JP5458536B2 - Method for producing lactic acid and additive for lactic acid fermentation - Google Patents

Description

  The present invention relates to the production of lactic acid by fermentation, and in particular, to the provision of a new additive for lactic acid fermentation in place of lactic acid fermentation using molasses and a method for producing lactic acid using the additive.

  In order to produce lactic acid, which is a raw material of polylactic acid, which is one of plant-derived plastics, at low cost by fermentation, the present applicants have already introduced an L-lactic acid dehydrogenase gene into wine yeast with high alcohol fermentation ability. Has succeeded in the development of a genetically modified yeast that produces L-lactic acid with high efficiency (Patent Document 1).

  In general, in fermentation on an industrial scale, nutrient efficiency such as vitamins and minerals is added to a medium to increase fermentation efficiency. Waste molasses are used in alcoholic fermentation. Waste molasses is a by-product of sugar production and is a dark brown viscous liquid containing components other than sugar such as vitamins and minerals. Therefore, it is an excellent medium additive for fermentation.

  On the other hand, in alcoholic fermentation using yeast, it is disclosed to use a defatted soybean-derived soybean peptide material as an additive (Patent Document 2).

  Furthermore, there is also a report describing that the resistance to freezing stress of yeast is improved by adding a soybean peptide material to the medium (Non-patent Document 1).

JP 2003-164295 A JP 2006-238877 Izawa et al., Appl. Microbiol. Biotechnol. Vol. 75 (2007), p533-537

  Since molasses is an excellent fermentation medium additive rich in components other than sugar, it can also be used as a medium additive for lactic acid fermentation. However, according to the present inventors, the molasses is a black-brown liquid, and when the content of the molasses is increased in order to obtain a high amount of lactic acid, the fermentation solution is colored or contains extra components. It has been found that the quality of polylactic acid may be reduced. For this reason, when waste molasses was used as a culture medium additive in lactic acid fermentation, it was found that an additional purification process such as electrodialysis and decolorization was required to remove colored components derived from waste molasses. The addition of such a process becomes a big bottleneck in efficiently producing polylactic acid. In addition, an increase in the amount of molasses used will greatly affect the lactic acid production cost.

  That is, in lactic acid fermentation, it was found that molasses is nutritionally excellent, but is not always preferable in view of process and cost. Therefore, it is required that lactic acid can be efficiently produced even in a medium mainly composed of general-purpose raw materials such as glucose and sucrose as a saccharide raw material by avoiding or suppressing the use of waste molasses.

  Moreover, although the soybean peptide raw material is used as an additive for various fermented foods such as beer-like beverages, the soy peptide material is used for improving the properties (such as flavor) of the obtained food due to the properties of the food. Whether soy peptide is used in an oligotrophic medium has no effect on fermentation acceleration.

  Furthermore, Non-Patent Document 1 only discloses the knowledge specialized in the refrigeration stress of baker's yeast by the soy peptide material, and does not mention fermentability or lactic acid fermentation.

  As described above, at present, a medium, a medium additive, and a lactic acid production method suitable for industrial lactic acid fermentation for obtaining lactic acid as an industrial raw material have not yet been provided.

  Then, in view of such a situation, the present invention aims to provide a lactic acid fermentation technique that is more practical as an industrial raw material by avoiding or suppressing the use of waste molasses.

  As a result of various studies on the soy peptide material, the present inventors have found that the specific soy peptide material has an excellent effect of promoting lactic acid fermentation. That is, it was found that high lactic acid productivity can be realized by adding an additive containing a fraction of a predetermined soybean peptide material according to the present invention to a medium consisting only of sugars such as glucose and sucrose. It has also been found that the fermentation broth thus obtained has high transparency and can simplify or omit a special purification process. The present inventors have completed the present invention based on these findings. That is, according to the present invention, the following means are provided.

  According to the present invention, there is provided a method for producing lactic acid comprising a carbon source and a soybean peptide material having a molecular weight distribution in which a fraction of 1000 Da or less is 98% or more, and the absorbance at 400 nm is 0.1 or less. There is provided a production method comprising a step of culturing lactic acid-producing microorganisms and lactic acid fermentation using a certain medium.

  In the present invention, the peptide means a peptide having 2 or more amino acids and 100 or less bonds. The soy peptide material is a hydrolyzate of soy protein material such as isolated soy protein and defatted soy milk, which contains soy-derived peptides, and depending on the degree of degradation and fraction, free amino acids derived from soy are used. Contains.

  In the lactic acid production method of the present invention, the medium may contain 0.01 wt / vol% or more and 1.0 wt / vol% or less of the soybean peptide material. The soybean peptide material may have a fraction of 500 Da or less in the molecular weight distribution of 96% or more. The soybean peptide material may contain 18% by mass or more of free amino acids. Furthermore, the lactic acid-producing microorganism may be a genetically modified lactic acid-producing yeast or a Saccharomyces yeast.

  According to the present invention, there is provided a medium additive for lactic acid fermentation for producing lactic acid for industrial raw materials, which contains a soybean peptide material having a molecular weight distribution in which a fraction of 1000 Da or less is 98% or more. In the culture medium additive of the present invention, the soybean peptide material may have a fraction of 500 Da or less in the molecular weight distribution of 95% or more, or may contain 18% by mass or more of free amino acids. Furthermore, the culture medium additive of the present invention may be obtained by protease treatment of a water-soluble component of defatted soybean. Moreover, the culture medium additive of the present invention may be used for producing polylactic acid.

