WO2009107328A1

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

Info

Publication number: WO2009107328A1
Application number: PCT/JP2009/000403
Authority: WO
Inventors: 松下一信; 足立収生
Original assignee: 国立大学法人山口大学
Priority date: 2008-02-29
Filing date: 2009-02-03
Publication date: 2009-09-03
Also published as: JP2009201473A

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

Process for producing quinic acid and / or caffeic acid

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.

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.

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).

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).

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.

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.

Japanese Patent No. 3532151 JP-A-10-95975 JP 2003-200138 A JP-A-8-224564 JP-A-6-38723 JP 2007-300809 A Japanese Patent Laid-Open No. 7-8169 Japanese Patent Laid-Open No. 11-140014 JP 11-263746 A JP 2000-86575 A JP 2001-316295 A JP 2005-204555 A JP 2005-298382 A JP 2006-213636 A JP 2006-241006 A JP 2004-315386 A JP 2004-350619 A

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

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.

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.

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.

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 .

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. 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. 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. 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). 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. 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). .

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

Hereinafter, in order to describe the present invention in more detail, examples will be given, but the present invention is not limited to them.

<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).

<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.

<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.

<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.

<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.

<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.

<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.

<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.

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

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.
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.
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.
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.
Coffee coffee having chlorogenic acid hydrolase activity.
6. The coffee koji according to claim 5, wherein the koji mold is Aspergillus sojae.