WO2009107328A1 - Process for production of quinic acid and/or caffeic acid - Google Patents

Process for production of quinic acid and/or caffeic acid Download PDF

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WO2009107328A1
WO2009107328A1 PCT/JP2009/000403 JP2009000403W WO2009107328A1 WO 2009107328 A1 WO2009107328 A1 WO 2009107328A1 JP 2009000403 W JP2009000403 W JP 2009000403W WO 2009107328 A1 WO2009107328 A1 WO 2009107328A1
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acid
coffee
koji
aspergillus
chlorogenic acid
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PCT/JP2009/000403
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French (fr)
Japanese (ja)
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松下一信
足立収生
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国立大学法人山口大学
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/40Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42Hydroxy-carboxylic acids

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  • the present invention relates to a method for producing quinic acid and / or caffeic acid using coffee lees as a raw material. More specifically, the present invention relates to a method for producing quinic acid and / or caffeic acid from chlorogenic acid in coffee koji, using a microbial catalyst comprising coffee koji obtained by growing filamentous fungi on coffee koji.
  • Patent Document 2 As a method for producing organic materials for agriculture (Patent Document 2), a method that can be converted to incineration by a method of treating coffee mash, a method of performing anaerobic digestion after treatment with acid or alkali (Patent Document 3), Although it is used as a decomposition accelerator for waste (Patent Document 4), there is no report of using it as a raw material for pharmaceuticals.
  • coffee beans contain chlorogenic acid having functionality such as antioxidant activity (Patent Document 5).
  • the aroma and taste of coffee beans change due to roasting, which is thought to be due to the decomposition of chlorogenic acid to produce quinic acid and caffeic acid (also called caffeic acid or caffeic acid).
  • quinic acid can be a raw material for shikimic acid used as a useful raw material for many antibiotics, alkaloids, herbicidal active substances, and the like (Patent Document 6).
  • Shikimic acid is an important raw material compound of Tamiflu (registered trademark) that is expected to be most effective against human influenza and avian influenza viruses, which are expected to cause a global epidemic in recent years. Yes.
  • WHO World Health Organization
  • quinic acid is contained in plants such as kina peel and sugar beet in addition to coffee beans, and extracted and purified from these materials.
  • coffee beans as a raw material, after alkali hydrolysis, after treatment with a strongly basic anion exchange resin, followed by treatment with alkali, and further desalting and purifying with an ion exchange membrane electrophoresis apparatus (Patent Document 7)
  • kina in the raw material liquid A method in which an acid is precipitated as a metal salt with a metal salt or metal hydroxide and separated, and then quinic acid is purified using an acid (Patent Document 8).
  • a raw material solution containing quinic acid is converted to ketones using an acid catalyst.
  • a method of obtaining a quinic acid acetal by reacting with an aldehyde and purifying it, followed by hydrolysis (Patent Document 9), or a plant containing a quinic acid derivative (kina peel, cod bean pod, coffee bean , Tobacco leaves, sweet potatoes, pear leaves, apples, etc.) or their processed products using an organic solvent to extract the quinic acid derivative, and the extract is purified by hydrolysis under an acid or base catalyst.
  • Patent Document 10 Coffee beans are hydrolyzed using enzymes, and purified by adsorbent, quinic acid as a vitamin odor inhibitor (Patent Document 11), extracted from tea leaves with water or polar organic solvent Or by hydrolyzing coffee beans with enzymes and purifying them to obtain a quinic acid derivative for enhancing pungent taste (Patent Document 12), extracting sweet potato leaves with alcohol, hydrous alcohol and water, and concentrating the extract Then, the method obtained by dissolving in water and partitioning with a polar organic solvent and purifying by column chromatography to obtain a quinic acid derivative (Patent Document 13), using propolis obtained from honeybee's nest as a raw material, A method of extracting a quinic acid derivative for cell growth inhibitor with a hydrophilic solvent (Patent Document 14), water extraction from green coffee beans, roasted coffee beans, pulverized products thereof, etc. Adsorbed onto arm, extracted with ethanol, and a method (Patent Document 15) to obtain a chlorogenic acids containing
  • caffeic acid has been recognized as an anti-oxidant ability and an effect of suppressing metastasis and proliferation of cancer cells, and is required as a functional material. Since the sugar transfer substance of caffeic acid has antimicrobial activity, its use in dentifrices, oral cleansing agents or oral bactericides containing the same has been disclosed (Patent Document 16).
  • Patent Document 16 As a method for producing caffeic acid, a method for purifying caffeic acid by removing mono- or dicaffeoylquinic acid as an impurity by allowing a rice bran extract to act on polyphenols in sweet potato shochu distilled spirit is disclosed. However, this does not suggest the production of quinic acid or caffeic acid using coffee koji using coffee koji as a raw material.
  • the main object of the present invention is to provide a method for producing quinic acid and / or caffeic acid, which is a raw material for pharmaceuticals and functional materials, from chlorogenic acid contained in coffee mash as an effective method of using coffee mash.
  • the present inventors produce quinic acid and / or caffeic acid from chlorogenic acid in coffee cake using a microbial catalyst comprising coffee cake obtained by growing koji molds (filamentous fungi) on coffee cake. The method has been found and the present invention has been completed.
  • the present invention includes (1) a step (A) of producing a coffee koji by growing a koji mold that produces chlorogenic acid hydrolase on the coffee koji, and the chlorogen extracted from the coffee koji or the coffee koji.
  • the main culture is performed for 5 to 10 days using a koji making tool while turning over at 25 to 37 ° C. (1) to (1) above, characterized in that it is a coffee grinder manufactured by Method for producing quinic acid and / or caffeic acid according to any one of 3) related.
  • the present invention relates to (5) a coffee koji having chlorogenic acid hydrolase activity and (6) a coffee koji described in (5) above, wherein the koji mold is Aspergillus sojae .
  • B is an enlarged photograph of A. It is a figure which shows the production
  • the method for producing quinic acid and / or caffeic acid of the present invention includes a step (A) of producing coffee koji by growing a koji mold that produces chlorogenic acid hydrolase in coffee koji, and the coffee koji or The method includes a step (B) of producing quinic acid and / or caffeic acid by reacting chlorogenic acid extracted from coffee koji as a catalyst with chlorogenic acid as a raw material.
  • the term “coffee mash” means an extraction residue of coffee beans after the roasted coffee beans for the production of coffee beverages are extracted with steam, hot water, water, etc.
  • the kind of bean is not particularly limited, and any kind of coffee bean may be used.
  • the coffee cake may be in any of wet and dry conditions. For example, a coffee cake discharged after instant coffee production that has been disposed of up to now, a coffee cake discharged from a coffee shop, and the like are suitable. Can be used.
  • Aspergillus genus Penicillium genus, Rhizopus genus, Mucor genus, Monascus genus that can produce chlorogenic acid hydrolase
  • 4033 NBRC 4033
  • the above-mentioned microorganisms include Kyoto University Faculty of Agriculture, graduate School of Agriculture (AKU: Faculty of Agriculture, Kita-Shirakawa Oiwake-machi, Sakyo-ku, Kyoto), Fermentation Institute (IFO), Institute for Formentation, Juso-cho, Sasagawa-ku, Osaka 2-17-85), National Institute of Biotechnology, Biotechnology Headquarters, National Institute of Technology and Evaluation (NBRC; National Institute of Technology and Evaluation, Biological Resource Center, 2-5-8, Kazusa Kamashizu, Kisarazu City, Chiba Prefecture) Can be sold at
  • the above coffee bowl can be manufactured as follows, for example. That is, excess water contained in the collected coffee cake is removed, and heat sterilized in a hermetic heat-resistant container. The heat sterilization is performed at 100 to 130 ° C. for about 3 to 10 minutes, preferably at 110 to 120 ° C. for 5 to 7 minutes. After cooling to room temperature, a culture solution (seed fungus) containing koji molds that have been cultured separately is added. The amount of inoculum per coffee lees varies depending on the amount of water in the coffee lees and the growth of the inoculum in the culture solution. Add about 70 to 200 ml of culture broth, preferably about 100 ml of broth.
  • any koji mold capable of growing on coffee koji may be used, and any microorganism capable of growing in a culture solution containing chlorogenic acid as a sole carbon source may be selected from chlorogenic acid hydrolase. It can be judged to produce.
  • the inoculum can be produced by a commonly used method for culturing filamentous fungi, which is produced by culturing for 1 to 2 days in a medium in which koji molds can grow.
  • the coffee koji with the inoculum be preliminarily cultured in a culture room or an incubator to promote the growth of mycelia and then perform the main culture.
  • the preculture is carried out at 25 to 37 ° C. for 12 to 24 hours, but can be transferred to the main culture if the mycelial growth can be confirmed.
  • the form of the main culture may be any form as long as the microorganisms attached to the coffee gruel can sufficiently grow, but a tool that can take a large surface area such as a straw lid for sake brewing is used. It is desirable.
  • the pre-cultured contents are transferred to a lid and cultured at 25-37 ° C., preferably 30-35 ° C., for 5-10 days, preferably 5-7 days.
  • the coffee koji produced in this way has a hydrolytic enzyme stably held on the outside of the hyphae of the elongated microorganism.
  • this coffee koji can be used as it is as a microbial catalyst, or chlorogenic acid hydrolase can be extracted from this coffee koji and used as a catalyst.
  • filamentous fungi can extend mycelia to the fine gaps in the coffee gruel, so that a microbial catalyst with high production efficiency can be produced.
  • Microorganisms cannot grow by heat treatment, but hydrolase activity, particularly chlorogenic acid hydrolase activity, is heat resistant and the enzyme activity is stably maintained even after heat treatment. Since the microbial catalyst is in the form of a kind of immobilized microbial catalyst, it can be used as it is suspended in water or physiological saline and added to the reaction solution without any special immobilization treatment. it can.