  According to the present invention, a method for producing a lactic acid fermentation medium additive for producing lactic acid for industrial raw materials, the step of recovering a water-soluble component from defatted soybean, and treating the water-soluble component with a protease, And obtaining a molecular weight distribution soybean peptide material having a fraction of 1000 Da or less of 98% or more as at least part of the lactic acid fermentation medium additive.

  The present invention relates to a method for producing lactic acid, a medium additive for lactic acid fermentation, and a method for producing the same. According to the present invention, by using a soy peptide material having a predetermined molecular weight distribution, lactic acid fermentation can be promoted even in a medium in which chromaticity is regulated so that the use of waste molasses or the like is suppressed or avoided. Can be increased. Moreover, according to this invention, since the fermented liquor by which chromaticity was suppressed can be obtained, it does not request | require an additional refinement | purification process about fermented liquor. Therefore, according to the present invention, it is possible to efficiently produce lactic acid while suppressing or avoiding mixing of coloring components, and to provide a lactic acid fermentation technique suitable for producing lactic acid as an industrial raw material such as a polylactic acid raw material. it can.

  In the present specification, “waste molasses” is a by-product during sugar production, and is a brown to black-brown viscous liquid containing components other than sugar such as vitamins and minerals. Further, “waste molasses” may contain a sugar component derived from raw material sugar. On the other hand, “molasses” refers to a liquid obtained by separating molasses other than sugar from raw sugar.

  Hereinafter, with respect to various embodiments of the present invention, a medium additive for lactic acid fermentation and a method for producing the same will be described first, and then a method for producing lactic acid will be described in detail.

(Medium additive for lactic acid fermentation and production method thereof)
The additive for lactic acid fermentation of the present invention can contain a predetermined soybean peptide material. Hereinafter, first, a preferable production method for obtaining the lactic acid fermentation additive of the present invention will be described, and then a medium additive for lactic acid fermentation will be described.

(Method for producing additive for lactic acid fermentation)
The method for producing an additive for lactic acid fermentation according to the present invention comprises a step of recovering a water-soluble component from a soy protein material such as separated soy protein or defatted soymilk, and subjecting the water-soluble component to a protease treatment to produce a predetermined soy peptide material. Obtaining at least a part of the medium additive.

(Recovery process of water-soluble components)
(Soy protein material)
The soybean used as a raw material for the soybean protein material in the present invention may be of any origin, kind, breeding method, presence / absence of genetic manipulation, presence / absence of germination treatment or fermentation treatment, and component composition. For example, any of yellow soybean, white soybean, green soybean, black soybean and the like can be used. Also used are soybeans lacking specific enzymes such as lipoxygenase by breeding and genetic engineering techniques, and soybeans enriched with specific components such as isoflavones, saponins, GABA, soybean β-conglycinin, soybean glycinin, and soybean lipid-associated protein it can.

  The soy protein material used in the present invention is separated soy protein or defatted soy milk, which is obtained through defatted soybean. The defatted soybean is obtained by removing lipids from whole fat soybean by pressing extraction or solvent extraction, and the lipid content when defatted with hexane is usually 2% by weight or less. At the time of degreasing, lipids are difficult to remove unless the oil body in full-fat soybeans is broken. Therefore, it is usually processed into a flake shape by physical treatment such as compression, and the cells are processed to some extent. In addition, in order to improve the flavor, defatted soybeans are subjected to heat treatment by steam or dry heat, alcohol washing treatment, acid washing treatment, and even if the NSI is reduced to 70% or less, according to the production method of the present invention. Although protein can be extracted with a sufficiently high yield, it is more preferable to use 70 or more low-denatured defatted soybeans, and 80 or more is even more preferable.

  In recovering the water-soluble component from the defatted soybean, it is preferable to extract the water-soluble component by solid-liquid separation after extraction from the defatted soybean. Extraction from defatted soybeans can be performed, for example, as follows. The amount of water added to the defatted soybean is preferably 5 to 10 times, more preferably 6 to 8 times. Although the temperature of the water to add is not specifically limited, It is preferable that it is 50 degreeC or more warm water. More preferably, it is 60 degreeC or more, Most preferably, it is 70 degreeC or more and 75 degrees C or less. Further, the extraction time is not particularly limited, but it can be about 30 minutes to 2 hours, preferably about 1 hour. In addition, when extracting with the said warm water, the defatted soybean dispersion which supplied warm water to the defatted soybean is maintained at 50 ° C. or higher, preferably 60 ° C. or higher, more preferably 70 ° C. or higher and 75 ° C. or lower. To. This is because a water-soluble component useful for promoting lactic acid fermentation can be effectively extracted and a soybean peptide material having a stable composition can be obtained.

  In order to solid-liquid separate the dispersion liquid containing the defatted soybean soaked in water, ordinary solid-liquid separation means such as centrifugation, filtration, and press can be used. A liquid fraction obtained by solid-liquid separation can be used as a water-soluble component.

  Prior to the subsequent protease treatment step, the water-soluble component is preferably heat-treated in advance. By doing so, the composition of the degradation product by protease can be made more uniform. The heat treatment conditions are not particularly limited, but heat treatment under pressure is preferable. For example, it can be heated to 120 ° C. to 140 ° C. under pressure using a steam direct introduction type sterilizer, and held for a certain period of time (typically about several seconds to 1 minute). In addition, after heat processing, you may actively cool after that. In consideration of protease treatment, the cooling arrival temperature is preferably close to the action temperature of the protease used in the subsequent protease treatment step.

