JP2019017370A - METHOD FOR PRODUCING COMPOSITION COMPRISING β-GLUCAN AND AN OXIDIZED DERIVATIVE OF UNSATURATED FATTY ACID FROM BREWER'S GRAIN - Google Patents

Abstract

To provide means capable of producing useful β-glucan and oxidized derivatives of unsaturated fatty acid by simple and inexpensive means.SOLUTION: The present invention relates to a method for producing a composition comprising β-glucan and a hydroxyl derivative of one or more unsaturated fatty acids, the method comprising an alkaline extraction step of subjecting brewer's grain to alkaline treatment to obtain an alkaline extract, and an insolubilization step of acidifying the alkaline extract to obtain an insoluble fraction comprising a hydroxyl derivative of at least one unsaturated fatty acid selected from the group consisting of acidic supernatant fraction as well as β-glucan and a compound represented by the formula (I-1) and a compound represented by the formula (I-2).SELECTED DRAWING: None

Description

  The present invention relates to a method for producing a composition comprising β-glucan and an oxidized derivative of an unsaturated fatty acid from beer lees.

  β-glucan is a polysaccharide whose basic skeleton is a sugar chain in which glucose is polymerized by β-1,3-glucoside bonds or β-1,4-glucoside bonds. β-glucan not only has functions such as intestinal regulation as dietary fiber, but may also have functions such as risk reduction of coronary heart disease and heart disease, suppression of increase in postprandial blood glucose level, and reduction in cholesterol level. Are known.

β-glucan is known to be contained in plants (for example, grains such as barley, oats and wheat), mushrooms, and yeasts. For example, Patent Document 1 describes a method of isolating β (1-3) β (1-4) glucan from a pulverized grain or a pulverized portion of the grain. The document includes a step of extracting a pulverized grain or a pulverized portion of the grain with an alkaline solution, and adding C _{1 to} C ₄ alcohol to the purified extract to add the β (1-3) β And (1-4) a step of precipitating glucan.

  In recent years, oxidative derivatives of linoleic acid, an unsaturated fatty acid (for example, linoleic acid peroxide (HPODE), hydroxylated derivative (HODE) and oxo derivatives) have attracted attention as natural physiologically active substances contained in plants. . These oxidized derivatives have been found as metabolites of oxidative metabolic pathways of unsaturated fatty acids in plants such as tomato, cucumber, rice and barley (Non-patent Document 1).

  Peroxide and hydroxylated derivatives of linoleic acid are peroxisome proliferator-activated receptors (PPARs) involved in anti-inflammatory, lipid metabolism, sugar metabolism, bone metabolism, anticancer action, anti-obesity, and intestinal action Have been reported to be endogenous agonists (Non-patent Documents 2 and 3). In addition, it has been reported that ketooctadecadienoic acid, which is an oxo derivative of linoleic acid, has fat burning activity (Non-patent Document 4).

  Since the useful biological activity of oxidized fatty acid derivatives such as linoleic acid peroxides, hydroxylated derivatives and oxo derivatives has been clarified, oxidized derivatives having useful physiological activities are produced from unsaturated fatty acids. A method was developed (Patent Documents 2 to 4).

Special Table 2006-525381 JP 2010-51174 A International Publication No. 2014/088002 JP-A-2015-186545

Hubke, H. et al., J. Agric. Food Chem, 2005, 53, p. 1556-1562 Silvia YMC. Et al., J Lipid Res, 1999, 40, p. 1426-1433 Obitani H. et al., Prostaglandins Other Lipid Mediat., 2009, Vol. 89, p. 66-72 Kim, Y. et al., PLos One, 2012, 7 (2), e31317

  Oxidized derivatives of β-glucan and unsaturated fatty acids are useful as naturally occurring physiologically active substances. However, although a method for separately producing an oxidized derivative of β-glucan and an unsaturated fatty acid from a specific material is known, a method for obtaining a composition containing the compound together from a single material is known. There wasn't.

  As a method for producing an oxidized derivative of an unsaturated fatty acid, for example, in the methods described in Patent Documents 2 to 4, the oxidation reaction of the unsaturated fatty acid proceeds mainly using a specific microorganism or enzyme. In the case of such a method, there was a need to prepare a specific microorganism or enzyme. In addition, depending on the type of microorganism or enzyme used, there are cases where it is difficult to add to the food or medicine as it is from the viewpoint of safety. In such a case, it has been necessary to carry out a post-treatment such as purification and / or isolation of the formed oxidized derivative from a reaction system containing microorganisms or enzymes.

  If it is possible to produce a composition containing these compounds together by separating and purifying oxidized derivatives of β-glucan and unsaturated fatty acid contained in a single material, it will be easier than conventional methods. In addition, the target compound can be produced at low cost. However, oxidized derivatives of unsaturated fatty acids are highly reactive and can be converted to other compounds in the material preparation stage and / or in the separation and purification stage. In plants and the like, the oxidized derivative of unsaturated fatty acid produced by an endogenous metabolic pathway is usually a trace amount. Thus, a material that can stably supply an oxidized derivative of an unsaturated fatty acid that is highly reactive and is produced only in a trace amount, and / or a means that can be stably separated and purified has not yet been found. .

  Therefore, an object of the present invention is to provide a means by which useful β-glucan and an oxidized derivative of unsaturated fatty acid can be produced by a simple and inexpensive means.

  The present inventors have studied various means for solving the above problems. The present inventors have found that beer koji discharged in large quantities in beer production contains oxidized derivatives of β-glucan and unsaturated fatty acids. The present inventors include an oxidized derivative of β-glucan and an unsaturated fatty acid by treating beer lees with an alkali to extract an oxidized derivative of β-glucan and an unsaturated fatty acid, and further acidifying the extract. It was found that an insoluble fraction was obtained. Based on the above findings, the present inventors have completed the present invention.

［式中、
R¹は、水素、置換若しくは非置換のC₁〜C₅アルキル、置換若しくは非置換のC₂〜C₅アルケニル、置換若しくは非置換のC₂〜C₅アルキニル、置換若しくは非置換のC₃〜C₆シクロアルキル、置換若しくは非置換のC₄〜C₆シクロアルケニル、置換若しくは非置換のC₄〜C₆シクロアルキニル、又は置換若しくは非置換のC₇〜C₁₁シクロアルキルアルキルであり、
R²は、非置換のC₁〜C₇アルキル、又は非置換のC₄〜C₁₆アルケニルであり、
L¹は、非置換のC₁〜C₆アルキレン、又は非置換のC₄〜C₁₆アルケニレンであり、
但し、R²及びL¹は、炭素数の合計が13〜17の範囲であり、二重結合の合計が0〜5の範囲であるとの条件を満たす化合物である。］
で表される化合物、及び
式（I-2）：

［式中、R¹、R²及びL¹は、前記と同義である。］
で表される化合物からなる群より選択される1種以上の不飽和脂肪酸のヒドロキシル誘導体を含む不溶画分を得る、不溶化工程
を含む、β-グルカン及び1種以上の不飽和脂肪酸のヒドロキシル誘導体を含む組成物の製造方法。
（2） アルカリ抽出工程が、0.1 M以上の濃度の水酸化ナトリウム又は水酸化カリウム水溶液を用いて実施される、前記実施形態（1）に記載の方法。
（3） アルカリ抽出工程が、メタノール、エタノール及び2-プロパノール、並びにそれらの混合物からなる群より選択される低級アルコール存在下で実施される、前記実施形態（1）又は（2）に記載の方法。
（4） R²がn-ブチルであり、
L¹が式（L¹-1）：

［式中、
*は、アルケニレン鎖との結合位置であり、
**は、カルボン酸基との結合位置である。］
で表される2価基である；又は
R²が式（R²-1）：

［式中、
*は、アルケニレン鎖との結合位置である。］
で表される2価基であり、
L¹がn-ヘキシレンである；
の条件を満たす、前記実施形態（1）〜（3）のいずれかに記載の方法。
（5） 不溶化工程で得られた酸性の上清画分を有機溶媒で抽出して、フェルラ酸及びクマル酸の少なくともいずれかを含む画分を得る、有機溶媒抽出工程をさらに含む、前記実施形態（1）〜（4）のいずれかに記載の方法。 That is, the gist of the present invention is as follows.
(1) Alkali extraction process for obtaining an alkaline extract by alkali treatment of beer lees,
Acidify the alkaline extract to obtain an acidic supernatant fraction, as well as β-glucan and formula (I-1):