  • chlorogenic acid hydrolase in the coffee mash can be confirmed by pulverizing the coffee mash and extracting with water or about 0.7 to 1.0% physiological saline. That is, after extracting from a coffee grinder soaked in the solution, it is filtered through filter paper to remove solids and mycelia, and the filtrate is dialyzed against saturated ammonium sulfate. During dialysis, the water in the enzyme solution decreases, and enzyme proteins and other proteins that coexist are precipitated. Collect the precipitate with a centrifuge, put the precipitate into a new dialysis membrane, and dialyze again with 0.7-1.0% saline. A concentrated enzyme solution can be obtained by removing the insoluble precipitate generated after dialysis by centrifugation.
  • One unit of chlorogenic acid hydrolase is defined as one unit of enzyme power capable of hydrolyzing chlorogenic acid by 1 ⁇ mole per minute.
  • the chlorogenic acid extracted from the coffee bean and the coffee koji can be used advantageously, and waste utilization From the viewpoint, chlorogenic acid extracted from coffee candy is particularly preferable.
  • Coffee beans contain a large amount of chlorogenic acid, the content of which is mostly raw beans, 5.5 to 8.0%, and 1.2 to 2.3% after roasting .
  • the coffee mash used as a raw material include a crushed mash obtained from roasted coffee beans, a mash after instant coffee production, and a mash after coffee extraction discharged at a coffee shop or the like.
  • the coffee cake has a concentration of 0.1 to 50%, preferably 1 to 20%.
  • a microbial catalyst comprising a coffee koji containing hydrolase and 10 to 50
  • the reaction can be carried out at 0.degree. C., preferably 30 to 40.degree. C. for 1 to 24 hours, preferably 3 to 8 hours.
  • the method for extracting coffee brewing chlorogenic acid can be performed in accordance with a general organic acid extraction method.
  • coffee mash is extracted with hot water, decolorized with activated carbon, adsorbed on a Dowex (registered trademark) column of anion exchange resin, and water-soluble organics such as methanol, ethanol, isopropanol, acetone, acetonitrile containing acetic acid
  • Chlorogenic acid can be easily obtained by elution with a solvent, separation and purification, and removal of the solvent.
  • the method for producing quinic acid and / or caffeic acid using chlorogenic acid thus prepared as a raw material can be carried out as follows, for example.
  • a buffer solution of pH 5-8, preferably pH 6-7 by adding to the chlorogenic acid a microbial catalyst consisting of coffee koji containing 0.5-10 units, preferably 1-3 units of chlorogenic acid hydrolase,
  • the reaction is carried out at 10 to 50 ° C., preferably 30 to 40 ° C. for 1 to 24 hours, preferably 3 to 8 hours with an acetate buffer or phosphate buffer.
  • the hydrolysis reaction can be performed by immobilizing a microbial catalyst in a column or the like and adsorbing and eluting chlorogenic acid.
  • the method for purifying quinic acid and / or caffeic acid produced using a microbial catalyst comprising coffee koji is not particularly limited, and a commonly used organic acid purification method can be used. , Using the method of Adachi et al. (Adachi, O., Ano, Y., Toyama, H., and Matsushita, Biosci. Biotechnol.
  • Biochem., 70: 3081-3083, 2006 Adsorbs a reaction mixture containing chlorogenic acid, quinic acid and caffeic acid on a Dowex (registered trademark) column of an anion exchange resin, and a suitable eluent such as methanol, ethanol, isopropanol, acetone, acetonitrile, etc. Acids such as acetic acid, formic acid and hydrochloric acid are added to a water-soluble organic solvent and separated and eluted.
  • Quinic acid can be easily eluted from the column with low-concentration sodium chloride and other salt solutions, caffeic acid can be eluted with 1M acetic acid-methanol solution, and unreacted chlorogenic acid can be obtained by increasing the acetic acid concentration. Can be eluted.
  • quinic acid, chlorogenic acid, and caffeic acid can be separated and eluted in this order by adding a low-concentration weak acid to the water-soluble organic solvent.
  • the coffee cake having chlorogenic acid hydrolase activity prepared for use in the method for producing quinic acid and / or caffeic acid described in the present invention is obtained by contacting the koji mold with chlorogenic acid contained in the coffee cake. Therefore, the chlorogenic acid hydrolase is always activated and has a high chlorogenic acid hydrolyzing activity.
  • coffee koji has higher thermal stability than the extracted enzyme, and the chlorogenic acid hydrolase activity is obtained by heat treating the coffee koji for 30 minutes at 60 ° C. in advance.
  • Those in which the proliferative ability of viable cells of the pupae is stopped without impairing can be put into an appropriate reaction vessel as it is and used as an immobilized enzyme. Specifically, for example, by filling the coffee koji after the above heat treatment into a column or the like, it can be used as it is as an immobilized enzyme that has excellent water permeability and can freely adjust the reaction rate.
  • Example 1 ⁇ Hydrolysis reaction of chlorogenic acid by coffee lees>
  • chlorogenic acid was hydrolyzed using coffee koji produced using Aspergillus soya (AKU 3312) and the enzyme solution prepared in Example 2 as a control. That is, 50 ⁇ mole of chlorogenic acid (corresponding to 17.7 mg): 1) coffee cake containing 1 unit of chlorogenic acid hydrolase that was heat-treated at 120 ° C. for 5 minutes; 2) chlorogenic acid hydrolase 1 without heat treatment Coffee cake containing units; 3) enzyme solution containing 1 unit of chlorogenic acid hydrolase that has been heat treated at 120 ° C.
  • Example 1 ⁇ Manufacture of quinic acid and caffeic acid from chlorogenic acid>
  • the hydrolysis reaction of chlorogenic acid was performed using a coffee koji containing chlorogenic acid hydrolase produced using Aspergillus soja AKU 3312. That is, add coffee mash containing 2 units of chlorogenic acid hydrolase to 100 ⁇ mole of chlorogenic acid (corresponding to 35.4 mg), and adjust the reaction volume to 1 ml with 0.1M acetic acid buffer (pH 6.5). And reacted at 30 ° C. for 5 hours.
  • Aspergillus soya (AKU 3312) was cultured in a culture solution containing instant coffee powder, and the effect of coffee powder on the activity of chlorogenic acid hydrolase was examined by reacting the bacterial extract after culture with chlorogenic acid. As shown in FIG. 4, the bacterial extract cultivated under the condition containing no coffee powder showed almost no chlorogenic acid hydrolase activity, but the bacterial extract cultivated under the condition containing coffee powder. Showed remarkable chlorogenic acid hydrolase activity. Such induction / activation effect of chlorogenic acid hydrolase was also observed when filamentous fungi were cultured in a culture solution containing 5 to 20% (w / v) coffee candy.
  • quinic acid and caffeic acid which are pharmaceuticals and functional materials
  • can be produced from coffee mash it becomes possible to effectively use coffee mash that is discarded in large quantities, and the shikimic acid and shikimic acid pathways.
  • Quinic acid which is a raw material for the metabolic intermediate, can be provided at a low cost.

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Abstract

Quinic acid or caffeic acid is a raw material for a pharmaceutical agent or a functional material. Disclosed is a process for producing quinic acid and/or caffeic acid from chlorogenic acid contained in coffee grounds, as an effective method for utilization of coffee grounds. Specifically disclosed is a process for producing quinic acid and/or caffeic acid, which is characterized by reacting chlorogenic acid contained in coffee grounds (as a raw material) with a microbial catalyst prepared from at least one microorganism selected from Aspergillus niger, Aspergillus awamori, Aspergillus sojae, Penicillium chrysogenum, Mucorales mucor, Mucorales rhyzopus and Monascus purpureus each of which has a chlorogenic acid hydrolase and has been grown in coffee grounds.

Description

キナ酸および/またはカフェ酸の製造法Process for producing quinic acid and / or caffeic acid
 本発明は、コーヒー粕を原料とした、キナ酸および/またはカフェ酸の製造法等に関する。より詳しくは、コーヒー粕に糸状菌を生育させて得られたコーヒー粕麹からなる微生物触媒を用いて、コーヒー粕中のクロロゲン酸より、キナ酸および/またはカフェ酸を製造する方法等に関する。 The present invention relates to a method for producing quinic acid and / or caffeic acid using coffee lees as a raw material. More specifically, the present invention relates to a method for producing quinic acid and / or caffeic acid from chlorogenic acid in coffee koji, using a microbial catalyst comprising coffee koji obtained by growing filamentous fungi on coffee koji.
 近年、コーヒーは嗜好品として広く世界中で消費されている。それに伴い大量に排出されるコーヒー粕は特段の有効な利用面のないまま廃棄されている。これまで、コーヒー粕の用途として提案されてきたことは、好気性菌により発酵させ、その発酵熱による乾燥、燃焼行程を経て素炭を製造する方法(特許文献1)、リグニン分解菌で処理して農業用有機資材を製造する方法(特許文献2)、コーヒー粕の処理法で焼却に変わり得る方法として、酸またはアルカリで処理した後、嫌気性消化処理を行う方法(特許文献3)、生ごみ用分解促進剤としての利用(特許文献4)などがあるが、医薬品の原料として利用する報告はない。 In recent years, coffee has been widely consumed around the world as a luxury product. Along with this, coffee mash that is discharged in large quantities is discarded without any particularly effective use. Until now, what has been proposed as a use of coffee koji is fermented by aerobic bacteria, dried by the heat of fermentation, and a process of producing raw coal through a combustion process (Patent Document 1), treated with lignin-degrading bacteria. As a method for producing organic materials for agriculture (Patent Document 2), a method that can be converted to incineration by a method of treating coffee mash, a method of performing anaerobic digestion after treatment with acid or alkali (Patent Document 3), Although it is used as a decomposition accelerator for waste (Patent Document 4), there is no report of using it as a raw material for pharmaceuticals.
 コーヒー豆には、抗酸化作用等の機能性を有するクロロゲン酸が含有されていることが知られている(特許文献5)。コーヒー豆は、焙煎によって香りと味が変わるが、これは、クロロゲン酸が分解されて、キナ酸とカフェ酸(コーヒー酸、カフェイン酸とも呼ばれる)が生成されるためと考えられている。 It is known that coffee beans contain chlorogenic acid having functionality such as antioxidant activity (Patent Document 5). The aroma and taste of coffee beans change due to roasting, which is thought to be due to the decomposition of chlorogenic acid to produce quinic acid and caffeic acid (also called caffeic acid or caffeic acid).
 キナ酸は、多数の抗生物質、アルカロイド、除草活性物質などの有用な原料として用いられるシキミ酸の原料となり得ることを、本発明者らは、先の出願で明らかにした(特許文献6)。シキミ酸は、特に近年になって、世界的な流行を引き起こすと懸念されているヒトインフルエンザ及び鳥インフルエンザウイルスに対し、最も効果が期待されているタミフル(登録商標)の重要な原料化合物となっている。WHO(世界保健機関)が、広く警告を出しているにもかかわらず、世界中で利用できる程充分な備蓄はない。その理由の1つとして、原料となるシキミ酸の調製が難しいことがあげられる。グルコースからシキミ酸に至るまでシキミ酸経路は、二つの異なった代謝経路より得られたホスホエノールピルビン酸とエリスロースー4-リン酸の縮合反応から始まる多段階の代謝経路を経由しなくてはならない。そのため、最新技術の代謝制御法や遺伝子増幅法を駆使してシキミ酸経路の代謝中間体を効率良く製造しても、シキミ酸まで到達するには障壁が大き過ぎるという欠点があった。本発明者らは特許文献6で、酢酸菌中の細胞膜結合型キナ酸脱水素酵素(Quinoprotein quinate dehydrogenase:QDH)を用いて、キナ酸からシキミ酸経路の中間体である3-デヒドロシキミ酸を製造する方法を明らかにし、また酢酸菌の単独培養系でキナ酸からシキミ酸を製造する方法も提供している(非特許文献1)。 The present inventors have clarified in a previous application that quinic acid can be a raw material for shikimic acid used as a useful raw material for many antibiotics, alkaloids, herbicidal active substances, and the like (Patent Document 6). Shikimic acid is an important raw material compound of Tamiflu (registered trademark) that is expected to be most effective against human influenza and avian influenza viruses, which are expected to cause a global epidemic in recent years. Yes. Despite wide warnings from the World Health Organization (WHO), there is not enough reserve available around the world. One of the reasons is that it is difficult to prepare shikimic acid as a raw material. From glucose to shikimic acid, the shikimic acid pathway must go through a multi-step metabolic pathway starting from the condensation reaction of phosphoenolpyruvate and erythrose-4-phosphate obtained from two different metabolic pathways. . Therefore, even if the metabolic intermediate of the shikimic acid pathway is efficiently produced using the latest metabolic control method and gene amplification method, there is a drawback that the barrier is too large to reach shikimic acid. In Patent Document 6, the inventors of the present invention used 3-membrane hydroquinone dehydrogenase (QDH) in acetic acid bacteria to convert 3-dehydroshikimate, which is an intermediate of the kimic acid to shikimate pathway, using quinoprotein quinate dehydrogenase (QDH). A method for producing shikimic acid from quinic acid in a single culture system of acetic acid bacteria has also been clarified (Non-patent Document 1).
 キナ酸は、コーヒー豆のほか、キナ皮、サトウダイコンなどの植物に含まれ、これらの材料から抽出・精製されることが報告されている。コーヒー豆を原料として、アルカリ加水分解後に強塩基性陰イオン交換樹脂で処理した後アルカリで処理し、さらにイオン交換膜電気泳動装置で脱塩精製する方法(特許文献7)、原料液中のキナ酸を金属塩または金属水酸化物により金属塩として析出させて分離後、酸を用いてキナ酸を精製する方法(特許文献8)、キナ酸を含有している原料液を酸触媒によってケトン類またはアルデヒド類と反応させて、キナ酸アセタール体にして精製した後、加水分解により得る方法(特許文献9)、あるいは、キナ酸誘導体を含有する植物体(キナ皮、タラ豆のさや、コーヒー豆、タバコ葉、サツマイモ、ナシ葉、リンゴ等)、又はその処理物より有機溶媒を用いて、キナ酸誘導体を抽出し、抽出物を酸または塩基触媒下で加水分解処理して精製する方法(特許文献10)、コーヒー豆を、酵素を用いて加水分解し、吸着剤により精製するビタミン臭抑制剤としてのキナ酸の製法(特許文献11)、茶葉から水または極性有機溶媒で抽出して、あるいはコーヒー豆から酵素を使って加水分解し、精製して辛味増強用のキナ酸誘導体を得る方法(特許文献12)、サツマイモ葉をアルコール、含水アルコール、水で抽出し、抽出液を濃縮後、水に溶解させ、極性有機溶媒で分配し得られた区分を、カラムクロマトグラフィーで精製してキナ酸誘導体を得る方法(特許文献13)、セイヨウミツバチの巣から得られるプロポリスを原料とし、親水性溶媒で細胞増殖抑制剤のためのキナ酸誘導体を抽出する方法(特許文献14)、コーヒー生豆、焙煎コーヒー豆、その粉砕物等から水抽出後、カラムに吸着させ、エタノールで抽出して、キナ酸誘導体を85%以上含むクロロゲン酸類を得る方法(特許文献15)などが知られているが、麹を使ってキナ酸を製造する方法はない。 It is reported that quinic acid is contained in plants such as kina peel and sugar beet in addition to coffee beans, and extracted and purified from these materials. Using coffee beans as a raw material, after alkali hydrolysis, after treatment with a strongly basic anion exchange resin, followed by treatment with alkali, and further desalting and purifying with an ion exchange membrane electrophoresis apparatus (Patent Document 7), kina in the raw material liquid A method in which an acid is precipitated as a metal salt with a metal salt or metal hydroxide and separated, and then quinic acid is purified using an acid (Patent Document 8). A raw material solution containing quinic acid is converted to ketones using an acid catalyst. Alternatively, a method of obtaining a quinic acid acetal by reacting with an aldehyde and purifying it, followed by hydrolysis (Patent Document 9), or a plant containing a quinic acid derivative (kina peel, cod bean pod, coffee bean , Tobacco leaves, sweet potatoes, pear leaves, apples, etc.) or their processed products using an organic solvent to extract the quinic acid derivative, and the extract is purified by hydrolysis under an acid or base catalyst. Method (Patent Document 10), Coffee beans are hydrolyzed using enzymes, and purified by adsorbent, quinic acid as a vitamin odor inhibitor (Patent Document 11), extracted from tea leaves with water or polar organic solvent Or by hydrolyzing coffee beans with enzymes and purifying them to obtain a quinic acid derivative for enhancing pungent taste (Patent Document 12), extracting sweet potato leaves with alcohol, hydrous alcohol and water, and concentrating the extract Then, the method obtained by dissolving in water and partitioning with a polar organic solvent and purifying by column chromatography to obtain a quinic acid derivative (Patent Document 13), using propolis obtained from honeybee's nest as a raw material, A method of extracting a quinic acid derivative for cell growth inhibitor with a hydrophilic solvent (Patent Document 14), water extraction from green coffee beans, roasted coffee beans, pulverized products thereof, etc. Adsorbed onto arm, extracted with ethanol, and a method (Patent Document 15) to obtain a chlorogenic acids containing quinic acid derivative 85% is known, there is no way to produce quinic acid with a koji.
 一方、カフェ酸も、抗酸化能や、癌細胞の転移や増殖を抑制する効果が認められており、機能性素材として求められているものである。カフェ酸の糖転移物質は、抗微生物活性を有するため、それを含有した歯磨剤、口腔清浄剤又は口腔殺菌剤への利用が開示されている(特許文献16)。カフェ酸の製造法としては、甘しょ焼酎蒸留粕中のポリフェノールに米麹抽出液を作用させて、不純物であるモノ又はジカフェオイルキナ酸を除去し、カフェ酸を精製する方法が開示されているが(特許文献17)、コーヒー粕を原料として、コーヒー粕麹によるキナ酸、あるいはカフェ酸の製造を示唆するものではない。 On the other hand, caffeic acid has been recognized as an anti-oxidant ability and an effect of suppressing metastasis and proliferation of cancer cells, and is required as a functional material. Since the sugar transfer substance of caffeic acid has antimicrobial activity, its use in dentifrices, oral cleansing agents or oral bactericides containing the same has been disclosed (Patent Document 16). As a method for producing caffeic acid, a method for purifying caffeic acid by removing mono- or dicaffeoylquinic acid as an impurity by allowing a rice bran extract to act on polyphenols in sweet potato shochu distilled spirit is disclosed. However, this does not suggest the production of quinic acid or caffeic acid using coffee koji using coffee koji as a raw material.