(Protease treatment process)
The protease treatment step is a step in which the water-soluble component is treated with a protease to obtain a soybean peptide material having predetermined characteristics as at least a part of the medium additive. The protease to be used is not particularly limited, and various commercially available proteases can be appropriately used. Proteases are roughly classified into exo-type proteases and endo-type proteases. Preferably, both endo-type proteases and exo-type proteases are used. By using both of these, both the molecular weight distribution and the free amino acid content can be adjusted.

  The endo-type protease that can be used in the present invention is not particularly limited, and examples include endo-proteases derived from animals and plants and microorganisms. Examples include proteases derived from bacteria, Neisseria gonorrhoeae and actinomycetes, plant-derived papain, bromelain, animal-derived trypsin, pepsin, and cathepsins. Specifically, for example, bacteria such as protease N (manufactured by Amano Enzyme), alcalase, neutralase (manufactured by Novo Nordisk Bioindustry), orientase 10NL (manufactured by HIBI), thermolysin (manufactured by Daiwa Kasei) Proteases derived from Aspergillus, such as Sumiteam FP (manufactured by Shin Nippon Chemical Industry Co., Ltd., including exo-type protease (both types)), orientase ONS (manufactured by HIBI), protease M (manufactured by Amano Enzyme) A protease derived from actinomycetes such as actinase AS (manufactured by Kaken Pharma); a protease derived from plants such as papain W-40 (manufactured by Amano Enzyme) and bromelain (manufactured by Nippon Biocon); trypsin PTN (Novo Nordisk Bio Industry) Company ), Protease of animal origin, such as pepsin (manufactured by Nippon Biocon Ltd.); mention may be made of cathepsin such as squid liver-derived cathepsin D.

  The exo-type protease that can be used in the present invention is not particularly limited and includes exo-type proteases derived from animals and plants and microorganisms. For example, acid carboxypeptidase from Aspergillus, acid carboxypeptidase from yeast, carboxypeptidase from squid liver, carboxypeptidase from wheat, aminopeptidase from Aspergillus, aminopeptidase from bacteria, aminopeptidase from Maitake, etc. be able to. Specifically, for example, carboxypeptidase P (manufactured by Sigma Aldrich), Sumiteam FLPA (manufactured by Shin Nippon Kagaku Kogyo Co., Ltd.), protease M (manufactured by Amano Enzyme) and other acid carboxypeptidase Y (sigma) Acid-derived carboxypeptidases derived from yeast such as Aldrich; acid-derived carboxypeptidases derived from animals such as squid liver carboxypeptidase; Examples include aminopeptidases derived from Aspergillus such as Pharma), and aminopeptidases derived from bacteria such as Accelase (available from Aritsu Trading Co., Ltd.).

  The conditions for the protease treatment step are appropriately set according to the characteristics of the soy peptide material to be obtained and the characteristics such as the optimum pH and temperature of the protease to be used. When a plurality of types of proteases having different optimum pH and optimum temperature are used, the protease treatment step includes a plurality of protease treatment steps having different pH and / or temperature. Usually, for example, a method in which treatment is performed at a pH of 2.5 to 9.5 and a temperature range of 20 to 70 ° C. for a total of 0.5 to 24 hours can be exemplified. The protease to be used may be one type or a combination of two or more types. Preferably, two or more kinds of proteases are used. This is because by using two or more kinds of proteases, the molecular weight of the degradation product tends to concentrate in a specific range compared to the case of using a single protease, resulting in a sharp molecular weight distribution. For example, as described above, it is preferable to use proteases that are different in heterogeneous biological origin and / or operating conditions in addition to the above-mentioned heterolytic types (endo type and exo type) such as alcalase and Sumiteam FP.

  As will be described later, the molecular weight distribution of the protease-treated product can be adjusted by adjusting the protease treatment time. For example, by extending the treatment time with exo-type protease, it becomes easy to increase the low molecular weight fraction of 1000 Da or less. In particular, it is possible to increase the fraction of 500 Da or less and the free amino acid content by reducing more than 500 Da and 1000 Da or less.

  The protease treatment step is preferably treated with an exo-type protease after the endo-type protease treatment step. The exo-type protease treatment step may be a treatment with both exo-type protease and endo-type protease. By performing the protease treatment in this order, it becomes easy to obtain a soybean peptide material having a desired molecular weight distribution and free amino acid content. In addition, although the usage-amount of protease cannot say unconditionally by titer etc., for example, illustrate the enzyme agent of the range of 0.01-100% of mass with respect to the mass of defatted soybean (solid content). Can do.

(Molecular weight distribution by gel filtration chromatography)
The protease treatment step is performed in gel filtration chromatography so that the soybean peptide material obtained by the protease treatment satisfies one or both of the following indices.
(A) The fraction of 1000 Da or less has a molecular weight distribution of 98% or more (b) The fraction of 500 Da or less has a molecular weight distribution of 96% or more

  When the soybean peptide material satisfies the index (a) above, an additive suitable for lactic acid fermentation can be obtained. In addition, when the soybean peptide material satisfies the above index (b), a high lactic acid production amount can be obtained particularly in the early stage of fermentation.

