of the transmembrane pressure difference is exceeded, clogging of microorganisms and medium components may occur rapidly, leading to a decrease in the amount of permeated water, which may cause problems in continuous fermentation operation.

  As a driving force for filtration, a transmembrane pressure difference can be generated in the porous membrane by a siphon utilizing the liquid level difference (water head difference) of the fermentation broth and the porous membrane treated water. Moreover, a suction pump may be installed on the porous membrane treated water side as a driving force for filtration, or a pressure pump may be installed on the fermentation culture solution side of the porous membrane. As a means for controlling the transmembrane pressure difference within the above range, it can be controlled by changing the liquid level difference between the fermentation broth and the porous membrane treated water as described above. When a pump is used to generate the pressure, the transmembrane pressure can be controlled by the suction pressure. Furthermore, the transmembrane pressure difference can be controlled by the pressure of the gas or liquid that introduces the pressure on the fermentation broth side. When these pressure controls are performed, the pressure difference between the pressure on the fermentation broth side and the pressure on the porous membrane treated water side can be used as the transmembrane pressure difference, which can be used to control the transmembrane pressure difference.

  Moreover, it is preferable that the porous membrane used in this invention has the performance which can be filtered in the range of 0.1 kPa or more and 20 kPa or less as a transmembrane differential pressure to filter.

  Among the continuous fermentation apparatus used in the method for producing lactic acid by continuous fermentation of the present invention, a typical example in which the separation element is installed inside the fermentation reaction tank is shown in the schematic diagram of FIG. FIG. 1 is a schematic side view for explaining one embodiment of a continuous fermentation apparatus used in the present invention.

  In FIG. 1, the continuous fermentation apparatus basically includes a fermentation reaction tank 1 provided with a separation membrane element 2 and a water head difference control device 3. A porous membrane is incorporated in the separation membrane element 2 in the fermentation reaction tank 1. As the porous membrane, for example, a separation membrane and a separation membrane element disclosed in International Publication No. 2002/064240 can be used. The separation membrane element will be described in detail later.

  Next, the form of continuous fermentation by the continuous fermentation apparatus of FIG. 1 will be described.

  The medium is continuously or intermittently charged into the fermentation reaction tank 1 by the medium supply pump 7. The medium can be sterilized by heating or sterilization using a filter as necessary before being put into the fermentation reaction tank 1. At the time of fermentation production, the fermentation broth in the fermentation reaction tank 1 is agitated with the agitator 5 as necessary, and the necessary gas is supplied with the gas supply device 4 as necessary. Performing highly productive fermentation production by adjusting the pH of the fermentation broth with the control device 9 and the pH adjusting solution supply pump 8, and adjusting the temperature of the fermentation broth with the temperature controller 10 as necessary. it can.

  Here, the pH and temperature are exemplified for the adjustment of the physicochemical conditions of the fermentation broth by the instrumentation / control device. However, if necessary, the dissolved oxygen and ORP can be controlled, and an analytical device such as an online chemical sensor can be used. The concentration of lactic acid in the fermentation broth can be measured, and physicochemical conditions can be controlled using the concentration as an index. Moreover, regarding the form of continuous or intermittent input of the medium, the amount and speed of the medium input can be appropriately adjusted using the measured value of the physicochemical environment of the fermentation broth by the instrumentation device as an index.

  The fermentation broth is filtered and separated into microorganisms and fermentation products by the separation membrane element 2 installed in the fermentation reaction tank 1, and the fermentation products are taken out from the apparatus system. Moreover, the microorganisms filtered and separated remain in the apparatus system, so that the microorganism concentration in the apparatus system can be maintained high, and fermentation production with high productivity is possible. Here, the filtration / separation by the separation membrane element 2 is performed by the water head differential pressure with the water surface of the fermentation reaction tank 1, and no special power is required. Further, if necessary, the filtration / separation speed of the separation membrane element 2 and the amount of the fermentation broth in the fermentation reaction tank can be appropriately adjusted by the level sensor 6 and the head differential pressure control device 3. The above-described filtration / separation by the separation membrane element is exemplified by the water head differential pressure. However, if necessary, the filtration / separation can be performed by suction filtration with a pump or the like or pressurizing the inside of the apparatus system.

  FIG. 2 is a schematic side view for explaining an example of another continuous fermentation apparatus used in the method for producing lactic acid by continuous fermentation of the present invention. FIG. 2 is a typical example of a continuous fermentation apparatus in which the separation membrane element is installed outside the fermentation reaction tank.

  In FIG. 2, the continuous fermentation apparatus basically includes a fermentation reaction tank 1, a membrane separation tank 12 including a membrane separation membrane element 2, and a water head difference control device 3. Here, a porous membrane is incorporated in the separation membrane element 2. For the porous membrane and the separation membrane element, it is preferable to use, for example, the separation membrane and the separation membrane element disclosed in WO 2002/064240. Moreover, the membrane separation tank 12 is connected to the fermentation reaction tank 1 via the fermentation culture solution circulation pump 11, and enables the circulation of the fermentation culture solution.

  In FIG. 2, the culture medium is fed into the fermentation reaction tank 1 by the medium supply pump 7, the fermentation culture solution in the fermentation reaction tank 1 is stirred by the stirrer 5 as necessary, and the gas supply device is used as necessary. 4 can supply the required gas. At this time, the supplied gas can be recovered and recycled and supplied again by the gas supply device 4. If necessary, the pH of the fermentation broth is adjusted by the pH sensor / control device 9 and the pH adjusting solution supply pump 8, and the temperature of the fermentation broth is adjusted by the temperature controller 10 as necessary. Thus, fermentative production with high productivity can be performed. Further, the fermentation broth in the apparatus is circulated between the fermentation reaction tank 1 and the membrane separation tank 12 by the fermentation broth circulation pump 11.

  The fermentation broth containing the fermentation product is filtered and separated into the microorganism and the fermentation product by the separation membrane element 2, and the fermentation product can be taken out from the apparatus system. Moreover, the microorganisms filtered and separated remain in the apparatus system, so that the microorganism concentration in the apparatus system can be maintained high, and fermentation production with high productivity is possible.

  Here, the filtration / separation by the separation membrane element 2 can be performed by the water head differential pressure with the water surface of the membrane separation tank 12, and no special power is required. If necessary, the filtration / separation speed of the separation membrane element 2 and the amount of culture solution in the apparatus system can be appropriately adjusted by the level sensor 6 and the head differential pressure control device 3. If necessary, the required gas can be supplied into the membrane separation tank 12 by the gas supply device 4. At this time, the supplied gas can be recovered and recycled and supplied again into the membrane separation tank 12 by the gas supply device 4.

  Filtration / separation by the separation membrane element 2 can be performed by suction filtration using a pump or the like, or by pressurizing the inside of the apparatus system as necessary. In addition, microorganisms or cultured cells can be cultured for continuous fermentation in a separate culture tank (not shown), and supplied to the fermentation reaction tank as necessary. By continuously culturing microorganisms or cultured cells for continuous fermentation in a separate culture tank, and supplying the resulting culture liquid to the fermentor as needed, continuous fermentation with microorganisms that are always fresh and have high lactic acid production capability is possible. Thus, continuous fermentation can be performed while maintaining high production performance for a long time.

  Next, the separation membrane element preferably used in the continuous fermentation apparatus used in the method for producing lactic acid according to the present invention will be described.

  The separation membrane element shown in FIG. 3 will be described. FIG. 3 is a schematic perspective view for illustrating the separation membrane element used in the present invention. In the continuous fermentation apparatus used in the method for producing lactic acid according to the present invention, the separation membrane and the separation membrane element disclosed in International Publication No. 2002/064240 can be preferably used. As shown in FIG. 3, the separation membrane element is configured by arranging a flow path material 14 and the separation membrane 15 (porous membrane) in this order on both surfaces of a rigid support plate 13. The support plate 13 has recesses 16 on both sides. The separation membrane 15 filters the culture solution. The flow path member 14 is for efficiently flowing the filtrate filtered by the separation membrane 15 to the support plate 13. The filtrate that has flowed to the support plate 13 passes through the recess 16 of the support plate 13 and is taken out of the continuous fermentation apparatus via the water collecting pipe 17. As a power for taking out the filtrate, a method such as a differential pressure at the water head, a pump, suction filtration with a liquid or gas, or pressurization in the apparatus system can be used.

  The separation membrane element shown in FIG. 4 will be described. FIG. 4 is a schematic perspective view for illustrating another separation membrane element used in the present invention. As shown in FIG. 4, the separation membrane element is mainly composed of a separation membrane bundle 18 composed of a hollow fiber membrane (porous membrane), an upper resin sealing layer 19, and a lower resin sealing layer 20. The separation membrane bundle 18 is bonded and fixed in a bundle by an upper resin sealing layer 19 and a lower resin sealing layer 20. Adhesion / fixation by the lower resin sealing layer 20 has a structure in which the hollow portion of the hollow fiber membrane (porous membrane) of the separation membrane bundle 18 is sealed to prevent leakage of the fermentation broth. On the other hand, the upper resin sealing layer 19 does not seal the inner hole of the hollow fiber membrane (porous membrane) of the separation membrane bundle 18 and has a structure in which the filtrate flows through the water collecting pipe 22. This separation membrane element can be installed in the continuous fermentation apparatus via the support frame 21. The filtrate filtered by the separation membrane bundle 18 passes through the hollow portion of the hollow fiber membrane and is taken out to the outside of the continuous fermentation apparatus via the water collecting pipe 22. As a power for taking out the filtrate, a method such as a differential pressure at the water head, a pump, suction filtration with a liquid or gas, or pressurization in the apparatus system can be used.

  The member constituting the separation membrane element of the continuous fermentation apparatus used in the method for producing lactic acid by continuous fermentation of the present invention is preferably a member resistant to high-pressure steam sterilization operation. If the inside of the continuous fermentation apparatus can be sterilized, it is possible to avoid the risk of contamination by undesirable microorganisms during continuous fermentation, and more stable continuous fermentation is possible. The member constituting the separation membrane element is preferably resistant under the condition of high-pressure steam sterilization operation at a temperature of 121 ° C. for 15 minutes. Separation membrane element members include, for example, metals such as stainless steel and aluminum, polyamide resins, fluorine resins, polycarbonate resins, polyacetal resins, polybutylene terephthalate resins, PVDF, modified polyphenylene ether resins, and polysulfone resins. The resin can be preferably selected.

  In the continuous fermentation apparatus used in the method for producing lactic acid by continuous fermentation according to the present invention, the separation membrane element may be installed in the fermentation reaction tank as shown in FIG. 1 or outside the fermentation reaction tank as shown in FIG. May be installed. When the separation membrane element is installed outside the fermentation reaction tank, a separate membrane separation tank can be provided and the separation membrane element can be installed inside it, and the culture fluid is circulated between the fermentation reaction tank and the membrane separation tank. The culture solution can be continuously filtered by the separation membrane element.

  In the continuous fermentation apparatus used in the method for producing lactic acid by continuous fermentation according to the present invention, the membrane separation tank is preferably capable of high-pressure steam sterilization. If the membrane separation tank can be autoclaved, it is easy to avoid contamination with germs.

  When continuous fermentation is performed according to the present invention, longer-term lactic acid production and a higher volumetric production rate can be obtained as compared with conventional continuous fermentation, and extremely efficient fermentation production becomes possible. Here, the production rate in continuous fermentation culture is calculated by the following (formula 3).

  Next, the nutritionally non-requiring yeast having the ability to produce lactic acid that can be used in the method for producing lactic acid of the present invention will be described with specific examples.

  Examples of yeast that can be used in the present invention include yeast such as baker's yeast. Yeast may be isolated from the natural environment, or may be partially modified in nature by mutation or genetic recombination.

  The yeast used in the present invention is a yeast having the ability to produce lactic acid. The yeast used in the present invention preferably has a high lactic acid production capacity. The yeast used in the present invention may be a yeast that originally has a high lactic acid production capacity, may be isolated from the natural world, or may be an artificially enhanced yeast. Specifically, the yeast used in the present invention may be a yeast whose properties are partially modified by mutation or gene recombination.

  Examples of the yeast suitably used in the present invention include yeast belonging to the genus Saccharomyces, the genus Schizosaccharomyces, or the genus Kluyveromyces. Among them, yeast belonging to the genus Saccharomyces is more preferable, and Saccharomyces cerevisiae is more preferable. Specifically, yeasts particularly preferably used in the present invention are NBRC10505 strain and NBRC10506 strain.

  In the method for producing lactic acid by continuous fermentation of the present invention, the yeast is preferably a yeast into which a lactic acid synthase gene has been introduced. In the present invention, the lactic acid synthase gene (ldh gene) means lactate dehydrogenation having an activity of converting reduced nicotinamide adenine dinucleotide (NADH) and pyruvate into lactic acid and oxidized nicotinamide adenine dinucleotide (NAD +). A gene encoding an enzyme. In the present invention, the lactate synthase gene (ldh gene) is a lactate dehydrogenase (ldh) gene.

  The lactate dehydrogenase gene has various homologues depending on the type of organism or in vivo. The lactate dehydrogenase gene in the present invention may be a lactate dehydrogenase gene artificially synthesized by chemical synthesis or genetic engineering techniques, in addition to a naturally occurring lactate synthase gene.

  When L-lactic acid is produced by the method for producing lactic acid according to the present invention, yeast imparted or enhanced with L-lactic acid production ability can be used. L-lactic acid production ability can be imparted or enhanced by introducing an L-lactic acid synthase gene. The gene encoding L-lactic acid synthase is preferably an L-lactic acid synthase gene having strong enzyme activity. Examples of naturally-occurring lactic acid synthase genes include prokaryotic organisms such as lactic acid bacteria or those derived from eukaryotic microorganisms such as mold, and lactate synthase genes derived from higher eukaryotes including mammals such as cattle and humans. Can do.

  Examples of L-lactate dehydrogenase genes preferably used in the present invention include those derived from frogs such as Rhocophoridae, Ranaidae, Hyridae, Microhylidae, Toad. Examples include the L-lactate dehydrogenase genes belonging to the family Buphonidae, Hyperoliidae, Pelobatinae, Discoglossidae, and Pipidae. Among these, it is preferable to use an L-lactate dehydrogenase gene belonging to the family Pipidae, and among the frogs belonging to the family Comidae, the L-lactate dehydrogenase gene derived from Xenopus laevis is preferably used.

  The L-lactate dehydrogenase gene preferably used in the present invention is an L-lactate dehydrogenase gene derived from Xenopus laevis. The L-lactate dehydrogenase gene used more preferably is a lactate dehydrogenase gene having the nucleic acid sequence shown in SEQ ID NO: 1, and the L-lactate dehydrogenase gene used more preferably is frog-derived L- A gene encoding lactate dehydrogenase, which has the nucleic acid sequence shown in SEQ ID NO: 1.

  In the method for producing lactic acid by continuous fermentation according to the present invention, the lactate dehydrogenase gene is preferably a gene encoding L-lactate dehydrogenase, and the gene encoding L-lactate dehydrogenase is L-lactate dehydrogenase. It is preferably introduced downstream of a promoter that enables expression of the gene encoding. Furthermore, in the method for producing lactic acid by continuous fermentation of the present invention, the gene encoding L-lactate dehydrogenase is introduced in a state that can be expressed downstream of the promoter of pyruvate decarboxylase 1 gene on the chromosome. It is preferable.

  Lactic acid dehydrogenase genes preferably used in the present invention include genetic polymorphisms and mutant genes caused by mutagenesis. Here, the genetic polymorphism means that the base sequence of a gene is partially changed due to a natural mutation on the gene. Mutagenesis refers to artificially introducing a mutation into a gene. For example, a method using a site-specific mutagenesis kit (Mutant-K (manufactured by Takara Bio Inc.)) or a random mutagenesis kit. And a method using (BD Diversity PCR Random Mutagenesis (manufactured by CLONTECH)).

  Furthermore, as DNA encoding lactic acid synthase with enhanced enzyme activity, for example, DNA encoding lactic acid synthase with strong enzyme activity obtained by a mutation technique using site-directed mutagenesis or a strong promoter And the like can be exemplified by DNA linked with a DNA encoding lactic acid synthase under the control of

  The yeast used in the method for producing lactic acid by continuous fermentation of the present invention is preferably produced by introducing a gene encoding lactic acid synthase (lactate dehydrogenase) using a technique such as genetic recombination. be able to. In addition, when yeast already has a gene encoding a target lactic acid synthase, it is not always necessary to introduce the gene.

  The yeast used in the present invention may be haploid but may be polyploid. It is haploid that one set of chromosomes is a polyploid. For example, if there are two pairs of chromosomes, it is called a diploid.

  In the method for producing lactic acid by continuous fermentation according to the present invention, as a method for introducing a gene encoding lactate dehydrogenase into yeast, for example, a lactate dehydrogenase gene is cloned, and an expression vector incorporating the cloned gene is used. Examples include, but are not limited to, a method of transforming into yeast and a method of inserting the cloned gene into a target site on a chromosome by homologous recombination.

  There is no restriction | limiting in particular as a method of cloning the lactate dehydrogenase gene preferably used by this invention, A known method can be used. For example, based on known gene information, a method of amplifying and acquiring a necessary genetic region using PCR (Polymerase Chain Reaction) method, a method of cloning from a genomic library or cDNA library using homology or enzyme activity as an index, etc. Is mentioned. A method of chemical synthesis or genetic engineering based on known protein information is also possible.

  As an expression vector into which the cloned lactate dehydrogenase gene is incorporated, an expression vector generally used in yeast can be used. An expression vector widely used in yeast has a sequence necessary for autonomous replication in yeast cells, a sequence necessary for autonomous replication in E. coli cells, a yeast selection marker, and an E. coli selection marker. Yes. Further, in order to express the incorporated lactate dehydrogenase gene, it is desirable to have a so-called regulatory sequence such as an operator, promoter, terminator or enhancer that regulates the expression.

  Here, the sequence necessary for autonomous replication in yeast cells is, for example, a yeast autonomous replication origin (ARS1) and a centromere sequence pair or the replication origin of a yeast 2 μm plasmid. The sequence necessary for autonomous replication is, for example, the ColE1 replication origin of Escherichia coli. Examples of yeast selection markers include auxotrophic complementary genes such as URA3 or TRP1, drug resistance genes such as G418 resistance gene and neomycin resistance gene, and selection markers for Escherichia coli include ampicillin resistance gene and kanamycin resistance gene. And antibiotic resistance genes such as Regulatory sequences include GAPDH (glyceraldehyde-3-phosphate dehydrogenase) promoter, ADH (alcohol dehydrogenase) promoter and GAPDH terminator. However, the expression vector is not limited to these.

  By introducing a lactate dehydrogenase gene downstream of the expression vector promoter, a vector capable of expressing the gene can be obtained. By transforming the obtained lactate dehydrogenase gene expression vector into yeast by the method described later, the lactate dehydrogenase gene can be introduced into yeast.

  As a method of inserting the lactate dehydrogenase gene into the target site on the chromosome, preferably downstream of the promoter of the pdc1 gene by homologous recombination, homologous recombination is possible upstream and downstream of the lactate dehydrogenase gene. Examples include a method in which PCR is performed using a primer designed to add a portion, and the obtained PCR fragment is transformed into yeast by the method described later. In order to facilitate selection of transformants, the PCR fragment preferably contains a yeast selection marker.

  The method of adjusting the PCR fragment used here can be performed, for example, by the steps 1 to 3 of the following (1) to (3). A schematic diagram thereof is shown in FIG. FIG. 5 is a schematic diagram for explaining a method of introducing a lactate dehydrogenase gene (ldh gene) downstream of the pdc1 gene promoter.

  (1) Step 1: Using a plasmid having a terminator connected downstream of a lactate dehydrogenase gene as a template and using primer 1 and primer 2 as a set, a fragment containing the lactate dehydrogenase gene and terminator is amplified by PCR. Primer 1 is designed so as to add a homologous sequence of 40 bp or more upstream of the target introduction site, and primer 2 is designed based on a sequence derived from a plasmid downstream from the terminator. Preferably, the sequence homologous to the upstream side of the introduction target site added to the primer 1 is a sequence homologous upstream of the pyruvate decarboxylase 1 gene (pdc1 gene).

  (2) Step 2: A plasmid containing a yeast selection marker is amplified by PCR using a plasmid having a yeast selection marker, for example, pRS424 or pRS426 as a template, primer 3 and primer 4 as a set. Primer 3 is designed so that a sequence homologous to the sequence downstream of the terminator of the PCR fragment in Step 1 above adds 30 bp or more, and primer 4 has a sequence homologous downstream of the target site of introduction. Design to add 40 bp or more. Preferably, the sequence homologous to the downstream side of the target site to be added to the primer 4 is a sequence homologous downstream of the pdc1 gene.

  (3) Step 3: Perform PCR using a mixture of the PCR fragments obtained in Step 1 and Step 2 above as a template and Primer 1 and Primer 4 as a set. And a PCR fragment containing a lactate dehydrogenase gene, a terminator, and a yeast selection marker, to which a homologous sequence is added on the downstream side. Preferably, the PCR fragment is a PCR fragment containing an ldh gene, a terminator, and a marker gene with homologous sequences added upstream and downstream of the pdc1 gene at both ends.

  In order to introduce the lactate dehydrogenase gene expression vector or PCR fragment obtained above into yeast, methods such as transformation, transduction, transfection, cotransfection and electroporation can be used. Specifically, for example, a method using lithium acetate or a protoplast method is used.

  As a method for culturing the obtained transformant, for example, a known method described in “MD Rose et al.,“ Methods In Yeast Genetics ”, Cold Spring Harbor Laboratory Press (1990)” is used. Can be used. Yeast into which a lactate dehydrogenase gene expression vector or PCR fragment has been introduced can be selected by culturing in a nutrient-free medium or a drug-added medium according to the yeast selection marker possessed by the expression vector or PCR fragment.

  Lactic acid can be produced in the medium by culturing the yeast introduced with the lactate dehydrogenase gene preferably used in the present invention.

  The method for measuring the obtained lactic acid is not particularly limited, and examples include a method using HPLC and a method using F-kit (Roche).

  In the present invention, lactate dehydrogenase activity indicates the activity of converting pyruvate and NADH into lactic acid and NAD +. Although not limited, lactate dehydrogenase activity can be compared using specific activity as an index. That is, yeast having the same ldh gene introduction method and the same genetic background are cultured under the same conditions, and the change in absorbance at 340 nm due to the decrease in NADH is measured using a protein extracted from the cultured cells. At that time, the specific activity of lactate dehydrogenase is expressed by the following (formula 4) by defining the amount of enzyme that decreases 1 μmol of NADH per minute at room temperature as 1 unit (Unit). Here, Δ340 is the amount of decrease in absorbance at 340 nm per minute, and 6.22 is the mmol molecular extinction coefficient of NADH.

  Lactic acid in the obtained fermentation broth can be purified by a conventionally known method. For example, the fermentation broth obtained by centrifuging microorganisms is adjusted to pH 1 or lower, extracted with diethyl ether or ethyl acetate, adsorbed on an ion exchange resin, washed and then eluted, and alcohol in the presence of an acid catalyst. It can be purified by a method of distillation after reacting as an ester, a method of crystallization as a calcium salt or a lithium salt, and the like.

  In the present invention, the fact that the total amino acid concentration in the fermentation raw material is 2 g / L or less means that the concentration of amino acids contained in the lactic acid fermentation medium used in the present invention is 2 g / L or less in total. . As a preferable specific example of the medium for lactic acid fermentation in which the total amino acid concentration in the fermentation raw material is 2 g / L or less, a medium having a composition containing 0.9 g / L of the total amino acid concentration shown in the following Table 1 (hereinafter referred to as the following) , Sometimes abbreviated as Synthetic Medium 1), a medium having a composition containing 45 mg / L of the total amino acid concentration shown in Table 2 (hereinafter sometimes abbreviated as Natural Medium 1), and Table 3 And a medium having a composition containing 1.5 g / L of the total amino acid concentration (hereinafter sometimes abbreviated as “natural medium 2”).

  The above synthetic medium 1 can be prepared as follows. For example, among the nutrients shown in Table 1, vitamins and metal salts are sterilized by filtering, and other nutrients are divided into sugar, amino acid, nucleic acid and inorganic salt groups, and autoclaves are grouped for each group. The solution is sterilized by treatment, cooled to a temperature below room temperature, and then adjusted to the concentration shown in Table 1. The amino acids shown in Table 1 are prepared by adding each amino acid to the concentration shown in Table 1 and adding it to the culture medium, or preparing a 10-fold concentration of each amino acid solution and diluting it and showing in Table 1. It can be added so that the concentration is as high as possible. Each amino acid such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or these One or more amino acids selected from the group such as hydrates may be used, and the necessary amount may be added so that the total amino acid concentration of the medium becomes 0.9 g / L.

  Moreover, said natural culture medium 1 can be prepared as follows. For example, among the nutrients shown in Table 2, the raw material sugar, amino acid and inorganic sulfate ammonium sulfate are divided into groups, sterilized by autoclaving for each group, cooled to a temperature below room temperature, and then the concentrations shown in Table 2 Prepare so that. Each amino acid solution can be added to the medium by preparing each amino acid solution to have a concentration shown in Table 2. Each amino acid solution can be made to a 10-fold concentration, diluted and added to the concentration shown in Table 2. As each amino acid, for example, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and these The hydrates can be used. As for the total amino acid concentration of the medium, one or more amino acids selected from each amino acid group may be used, and a necessary amount may be added so that the total amino acid concentration of the medium is 45 mg / L.

  Moreover, said natural culture medium 2 can be prepared as follows. For example, among the nutrients shown in Table 3, the raw sugar, amino acid and inorganic salt ammonium sulfate are divided into groups, sterilized by autoclaving for each group, cooled to a temperature below room temperature, and then the concentrations shown in Table 3 Prepare so that. Each amino acid solution can be added to the medium by preparing each amino acid solution to have the concentration shown in Table 3. Each amino acid solution can be made to a 10-fold concentration, diluted and added to the concentrations shown in Table 3. As each amino acid, for example, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and these The hydrates can be used. As for the total amino acid concentration of the medium, one or more amino acids selected from each amino acid group may be used, and a necessary amount may be added so that the total amino acid concentration of the medium becomes 1.5 g / L.

  By making the total amino acid concentration in the fermentation raw material 2 g / L or less, the growth rate of yeast having the ability to produce lactic acid from the start of lactic acid fermentation to the end of fermentation can be delayed and densified over a long period of time, Lactic acid productivity can be improved. In addition, by delaying the death cycle of yeast and reducing the concentration of by-products generated at the time of death, the clogging of the porous membrane used as a separation membrane for fermentation broth can be suppressed, and the lactic acid fermentation can be further prolonged. It is possible to increase the production efficiency of lactic acid fermentation.

In the present invention, the amino acid concentration in the fermentation raw material or the medium for lactic acid fermentation can be measured, for example, using the following amino acid analyzer under the following 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 ° C
・ Detection wavelength: 570 nm, 440 nm
Sample injection volume: 25 μl.

  In the present invention, the fermentation raw material having a total amino acid concentration of 2 g / L or less is a fermentation raw material for all the media introduced from the start of lactic acid fermentation to the end of the fermentation, and is newly added to the fermentation broth. The fermentation raw material also has a total amino acid concentration of 2 g / L or less.

  The production method of lactic acid by continuous fermentation of the present invention will be specifically described based on examples.

  In the examples of the present invention, a frog lactate dehydrogenase gene was selected as an example of a lactate dehydrogenase gene, and Saccharomyces cerevisiae produced in Reference Example 1 was selected as an example of yeast. Continuous fermentation of lactic acid was performed using the continuous fermentation apparatus shown in FIG. As the separation membrane, the separation membrane elements shown in FIGS. 3 and 4 using the separation membranes prepared in Reference Example 2 and Reference Example 3 were used.

(Reference Example 1) (Generation of yeast having lactic acid synthesis ability)
As the lactate dehydrogenase (ldh) gene, a lactate dehydrogenase gene derived from Xenopus laevis having the nucleic acid sequence shown in SEQ ID NO: 1 was used. The lactate dehydrogenase gene was cloned by the PCR method. For PCR, phagemid DNA prepared from a Xenopus laevis kidney-derived cDNA library (Stratagene) according to the attached protocol was used as a template.

  For the PCR amplification reaction, KOD-Plus polymerase (manufactured by Toyobo Co., Ltd.) was used, and the attached reaction buffer and dNTPmix were used. As described above, a 50 μl reaction system was prepared so that the phagemid DNA prepared according to the attached protocol was 50 ng / sample, the primer was 50 pmol / sample, and the KOD-Plus polymerase was 1 unit / sample. The prepared reaction solution was thermally denatured at 94 ° C. for 5 minutes with a PCR amplification device iCycler (manufactured by BIO-RAD), then 94 ° C. (thermal denature): 30 seconds, 55 ° C. (primer annealing): 30 Second, 68 ° C. (extension of complementary strand): 1 cycle was performed for 30 cycles, and then cooled to a temperature of 4 ° C. The gene amplification primer (SEQ ID NO: 2 and 3) was prepared such that a SalI recognition sequence was added to the 5 terminal side and a NotI recognition sequence was added to the 3 terminal side.

  The PCR amplified fragment was purified, and the end was phosphorylated with T4 polynucleotide Kinase (manufactured by Takara Bio Inc.), and then ligated to the pUC118 vector (which was cleaved with the restriction enzyme HincII and the cut surface was dephosphorylated). Ligation was performed using DNA Ligation Kit Ver. 2 (manufactured by Takara Bio Inc.). The ligation solution was transformed into E. coli DH5α competent cells (Takara Bio Inc.), plated on an LB plate containing 50 μg / mL of antibiotic ampicillin, and cultured overnight. For the grown colonies, plasmid DNA was collected with a miniprep, cut with restriction enzymes SalI and NotI, and a plasmid into which the ldh gene was inserted was selected. All of these series of operations were performed according to the attached protocol.

  The pUC118 vector in which the lactate dehydrogenase gene is inserted is cleaved with the restriction enzymes SalI and NotI, the DNA fragments are separated by 1% agarose gel electrophoresis, and the fragment containing the lactate dehydrogenase gene is purified according to a conventional method. did. The fragment containing the obtained lactate dehydrogenase gene was ligated to the XhoI / NotI cleavage site of the yeast expression vector pTRS11 shown in FIG. 6, and the plasmid DNA was recovered in the same manner as described above, and digested with the restriction enzymes XhoI and NotI. By doing so, an expression vector into which the lactate dehydrogenase gene was inserted was selected. Hereinafter, an expression vector incorporating the lactate dehydrogenase gene prepared in this manner was designated as pTRS102.

  A 1.3 kb DNA fragment containing a lactate dehydrogenase gene and a GAPDH terminator sequence was amplified by PCR using the obtained expression vector pTS102 as an amplification template and oligonucleotides (SEQ ID NOs: 4 and 5) as primer sets (FIG. 5 corresponds to step 1). Here, SEQ ID NO: 4 was designed such that a sequence having homology was added to 65 bp upstream of the pdc1 gene.

  Next, a 1.2 kb DNA fragment containing the TRP1 gene as a yeast selection marker was obtained by PCR using plasmid pRS424 having a plasmid yeast selection marker as an amplification template and oligonucleotides (SEQ ID NOs: 6 and 7) as primer sets. Amplified. Here, SEQ ID NO: 7 was designed so that a sequence having homology was added to 65 bp downstream of the pdc1 gene.

  Each DNA fragment was separated by 1.5% agarose gel electrophoresis and purified according to a conventional method. A mixture of each 1.3 kb fragment and 1.2 kb fragment obtained here was used as an amplification template, and a PCR method using oligonucleotides (SEQ ID NOs: 4 and 7) as a primer set was used for the lactate dehydrogenase gene, GAPDH. A DNA fragment of about 2.5 kb to which the terminator and TRP1 gene were linked was amplified.

  The above DNA fragments are separated by 1.5% agarose gel electrophoresis, purified according to a conventional method, transformed into yeast Saccharomyces cerevisiae NBRC10505, and cultured in a medium without tryptophan, whereby the ldh gene is chromosomally A transformant introduced downstream of the pdc1 gene promoter was selected.

  Confirmation that the transformant obtained as described above was a yeast in which the lactate dehydrogenase gene was introduced downstream of the pdc1 gene promoter on the chromosome was performed as follows. First, the genomic DNA of the transformant was prepared using a genomic DNA extraction kit “Gentori-kun” (registered trademark) (manufactured by Takara Bio Inc.), which was used as an amplification template, and oligonucleotides (SEQ ID NOs: 7 and 8) were used as primer sets. As a result of PCR, it was confirmed that an amplified DNA fragment of about 2.8 kb was obtained. In the non-transformed strain, an amplified DNA fragment of about 2.1 kb is obtained by the PCR. Hereinafter, the transformed strain in which the lactate dehydrogenase gene was introduced downstream of the pdc1 gene promoter on the chromosome was used as a B2 strain in the subsequent examples.

(Reference Example 2) Production of porous membrane (Part 1)
Polyvinylidene fluoride (PVDF) resin was used as the resin and N, N-dimethylacetamide (DMAc) was used as the solvent, and these were sufficiently stirred at a temperature of 90 ° C. to obtain a stock solution having the following composition.
[Undiluted solution]
・ PVDF: 13.0% by weight
DMAc: 87.0% by weight
Next, after cooling the above stock solution to a temperature of 25 ° C., a polyester fiber non-woven fabric (porous substrate) having a density of 0.48 g / cm ³ and a thickness of 220 μm, which was previously pasted on a glass plate. And immediately immersed in a coagulation bath at 25 ° C. having the following composition for 5 minutes to obtain a porous film having a porous resin layer formed on a porous substrate.
[Coagulation bath]
-Water: 30.0% by weight
DMAc: 70.0% by weight
After peeling this porous membrane from the glass plate, it was immersed in hot water at a temperature of 80 ° C. three times to wash out DMAc to obtain a separation membrane. When the surface of the porous resin layer was observed with a scanning electron microscope at a magnification of 10,000 within the range of 9.2 μm × 10.4 μm, the average diameter of all observable pores was 0.1 μm. . Next, when the pure water permeability coefficient was evaluated about the said separation membrane, it was 50 * 10 < ^-9 > m < ³ > / m < ² > / s / Pa. The pure water permeation amount was measured using purified water at a temperature of 25 ° C. by a reverse osmosis membrane at a head height of 1 m. The standard deviation of the average pore diameter was 0.035 μm, and the membrane surface roughness was 0.06 μm.

(Reference Example 3) Production of porous membrane (part 2)
A vinylidene fluoride homopolymer having a weight average molecular weight of 41,000 and γ-butyrolactone were dissolved at a temperature of 170 ° C. at a ratio of 38% by weight and 62% by weight, respectively, to prepare a stock solution. This stock solution was discharged from the base while stirring γ-butyrolactone as a hollow portion forming liquid, and solidified in a cooling bath composed of an 80% by weight aqueous solution of γ-butyrolactone at a temperature of 20 ° C. to produce a hollow fiber membrane.

Next, 14% by weight of vinylidene fluoride homopolymer having a weight average molecular weight of 284,000, 1% by weight of cellulose acetate propionate (Eastman Chemical Co., CAP482-0.5), and N-methyl-2-pyrrolidone 77% by weight, polyoxyethylene coconut oil fatty acid sorbitan (manufactured by Sanyo Kasei Co., Ltd., trade name Ionette T-20C) at 5% by weight, and 3% by weight of water are mixed and dissolved at a temperature of 95 ° C. It was adjusted. This undiluted solution was uniformly applied to the surface of the hollow fiber membrane obtained above, and a hollow fiber porous membrane used in the present invention was immediately solidified in a water bath. The average pore diameter of the treated water side surface of the obtained hollow fiber porous membrane was 0.05 μm. Next, the pure water permeation rate of the hollow fiber porous membrane as the separation membrane was evaluated and found to be 5.5 × 10 ⁻⁹ m ³ / m ² · s · Pa. The amount of water permeation was measured using purified water at a temperature of 25 ° C. by a reverse osmosis membrane at a head height of 1 m. The standard deviation of the average pore diameter was 0.006 μm.

(Example 1) Production of L-lactic acid by continuous fermentation (part 1)
In order to investigate whether 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. As the medium, the synthetic medium 1 for lactic acid fermentation shown in Table 1 above adjusted to a total amino acid concentration of 0.9 g / L was used after being subjected to high-pressure steam sterilization at a temperature of 121 ° C. for 15 minutes.

  The above synthetic medium 1 was prepared as follows. Of the nutrients shown in Table 1, vitamins and metal salts are sterilized by filter treatment, and other nutrients are divided into sugar, amino acid, nucleic acid and inorganic salt groups, and each group is autoclaved. After sterilization and cooling to room temperature or lower, the total amino acid concentration shown in Table 1 was adjusted to 0.9 g / L. The amino acids shown in Table 1 were each added to a concentration shown in Table 1 by preparing a 10-fold concentrated amino acid solution, diluting it. Each amino acid includes alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or these The hydrate of was used.

For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, the porous membrane produced in the above-mentioned Reference Example 2 containing polyvinylidene fluoride (PVDF) as a main component was used. 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 1 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 1.5 (L)
-Separation membrane used: PVDF filtration membrane-Membrane separation element Effective filtration area: 120 square cm
・ Temperature adjustment: 30 (℃)
・ Aeration volume of fermentation reaction tank: 200 (mL / min)
・ Fermentation reactor stirring speed: 800 (rpm)
Sterilization: All the culture tanks containing the separation membrane element and the working medium were autoclaved at 121 ° C. for 20 minutes under high pressure steam sterilization.
・ PH adjustment: Adjust pH to 5 with 8N NaOH ・ Membrane permeate flow control: Flow rate is controlled by the water separation of the membrane separation tank (the water head difference is controlled within 2m)
The lactic acid contained in the fermentation broth was confirmed by measuring the amount of lactic acid by the HPLC method under the conditions shown below.
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, and 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min)
-Detection method: electrical conductivity-Temperature: 45 (° C).

The optical purity of L-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 copper sulfate aqueous solution-Flow rate: 1.0 ml / min-Detection method: UV254 nm
-Temperature: 30 degreeC.

Moreover, the optical purity of L-lactic acid is calculated by the following formula.
Optical purity (%) = 100 × (LD) / (L + D)
Here, L represents the concentration of L-lactic acid, and D represents the concentration of D-lactic acid. For measurement of glucose concentration, “Glucose Test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used.

  First, the B2 strain was statically cultured at a temperature of 30 ° C. for 24 hours in a synthetic medium for lactic acid fermentation purged with 5 ml of nitrogen gas shown in Table 1 in a test tube (cultured in advance). The obtained culture solution was inoculated into 50 ml of a synthetic medium for fresh lactic acid fermentation purged with nitrogen gas, and statically cultured at a temperature of 30 ° C. for 24 hours (pre-culture). The culture broth was inoculated into 1 L of lactic acid fermentation medium purged with nitrogen gas from the continuous fermentation apparatus shown in FIG. The amount was adjusted and the temperature was adjusted to a temperature of 30 ° C., and cultured for 24 hours (pre-culture). Immediately after the completion of the pre-culture, the lactic acid fermentation medium is continuously supplied, and the continuous culture is performed while controlling the amount of permeated water so that the fermentation broth of the continuous fermentation apparatus is 1.5 L. went. 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 lactic acid concentration and residual glucose concentration in the membrane permeation fermentation broth were measured appropriately. The lactic acid production rates calculated from the input glucose calculated from the lactic acid and glucose concentrations are shown in Table 4 together with the results of Examples 2 to 8 and Comparative Example 1 described later. As a result of performing a fermentation test for 680 hours, by using this synthetic medium for lactic acid fermentation and a continuous fermentation apparatus, stable lactic acid having a longer time and higher productivity than the previous excellent lactic acid continuous fermentation (Comparative Example 1). It was confirmed that production by continuous fermentation was possible.

(Example 2) Production of L-lactic acid by continuous fermentation (part 2)
From the synthetic medium 1 for lactic acid fermentation in which the medium was adjusted so that the total amino acid concentration was 0.9 g / L using the porous membrane prepared in Reference Example 2, the total amino acid concentrations shown in Table 2 above L-lactic acid continuous fermentation was carried out in the same manner as in Example 1 except that the natural medium 1 containing 45 mg / L was changed.

  The natural medium 1 was prepared as follows. Among the nutrients shown in Table 2, the raw material sugar, amino acid, and ammonium sulfate, which is an inorganic salt, are divided into groups, sterilized by autoclaving for each group, cooled to a temperature below room temperature, and then total amino acid concentrations shown in Table 2 Was adjusted to 45 mg / L. The amino acids shown in Table 2 are shown in Table 2 by measuring the amino acid components and amino acid concentrations contained in the raw sugar using an amino acid analyzer, and diluting the 10-fold concentrated amino acid solution for the shortage of amino acids. The total amino acid concentration was added. Each amino acid includes alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or these The hydrate of was used. If the concentration of all amino acids contained in the raw sugar is higher than the concentration shown in Table 2, dilute the raw sugar to reduce the total amino acid concentration to the concentration shown in Table 2, and The sugar concentration deficient there was adjusted by adding sucrose.

  As a result of performing a fermentation test for 1120 hours, as shown in Table 4, by using this natural medium 1 and the continuous fermentation apparatus shown in FIG. 1, a longer time than the previous excellent continuous lactic acid fermentation (Comparative Example 1). It was also confirmed that stable production of lactic acid with high productivity was possible by continuous fermentation.

(Example 3) Production of L-lactic acid by continuous fermentation (part 3)
In order to investigate whether or not lactic acid can be obtained by operating the continuous fermentation apparatus shown in FIG. 2, a continuous fermentation test using the same apparatus was performed. Synthetic medium for lactic acid fermentation shown in Table 1 as in Example 1 wherein the yeast B2 strain prepared in Reference Example 1 described above was used as the microorganism, and the total amino acid concentration was adjusted to 0.9 g / L. 1 was used after high-pressure steam sterilization at a temperature of 121 ° C. for 15 minutes. For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. Moreover, the same porous membrane as Example 1 was used for the separation membrane. 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 3 are as follows unless otherwise specified.
・ Fermentation reactor capacity: 1.5 (L)
・ Membrane separation tank capacity: 0.5 (L)
-Separation membrane used: PVDF filtration membrane-Membrane separation element Effective filtration area: 120 square cm
・ Temperature adjustment: 30 (℃)
・ Aeration volume of fermentation reaction tank: 200 (mL / min)
・ Fermentation reactor stirring speed: 800 (rpm)
-PH adjustment: pH 5.0 adjusted with 8N NaOH-Sterilization: All the culture tanks containing the separation membrane element and the used medium were autoclaved at 121 ° C for 20 minutes under high pressure steam sterilization.
・ Membrane permeate control: Flow rate is controlled by the difference in the water head of the membrane separation tank (the head difference is controlled within 2m)
Evaluation of the concentration of L-lactic acid as a product is the same as the method for measuring lactic acid by HPLC shown in Example 1 above.

  Using the porous membrane prepared in Reference Example 2, L-lactic acid continuous fermentation was performed in the same medium and culture environment as in Example 1. As a result of conducting a 640 hour fermentation test, as shown in Table 4, by using this synthetic medium 1 for lactic acid fermentation and the continuous fermentation apparatus shown in FIG. 2, the previous excellent lactic acid continuous fermentation (Comparative Example 1) As a result, it was confirmed that production of stable lactic acid with a long time and high productivity was possible by continuous fermentation.

(Example 4) Production of L-lactic acid by continuous fermentation (part 4)
L-lactic acid continuous fermentation was performed in the same manner as in Example 3, using the porous membrane prepared in Reference Example 2 and the natural medium 1 shown in Table 2 as in Example 2. As a result of conducting the fermentation test for 1050 hours, by using this natural medium 1 and continuous fermentation apparatus as shown in Table 4, it is longer and has higher productivity than the previous excellent lactic acid continuous fermentation (Comparative Example 1). It was confirmed that production of stable lactic acid by continuous fermentation was possible.

(Example 5) Production of L-lactic acid by continuous fermentation (part 5)
Instead of the porous membrane mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 2, a porous membrane (hollow fiber) mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 3 as a separation membrane A continuous fermentation test was conducted in the same manner as in Example 1 except that the membrane) was used. As shown in Table 4, it was confirmed that production of stable lactic acid for a long time and high productivity by continuous fermentation was possible compared to the previous excellent lactic acid continuous fermentation (Comparative Example 1).

(Example 6) Production of L-lactic acid by continuous fermentation (part 6)
Instead of the porous membrane mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 2, a porous membrane (hollow fiber) mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 3 as a separation membrane A continuous fermentation test was conducted in the same manner as in Example 2 except that the membrane) was used. As shown in Table 4, it was confirmed that production of stable lactic acid for a long time and high productivity by continuous fermentation was possible compared to the previous excellent lactic acid continuous fermentation (Comparative Example 1).

(Example 7) Production of L-lactic acid by continuous fermentation (part 7)
Instead of the porous membrane mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 2, a porous membrane (hollow fiber) mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 3 as a separation membrane A continuous fermentation test was conducted in the same manner as in Example 3 except that the membrane) was used. As shown in Table 4, it was confirmed that production of stable lactic acid for a long time and high productivity by continuous fermentation was possible compared to the previous excellent lactic acid continuous fermentation (Comparative Example 1).

(Example 8) Production of L-lactic acid by continuous fermentation (part 8)
Instead of the porous membrane mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 2, a porous membrane (hollow fiber) mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 3 as a separation membrane A continuous fermentation test was conducted in the same manner as in Example 4 except that the membrane) was used. As shown in Table 4, it was confirmed that production of stable lactic acid for a long time and high productivity by continuous fermentation was possible compared to the previous excellent lactic acid continuous fermentation (Comparative Example 1).

(Example 9) Production of L-lactic acid by continuous fermentation (part 9)
Instead of the porous membrane mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 2, a porous membrane (hollow fiber) mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 3 as a separation membrane In the same manner as in Example 8, except that the medium was adjusted so that the total amino acid concentration was 1.5 g / L and the natural medium 2 for lactic acid fermentation shown in Table 3 was used. A continuous fermentation test was conducted.

  The natural medium 2 in Table 3 above was prepared as follows. Among the nutrients shown in Table 3, the raw material sugar, amino acid and inorganic salt ammonium sulfate are divided into groups, sterilized by autoclaving for each group, cooled to a temperature below room temperature, and then the total amino acid concentration shown in Table 3 Was adjusted to 1.5 g / L. The amino acids shown in Table 3 are shown in Table 3 by measuring the amino acid components and amino acid concentrations contained in the raw sugar using an amino acid analyzer, and diluting each 10-fold concentrated amino acid solution for the shortage of amino acids. The total amino acid concentration was added. Each amino acid includes alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or these The hydrate of was used. If the concentration of all amino acids contained in the raw sugar is higher than the concentration shown in Table 3, dilute the raw sugar to reduce the total amino acid concentration to the concentration shown in Table 3, and The sugar concentration deficient there was adjusted by adding sucrose.

  As shown in Table 4, it was confirmed that production of stable lactic acid for a long time and high productivity by continuous fermentation was possible compared to the previous excellent lactic acid continuous fermentation (Comparative Example 1).

(Comparative Example 1) Production of L-lactic acid by continuous fermentation (part 10)
L-lactic acid continuous fermentation was carried out in the same manner as in Example 3 except that the medium was a synthetic medium for lactic acid fermentation adjusted so that the total amino acid concentration shown in the following Table 5 was 3.6 g / L. It was.

  Synthetic medium 2 in Table 5 above was prepared as follows. Of the nutrients shown in Table 5, vitamins and metal salts are sterilized by filter treatment, and other nutrients are divided into sugar, amino acid, nucleic acid and inorganic salt groups, and each group is autoclaved. After sterilization and cooling to room temperature or lower, the total amino acid concentration shown in Table 5 was adjusted to 3.6 g / L. The amino acids shown in Table 5 were each added to a concentration of each amino acid shown in Table 5 by preparing a 10-fold concentration of each amino acid solution and diluting it. The core amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or these The hydrate of was used.

  As a result of performing a 300-hour fermentation test, stable production of lactic acid by continuous fermentation was possible by using this synthetic medium 2 for lactic acid fermentation and a continuous fermentation apparatus. However, as shown in Table 4, the amount and yield of lactic acid and the production rate were inferior to those of the examples of the present invention.

(Comparative Example 2) Production of L-lactic acid by continuous fermentation (part 11)
L-lactic acid continuous fermentation was carried out in the same manner as in Example 3 except that the medium was synthetic medium 3 for lactic acid fermentation adjusted so that the total amino acid concentration shown in the following Table 6 was 2.3 g / L. went.

  The above synthetic medium 3 in Table 6 was prepared as follows. Among the nutrients shown in Table 6, vitamins and metal salts are sterilized by filtering, and other nutrients are divided into sugar, amino acid, nucleic acid and inorganic salt groups, and each group is subjected to autoclaving. After sterilizing and cooling to a temperature below room temperature, the total amino acid concentration shown in Table 6 was adjusted to 2.3 g / L. The amino acids shown in Table 6 were each added to a concentration of each amino acid shown in Table 6 by preparing a 10-fold concentration of each amino acid solution, diluting it. The core amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or these The hydrate of was used.

  As a result of performing a 300-hour fermentation test, stable production of lactic acid by continuous fermentation was possible by using this synthetic medium 3 for lactic acid fermentation and a continuous fermentation apparatus. However, as shown in Table 4, the amount and yield of lactic acid and the production rate were inferior to those of the examples of the present invention.

FIG. 1 is a schematic side view for explaining one embodiment of the continuous fermentation apparatus of the present invention. FIG. 2 is a schematic side view for explaining an example of another continuous fermentation apparatus used in the present invention. FIG. 3 is a schematic perspective view for explaining one embodiment of the separation membrane element used in the present invention. FIG. 4 is a schematic cross-sectional explanatory diagram for explaining an example of another separation membrane element used in the present invention. FIG. 5 is a schematic diagram for explaining a method of introducing a lactate dehydrogenase gene (ldh gene) downstream of the pdc1 gene promoter. FIG. 6 is a diagram showing a physical map of the yeast expression vector pTRS11 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 (13)

The yeast fermentation broth having the ability to produce lactic acid is filtered through a separation membrane, the product is recovered from the filtrate, and the unfiltrated liquid is retained or refluxed in the fermentation broth, and the fermentation raw material is fed into the fermentation broth. A method for producing lactic acid by continuous fermentation added to a fermentation broth, wherein a porous membrane having an average pore diameter of 0.01 μm or more and less than 1 μm is used as the separation membrane, and the transmembrane pressure difference is 0.1 or more and 20 kPa. A method for producing lactic acid by continuous fermentation, wherein the total amino acid concentration in the fermentation raw material is adjusted to 2 g / L or less while being subjected to filtration treatment within a range of less than the range. 2. The pure water permeability coefficient of the porous membrane is 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 6 × 10 ⁻⁷ m ³ / m ² / s / pa or less. A method for producing lactic acid by continuous fermentation. 3. The continuous fermentation according to claim 1, wherein the porous membrane has an average pore diameter of 0.01 μm or more and less than 0.2 μm, and a standard deviation of the average pore diameter of 0.1 μm or less. A method for producing lactic acid. The method for producing lactic acid by continuous fermentation according to any one of claims 1 to 3, wherein the membrane surface roughness of the porous membrane is 0.1 µm or less. The method for producing lactic acid by continuous fermentation according to any one of claims 1 to 4, wherein the porous membrane is a porous membrane comprising a porous resin layer. The method for producing lactic acid by continuous fermentation according to any one of claims 1 to 5, wherein a polyvinylidene fluoride resin is used as a membrane material of the porous membrane. The method for producing lactic acid according to claim 1, wherein the fermentation raw material is a medium containing at least one selected from the group consisting of sucrose, fructose, glucose, galactose, lactose and maltose. Fermentation raw material is starch, starch hydrolysate, sugarcane molasses, sugar beet molasses, cane juice, sugar beet molasses or cane juice extract or concentrate, sugar beet molasses or cane juice filtrate, syrup (high test molasses), sugar beet 2. The medium comprising at least one selected from the group consisting of raw sugar refined or crystallized from molasses or cane juice and refined sugar crystallized from sugar beet molasses or cane juice or crystallized refined sugar A method for producing lactic acid. Lactic acid is L-lactic acid, The manufacturing method in any one of Claims 1-8. The method for producing lactic acid according to claim 1, wherein the yeast belongs to the genus Saccharomyces. The method for producing lactic acid according to claim 1 or 10, wherein the yeast is a yeast into which a lactic acid synthase gene has been introduced. The method for producing lactic acid according to claim 11, wherein the lactic acid synthase gene is a gene encoding an L-lactic acid dehydrogenase derived from Xenopus laevis. The method for producing lactic acid according to claim 12, wherein the gene encoding L-lactate dehydrogenase is a gene having the nucleic acid sequence shown in SEQ ID NO: 1.

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