特許第3532151号公報Japanese Patent No. 3532151 特開平10-95975号公報JP-A-10-95975 特開2003-200138号公報JP 2003-200138 A 特開平8-224564号公報JP-A-8-224564 特開平6-38723号公報JP-A-6-38723 特開2007-300809号公報JP 2007-300809 A 特開平7-8169号公報Japanese Patent Laid-Open No. 7-8169 特開平11-140014号公報Japanese Patent Laid-Open No. 11-140014 特開平11-263746号公報JP 11-263746 A 特開2000-86575号公報JP 2000-86575 A 特開2001-316295号公報JP 2001-316295 A 特開2005-204555号公報JP 2005-204555 A 特開2005-298382号公報JP 2005-298382 A 特開2006-213636号公報JP 2006-213636 A 特開2006-241006号公報JP 2006-241006 A 特開2004-315386号公報JP 2004-315386 A 特開2004-350619号公報JP 2004-350619 A
 本発明は、コーヒー粕の有効な利用法として、コーヒー粕中に含まれるクロロゲン酸より、医薬品や機能性素材の原料となるキナ酸および/またはカフェ酸の製造法を提供することを主な課題とする。 The main object of the present invention is to provide a method for producing quinic acid and / or caffeic acid, which is a raw material for pharmaceuticals and functional materials, from chlorogenic acid contained in coffee mash as an effective method of using coffee mash. And
 本発明者らは、コーヒー粕に麹菌(糸状菌)を生育させて得られたコーヒー粕麹からなる微生物触媒を用いて、コーヒー粕中のクロロゲン酸より、キナ酸および/またはカフェ酸を製造する方法を見出し、本発明を完成するに至った。 The present inventors produce quinic acid and / or caffeic acid from chlorogenic acid in coffee cake using a microbial catalyst comprising coffee cake obtained by growing koji molds (filamentous fungi) on coffee cake. The method has been found and the present invention has been completed.
 すなわち本発明は、(1)コーヒー粕にクロロゲン酸加水分解酵素を産生する麹菌を生育させることによりコーヒー粕麹を作製する工程(A)と、前記コーヒー粕麹又はコーヒー粕麹から抽出されたクロロゲン酸加水分解酵素を触媒として、クロロゲン酸を原料として反応させることにより、キナ酸および/またはカフェ酸を生成させる工程(B)とを含むことを特徴とする、キナ酸および/またはカフェ酸の製造方法や、(2)コーヒー粕から抽出されたクロロゲン酸を原料として反応させることを特徴とする上記(1)に記載のキナ酸および/またはカフェ酸の製造方法に関する。 That is, the present invention includes (1) a step (A) of producing a coffee koji by growing a koji mold that produces chlorogenic acid hydrolase on the coffee koji, and the chlorogen extracted from the coffee koji or the coffee koji. A step (B) of producing quinic acid and / or caffeic acid by reacting chlorogenic acid as a raw material with acid hydrolase as a catalyst, and producing quinic acid and / or caffeic acid And (2) a method for producing quinic acid and / or caffeic acid as described in (1) above, wherein chlorogenic acid extracted from coffee lees is reacted as a raw material.
 また本発明は、(3)麹菌が、アスペルギルス ニガー(Aspergillus niger)、アスペルギルス アワモリ(Aspergillus awamori)、アスペルギルス ソーヤ(Aspergillus sojae)、アスペルギウス オリゼ(Aspergillus oryzae)、アスペルギウス パラスティカス(Aspergillus parasiticus)、ペニシリウム クリソジェナム(Penicillium chrysogenum)から選択される少なくとも1以上の微生物であることを特徴とする上記(1)又は(2)に記載のキナ酸および/またはカフェ酸の製造方法や、(4)コーヒー粕麹が、コーヒー粕1部に対し、麹菌を0.001~0.1部含む培養液と共に予備培養後、製麹用道具を用いて、25~37℃で切り返しを行いながら5~10日間本培養を行うことにより製造されたコーヒー粕麹であることを特徴とする上記(1)~(3)のいずれかに記載のキナ酸および/またはカフェ酸の製造方法に関する。 In the present invention, (3) Aspergillus niger, Aspergillus awamori, Aspergillus sojae, Aspergillus olizae, Aspergillus paliscus, Aspergillus pallicus chrysogenum), at least one microorganism selected from the group consisting of quinic acid and / or caffeic acid as described in (1) or (2) above; After pre-cultured with 1 to 1 part of the culm together with a culture solution containing 0.001 to 0.1 part of Aspergillus, the main culture is performed for 5 to 10 days using a koji making tool while turning over at 25 to 37 ° C. (1) to (1) above, characterized in that it is a coffee grinder manufactured by Method for producing quinic acid and / or caffeic acid according to any one of 3) related.
 さらに本発明は、(5)クロロゲン酸加水分解酵素活性を有するコーヒー粕麹や、(6)麹菌がアスペルギルス ソーヤ(Aspergillus sojae)であることを特徴とする上記(5)に記載のコーヒー粕麹に関する。 Furthermore, the present invention relates to (5) a coffee koji having chlorogenic acid hydrolase activity and (6) a coffee koji described in (5) above, wherein the koji mold is Aspergillus sojae .
酒造用の麹蓋に製造したコーヒー粕麹を示した図面に代わる写真である。BはAを拡大した写真である。It is the photograph replaced with drawing which showed the coffee pot manufactured in the lid for brewing. B is an enlarged photograph of A. クロロゲン酸の加水分解反応によるカフェ酸の生成を示す図である。1)加熱したコーヒー粕麹;2)非加熱のコーヒー粕麹;3)加熱した酵素液;4)非加熱の酵素液;をそれぞれ使用して加水分解反応を行っている。It is a figure which shows the production | generation of caffeic acid by the hydrolysis reaction of chlorogenic acid. Hydrolysis reaction is carried out using 1) heated coffee cake; 2) non-heated coffee cake; 3) heated enzyme solution; 4) non-heated enzyme solution. クロロゲン酸の加水分解反応によるキナ酸とカフェ酸の生成を示す液体クロマトグラフィーの図である。It is a figure of the liquid chromatography which shows the production | generation of quinic acid and caffeic acid by the hydrolysis reaction of chlorogenic acid. コーヒー粉末(グラム重量/培養液容量)を含む培養液を用いてアスペルギルス ソーヤを培養することにより、クロロゲン酸加水分解酵素が活性化されることを示す図である。It is a figure which shows that a chlorogenic acid hydrolase is activated by culture | cultivating Aspergillus soya using the culture solution containing coffee powder (gram weight / culture solution volume). コーヒー粕麹は抽出された酵素に比べて熱安定性が高いことを示す図である。図中、Eはクロロゲン酸加水分解酵素液を、Mはコーヒー粕麹に付着した麹菌のクロロゲン酸加水分解酵素をそれぞれ示す。It is a figure which shows that coffee mash is high in heat stability compared with the extracted enzyme. In the figure, E indicates a chlorogenic acid hydrolase solution, and M indicates a chlorogenic acid hydrolase of Aspergillus adhering to the coffee koji. コーヒー粉末0.5%(グラム重量/培養液容量)を含む培養液を用いて培養することにより、数種類のアスペルギルス属の糸状菌においてクロロゲン酸加水分解酵素が活性化されることを示す図である。It is a figure which shows that chlorogenic acid hydrolase is activated in several types of Aspergillus filamentous fungi by culture | cultivating using the culture solution containing 0.5% of coffee powder (gram weight / culture solution volume). .
 本発明のキナ酸および/またはカフェ酸の製造方法としては、コーヒー粕にクロロゲン酸加水分解酵素を産生する麹菌を生育させることによりコーヒー粕麹を作製する工程(A)と、前記コーヒー粕麹又はコーヒー粕麹から抽出されたクロロゲン酸加水分解酵素を触媒として、クロロゲン酸を原料として反応させることにより、キナ酸および/またはカフェ酸を生成させる工程(B)とを含む方法であれば特に制限されるものではなく、本明細書において「コーヒー粕」とは、コーヒー飲料製造のために焙煎したコーヒー豆を蒸気、熱湯、水等で抽出した後のコーヒー豆の抽出残滓を意味し、上記コーヒー豆の種類は特に制限されるものではなく、どのような種類のコーヒー豆であってもよい。また、上記コーヒー粕は、湿性、乾性、いずれの状態であってもよく、例えば、これまで廃棄されていたインスタントコーヒー製造後に排出されるコーヒー粕や、コーヒーショップから排出されるコーヒー粕等を好適に用いることができる。 The method for producing quinic acid and / or caffeic acid of the present invention includes a step (A) of producing coffee koji by growing a koji mold that produces chlorogenic acid hydrolase in coffee koji, and the coffee koji or The method includes a step (B) of producing quinic acid and / or caffeic acid by reacting chlorogenic acid extracted from coffee koji as a catalyst with chlorogenic acid as a raw material. In the present specification, the term “coffee mash” means an extraction residue of coffee beans after the roasted coffee beans for the production of coffee beverages are extracted with steam, hot water, water, etc. The kind of bean is not particularly limited, and any kind of coffee bean may be used. Further, the coffee cake may be in any of wet and dry conditions. For example, a coffee cake discharged after instant coffee production that has been disposed of up to now, a coffee cake discharged from a coffee shop, and the like are suitable. Can be used.
 上記工程(A)における麹菌としては、クロロゲン酸加水分解酵素を産生することができるアスペルギルス(Aspergillus)属、ペニシリウム(Penicillium)属、リゾップス(Rhizopus)属、ムコール(Mucor)属、モナスカス(Monascus)属等の糸状菌であれば特に制限されるものではなく、具体的には、例えば、アスペルギルス ニガー(A.niger;AKU 3333 = IFO 4416 = NBRC 4416)、アスペルギルス アワモリ(A.awamori;AKU 3306 = IFO 4033 = NBRC 4033)、アスペルギルス ソーヤ(A.sojae;AKU 3312 = IFO 4386 = NBRC = NBRC 4386)、アスペルギウス オリゼ(Aspergillus oryzae;AKU 3382)、アスペルギウス パラスティカス(Aspergillus parasiticus;AKU3352)、等のアスペルギルス属の糸状菌や、ペニシリウム クリソジェナム(P.chrysogenum;AKU 3407 = IFO 4897 = NBRC 4897)等のペニシリウム属の糸状菌や、ムコール ラセモサス(M.racemosus;AKU 3002= IFO 4581 = NBRC 4581)、ムコール ジャバニクス(M.javanicus;AKU 3009 = IFO 4570 = NBRC 4570)等のケカビ属の糸状菌や、リゾップス オリゼ(R.oryzae;AKU 3119 = IFO 4705 = NBRC 4705)、リゾップス ジャバニクス(R.javanicus;AKU 3110 = IFO 4737)等のクモノスカビ属の糸状菌や、モナスカス パープレウス(M.purpureus;AKU 3503 = IFO 5965 = NBRC 5965)等のベニコウジカビ属の糸状菌を好適に挙げることができるが、なかでもアスペルギルス属の糸状菌であることが好ましく、アスペルギルス ニガー又はアスペルギルス ソーヤであることが特に好ましい。上記の微生物は京都大学農学部・農学研究科(AKU;Faculty of Agriculture, Kyoto University、京都市左京区北白川追分町)や、財団法人発酵研究所(IFO;Institute for Fermentation、大阪市淀川区十三本町2丁目17番85号)や、独立行政法人製品評価技術基盤機構バイオテクノロジー本部生物遺伝資源部門(NBRC;National Institute of Technology and Evaluation Biological Resource Center、千葉県木更津市かずさ鎌足2-5-8)にて分譲を受けることが可能である。 As the koji mold in the above-mentioned step (A), Aspergillus genus, Penicillium genus, Rhizopus genus, Mucor genus, Monascus genus that can produce chlorogenic acid hydrolase For example, Aspergillus niger (A.niger; AKU 3333 = IFO 4416 = NBRC 4416), Aspergillus awamori (A.awamori; AKU 3306 = IFO) 4033 = NBRC 4033), Aspergillus sojae (A.sojae; AKU 3312 = IFO 4386 = NBRC = NBRC 4386), Aspergillus oryzae (AKU 3382), Aspergillus sp. Fungi and Penicillium chrysogenum (P.chrysogenum; AKU 3407 = IFO 4897 = NBRC 4897) Penicillium spp., Mucor racemosus (M.racemosus; AKU 3002 = IFO 4581 = NBRC 4581), Mucor jabnicus (M.javanicus; AKU 3009 = IFO 4570 = NBRC 4570), Rhizopus oryzae (R.oryzae; AKU 3119 = IFO 4705 = NBRC 4705), Rhizopus jabanix (R.javanicus; AKU 3110 = IFO 4737), etc., and Monascus perpreus (M.purpureus = AKU 350 5965 = NBRC 5965) and the like, preferably, Aspergillus filamentous fungi are preferable, and Aspergillus niger or Aspergillus soya is particularly preferable. The above-mentioned microorganisms include Kyoto University Faculty of Agriculture, Graduate School of Agriculture (AKU: Faculty of Agriculture, Kita-Shirakawa Oiwake-machi, Sakyo-ku, Kyoto), Fermentation Institute (IFO), Institute for Formentation, Juso-cho, Sasagawa-ku, Osaka 2-17-85), National Institute of Biotechnology, Biotechnology Headquarters, National Institute of Technology and Evaluation (NBRC; National Institute of Technology and Evaluation, Biological Resource Center, 2-5-8, Kazusa Kamashizu, Kisarazu City, Chiba Prefecture) Can be sold at
 上記コーヒー粕麹は、例えば以下のようにして製造することができる。すなわち、集めたコーヒー粕に含まれる過剰の水分を除去し、密閉性の耐熱性容器で加熱殺菌する。加熱殺菌は、100~130℃で、3~10分程度行うが、望ましくは、110~120℃で、5~7分行う。室温まで放冷後、別途培養しておいた麹菌を含む培養液(種菌)を加える。コーヒー粕あたりの種菌の量としては、コーヒー粕中の水分量、培養液中の種菌の生育量により変わるが、コーヒー粕1部に対し、種菌となる麹菌を0.001~0.1部含む培養液70~200ml、好ましくは100ml前後の培養液を加える。種菌として使用するのは、コーヒー粕に生育可能な麹菌であればいかなるものでも良く、クロロゲン酸を唯一の炭素源とした培養液に生育できる微生物であれば、該微生物はクロロゲン酸加水分解酵素を産生すると判断できる。種菌の製法は、一般的に行われる糸状菌の培養法で製造することができ、麹菌が生育可能な培地で1~2日間培養して製造する。 The above coffee bowl can be manufactured as follows, for example. That is, excess water contained in the collected coffee cake is removed, and heat sterilized in a hermetic heat-resistant container. The heat sterilization is performed at 100 to 130 ° C. for about 3 to 10 minutes, preferably at 110 to 120 ° C. for 5 to 7 minutes. After cooling to room temperature, a culture solution (seed fungus) containing koji molds that have been cultured separately is added. The amount of inoculum per coffee lees varies depending on the amount of water in the coffee lees and the growth of the inoculum in the culture solution. Add about 70 to 200 ml of culture broth, preferably about 100 ml of broth. As the inoculum, any koji mold capable of growing on coffee koji may be used, and any microorganism capable of growing in a culture solution containing chlorogenic acid as a sole carbon source may be selected from chlorogenic acid hydrolase. It can be judged to produce. The inoculum can be produced by a commonly used method for culturing filamentous fungi, which is produced by culturing for 1 to 2 days in a medium in which koji molds can grow.
 種菌を加えたコーヒー粕は、培養室または孵卵器内で予備的に培養して菌糸の伸長を促したのち、本培養を行うことが望ましい。予備培養は、25~37℃で、12~24時間行うが、菌糸の成長を確認できれば本培養へ移すことができる。本培養の形態は、コーヒー粕に付着した微生物が充分に生育できる状態であれば、どのような形であっても良いが、酒造用の麹蓋など表面積を広くとることができる道具を使用することが望ましい。麹蓋に予備培養した内容物を移して、25~37℃で、好ましくは30~35℃で、5~10日間、好ましくは5~7日間培養する。培養中には、胞子の着生を妨げ、菌糸の伸長を促すために、切り返しを一日数回行うことが望ましい。培養経過とともに水分の蒸散、菌糸の伸長が起こり、コーヒー粕の塊から、さらさらしたコーヒー粕麹を製造することができる。このようにして製造したコーヒー粕麹は、自然乾燥により長期保存が可能である。また、コーヒー粕麹製造に際し、製麹を容易にするために一部を仕込みに加えることもできる。 It is desirable that the coffee koji with the inoculum be preliminarily cultured in a culture room or an incubator to promote the growth of mycelia and then perform the main culture. The preculture is carried out at 25 to 37 ° C. for 12 to 24 hours, but can be transferred to the main culture if the mycelial growth can be confirmed. The form of the main culture may be any form as long as the microorganisms attached to the coffee gruel can sufficiently grow, but a tool that can take a large surface area such as a straw lid for sake brewing is used. It is desirable. The pre-cultured contents are transferred to a lid and cultured at 25-37 ° C., preferably 30-35 ° C., for 5-10 days, preferably 5-7 days. During culturing, it is desirable to cut back several times a day to prevent spore formation and promote hyphal elongation. Moisture transpiration and hyphal elongation occur as the culture progresses, and a smooth coffee candy can be produced from the coffee candy mass. The coffee cake produced in this way can be stored for a long time by natural drying. In addition, when making coffee koji, a part can be added to the preparation in order to facilitate koji making.
 このようにして作製されたコーヒー粕麹は、伸長した微生物の菌糸の外側に加水分解酵素が安定に保持されているものである。このため、上記工程(B)においては、該コーヒー粕麹を微生物触媒としてそのまま使用することも、該コーヒー粕麹からクロロゲン酸加水分解酵素を抽出して触媒として使用することもできる。とくに糸状菌は、コーヒー粕の微細な間隙にまで菌糸を伸長させることができるので、生産効率の良い微生物触媒を作製することができる。微生物触媒として使用する場合は、例えば、50~60℃で、約30分間の熱処理を行い、微生物の生育能力を喪失させてから行うことが望ましい。微生物は、熱処理によって増殖できなくなるが、加水分解酵素活性、特にクロロゲン酸加水分解酵素活性は、耐熱性であり加熱処理後も酵素活性は安定に保持されている。該微生物触媒は、一種の固定化微生物触媒の形態を保有しているので、さらに特別な固定化処理をすることなく、そのまま水または生理食塩水に懸濁して反応液に加えて使用することもできる。 The coffee koji produced in this way has a hydrolytic enzyme stably held on the outside of the hyphae of the elongated microorganism. For this reason, in the said process (B), this coffee koji can be used as it is as a microbial catalyst, or chlorogenic acid hydrolase can be extracted from this coffee koji and used as a catalyst. In particular, filamentous fungi can extend mycelia to the fine gaps in the coffee gruel, so that a microbial catalyst with high production efficiency can be produced. When used as a microbial catalyst, for example, it is desirable to carry out heat treatment at 50 to 60 ° C. for about 30 minutes to lose the ability to grow microorganisms. Microorganisms cannot grow by heat treatment, but hydrolase activity, particularly chlorogenic acid hydrolase activity, is heat resistant and the enzyme activity is stably maintained even after heat treatment. Since the microbial catalyst is in the form of a kind of immobilized microbial catalyst, it can be used as it is suspended in water or physiological saline and added to the reaction solution without any special immobilization treatment. it can.
 クロロゲン酸加水分解酵素が、コーヒー粕麹に存在することは、コーヒー粕麹を粉砕し、水または0.7~1.0%程度の生理食塩水で抽出して確認することができる。すなわち、溶液に浸漬したコーヒー粕麹から抽出した後、固形物や菌糸を除くためにろ紙で濾過し、ろ液を飽和硫安水で透析する。透析中に酵素液の水分は減少するとともに、酵素タンパク質や共存する他のタンパク質などは沈殿する。遠心分離器で沈殿をあつめ、新たな透析膜に沈殿を入れて、再度0.7~1.0%の生理食塩水で透析する。透析後に発生している不溶性の沈殿を遠心分離して除去することにより濃縮された酵素液が得られる。クロロゲン酸加水分解酵素の1単位(unit)は、1分間にクロロゲン酸を1μmole加水分解できる酵素力を1単位とする。 The presence of chlorogenic acid hydrolase in the coffee mash can be confirmed by pulverizing the coffee mash and extracting with water or about 0.7 to 1.0% physiological saline. That is, after extracting from a coffee grinder soaked in the solution, it is filtered through filter paper to remove solids and mycelia, and the filtrate is dialyzed against saturated ammonium sulfate. During dialysis, the water in the enzyme solution decreases, and enzyme proteins and other proteins that coexist are precipitated. Collect the precipitate with a centrifuge, put the precipitate into a new dialysis membrane, and dialyze again with 0.7-1.0% saline. A concentrated enzyme solution can be obtained by removing the insoluble precipitate generated after dialysis by centrifugation. One unit of chlorogenic acid hydrolase is defined as one unit of enzyme power capable of hydrolyzing chlorogenic acid by 1 μmole per minute.
 また、上記工程(B)において、原料となるクロロゲン酸の由来としては特に制限されるものではないが、コーヒー豆やコーヒー粕から抽出されたクロロゲン酸を有利に用いることができ、廃棄物利用の観点からするとコーヒー粕から抽出されたクロロゲン酸であることが特に好ましい。コーヒー豆にはクロロゲン酸が多く含有されており、その含有量は生豆時が多く、5.5~8.0%であり、焙煎後で1.2~2.3%とされている。原料として使用するコーヒー粕としては、焙煎コーヒー豆から得られた粉砕粕、インスタントコーヒー製造後の粕、コーヒー店等で排出されるコーヒー抽出後の粕があげられる。 Moreover, in the said process (B), although it does not restrict | limit especially as origin of the chlorogenic acid used as a raw material, the chlorogenic acid extracted from the coffee bean and the coffee koji can be used advantageously, and waste utilization From the viewpoint, chlorogenic acid extracted from coffee candy is particularly preferable. Coffee beans contain a large amount of chlorogenic acid, the content of which is mostly raw beans, 5.5 to 8.0%, and 1.2 to 2.3% after roasting . Examples of the coffee mash used as a raw material include a crushed mash obtained from roasted coffee beans, a mash after instant coffee production, and a mash after coffee extraction discharged at a coffee shop or the like.
 コーヒー粕に、加水分解活性を有する微生物触媒を作用させて、キナ酸および/またはカフェ酸を製造するためには、例えば、コーヒー粕を0.1~50%、好ましくは1~20%の濃度になるように、水、生理食塩水、バッファーなどを加えて懸濁溶液にして、あるいは熱水で抽出した後に、加水分解酵素を含有するコーヒー粕麹からなる微生物触媒に接触させ、10~50℃、好ましくは30~40℃で、1~24時間、好ましくは3~8時間反応させることもできるが、コーヒー粕からクロロゲン酸を抽出して、クロロゲン酸を原料とすることもできる。クロロゲン酸を原料とする場合は、酵素の必要量が明確であり、生成したキナ酸やカフェ酸の精製が容易であることから、クロロゲン酸を用いることが望ましい。コーヒー粕クロロゲン酸を抽出する方法は、一般的な有機酸の抽出法に準じて行うことができる。例えば、コーヒー粕を熱水抽出して、活性炭で脱色し、陰イオン交換樹脂のダウエックス(登録商標)カラムに吸着させ、酢酸を含有するメタノール、エタノール、イソプロパノール、アセトン、アセトニトリルなどの水溶性有機溶媒で溶出して分離精製し、溶媒を除去することでクロロゲン酸を容易に得ることができる。 In order to produce a quinic acid and / or caffeic acid by allowing a microbial catalyst having hydrolysis activity to act on the coffee cake, for example, the coffee cake has a concentration of 0.1 to 50%, preferably 1 to 20%. After adding water, physiological saline, buffer, etc. to make a suspension solution or extracting with hot water, it is contacted with a microbial catalyst comprising a coffee koji containing hydrolase and 10 to 50 The reaction can be carried out at 0.degree. C., preferably 30 to 40.degree. C. for 1 to 24 hours, preferably 3 to 8 hours. When chlorogenic acid is used as a raw material, it is desirable to use chlorogenic acid because the required amount of enzyme is clear and the produced quinic acid and caffeic acid are easily purified. The method for extracting coffee brewing chlorogenic acid can be performed in accordance with a general organic acid extraction method. For example, coffee mash is extracted with hot water, decolorized with activated carbon, adsorbed on a Dowex (registered trademark) column of anion exchange resin, and water-soluble organics such as methanol, ethanol, isopropanol, acetone, acetonitrile containing acetic acid Chlorogenic acid can be easily obtained by elution with a solvent, separation and purification, and removal of the solvent.
 このようにして調製されたクロロゲン酸を原料として、キナ酸および/またはカフェ酸を製造する方法は、例えば、以下のように行うことができる。クロロゲン酸に、クロロゲン酸加水分解酵素0.5~10単位、好ましくは1~3単位を含んでいるコーヒー粕麹からなる微生物触媒を加えて、pH5~8、好ましくはpH6~7の緩衝液、例えば酢酸緩衝液、リン酸緩衝液で、10~50℃、好ましくは30~40℃で、1~24時間、好ましくは3~8時間反応させる。微生物触媒の量が多い場合は反応時間を短くすることができる。また、微生物触媒をカラム等に入れて固定化して、クロロゲン酸を吸着溶出させて加水分解反応を行うこともできる。 The method for producing quinic acid and / or caffeic acid using chlorogenic acid thus prepared as a raw material can be carried out as follows, for example. A buffer solution of pH 5-8, preferably pH 6-7, by adding to the chlorogenic acid a microbial catalyst consisting of coffee koji containing 0.5-10 units, preferably 1-3 units of chlorogenic acid hydrolase, For example, the reaction is carried out at 10 to 50 ° C., preferably 30 to 40 ° C. for 1 to 24 hours, preferably 3 to 8 hours with an acetate buffer or phosphate buffer. When the amount of the microbial catalyst is large, the reaction time can be shortened. Alternatively, the hydrolysis reaction can be performed by immobilizing a microbial catalyst in a column or the like and adsorbing and eluting chlorogenic acid.
 コーヒー粕麹からなる微生物触媒を用いて製造したキナ酸および/またはカフェ酸を精製する方法は特に制限されるものではなく、一般的に行われる有機酸の精製法を用いることができるが、例えば、イオン交換樹脂を使用した足立らの方法(Adachi, O., Ano, Y., Toyama, H., and Matsushita, Biosci. Biotechnol. Biochem., 70: 3081-3083, 2006)に準じて行う場合は、クロロゲン酸、キナ酸及びカフェ酸が混在する反応液を陰イオン交換樹脂のダウエックス(登録商標)カラムに吸着させ、適切な溶出液、例えば、メタノール、エタノール、イソプロパノール、アセトン、アセトニトリルなどの水溶性有機溶媒に、酢酸、ギ酸、塩酸などの酸を加え、分離して溶出させる。キナ酸は低濃度の塩化ナトリウムなどの塩類溶液で容易にカラムから溶出され、カフェ酸は、1M 酢酸-メタノール溶液等で溶出させることができ、未反応のクロロゲン酸は、酢酸濃度をあげることにより溶出させることができる。あるいは、逆相クロマトグラフィーを用いる場合は、上記水溶性有機溶媒に低濃度の弱酸を加えることにより、キナ酸、クロロゲン酸、カフェ酸の順に分離して溶出させることができる。 The method for purifying quinic acid and / or caffeic acid produced using a microbial catalyst comprising coffee koji is not particularly limited, and a commonly used organic acid purification method can be used. , Using the method of Adachi et al. (Adachi, O., Ano, Y., Toyama, H., and Matsushita, Biosci. Biotechnol. Biochem., 70: 3081-3083, 2006) Adsorbs a reaction mixture containing chlorogenic acid, quinic acid and caffeic acid on a Dowex (registered trademark) column of an anion exchange resin, and a suitable eluent such as methanol, ethanol, isopropanol, acetone, acetonitrile, etc. Acids such as acetic acid, formic acid and hydrochloric acid are added to a water-soluble organic solvent and separated and eluted. Quinic acid can be easily eluted from the column with low-concentration sodium chloride and other salt solutions, caffeic acid can be eluted with 1M acetic acid-methanol solution, and unreacted chlorogenic acid can be obtained by increasing the acetic acid concentration. Can be eluted. Alternatively, when reverse-phase chromatography is used, quinic acid, chlorogenic acid, and caffeic acid can be separated and eluted in this order by adding a low-concentration weak acid to the water-soluble organic solvent.
 本発明の記載のキナ酸および/またはカフェ酸の製造方法に用いるために調製されたクロロゲン酸加水分解酵素活性を有するコーヒー粕麹は、麹菌がコーヒー粕中に含まれるクロロゲン酸と常時接触している状態にあるため、クロロゲン酸加水分解酵素は常時活性化された状態にあり、高いクロロゲン酸加水分解活性を有している。また、以下の実施例に示すように、コーヒー粕麹は抽出された酵素に比べて熱安定性が高く、あらかじめコーヒー粕麹を60℃で30分程度熱処理することにより、クロロゲン酸加水分解酵素活性を損なうことなく麹の生細胞の繁殖力を停止したものを、そのまま適当な反応容器に入れ、固定化酵素として使用することができる。具体的には、例えば、上記熱処理後のコーヒー粕麹を、カラム等に充填してすることにより、通水性に優れ、反応速度を自在に調節できる固定化酵素としてそのまま使用することができる。 The coffee cake having chlorogenic acid hydrolase activity prepared for use in the method for producing quinic acid and / or caffeic acid described in the present invention is obtained by contacting the koji mold with chlorogenic acid contained in the coffee cake. Therefore, the chlorogenic acid hydrolase is always activated and has a high chlorogenic acid hydrolyzing activity. In addition, as shown in the following examples, coffee koji has higher thermal stability than the extracted enzyme, and the chlorogenic acid hydrolase activity is obtained by heat treating the coffee koji for 30 minutes at 60 ° C. in advance. Those in which the proliferative ability of viable cells of the pupae is stopped without impairing can be put into an appropriate reaction vessel as it is and used as an immobilized enzyme. Specifically, for example, by filling the coffee koji after the above heat treatment into a column or the like, it can be used as it is as an immobilized enzyme that has excellent water permeability and can freely adjust the reaction rate.
 以下、本発明を更に詳しく説明するため、実施例を挙げるが本発明はこれに限定されない。 Hereinafter, in order to describe the present invention in more detail, examples will be given, but the present invention is not limited to them.
 <コーヒー粕麹の製造>
 コーヒー粕4.5kgを集め、布袋に入れて洗濯機の脱水槽で1~2分脱水して過剰の水分を除去した。これを密閉できる耐熱性容器(3リットル容フラスコ)3個(1個あたり約1.5kgのコーヒー粕)に入れて、口を綿栓とアルミホイルで覆い、1気圧120℃で、約5分間殺菌した。室温まで放冷後、別途、Czapek培地で培養しておいたアスペルギルス ソーヤ(AKU 3312)を含む培養液を1kgのコーヒー粕あたりそれぞれ100ml加えた。培養液を付着させたコーヒー粕の入ったフラスコを、30℃の培養室内で一夜放置して菌糸の伸長を促したのち、酒造用の麹蓋(縦×横×高さ=29.0cm×44.0cm×5.0cm)3枚に各フラスコ内容物を移して、30℃で培養を継続した。子嚢胞子の着生を妨げ、菌糸の伸長を促すために、米麹の製造と同様に切り返しを1日あたり2~3度行った。菌糸の伸長にともない培養経過とともに水分の蒸散もあり、当初見られたコーヒー粕の塊は徐々に小さくなり、7日目でさらさらしたコーヒー粕麹ができた(図1)。
<Manufacture of coffee candy>
4.5 kg of coffee mash was collected, put in a cloth bag and dehydrated in a washing machine dehydration tank for 1-2 minutes to remove excess water. Put this in three heat-resistant containers (3 liter flasks) that can be sealed (about 1.5 kg of coffee per cup), cover the mouth with a cotton plug and aluminum foil, and 1 atmosphere at 120 ° C for about 5 minutes Sterilized. After allowing to cool to room temperature, 100 ml of a culture solution containing Aspergillus soya (AKU 3312) that had been cultured in a Czapek medium separately was added to each 1 kg of coffee grounds. The flask containing the coffee cake with the culture solution attached was allowed to stand overnight in a 30 ° C. culture chamber to promote the growth of the mycelium, and then the brewery lid (length × width × height = 29.0 cm × 44) The contents of each flask were transferred to 3 sheets (0.0 cm × 5.0 cm), and the culture was continued at 30 ° C. In order to prevent the growth of ascospores and promote the growth of hyphae, turning was performed 2-3 times per day in the same manner as rice bran production. As the mycelium stretched, there was a transpiration of water with the progress of cultivation, and the initially observed coffee cake lump gradually became smaller, and a smooth coffee cake was formed on the seventh day (FIG. 1).
 <クロロゲン酸加水分解酵素液の調製>
 実施例1で得られたコーヒー粕麹の酵素活性を確認するための対照として、あらかじめコーヒー粕麹の表面についている糸状菌からの酵素液を調製した。すなわち、コーヒー粕麹50gを家庭用のジューサーに入れて、0.7%生理食塩水に浸漬して撹拌し、固形物や菌糸を除くためにろ紙で濾過した。これを、塩は除去できるがタンパク質は通過できない構造を有する透析膜でできた袋に入れ、飽和硫安水(pH7.0)に一夜透析した。透析中に酵素液の水分は減少するとともに、酵素タンパク質や共存する他のタンパク質などは沈殿する。沈殿を集めることができる遠心分離器で沈殿を集め、新たな透析膜に沈殿を入れて1.0%の生理食塩水に一夜透析した。透析後に発生している不溶性の沈殿を遠心分離で除去し、透明な上清を取り、濃縮された酵素液を得た。
<Preparation of chlorogenic acid hydrolase solution>
As a control for confirming the enzyme activity of the coffee koji obtained in Example 1, an enzyme solution from a filamentous fungus previously attached to the surface of the coffee koji was prepared. That is, 50 g of coffee koji was put in a household juicer, immersed in 0.7% physiological saline and stirred, and filtered with a filter paper to remove solids and mycelia. This was put in a bag made of a dialysis membrane having a structure capable of removing salts but not allowing protein to pass through, and dialyzed overnight against saturated ammonium sulfate (pH 7.0). During dialysis, the water in the enzyme solution decreases, and enzyme proteins and other proteins that coexist are precipitated. The precipitate was collected with a centrifuge capable of collecting the precipitate, placed in a new dialysis membrane, and dialyzed overnight against 1.0% physiological saline. Insoluble precipitate generated after dialysis was removed by centrifugation, and a clear supernatant was taken to obtain a concentrated enzyme solution.
 <コーヒー粕麹によるクロロゲン酸の加水分解反応>
 実施例1で、アスペルギルス ソーヤ(AKU 3312)を用いて製造したコーヒー粕麹、および対照とする実施例2で調製した酵素液を用いてクロロゲン酸の加水分解反応を行った。すなわち、クロロゲン酸50μmole(17.7mg相当)に、1)120℃で5分間の熱処理をしたクロロゲン酸加水分解酵素1単位を含んでいるコーヒー粕麹;2)熱処置しないクロロゲン酸加水分解酵素1単位を含んでいるコーヒー粕麹;3)120℃で5分間の熱処理をしたクロロゲン酸加水分解酵素1単位を含んでいる酵素液;4)熱処置しないクロロゲン酸加水分解酵素1単位を含んでいる酵素液;をそれぞれ加え、0.1M 酢酸緩衝液(pH6.5)で反応液量を1mlに調節し、30℃で3時間反応させた。反応液の一部(5μl)を薄層にスポットして、n-ブタノール:酢酸:水=4:2:1の展開溶液で薄層クロマトグラフィーを行った。図2に示したように、熱処理しないで行った反応液2)と4)中には、カフェ酸(Rf=0.85)の存在を示すスポットが見られ、酵素液と同様に、コーヒー粕麹中にクロロゲン酸加水分解酵素が存在することが明らかになった。また、熱処理した反応液1)と3)中にはクロロゲン酸(Rf=0.65)が分解されずに残存し、酵素液、コーヒー粕麹、両者共に、クロロゲン酸加水分解酵素活性が失われていることを示している。また、足立らの方法(Adachi O. et al. Biosci. Biotecnol. Biochem., 67: 2124-2131, 2003)に従って標準物のキナ酸から位置を特定して、反応液2)と4)中には、キナ酸(Rf=0.15)の存在を示すスポットが確認できた。なお、キナ酸はフェノール性化合物でないことから空気にさらされて黒化しないために、カフェ酸と比較してスポットが薄くなっている。
<Hydrolysis reaction of chlorogenic acid by coffee lees>
In Example 1, chlorogenic acid was hydrolyzed using coffee koji produced using Aspergillus soya (AKU 3312) and the enzyme solution prepared in Example 2 as a control. That is, 50 μmole of chlorogenic acid (corresponding to 17.7 mg): 1) coffee cake containing 1 unit of chlorogenic acid hydrolase that was heat-treated at 120 ° C. for 5 minutes; 2) chlorogenic acid hydrolase 1 without heat treatment Coffee cake containing units; 3) enzyme solution containing 1 unit of chlorogenic acid hydrolase that has been heat treated at 120 ° C. for 5 minutes; 4) containing 1 unit of chlorogenic acid hydrolase without heat treatment Each of the enzyme solutions was added, and the volume of the reaction solution was adjusted to 1 ml with 0.1 M acetate buffer (pH 6.5), and reacted at 30 ° C. for 3 hours. A part (5 μl) of the reaction solution was spotted on a thin layer, and thin layer chromatography was performed with a developing solution of n-butanol: acetic acid: water = 4: 2: 1. As shown in FIG. 2, spots indicating the presence of caffeic acid (Rf = 0.85) were observed in the reaction solutions 2) and 4) that were not heat-treated. It was revealed that chlorogenic acid hydrolase was present in the straw. In addition, chlorogenic acid (Rf = 0.65) remains in the heat-treated reaction liquids 1) and 3) without being decomposed, and the chlorogenic acid hydrolase activity is lost in both the enzyme liquid and coffee cake. It shows that. Also, according to the method of Adachi et al. (Adachi O. et al. Biosci. Biotecnol. Biochem., 67: 2124-2131, 2003), the position was determined from the standard quinic acid, and during the reaction solutions 2) and 4) Confirmed a spot indicating the presence of quinic acid (Rf = 0.15). In addition, since quinic acid is not a phenolic compound, it is exposed to air and does not darken, so the spot is thinner than caffeic acid.
 <クロロゲン酸を原料とするキナ酸とカフェ酸の製造>
 実施例1で、アスペルギルス ソーヤ AKU 3312を用いて製造したクロロゲン酸加水分解酵素を含んでいるコーヒー粕麹を用いてクロロゲン酸の加水分解反応を行った。すなわち、クロロゲン酸100μmole(35.4mg相当)に、クロロゲン酸加水分解酵素2単位を含んでいるコーヒー粕麹を加え、0.1M 酢酸緩衝液(pH6.5)で反応液量を1mlに調節して、30℃で5時間反応させた。反応液の1μlを液体クロマトグラフィー(装置 Shimadzu LC-20AD、カラム Mightysil RP-18(4.6x150mm)、溶媒 50mM酢酸:アセトニトリル=8:2、流速 0.8ml/分、検出器 RID)で分析した。得られたチャートを図3に示した。反応前に存在したクロロゲン酸(100μmole/ml)は完全に消滅して、化学量論的にカフェ酸とキナ酸が生成された。反応液中にはカフェ酸と等量のキナ酸が生成されていることが証明された。
<Manufacture of quinic acid and caffeic acid from chlorogenic acid>
In Example 1, the hydrolysis reaction of chlorogenic acid was performed using a coffee koji containing chlorogenic acid hydrolase produced using Aspergillus soja AKU 3312. That is, add coffee mash containing 2 units of chlorogenic acid hydrolase to 100 μmole of chlorogenic acid (corresponding to 35.4 mg), and adjust the reaction volume to 1 ml with 0.1M acetic acid buffer (pH 6.5). And reacted at 30 ° C. for 5 hours. 1 μl of the reaction solution was analyzed by liquid chromatography (equipment Shimadzu LC-20AD, column Mightysil RP-18 (4.6 × 150 mm), solvent 50 mM acetic acid: acetonitrile = 8: 2, flow rate 0.8 ml / min, detector RID). . The obtained chart is shown in FIG. Chlorogenic acid (100 μmole / ml) present before the reaction was completely extinguished, and stoichiometrically produced caffeic acid and quinic acid. It was proved that quinic acid equivalent to caffeic acid was produced in the reaction solution.
 <コーヒー粕中のクロロゲン酸を原料とするキナ酸とカフェ酸の製造>
 コーヒー粕1kgを1時間熱水抽出して、ろ過し、ろ液を活性炭で脱色して陰イオン交換樹脂のダウエックス(登録商標)カラムにかけた。塩化ナトリウム水溶液、メタノール、1M酢酸-メタノールの順に溶出溶媒を変えて不純物を溶出させた後、5M酢酸-メタノール溶液でクロロゲン酸をカラムから溶出し、溶媒を除去してクロロゲン酸得た。このクロロゲン酸を原料として、実施例4と同様にしてカフェ酸とキナ酸を製造し、反応液を液体クロマトグラフィーにかけカフェ酸とキナ酸の生成を確認した。
<Manufacture of quinic acid and caffeic acid from chlorogenic acid in coffee cake>
1 kg of coffee grounds was extracted with hot water for 1 hour and filtered, and the filtrate was decolorized with activated carbon and applied to a Dowex (registered trademark) column of anion exchange resin. Impurities were eluted by changing the elution solvent in the order of sodium chloride aqueous solution, methanol and 1M acetic acid-methanol, and then chlorogenic acid was eluted from the column with a 5M acetic acid-methanol solution to obtain chlorogenic acid. Using this chlorogenic acid as a raw material, caffeic acid and quinic acid were produced in the same manner as in Example 4, and the reaction solution was subjected to liquid chromatography to confirm the formation of caffeic acid and quinic acid.
 <クロロゲン酸加水分解酵素の活性化>
 アスペルギルス ソーヤ(AKU 3312)をインスタントコーヒー粉末を含む培養液で培養し、培養後の菌抽出液をクロロゲン酸と反応させることにより、クロロゲン酸加水分解酵素の活性に及ぼすコーヒー粉末の影響について検討した。図4に結果を示すように、コーヒー粉末を含まない条件下で培養した菌抽出液にはクロロゲン酸加水分解酵素活性がほとんど確認できなかったが、コーヒー粉末を含む条件下で培養した菌抽出液では顕著なクロロゲン酸加水分解酵素の活性が認められた。このような、クロロゲン酸加水分解酵素の誘導・活性化効果は5~20%(w/v)のコーヒー粕を含む培養液で糸状菌を培養した場合にも同様に認められた。
<Activation of chlorogenic acid hydrolase>
Aspergillus soya (AKU 3312) was cultured in a culture solution containing instant coffee powder, and the effect of coffee powder on the activity of chlorogenic acid hydrolase was examined by reacting the bacterial extract after culture with chlorogenic acid. As shown in FIG. 4, the bacterial extract cultivated under the condition containing no coffee powder showed almost no chlorogenic acid hydrolase activity, but the bacterial extract cultivated under the condition containing coffee powder. Showed remarkable chlorogenic acid hydrolase activity. Such induction / activation effect of chlorogenic acid hydrolase was also observed when filamentous fungi were cultured in a culture solution containing 5 to 20% (w / v) coffee candy.
 <クロロゲン酸加水分解酵素の耐熱性>
 実施例1で製造したコーヒー粕麹と、実施例2で調製した酵素液を、50℃~70℃でそれぞれ30分間処理することにより、クロロゲン酸加水分解酵素の耐熱性について検討した。図5に結果を示すように、実施例2で調製した酵素液の安定性は50℃の処理では保たれていたが、60℃の処理後には酵素活性が確認できなかった。一方、菌体の酵素は60℃で30分の処理の後でも酵素活性が確認された。
<Heat resistance of chlorogenic acid hydrolase>
The heat resistance of the chlorogenic acid hydrolase was examined by treating the coffee lees produced in Example 1 and the enzyme solution prepared in Example 2 for 30 minutes at 50 ° C. to 70 ° C., respectively. As shown in FIG. 5, the stability of the enzyme solution prepared in Example 2 was maintained by the treatment at 50 ° C., but the enzyme activity could not be confirmed after the treatment at 60 ° C. On the other hand, the enzyme activity of the bacterial cell enzyme was confirmed even after treatment at 60 ° C. for 30 minutes.
 <アスペルギルス属のクロロゲン酸加水分解酵素の活性化>
 数種類のアスペルギルス属の糸状菌をインスタントコーヒー粉末を含む培養液で培養し、培養後の菌抽出液をクロロゲン酸と反応させることにより、アスペルギルス ソーヤ以外のアスペルギルス属の糸状菌のクロロゲン酸加水分解酵素の活性について検討した。図6に結果を示すように、アスペルギルス ソーヤ以外のアスペルギルス属の糸状菌においても、クロロゲン酸加水分解酵素の活性が認められた。
<Activation of Aspergillus chlorogenic acid hydrolase>
By culturing several types of Aspergillus fungi in a culture solution containing instant coffee powder, and reacting the bacterial extract after incubation with chlorogenic acid, chlorogenic acid hydrolase of Aspergillus fungi other than Aspergillus sojae The activity was examined. As shown in FIG. 6, the activity of chlorogenic acid hydrolase was also observed in filamentous fungi of the genus Aspergillus other than Aspergillus soya.
 本発明により、医薬品や機能性素材となるキナ酸やカフェ酸を、コーヒー粕から製造することができるため、大量に廃棄されるコーヒー粕の有効利用が可能になるとともに、シキミ酸およびシキミ酸経路の代謝中間体の原料となるキナ酸を安価で提供することができる。 According to the present invention, since quinic acid and caffeic acid, which are pharmaceuticals and functional materials, can be produced from coffee mash, it becomes possible to effectively use coffee mash that is discarded in large quantities, and the shikimic acid and shikimic acid pathways. Quinic acid, which is a raw material for the metabolic intermediate, can be provided at a low cost.

Claims (6)

  1.  コーヒー粕にクロロゲン酸加水分解酵素を産生する麹菌を生育させることによりコーヒー粕麹を作製する工程(A)と、前記コーヒー粕麹又はコーヒー粕麹から抽出されたクロロゲン酸加水分解酵素を触媒として、クロロゲン酸を原料として反応させることにより、キナ酸および/またはカフェ酸を生成させる工程(B)とを含むことを特徴とする、キナ酸および/またはカフェ酸の製造方法。 Step (A) for producing coffee koji by growing a koji mold producing chlorogenic acid hydrolase on coffee koji, and using as a catalyst the chlorogenic acid hydrolase extracted from the coffee koji or coffee koji, A step (B) of producing quinic acid and / or caffeic acid by reacting chlorogenic acid as a raw material, and a method for producing quinic acid and / or caffeic acid.
  2.  コーヒー粕から抽出されたクロロゲン酸を原料として反応させることを特徴とする請求項1に記載のキナ酸および/またはカフェ酸の製造方法。 The method for producing quinic acid and / or caffeic acid according to claim 1, wherein chlorogenic acid extracted from coffee mash is reacted as a raw material.
  3.  麹菌が、アスペルギルス ニガー(Aspergillus niger)、アスペルギルス アワモリ(Aspergillus awamori)、アスペルギルス ソーヤ(Aspergillus sojae)、アスペルギウス オリゼ(Aspergillus oryzae)、アスペルギウス パラスティカス(Aspergillus parasiticus)、ペニシリウム クリソジェナム(Penicillium chrysogenum)から選択される少なくとも1以上の微生物であることを特徴とする請求項1又は2に記載のキナ酸および/またはカフェ酸の製造方法。 Aspergillus niger (Aspergillus niger), Aspergillus awamori, Aspergillus sojae, Aspergillus olizae (Aspergillus oryzae), Aspergillus ジ ェ silisum (Principalum cerium) The method for producing quinic acid and / or caffeic acid according to claim 1 or 2, wherein the microorganism is the above microorganism.
  4.  コーヒー粕麹が、コーヒー粕1部に対し、麹菌を0.001~0.1部含む培養液と共に予備培養後、製麹用道具を用いて、25~37℃で切り返しを行いながら5~10日間本培養を行うことにより製造されたコーヒー粕麹であることを特徴とする請求項1~3のいずれかに記載のキナ酸および/またはカフェ酸の製造方法。 The coffee koji is precultured with a culture solution containing 0.001 to 0.1 parts of koji molds per 1 knot of coffee koji, and then turned over at 25 to 37 ° C. using a koji making tool. The method for producing quinic acid and / or caffeic acid according to any one of claims 1 to 3, which is a coffee koji produced by performing main culture for a day.
  5.  クロロゲン酸加水分解酵素活性を有するコーヒー粕麹。 Coffee coffee having chlorogenic acid hydrolase activity.
  6.  麹菌がアスペルギルス ソーヤ(Aspergillus sojae)であることを特徴とする請求項5に記載のコーヒー粕麹。 6. The coffee koji according to claim 5, wherein the koji mold is Aspergillus sojae.
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