Gel filtration chromatography conditions are not particularly limited. For example, molecular weight distribution can be obtained by the following method. Gel filtration chromatography preferably uses HPLC. Typical measurement conditions are shown below, but conditions under which molecular weight distribution can be measured with the same degree of accuracy and accuracy as the following conditions can also be adopted.
Column: Column TSK gel G3000PWXL + TSK gel G2500PWXL manufactured by Tosoh Corporation is used. Eluent: 45% acetonitrile + 0.05% TFA
Flow rate: 0.3ml / min
Detection: 220nm
Molecular weight markers: Albmin 67000, Myoglobin 18000, Cytochrome-C12270, Insulin 5807, GlyGlyGlyGly246, GlyGlyGly189 and Gly75 are used.

(Free amino acid content)
The protease treatment step is also preferably carried out so that the obtained soybean peptide material contains (c) 18% by mass or more of free amino acids. More preferably, the free amino acid is less than 50% of the mass of the soy peptide material. When the obtained soybean peptide material satisfies the characteristics (c), lactic acid production by lactic acid-producing microorganisms can be promoted at an early stage. The free amino acid content can be measured using a known method using HPLC or the like.

  The protease treatment step is preferably carried out after presetting the protease treatment conditions necessary for obtaining the above-mentioned indicators for molecular weight distribution and free amino acid content. For example, it is treated with an endo-type protease such as alcalase (typically about 30 minutes to 1 hour), and then 5 to 30 hours using an endo-type protease such as Sumiteam FP, an exo-type protease, or an exo-type protease. By processing to a certain extent, a protease-treated product that satisfies the above-described index, that is, a soybean peptide material can be obtained.

  After the protease treatment step, the enzyme solution is heated to about 80 ° C. to 90 ° C. or higher to deactivate the enzyme. At the same time, it is preferable that the temperature and time are intended for sterilization.

  The enzyme solution containing the soy peptide material thus obtained can be used as a medium additive for lactic acid fermentation with the solution fraction as it is after performing solid-liquid separation as necessary, spray drying, freeze drying method, etc. It can be dried and solidified (powdered).

(Medium additive for lactic acid fermentation)
The lactic acid fermentation additive of the present invention contains a soy peptide material that satisfies any of the following indices used as an index in the protease treatment step of the method for producing a medium additive for lactic acid fermentation of the present invention. That is, it can have any of the following conditions.
(A) The fraction of 1000 Da or less has a molecular weight distribution of 98% or more (b) The fraction of 500 Da or less has a molecular weight distribution of 96% or more (c) 18% by mass or more of free amino acids

  Therefore, it can use as an additive which accelerates | stimulates lactic acid fermentation by adding to a culture medium using only glucose and sucrose as a saccharide | sugar raw material, As a result, use of waste molasses can be suppressed or avoided. Furthermore, the protease-treated product obtained does not contain a coloring substance such as caramel, especially like molasses, so even if it is added to the culture medium, the fermentation broth is not colored by the addition. Thus, the addition of the additive of the present invention does not require an additional purification process. For this reason, it can be preferably used for lactic acid fermentation as a raw material for polylactic acid.

  The culture medium additive of the present invention may contain various components that can be appropriately added in fermentation within a range that does not interfere with the action of the culture medium additive of the present invention, in addition to the soybean peptide material having predetermined characteristics.

(Production method of lactic acid)
The method for producing lactic acid according to the present invention comprises culturing a lactic acid-producing microorganism using a medium obtained by mixing a carbon source and a soybean peptide material having a molecular weight distribution with a fraction of 1000 Da or less of 98% or more. And lactic acid fermentation. According to the lactic acid production method of the present invention, even if the use of waste molasses is suppressed or avoided as a saccharide raw material, lactic acid fermentation can be promoted and a high amount of lactic acid can be obtained. The purification process can be avoided. For this reason, it is preferable as a method for producing lactic acid, which is a raw material for polylactic acid.

(Lactic acid fermentation process)
It is preferable that the soybean peptide material used in the lactic acid production method of the present invention has an index for the molecular weight distribution and / or free amino content of the soybean peptide material of the additive for lactic acid fermentation of the present invention.

  The addition amount (content) of the soybean peptide material in the medium is not particularly limited, but preferably 0.01% wt / vol% to 1.0% wt / vol% in the medium. Within this range, it is possible to obtain a fermentation broth that promotes lactic acid fermentation and has suppressed chromaticity. More preferably, it is 0.1% wt / vol% or more and 0.5% wt / vol% or less.

  The carbon source of the medium used in the lactic acid production method of the present invention may be a saccharide raw material mainly composed of monosaccharides or disaccharides such as glucose, fructose and sucrose, or a starchy raw material. Examples of the sugar raw material include cane (sugar cane) juice, beet (sugar beet) juice, and the like. Waste molasses is also included. Examples of the starchy material include corn, wheat, sweet potato, and potato.

  The carbon source is preferably composed of one or more selected from glucose, fructose and sucrose as a main component (70% by mass or more, preferably 80% by mass or more, more preferably 90% by mass or more, more preferably It is preferable to use a carbohydrate raw material of 95% by mass or more. The medium may be a eutrophic medium containing peptone or yeast extract in addition to the carbon source, but it is preferable not to include such additives in order to omit or maximize the purification process. . Moreover, it is preferable that waste molasses is not included substantially as a nutrient source. In other words, it is preferable that a coloring substance such as a caramel substance produced as a by-product in a sugar crystallization step or the like is substantially not included. As described above, the method for producing lactic acid of the present invention can obtain a higher effect when avoiding the use of molasses.

