JP5262004B2 - Process for producing lactic acid by continuous fermentation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing lactic acid by such a continuous fermentation process that its high productivity can be maintained stably over a long period of time under simple operating conditions. <P>SOLUTION: The method comprises separating a fermented culture liquid of prokaryotic microorganisms having the ability to produce lactic acid through a separation membrane into a filtrate and an unfiltrate, and retrieving lactic acid as the desired fermentation product from the filtrate while the unfiltrate is held in or refluxed into the fermented culture liquid. As the separation membrane, a hard-to-close specific porous membrane having both high permeability and cell-blocking ability is used, and a filtration treatment is conducted under a specific low intermembrane differential pressure, thus enabling the production efficiency of the objective lactic acid by fermentation to be markedly improved stably at low cost. <P>COPYRIGHT: (C)2008,JPO&amp;INPIT

Description

  The present invention relates to an improved method for culturing prokaryotic microorganisms capable of producing lactic acid. More specifically, the present invention efficiently filters and collects a liquid containing lactic acid from a fermentation culture solution of a prokaryotic microorganism capable of producing lactic acid through a porous separation membrane that is less likely to be clogged. The present invention relates to a method for producing lactic acid by continuous fermentation, which can improve the concentration of microorganisms involved in fermentation by returning to a fermentation broth and obtain high productivity.

  Fermentation methods, which are substance production methods that involve the cultivation of microorganisms and cultured cells, are largely divided into (1) batch fermentation methods (Batch fermentation methods) and fed-batch fermentation methods (Fed-Batch fermentation methods), and (2) continuous fermentation methods. Can be classified.

  The batch fermentation method and fed-batch fermentation method (1) are simple in terms of equipment, have an advantage that the culture is completed in a short time, and there is little damage due to contamination with bacteria. However, as the time elapses, the concentration of the product in the culture solution increases, 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 (2) can maintain high yield and high productivity over a long period of time by avoiding accumulation of the target substance at a high concentration in the fermenter. There are advantages.

  Next, the technical background regarding the method for producing lactic acid will be described. Lactic acid is mainly used as a substitute for conventional citric acid and tartaric acid as an acidulant, and is also used as an acidic detergent. In addition, lactic acid has uses such as pharmacopoeia acid drugs and skin antiseptics as pharmaceuticals. In addition, lactic acid has derivatives such as sodium salts (use for moisturizing agents such as cosmetics and foods) and calcium salts (use for calcium agents and tonics). In addition, lactic acid can be used as an alternative to ozone depleting solvents such as chlorofluorocarbon and trichloroethane as esters such as methyl ester and ethyl ester. Recently, the use of polylactic acid as a raw material has further expanded. Therefore, if this lactic acid can be produced efficiently, it can be expected that the supply of lactic acid to the above-mentioned various uses becomes easier.

  Currently, lactic acid is industrially produced mainly by batch fermentation using prokaryotic microorganisms called lactic acid bacteria or fungi of the genus Rhizopus, but the yield of lactic acid against sugar is higher when lactic acid bacteria are used. Is known (see Non-Patent Document 1).

  On the other hand, in order to further improve the productivity of lactic acid, studies on continuous lactic acid fermentation are being conducted. For example, Kobayashi et al. Are investigating continuous lactic acid fermentation by lactic acid bacteria using a ceramic filter. In order to ensure sufficient permeation performance in filtration of lactic acid bacteria, the filtration pressure is about 90 kPa. Reported the necessity of high transmembrane pressure and backwashing. Further, it is disclosed that the permeability is improved by increasing the circulation speed on the membrane surface, but at the same time, the shearing force on the membrane surface causes problems such as a decrease in the activity of microorganisms and rupture (Non-Patent Document 2). reference.).

  Similarly, an apparatus for performing continuous lactic acid fermentation using a ceramic filter has been proposed. In this apparatus, a gas such as air or nitrogen, water, Since liquids such as permeated membrane permeated water are added to the culture solution, it is difficult to maintain optimal culture conditions due to changes in the ability of microorganisms and cultured cells to produce substances. For this reason, there is a problem that the filtration membrane device for this is complicated (see Patent Document 1). Furthermore, a technique for cultivating lactic acid bacteria using a filtration membrane has also been proposed, but there are problems associated with back-washing operations as in the above-mentioned device, and suction by a pump is necessary during filtration, which complicates the filtration membrane device. There was a problem (see Patent Document 2).

That is, in the continuous fermentation method of lactic acid, microorganisms and cells are filtered through a separation membrane, the product is recovered from the filtrate, and at the same time, the filtered microorganisms and cells are refluxed to the fermentation broth, and the microorganisms and cell concentration in the fermentation broth It is still difficult to obtain high material productivity by improving and maintaining high, and technological innovation has been desired.
Bioindustry Association, Fermentation and Metabolism Study Group, “Fermentation Handbook”, 64-65, Kyoritsu Publishing (2001) Takeshi Kobayashi, Chemical Engineer, 12, 49 (1988) Japanese Patent Laid-Open No. 62-138184 Japanese Patent Laid-Open No. 2-174673

  Accordingly, an object of the present invention is to provide a method for producing lactic acid by a continuous fermentation method that can maintain high productivity stably over a long period of time with a simple operation method.

  As a result of diligent research, the present inventors have found that the separation membrane is clogged when the fermentation broth is filtered under the condition that the microorganisms and the cells enter the separation membrane and the transmembrane pressure is low. Has been found to be remarkably suppressed, and it has been possible to stably maintain the filtration of high concentration microorganisms, which has been a problem, for a long period of time, and the present invention has been completed.

The method for producing lactic acid of the present invention is a fermentation of prokaryotic microorganisms capable of producing lactic acid (excluding microorganisms belonging to the genus Bacillus and having the ability to produce D-lactic acid). The culture solution 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 raw material of the prokaryotic microorganism is added to the fermentation culture solution by continuous fermentation. In the method for producing lactic acid, 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 a filtration treatment is performed with a transmembrane pressure difference of 0.1 to 20 kPa. It is the manufacturing method of the lactic acid by the continuous fermentation characterized.

  Specifically, the present invention separates the fermentation broth into a filtrate and an unfiltered liquid through a separation membrane, collects lactic acid, which is a desired fermentation product, from the filtrate and holds the unfiltered liquid in the fermented broth or A method for producing lactic acid by a continuous fermentation method for refluxing, which uses the above-mentioned specific porous separation membrane that has high permeability and high cell blocking rate and is difficult to block, and performs filtration with the above-mentioned specific low transmembrane pressure. By this, the manufacturing method of lactic acid which improves fermentation production efficiency remarkably stably at low cost is provided.

