JP6343967B2 - Method for producing ferulic acid - Google Patents

Description

  The present invention relates to a method for producing ferulic acid from a lignocellulosic biomass raw material.

  The technology for producing sugar from lignocellulose raw materials can produce chemical raw materials such as alcohol, succinic acid and lactic acid as alternative fuels for gasoline by using this sugar as a fermentation substrate for microorganisms. This technology is useful for the formation of society. As a method for producing monosaccharides and small saccharides as fermentation substrates from polysaccharides contained in plant biomass, there is an enzymatic saccharification method in which hydrolysis is performed using an enzyme or a microorganism that produces the enzyme. Various techniques have been developed for efficient enzymatic saccharification. For example, Patent Document 1 proposes a method for efficiently recovering an enzyme from a residue on which the enzyme is adsorbed by a simple method without affecting downstream processes such as fermentation by microorganisms.

  For the purpose of producing alcohol as an alternative fuel, it is also important that enzymatic saccharification treatment can be carried out at a very low cost, but if valuable materials can be produced from waste after the production of the target substance, the entire process It can contribute to economic improvement. For example, Patent Document 2 proposes that after an alkali treatment with calcium hydroxide is performed on a cellulosic biomass raw material, the solid content is neutralized and subjected to an enzymatic saccharification treatment step. On the other hand, it proposes to collect ferulic acid etc. by performing the production | generation with a hydrophobic resin or an anion exchange resin with respect to the liquid component isolate | separated from solid content. Further, Patent Document 3 proposes to obtain a bast fiber from a residue by subjecting a bark of a woody plant to a saccharification treatment to perform a solid-liquid separation treatment for separating a liquid component containing sugar and a solid residue. . Furthermore, Patent Document 4 discloses a method in which lignocellulose subjected to coarse crushing and / or grinding treatment is subjected to grinding treatment on a residue obtained from the first saccharification step of removing sugar by polysaccharide degrading enzyme, and then subjected to polysaccharide degrading enzyme. The present invention proposes a method for producing lignin characterized by being obtained by a second saccharification step for removing sugar.

  On the other hand, ferulic acid (3-methoxy-4-hydroxycinnamic acid) is a precursor of lignin and is known to be abundant in herbaceous plants (such as rice bran). Ferulic acid has a radical scavenging action and an active oxygen scavenging action, and is used as a food antioxidant. It is also used for cosmetics as a naturally-occurring UV absorber. Ferulic acid has recently been reported to improve hypertension, improve endurance and anti-fatigue, cure hepatitis ethanol, and improve symptoms in patients with Alzheimer's disease.

JP 2013-188202 A JP 2013-220067 A JP 2011-47083 A JP2012-16285A

The subject of this invention is providing the method of utilizing the waste material from the process including the enzyme saccharification process of a lignocellulosic biomass raw material as a raw material for production of a useful substance.
In addition, ferulic acid is also contained in woody plants, although the amount is small compared to herbaceous plants, and if there is a practical method for obtaining ferulic acid from woody plants, securing raw materials There is a merit that becomes easy.

  The inventors of the present invention have examined the effective utilization of fermentation residues by-produced in bioethanol production. And if it was the pre-processing raw material which contains hemicellulose comparatively much, it thought that the substance derived from lignin might be contained in the saccharification fermentation residue. Therefore, as a result of solid-liquid separation of the treated suspension obtained by subjecting the pretreated lignocellulosic raw material to parallel saccharification and fermentation, the solid content (residue) was examined in detail. The inventors found that an acid can be obtained and completed the present invention. The present invention provides the following.

[1] A method for producing ferulic acid, comprising solid-liquid separation of a treated suspension from a lignocellulosic raw material that has undergone a saccharification treatment with an enzyme, and separating ferulic acid from the obtained solid content.
[2] The method for producing ferulic acid according to [1], wherein the solid content contains lignin.
[3] The method for producing ferulic acid according to [1] or [2], wherein the treated suspension has undergone a pretreatment step before the enzymatic saccharification treatment.

[4] The method for producing ferulic acid according to [3], wherein the pretreatment is a chemical treatment.
[5] The method for producing ferulic acid according to any one of [1] to [4], wherein the treated suspension is fermented together with the enzymatic saccharification treatment or after the enzymatic saccharification treatment.
[6] The method for producing ferulic acid according to any one of [1] to [5], wherein the lignocellulose-based material is obtained from hardwood.

  According to the present invention, ferulic acid can be efficiently obtained from a process that undergoes a saccharification treatment step of a lignocellulosic raw material.

FIG. 1 is a diagram showing one embodiment of a method for producing ferulic acid according to the present invention.

I: Uniaxial crusher CO: Heat crusher R: Refiner BR: Parallel saccharification and fermentation tank S: Solid-liquid separator

Hereinafter, the present invention will be described in more detail.

<Lignocellulose raw material>
Examples of the lignocellulosic raw material used as a raw material in the method of the present invention include the following. Woody materials: Chips or bark for paper-making trees, forest residue, thinned wood, sprouting from stumps of woody plants, sawdust or sawdust from lumber mills, pruned branches of street trees, construction waste, etc. Herbaceous: Agricultural waste such as kenaf, rice straw, straw, corn cob, bagasse, residue and waste of industrial crops such as oil crops and rubber (eg, EFB: Empty Fruit Bunch), Eliansus of herbaceous energy crops , Miscanthus, Napiergrass etc. In particular, it is not limited to these. In addition, food wastes such as wood-derived paper, waste paper, pulp, pulp sludge, sludge, sewage sludge and the like can be used as raw materials, but are not particularly limited thereto. These raw materials can be used alone or in combination. The raw material may be a dry solid, a solid containing water, or a slurry.

The woody lignocellulosic material is not particularly limited, but includes Eucalyptus genus plants, Acacia genus plants, Salix genus plants, Poplar genus plants, and Cryptomeria genus plants. Etc. are available. This is because Eucalyptus plants, Acacia plants, and Willow plants are easily collected in large quantities as raw materials. In particular, Eucalyptus globulus and Eucalyptus pelleta are used as Eucalyptus plants, Acacia mangium and Acacia auris are used as Acacia species, and Acacia mangium and Acaciaurium species are used preferable.
Among the lignocellulosic raw materials derived from woody plants, it is preferable to use forest residue (including bark and leaves) and bark. For example, bark of tree species such as Eucalyptus genus or Acacia genus commonly used for papermaking raw materials can be obtained in large quantities stably from lumber mills and chip factories for papermaking raw materials. Preferably used.
As a suitable example of the woody lignocellulosic material used in the present invention from another viewpoint, there may be mentioned hardwood materials such as eucalyptus, oak, acacia, beach, tan oak, and alder. The hardwood used may contain some softwood.

<Pretreatment>
In the present invention, the lignocellulose raw material can be pretreated for enzymatic saccharification. The pretreatment applied to the present invention is preferably carried out so that the solid content obtained by solid-liquid separation of the treated suspension subjected to the saccharification treatment described below contains a sufficient amount of lignin or ferulic acid. This is because it is presumed that lignin or the like does not remain in the solid content when pretreatment is performed under strong conditions. As long as the pretreatment satisfies such conditions, ferulic acid, lignin and the like can be applied to the present invention.

(Mechanical processing)
In the present invention, the lignocellulose raw material can be subjected to a mechanical treatment as a pretreatment for enzymatic saccharification. Examples of the mechanical treatment include arbitrary mechanical means such as crushing, cutting, and grinding, and making lignocellulose easy to be saccharified in the next chemical treatment step. Although it does not specifically limit about the mechanical apparatus to be used, For example, a uniaxial crusher, a biaxial crusher, a hammer crusher, a refiner, a kneader etc. can be used.

As a pre-process or post-process of the mechanical treatment, a foreign matter removing step by washing or the like for removing foreign matter (foreign matter other than lignocellulose such as stone, dust, metal, plastic) can be introduced.
Examples of the method for washing the raw material include a method of removing water from the foreign material mixed with the raw material by spraying water on the raw material, or a method of removing the foreign material by immersing the raw material in water and sedimenting the foreign material. Moreover, the method of isolate | separating a foreign material from a raw material using apparatuses, such as a metal trap, is mentioned.
If foreign materials are included in the raw materials, there is a possibility of causing damage to equipment parts used in mechanical processing such as refiner discs (plates), and causing problems in the manufacturing process such as clogging of piping. Therefore, it is desirable to introduce a foreign substance removing step.

(Chemical treatment)
The lignocellulose raw material may be subjected to chemical treatment. An example of the chemical treatment is immersion in a solution containing one or more alkaline chemicals selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate and sodium hydrogen carbonate. Another example of the chemical treatment is immersion in a solution containing sodium sulfite and one or more alkaline chemicals selected from the above alkaline chemicals. Further, chemical treatment with an oxidizing agent such as ozone or chlorine dioxide is also possible.
The chemical treatment is preferably performed as a treatment after the pretreatment in combination with the mechanical treatment.

  The amount of chemicals used in the chemical treatment can be arbitrarily adjusted depending on the situation, but lignocellulosic from the standpoint of reducing chemical costs and preventing yield loss due to elution and overdegradation of cellulose. It is desirable that it is 70 mass parts or less with respect to 100 mass parts of absolutely dry materials. The immersion time and the treatment temperature of the chemical in the chemical treatment can be arbitrarily set depending on the raw materials and chemicals to be used, but a treatment time of 20 to 90 minutes and a treatment temperature of 80 to 200 ° C. are preferable. By tightening the processing conditions, elution or excessive decomposition of cellulose in the raw material may occur, so that the processing time is preferably 70 minutes or less and the processing temperature is preferably 180 ° C. or less.

  As the chemical treatment, 10 to 70% by mass of sodium sulfite and 0.1 to 5% by mass of alkali as a pH adjuster can be added to the lignocellulose raw material (dry weight). When sodium sulfite is added alone to the lignocellulose in the above-mentioned addition amount and heat-treated, an organic acid such as acetic acid is generated during hydrolysis, so that the pH is lowered and the hydrolyzed solution becomes acidic. When hydrolysis is continued under acidic conditions, the problem arises that xylose produced by hydrolysis is converted to furfural. Since furfural is an inhibitor of ethanol fermentation, it is desirable not to produce it as much as possible. Moreover, since the yield of xylose which is a fermentation substrate falls, ethanol production efficiency falls as a result. By adding sodium sulfite and alkali as a pH adjuster to the lignocellulose raw material in the above-mentioned amount and heat-treating, the pH during hydrolysis is maintained from neutral to weakly alkaline. Yield reduction can be suppressed. Moreover, since the pH of the aqueous solution containing lignocellulose after heat treatment (after hydrolysis) is 4.0 to 7.0 (neutral to weakly alkaline), the waste liquid or hydrolyzate after the hydrolysis treatment is neutralized. There is a merit that the amount of chemicals used for the reduction can be reduced.

  Examples of the alkali used as the pH adjuster include sodium hydroxide, potassium hydroxide, sodium carbonate and the like, but are not particularly limited to these chemicals.

  The heat treatment temperature when the heat treatment is performed by adding 10 to 70% by mass of sodium sulfite and 0.1 to 5% by mass of alkali as a pH adjuster with respect to the lignocellulose raw material (dry weight) is 80 -200 degreeC is preferable and 120-180 degreeC is more preferable. Moreover, although heat processing time can be performed in 10 to 300 minutes, 30 to 120 minutes are preferable. By tightening the processing conditions, elution or excessive decomposition of cellulose in the raw material may occur, so that the processing temperature is preferably 180 ° C. or lower and the processing time is 120 minutes or shorter.

(Grinding treatment)
In the present invention, the lignocellulosic raw material obtained by the chemical treatment is preferably ground in a refiner disk (plate) clearance of 0.1 to 2.0 mm, preferably in the range of 0.1 to 1.0 mm. Is more preferable. As a refiner to be used, a single disk refiner, a double disk refiner, or the like can be used, and any refiner that can set the clearance of the opposing disk within a range of 0.1 to 2.0 mm can be used without particular limitation. it can. If the disc clearance exceeds 2.0 mm, the sugar yield obtained by saccharification or parallel saccharification and fermentation is added, which is not preferable. On the other hand, if the disc clearance is lower than 0.1 mm, the yield of the hydrolyzate (solid content) after grinding with a refiner is not preferable. Also, if the disc clearance is lower than 0.1 mm, the electricity consumption required for the operation of the refiner increases, which is not preferable.

  The material of the refiner disk (plate), the disk mold, the disk surface blade mold, the direction of the blade with respect to the disk surface, etc. should be used without any limitation as long as the material and shape are effective. Can do.

  The lignocellulosic raw material that has been subjected to the above grinding treatment is solid-liquid separated into an aqueous solution and a solid content, and the solid content is used as a raw material for saccharification or concurrent saccharification and fermentation. As a method for solid-liquid separation, for example, an apparatus that can be separated into an aqueous solution and a solid content using a screw press or the like and can be used without limitation as long as it can be separated into an aqueous solution and a solid content.

(Sterilization treatment)
Before performing saccharification or concurrent saccharification fermentation using the raw material after solid separation, it is preferable to perform sterilization treatment. When miscellaneous bacteria are mixed in the lignocellulosic biomass raw material, there is a problem that the miscellaneous bacteria consume sugar when the enzyme is saccharified and the yield of the product decreases.
The sterilization treatment may be a method in which the raw material is exposed to a pH at which bacteria are difficult to grow, such as acid or alkali, but may be a method in which treatment is performed at a high temperature, or a combination of both. About the raw material after an acid and an alkali treatment, it is preferable to use as a raw material, after adjusting to neutrality vicinity or pH suitable for a saccharification and / or saccharification fermentation process. In addition, even when pasteurized at a high temperature, it is preferably used as a raw material after the temperature is lowered to room temperature or a temperature suitable for the saccharification and fermentation process. Thus, by feeding out the raw material after adjusting the temperature and pH, it is possible to prevent the enzyme from being exposed to the outside of the preferred pH and the preferred temperature and being deactivated.

  The lignocellulose raw material that has been subjected to the pretreatment as the case may be supplied to the saccharification step or the concurrent saccharification and fermentation step. Hereinafter, the present invention may be described by way of an example using a pretreated lignocellulose raw material, but the present invention is not limited by the embodiment.

<Saccharification process>
In the present invention, a lignocellulosic raw material that is optionally pretreated is mixed with an appropriate amount of water and an enzyme to form a raw material suspension, which is supplied to the enzymatic saccharification step. Lignocellulose-based raw materials are saccharified (cellulose → glucose, hemicellulose → glucose, xylose) by enzymes (cellulase, hemicellulase). The enzyme treatment is considered to work advantageously for the extraction of ferulic acid, which is a lignin component, by partially decomposing the raw material.

(Saccharification process and concurrent saccharification and fermentation process)
The saccharification process of the lignocellulosic material may be a concurrent saccharification and fermentation process. In the present invention, the saccharification step or enzyme saccharification step includes a concurrent saccharification and fermentation step. The concurrent saccharification and fermentation process can be carried out by mixing a lignocellulosic raw material with an appropriate amount of water and an enzyme to form a raw material suspension, and further mixing with a microorganism such as yeast. The lignocellulosic material is saccharified by an enzyme, and the produced saccharide is converted into a product such as ethanol by a fermenting microorganism (such as yeast).

  It is preferable that the suspension concentration of the lignocellulosic material used in the saccharification process or the concurrent saccharification and fermentation process is 1 to 30% by mass. If it is less than 1% by mass, there is a problem in that the concentration of the product is ultimately too low and the cost for concentrating the product becomes high. Moreover, as the concentration exceeds 30% by mass, it becomes difficult to stir the raw materials, resulting in a problem that productivity is lowered.

Cellulolytic enzymes used in the saccharification step or the concurrent saccharification and fermentation are enzymes collectively called cellulases having cellobiohydrolase activity, endoglucanase activity, and betaglucosidase activity.
Each cellulolytic enzyme may be added with an appropriate amount of an enzyme having the respective activity. However, commercially available cellulase preparations have the above-mentioned various cellulase activities and also have hemicellulase activity. Since many products are available, a commercially available cellulase preparation may be used.

Commercial cellulase preparations include the genus Trichoderma, the genus Acremonium, the genus Aspergillus, the genus Phanerochaete, the genus Trametes, the genus Humicola, and the like. There are cellulase formulations derived from Examples of such commercially available cellulase preparations include cellulosin T2 (manufactured by HIPI), mecerase (manufactured by Meiji Seika Co., Ltd.), Novozyme 188 (manufactured by Novozyme), multifect CX10L (manufactured by Genencor), and GC220 (Genencor). Manufactured) and the like.
0.5-100 mass parts is preferable and, as for the usage-amount of the cellulase formulation with respect to 100 mass parts of raw material solid content, 1-50 mass parts is especially preferable.

  The pH of the reaction solution in the saccharification step or the concurrent saccharification and fermentation step is preferably maintained in the range of 3.5 to 10.0, more preferably in the range of 4.0 to 7.5.

  The temperature of the reaction solution in the saccharification step or the simultaneous saccharification and fermentation step is not particularly limited as long as it is within the optimum temperature range of the enzyme, preferably 25 to 50 ° C, more preferably 30 to 40 ° C. The reaction is preferably continuous, but may be semi-batch or batch. The reaction time varies depending on the enzyme concentration, but in the case of a batch system, it is 10 to 240 hours, more preferably 15 to 160 hours. Also in the case of a continuous type, the average residence time is 10 to 150 hours, more preferably 15 to 100 hours.

  In the concurrent saccharification and fermentation step, it is preferable to use a fermentation microorganism capable of fermenting saccharides (hexose sugar, pentose sugar). Examples of fermenting microorganisms include Saccharomyces cerevisiae, Pichia stipitis, Candida shiientais, and Pachisolen sapiens. -Bacteria such as mobilis (Zymomonas mobilis). In addition, genetically modified microorganisms (yeast, bacteria, etc.) produced using genetic recombination techniques can also be used. As the genetically modified microorganism, a microorganism capable of simultaneously fermenting hexose and pentose can be used without particular limitation.

  Microorganisms may be immobilized. Immobilizing microorganisms can eliminate the step of separating and re-recovering microorganisms in the next step, so that at least the burden required for the recovery step can be reduced, and the loss of microorganisms can be reduced. . Moreover, the collection of microorganisms can be facilitated by selecting microorganisms having aggregating properties.

<Solid-liquid separation process>
The culture solution discharged from the saccharification step or the concurrent saccharification and fermentation step can be transferred to a solid-liquid separator and separated into a liquid component (filtrate) and a solid component (residue). As an apparatus for performing solid-liquid separation, a screw press, a screen, a filter press, a belt press, a rotary press, or the like can be used. As the screen, a vibrating screen to which a vibrating device is added can be used.
The recovered solid content (residue) can be further transferred to a saccharification process or a concurrent saccharification and fermentation process and used as a raw material for saccharification or saccharification and fermentation.

<Step of obtaining ferulic acid>
In the present invention, the lignocellulosic raw material treatment suspension that has undergone at least the saccharification treatment step is subjected to solid-liquid separation, and the solid content (residue) becomes a raw material for obtaining ferulic acid. In addition, process suspension may be called culture solution, when referring to what was discharged | emitted from the saccharification process or the parallel saccharification fermentation process. Moreover, solid content (residue) here may contain what was obtained from the primary residue separation process or secondary residue separation process mentioned later. In the present invention, the term ferulic acid includes not only free ferulic acid but also glycoside or ester forms unless otherwise specified.

  In order to obtain ferulic acid from the solid content, the solid content may be mixed with water containing alkali and processed. The amount of alkali added can be arbitrarily adjusted according to the situation. For example, in order to obtain ferulic acid with a high yield, 0.5% by mass or more, preferably with respect to the solid content (absolute dry weight) Can be used in an amount of 1% by mass or more, more preferably 1.25% by mass or more. The upper limit of the amount of alkali added can be determined as appropriate, and can be, for example, 50% by mass or less, 30% by mass or less, and 3% by mass or less with respect to the solid content (absolute dry weight). Examples of the alkali include sodium hydroxide, potassium hydroxide, sodium carbonate and the like, but are not particularly limited to these chemicals. Water can be used in an amount of 1 to 100% by mass, preferably 5 to 50% by mass, based on the solid content (absolute dry weight). In the treatment, isopropyl alcohol may be added.

  The mixed solution can be heated to 60 ° C. or higher, preferably 80 ° C. or higher, more preferably 90 ° C. or higher, and allowed to react for an hour with stirring as necessary. In order to obtain ferulic acid with a high yield, the reaction may be performed for 4 hours or longer, preferably 6 hours or longer, more preferably 8 hours or longer. The upper limit of the reaction time can be determined as appropriate, and can be, for example, 24 hours or shorter, 16 hours or shorter, or 12 hours or shorter. Thereafter, the mixture is cooled as necessary, and an organic solvent such as hexane is added to extract and remove undesired components from the oil layer. Then, an aqueous layer is obtained, and the aqueous layer is filtered to obtain a solid content (insoluble matter). It can be removed to obtain a filtrate. Ferulic acid can be precipitated by adding an acid to the filtrate. The obtained precipitate may be purified by usual means such as recrystallization.

  The obtained ferulic acid can be identified and quantified by a conventional method, for example, by HPLC or UPLC (wavelength 320 nm).

  The method for producing ferulic acid of the present invention will be described with reference to the example shown in FIG. The raw material is mechanically processed by I (uniaxial crusher), then transferred to BR (parallel saccharification and fermentation tank) through CO (heat treatment device) and R (refiner). The treated suspension through the saccharification and fermentation treatment step is separated into a liquid component and a solid component (residue) by S (solid-liquid separator). The obtained solid content is transferred to a reaction step for obtaining ferulic acid.

<Other processes>
(Fermentation process)
In the embodiment of the present invention, the saccharification step and the fermentation step may be performed in separate reaction tanks. The liquid component (filtrate) separated in the solid-liquid separation step is transferred to a fermentation step and fermented using a fermentation microorganism. Examples of fermenting microorganisms include Saccharomyces cerevisiae, Pichia stipitis, Candida shiientais, and Pachisolen sapiens. -Bacteria, such as mobilis (Zymomonas mobilis), etc. are mentioned. In addition, genetically modified microorganisms (yeast, bacteria, etc.) produced using genetic recombination techniques can also be used. As the genetically modified microorganism, a microorganism capable of simultaneously fermenting hexose and pentose can be used without particular limitation.
Microorganisms may be immobilized. By immobilizing microorganisms, the process of separating and re-recovering microorganisms in the next step can be omitted, so that at least the burden required for the recovery process can be reduced and the loss of microorganisms can be reduced. is there. Moreover, the collection of microorganisms can be facilitated by selecting microorganisms having aggregating properties.

(Distillation process)
The processing liquid fermented in the fermentation process or the processing liquid processed in the concurrent saccharification and fermentation process (if the solid-liquid separation process is performed, the liquid component separated in the solid-liquid separation process) is transferred to the distillation process Then, the fermentation product (ethanol or the like) can be distilled and separated by a vacuum distillation apparatus. Since the fermentation product can be separated at a low temperature under reduced pressure, inactivation of the enzyme can be prevented. As the vacuum distillation apparatus, a rotary evaporator, a flash evaporator, or the like can be used.
In the case of ethanol, the distillation temperature is preferably 25 to 60 ° C. If it is lower than 25 ° C., it takes time to distill the product, and the productivity is lowered. On the other hand, when the temperature is higher than 60 ° C., the enzyme is heat-denatured and deactivated, and the amount of newly added enzyme increases, resulting in poor economic efficiency.
The concentration of the fermentation product remaining in the distillation residue fraction after distillation is preferably 0.1% by mass or less. By setting it as such a density | concentration, the amount of fermentation products discharged | emitted with a solid substance in a subsequent solid-liquid separation process can be reduced, and a yield can be improved.

(Primary residue separation process)
In the embodiment of the present invention, the entire process may be configured as in the above-mentioned Patent Document 1. In the manufacturing process shown in FIG. 1 and FIG. 2 of the above-mentioned Patent Document 1, the distillation residue after separation of the fermentation product after distillation (ethanol and the like) is transferred to the primary residue separation step, and in the primary residue separation device C1. Separated into liquid fraction and residue. The liquid fraction separated by the primary residue separator C1 is circulated to the primary parallel saccharification and fermentation tank BR1 via the line 7 (and the culture solution storage tank T). Further, in the production process shown in FIG. 3, the liquid fraction separated in the primary residue separation process (residue separation apparatus C1) passes through the line 3 to the secondary parallel saccharification and fermentation tank BR2 (the primary parallel saccharification and fermentation process and Are transferred to different parallel saccharification and fermentation processes). In the secondary concurrent saccharification and fermentation step, a new lignocellulose raw material can be added to cause saccharification and fermentation, or fermentation for the purpose of fermentation of pentoses such as xylose can be performed.

  The liquid fraction separated in the primary residue separation step contains an enzyme, and can be recycled to the saccharification step, primary parallel saccharification and fermentation step, or secondary parallel saccharification and fermentation step and reused as an enzyme-containing liquid. The residue separated in the primary residue separation step can be subjected to the ferulic acid recovery step described above. On the other hand, since the residue separated in the primary residue separation step contains an enzyme, it may be transferred to the enzyme recovery step.

(Secondary residue separation step)
In an embodiment of the present invention, the entire process is configured as described in Patent Document 1, and the residue suspension after being treated with water-soluble salts in the enzyme recovery step is transferred to a secondary residue separation step. You may isolate | separate into a residue and a liquid fraction by the next residue separation apparatus C2. In the manufacturing process shown in FIG. 1 and FIG. 2 of Patent Document 1, the liquid fraction containing the enzyme can be transferred to the primary saccharification and fermentation tank BR1 via the line 8 (and the culture solution storage tank T). In the production process shown in FIG. 3, the liquid fraction separated in the secondary residue separation step (secondary residue separation device C2) passes through the line 8a to the secondary parallel saccharification and fermentation tank BR2 (the primary parallel saccharification and fermentation step). To a different saccharification and fermentation process). In the secondary concurrent saccharification and fermentation step, a new lignocellulose raw material can be added to cause saccharification and fermentation, or fermentation for the purpose of fermentation of pentoses such as xylose can be performed. The residue separated in the secondary residue separation step can be subjected to the ferulic acid recovery step described above.
By continuously circulating the liquid fraction (filtrate) containing the enzyme in the process, the enzyme activity in the process can be maintained at a high level over a long period of time.

  As a residue separator (primary residue separator, secondary residue separator) used in the primary residue separation process or the secondary residue separation process, a centrifuge, a filter press, a belt press, a rotary press, a screen, or the like may be used. it can. As the screen, a vibrating screen to which a vibrating device is added can be used.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited by these Examples.

[Example 1]
Chip-like eucalyptus and globula woodland residues (70% bark, 30% branches and leaves) were crushed with a uniaxial crusher (SC-15, manufactured by Saiho Kiko Co., Ltd.) equipped with a 20 mm round hole screen and used as a raw material.
After adding 200 g of 97% sodium sulfite and 10 g of sodium hydroxide to 1 kg (absolute dry weight) of the above raw material, water was added to adjust the volume of the aqueous solution to 8 L. The raw material suspension was mixed and then heated at 170 ° C. for 1 hour. The raw material suspension after the heat treatment was ground to a disc (plate) clearance of 1.0 mm with a refiner (KRK high concentration disc refiner). Next, the raw material suspension after the grinding treatment was subjected to solid-liquid separation (dehydration) using a 20 mesh (847 μm) screen, and washed with water until the electric conductivity of the solution reached 30 μS / cm. Simultaneous saccharification and fermentation was carried out using the solid content after solid-liquid separation as a raw material (hereinafter referred to as “pretreatment raw material”).

<Concurrent saccharification and fermentation>
In advance, in a liquid medium (glucose 30 g / L, polypeptone 5 g / L, yeast extract 3 g / L, malt extract 3 g / L, pH 5.6) 50 L, yeast Saccharomyces cerevisiae (commercially available yeast, trade name: trade name: Maurivin: Maurii) Yeast Australia Pty Limited) was cultured at 30 ° C. for 24 hours. After adding each to a saccharification and fermentation tank so that it might become 5 g / L of polypeptone, 3 g / L of yeast extract, and 3 g / L of malt extract, water was added to adjust the final volume to 0.8 m ³ . A culture solution containing yeast cells was added to the saccharification and fermentation tank and cultured for 24 hours. When the density of the yeast grew to 1 × 10 ⁸ / ml, 50 L of a commercially available cellulase solution (Multifect CX10L) and 100 kg (dry weight) of the raw material were added to the saccharification and fermentation tank. Next, water was added to the saccharification and fermentation tank to adjust the final volume of the culture solution to 1 m ³ . The pH of the culture solution was adjusted to 5.0, and concurrent saccharification and fermentation was started at 30 ° C. Saccharification and fermentation were carried out by setting the retention time of the culture solution in the saccharification and fermentation tank BR (time for the raw material suspension to pass through the saccharification and fermentation tank: capacity / flow rate of the saccharification and fermentation tank) to 20 hours. That is, from the start of saccharification and fermentation, a raw material suspension (raw material is suspended in water) having a solid content concentration (per dry weight) of 10% by mass is continuously supplied from the raw material supply port of the saccharification and fermentation tank at a flow rate of 50 L / h. Was added. On the other hand, the raw material suspension was discharged at 50 L / h from the culture liquid outlet of the saccharification and fermentation tank simultaneously with the start of the raw material supply, and transferred to the solid-liquid separation step. The cellulase solution was continuously added to the saccharification and fermentation tank at 2.5 L / h. When the culture solution decreased during continuous operation, the final volume of the culture solution was maintained at 1 m ³ by automatically adding a medium. The pH of the culture medium during the culture was maintained at 5.0.

<Solid-liquid separation>
The raw material suspension discharged from the parallel saccharification and fermentation step is solid-liquid separated by a screw press (SHX-200 x 1500 L, screen size 1.2 mm, manufactured by Togoku Industry Co., Ltd.) to obtain a solid (residue) and a liquid ( The filtrate) was separated.

  The solid content (residue) was used as a sample. After adding 10 g of sample (absolute dry weight), 1.5 g of sodium hydroxide (absolute dry weight), 20 ml of water and 20 ml of isopropyl alcohol to a three-necked flask, the mixture was heated to 90-100 ° C. and stirred for 8 hours to react. Thereafter, the mixture was cooled to room temperature. Next, hexane was added to the mixture, and after stirring, the mixture was fractionated into an aqueous layer and a hexane layer. The aqueous layer was filtered to remove solid content (insoluble matter) to obtain a filtrate. 5 ml of 7.5N sulfuric acid was added to the filtrate to crystallize ferulic acid. The obtained crystals were filtered, and the crystals were dissolved again with hot water and then recrystallized to purify ferulic acid. The ferulic acid purified from the sample was analyzed by HPLC (column / CADENZA CD-C18, column temperature / 30 ° C, eluent / MeOH (10 → 60%) + acetic acid aqueous solution (90 → 40%), measured at a wavelength of 320 nm). The ferulic acid contained in the sample was measured. The results are shown in Table 1.

[Example 2]
In Example 1, it tested by the method similar to Example 1 except having changed the sample used for reaction into 3g. Although it could not be isolated by recrystallization, fine crystals were formed. The results are shown in Table 1.

[Comparative Example 1]
In Example 1, it tested by the method similar to Example 1 except having used the pre-processing raw material before saccharification and fermentation instead of the residue as a sample. Crystals did not form. The results are shown in Table 1.

[Comparative Example 2]
In Example 1, it tested by the method similar to Example 1 except having used the solid content (solid content A) obtained by concentrating and drying the liquid component after solid-liquid separation instead of the residue as a sample. Crystals did not form. The results are shown in Table 1.

  As shown in Table 1, it was found that ferulic acid can be efficiently produced from the solid content obtained by solid-liquid separation of the culture solution after saccharification and fermentation into a solid content and a liquid content.

  According to the present invention, ferulic acid can be produced from a solid content (residue) after saccharification and fermentation in the production of useful substances such as ethanol using lignocellulosic biomass as a raw material. Ferulic acid is useful in fields such as food, cosmetics and medicine.

Claims (7)

Manufacture ferulic acid, including a step of solid-liquid separation of a suspension of lignocellulosic raw material that has undergone a saccharification treatment with an enzyme, and separating the ferulic acid by subjecting the resulting solid to an alkali treatment Method.   2. The method for producing ferulic acid according to claim 1, wherein the solid content contains lignin.   The method for producing ferulic acid according to claim 1 or 2, wherein the treated suspension has undergone a pretreatment step before the enzymatic saccharification treatment.   The method for producing ferulic acid according to claim 3, wherein the pretreatment is a chemical treatment.   The method for producing ferulic acid according to any one of claims 1 to 4, wherein the treated suspension is fermented together with the enzymatic saccharification treatment or after the enzymatic saccharification treatment.   The method for producing ferulic acid according to any one of claims 1 to 5, wherein the lignocellulosic material is obtained from hardwood. The method for producing ferulic acid according to any one of claims 1 to 6, wherein the alkali is sodium hydroxide.

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