  The medium used in the lactic acid production method of the present invention may be any medium obtained by mixing a carbon source and the soybean peptide material. Therefore, it may be obtained by mixing only the carbon source and the soybean peptide material, or may contain other components separately. Moreover, the preparation form is also not ask | required. A mixture containing a carbon source and the soy peptide material in advance may be dissolved or suspended in a suitable medium at the time of use to prepare a medium, or it may be prepared by separately adding the carbon source and the soy peptide material to the medium. There may be.

  In addition, since the soy peptide material may be obtained by mixing a carbon source and the soy peptide material, at least the predetermined molecular weight distribution and / or free amino acid content is satisfied at the time of medium preparation. However, it does not require that the soy peptide material in the medium satisfies the predetermined molecular weight distribution and / or free amino acid content at any time after the medium has been prepared. That is, changes over time in the molecular weight distribution and free amino acid content of the soybean peptide material in the medium are allowed. The medium may be liquid or solid, but is preferably liquid.

  The finally obtained soybean peptide material-containing medium preferably has an absorbance at 400 nm of 0.1 or less. By using soy peptide material as a medium additive, a medium having an effect of promoting lactic acid fermentation can be easily obtained even if the absorbance is 0.1 or less (for example, a poor nutrient medium). . In addition, if the absorbance at 400 nm is 0.1 or less, it is markedly colorless compared to the waste molasses-containing medium, so that the purification process of the colored components can be considerably simplified and may be omitted depending on the application. Can do. More preferably, the absorbance is 0.05 or less. If the absorbance is 0.05 or less, the purification process can be further simplified or omitted. More preferably, the absorbance is 0.02 or less.

  From the above, as another embodiment of the present invention, there is provided a composition for a lactic acid fermentation medium containing a carbon source and a soybean peptide material having the predetermined molecular weight distribution and / or free amino acid content. . According to this culture medium composition for lactic acid fermentation, it can be set as the powder or solid which mix | blended the carbon source and the said soybean peptide raw material previously. A medium for lactic acid fermentation can be obtained by dissolving in an appropriate medium when necessary. Moreover, the kit for lactic acid fermentation culture media provided with the carbon source and the soybean peptide raw material which has the said predetermined molecular weight distribution and / or free amino acid content as another form of this invention is also provided. A lactic acid fermentation medium can be obtained by dissolving the carbon source and soybean peptide material of this kit in an appropriate medium when necessary.

  The lactic acid-producing microorganism used in the production method of the present invention is not particularly limited, but is preferably a genetically modified lactic acid-producing yeast. Examples of the genetically modified yeast include, for example, genetic recombinants that use the genus Saccharomyces such as Saccharomyces cerevisiae, genetic recombinants such as Candida sonorensis, Kluyveromyces lactis, Kluyveromyces Examples include genetic recombinants of the genus Kluyveromyces such as thermotolerance and Kluyveromyces marcianus. Among these, a genetically modified yeast obtained by imparting a high lactic acid-producing ability to a yeast convenient for industrial fermentation production can be preferably used. As a genetically modified yeast having a high ability to produce lactic acid, a gene group comprising an exogenous lactate anhydrase (LDH) gene such as bovine under the control of a pyruvate decarboxylase (PDC) gene (preferably PDC1 gene) promoter Replacement yeast is preferred.

  As described above, lactic acid-producing microorganisms that produce lactic acid by gene recombination are disclosed in JP 2001-204468, JP 2003-93060, JP 2003-334092, JP 2003-259878, JP 2003-093060, JP 2003-2003. 259878, JP-A-2005-137306, JP-A-2006-42719, JP-A-2006-20602, JP-A-2006-288318, JP-A-2006-296377, JP-T-2005-528106, JP-T-2006-52525, etc. . Examples of lactic acid-producing microorganisms that are gene recombinants that predominantly produce D-lactic acid or L-lactic acid include JP 2005-528112, Table 2004-104202, JP-A 2005-187743, JP-A 2007-074939. Already disclosed.

  Lactic acid fermentation conditions are not particularly limited. The culture method in the present invention is not particularly limited, and a conventionally known culture method can be used. For example, any one of batch culture, semi-batch culture, continuous culture, or a combination thereof can be performed. The oxygen supply method and the stirring method can also be selected according to the type of lactic acid-producing microorganism to be used. In the case of yeasts such as Saccharomyces cerevisiae, the reaction is usually performed at about 25 to 35 ° C. for about 12 to 80 hours under aerobic conditions such as shaking culture or aeration and agitation culture. Moreover, during culture | cultivation, antibiotics, such as an ampicillin and a tetracycline, can be added to a culture medium as needed.

  The pH varies depending on the acid resistance of the lactic acid-producing microorganism to be used, but can be set to about pH 4.0 to 8.0 in the case of neutralization conditions involving neutralization with alkali. Moreover, as acidic conditions or non-neutralizing conditions which can acquire free lactic acid, it can also set suitably in pH 1.0-4.5.

(Fermentation liquid separation process)
After the culture step, a step of separating the fermentation broth can be performed by removing lactic acid-producing microorganisms from the fermentation broth. According to the present invention, since the absorbance of the culture medium is limited to 400 nm, the degree of coloring of the finally obtained lactic acid fermentation broth is also maintained. For this reason, in the subsequent lactic acid separation step, the purification step intended to separate the colored components can be omitted, and lactic acid can be obtained efficiently. Lactic acid producing microorganisms are removed by a known solid-liquid separation method such as filtration or centrifugation.