[Where:
R ¹ is hydrogen, substituted or unsubstituted C ₁ -C ₅ alkyl, substituted or unsubstituted C ₂ -C ₅ alkenyl, substituted or unsubstituted C ₂ -C ₅ alkynyl, substituted or unsubstituted C _3- C ₆ cycloalkyl, substituted or unsubstituted C ₄ -C ₆ cycloalkenyl, substituted or unsubstituted C ₄ -C ₆ cycloalkenyl, or substituted or unsubstituted C ₇ -C ₁₁ cycloalkylalkyl,
R ² is unsubstituted C ₁ -C ₇ alkyl, or unsubstituted C ₄ -C ₁₆ alkenyl,
L ¹ is unsubstituted C ₁ -C ₆ alkylene, or unsubstituted C ₄ -C ₁₆ alkenylene,
However, R ² and L ¹ are compounds that satisfy the condition that the total number of carbon atoms is in the range of 13 to 17 and the total number of double bonds is in the range of 0 to 5. ]
And a compound represented by formula (I-2):

[Wherein, R ¹ , R ² and L ¹ have the same meanings as described above. ]
A β-glucan and a hydroxyl derivative of one or more unsaturated fatty acids, including an insolubilization step, obtaining an insoluble fraction containing one or more unsaturated fatty acid hydroxyl derivatives selected from the group consisting of the compounds represented by: A method for producing a composition comprising the same.
(2) The method according to the embodiment (1), wherein the alkali extraction step is performed using a sodium hydroxide or potassium hydroxide aqueous solution having a concentration of 0.1 M or more.
(3) The method according to the embodiment (1) or (2), wherein the alkali extraction step is performed in the presence of a lower alcohol selected from the group consisting of methanol, ethanol and 2-propanol, and mixtures thereof. .
(4) R ² is n-butyl,
L ¹ is the formula (L ¹ -1):

[Where:
* Is the bonding position with the alkenylene chain,
** is a bonding position with a carboxylic acid group. ]
A divalent group represented by: or
R ² is the formula (R ² -1):

[Where:
* Is the bonding position with the alkenylene chain. ]
A divalent group represented by
L ¹ is n-hexylene;
The method according to any one of the embodiments (1) to (3), wherein the condition is satisfied.
(5) The embodiment further comprising the organic solvent extraction step of extracting the acidic supernatant fraction obtained in the insolubilization step with an organic solvent to obtain a fraction containing at least one of ferulic acid and coumaric acid The method according to any one of (1) to (4).

  According to the present invention, it is possible to provide a means by which useful β-glucan and an oxidized derivative of unsaturated fatty acid can be produced by a simple and inexpensive means.

  Problems, configurations, and effects other than those described above will be clarified by the following description of embodiments.

FIG. 1 shows the relationship between the NaOH concentration in alkali extraction of beer koji powder and the amounts of oxidized derivatives, ferulic acid and β-glucan contained in the extract. A: amount of oxidized derivative contained in the extract, B: amount of ferulic acid and β-glucan contained in the extract. FIG. 2 shows the relationship between the alkali extraction time in the alkali extraction of beer koji powder and the amounts of oxidized derivatives, ferulic acid and β-glucan contained in the extract. A: amount of oxidized derivative contained in the extract, B: amount of ferulic acid and β-glucan contained in the extract. FIG. 3 shows the relationship between the alkali extraction temperature in the alkali extraction of beer koji powder and the amounts of oxidized derivatives, ferulic acid and β-glucan contained in the extract. A: amount of oxidized derivative contained in the extract, B: amount of ferulic acid and β-glucan contained in the extract. FIG. 4 shows the relationship between the lower alcohol in the alkaline extraction of beer koji powder and the amounts of oxidized derivatives, ferulic acid and β-glucan contained in the extract. A: amount of oxidized derivative contained in the extract, B: amount of ferulic acid and β-glucan contained in the extract. FIG. 5 shows the amounts of oxidized derivatives, ferulic acid, coumaric acid, and β-glucan contained in the ethyl acetate fraction or insoluble fraction when alkali extracted under conditions of 0.3 or 1.0 M NaOH. A: Amount of oxidized derivative contained in each fraction, B: Amount of ferulic acid, coumaric acid and β-glucan contained in each fraction.

  Hereinafter, preferred embodiments of the present invention will be described in detail.

<1: Definition of compounds>
In this specification, the prefix “n” used for a group in a chemical formula is normal, “i” or “iso” is iso, “s” or “sec” is secondary, “t” or “tert” "Means tertiary," c "means cyclo," o "means ortho," m "means meta, and" p "means para.

As used herein, “alkyl” means a straight or branched chain saturated aliphatic hydrocarbon group containing the specified number of carbon atoms. For example, “C ₁ -C ₅ alkyl” means a straight or branched chain saturated aliphatic hydrocarbon group containing at least 1 and at most 5 carbon atoms. Suitable alkyls include, but are not limited to, linear or branched C, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl and n-pentyl. it can be mentioned ₁ -C ₅ alkyl.

In the present specification, “alkenyl” means a group in which one or more CC single bonds of the alkyl are substituted with double bonds. Suitable alkenyls include, but are not limited to, vinyl, 1-propenyl, allyl, 1-methylethenyl (isopropenyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-2-propenyl, 2 Mention may be made of straight-chain or branched C ₂ -C ₅ alkenyl such as -methyl-2-propenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl and 1-pentenyl.

In the present specification, “alkynyl” means a group in which one or more CC single bonds of the alkyl are substituted with triple bonds. Suitable alkynyls include, but are not limited to, straight chain such as ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl and 1-pentynyl. or it can be given C ₂ -C ₅ alkynyl branched.

As used herein, “cycloalkyl” means an alicyclic alkyl containing the specified number of carbon atoms. For example, “C ₃ -C ₆ cycloalkyl” means a cyclic hydrocarbon group containing at least 3 and at most 6 carbon atoms. Suitable cycloalkyls include, but are not limited to, C ₃ -C ₆ cycloalkyls such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

In the present specification, “cycloalkenyl” means a group in which one or more CC single bonds of the cycloalkyl are substituted with double bonds. Suitable cycloalkenyl includes, but is not limited to, C ₄ -C ₆ cycloalkenyl such as cyclobutenyl, cyclopentenyl, and cyclohexenyl.

In the present specification, “cycloalkynyl” means a group in which one or more CC single bonds of the cycloalkyl are substituted with triple bonds. Suitable cycloalkynyl includes, but is not limited to, C ₄ -C ₆ cycloalkynyl such as cyclobutynyl, cyclopentynyl and cyclohexynyl.

  In the present specification, “heterocycloalkyl” means that one or more carbon atoms of the cycloalkyl, cycloalkenyl or cycloalkynyl are each independently selected from nitrogen (N), sulfur (S) and oxygen (O). Means a group substituted with one or more heteroatoms. In this case, substitution with N or S includes substitution with N-oxide or S oxide or dioxide, respectively. Suitable heterocycloalkyl include, but are not limited to, eg, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, and piperazinyl. Mention may be made of ˜6-membered heterocycloalkyl.

In the present specification, “cycloalkylalkyl” means a group in which one of the hydrogen atoms of the alkyl, alkenyl or alkynyl is substituted with the cycloalkyl, cycloalkenyl or cycloalkynyl. Suitable cycloalkylalkyl, but are not limited to, may be, for example, a C ₇ -C ₁₁ cycloalkylalkyl such as cyclohexylmethyl and cyclohexenyl methyl.

As used herein, “heterocycloalkylalkyl” means a group in which one of the alkyl, alkenyl or alkynyl hydrogen atoms has been replaced with the heterocycloalkyl. Suitable heterocycloalkylalkyls include, but are not limited to, 3-6 membered heterocycloalkyl-C ₁ -C ₅ alkyl.

