JP2009201473A - Process for production of quinic acid and/or caffeic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide 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, wherein quinic acid or caffeic acid is a raw material for a pharmaceutical agent or a functional material. <P>SOLUTION: 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. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for producing quinic acid and / or caffeic acid using coffee mash 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.

  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, a method 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 an antioxidant effect (Patent Document 5). Coffee beans change in aroma and taste by roasting, which is thought to be due to the decomposition of chlorogenic acid to produce quinic acid and 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 dehydroshikimate, which is an intermediate of the shikimate pathway, from quinic acid using cell membrane-bound quinate dehydrogenase (QDH) in acetic acid bacteria. 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 has been reported that quinic acid is contained in plants such as kina peel and sugar beet, as well as coffee beans, and extracted and purified from these materials. Using coffee beans as a raw material, alkali hydrolysis followed by treatment with a strongly basic anion exchange resin, followed by treatment with alkali, and further desalting and purification 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. (Patent Document 10), a method for producing quinic acid as a vitamin odor inhibitor that hydrolyzes coffee beans using an enzyme and purifies them with an adsorbent (Patent Document 11), and is extracted from tea leaves with water or a 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 a 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, 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. (Patent Document 17) does not suggest the production of quinic acid or caffeic acid using coffee cake as a raw material.
Japanese Patent No. 3532151 Japanese Patent Laid-Open No. 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 Adachi O. et al. Biosci. Biotecnol. Biochem. 67: 2124-2131, 2003.

  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 have found 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, and the present invention. It came to complete.

  That is, the present invention provides the following (1) to (4).

  (1) In the production of quinic acid and / or caffeic acid, a kina characterized by reacting, as a microbial catalyst, a microorganism having a chlorogenic acid hydrolase grown on coffee mash using chlorogenic acid in coffee mash as a raw material Method for producing acid and / or caffeic acid.

  (2) microbial catalyst, Aspergillus niger (Aspergillus niger), Aspergillus awamori (Aspergillus awamori), Aspergillus sojae (Aspergillus sojae), Penicillium Kurisojenamu (Penicillium chrysogenum), Mucor (Mucorales Mucor), Rhizopus (Mucorales rhyzopus), Monascus purpureus ( The method for producing quinic acid and / or caffeic acid according to the above (1), which is prepared from at least one microorganism selected from Monascus purpureus).

  (3) The microorganism catalyst is cut back at 25-37 ° C. using a koji-making tool after pre-culture with a culture solution containing 0.001-0.1 part of a microorganism serving as an inoculum for 1 part of coffee mash. The method for producing quinic acid and / or caffeic acid according to (1) or (2) above, wherein the coffee koji is produced by performing main culture for 5 to 10 days while carrying out the above.

  (4) Chlorogen, which is a coffee koji in which a microorganism having a hydrolase is grown, and is prepared for producing the quinic acid and / or caffeic acid according to any one of (1) to (3) above Coffee mash having acid hydrolase activity.

  According to the present invention, since coffee mash that is discarded in large quantities can be used for the production of quinic acid and caffeic acid, it is possible to produce useful pharmaceuticals and functional materials with inexpensive materials.

  The present invention relates to a microbial catalyst comprising a coffee koji produced by inoculating microorganisms into coffee koji discharged after instant coffee production or coffee koji discharged from a coffee shop and growing hyphae on the entire surface of the coffee koji. It is used to produce quinic acid and / or caffeic acid by hydrolyzing the chlorogenic acid contained in the coffee gruel.

  The coffee cake that is the microbial catalyst of the present invention can be produced as follows. 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 allowing to cool to room temperature, a culture solution (seed fungus) containing separately cultured microorganisms 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 70-200 ml, preferably around 100 ml of culture broth. Any microorganism can be used as an inoculum as long as it is capable of growing in a coffee koji, but a filamentous fungus is desirable. The inoculum can be produced by a commonly used (microorganism) culture method, which is produced by culturing in a medium capable of growing microorganisms for 1 to 2 days.

  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. Preliminary culture is performed at 25 to 37 ° C. for 12 to 24 hours, and can be transferred to main culture if hyphal 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 lees of the present invention are those in which the hydrolase is stably held outside the hyphae of the elongated microorganisms. For this reason, the coffee cake can be used as a microbial catalyst. In particular, when filamentous fungi are used, the mycelium can be extended to the fine gaps of 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 stably maintained 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 hydrolase in the coffee mash can be confirmed by pulverizing the coffee mash and extracting it with water or physiological saline of about 0.7 to 1.0%. 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. The precipitate is collected with a centrifuge, put into a new dialysis membrane, and dialyzed again with 0.7-1.0% physiological 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.

  Although any microorganism can be used as the microbial catalyst comprising the coffee koji of the present invention as long as it can produce chlorogenic acid hydrolase, it is desirable to use filamentous fungi. The filamentous fungi, Aspergillus niger (Aspergillus niger), Aspergillus awamori (Aspergillus awamori), Aspergillus sojae (Aspergillus sojae), Penicillium Kurisojenamu (Penicillium chrysogenum), Mucor (Mucorales mucor), Rhizopus (Mucorales rhyzopus), Monascus purpureus (Monascus purpureus ).

  Specific examples include Aspergillus niger AKU 3333, Aspergillus awamori AKU 3306, Aspergillus soya AKU 3312, Penicillium chrysogenum AKU3407, and the like.

  Coffee mash serving as a raw material for producing quinic acid and / or caffeic acid of the present invention contains a large amount of chlorogenic acid which is a precursor of quinic acid and caffeic acid. Its content is often at the time of green beans and is 5.5 to 8.0%, and is 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 brought into contact with a microbial catalyst consisting of coffee koji containing hydrolase, and 10 to 50 The reaction can be carried out at 0 ° C., preferably 30 to 40 ° C., for 1 to 24 hours, preferably 3 to 8 hours. However, chlorogenic acid can be extracted from coffee grounds to use chlorogenic acid as a raw material. 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 of extracting chlorogenic acid from coffee mash can be performed according to a general organic acid extraction method. For example, coffee mash is extracted with hot water, decolorized with activated carbon, adsorbed on Dowex (registered trademark) column of anion exchange resin, water-soluble organic 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.

  A method for producing quinic acid and / or caffeic acid using chlorogenic acid as a raw material can be performed, for example, as follows. A chlorogenic acid to which a microbial catalyst consisting of coffee cake containing 0.5 to 10 units, preferably 1 to 3 units of chlorogenic acid hydrolase is added, and a buffer solution of pH 5 to 8, preferably pH 6 to 7, For example, the reaction is performed with an acetate buffer and a phosphate buffer at 10 to 50 ° C., preferably 30 to 40 ° C., for 1 to 24 hours, preferably 3 to 8 hours. When the amount of the microbial catalyst is large, the reaction time can be shortened. Alternatively, the hydrolysis reaction may be performed by immobilizing microbial catalyst in a column or the like and adsorbing and eluting chlorogenic acid.

  As a method for purifying quinic acid and / or caffeic acid produced using a microbial catalyst made of coffee koji, a generally used organic acid purification method can be used. For example, an ion exchange resin is used. When performed according to the method of Adachi et al. (Adachi, O., Ano, Y., Toyama, H., and Matsushita, Biosci. Biotechnol. Biochem., 70: 3081-3083, 2006), chlorogenic acid, quinic acid And adsorbed on a Dowex (registered trademark) column of anion exchange resin and mixed with a suitable eluent, for example, water-soluble organic solvent such as methanol, ethanol, isopropanol, acetone, acetonitrile, acetic acid. Add acid such as formic acid, hydrochloric acid or nitric acid, and separate and elute. Quinic acid can be easily eluted from the column with a salt solution such as low-concentration sodium chloride, caffeic acid can be eluted with a 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.

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

<Manufacture of coffee candy>
4.5 kg of coffee mash was collected, put in a cloth bag and dehydrated for 1-2 minutes in a washing machine dewatering tank 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 each of a culture solution containing Aspergillus soya AKU 3312 that had been cultured in a Czapek medium was separately added. 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 mycelia, cutting was performed 2-3 times a day in the same manner as in the production of rice bran. 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 content of 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 candy produced using Aspergillus soya AKU 3312 and the enzyme solution prepared in Example 2 as a control. In other words, 50 μmole of chlorogenic acid (corresponding to 17.7 mg): 1) coffee koji 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 The enzyme solution was added, the amount of the reaction solution was adjusted to 1 ml with 0.1 M acetate buffer (pH 6.5), and the mixture was reacted at 30 ° C. for 3 hours. A part of the reaction solution (5 μl) 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. Further, the position was identified from the standard quinic acid, and spots indicating the presence of quinic acid (Rf = 0.15) were confirmed in the reaction solutions 2) and 4).

<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 (apparatus Shimadzu LC-20AD, column Lighttysil 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 the same amount of quinic acid as 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 aqueous sodium chloride 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.

  According to the present invention, since quinic acid and caffeic acid, which are pharmaceuticals and functional materials, can be produced from discarded coffee mash, the coffee mash can be effectively used, and the shikimic acid and shikimic acid pathways can be used. Quinic acid as a raw material for metabolic intermediates can be provided at a low cost.

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. 1: Heated coffee cake, 2: Unheated coffee cake, 3: Heated enzyme solution, 4: Hydrolysis reaction using 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.

Claims (4)

  In the production of quinic acid and / or caffeic acid, a quinic acid and / or a microorganism having a chlorogenic acid-hydrolyzing enzyme grown on coffee mash is reacted as a microbial catalyst using chlorogenic acid in coffee mash as a raw material. Or a method for producing caffeic acid.   Microbial catalyst, Aspergillus niger (Aspergillus niger), Aspergillus awamori (Aspergillus awamori), Aspergillus sojae (Aspergillus sojae), Penicillium Kurisojenamu (Penicillium chrysogenum), Mucor (Mucorales mucor), Rhizopus (Mucorales rhyzopus), Monascus purpureus (Monascus purpureus) The method for producing quinic acid and / or caffeic acid according to claim 1, wherein the quinic acid and / or caffeic acid is prepared from at least one microorganism selected from the group consisting of:   The microorganism catalyst is pre-cultured with a culture solution containing 0.001 to 0.1 part of a microorganism serving as an inoculum for 1 part 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 claim 1 or 2, wherein the coffee mash is produced by performing main culture for 5 to 10 days. A coffee koji grown with a microorganism having a hydrolase, wherein the chlorogenic acid hydrolase activity prepared for producing quinic acid and / or caffeic acid according to any one of claims 1 to 3 Having coffee candy.