  Furthermore, lactic acid can be obtained by implementing the process of isolate | separating lactic acid from the fermentation liquid after isolate | separating microorganisms. In separating lactic acid, an appropriate separation step can be performed depending on the form of lactic acid (whether it is free or salt).

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the following examples do not limit the present invention.

(Preparation of soybean peptide material)
The defatted soybeans obtained by squeezing the whole soybeans were dissolved in water and then centrifuged to obtain a supernatant solution. The supernatant solution was treated with a hydrolase, and the resulting solution was dried with a freeze-drying apparatus to prepare a powder sample of soybean peptide material. Specifically, the following operations were performed.

First, 10 kg of defatted soybeans obtained by squeezing whole soybeans were prepared by adding 70 ° C. warm water to 70 kg, then holding at 60 to 70 ° C. for 1 hour, and a continuous centrifuge capable of continuously processing the dispersion (Ishikawajima The product was separated into a soluble component and an insoluble component using Harima Heavy Industries). A steam of 8 kg / cm ² pressure was blown into this soluble component, the temperature was raised to 142 ° C., held at that temperature for 7 seconds, and then the treated liquid obtained by cooling to 50 ° C. with a plate through which cooling water passed was obtained. A starting solution for enzymatic degradation was used. 30 kg of this starting solution (solid content 7%) was transferred to a 50 liter container capable of adjusting the temperature and kept at 50 ° C. The starting solution was adjusted to pH 7.0 with 40% sodium hydroxide solution, 21 g of Alcalase 2.4 L (Novozyme) was added (1% of the solid content), and after 30 minutes of enzymatic degradation, then continued. Powdered citric acid was added to adjust the pH to 5.3, 63 g of Sumiteam FP (Shin Nippon Chemical Industry Co., Ltd.) was added (3% added to the solid content), and enzymatic degradation was performed at 50 ° C. for 5 hours. Then, this enzyme digestion solution is held at 85 ° C. for 30 minutes, and after enzyme inactivation and sterilization, the treatment solution is passed through a high-speed centrifuge (manufactured by Westphaliaseparator) to remove insoluble matters. Thereafter, the obtained clear liquid after centrifugation was powder-dried with a spray dryer to prepare soybean peptide (A44C).

(Examination of soybean peptide addition effect to eutrophic broth)
Previously filed lactic acid-producing yeast strain TC38 (see JP2003-164295, JP2003-334092, and JP2006-75133. This lactic acid-producing yeast uses a lactate dehydrogenase gene derived from bovine as a promoter of the PDC1 gene. The yeast IFO2260 strain introduced into the yeast chromosome under control and disrupting the host GPD1 gene and GPD2 gene) was used to test the effect of adding soybean peptide in the culture solution. The lactic acid-producing yeast was inoculated into a 5 ml test tube of YPD culture solution, and cultured by shaking culture at 30 ° C. for 18 hours as the initial cell. Add 40 ml culture solution and 5% (wt./vol.) Calcium carbonate (Wako Pure Chemicals) as a neutralizing agent to a 100 ml Erlenmeyer flask, and add 0.3% in volume ratio (PCV). The above initial bacterial cells were inoculated. Two types of medium were used: YPD broth containing 10% (wt./vol.) Glucose (Wako Pure Chemical) and liquid molasses medium adjusted to 10% sugar concentration. . Next, to these fermentation solutions, 0.5% (wt./vol.) Of the soybean peptide material (A44C) prepared in Example 1 above was added and an unadded sample was prepared. Lactic acid fermentation was carried out at a shaking speed of 80 rpm and a fermentation temperature of 30 ° C. for 72 hours. 1 ml of the supernatant was collected every 24 hours from the start of fermentation, and the amount of L-lactic acid in the solution was measured. For the measurement of lactic acid, Biosensor BF-5 (manufactured by Oji Scientific Instruments) was used, and the detailed measurement method was in accordance with the attached instruction manual. The result is shown in FIG.

  As shown in FIG. 1, it was confirmed that the lactic acid production rate and the lactic acid production amount were improved by adding the present soybean peptide material even in the eutrophic culture solution (YPD). Moreover, even if it was a liquid molasses culture medium with a low nutrient source compared with a YPD culture solution, the high lactic acid fermentation effect was confirmed by adding a soybean peptide raw material. It was found that by using this soy peptide material, lactic acid fermentation can be promoted even in a culture solution having a relatively poor nutrient source.

(Additive effect of soy peptide material to oligotrophic culture and comparison with other soy peptides)
Next, the effect at the time of adding this soybean peptide raw material to a poor nutrient medium and the comparison with another soybean peptide raw material were examined. The cells grown under the same conditions as in Example 2 were used as the initial cells, and 40 ml of the culture solution and 5% (wt./vol.) Of calcium carbonate (sum) as a neutralizer in a 100 ml Erlenmeyer flask. The first bacterial cells were inoculated to a volume ratio (PCV) of 0.3%. As the culture solution, a culture solution containing only 12% (wt./vol.) Sucrose (Wako Pure Chemical Industries) was used. Subsequently, for the main fermentation solution, the soybean peptide material (A44C) prepared in Example 1 above, and four other types of soybean peptide materials (soy peptide materials A, B, C, Samples with and without addition of 0.5% (wt./vol.) Of D) were prepared, and these were subjected to lactic acid fermentation for 72 hours at a shaking speed of 80 rpm and a fermentation temperature of 30 ° C. 1 ml of the supernatant was collected every 24 hours from the start of fermentation, and the amount of L-lactic acid in the solution was measured. For the measurement of lactic acid, Biosensor BF-5 (manufactured by Oji Scientific Instruments) was used, and the detailed measurement method was in accordance with the attached instruction manual. The result is shown in FIG.