According to the preferable aspect of the manufacturing method of lactic acid by continuous fermentation of this invention, the pure water permeability coefficient of the said porous membrane is 2 * 10 < ^-9 > m < ³ > / m < ² > / s / pa or more 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 0.1 μm or less. The film surface roughness is 0.1 μm or less.

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

  According to a preferred embodiment of the method for producing lactic acid by continuous fermentation of the present invention, the prokaryotic microorganism is a prokaryotic microorganism capable of producing lactic acid, and the prokaryotic microorganism capable of producing lactic acid is a lactic acid bacterium.

  According to a preferred embodiment of the method for producing lactic acid by continuous fermentation of the present invention, the lactic acid bacterium is a lactic acid bacterium belonging to the genus Lactococcus or Genus Lactobacillus, and the particularly preferred lactic acid bacterium is Lactococcus lactis. (Lactococcus lactis) and Lactobacillus casei.

  According to the preferable aspect of the manufacturing method of lactic acid by continuous fermentation of this invention, the said lactic acid is L-lactic acid.

  According to the present invention, continuous fermentation that maintains the high productivity of lactic acid that is a desired fermentation product stably over a long period of time under simple operation conditions is possible, and lactic acid that is a fermentation product widely in the fermentation industry. Can be stably produced at low cost. Lactic acid obtained by the method for producing lactic acid by continuous fermentation of the present invention is useful as a raw material for, for example, acidulants, detergents and pharmaceuticals, and polylactic acid resins.

  The present invention provides a fermentation culture solution of a prokaryotic microorganism capable of producing lactic acid through a separation membrane to collect a product from the filtrate, hold or reflux the unfiltrated liquid in the fermentation culture solution, and the prokaryotic microorganism. In which the fermentation raw material is added to the fermentation broth, 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 less than 20 kPa. It is a manufacturing method of lactic acid by continuous fermentation characterized by carrying out filtration processing.

  The porous membrane used as a separation membrane in the present invention is desirably one that is not easily clogged by prokaryotic microorganisms used for fermentation and has a performance that allows filtration performance to continue 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 structure of the porous membrane used as a separation membrane in the present invention will be described. The porous membrane in the present invention has separation performance and water permeation performance according to the quality of water to be treated and its use, and in terms of separation performance such as blocking performance, water permeation performance and dirt resistance, the porous resin layer It is preferable that the film contains a porous film. 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 organic fiber is preferably used among them. Preferred porous substrates are woven and non-woven fabrics using organic fibers such as cellulose fibers, cellulose triacetate fibers, polyester fibers, polypropylene fibers and polyethylene fibers. Among them, the density control is relatively easy and the production is also easy. An easy and inexpensive nonwoven fabric is 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. A cellulose triacetate-based resin may be used, and a mixture of these resins as a main component may be used. 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, the organic polymer membrane materials include polyvinyl chloride resins, polyvinylidene fluoride resins, polysulfone resins, polyethers that are easy to form in solution and have excellent physical durability and chemical resistance. A sulfone-based resin and a polyacrylonitrile-based resin are preferable, and a polyvinylidene fluoride-based resin or a resin containing the same as a main component is most preferably used.

  Here, as the polyvinylidene fluoride-based resin, a homopolymer of vinylidene fluoride is preferably used. In addition to a homopolymer of vinylidene fluoride, a copolymer of a vinyl monomer copolymerizable with vinylidene fluoride is used. A polymer is also preferably used. Examples of vinyl monomers copolymerizable with vinylidene fluoride include tetrafluoroethylene, hexafluoropropylene, and ethylene trichloride fluoride.

  An outline of a method for producing a porous membrane used in the present invention will be described by way of example.

  First, of the porous membrane, a flat membrane can be prepared as follows. A film of a stock solution containing the above-described resin and 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 may further contain a non-solvent. The temperature of the stock solution is usually preferably selected within the range of 15 to 120 ° C. from the viewpoint of film forming properties.

  Here, 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, and glycerin can be used.

  The solvent dissolves the resin. The solvent acts on the resin and the pore-opening agent to promote the action of them forming a porous resin layer. As the solvent, N-methylpyrrolidinone (NMP), N, N-dimethylacetamide (DMAc), N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone, methyl ethyl ketone, and the like are used. Among these, NMP, DMAc, DMF and DMSO, which have high resin solubility, are preferably used.

  Furthermore, a non-solvent can be added to the stock solution. 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 and methanol are preferably used from the viewpoint of price. The non-solvent may be a mixture thereof.

  Moreover, a hollow fiber membrane can be produced as follows among porous membranes. 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 at 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 adhering / sealing the hollow portion of the hollow fiber membrane using a member such as a resin and setting it on a support.

  As described above, the porous membrane used in the present invention may be a flat membrane or a hollow fiber membrane. In the case of a flat membrane, the average thickness is selected according to the application, but is preferably 20 μm or more and 5000 μm or less, and 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 from 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.

  The porous membrane used in the present invention can be made into a separation membrane element by combining with a support. The form of the separation membrane element having a porous membrane is not particularly limited, but the 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 the present invention. It is one of the suitable forms of the separation membrane element which has a porous membrane used by 1). In this embodiment, when it is difficult to increase the membrane area, it is also 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 above range, it is possible to achieve both a high exclusion rate without causing leakage of bacterial cells and sludge and high water permeability, and it is difficult to cause clogging. Can be carried out with higher accuracy and reproducibility. When bacteria are used, 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. Furthermore, since the standard deviation of the average pore diameter is small, that is, when the pore diameters are uniform, a uniform permeate can be obtained and the fermentation operation management becomes easy. Smaller is desirable if it is small.

  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, and the pure water permeability coefficient should be 2 × 10 ⁻⁹ m ³ / m ² / s / pa or more and 6 × 10 ⁻⁷ m ³ / m ² / s / pa or less. Thus, a practically sufficient amount of permeate can be obtained.

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

  In addition, since the membrane surface roughness of the porous membrane is low, it can be expected that the shearing force generated on the membrane surface will be reduced in the filtration of microorganisms, the destruction of microorganisms is suppressed, and the clogging of the porous membrane is also suppressed. By doing so, it is considered that stable filtration is possible for a long period of time.

Here, the film surface roughness is an average value of the height in the direction perpendicular to the film surface, and can be measured using the following atomic force microscope (AFM) under the following conditions.
・ Atomic force microscope (Nanoscope IIIa manufactured by Digital Instruments)
・ Conditions Tip SiN cantilever (Digital Instruments)
Scan 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).

  In the present invention, the transmembrane pressure difference when the prokaryotic microorganism is filtered through the separation membrane may be any condition as long as the prokaryotic microorganism and the medium components are not easily clogged, but the transmembrane pressure difference is 0.1 kPa or more and 20 kPa or less. It is important to carry out the filtration treatment within the range, and the transmembrane pressure difference is preferably in the range of 0.1 kPa to 10 kPa, more preferably in the range of 0.1 kPa to 5 kPa. When the range of the transmembrane pressure difference is exceeded, clogging of prokaryotic microorganisms and medium components occurs rapidly, leading to a decrease in the amount of permeated water, and may cause problems in continuous fermentation operation.

  As a driving force for filtration, it is possible to generate a transmembrane pressure difference in the porous membrane by a siphon utilizing a 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. The transmembrane pressure difference can be controlled by changing the liquid level difference between the fermentation broth and the porous membrane treated water, so there is no need to keep the inside of the fermentation reactor in a pressurized state, producing lactic acid. The ability to do is not reduced. Moreover, since there is no need to keep the inside of the fermentation reaction tank in a pressurized state, a power means for circulating the fermentation broth between the membrane separation tank and the fermentation reaction tank becomes unnecessary, and a porous membrane is installed in the fermentation reaction tank. This also makes it possible to reduce the size of the continuous fermentation apparatus.

  In addition, when a pump is used to generate the transmembrane pressure, the transmembrane pressure can be controlled by the suction pressure, and also by the gas or liquid pressure that introduces the pressure on the fermentation broth side. The transmembrane pressure can be controlled. 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.

  The prokaryotic fermentation raw material used in the present invention is not limited as long as it promotes the growth of the prokaryotic microorganism to be cultured and can satisfactorily produce lactic acid, which is the target fermentation product. In addition, a liquid medium or the like appropriately containing inorganic salts and, if necessary, organic micronutrients such as amino acids and vitamins are preferably used.

  Examples of the carbon source include sugars such as glucose, sucrose, fructose, galactose, lactose and maltose, starch saccharified liquid containing these sugars, sweet potato molasses, sugar beet molasses, high test molasses, cane juice, and cane juice extraction Product or concentrate, raw sugar purified or crystallized from cane juice, purified sugar crystallized or crystallized from cane juice, organic acids such as acetic acid and fumaric acid, alcohols such as ethanol, and glycerin Etc. 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.

  The carbon source may be added all at once at the start of culture, or may be added in portions during culture or continuously.

  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.

  Moreover, as said inorganic salt, a phosphate, magnesium salt, calcium salt, iron salt, manganese salt etc. can be suitably added and used, 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, a more preferable lactic acid production medium used in the method for producing lactic acid is a lactic acid production medium containing at least one selected from the group consisting of glucose, sucrose, fructose, galactose, lactose and maltose. More preferable lactic acid production medium used in the method for producing lactic acid is starch saccharified solution containing these sugars, sweet potato molasses, sugar beet molasses, high test molasses, cane juice, cane juice extract or concentrated solution, cane juice Selected from the group consisting of raw material sugar purified or crystallized from sugar, purified sugar crystallized or crystallized from cane juice, organic acids such as acetic acid and fumaric acid, alcohols such as ethanol, glycerin and the like More preferably, the medium is a lactic acid production medium containing at least one kind. In the present invention, these fermentation raw materials can be diluted as necessary.

  In the present invention, the fermentation broth refers to a broth obtained as a result of growth of microorganisms on the fermentation raw material, and the composition of the fermentation raw material to be added may be appropriately changed from the fermentation raw material composition at the start of the culture. When changing from the fermentation raw material composition to the composition of the fermentation raw material to be added, it is preferable to change the composition so as to increase the productivity of the target lactic acid. For example, by reducing the weight ratio of organic micronutrients such as nitrogen sources, inorganic salts, amino acids, and vitamins to the carbon source, it may be possible to reduce lactic acid production costs, that is, improve lactic acid productivity in a broad sense. is there. On the other hand, by increasing the weight ratio of organic micronutrients such as nitrogen sources, inorganic salts, amino acids and vitamins to the carbon source, lactic acid productivity may be improved.

  In the present invention, the concentration of fermentation raw materials such as sugars in the fermentation broth is preferably maintained at 5 g / l or less. The reason is to minimize the loss of the fermentation raw material due to the withdrawal of the culture solution. Therefore, the concentration of the fermentation raw material in the fermentation broth is desirably as small as possible.

  Prokaryotic fermentation is usually carried out in a range of pH 4 to 8 and temperature 20 to 40 ° C. The pH of the fermentation broth is adjusted to a predetermined value within the above range with an inorganic or organic acid, an alkaline substance, urea, calcium carbonate, ammonia gas, and the like.

  If it is necessary to increase the oxygen supply rate in prokaryotic fermentation, the oxygen concentration is maintained at 21% or higher by adding oxygen to the air, the culture solution is pressurized, the stirring speed is increased, or the aeration rate is increased. The following means 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 at the initial stage of culture to increase the microorganism concentration, and then continuous culture (pullout) may be started. Culture may be performed. It is possible to supply the fermentation raw material liquid and extract the culture from an appropriate time. The start time of the fermentation raw material supply and the withdrawal of the culture are not necessarily the same. Further, the supply of the fermentation raw material liquid and the withdrawal of the culture may be continuous or intermittent. What is necessary is just to add a nutrient required for microbial cell growth as shown above to a fermentation raw material liquid so that microbial cell growth may be performed continuously.

  The concentration of prokaryotic microorganisms in the fermentation broth should be maintained at a high level within the range where the environment of the fermentation broth is inappropriate for the growth of the microorganisms and the rate of death does not increase. Preferably, as an example, good production efficiency can be obtained by maintaining the dry weight at 5 g / L or more. In addition, the upper limit value of the concentration of prokaryotic microorganisms is not particularly limited as long as it does not cause problems in operation of the continuous fermentation apparatus or decrease in production efficiency.

  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 culture method for producing a product while growing cells. A plurality of fermentation reaction tanks may be used because the capacity of the fermentation reaction tank is small. In this case, 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, it is possible to continuously and efficiently extract the fermentation broth from the fermentation reaction tank while maintaining the prokaryotic microorganism in the fermentation reaction tank. It is also possible to efficiently produce the target chemical by changing the composition of the fermentation raw material solution after ensuring sufficient growth.

The prokaryotic microorganism used in the present invention has an ability to produce lactic acid (excluding microorganisms belonging to the genus Bacillus and having the ability to produce D-lactic acid) . Examples of such prokaryotic microorganisms include bacteria such as Escherichia coli and coryneform bacteria often used in the fermentation industry. The prokaryotic microorganism used may be isolated from the natural environment, or may be partially modified in nature by mutation or genetic recombination.

  The prokaryotic microorganism used in the present invention is a microorganism composed of prokaryotic cells. The most prominent feature of prokaryotic cells is that they do not have a structure called a cell nucleus (nucleus) in the cell. In that sense, it is clearly distinguished from a eukaryote having a cell nucleus (nucleus). In the present invention, among the prokaryotic microorganisms, lactic acid bacteria can be used more preferably. Here, lactic acid bacteria can be defined as prokaryotic microorganisms that produce 50% or more of lactic acid in terms of sugar yield with respect to consumed glucose.

Preferred lactic acid bacteria, for example, Lactobacillus (Genus Lactobacillus), the genus Pediococcus (Genus Pediococcus), tetra genomic genus (Genus Tetragenococcus), Carnot genus (Genus Carnobacterium), Ba Gokokkasu genus (Genus Vagococcus), leuco Genus Leuconostoc, Genus Oenococcus, Genus Atopobium, Streptococcus, Genus Enterococcus, Genus Enterococcus, Genus Enterococcus, Genus Enterococcus Lactic acid bacteria belonging to the genus Bacillus fine (Genus Bacillus) and the like.

  Among them, a lactic acid bacterium having a high yield of lactic acid with respect to sugar can be selected and preferably used for the production of lactic acid. Furthermore, among lactic acid bacteria, lactic acid bacteria having a high yield of L-lactic acid with respect to sugars can be selected and preferably used for the production of lactic acid. L-lactic acid is a kind of optical isomer of lactic acid and can be clearly distinguished from D-lactic acid which is an enantiomer thereof.

Examples of lactic acid bacteria having a high yield of L-lactic acid to saccharide include, for example, Lactobacillus yamanasiensis, Lactobacillus animalis, Lactobacillus bilis Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Casei (Lactobacillus casei), Lactobacillus delbruecki, Lactobacillus paracasei, Lactobacillus rhamnosus ractobacillus rhactus Shirasu-Ruminisu (Lactobacillus ruminis), Lactobacillus Saribarisu (Lactobacillus salivarius), Lactobacillus Shapii (Lactobacillus sharpeae), Pediococcus dexfenfluramine tri Nix (Pediococcus dextrinicus), and Lactococcus lactis (Lactococcus lactis) and the like, These can be selected and used for the production of L-lactic acid.

  Separation / purification of lactic acid contained in the filtration / separation fermentation broth produced by the method for producing lactic acid by continuous fermentation according to the present invention may be performed by combining conventionally known methods such as concentration, distillation and crystallization. it can.

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

  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 a separation membrane 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 membrane separation type continuous fermentation apparatus used in the present invention. In FIG. 1, a membrane separation type continuous fermentation apparatus basically includes a fermentation reaction tank 1 and a water head difference control apparatus 3. A porous membrane is incorporated in the separation membrane element 2 in the fermentation reaction tank 1. As the porous membrane and the separation membrane element, for example, the separation membrane and the separation membrane element disclosed in WO2002 / 064240 can be used. The separation membrane element will be described in detail later.

Next, the form of continuous fermentation by the membrane separation type continuous fermentation apparatus of FIG. 1 will be described. The culture medium is pumped continuously or intermittently into the fermentation reaction tank 1 by the culture medium supply pump 7. For medium-throw input before heat sterilization if necessary, it is possible to perform sterilization using heat sterilization or filter. At the time of fermentation production, the fermentation culture liquid in the fermentation reaction tank 1 is agitated with the agitator 5 in the fermentation reaction tank 1 as necessary, and the necessary gas is supplied from the gas supply device 4 to the fermentation reaction tank as necessary. 1 can be supplied. If necessary, the pH of the fermentation broth in the fermentation reaction tank 1 is adjusted by the pH sensor / control device 9 and the pH adjustment solution supply pump 8, and if necessary, the fermentation reaction tank 1 is adjusted by the temperature controller 10. The temperature of the fermentation broth inside can be adjusted. By doing in this way, fermentation production with higher productivity can be performed.

  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 control of dissolved oxygen and ORP, and the analysis device such as an online chemical sensor Thus, the concentration of lactic acid in the fermentation broth can be measured, and the physicochemical conditions can be controlled using it as an index. Further, the form of continuous or intermittent addition of the medium is not particularly limited, but the medium input amount and speed are appropriately determined using the measured value of the physicochemical environment of the fermentation broth by the instrumentation device as an index. Can be adjusted.

  The fermentation broth is filtered and separated into prokaryotic microorganisms and fermentation products by the separation membrane element 2 installed in the fermentation reaction tank 1 and taken out from the apparatus system. Further, the prokaryotic microorganisms filtered and separated can remain in the apparatus system to maintain a high concentration of prokaryotic microorganisms in the apparatus system, thereby enabling a highly productive fermentation production. Here, the filtration / separation by the separation membrane element 2 is performed by the water head differential pressure with respect to the water surface of the fermentation reaction tank 1, and no special power is required. Moreover, the filtration / separation speed of the separation membrane element 2 and the amount of the fermentation liquid in the fermentation reaction tank 1 can be appropriately adjusted by the level sensor 6 and the head differential pressure control device 3 as necessary. If necessary, the amount of the fermentation raw material added to the apparatus system is appropriately adjusted according to the weight of the filtrate from the separation membrane element 2, and the amount of the fermentation broth in the fermentation reaction tank 1 is appropriately adjusted. be able to. Although the filtration / separation by the separation membrane element 2 is exemplified by the water head differential pressure, the filtration / separation can be performed by suction filtration with a pump or the like or pressurizing the inside of the apparatus system as necessary.

  Moreover, in FIG. 1, a prokaryotic microorganism can be cultured by continuous fermentation in another culture tank (not shown), and can be supplied into the fermentation reaction tank 1 as necessary. By culturing prokaryotic microorganisms by continuous fermentation in a separate culture tank and supplying the culture solution to the fermentation reaction tank 1 as necessary, continuous fermentation with prokaryotic microorganisms that are always fresh and have a high production capacity for lactic acid becomes possible. , Continuous fermentation with high production performance can be maintained for a long time.

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

  In FIG. 2, the membrane separation type continuous fermentation apparatus basically includes a fermentation reaction tank 1, a membrane separation tank 12, and a water head difference control device 3. And the membrane separation tank 12 is connected to the fermentation reaction tank 1 via the fermentation liquid circulation pump 11, and the separation membrane element 2 is arrange | positioned in the inside. Here, a porous membrane is incorporated in the separation membrane element 2. As the porous membrane and the separation membrane element, for example, the separation membrane and the separation membrane element disclosed in International Publication No. 2002/064240 are suitably used.

  In FIG. 2, the 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 4 as needed. 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. Therefore, 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 liquid circulation pump 11. The fermentation broth containing the fermentation product is filtered and separated into the prokaryotic microorganism and the fermentation product by the separation membrane element 2 and can be taken out from the apparatus system. Further, the prokaryotic microorganisms filtered and separated can remain in the apparatus system to maintain a high concentration of prokaryotic microorganisms in the apparatus system, thereby enabling a highly productive fermentation production.

  Here, the filtration / separation by the separation membrane element 2 is performed by the water head differential pressure with the water surface of the membrane separation tank 12, and no special power is required. Further, if necessary, the filtration / separation speed of the separation membrane element 2 and the amount of fermentation liquid in the apparatus system can be appropriately adjusted by the level sensor 6 and the head differential pressure control device 3. Moreover, the gas required by the gas supply apparatus 4 can be supplied in the membrane separation tank 12 as needed. 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.

  As described above, the filtration / separation by the separation membrane element 2 is exemplified by the hydraulic head differential pressure. However, if necessary, the filtration / separation may be performed by suction filtration with a pump or the like, or pressurizing the apparatus system. it can.

  Moreover, in FIG. 2, a prokaryotic microorganism can be cultured by continuous fermentation in another culture tank (not shown), and can be supplied into the fermentation reaction tank 1 as necessary. By culturing prokaryotic microorganisms by continuous fermentation in a separate culture tank and supplying the culture solution to the fermentation reaction tank 1 as necessary, continuous fermentation with prokaryotic microorganisms that are always fresh and have a high production capacity for lactic acid becomes possible. , Continuous fermentation with high production performance can be maintained for a long time.

  Next, with respect to the separation membrane element used in the present invention, the separation membrane and the separation membrane element disclosed in the pamphlet of International Publication No. 2002/064240, which is an example of a preferred embodiment, are outlined below with reference to the drawings. explain. FIG. 3 is a schematic perspective view for explaining one embodiment of the separation membrane element used in the present invention.

  As shown in FIG. 3, the separation membrane element is configured by arranging a flow path material 14 and the separation membrane 15 (flat 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 fermentation broth. The flow path member 14 is for efficiently flowing the permeated water filtered by the separation membrane 15 to the support plate 13. The permeated water 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 through the water collecting pipe 17. As power for extracting the permeated water, a method such as a water head differential pressure, 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 permeate 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 permeated water filtered by the separation membrane bundle 18 passes through the hollow portion of the hollow fiber membrane and is taken out of the continuous fermentation apparatus via the water collecting pipe 22. As power for extracting the permeated water, a method such as a water head differential pressure, 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 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 members constituting the separation membrane element are preferably resistant to 15 minutes at 121 ° C., which is the condition of the high-pressure steam sterilization operation. 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 according to the present invention, the separation membrane element may be installed in the fermentation reaction tank as shown in FIG. 1 or installed outside the fermentation reaction tank as shown in FIG. Also good. 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, and the fermentation culture solution is placed between the fermentation reaction tank and the membrane separation tank. While circulating, the fermentation broth can be continuously filtered by the separation membrane element.

  In the continuous fermentation apparatus used in 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 method for producing lactic acid by continuous fermentation of the present invention, a high volumetric production rate is obtained and extremely efficient fermentation production is possible as compared with conventional batch fermentation. Here, the fermentation production rate in continuous culture is calculated by the following (Equation 3).
Fermentation production rate (g / L / hr)
= Product concentration in the extracted liquid (g / L) × fermentation liquid extraction speed (L / hr)
÷ Operating fluid volume of the device (L) (Equation 3)
The fermentation production rate by batch culture is calculated by dividing the amount of product (g) at the time of consumption of the raw carbon source by the time (h) required for consumption of the carbon source and the amount of culture solution (L) at that time. Is required.

  Lactic acid obtained by the method for producing lactic acid by continuous fermentation of the present invention is mainly used as an acidulant as a substitute for conventional citric acid or tartaric acid, and is also used as an acidic detergent. In addition, lactic acid has uses such as pharmacopeia acid drugs and skin antiseptics as pharmaceuticals. Lactic acid also has applications as derivatives such as sodium salts (for moisturizing agents such as cosmetics and foods) and calcium salts (for calcium agents and tonics). In addition, lactic acid also has uses such as methyl ester and ethyl ester as substitutes for ozone depleting solvents such as chlorofluorocarbon and trichloroethane. Recently, lactic acid is widely used as a raw material for polylactic acid. By using the method for producing lactic acid by continuous fermentation according to the present invention, it is possible to efficiently produce lactic acid with a wide range of uses, and thus it is possible to provide lactic acid at a lower cost.

  Hereinafter, in order to explain the production method of lactic acid by continuous fermentation of the present invention in more detail, examples of continuous fermentation of lactic acid by using the continuous fermentation apparatus shown in the schematic diagrams of FIG. 1 and FIG. Will be described. The present invention is not limited to these examples.

  In the following examples relating to the method for producing lactic acid, among the Lactococcus lactis, among the Lactococcus lactis, the lactic acid bacteria Lactococcus lactis JCM7638 strain and Lactobacillus casei Lactobacillus casei NRIC1941 strain, which is a lactic acid bacterium, was used. Moreover, glucose or cane juice was used as the carbon source among the fermentation raw materials, and the raw materials described in the following examples were used for the nitrogen source and inorganic salts.

(Example 1) Production of 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. The lactic acid fermentation medium for Lactococcus lactis shown in Table 1 was used as the medium, and it was used after being subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes. For the separation membrane element member, a molded product of stainless steel and polysulfone resin was used. As the separation membrane, a porous membrane made of polyvinylidene fluoride (PVDF) as a main component and produced in Reference Example 1 described later 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: 37 (℃)
・ Aeration volume of fermentation reaction tank: 200 (mL / min)
・ Fermentation reactor stirring speed: 600 (rpm)
・ Sterilization: The culture tank containing the separation membrane element and the medium used are all autoclaved at 121 ° C for 20 minutes in an autoclave.
-PH adjustment: pH adjusted to 6.5 with 8N ammonia aqueous solution.-Membrane permeated water amount control: Flow rate was controlled by membrane separation tank water head difference (water head difference was controlled within 2 m).

Lactococcus lactis JCM7638 strain was used as a prokaryotic microorganism, and a lactic acid fermentation medium for Lactococcus lactis having the composition shown in Table 1 was used as the medium. 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 Lactococcus lactis JCM7638 strain was statically cultured at a temperature of 37 ° C. for 24 hours in a lactic acid fermentation medium for Lactococcus lactis purged with 5 ml of nitrogen gas shown in Table 1 in a test tube (pre-culture). . The obtained culture solution was inoculated into 50 ml of a fresh lactic acid fermentation medium purged with nitrogen gas, and statically cultured at a temperature of 37 ° C. for 48 hours (pre-culture). The culture broth was inoculated in 1 L of lactic acid fermentation medium purged with nitrogen gas of the continuous fermentation apparatus shown in FIG. 1, the fermentation reaction tank 1 was stirred at 600 rpm by the attached stirrer 5, and the fermentation reaction tank 1 was aerated. The amount was adjusted and the temperature was adjusted to a temperature of 37 ° 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. Table 2 shows the lactic acid production rate calculated from the input glucose calculated from the lactic acid and glucose concentrations. As a result of conducting a fermentation test for 400 hours, it was confirmed that stable production of lactic acid by continuous fermentation was possible by using this continuous fermentation apparatus.

(Example 2) Production of lactic acid by continuous fermentation (part 2)
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. The lactic acid fermentation medium for Lactococcus lactis shown in Table 1 was used as the medium, and it was used after being subjected to high-pressure steam sterilization at 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 2 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: 37 (℃)
-Aeration volume of fermentation reaction tank: 200 (L / min)
・ Fermentation reactor stirring speed: 600 (rpm)
・ Sterilization: The culture tank containing the separation membrane element and the medium used are all autoclaved at 121 ° C for 20 minutes in an autoclave.
-PH adjustment: pH adjusted to 6.5 with 8N ammonia aqueous solution.-Membrane permeated water amount control: Flow rate was controlled by membrane separation tank water head difference (water head difference was controlled within 2 m).

  As a prokaryotic microorganism, Lactococcus lactis JCM7638 strain is used, and a lactic acid fermentation medium for Lactococcus lactis having the composition shown in Table 1 is used as a medium, and lactic acid and glucose as products are the method shown in Example 1. Measured with

  First, the Lactococcus lactis JCM7638 strain was statically cultured at a temperature of 37 ° C. for 24 hours in a lactic acid fermentation medium for Lactococcus lactis purged with 5 ml of nitrogen gas shown in Table 1 in a test tube (pre-culture). . The obtained culture solution was inoculated into 50 ml of a fresh lactic acid fermentation medium purged with nitrogen gas, and statically cultured at a temperature of 37 ° C. for 48 hours (pre-culture). The culture broth was inoculated in 1 L of lactic acid fermentation medium purged with nitrogen gas from the membrane separation type continuous fermentation apparatus shown in FIG. 2, and the fermentation reaction tank 1 was stirred at 600 rpm by the attached stirrer 5 to give a fermentation reaction tank. The aeration rate was adjusted to 1 and the temperature was adjusted to 37 ° C., followed by culturing for 24 hours (pre-culture). Immediately after the completion of the pre-culture, the continuous and batch fermentation medium is continuously supplied, and the continuous fermentation apparatus is continuously cultured while controlling the amount of permeated water so that the amount of the fermentation broth is 1.5 L. Manufactured. 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. Table 2 shows the lactic acid production rate calculated from the input glucose calculated from the lactic acid and glucose concentrations. As a result of conducting a fermentation test for 400 hours, it was confirmed that stable production of lactic acid by continuous fermentation was possible by using this continuous fermentation apparatus.

(Example 3) Production of lactic acid by continuous fermentation (part 3)
In order to examine whether lactic acid can be obtained by continuous fermentation, a continuous fermentation test was conducted using the secondary fermentation apparatus shown in FIG. The lactic acid fermentation medium for Lactobacillus casei shown in Table 3 was used as the medium, and it was used after being subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes. The characteristics of the separation membrane element member, the separation membrane, and the porous membrane are the same as those described in Example 1. The operating conditions in Example 3 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: 100 (mL / min)
・ Fermentation reactor stirring speed: 800 (rpm)
・ Sterilization: The culture tank containing the separation membrane element and the medium used are all autoclaved at 121 ° C for 20 minutes in an autoclave.
-PH adjustment: pH adjusted to 6.0 with 8N calcium hydroxide aqueous solution.-Membrane permeated water amount control: Flow rate was controlled by membrane separation tank water head difference (water head difference was controlled within 2 m).

  As a prokaryotic microorganism, Lactobacillus casei NRIC1941 strain was used, and a lactic acid fermentation medium for Lactobacillus casei having the composition shown in Table 3 was used as a medium. Lactic acid contained in the fermentation broth was confirmed by measuring under the same conditions as in Example 1.

  The optical purity of L-lactic acid is calculated in the same manner as in Example 1. Further, “glucose test Wako C” (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used for measuring the glucose concentration, and F-kit (manufactured by Roche) was used for measuring the sucrose concentration and fructose concentration. .

  First, the Lactobacillus casei NRIC1941 strain was statically cultured at a temperature of 30 ° C. for 24 hours in a lactic acid fermentation medium for Lactobacillus casei purged with 5 ml of nitrogen gas shown in Table 3 in a test tube (pre-culture). The obtained culture broth was inoculated into 50 ml of a fresh lactic acid fermentation medium purged with nitrogen gas and statically cultured at a temperature of 30 ° C. for 24 hours (pre-culture). The culture broth was inoculated in 1 L of lactic acid fermentation medium purged with nitrogen gas from the continuous fermentation apparatus shown in FIG. 1, and the fermentation reaction tank 1 was stirred at 800 rpm with the attached stirrer 5. 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. Where appropriate, the produced lactic acid concentration and residual sucrose, fructose and glucose concentrations in the membrane permeation fermentation broth were measured. The measured total sucrose, fructose and glucose concentrations were converted to total glucose concentrations. Table 2 shows the lactic acid production rate calculated from the input glucose calculated from the lactic acid and residual glucose concentrations. As a result of conducting a fermentation test for 500 hours, it was confirmed that stable production of lactic acid by continuous fermentation was possible by using this continuous fermentation apparatus.

(Example 4) Production of lactic acid by continuous fermentation (part 4)
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. The lactic acid fermentation medium for Lactobacillus casei shown in Table 3 was used as the medium, and it was used after being subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes. The characteristics of the separation membrane element member, the separation membrane, and the porous membrane are the same as those described in Example 2. The operating conditions in Example 4 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: 100 (L / min)
・ Fermentation reactor stirring speed: 800 (rpm)
・ Sterilization: The culture tank containing the separation membrane element and the medium used are all autoclaved at 121 ° C for 20 minutes in an autoclave.
-PH adjustment: pH adjusted to 6.0 with 8N calcium hydroxide aqueous solution-Membrane permeate flow rate control: Flow rate is controlled by membrane separation tank water head difference (water head difference was controlled within 2 m).

As prokaryotic microorganism, using La Kutobashirasu casei NRIC1941 strain, using the Lactobacillus casei for lactic acid fermentation medium having the composition shown in Table 3 as a medium, lactic acid and glucose is the product was measured by the method shown in Example 1.

  Moreover, F-kit (made by Roche) was used for the measurement of sucrose concentration and fructose concentration.

First, La Kutobashirasu casei NRIC1941 strain, 24 hours Lactobacillus casei for lactic acid fermentation medium was purged with nitrogen gas 5ml shown in Table 3 in a test tube, and static culture at a temperature of 30 ° C. (before last s culture) . The obtained culture broth was inoculated into 50 ml of a fresh lactic acid fermentation medium purged with nitrogen gas and statically cultured at a temperature of 30 ° C. for 24 hours (pre-culture). The culture broth was inoculated in 1 L of lactic acid fermentation medium purged with nitrogen gas from the continuous fermentation apparatus shown in FIG. 2, the fermentation reaction tank 1 was stirred at 800 rpm by the attached stirrer 5, and the fermentation reaction tank 1 was aerated. 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 continuous and batch fermentation medium is continuously supplied, and the continuous fermentation apparatus is continuously cultured while controlling the amount of permeated water so that the amount of the fermentation broth is 1.5 L. Manufactured. 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. Where appropriate, the produced lactic acid concentration and residual sucrose, fructose and glucose concentrations in the membrane permeation fermentation broth were measured. The measured total sucrose, fructose and glucose concentrations were converted to total glucose concentrations. Table 2 shows the lactic acid production rate calculated from the input glucose calculated from the lactic acid and residual glucose concentrations. As a result of conducting a fermentation test for 500 hours, it was confirmed that stable production of lactic acid by continuous fermentation was possible by using this continuous fermentation apparatus.

(Example 5) Production of lactic acid by continuous fermentation (part 5)
Instead of the porous membrane mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 1, a porous membrane (hollow fiber) mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 2 as a separation membrane A continuous fermentation test was conducted in the same manner as in Example 3 except that the membrane) was used. Table 2 shows the lactic acid production rate calculated from the input glucose calculated from the lactic acid and the total sugar concentration. As a result of conducting a fermentation test for 500 hours, it was confirmed that stable production of lactic acid by continuous fermentation was possible by using this continuous fermentation apparatus.

(Example 6) Production of lactic acid by continuous fermentation (part 6)
Instead of the porous membrane mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 1, a porous membrane (hollow fiber) mainly composed of polyvinylidene fluoride (PVDF) prepared in Reference Example 2 as a separation membrane A continuous fermentation test was conducted in the same manner as in Example 4 except that the membrane) was used. Table 2 shows the lactic acid production rate calculated from the input glucose calculated from the lactic acid and the total sugar concentration. As a result of conducting a fermentation test for 500 hours, it was confirmed that stable production of lactic acid by continuous fermentation was possible by using this continuous fermentation apparatus.

(Comparative Example 1) Production of lactic acid by batch fermentation The most typical batch fermentation as a fermentation form using prokaryotic microorganisms was performed using a 2 L jar fermenter, and the lactic acid productivity was evaluated. The lactic acid fermentation medium for Lactococcus lactis shown in Table 1 was used as the medium, and it was used after being subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes. In Comparative Example 1, Lactococcus lactis JCM7638 strain was used as a prokaryotic microorganism, and the concentration of lactic acid as a product was evaluated using the method shown in Example 1. Test Wako C "(registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used. The operating conditions of Comparative Example 1 are as follows.
-Fermentation reaction tank capacity (lactic acid fermentation medium amount): 1.5 (L)
・ Temperature adjustment: 37 (℃)
・ Aeration volume of fermentation reaction tank: 200 (mL / min)
・ Fermentation reactor stirring speed: 200 (rpm)
-PH adjustment: adjusted to pH 6.5 with 8N aqueous ammonia solution First, Lactococcus lactis JCM7638 strain was purged with 5 ml of nitrogen gas shown in Table 1 in a test tube in a lactic acid fermentation medium for Lactococcus lactis for 24 hours at 37 ° C. The culture was stationary at the temperature of (pre-culture). The obtained culture solution was inoculated into 50 ml of a fresh lactic acid fermentation medium purged with nitrogen gas, and statically cultured at a temperature of 37 ° C. for 48 hours (preculture). The preculture was inoculated into a 1.5 L jar fermenter continuous / batch fermentation medium shown in Table 1 and batch fermentation was performed. The results of batch fermentation are shown in Table 2 in comparison with the lactic acid fermentation productivity obtained in the continuous fermentation test of Example 1 and Example 2 above.

(Comparative example 2) Production of lactic acid by batch fermentation The most typical batch fermentation as a fermentation form using prokaryotic microorganisms was performed using a 2 L jar fermenter, and the lactic acid productivity was evaluated. The lactic acid fermentation medium for Lactobacillus casei shown in Table 3 was used as the medium, and it was used after being subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes. In Comparative Example 2, Lactobacillus casei NRIC1941 strain was used as a prokaryotic microorganism, and the concentration of lactic acid as a product was evaluated using the method shown in Example 1. The glucose concentration was measured using “Glucose Test Wako”. C ″ (registered trademark) (manufactured by Wako Pure Chemical Industries, Ltd.) was used. Further, F-kit (Roche) was used for measurement of sucrose concentration and fructose concentration. The operating conditions of Comparative Example 2 are as follows.
-Fermentation reaction tank capacity (lactic acid fermentation medium amount): 1.5 (L)
・ Temperature adjustment: 30 (℃)
・ Aeration volume of fermentation reaction tank (nitrogen gas): 100 (mL / min)
・ Fermentation reactor stirring speed: 120 (rpm)
-PH adjustment: pH is adjusted to 6.0 with 8N calcium hydroxide aqueous solution.

  First, Lactobacillus casei NRIC1941 strain was statically cultured at a temperature of 30 ° C. for 24 hours in a lactic acid fermentation medium for Lactobacillus casei purged with 5 ml of nitrogen gas shown in Table 3 in a test tube (pre-culture). The obtained culture broth was inoculated into 50 ml of a fresh lactic acid fermentation medium purged with nitrogen gas, and statically cultured at a temperature of 30 ° C. for 24 hours (preculture). The preculture was inoculated into a lactic acid fermentation medium for Lactobacillus casei shown in Table 3 of 1.5 L of jar fermenter, and batch fermentation was performed. The results of batch fermentation are shown in Table 2 in comparison with the lactic acid fermentation productivity obtained in the continuous fermentation test of Examples 3 to 6 above.

  As a result of comparison, it was revealed that the production rate of lactic acid was greatly improved by using the membrane-separated continuous fermentation apparatus of FIGS. That is, using a membrane separation type continuous fermentation apparatus incorporating a porous membrane disclosed by the present invention, by controlling the transmembrane pressure difference, the fermentation broth is separated into a filtrate and an unfiltered liquid by the separation membrane, It is clear that a desired fermentation product can be recovered from the filtrate and a continuous fermentation method in which the unfiltrated liquid is returned to the fermentation broth can be produced, and lactic acid can be produced by continuous fermentation while maintaining a high amount of microorganisms. became.

Reference Example 1 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.
・ PVDF: 13.0% by weight
DMAc: 87.0% by weight
Next, after cooling the undiluted solution to a temperature of 25 ° C., it was applied to a non-woven fabric made of polyester fiber having a density of 0.48 g / cm ³ and a thickness of 220 μm. It was immersed in a coagulation bath having the following composition at a temperature of 25 ° C. for 5 minutes to obtain a porous substrate on which a porous resin layer was formed.
-Water: 30.0% by weight
DMAc: 70.0% by weight
The porous substrate was peeled off from the glass plate and then immersed in hot water at a temperature of 80 ° C. three times to wash out DMAc to obtain a separation membrane (flat membrane porous membrane) having a total film thickness of 350 μm. When the surface of the porous resin layer on the side of the separation membrane in contact with the coagulation bath is observed with a scanning electron microscope at a magnification of 10,000 times within the range of 9.2 μm × 10.4 μm, the pores that can be observed The average of all the diameters was 2.0 μm, and when the scanning electron microscope observation was performed at a magnification of 10,000 times within the range of 9.2 μm × 10.4 μm of the porous resin layer surface on the opposite side, The average diameter of all the observable pores was 0.1 μm. Next, when the permeation amount of pure water was evaluated for the separation membrane, it was 50 × 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.

(Reference Example 2) Production of porous membrane (part 2)
A vinylidene fluoride homopolymer having a weight average molecular weight of 41,000 and γ-butyrolactone were blended in a proportion of 38% by weight and 62% by weight, respectively, and mixed and dissolved at a temperature of 170 ° C. to prepare a stock solution. The stock solution thus obtained 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. A membrane was prepared.

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. The stock solution thus obtained was uniformly applied to the surface of the hollow fiber membrane obtained above, and a porous membrane (hollow fiber membrane) used in the present invention was immediately solidified in a water bath. The hollow fiber porous membrane thus obtained had an inner diameter of 900 μm and a film thickness of 600 μm, and the average pore size of the surface of the hollow fiber porous membrane on the water to be treated was 0.05 μm. Next, the pure water permeation rate of the hollow fiber membrane (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.

  According to the method for producing lactic acid by the continuous fermentation method of the present invention, it is possible to perform continuous fermentation that maintains high productivity of lactic acid, which is a desired fermentation product, stably over a long period of time under simple operation conditions. In industry, lactic acid, which is a fermentation product, can be stably produced at low cost. Lactic acid obtained by the method for producing lactic acid by continuous fermentation of the present invention is useful, for example, as a food additive or a raw material for polylactic acid resin.

FIG. 1 is a schematic side view for explaining one embodiment of a membrane separation type continuous fermentation apparatus used in the present invention. FIG. 2 is a schematic side view for explaining one embodiment of another membrane separation type continuous fermentation apparatus used in the present invention. FIG. 3 is a schematic perspective view for explaining one embodiment of the separation membrane element used in the present invention. FIG. 4 is a schematic perspective view for explaining an example of another separation membrane element 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 (10)

Filtering the fermentation broth of prokaryotic microorganisms capable of producing lactic acid (except microorganisms belonging to the genus Bacillus and having the ability to produce D-lactic acid) through a separation membrane, A method for producing lactic acid by continuous fermentation in which a product is recovered from a filtrate and an unfiltrated liquid is retained or refluxed in the fermentation broth, and a fermentation raw material of the prokaryotic microorganism is added to the fermentation broth, Production of lactic acid by continuous fermentation, characterized in that 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 filtration is performed with a transmembrane pressure difference in the range of 0.1 to 20 kPa. Method. The continuous water fermentation according to claim 1, wherein the porous membrane has a pure water permeability coefficient of 2 × 10 ⁻⁹ m ³ / m ² / s / pa to 6 × 10 ⁻⁷ m ³ / m ² / s / pa. A method for producing lactic acid.   The method for producing lactic acid by continuous fermentation according to claim 1 or 2, 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 is 0.1 µm or less.   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 includes a porous resin layer, and the material of the porous resin layer is a polyvinylidene fluoride resin. 6. The method for producing lactic acid by continuous fermentation according to any one of claims 1 to 5 , wherein the prokaryotic microorganism is a lactic acid bacterium. The method for producing lactic acid by continuous fermentation according to claim 6, wherein the lactic acid bacterium is a lactic acid bacterium belonging to the genus Lactococcus or Genus Lactobacillus. The method for producing lactic acid by continuous fermentation according to claim 6 or 7, wherein the lactic acid bacterium is Lactococcus lactis. The method for producing lactic acid by continuous fermentation according to claim 6 or 7, wherein the lactic acid bacterium is Lactobacillus casei. The method for producing lactic acid by continuous fermentation according to any one of claims 1 to 9 , wherein the lactic acid is L-lactic acid.

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