In the present specification, “alkoxy” means a group in which a hydrogen atom of hydroxyl is substituted with the alkyl, alkenyl or alkynyl. Suitable alkoxy includes, but is not limited to, C ₁ -C ₅ alkoxy such as methoxy, ethoxy, propoxy, butoxy and pentoxy.

In the present specification, “cycloalkoxy” means a group in which a hydrogen atom of hydroxyl is substituted with the above cycloalkyl, cycloalkenyl or cycloalkynyl. Suitable cycloalkoxy includes, but is not limited to, C ₃ -C ₆ cycloalkoxy such as cyclopropoxy, cyclobutoxy and cyclopentoxy.

  In the present specification, “heterocycloalkoxy” means a group in which a hydrogen atom of hydroxyl is substituted with the heterocycloalkyl. Suitable cycloalkoxy includes, but is not limited to, for example, 3-6 membered heterocycloalkoxy.

In the present specification, “aryl” means an aromatic ring group. Suitable aryls include, but are not limited to, C ₆ -C ₁₈ aryls such as phenyl, biphenyl, terphenyl, naphthyl and anthracenyl.

In the present specification, “arylalkyl” means a group in which one of hydrogen atoms of the alkyl, alkenyl or alkynyl is substituted with the aryl. Preferred arylalkyl include, but are not limited to, for example, benzyl, 1-phenethyl, 2-phenethyl, biphenylmethyl, can be exemplified terphenyl methyl and C ₇ -C ₂₀ arylalkyl styryl like.

  In the present specification, “heteroaryl” means a group in which one or more carbon atoms of the aryl are each independently substituted with one or more heteroatoms selected from N, S and O. In this case, substitution with N or S includes substitution with N-oxide or S oxide or dioxide, respectively. Suitable heteroaryl include, but are not limited to, furanyl, thienyl (thiophenyl), pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridyl, pyridazinyl, pyrazinyl, Mention may be made of 5- to 15-membered heteroaryl such as pyrimidinyl, quinolinyl, isoquinolinyl and indolyl.

In the present specification, “heteroarylalkyl” means a group in which one of the alkyl, alkenyl or alkynyl hydrogen atoms is substituted with the heteroaryl. Suitable heteroarylalkyl includes, but is not limited to, 5- to 15-membered heteroaryl-C ₁ -C ₅ alkyl such as pyridylmethyl.

In the present specification, “aryloxy” means a group in which a hydroxyl hydrogen atom is substituted with the aryl. Suitable aryloxy include, but are not limited to, e.g., phenoxy, biphenyloxy, mention may be made of C ₆ -C ₁₅ aryloxy such as naphthyloxy or anthryloxy (anthracenyloxy).

In the present specification, “arylalkyloxy” means a group in which a hydrogen atom of hydroxyl is substituted with the arylalkyl. Preferred arylalkyloxy include, without limitation, may include, for example, benzyloxy, 1-phenethyloxy, a C ₇ -C ₂₀ arylalkyloxy and 2-phenethyloxy and styryloxy.

  In the present specification, “heteroaryloxy” means a group in which a hydrogen atom of hydroxyl is substituted with the heteroaryl. Suitable heteroaryloxy include, but are not limited to, furanyloxy, thienyloxy (thiophenyloxy), pyrrolyloxy, imidazolyloxy, pyrazolyloxy, triazolyloxy, tetrazolyloxy, thiazolyloxy, oxazolyloxy, iso 5-15 such as oxazolyloxy, oxadiazolyloxy, thiadiazolyloxy, isothiazolyloxy, pyridyloxy, pyridazinyloxy, pyrazinyloxy, pyrimidinyloxy, quinolinyloxy, isoquinolinyloxy and indolyloxy Mention may be made of member heteroaryloxy.

In the present specification, “heteroarylalkyloxy” means a group in which a hydrogen atom of hydroxyl is substituted with the heteroarylalkyl. Preferred heteroarylalkyloxy include, without limitation, mention may be made of heteroaryl -C ₁ -C ₅ alkyloxy, for example 5 to 15 members.

In the present specification, “acyl” means a group in which a monovalent group selected from the groups described above and carbonyl are linked. Suitable acyls include, but are not limited to, C ₁ -C ₅ aliphatic acyls such as formyl, acetyl and propionyl, and C ₇ -C ₁₆ aromatic acyls such as benzoyl.

  The groups described above are each independently unsubstituted or can be further substituted with one or more of the monovalent groups described above.

  In the present specification, “halogen” or “halo” means fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

<2: Manufacturing method>
The present inventors have found that beer koji discharged in large quantities in beer production contains oxidized derivatives of β-glucan and unsaturated fatty acids. The present inventors include an oxidized derivative of β-glucan and an unsaturated fatty acid by treating beer lees with an alkali to extract an oxidized derivative of β-glucan and an unsaturated fatty acid, and further acidifying the extract. It was found that an insoluble fraction was obtained. Therefore, one embodiment of the present invention relates to a method for producing a composition comprising β-glucan and a hydroxyl derivative of one or more unsaturated fatty acids. In the present invention, the oxidized derivative of unsaturated fatty acid means at least one compound selected from the group consisting of peroxides of unsaturated fatty acids, hydroxyl derivatives of unsaturated fatty acids, and oxo derivatives of unsaturated fatty acids, In particular, it means a hydroxyl derivative of an unsaturated fatty acid.

で表される化合物、及び
式（I-2）：

で表される化合物からなる群より選択される1種以上の化合物である。 In each embodiment of the present invention, the hydroxyl derivative of the unsaturated fatty acid has the formula (I-1):

And a compound represented by formula (I-2):

One or more compounds selected from the group consisting of compounds represented by:

In formulas (I-1) and (I-2),
R ¹ is hydrogen, substituted or unsubstituted C ₁ -C ₅ alkyl, substituted or unsubstituted C ₂ -C ₅ alkenyl, substituted or unsubstituted C ₂ -C ₅ alkynyl, substituted or unsubstituted C _3- C ₆ cycloalkyl, substituted or unsubstituted C ₄ -C ₆ cycloalkenyl, substituted or unsubstituted C ₄ -C ₆ cycloalkenyl, substituted or unsubstituted 3-6 membered heterocycloalkyl, substituted or unsubstituted C ₇ -C ₁₁ cycloalkylalkyl, substituted or unsubstituted 3-6 membered heterocycloalkyl -C ₁ -C ₅ alkyl, substituted or unsubstituted C ₆ -C ₁₅ aryl, substituted or unsubstituted C ₇ ~ C ₂₀ arylalkyl, substituted or unsubstituted 5-15 membered heteroaryl, or substituted or unsubstituted 5-15 membered heteroaryl-C ₁ -C ₅ alkyl;
R ² is a substituted or unsubstituted C ₁ -C ₂₀ alkyl, substituted or unsubstituted C ₂ -C ₂₀ alkenyl, or substituted or unsubstituted C ₂ -C ₂₀ alkynyl,
L ¹ is a single bond, substituted or unsubstituted C ₁ -C ₂₀ alkylene, substituted or unsubstituted C ₂ -C ₂₀ alkenylene, or substituted or unsubstituted C ₂ -C ₂₀ alkynylene.

R ¹ is hydrogen, substituted or unsubstituted C ₁ -C ₅ alkyl, substituted or unsubstituted C ₂ -C ₅ alkenyl, substituted or unsubstituted C ₂ -C ₅ alkynyl, substituted or unsubstituted C _3- C ₆ cycloalkyl, substituted or unsubstituted C ₄ -C ₆ cycloalkenyl, preferably a substituted or unsubstituted C ₄ -C ₆ cycloalkenyl, or substituted or unsubstituted C ₇ -C ₁₁ cycloalkylalkyl hydrogen, substituted or unsubstituted C ₁ -C ₅ alkyl, more preferably a substituted or unsubstituted C ₂ -C ₅ alkenyl, or substituted or unsubstituted C ₂ -C ₅ alkynyl, hydrogen, methyl, More preferred is ethyl, propyl, or glyceryl, and particularly preferred is hydrogen. When R ¹ is glyceryl, at least one of the 2-position and 3-position hydroxyls of glyceryl may form an ester bond with another unsaturated fatty acid represented by formula (I) or another fatty acid. Good.

R ² is preferably unsubstituted C ₁ -C ₂₀ alkyl, unsubstituted C ₂ -C ₂₀ alkenyl, or unsubstituted C ₂ -C ₂₀ alkynyl, unsubstituted C ₁ -C ₁₀ alkyl, Or more preferably unsubstituted C ₄ -C ₂₀ alkenyl, more preferably unsubstituted C ₁ -C ₇ alkyl, or unsubstituted C ₄ -C ₁₆ alkenyl, methyl, n-butyl, n-heptyl, n-but-1-en-yl, n-hept-1-en-yl, n-hepta-1,4-dien-yl, n-deca-1,4-dien-yl, n- Deca-1,4,7-trien-yl, n-trideca-1,4,7-trien-yl, n-trideca-1,4,7,10-tetraen-yl, or n-hexadeca-1,4 7,7,10,13-pentaen-yl is particularly preferred.

L ¹ is preferably a single bond, unsubstituted C ₁ -C ₂₀ alkylene, unsubstituted C ₂ -C ₂₀ alkenylene, or unsubstituted C ₂ -C ₂₀ alkynylene, single bond, unsubstituted C 1 ₁ -C ₁₀ alkylene, or more preferably an unsubstituted C ₄ -C ₂₀ alkenylene, more preferably an unsubstituted C ₁ -C ₆ alkylene, or unsubstituted C ₄ -C ₁₆ alkenylene, Methylene, ethylene, n-hexylene, n-but-1-ene-1,4-diyl, n-pent-1-ene-1,5-diyl, n-hepta-1,4-diene-1,7- Diyl, n-octa-1,4-diene-1,8-diyl, n-non-1-ene-1,9-diyl, n-deca-1,4,7-triene-1,10-diyl, n-Undeca-1,4,7-triene-1,11-diyl, n-dodeca-1,4-diene-1,12-diyl, n-trideca-1,4,7,10-tetraene-1, 13-diyl, n-tetradeca-1,4,7,10-tetraene-1,14-diyl or n-hexadeca-1,4,7,10,13-pentaene Particularly preferred is -1,16-diyl.

In the definition of R ¹ , R ² and L ¹ , when said group is substituted, said substituent is independently halogen, cyano, nitro, amino, hydroxyl, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₆ cycloalkyl, C ₃ -C ₆ cycloalkenyl, C ₃ -C ₆ cycloalkenyl, 3-6 membered heterocycloalkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₆ cycloalkoxy, 3-6 membered heterocycloalkyl alkoxy, C ₄ -C ₂₀ aryl, C ₆ -C ₁₅ aryloxy, C ₇ -C ₂₀ arylalkyloxy, 5-15 membered heteroaryl , 5-15 membered heteroaryloxy, 5-15 membered heteroaryl -C ₁ -C ₉ alkyloxy, C ₁ -C ₆ alkoxycarbonyl, C ₃ -C ₆ cycloalkoxy carbonyl, and C ₁ -C ₆ acyloxy Is at least one monovalent group selected from the group consisting of Door is preferable.

The hydroxyl derivative of one or more unsaturated fatty acids selected from the group consisting of the compound represented by the formula (I-1) and the compound represented by the formula (I-2) is R ¹ exemplified above. Compounds defined by any combination of R ² and L ¹ can be included.

［式中、
*は、アルケニレン鎖との結合位置であり、
**は、カルボン酸基との結合位置である。］
で表される2価基である；又は
R²が式（R²-1）：

［式中、
*は、アルケニレン鎖との結合位置である。］
で表される2価基であり、
L¹がn-ヘキシレンである；
条件（ii）：
R²がn-ヘプチルであり、
L¹がn-ヘキシレンである；
条件（iii）：
R²がメチルであり、
L¹が式（L¹-2）：

［式中、
*は、アルケニレン鎖との結合位置であり、
**は、カルボン酸基との結合位置である。］
で表される2価基である；又は
R²が式（R²-2）：

［式中、
*は、アルケニレン鎖との結合位置である。］
で表される2価基であり、
L¹がn-ヘキシレンである；
条件（iv）：
R²がメチルであり、
L¹が式（L¹-3）：

［式中、
*は、アルケニレン鎖との結合位置であり、
**は、カルボン酸基との結合位置である。］
で表される2価基である；又は
R²が式（R²-3）：

［式中、
*は、アルケニレン鎖との結合位置である。］
で表される2価基であり、
L¹がエチレンである；
条件（v）：
R²がメチルであり、
L¹が式（L¹-4）：

［式中、
*は、アルケニレン鎖との結合位置であり、
**は、カルボン酸基との結合位置である。］
で表される2価基である；又は
R²が式（R²-4）：

［式中、
*は、アルケニレン鎖との結合位置である。］
で表される2価基であり、
L¹がメチレンである；及び
条件（vi）：
R²がn-ブチルであり、
L¹が式（L¹-5）：

［式中、
*は、アルケニレン鎖との結合位置であり、
**は、カルボン酸基との結合位置である。］
で表される2価基である；又は
R²が式（R²-5）：

［式中、
*は、アルケニレン鎖との結合位置である。］
で表される2価基であり、
L¹がエチレンである；
からなる群より選択される条件を満たす化合物であり、
さらに好ましくは、R²及びL¹が、条件（i）を満たす化合物である。前記条件（i）〜（vi）で定義される、式（I-1）で表される化合物及び式（I-2）で表される化合物からなる群より選択される1種以上の不飽和脂肪酸のヒドロキシル誘導体は、リノール酸、オレイン酸、α-リノレン酸、エイコサペンタエン酸（EPA）、ドコサヘキサエン酸（DHA）及びアラキドン酸のヒドロキシル誘導体にそれぞれ対応する。これらの不飽和脂肪酸は、いずれも天然に存在することが知られている。また、ある種の不飽和脂肪酸の酸化誘導体は、有用な生理活性を有することが知られている（非特許文献2〜4）。それ故、本発明の一態様に係る方法により、有用な生理活性を有する、式（I-1）で表される化合物及び式（I-2）で表される化合物からなる群より選択される1種以上の不飽和脂肪酸のヒドロキシル誘導体を提供することができる。 The hydroxyl derivative of one or more unsaturated fatty acids selected from the group consisting of the compound represented by the formula (I-1) and the compound represented by the formula (I-2),
Preferably R ² and L ¹ are:
R ² is unsubstituted C ₁ -C ₇ alkyl, or unsubstituted C ₄ -C ₁₆ alkenyl,
L ¹ is unsubstituted C ₁ -C ₆ alkylene, or unsubstituted C ₄ -C ₁₆ alkenylene,
However, R ² and L ¹ are compounds that satisfy the condition that the total number of carbon atoms is in the range of 13 to 17 and the total number of double bonds is in the range of 0 to 5,
More preferably, R ² and L ¹ are:
Condition (i):
R ² is n-butyl,
L ¹ is the formula (L ¹ -1):

[Where:
* Is the bonding position with the alkenylene chain,
** is a bonding position with a carboxylic acid group. ]
A divalent group represented by: or
R ² is the formula (R ² -1):

[Where:
* Is the bonding position with the alkenylene chain. ]
A divalent group represented by
L ¹ is n-hexylene;
Condition (ii):
R ² is n-heptyl,
L ¹ is n-hexylene;
Condition (iii):
R ² is methyl,
L ¹ is the formula (L ¹ -2):

[Where:
* Is the bonding position with the alkenylene chain,
** is a bonding position with a carboxylic acid group. ]
A divalent group represented by: or
R ² is the formula (R ² -2):

[Where:
* Is the bonding position with the alkenylene chain. ]
A divalent group represented by
L ¹ is n-hexylene;
Condition (iv):
R ² is methyl,
L ¹ is the formula (L ¹ -3):

[Where:
* Is the bonding position with the alkenylene chain,
** is a bonding position with a carboxylic acid group. ]
A divalent group represented by: or
R ² is the formula (R ² -3):

[Where:
* Is the bonding position with the alkenylene chain. ]
A divalent group represented by
L ¹ is ethylene;
Condition (v):
R ² is methyl,
L ¹ is the formula (L ¹ -4):

[Where:
* Is the bonding position with the alkenylene chain,
** is a bonding position with a carboxylic acid group. ]
A divalent group represented by: or
R ² is the formula (R ² -4):

[Where:
* Is the bonding position with the alkenylene chain. ]
A divalent group represented by
L ¹ is methylene; and condition (vi):
R ² is n-butyl,
L ¹ is the formula (L ¹ -5):

[Where:
* Is the bonding position with the alkenylene chain,
** is a bonding position with a carboxylic acid group. ]
A divalent group represented by: or
R ² is the formula (R ² -5):

[Where:
* Is the bonding position with the alkenylene chain. ]
A divalent group represented by
L ¹ is ethylene;
A compound satisfying a condition selected from the group consisting of:
More preferably, R ² and L ¹ are compounds that satisfy the condition (i). One or more types of unsaturation selected from the group consisting of the compound represented by the formula (I-1) and the compound represented by the formula (I-2) defined by the above conditions (i) to (vi) The hydroxyl derivatives of fatty acids correspond to the hydroxyl derivatives of linoleic acid, oleic acid, α-linolenic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid, respectively. All of these unsaturated fatty acids are known to exist in nature. Moreover, it is known that the oxidation derivative of a certain kind of unsaturated fatty acid has useful physiological activity (nonpatent literatures 2-4). Therefore, the method according to one embodiment of the present invention is selected from the group consisting of the compound represented by formula (I-1) and the compound represented by formula (I-2) having useful physiological activity. Hydroxyl derivatives of one or more unsaturated fatty acids can be provided.

  In each embodiment of the present invention, β-glucan means a polysaccharide having a sugar chain in which glucose is polymerized by β-1,3-glucoside bond or β-1,4-glucoside bond as a basic skeleton. The beer lees used in the method according to one embodiment of the present invention mainly contains β-glucan derived from barley malt which is a raw material for producing beer. Therefore, the β-glucan obtained by the method according to one embodiment of the present invention usually contains β-glucan derived from barley malt contained in beer lees as a main component, and optionally in other materials. The derived β-glucan may also be included. Β-glucan derived from barley malt usually has a structure in which β-1,3-glucoside bonds and β-1,4-glucoside bonds are mixed. The β-glucan obtained by the method according to one embodiment of the present invention usually contains β-glucan having the above-exemplified properties as a main component, and may optionally contain β-glucan having other properties. .

  One or more selected from the group consisting of β-glucan obtained in the method according to one embodiment of the present invention, the compound represented by formula (I-1), and the compound represented by formula (I-2) Hydroxyl derivatives of unsaturated fatty acids can be identified and / or quantified by means of instrumental analysis such as, for example, high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrum (LC-MS). Further, β-glucan, and the chemical structure of one or more unsaturated fatty acid hydroxyl derivatives selected from the group consisting of the compound represented by formula (I-1) and the compound represented by formula (I-2) Can be confirmed, for example, by means of instrumental analysis such as nuclear magnetic resonance spectrum (NMR), infrared absorption spectrum (IR), ultraviolet absorption spectrum (UV), or MS.

  In each embodiment of the present invention, β-glucan, a hydroxyl group of one or more unsaturated fatty acids selected from the group consisting of a compound represented by formula (I-1) and a compound represented by formula (I-2) Derivatives and ferulic acid and coumaric acid described below include not only the compounds themselves, but also their salts, if present. Examples of the salt of the compound include, but are not limited to, a salt with a cation such as sodium ion, potassium ion, calcium ion, magnesium ion, or substituted or unsubstituted ammonium ion, or hydrochloric acid or hydrogen bromide. Inorganic acids such as acid, sulfuric acid, nitric acid, carbonic acid or phosphoric acid, or formic acid, acetic acid, maleic acid, fumaric acid, benzoic acid, ascorbic acid, succinic acid, bismethylenesalicylic acid, methanesulfonic acid, ethanedisulfonic acid, propionic acid , Tartaric acid, salicylic acid, citric acid, gluconic acid, aspartic acid, stearic acid, palmitic acid, itaconic acid, glycolic acid, p-aminobenzoic acid, glutamic acid, benzenesulfonic acid, cyclohexylsulfamic acid, methanesulfonic acid, ethanesulfonic acid, Isethonic acid, p-tolue Preferred are salts with organic acid anions such as sulfonic acid or naphthalenesulfonic acid. Each aspect of the present invention can be applied even when the compound is in the form of the salt.

  In each embodiment of the present invention, β-glucan, a hydroxyl group of one or more unsaturated fatty acids selected from the group consisting of a compound represented by formula (I-1) and a compound represented by formula (I-2) Derivatives and ferulic acid and coumaric acid described below include not only the compound itself, but also solvates of the compound or salts thereof, if present. The solvent that can form a solvate with the compound or a salt thereof is not limited. For example, water or a lower alcohol (for example, 1 to 2 such as methanol, ethanol, or 2-propanol (isopropyl alcohol)). Alcohols having 6 carbons), higher alcohols (eg alcohols having 7 or more carbons such as 1-heptanol or 1-octanol), dimethyl sulfoxide (DMSO), acetic acid, ethanolamine or ethyl acetate Organic solvents are preferred. Each embodiment of the present invention can be applied even when the compound or a salt thereof is in the form of a solvate with the solvent.

  In each embodiment of the present invention, β-glucan, a hydroxyl group of one or more unsaturated fatty acids selected from the group consisting of a compound represented by formula (I-1) and a compound represented by formula (I-2) Where the derivatives and ferulic acid and coumaric acid described below have one or more tautomers, the compounds also include the individual tautomeric forms of the compounds.

  In each embodiment of the present invention, β-glucan, a hydroxyl group of one or more unsaturated fatty acids selected from the group consisting of a compound represented by formula (I-1) and a compound represented by formula (I-2) When the derivatives, and ferulic acid and coumaric acid described below have one or more stereocenters (chiral centers), the compounds are the individual enantiomers and diastereomers of the compounds and their racemates such as racemates. Also includes mixtures.

  In each embodiment of the present invention, one or more unsaturated fatty acids selected from the group consisting of β-glucan, a compound represented by formula (I-1), and a compound represented by formula (I-2) If the geometry of one or more double bonds present in the ferulic acid and coumaric acid described below is not specified, the compound is an individual geometric isomer of the compound and mixtures thereof Is also included.

  The method according to this aspect includes an alkali extraction step and an insolubilization step. Each step will be described in more detail below.

[2-1: Alkali extraction process]
The method according to this embodiment includes an alkali extraction step in which beer lees are alkali-treated to obtain an alkaline extract.

  In this embodiment, “beer lees” means wort lees produced in beer production. Beer lees are discharged in large quantities in beer production, and are currently reused for livestock feed and fertilizer, or disposed of by incineration or the like.

  The beer lees used in this step are not particularly limited as long as they are produced in normal beer production, and the type and production method of beer and the type of malt used for beer production are not particularly limited. Further, the water content of the beer koji may be either a dehydrated state or a dried state generated in normal beer production, and is preferably a dried state. By using the beer lees having the above characteristics, the method according to this aspect can be easily carried out.

  Although the form of the beer lees used in this step is not particularly limited, it is preferably a powder form. The beer cake in the form of a powder can be obtained using, for example, a mill or a mixer that is usually used in the technical field. By using beer lees in the form of powder, alkali extraction can be carried out more efficiently.

  The alkali used in this step is preferably sodium hydroxide, potassium hydroxide, calcium hydroxide or calcium carbonate, or a mixture thereof, more preferably sodium hydroxide or potassium hydroxide, More preferably, it is sodium. Hydroxyl derivative of one or more unsaturated fatty acids selected from the group consisting of β-glucan, a compound represented by formula (I-1) and a compound represented by formula (I-2) , And the ferulic acid and coumaric acid described below, can exist not only in the free form, but also in a modified form such as an ester. In this step, the modified form of the compound can be converted to a free form by subjecting beer lees to an alkali treatment. Alkali extraction can be carried out more efficiently by subjecting beer lees to an alkali treatment using the alkali exemplified above.

  In this step, the alkali concentration in the alkaline extract is preferably 0.1 M or more, more preferably in the range of 0.1 to 1.5 M, still more preferably in the range of 0.1 to 1.0 M, 0.3 A range of ˜1.0 M is particularly preferred. When an alkali having a concentration lower than the lower limit is used, a desired compound may not be sufficiently extracted. Alkali extraction can be carried out more efficiently by subjecting beer lees to alkali treatment using the alkali in the concentration range exemplified above.

  In this step, the time for alkali treatment of the beer cake is preferably several minutes or more, more preferably 30 minutes or more, and further preferably 60 minutes or more. The upper limit of the alkali treatment time for beer koji is not particularly limited, but is, for example, several hours or less, typically 2 hours or less. Alkali extraction can be carried out rapidly by subjecting beer lees to alkali treatment for the time in the range exemplified above.

  In this step, the temperature at which the beer lees are alkali-treated is preferably room temperature or higher, more preferably 60 ° C. or higher, and further preferably 90 ° C. or higher. By subjecting beer lees to an alkali treatment at a temperature equal to or higher than the lower limit value, selected from the group consisting of β-glucan, a compound represented by formula (I-1), and a compound represented by formula (I-2) One or more hydroxyl derivatives of unsaturated fatty acids can be efficiently extracted. Moreover, the temperature at which the beer lees are alkali-treated is preferably 100 ° C. or lower, and more preferably 90 ° C. or lower. When beer lees are alkali-treated at a temperature exceeding the upper limit, the extraction efficiency of β-glucan may be reduced. Alkali extraction can be carried out more efficiently by subjecting beer lees to an alkali treatment at a temperature in the range exemplified above.

In one embodiment of this step, the alkali extraction is preferably performed in the presence of a lower alcohol. In the case of this embodiment, the lower alcohol is preferably selected from the group consisting of methanol, ethanol, propanol (for example, 1-propanol or 2-propanol) and butanol, and a mixture thereof, methanol, ethanol and 2-propanol. , As well as a mixture thereof, more preferably selected from the group consisting of ethanol. When an alcohol having a carbon chain length exceeding C ₄ is used, selected from the group consisting of β-glucan and / or a compound represented by formula (I-1) and a compound represented by formula (I-2) The extraction efficiency of one or more unsaturated fatty acid hydroxyl derivatives may be reduced. Alkali extraction can be carried out more efficiently by subjecting beer lees to alkali treatment in the presence of the lower alcohol exemplified above.

  In the embodiment, the amount of the lower alcohol is preferably in the range of 10 to 50% by volume and more preferably in the range of 40 to 50% by volume with respect to the total volume of the alkaline extract. When the lower alcohol in an amount exceeding the upper limit is present, it is selected from the group consisting of β-glucan and / or the compound represented by formula (I-1) and the compound represented by formula (I-2). The extraction efficiency of the hydroxyl derivative of one or more unsaturated fatty acids may be reduced. Alkali extraction can be carried out more efficiently by subjecting beer lees to alkali treatment in the presence of the lower alcohol exemplified above.

  In this step, beer lees are separated to obtain an alkaline extract. As means for separating beer lees from the alkaline extract, means usually used in the art such as centrifugation and filtration can be applied.

  By carrying out this step based on the conditions described above, an alkaline extract containing β-glucan and a hydroxyl derivative of an unsaturated fatty acid can be obtained.

[2-2: Insolubilization process]
The method according to this embodiment is obtained by acidifying an alkaline extract, expressing an acidic supernatant fraction, β-glucan, a compound represented by formula (I-1), and formula (I-2). An insolubilization step of obtaining an insoluble fraction containing a hydroxyl derivative of one or more unsaturated fatty acids selected from the group consisting of:

  In this step, the acid used for acidifying the alkaline extract is preferably hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, acetic acid or citric acid, or a mixture thereof, more preferably hydrochloric acid. A group consisting of β-glucan, a compound represented by formula (I-1) and a compound represented by formula (I-2) by acidifying an alkaline extract using the acid exemplified above More selected hydroxyl derivatives of one or more unsaturated fatty acids can be insolubilized.

  In this step, the concentration of the acid used for acidification is preferably 0.1 M or more, more preferably in the range of 0.1 to 6 M, and even more preferably in the range of 1 to 6 M. By acidifying an alkaline extract using an acid in the concentration range exemplified above, β-glucan, a compound represented by formula (I-1), and a compound represented by formula (I-2) One or more hydroxyl derivatives of unsaturated fatty acids selected from the group consisting of can be insolubilized.

  In this step, using the acid described above, the alkaline extract is preferably acidified to a pH of less than 7, more preferably acidified to a pH of 1 to 4, and a pH of 1 to 2 It is further preferred to acidify to the range. When acidified to a pH exceeding the above upper limit, 1 is selected from the group consisting of β-glucan and / or a compound represented by formula (I-1) and a compound represented by formula (I-2) More than one kind of unsaturated fatty acid hydroxyl derivative may not be sufficiently insolubilized, resulting in a decrease in yield of the desired compound. From the group consisting of β-glucan, a compound represented by formula (I-1) and a compound represented by formula (I-2) by acidifying an alkaline extract to the pH range exemplified above An insoluble fraction containing one or more selected unsaturated fatty acid hydroxyl derivatives can be obtained in high yield.

  In this step, the alkaline extract is acidified to the pH range and held for a predetermined time, whereby β-glucan, the compound represented by formula (I-1), and the formula (I-2) are represented. And insolubilizing one or more hydroxyl derivatives of unsaturated fatty acids selected from the group consisting of: The time required for this insolubilization is usually several minutes or more, for example, in the range of 1 minute to 2 hours. By maintaining the acidified extract for a time in the above range, it is selected from the group consisting of β-glucan, a compound represented by formula (I-1), and a compound represented by formula (I-2) Insoluble fractions containing one or more hydroxyl derivatives of unsaturated fatty acids can be obtained in high yield.

  In this step, the temperature for acidifying the alkaline extract is preferably 50 ° C. or lower, more preferably in the range of 0 to 50 ° C., and further preferably in the range of 0 to 25 ° C. One selected from the group consisting of the compound represented by formula (I-1) and the compound represented by formula (I-2) when the temperature when acidifying the alkaline extract exceeds 50 ° C The above hydroxyl derivatives of unsaturated fatty acids may isomerize. By acidifying the alkaline extract at a temperature in the above range, selected from the group consisting of β-glucan, a compound represented by formula (I-1), and a compound represented by formula (I-2) One or more hydroxyl derivatives of unsaturated fatty acids can be insolubilized.

  In each embodiment of the present invention, “insoluble” means a state in which a solute of a specific ratio or more is not mixed with a solvent to form a substantially homogeneous liquid phase. The insolubilized state may include not only the insolubilized solute in the system but also a solute that is mixed with a solvent to form a substantially homogeneous liquid phase (ie, dissolved). The insolubilized solute aggregates in the solvent to form particles, lumps or precipitates.

  In this step, from the acidified extract, the acidic supernatant fraction, the insolubilized β-glucan, the compound represented by formula (I-1) and the compound represented by formula (I-2) The insoluble fraction containing the desired compound is obtained by separating from one or more hydroxyl derivatives of unsaturated fatty acids selected from the group consisting of: As means for separating the acidic supernatant fraction and insoluble fraction from the acidified extract, means commonly used in the art such as centrifugation and filtration can be applied. By carrying out this step by applying these means, selected from the group consisting of β-glucan, a compound represented by formula (I-1), and a compound represented by formula (I-2) An insoluble fraction containing one or more unsaturated fatty acid hydroxyl derivatives can be obtained, for example, in the form of particles, lumps or precipitates.

  By performing this step based on the conditions described above, it is selected from the group consisting of β-glucan, a compound represented by formula (I-1), and a compound represented by formula (I-2) An insoluble fraction comprising one or more unsaturated fatty acid hydroxyl derivatives can be obtained.

  In addition, as described below, by performing this step, β-glucan is selected from the group consisting of the compound represented by formula (I-1) and the compound represented by formula (I-2). In addition to the hydroxyl derivative of one or more unsaturated fatty acids, an insoluble fraction further containing at least one of ferulic acid and coumaric acid can be obtained.

[2-3: Organic solvent extraction process]
In the method according to this aspect, an organic solvent extraction step is performed in which the acidic supernatant fraction obtained in the insolubilization step is extracted with an organic solvent to obtain a fraction containing at least one of ferulic acid and coumaric acid. Can further be included.

  Ferulic acid and coumaric acid are both known as naturally occurring aromatic compounds. There are three positional isomers of coumaric acid: o-, m-, and p-coumaric acid. In the present specification, “coumaric acid” includes any of these three positional isomers. Ferulic acid and coumaric acid have physiological activities such as immunopotentiating action, blood pressure lowering action, blood flow improving action, angiotensin I converting enzyme (ACE) inhibitory action, antibacterial action and antioxidant action. (For example, JP 2002-371002 A, JP 2013-53126 A, and JP 2014-40375 A).

  The inventors include at least one of ferulic acid and coumaric acid in the alkaline extract obtained in the alkali extraction step and the insoluble fraction obtained in the insolubilization step by carrying out the method according to this aspect. I found out. Moreover, the present inventors have found that a fraction containing at least one of ferulic acid and coumaric acid can be obtained by extracting the acidic supernatant fraction obtained in the insolubilization step with an organic solvent.

  Ferulic acid and coumaric acid obtained in the method according to this embodiment can be identified and / or quantified by means of instrumental analysis such as HPLC or LC-MS, for example. The chemical structures of ferulic acid and coumaric acid can be confirmed by instrumental analysis means such as NMR, IR, UV, or MS.

  In this step, the organic solvent used for extraction is preferably ethyl acetate or chloroform, or a mixture thereof, more preferably ethyl acetate. By extracting the acidic supernatant fraction obtained in the insolubilization step using the organic solvent exemplified above, a fraction containing at least one of ferulic acid and coumaric acid can be efficiently obtained.

  In this step, the temperature at which the acidic supernatant fraction obtained in the insolubilization step is extracted with an organic solvent is preferably room temperature or higher, more preferably in the range of room temperature to 40 ° C, more preferably 20 to 40 ° C. More preferably, it is the range. By extracting an acidic supernatant fraction with an organic solvent at a temperature in the above range, a fraction containing at least one of ferulic acid and coumaric acid can be efficiently obtained.

  By performing this step based on the conditions described above, a fraction containing at least one of ferulic acid and coumaric acid can be efficiently obtained.

  1 is selected from the group consisting of β-glucan, a compound represented by formula (I-1), and a compound represented by formula (I-2) by carrying out the method according to one embodiment of the present invention. Compositions comprising hydroxyl derivatives of more than one unsaturated fatty acid, and optionally ferulic acid and coumaric acid can be obtained. In the composition obtained by the method according to this embodiment, one or more selected from the group consisting of β-glucan, a compound represented by formula (I-1), and a compound represented by formula (I-2) The unsaturated fatty acid hydroxyl derivatives, and optionally ferulic acid and coumaric acid, can be present in any form and quantity ratio.

  In the present specification, as described in detail, according to each aspect of the present invention, from beer lees, β-glucan, a compound represented by formula (I-1), and a formula (I-2) are represented. One or more hydroxyl derivatives of unsaturated fatty acids selected from the group consisting of compounds, and optionally ferulic acid and coumaric acid can be obtained. β-glucan is known to exhibit functions such as reducing the risk of coronary heart disease and heart disease, suppressing the increase in postprandial blood glucose level, and lowering the cholesterol level. In addition, oxidized derivatives such as hydroxyl derivatives of unsaturated fatty acids are known to exhibit actions such as anti-inflammatory, lipid metabolism, sugar metabolism, bone metabolism, anti-cancer action, anti-obesity, and intestinal regulation. . Therefore, according to the method of one embodiment of the present invention, various supplements or health foods containing these compounds as active ingredients can be provided. Also, beer lees is a safe material that is discharged in the process of brewing beer, but at present, most of it is treated as industrial waste. Therefore, the method according to one embodiment of the present invention can provide a safe supplement or health food by effectively using beer lees.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

<Experiment I: Examination of alkali extraction conditions>
[I-1: Method]
The most squeezed dried beer koji (Kirin beer) was pulverized with a coffee mill to obtain a beer koji powder. In a 10 mL small test tube, 250 mg of beer koji powder was placed, and 5 mL of lower alcohol (methanol (MeOH), ethanol (EtOH), 2-propanol (2-PrOH) or butanol (BuOH)) was added. This mixture was sonicated for 5 minutes to evenly wet the lower alcohol into the beer koji powder. To the resulting suspension of beer koji powder, 5 mL of a NaOH aqueous solution having a predetermined concentration was added and further suspended. The suspension of beer koji powder was alkali-treated under predetermined conditions to obtain an extract. The obtained extract was centrifuged at 4000 rpm for 10 minutes. The supernatant was transferred to a 15 mL tube (Corning). The supernatant was adjusted to pH 8 with 6 M HCl and used as an analytical sample.

The hydroxyl derivative (HODE) of unsaturated fatty acid and ferulic acid in the analysis sample were analyzed by high performance liquid chromatography (HPLC) under the following conditions.
[HPLC conditions]
○ HODE analytical HPLC
Column: TSK ODS 100V (3μm) 4.6 x 150 mm (Tosoh Corporation)
Column temperature: 40 ° C
Sample injection volume: 2 μL
Solvent: A: 1/100 M HCl
B: CH ₃ CN
C: CH ₃ OH
Mixing ratio A: B: C = 48: 44: 8
Flow rate: 1.0 mL / min Detection: 235 nm, 0.01 range ○ Analytical HPLC of ferulic acid
Column: TSK ODS 100V (3μm) 4.6 x 150 mm (Tosoh Corporation)
Column temperature: 40 ° C
Sample injection volume: 5 μL
Solvent: A: 1/100 M HCl
B: CH ₃ CN
Mixing ratio A: B = 75: 25
Flow rate: 1 mL / min Detection: 235 nm, 0.04 range

In this example, Fr. 1 to Fr. 4 shown in the tables and figures represent HODE of linoleic acid shown in the following scheme.

  Β-glucan in the analytical sample was analyzed by the Congo red method (Naoyuki Ishikawa et al., Tochigi Agricultural Experiments, Vol. 47, p. 57-64 (1998); MCSemedo et al., Journal of Microbiological Methods, Vol. 109) , p. 140-148 (2015) modified). Curdlan was used as a standard sample. It is known that the low molecular weight glucan cannot be detected by the Congo Red method. For this reason, analysis values may differ due to causes such as differences in sample preparation methods.

[I-2: Results]
Alkaline extraction of beer koji powder was carried out in a NaOH solution having a concentration in the range of 0.1 to 1.0 M under conditions of 60 ° C. for 1 hour. FIG. 1 shows the relationship between the NaOH concentration in alkali extraction of beer koji powder and the amounts of oxidized derivatives, ferulic acid and β-glucan contained in the extract. In the figure, A shows the amount of oxidized derivative contained in the extract, and B shows the amounts of ferulic acid and β-glucan contained in the extract, respectively. The amounts of the oxidized derivative, ferulic acid and β-glucan in the figure are calculated values when 1 g of beer koji powder is used, calculated based on the analysis value determined by the analysis means.

  As shown in FIG. 1, HODE and β-glucan could be efficiently extracted by alkaline extraction of beer koji powder in a NaOH solution having a concentration in the range of 0.1 to 1.0 M.

  Alkaline extraction of beer lees powder was carried out in NaOH solutions with concentrations ranging from 0.3 M under conditions of 60 ° C., 30, 60 or 120 minutes. FIG. 2 shows the relationship between the alkali extraction time in the alkali extraction of beer koji powder and the amounts of oxidized derivatives, ferulic acid and β-glucan contained in the extract. In the figure, A shows the amount of oxidized derivative contained in the extract, and B shows the amounts of ferulic acid and β-glucan contained in the extract, respectively. The amounts of the oxidized derivative, ferulic acid and β-glucan in the figure are calculated values when 1 g of beer koji powder is used, calculated based on the analysis value determined by the analysis means.

  As shown in FIG. 2, HODE and β-glucan could be efficiently extracted by alkaline extraction of beer koji powder for 30 minutes or more.

  Alkaline extraction of beer koji powder was carried out in NaOH solution with a concentration in the range of 0.3 M under conditions of room temperature, 60 ° C. or 90 ° C. for 60 minutes. FIG. 3 shows the relationship between the alkali extraction temperature in the alkali extraction of beer koji powder and the amounts of oxidized derivatives, ferulic acid and β-glucan contained in the extract. In the figure, A shows the amount of oxidized derivative contained in the extract, and B shows the amounts of ferulic acid and β-glucan contained in the extract, respectively. The amounts of the oxidized derivative, ferulic acid and β-glucan in the figure are calculated values when 1 g of beer koji powder is used, calculated based on the analysis value determined by the analysis means.

  As shown in FIG. 3, HODE and β-glucan could be efficiently extracted by alkali-extracting beer koji powder at room temperature or higher. HODE was extracted at higher temperatures, whereas β-glucan was extracted more in the case of alkaline extraction at room temperature than at 60 ° C or 90 ° C. .

  Alkaline extraction of beer lees powder was carried out in the presence of lower alcohols of MeOH, EtOH, 2-PrOH or BuOH in NaOH solution with a concentration in the range of 0.3 M at 60 ° C. for 60 minutes. FIG. 4 shows the relationship between the lower alcohol in the alkaline extraction of beer koji powder and the amounts of oxidized derivatives, ferulic acid and β-glucan contained in the extract. In the figure, A shows the amount of oxidized derivative contained in the extract, and B shows the amounts of ferulic acid and β-glucan contained in the extract, respectively. The amounts of the oxidized derivative, ferulic acid and β-glucan in the figure are calculated values when 1 g of beer koji powder is used, calculated based on the analysis value determined by the analysis means.

  As shown in FIG. 4, HODE and β-glucan could be efficiently extracted by alkaline extraction of beer koji powder in the presence of MeOH, EtOH or 2-PrOH. On the other hand, when the beer koji powder was alkali extracted in the presence of BuOH, HODE was hardly extracted.

<Experiment II: Examination of precipitation conditions>
[II-1: Method]
Dry beer lees (300 g) was pulverized with a mixer to obtain beer lees powder. In a reaction vessel, 100 g of beer koji powder was placed and suspended in 1 L of an aqueous solution of 0.3 or 1.0 M NaOH (containing 50% by volume EtOH). The mixture was sonicated for 20 minutes. The obtained beer koji powder suspension was alkali-treated at 60 ° C. for 60 minutes to obtain an alkali extract. The obtained alkaline extract was centrifuged at 8000 rpm for 20 minutes. The supernatant was transferred to another container. To the precipitate, 500 mL of pure water was added and sonicated for 20 minutes. The suspension was centrifuged at 8000 rpm for 20 minutes. The supernatant was combined with the previously collected supernatant. The treatment was repeated twice to wash the precipitate. The combined supernatants were adjusted to pH 2 with 6 M hydrochloric acid at room temperature. The acidified extract was kept at the same temperature for 1 hour, and then centrifuged at 8000 rpm for 20 minutes to obtain an acidic supernatant fraction and an insoluble fraction precipitate. The acidic supernatant fraction was extracted with ethyl acetate under room temperature conditions to obtain an ethyl acetate fraction. The precipitate of insoluble fraction was lyophilized. HODE, ferulic acid and β-glucan contained in the ethyl acetate fraction and the insoluble fraction were analyzed in the same procedure as in Experiment I. The coumaric acid contained in the ethyl acetate fraction and the insoluble fraction was analyzed by the same procedure as the ferulic acid analysis shown in Experiment I. The analysis value of coumaric acid was calculated from the peak height of coumaric acid in the obtained HPLC chromatogram as a converted value based on the peak height of the ferulic acid sample.

[II-2: Results]
The lyophilized insoluble fraction was 26.65 g or 38.52 g when alkali extracted under conditions of 0.3 or 1.0 M NaOH, respectively. FIG. 5 shows the amounts of oxidized derivatives, ferulic acid, coumaric acid and β-glucan contained in the ethyl acetate fraction or insoluble fraction when alkali extraction is performed under the condition of 0.3 or 1.0 M NaOH. In the figure, A is the amount of oxidized derivative contained in the extract, B is ferulic acid (left vertical axis), coumaric acid (left vertical axis) and β-glucan (right vertical axis) contained in the extract. The amount is indicated respectively.

  As shown in FIG. 5, HODE and β-glucan were obtained from the insoluble fraction, but were not substantially detected from the ethyl acetate fraction. Ferulic acid and coumaric acid were obtained from both the ethyl acetate fraction and the insoluble fraction. In this experiment, since alkaline extraction was carried out in the presence of 50% EtOH, it is presumed that the extraction efficiency of ferulic acid and coumaric acid into the ethyl acetate fraction was lowered.

  In addition, this invention is not limited to an above-described Example, Various modifications are included. For example, the above-described embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described. Moreover, it is possible to add, delete, and / or replace another configuration with respect to a part of the configuration of each embodiment.

Claims (5)

An alkali extraction step to obtain an alkaline extract by alkali treatment of beer lees,
Acidify the alkaline extract to obtain an acidic supernatant fraction, as well as β-glucan and formula (I-1):

[Where:
R ¹ is hydrogen, substituted or unsubstituted C ₁ -C ₅ alkyl, substituted or unsubstituted C ₂ -C ₅ alkenyl, substituted or unsubstituted C ₂ -C ₅ alkynyl, substituted or unsubstituted C _3- C ₆ cycloalkyl, substituted or unsubstituted C ₄ -C ₆ cycloalkenyl, substituted or unsubstituted C ₄ -C ₆ cycloalkenyl, or substituted or unsubstituted C ₇ -C ₁₁ cycloalkylalkyl,
R ² is unsubstituted C ₁ -C ₇ alkyl, or unsubstituted C ₄ -C ₁₆ alkenyl,
L ¹ is unsubstituted C ₁ -C ₆ alkylene, or unsubstituted C ₄ -C ₁₆ alkenylene,
However, R ² and L ¹ are compounds that satisfy the condition that the total number of carbon atoms is in the range of 13 to 17 and the total number of double bonds is in the range of 0 to 5. ]
And a compound represented by formula (I-2):

[Wherein, R ¹ , R ² and L ¹ have the same meanings as described above. ]
A β-glucan and a hydroxyl derivative of one or more unsaturated fatty acids, including an insolubilization step, obtaining an insoluble fraction containing one or more unsaturated fatty acid hydroxyl derivatives selected from the group consisting of the compounds represented by: A method for producing a composition comprising the same.   2. The method according to claim 1, wherein the alkali extraction step is carried out using a sodium hydroxide or potassium hydroxide aqueous solution having a concentration of 0.1 M or more.   The method according to claim 1 or 2, wherein the alkali extraction step is carried out in the presence of a lower alcohol selected from the group consisting of methanol, ethanol and 2-propanol, and mixtures thereof. R ² is n-butyl,
L ¹ is the formula (L ¹ -1):

[Where:
* Is the bonding position with the alkenylene chain,
** is a bonding position with a carboxylic acid group. ]
A divalent group represented by: or
R ² is the formula (R ² -1):

[Where:
* Is the bonding position with the alkenylene chain. ]
A divalent group represented by
L ¹ is n-hexylene;
The method of any one of Claims 1-3 which satisfy | fills these conditions.   The acidic supernatant fraction obtained in the insolubilization step is extracted with an organic solvent to further obtain an organic solvent extraction step of obtaining a fraction containing at least one of ferulic acid and coumaric acid. The method according to any one of the above.

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