  As shown in Fig. 2, in the case of an oligotrophic medium consisting only of 12% (wt./vol.) Sucrose, fermentation does not proceed with lactic acid-producing yeast, whereas 0.5% (wt./vol.) Soy peptide material It was confirmed that the production rate similar to that of the eutrophic medium represented by YPD medium and the like was exhibited by simply adding. In addition, as a result of tests using a plurality of types of soybean peptide materials with different production methods, the same effect could not be obtained with any soybean peptide material. From the above, it was confirmed that a high effect was recognized only with the soybean peptide (A44C) prepared this time.

(Molecular weight distribution and free amino acid content of various soybean peptide materials)
The soy peptide material A44C and six kinds of soy peptide materials were prepared under the enzymatic degradation conditions of the soy peptide material A44C prepared in Example 1 in which the effect of improving lactic acid production was recognized and conditions obtained by partially changing this. About these seven kinds of soybean peptide raw materials, molecular weight distribution and free amino acid content were measured using gel filtration chromatography.

  The methods for preparing seven kinds of soybean peptide materials (RE00, RE01, RE05 (A44C) RE03, RE07, RE10, RE24) newly prepared in this example were as follows.

10 kg of the enzymatic decomposition start solution obtained in the same manner as in Example 1 (solid content: 7%) was adjusted to pH 7.0 with 40% sodium hydroxide solution and 7 g of alcalase 2.4 L (Novozyme) was added (solid content) 1 kg from the enzyme-decomposed solution for 30 minutes, immediately heated at 85 ° C. for 30 minutes, and centrifuged to obtain a test sample (RE00). .
The remaining 9 kg of liquid was continuously adjusted to pH 5.3 by adding powdered citric acid, and then 18.9 g of Sumiteam FP (Shin Nihon Chemical Industry Co., Ltd.) was added (3% added to the solid content). Enzymatic degradation was carried out at 24 ° C. until 24 hours later. On the way, after adding Sumiteam FP, after 1, 3, 5, 7, 10, 24 hours, a part of the enzyme digestion solution is extracted, immediately heated at 85 ° C. for 30 minutes, and then centrifuged. The supernatant was freeze-dried, and test samples RE01 (1 hour after FP addition), RE03 (3 hours after FP addition), RE05 (A44C) (5 hours after FP addition), RE07 (7 hours after FP addition), RE10 (10 hours after FP addition) and RE24 (24 hours after FP addition) were prepared.

(Measurement of molecular weight distribution by gel filtration chromatography)
The molecular weight was measured by HPLC (manufactured by JASCO Corporation). The measurement conditions were as follows. The molecular weight was calculated by JASCO's chromatographic data processing. The results are shown in Table 1.
Column: Tosoh Co., Ltd. column TSK gel G3000PWXL + TSK gel G2500PWXL is used. Eluent: 45% acetonitrile + 0.05% TFA
Flow rate: 0.3ml / min
Detection: 220nm
Molecular weight markers: Albmin 67000, Myoglobin 18000, Cytochrome-C12270, Insulin 5807, GlyGlyGlyGly246, GlyGlyGly189 and Gly75 are used.

(Measurement of free amino acid content)
The free amino acid content of various soybean peptide materials was measured using a Hitachi amino acid analyzer (L8900). The detailed measurement method was in accordance with the attached instruction manual. The results are also shown in Table 1.

  As shown in Table 1, as the treatment time with Sumiteam FP, which is a mixture of endo-type protease and exo-type protease, becomes longer, the fraction of 500 Da or less gradually increased and the free amino acids also increased. In RE00 to RE03, the fraction of 1000 Da or less was less than 97% (the fraction of 500 Da or less was less than 97%) and the free amino acid content was 17% or less, whereas RE05 (A44C) to RE24 As for the same fraction, the fraction was 98% or more (the fraction of 500 Da or less was 96% or more), and the free amino acid content was 18% or more.

(Examination of the effect of various proteolysate A44C fraction samples on lactic acid fermentation)
Using the soybean peptide material prepared in Example 4, the effect on lactic acid production was examined. The cells grown under the same conditions as in Example 2 were used as the initial cells, and 40 ml of the culture solution and 5% (wt./vol.) Of calcium carbonate (sum) as a neutralizer in a 100 ml Erlenmeyer flask. The first bacterial cells were inoculated so that the volume ratio (PCV) was 0.25%. As in Example 3, a culture solution containing only 12% (wt./vol.) Sucrose (Wako Pure Chemical Industries) was used. Subsequently, a sample in which 0.5% (wt./vol.) Each of the seven types of soybean peptide materials prepared in Example 4 were added to this fermentation solution and a sample to which no soybean peptide material was added were prepared. These were subjected to lactic acid fermentation at a shaking speed of 80 rpm and a fermentation temperature of 30 ° C. for 72 hours. 1 ml of the supernatant was collected every 24 hours from the start of fermentation, and the amount of L-lactic acid in the solution was measured. For the measurement of lactic acid, Biosensor BF-5 (manufactured by Oji Scientific Instruments) was used, and the detailed measurement method was in accordance with the attached instruction manual. The results are shown in Table 2 and FIG.

  As shown in FIG. 3, high lactic acid production especially after 24 and 48 hours for the samples to which soybean peptide materials RE05 (A44C), RE07, RE10, and RE24 (processing time with Sumiteam FP were added for 5 to 24 hours) were added. showed that. On the other hand, regarding the sample to which the soybean peptide materials RE00, RE01 and RE03 (treatment time of Sumiteam FP was 0 hours to 3 hours), the above four kinds of trends were not recognized. Moreover, lactic acid production was not recognized at all in the sample to which the soybean peptide material was not added. From the above, it is characterized by fractions containing low molecular weight peptides and free amino acids (fractions with a molecular weight of 1,000 Da or less are 98% or more, and fractions with 500 Da or less are 96% or more) and free amino acids are 18% or more. It was found that the soy peptide material shown has the effect of promoting lactic acid fermentation.

(Confirmation of degree of medium coloring)
In this example, a medium using waste molasses, a YPD medium, and a soy peptide material-added oligotrophic medium (all liquids) were prepared according to the following compositions. In addition, as a molasses and YPD culture medium, it can obtain commercially by a very general method, and used the general purpose product. About these culture media, while confirming the coloring degree visually, the spectrum was measured over the visible region from the ultraviolet region. The results are shown in FIG.

(Waste molasses-containing medium,% represents mass / volume%)
Glucose 12%
Waste molasses 0.25%
(Enriched medium,% represents mass / volume%)
Glucose 12%
East Extract 1%
Bactotripton 2%
(Soybean peptide-containing oligotrophic medium,% represents mass / volume%)
Glucose 12%
Soy peptide material 0.5%

    As shown in the upper part of FIG. 4, according to visual observation, the medium prepared by adding the soybean peptide material to the poor nutrient medium is colorless and almost clear, and does not require a purification process to remove coloring components and the like. It was a thing. On the other hand, the waste molasses-containing medium and eutrophic medium were brown and yellow, respectively, and both were colored amounts that required a purification step after lactic acid fermentation. In addition, since the nutrient rich medium has a coloring degree of about one-tenth compared with the waste molasses-containing medium, the coloring component can be removed by a simple purification process. In addition, as shown in the lower part of FIG. 4, other than the soy peptide-containing oligotrophic medium was diluted (10-fold diluted molasses-containing medium and 3-fold diluted rich medium), but the molasses-containing medium was visible. At 300 to 400 nm of light, the absorbance was significantly higher than that of the soy peptide material-containing medium, and the rich medium showed a higher absorbance than the soy peptide-containing oligotrophic medium, although it was lower than the waste molasses-containing medium. At 400 nm, the absorbance at 400 nm of the soybean peptide material-containing medium was 0.018, whereas that of the waste molasses-containing medium was 1.0, and that of the rich medium was 0.097.

It is a figure which shows the soybean peptide raw material addition effect to a eutrophic culture medium (YPD culture medium and liquid molasses culture medium). It is a figure which shows the soybean peptide raw material addition effect to an oligotrophic culture medium (medium which contains only 12% sucrose as a saccharide raw material). It is a figure which shows the various soybean peptide raw material addition effect to an oligotrophic culture medium (medium which contains only 12% sucrose as a saccharide raw material). It is a figure which shows the external appearance and absorption spectrum of a waste molasses containing culture medium, a YPD culture medium, and a soybean peptide raw material containing culture medium.

Claims (10)

A method for producing lactic acid,
A soy peptide material having a carbon source and a molecular weight distribution in which a fraction of 500 Da or less is 96% or more and containing a free amino acid of 18% by mass or more and less than 50% by mass, and having an absorbance at 400 nm at the time of medium preparation 0 . A step of culturing a genetically modified lactic acid-producing yeast using a medium that is no greater than 05 and lactic acid fermentation,
A manufacturing method comprising:   The production method according to claim 1, wherein the genetically modified lactic acid-producing yeast is a genetically modified yeast comprising an exogenous lactate dehydrogenase gene under the control of a pyruvate dehydrogenase (PDC) gene promoter.   The method according to claim 1 or 2, wherein the lactic acid-producing yeast is a Saccharomyces yeast.   In addition to the soybean peptide material, the medium has a medium composition component selected from the group consisting of a YPD medium, a molasses medium, and an oligotrophic medium having only sucrose as a carbon source. The manufacturing method as described. The manufacturing method according to any one of claims 1 to 4 , wherein the absorbance at 400 nm during the medium preparation is 0.02 or less. The said culture medium is a manufacturing method in any one of Claims 1-5 which contains the said soybean peptide raw material 0.01 wt / vol% or more and 1.0 wt / vol% or less. 500Da following fractions is soybean peptide material comprising less than a having and free amino acids of molecular weight distribution is 96% or more 18 wt% to 50 wt%, the absorbance at 400nm during medium preparation is 0.05 or less, Medium additive for production of lactic acid for industrial raw materials by genetically modified lactic acid producing yeast. The additive according to claim 7 , which is obtained by protease treatment of a water-soluble component of defatted soybean. The additive according to claim 7 or 8 , which is used for producing polylactic acid. A method for producing the culture medium additive according to any one of claims 7 to 9 ,
Recovering water-soluble components from defatted soybean;
A soy peptide material having a molecular weight distribution in which a fraction of 500 Da or less is 96% or more obtained by subjecting the water-soluble component to protease treatment and containing 18% by mass or more and less than 50% by mass of a free amino acid is at least one of the medium additives. A process of acquiring as a part,
A manufacturing method comprising: