EP4655410A2

EP4655410A2 - O-methylation of phenolic substances via fungal o-methyltransferase

Info

Publication number: EP4655410A2
Application number: EP24702333.6A
Authority: EP
Inventors: Holger Zorn; Jean-Philip KANTER; Jakob Peter Ley; Lars MILKE; Bastian ZIRPEL
Original assignee: Symrise AG
Current assignee: Symrise AG
Priority date: 2023-01-27
Filing date: 2024-01-26
Publication date: 2025-12-03
Also published as: WO2024156879A2; WO2024156879A3; WO2024156359A1

Abstract

The present invention relates to a method for producing an O-methylated phenolic substance from the corresponding phenolic substance, to a mixture comprising an O-methyltransferase of a fungus, wherein the mixture is obtained or obtainable by a method according to the invention, to the use of such a mixture for producing an O-methylated phenolic substance, and to the use of one or more fungi for producing an O-methylated phenolic substance.

Description

O-methylation of phenolic substances via fungal O-methyltransferase

The present invention relates to a method for producing an O-methylated phenolic substance from the corresponding phenolic substance, to a mixture comprising an O-methyl- transferase of a fungus, wherein the mixture is obtained or obtainable by a method according to the invention, to the use of such a mixture for producing an O-methylated phenolic substance, and to the use of one or more fungi for producing an O-methylated phenolic substance.

(Poly)phenolic substances are frequently present in plants and are thus also found in human nutrition. In particular, the class of (phenolic) flavonoids and particularly O-methylated flavonoids may be phenolic acids such as vanillic acid, isovanillic acid, homovanillic acid, isohomovanillic acid, vanillomandelic acid, isovanillomandelic acid, p-methoxycinnamic acid, isoferulic acid, ferulic acid, and sinapic acid. Particularly, O-methylated phenolic substances, particularly O-methylated flavonoids, provide a large number of advantageous effects, ranging from their use in food products to medical applications. Particularly in the food and flavour industry, phenolic substances such as flavonoids are important flavour ingredients, which are frequently used. Among such flavonoids, a particular focus is set to the O-methylated flavonoids such as hesperetin, homoeriodictyol, hesperetin dihydrochal- cone, homoeriodictyol dihydrochalcone, isosakuranetin, isosakuranetin dihydrochalcone, 2',4'-Dihydroxy-4-methoxychalcone, homobutein, (2E)-1-(2,4-Dihydroxyphenyl)-3-(3-hy- droxy-4-methoxyphenyl)-2-propen-1-one, 2,3-Dihydro-7-hydroxy-2-(4-methoxyphenyl)- 4H-1-benzopyran-4-one, 2,3-Dihydro-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-1- benzopyran-4-one, 2,3-Dihydro-7-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopy- ran-4-one, 3'-O-Methyl-(-)-epicatechin, 4'-O-Methyl-(-)-epicatechin, 3,4-Dihydro-8-hy- droxy-3-(4-methoxyphenyl)-1 H-2-benzopyran-1-one, 3,4-Dihydro-8-hydroxy-3-(4-hydroxy- 3-methoxyphenyl)-1 H-2-benzopyran-1-one, stilbene derivatives, dihydrostilbene deriva- tives such as oxyrhapontigenin, isorhapontigenin, rhapontigenin, or phyllodulcin, their glycosides, and mixtures thereof. Particularly, stilbene or dihydrostilbene derivatives such as desoxyrhapontigenin, isorhapontigenin, rhapontigenin, or phyllodulcin are interesting as actives for food and flavour applications. As long as the O-methylated phenolic substances mentioned bear at least an additional hydroxyl group they can occur, be prepared and be used as glycosides.

O-methylated phenolic substances may be obtained by methylating the corresponding phenolic substance, wherein the methylation takes place at the I a hydroxyl group of the phenolic substance. For example, the O-methylated flavonoid hesperetin can be obtained by methylating the flavonoid eriodictyol. Likewise, hesperetin dihydrochalcone can be ob- tained by methylating by methylating 3-hydroxyphloretin: In such methods, the methylation is performed by O-methyltransferases. Thus, for producing O-methylated phenolic substances on industrial scale, large amounts of O-methyltrans- ferases are needed.

Methods for heterologous expression of genes encoding O-methyltransferases are already known. In these methods, genetically modified organisms (GMOs) are used. However, for several applications, a GMO strain shall or must not be used. Frequently, many consumers have concerns or reject products obtained from or with the participation of GMOs.

Thus, it is preferred to produce O-methylated phenolic substances without utilization of GMOs for such cases and applications.

Genes encoding O-methyltransferases have been found in some organisms, such as a small number of particular fungi. However, if at all - or under which conditions - the respective genes are expressed in these fungi has not been reported yet. The mere presence of a gene in the genome of an organism does not guarantee that the gene is expressed. For example, the expression of a gene may depend on the developmental stage of an organism or on the presence of particular substances in the organism’s surrounding.

Furthermore, how to achieve an expression of at least one endogenous gene encoding for an enzyme with O-methyltransferase activity, which is satisfying the needs of producing O- methyltransferases on industrial scale, has not been described. Many factors may influence the expression levels of a gene, if expressed at all.

Thus, a primary object of the present invention was to provide methods for producing an O-methylated phenolic substance from a phenolic substance without being dependent on GMOs in such methods, preferably such that high levels of O-methyltransferases and O- methylated phenolic substances are obtained for applying the method on industrial scale.

The primary object of the present invention is solved by a for producing an O-methylated phenolic substance from the corresponding phenolic substance, the method comprising the steps i) providing one or more phenolic substance(s), ii) providing one or more fungi, wherein the, one, two, three or more fungi is/are of a genus selected from the group consisting of Lentinus, Lentinula and Hypsizygus, iii) optionally: providing one or more methyl donors, iv) mixing the one or more phenolic substance(s) provided in step i), the one or more fungi provided in step ii) and, if present, the one or more methyl donor provided in step iii) to obtain a reaction mixture, and reacting the reaction mixture under conditions allowing the O-methylation of the phenolic substance(s).

Several genera of fungi have been tested, which potentially inherit a gene encoding an O- methyltransferase. However, it was surprisingly found that only the fungi of the genera Len- tinus, Lentinula or Hypsizygus were able to provide an O-methyltransferase and to producing an O-methylated phenolic substance from a phenolic substance (see Example 1).

The term “phenolic substance”, as used herein, preferably refers to a compound, which possesses one or more, preferably at least one, preferably at least two, preferably two, hydroxyl groups, preferably wherein the hydroxyl group(s) is/are bound to a phenyl group or a derivative thereof. Preferably, the term refers to a compound, which possesses a catechol group. Particularly preferably, the term relates to a compound selected from the group consisting of benzoic acids (preferably hydroxy benzoic acids and dihydroxybenzoic acids), benzaldehydes (preferably hydroxybenzaldehydes and dihydroxybenzaldehydes), dihydroxybenzenes (preferably 1 ,2-dihydroxybenzenes) phenylacetic acids, mandelic acids, cinnamic acids, cinnamic acid derivatives, dihydrocinnamic acids, dihydrocinnamic acid derivatives, chaicones, dihydrochalcones, flavanes, catechins, flavanones, flavones, 3-hydroxyflavones, anthocyanes, stilbenes, dihydrostilbenes, dihydroisocoumarins, isocoumarins, phenylpropanoids, flavanols, their glycosides, particularly O-glycosides, and mixtures thereof. Further preferably, the term relates to a compound selected from the group consisting of benzoic acids, phenylacetic acids, mandelic acids, cinnamic acids, dihydrocinnamic acids, chaicones, dihydrochalcones, flavanes, catechins, flavanones, flavones, 3-hydroxyflavones, anthocyanes, stilbenes, dihydrostilbenes, dihydroisocoumarins, isocoumarins, phenylpropanoids, flavanols, their glycosides, and mixtures thereof.

Furthermore, when a “phenolic substance”, as described herein, is methylated at the or one of the hydroxyl groups, an “O-methylated phenolic substance”, as described herein, is obtained. Thus, the term “the corresponding phenolic substance” refers to a phenolic substance, which can be reacted to the respective O-methylated phenolic substance by methylation, preferably by O-methylation. The term “O-methylated phenolic substance”, as used herein, refers to a phenolic substance, as described herein, which possesses a methyl group bound by an oxygen atom to the remaining structure, thus, the O-methylated phenolic substance possesses one or more methoxy groups. Particularly, in the case of O-glycosides, the corresponding phenolic substance preferably contains at least one unsubstituted hydroxyl group.

Preferably, the term “O-methylated phenolic substance”, as used herein, refers to a compound selected from the group consisting of Ferulic acid, Dihydroferulic acid, Homoeriodictyol dihydrochalcone, Homobutein, Homoeriodictyol, 2,3-Dihydro-7-hydroxy-2-(4-hydroxy- 3-methoxyphenyl)-4H-1-benzopyran-4-one, Isorhapontigenin, 3'-O-Methyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-hydroxy-3-methoxyphenyl)-1 H-2-benzopyran-1-one, Isorham- netin, 1-[2,6-dihydroxy-4-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]-3-(4-hy- droxy-3-methoxyphenyl)propan-1-one, 5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-7- [3,4,5-trihydroxy-6-[(3,4,5-trihydroxy-6-methyloxan-2-yl)oxymethyl]oxan-2-yl]oxy-2,3-dihy- drochromen-4-one, Narcissin, p-Methoxycinnamic acid, Isoferulic acid, Dihydroisoferulic acid, Isosakuranetin dihydrochalcone, Hesperetin dihydrochalcone, 2',4'-Dihydroxy-4- methoxychalcone, (2E)-1-(2,4-Dihydroxyphenyl)-3-(3-hydroxy-4-methoxyphenyl)-2-pro- pen-1-one, Isosakuranetin, Hesperetin, 2,3-Dihydro-7-hydroxy-2-(4-methoxyphenyl)-4H-1- benzopyran-4-one, 2,3-Dihydro-7-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopy- ran-4-one, Desoxyrhapontigenin, Rhapontigenin, (2R,3R)-3,4-Dihydro-2-(4-methoxy- phenyl)-2H-1-benzopyran-3,5,7-triol, 4'-O-Methyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy- 3-(4-methoxyphenyl)-1 H-2-benzopyran-1-one, stilbene derivatives, dihydrostilbene derivatives, Phyllodulcin, Tamarixetin, Kaempferid, Hesperetin dihydrochalcone glucoside, Hes- peridin, 1-[4-(p-D-glucopyranosyloxy)-2,6-dihydroxyphenyl]-3-(4-methoxyphenyl)-1 -Propanone, 3-[[6-0-(6-deoxy-a-L-mannopyranosyl)-p-D-glucopyranosyl]oxy]-5,7-dihydroxy-2- (3-hydroxy-4-methoxyphenyl)-4H-1-Benzopyran-4-one, 3',4'-Dimethoxycinnamic acid, 3- (3,4-Dimethoxyphenyl)-1 -(2, 4, 6-trihydroxyphenyl)-1 -propanone, (2E)-1-(2,4-Dihydroxy- phenyl)-3-(3,4-dimethoxyphenyl)-2-propen-1-one, 2-(3,4-Dimethoxyphenyl)-2,3-dihydro- 5,7-dihydroxy-4H-1-benzopyran-4-one, 2-(3,4-Dimethoxyphenyl)-2,3-dihydro-7-hydroxy- 4H-1-benzopyran-4-one, 5-[(1 E)-2-(3,4-Dimethoxyphenyl)ethenyl]-1 ,3-benzenediol, (2R,3R)-2-(3,4-Dimethoxyphenyl)-3,4-dihydro-2H-1-benzopyran-3,5,7-triol, Thunberginol H, Dillenetin, Calomelanone, vanillin, isovanillin, vanillic acid, isovanillic acid, homovanillic acid, isohomovanillic acid, vanillomandelic acid, isovanillomandelic acid, sinapic acid, eugenol, chavibetol, 4-ethylguaiacol, 5-ethylguaiacol, their glycosides, and mixtures thereof. Preferably, the term “O-methylated phenolic substance”, as used herein, refers to a compound selected from the group consisting of Ferulic acid, Dihydroferulic acid, Homoeriodictyol dihydrochalcone, Homobutein, Homoeriodictyol, 2,3-Dihydro-7-hydroxy-2-(4-hydroxy- 3-methoxyphenyl)-4H-1-benzopyran-4-one, Isorhapontigenin, 3'-O-Methyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-hydroxy-3-methoxyphenyl)-1 H-2-benzopyran-1-one, Isorham- netin, 1-[2,6-dihydroxy-4-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]-3-(4-hy- droxy-3-methoxyphenyl)propan-1-one, 5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-7- [3,4,5-trihydroxy-6-[(3,4,5-trihydroxy-6-methyloxan-2-yl)oxymethyl]oxan-2-yl]oxy-2,3-dihy- drochromen-4-one, Narcissin, p-Methoxycinnamic acid, Isoferulic acid, Dihydroisoferulic acid, Isosakuranetin dihydrochalcone, Hesperetin dihydrochalcone, 2',4'-Dihydroxy-4- methoxychalcone, (2E)-1-(2,4-Dihydroxyphenyl)-3-(3-hydroxy-4-methoxyphenyl)-2-pro- pen-1-one, Isosakuranetin, Hesperetin, 2,3-Dihydro-7-hydroxy-2-(4-methoxyphenyl)-4H-1- benzopyran-4-one, 2,3-Dihydro-7-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopy- ran-4-one, Desoxyrhapontigenin, Rhapontigenin, (2R,3R)-3,4-Dihydro-2-(4-methoxy- phenyl)-2H-1-benzopyran-3,5,7-triol, 4'-O-Methyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy- 3-(4-methoxyphenyl)-1 H-2-benzopyran-1-one, stilbene derivatives, dihydrostilbene derivatives, Phyllodulcin, Tamarixetin, Kaempferid, Hesperetin dihydrochalcone glucoside, Hes- peridin, 1-[4-(p-D-glucopyranosyloxy)-2,6-dihydroxyphenyl]-3-(4-methoxyphenyl)-1 -Propanone, 3-[[6-0-(6-deoxy-a-L-mannopyranosyl)-p-D-glucopyranosyl]oxy]-5,7-dihydroxy-2- (3-hydroxy-4-methoxyphenyl)-4H-1-Benzopyran-4-one, 3',4'-Dimethoxycinnamic acid, 3- (3,4-Dimethoxyphenyl)-1 -(2, 4, 6-trihydroxyphenyl)-1 -propanone, (2E)-1-(2,4-Dihydroxy- phenyl)-3-(3,4-dimethoxyphenyl)-2-propen-1-one, 2-(3,4-Dimethoxyphenyl)-2,3-dihydro- 5,7-dihydroxy-4H-1-benzopyran-4-one, 2-(3,4-Dimethoxyphenyl)-2,3-dihydro-7-hydroxy- 4H-1-benzopyran-4-one, 5-[(1 E)-2-(3,4-Dimethoxyphenyl)ethenyl]-1 ,3-benzenediol, (2R,3R)-2-(3,4-Dimethoxyphenyl)-3,4-dihydro-2H-1-benzopyran-3,5,7-triol, Thunberginol H, Dillenetin, Calomelanone, vanillic acid, isovanillic acid, homovanillic acid, isohomovanillic acid, vanillomandelic acid, isovanillomandelic acid, sinapic acid, their glycosides, and mixtures thereof.

The term “providing a fungus”, as used herein, refers to providing the fungus or cells of the fungus, preferably including providing fungal mycelium. Preferably, the term also refers to providing parts or fragments of the fungus.

The term “methyl donor”, as used herein refers to a chemical structure, which readily donate a methyl group to another substance in a chemical or enzymatic reaction. A methyl donor, as described herein, can also be a mixture of several of such chemical structures. The methylation as described herein is typically achieved by an O-methyltransferase. Thus, a methyl donor is frequently required for such a reaction. Typically, such methyl donors are present in the one or more fungi provided in the method according to the invention. Nevertheless, it may also be advantageous to provide (additional) methyl donor(s). Thus, step iii) of the method according to the invention is optional. In case step iii) is present in the method, the methyl donor provided in step iii) thus refers to a methyl donor from a different source than the one or more fungi provided in step ii), if the one or more fungi contain a methyl donor. The methyl donor provided in step iii) and the methyl donor present in the one or more fungi provided in step ii) may be the same compound or may comprise the same compound(s).

Preferably, a methyl donor is present in the, one or more or all fungi provided in step ii).

Preferably, a methyl donor is present in the, one or more or all fungi provided in step ii), wherein the methyl donor is or comprises S-adenosylmethionine (SAM).

Preferably, a methyl donor is provided in step iii), i.e. step iii) is present, wherein the methyl donor is or comprises S-adenosylmethionine (SAM).

Furthermore, sufficient amounts of methyl donor should be present for the production for the O-methylated phenolic substance in the method according to the invention on industrial scale. It is thus preferred that in the mixture obtained in step iv) of the method according to the invention, the ratio of the amount of substance of the phenolic substance(s) and the amount of substance of the methyl donor(s) is in a range of from 10:1 to 1 :10, preferably in a range of from 5:1 to 1 :7.5, preferably in a range of from 2.5 to 1 to 1 :5, preferably in a range of from 1 :1 to 1 :2.5. As described above, the one or more fungi may also contain methyl donor(s). Thus, the methyl donor(s) in the mixture obtained in step iv) may be provided in step ii) and/or step iii) of the method according to the invention.

Similarly, it is preferred that the ratio of the amount of substance of the provided phenolic substance(s) in step i) and the amount of substance of provided methyl donor(s) in step iii) is in a range of from 10:1 to 1 :10, preferably in a range of from 5:1 to 1 :7.5, preferably in a range of from 2.5 to 1 to 1 :5, preferably in a range of from 1 :1 to 1 :2.5.

Similarly, it is preferred that the ratio of the amount of substance of the provided phenolic substance(s) in step i) and the amount of substance of provided methyl donor(s) in step ii) is in a range of from 10:1 to 1 :10, preferably in a range of from 5:1 to 1 :7.5, preferably in a range of from 2.5 to 1 to 1 :5, preferably in a range of from 1 :1 to 1 :2.5.

Similarly, it is preferred that the ratio of the amount of substance of the provided phenolic substance(s) in step i) and the amount of substance of provided methyl donor(s) in steps ii) and iii) is in a range of from 10:1 to 1 :10, preferably in a range of from 5:1 to 1 :7.5, preferably in a range of from 2.5 to 1 to 1 :5, preferably in a range of from 1 :1 to 1 :2.5.

It is further preferred in the method according to the invention that the, one, two, three or more or all phenolic substances provided in step i) is/are selected from the group consisting of benzoic acids (preferably hydroxy benzoic acids and dihydroxybenzoic acids), benzaldehydes (preferably hydroxybenzaldehydes and dihydroxybenzaldehydes), dihydroxybenzenes (preferably 1 ,2-dihydroxybenzenes) phenylacetic acids, mandelic acids, cinnamic acids, cinnamic acid derivatives, dihydrocinnamic acids, dihydrocinnamic acid derivatives, chaicones, dihydrochalcones, flavanes, catechins, flavanones, flavones, 3-hydroxyfla- vones, anthocyanes, stilbenes, dihydrostilbenes, dihydroisocoumarins, isocoumarins, phe- nylpropanoids, flavanols, their glycosides, particularly O-glycosides, and mixtures thereof, preferably selected from the group consisting of benzoic acids, phenylacetic acids, mandelic acids, cinnamic acids, dihydrocinnamic acids, chaicones, dihydrochalcones, flavanes, catechins, flavanones, flavones, 3-hydroxyflavones, anthocyanes, stilbenes, dihydrostilbenes, dihydroisocoumarins, isocoumarins, phenylpropanoids, flavanols, their glycosides, particularly O-glycosides, and mixtures thereof, preferably selected from the group consisting of flavanones, chaicones, dihydrochalcones, phenylpropanoids, flavanols, dihydroisocoumarines, stilbenes, their glycosides, and mixtures thereof.

Particularly preferably, the, one, two, three or more or all phenolic substance(s) provided in step i) is/are selected from the group consisting of naringenin, eriodictyol, homoeriodictyol, 3-hydroxyphloretin, phloretin, phloridzin, 3-hydroxyphloridzin, sieboldin, p-coumaric acid, caffeic acid, dihydrocaffeic acid, isoliquiritigenin, butein, liquiritigenin, butein, resveratrol, piceatannol, afzelechin, epiafzelechin, catechin, epicatechin, quercetin, kaempferol, eriocitrin, trilobatin, rutin, luteolin, hydrangenol, thunberginol G, flavonols, protocatechuic acid, protocatechuic aldehyde, hydroxychavicol, 4-ethylcatechol, and their glycosides. Particularly preferably, the, one, two, three or more or all phenolic substance(s) provided in step i) is/are selected from the group consisting of naringenin, eriodictyol, homoeriodictyol, 3-hydroxyphloretin, phloretin, phloridzin, 3-hydroxyphloridzin, sieboldin, p-coumaric acid, caffeic acid, dihydrocaffeic acid, isoliquiritigenin, butein, liquiritigenin, butein, resveratrol, piceatannol, afzelechin, epiafzelechin, catechin, epicatechin, quercetin, kaempferol, eriocitrin, trilobatin, rutin, luteolin, hydrangenol, thunberginol G, flavonols, and their glycosides.

The term “their glycosides”, as used herein, refers to one or more glycosides of a compound, wherein the glycoside(s) may be mono-, di-, tri, or poly-glycosides of the compound or a mixture of mono-, di-, tri, or poly-glycosides of the compound. Preferably, the glycosides, as described herein, are or comprise glucosides.

It was found that fungi of the group consisting of Lentinula edodes, Lentinula lateritia, Len- tinus squarrosulus, Lentinus cladopus, and Hypsizygus tessulatus provided the best results when performing the method according to the invention.

Thus, it is further preferred that the, one, two, three or more or all fungi provided in step ii) is/are selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus, Lentinus cladopus, and Hypsizygus tessulatus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus and Lentinus cladopus.

Furthermore, it is preferred that providing one or more fungi includes a step of providing a cell free extract of said one or more fungi, which is then admixed in step iv) of the method according to the invention. A cell free extract, as used herein, refers to a cell extract-based cell free system and is known to a skilled person. A cell free extract may be provided by e.g. extraction and/or centrifugation, preferably ultracentrifugation.

It was surprisingly found that the method according to the invention provided particularly high amounts of the O-methylated phenolic substance, if the reaction in step iv) was performed at a pH in the range of from 7 to 8.5, particularly advantageously at a pH in the range of from 7.5 to 8.

It is thus preferred that the conditions allowing the O-methylation of the phenolic substance^) in step iv) include a pH in a range of from 6.5 to 9.5, preferably in a range of from 6.75 to 9, preferably in a range of from 7 to 8.5. Moreover, it was surprisingly found that the method according to the invention provided particularly high amounts of the O-methylated phenolic substance, if the reaction in step iv) was performed for a time in the range of from 45 to 180 h. For stopping the reaction, the pH value may be decreased.

It is thus preferred that the conditions allowing the O-methylation of the phenolic substance^) in step iv) include a reaction time in the range of from 45 to 180 h, preferably in the range of from 60 to 150 h. Preferably, the reaction is stopped by acidification, preferably by acidification with 4 M HCI. It is preferred that for acidification, 10 pl of 4 M HCI are added per 1 mL of the reaction volume obtained in step iv) of the method according to the invention.

Preferably, the O-methyltransferase, as described herein, is a 4’-O-methyltransferase.

Additionally or alternatively, it is preferred that the O-methyltransferase, as described herein, comprises or consists of an amino acid sequence according to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or an amino acid sequence having at least 90%, preferably at least 91 %, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, sequence identity to the amino acid sequence according to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

Preferably, whenever the present disclosure refers to sequence identities of amino acid sequences in terms of percentages, such references are to values as can be calculated using EMBOSS Water Pairwise Sequence Alignments (proteins) (http://www.ebi.ac.uk/Tools/psa/emboss_water/) for amino acid sequences, respectively. Local sequence alignment tools provided by the European Molecular Biology Laboratory (EMBL) European Bioninformatics Institute (EBI) use a modified Smith-Waterman algorithm (see http://www.ebi.ac.uk/Tools/psa/ and Smith, T.F. & Waterman, M.S. "Identification of common molecular subsequences" Journal of Molecular Biology, 1981 147 (1):195- 197). Furthermore, here, when performing the respective pairwise alignment of two sequences using the modified Smith-Waterman algorithm, reference is made to the default parameters currently given by EMBL-EBI. These are for amino acid sequences: Matrix = BLOSUM62, Gap open penalty = 10 and Gap extend penalty = 0.5. Moreover, the present invention relates to a mixture comprising a) a fungus of a genus selected from the group consisting of Lentinus, Lentinula and Hypsizygus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus, Lentinus cladopus, and Hypsizygus tessulatus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus and Lentinus cladopus, wherein the fungus (homologously) expresses an O-methyltransferase, preferably an O-methyltransferase comprising or consisting of an amino acid sequence according to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or an amino acid sequence having at least 90%, preferably at least 91 %, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, sequence identity to the amino acid sequence according to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, and b) one or more compound(s) selected from the group consisting of naringenin, eriodictyol, homoeriodictyol, 3-hydroxyphloretin, phloretin, phloridzin, 3-hydroxyphloridzin, sieboldin, p-coumaric acid, caffeic acid, dihydrocaffeic acid, isoliquiritigenin, butein, liquiritigenin, resveratrol, piceatannol, afzelechin, epiafzelechin, catechin, epicatechin, quercetin, kaempferol, eriocitrin, trilobatin, rutin, luteolin, hydrangenol, thun- berginol G, flavonols, protocatechu ic acid, protocatechu ic aldehyde, hydroxychavicol, 4-ethylcatechol, their glycosides, and mixtures thereof, preferably one or more compound(s) selected from the group consisting of naringenin, eriodictyol, homoeriodictyol, 3-hydroxyphloretin, phloretin, phloridzin, 3- hydroxyphloridzin, sieboldin, p-coumaric acid, caffeic acid, dihydrocaffeic acid, isoliquiritigenin, butein, liquiritigenin, resveratrol, piceatannol, afzelechin, epiafzelechin, catechin, epicatechin, quercetin, kaempferol, eriocitrin, trilobatin, rutin, luteolin, hydrangenol, thunberginol G, flavonols, their glycosides, and mixtures thereof, Preferably, the mixture according to the invention comprises one or more methyl donors, as described herein. Particularly preferably, the methyl donor is or comprises S-adenosyl- methionine (SAM).

Moreover, the present invention relates to a mixture, preferably a mixture as described above, comprising a) an O-methyltransferase of a fungus of a genus selected from the group consisting of Lentinus, Lentinula and Hypsizygus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus, Lentinus cladopus, and Hypsizygus tessulatus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus and Lentinus cladopus, preferably an O-methyltransferase comprising or consisting of an amino acid sequence according to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or an amino acid sequence having at least 90%, preferably at least 91 %, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, sequence identity to the amino acid sequence according to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, and b) one or more compound(s) selected from the group consisting of naringenin, eriodictyol, homoeriodictyol, 3-hydroxyphloretin, phloretin, phloridzin, 3-hydroxyphloridzin, sieboldin, p-coumaric acid, caffeic acid, dihydrocaffeic acid, isoliquiritigenin, butein, liquiritigenin, butein, resveratrol, piceatannol, afzelechin, epiafzelechin, catechin, epicatechin, quercetin, kaempferol, eriocitrin, trilobatin, rutin, luteolin, hydrangenol, thunberginol G, flavonols, protocatechu ic acid, protocatechuic aldehyde, hydroxychavicol, 4-ethylcatechol, their glycosides, and mixtures thereof, preferably one or more compound(s) selected from the group consisting of naringenin, eriodictyol, homoeriodictyol, 3-hydroxyphloretin, phloretin, phloridzin, 3- hydroxyphloridzin, sieboldin, p-coumaric acid, caffeic acid, dihydrocaffeic acid, isoliquiritigenin, butein, liquiritigenin, butein, resveratrol, piceatannol, afzelechin, epiafzelechin, catechin, epicatechin, quercetin, kaempferol, eriocitrin, trilobatin, rutin, luteolin, hydrangenol, thunberginol G, flavonols, their glycosides, and mixtures thereof, wherein the mixture is obtained or obtainable by a method according to the invention.

Preferably, the term “methyltransferase of a fungus”, as used herein, refers to a methyltransferase, which is obtained from the fungus or which corresponds to a methyltransferase of the fungus, by sequence identity.

Preferably, the mixture according to the invention comprises one or more methyl donors, as described herein. Particularly preferably, the methyl donor is or comprises S-adenosyl- methionine (SAM).

What was said herein with regard to the method according to the invention applies accordingly to the mixtures according to the invention, where applicable. What was said herein with regard to the mixtures according to the invention applies accordingly to the method according to the invention, where applicable.

Furthermore, the present invention relates to the use of a mixture according to the invention for producing an O-methylated phenolic substance, preferably wherein the O-methylated phenolic substance is selected from the group consisting of Ferulic acid, Dihydroferulic acid, Homoeriodictyol dihydrochalcone, Homobutein, Homoeriodictyol, 2,3-Dihydro-7-hy- droxy-2-(4-hydroxy-3-methoxyphenyl)-4H-1-benzopyran-4-one, Isorhapontigenin, 3'-O- Methyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-hydroxy-3-methoxyphenyl)-1 H-2-ben- zopyran-1-one, Isorhamnetin, 1-[2,6-dihydroxy-4-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan- 2-yl]oxyphenyl]-3-(4-hydroxy-3-methoxyphenyl)propan-1-one, 5-hydroxy-2-(4-hydroxy-3- methoxyphenyl)-7-[3,4,5-trihydroxy-6-[(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyme- thyl]oxan-2-yl]oxy-2,3-dihydrochromen-4-one, Narcissin, p-Methoxycinnamic acid, Isoferulic acid, Dihydroisoferulic acid, Isosakuranetin dihydrochalcone, Hesperetin dihydrochalcone, 2',4'-Dihydroxy-4-methoxychalcone, (2E)-1 -(2,4-Dihydroxyphenyl)-3-(3-hydroxy-4- methoxyphenyl)-2-propen-1-one, Isosakuranetin, Hesperetin, 2,3-Dihydro-7-hydroxy-2-(4- methoxyphenyl)-4H-1-benzopyran-4-one, 2,3-Dihydro-7-hydroxy-2-(3-hydroxy-4-methoxy- phenyl)-4H-1-benzopyran-4-one, Desoxyrhapontigenin, Rhapontigenin, (2R,3R)-3,4-Dihy- dro-2-(4-methoxyphenyl)-2H-1-benzopyran-3,5,7-triol, 4'-O-Methyl-(-)-epicatechin, 3,4-Di- hydro-8-hydroxy-3-(4-methoxyphenyl)-1 H-2-benzopyran-1-one, stilbene derivatives, dihy- drostilbene derivatives, Phyllodulcin, Tamarixetin, Kaempferid, Hesperetin dihydrochal- cone glucoside, Hesperidin, 1-[4-(p-D-glucopyranosyloxy)-2,6-dihydroxyphenyl]-3-(4- methoxyphenyl)-1 -Propanone, 3-[[6-0-(6-deoxy-a-L-mannopyranosyl)-p-D-glucopyra- nosyl]oxy]-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-Benzopyran-4-one, 3', 4'- Dimethoxycinnamic acid, 3-(3,4-Dimethoxyphenyl)-1 -(2,4, 6-trihydroxyphenyl)-1 -propanone, (2E)-1-(2,4-Dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-2-propen-1-one, 2-(3,4- Dimethoxyphenyl)-2,3-dihydro-5,7-dihydroxy-4H-1-benzopyran-4-one, 2-(3,4-Dimethoxy- phenyl)-2,3-dihydro-7-hydroxy-4H-1 -benzopyran-4-one, 5-[(1 E)-2-(3,4-Dimethoxy- phenyl)ethenyl]-1 ,3-benzenediol, (2R,3R)-2-(3,4-Dimethoxyphenyl)-3,4-dihydro-2H-1 -ben- zopyran-3,5,7-triol, Thunberginol H, Dillenetin, Calomelanone, vanillin, isovanillin, vanillic acid, isovanillic acid, homovanillic acid, isohomovanillic acid, vanillomandelic acid, isovanil- lomandelic acid, sinapic acid, eugenol, chavibetol, 4-ethylguaiacol, 5-ethylguaiacol, their glycosides, and mixtures thereof, preferably wherein the O-methylated phenolic substance is selected from the group consisting of Ferulic acid, Dihydroferulic acid, Homoeriodictyol dihydrochalcone, Homobutein, Homoeriodictyol, 2,3-Dihydro-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-1-benzopy- ran-4-one, Isorhapontigenin, 3'-O-Methyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-hy- droxy-3-methoxyphenyl)-1 H-2-benzopyran-1-one, Isorhamnetin, 1-[2,6-dihydroxy-4-[3,4,5- trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]-3-(4-hydroxy-3-methoxyphenyl)propan-

1-one, 5-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-7-[3,4,5-trihydroxy-6-[(3,4,5-trihydroxy- 6-methyloxan-2-yl)oxymethyl]oxan-2-yl]oxy-2,3-dihydrochromen-4-one, Narcissin, p-Meth- oxycinnamic acid, Isoferulic acid, Dihydroisoferulic acid, Isosakuranetin dihydrochalcone, Hesperetin dihydrochalcone, 2',4'-Dihydroxy-4-methoxychalcone, (2E)-1-(2,4-Dihydroxy- phenyl)-3-(3-hydroxy-4-methoxyphenyl)-2-propen-1-one, Isosakuranetin, Hesperetin, 2,3- Dihydro-7-hydroxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one, 2,3-Dihydro-7-hydroxy-

2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, Desoxyrhapontigenin, Rhaponti- genin, (2R,3R)-3,4-Dihydro-2-(4-methoxyphenyl)-2H-1-benzopyran-3,5,7-triol, 4'-O-Me- thyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-methoxyphenyl)-1 H-2-benzopyran-1-one, stilbene derivatives, dihydrostilbene derivatives, Phyllodulcin, Tamarixetin, Kaempferid, Hesperetin dihydrochalcone glucoside, Hesperidin, 1-[4-(p-D-glucopyranosyloxy)-2,6-dihy- droxyphenyl]-3-(4-methoxyphenyl)-1 -Propanone, 3-[[6-0-(6-deoxy-a-L-mannopyranosyl)- p-D-glucopyranosyl]oxy]-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-Benzopy- ran-4-one, 3',4'-Dimethoxycinnamic acid, 3-(3,4-Dimethoxyphenyl)-1-(2,4,6-trihydroxy- phenyl)-1 -propanone, (2E)-1-(2,4-Dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-2-propen-1- one, 2-(3,4-Dimethoxyphenyl)-2,3-dihydro-5,7-dihydroxy-4H-1-benzopyran-4-one, 2-(3,4- Dimethoxyphenyl)-2,3-dihydro-7-hydroxy-4H-1-benzopyran-4-one, 5-[(1 E)-2-(3,4-Di- methoxyphenyl)ethenyl]-1 ,3-benzenediol, (2R,3R)-2-(3,4-Dimethoxyphenyl)-3,4-dihydro- 2H-1-benzopyran-3,5,7-triol, Thunberginol H, Dillenetin, Calomelanone, vanillic acid, isovanillic acid, homovanillic acid, isohomovanillic acid, vanillomandelic acid, isovanillo- mandelic acid, sinapic acid, their glycosides, and mixtures thereof.

Moreover, the present invention relates to the use one or more fungi for producing an O- methylated phenolic substance, wherein the, one, two, three or more fungi is/are selected from a genus selected from the group consisting of Lentinus, Lentinula and Hypsizygus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus, Lentinus cladopus, and Hypsizygus tessulatus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus and Lentinus cladopus, preferably wherein the O-methylated phenolic substance is selected from the group consisting of Ferulic acid, Dihydroferulic acid, Homoeriodictyol dihydrochalcone, Homobutein, Homoeriodictyol, 2,3-Dihydro-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-1-benzopy- ran-4-one, Isorhapontigenin, 3'-O-Methyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-hy- droxy-3-methoxyphenyl)-1 H-2-benzopyran-1-one, Isorhamnetin, 1-[2,6-dihydroxy-4-[3,4,5- trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]-3-(4-hydroxy-3-methoxyphenyl)propan-

2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, Desoxyrhapontigenin, Rhaponti- genin, (2R,3R)-3,4-Dihydro-2-(4-methoxyphenyl)-2H-1-benzopyran-3,5,7-triol, 4'-O-Me- thyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-methoxyphenyl)-1 H-2-benzopyran-1-one, stilbene derivatives, dihydrostilbene derivatives, Phyllodulcin, Tamarixetin, Kaempferid, Hesperetin dihydrochalcone glucoside, Hesperidin, 1-[4-(p-D-glucopyranosyloxy)-2,6-dihy- droxyphenyl]-3-(4-methoxyphenyl)-1 -Propanone, 3-[[6-0-(6-deoxy-a-L-mannopyranosyl)- p-D-glucopyranosyl]oxy]-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-Benzopy- ran-4-one, 3',4'-Dimethoxycinnamic acid, 3-(3,4-Dimethoxyphenyl)-1-(2,4,6-trihydroxy- phenyl)-1 -propanone, (2E)-1-(2,4-Dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-2-propen-1- one, 2-(3,4-Dimethoxyphenyl)-2,3-dihydro-5,7-dihydroxy-4H-1-benzopyran-4-one, 2-(3,4- Dimethoxyphenyl)-2,3-dihydro-7-hydroxy-4H-1-benzopyran-4-one, 5-[(1 E)-2-(3,4-Di- methoxyphenyl)ethenyl]-1 ,3-benzenediol, (2R,3R)-2-(3,4-Dimethoxyphenyl)-3,4-dihydro- 2H-1-benzopyran-3,5,7-triol, Thunberginol H, Dillenetin, Calomelanone, vanillin, isovanillin, vanillic acid, isovanillic acid, homovanillic acid, isohomovanillic acid, vanillomandelic acid, isovanillomandelic acid, sinapic acid, eugenol, chavibetol, 4-ethylguaiacol, 5-ethylguaiacol, their glycosides, and mixtures thereof, preferably wherein the O-methylated phenolic substance is selected from the group consisting of Ferulic acid, Dihydroferulic acid, Homoeriodictyol dihydrochalcone, Homobutein, Homoeriodictyol, 2,3-Dihydro-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-1-benzopy- ran-4-one, Isorhapontigenin, 3'-O-Methyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-hy- droxy-3-methoxyphenyl)-1 H-2-benzopyran-1-one, Isorhamnetin, 1-[2,6-dihydroxy-4-[3,4,5- trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]-3-(4-hydroxy-3-methoxyphenyl)propan-

2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, Desoxyrhapontigenin, Rhaponti- genin, (2R,3R)-3,4-Dihydro-2-(4-methoxyphenyl)-2H-1-benzopyran-3,5,7-triol, 4'-O-Me- thyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-methoxyphenyl)-1 H-2-benzopyran-1-one, stilbene derivatives, dihydrostilbene derivatives, Phyllodulcin, Tamarixetin, Kaempferid, Hesperetin dihydrochalcone glucoside, Hesperidin, 1-[4-(p-D-glucopyranosyloxy)-2,6-dihy- droxyphenyl]-3-(4-methoxyphenyl)-1 -Propanone, 3-[[6-0-(6-deoxy-a-L-mannopyranosyl)- p-D-glucopyranosyl]oxy]-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-Benzopy- ran-4-one, 3',4'-Dimethoxycinnamic acid, 3-(3,4-Dimethoxyphenyl)-1-(2,4,6-trihydroxy- phenyl)-1 -propanone, (2E)-1-(2,4-Dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-2-propen-1- one, 2-(3,4-Dimethoxyphenyl)-2,3-dihydro-5,7-dihydroxy-4H-1-benzopyran-4-one, 2-(3,4- Dimethoxyphenyl)-2,3-dihydro-7-hydroxy-4H-1-benzopyran-4-one, 5-[(1 E)-2-(3,4-Di- methoxyphenyl)ethenyl]-1 ,3-benzenediol, (2R,3R)-2-(3,4-Dimethoxyphenyl)-3,4-dihydro- 2H-1-benzopyran-3,5,7-triol, Thunberginol H, Dillenetin, Calomelanone, vanillic acid, isovanillic acid, homovanillic acid, isohomovanillic acid, vanillomandelic acid, isovanillomandelic acid, sinapic acid, their glycosides, and mixtures thereof.

Further, described herein is the use of an O-methyltransferase for producing an O-methyl- ated phenolic substance, wherein the O-methyltransferase is an O-methyltransferase of a fungus of a genus selected from the group consisting of Lentinus, Lentinula and Hypsizygus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus, Lentinus cladopus, and Hypsizygus tessulatus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus and Lentinus cladopus, preferably wherein the O-methyltransferase comprises or consists of an amino acid sequence according to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or an amino acid sequence having at least 90%, preferably at least 91 %, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, sequence identity to the amino acid sequence according to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, preferably wherein the O-methylated phenolic substance is selected from the group consisting of Ferulic acid, Dihydroferulic acid, Homoeriodictyol dihydrochalcone, Homobutein, Homoeriodictyol, 2,3-Dihydro-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-1-benzopy- ran-4-one, Isorhapontigenin, 3'-O-Methyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-hy- droxy-3-methoxyphenyl)-1 H-2-benzopyran-1-one, Isorhamnetin, 1-[2,6-dihydroxy-4-[3,4,5- trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]-3-(4-hydroxy-3-methoxyphenyl)propan-

2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, Desoxyrhapontigenin, Rhaponti- genin, (2R,3R)-3,4-Dihydro-2-(4-methoxyphenyl)-2H-1-benzopyran-3,5,7-triol, 4'-O-Me- thyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-methoxyphenyl)-1 H-2-benzopyran-1-one, stilbene derivatives, dihydrostilbene derivatives, Phyllodulcin, Tamarixetin, Kaempferid, Hesperetin dihydrochalcone glucoside, Hesperidin, 1-[4-(p-D-glucopyranosyloxy)-2,6-dihy- droxyphenyl]-3-(4-methoxyphenyl)-1 -Propanone, 3-[[6-Q-(6-deoxy-a-L-mannopyranosyl)- p-D-glucopyranosyl]oxy]-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-Benzopy- ran-4-one, 3',4'-Dimethoxycinnamic acid, 3-(3,4-Dimethoxyphenyl)-1-(2,4,6-trihydroxy- phenyl)-1 -propanone, (2E)-1-(2,4-Dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-2-propen-1- one, 2-(3,4-Dimethoxyphenyl)-2,3-dihydro-5,7-dihydroxy-4H-1-benzopyran-4-one, 2-(3,4- Dimethoxyphenyl)-2,3-dihydro-7-hydroxy-4H-1-benzopyran-4-one, 5-[(1 E)-2-(3,4-Di- methoxyphenyl)ethenyl]-1 ,3-benzenediol, (2R,3R)-2-(3,4-Dimethoxyphenyl)-3,4-dihydro- 2H-1-benzopyran-3,5,7-triol, Thunberginol H, Dillenetin, Calomelanone, vanillin, isovanillin, vanillic acid, isovanillic acid, homovanillic acid, isohomovanillic acid, vanillomandelic acid, isovanillomandelic acid, sinapic acid, eugenol, chavibetol, 4-ethylguaiacol, 5-ethylguaiacol, their glycosides, and mixtures thereof, preferably wherein the O-methylated phenolic substance is selected from the group consisting of Ferulic acid, Dihydroferulic acid, Homoeriodictyol dihydrochalcone, Homobutein, Homoeriodictyol, 2,3-Dihydro-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-1-benzopy- ran-4-one, Isorhapontigenin, 3'-O-Methyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-hy- droxy-3-methoxyphenyl)-1 H-2-benzopyran-1-one, Isorhamnetin, 1-[2,6-dihydroxy-4-[3,4,5- trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]-3-(4-hydroxy-3-methoxyphenyl)propan-

2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, Desoxyrhapontigenin, Rhaponti- genin, (2R,3R)-3,4-Dihydro-2-(4-methoxyphenyl)-2H-1-benzopyran-3,5,7-triol, 4'-O-Me- thyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-methoxyphenyl)-1 H-2-benzopyran-1-one, stilbene derivatives, dihydrostilbene derivatives, Phyllodulcin, Tamarixetin, Kaempferid, Hesperetin dihydrochalcone glucoside, Hesperidin, 1-[4-(p-D-glucopyranosyloxy)-2,6-dihy- droxyphenyl]-3-(4-methoxyphenyl)-1 -Propanone, 3-[[6-0-(6-deoxy-a-L-mannopyranosyl)- p-D-glucopyranosyl]oxy]-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-Benzopy- ran-4-one, 3',4'-Dimethoxycinnamic acid, 3-(3,4-Dimethoxyphenyl)-1-(2,4,6-trihydroxy- phenyl)-1 -propanone, (2E)-1-(2,4-Dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-2-propen-1- one, 2-(3,4-Dimethoxyphenyl)-2,3-dihydro-5,7-dihydroxy-4H-1-benzopyran-4-one, 2-(3,4- Dimethoxyphenyl)-2,3-dihydro-7-hydroxy-4H-1-benzopyran-4-one, 5-[(1 E)-2-(3,4-Di- methoxyphenyl)ethenyl]-1 ,3-benzenediol, (2R,3R)-2-(3,4-Dimethoxyphenyl)-3,4-dihydro- 2H-1-benzopyran-3,5,7-triol, Thunberginol H, Dillenetin, Calomelanone, vanillic acid, isovanillic acid, homovanillic acid, isohomovanillic acid, vanillomandelic acid, isovanillomandelic acid, sinapic acid, their glycosides, and mixtures thereof. What was said herein with regard to the method and/or mixture according to the invention applies accordingly to the uses according to the invention, where applicable. What was said herein with regard to the uses according to the invention applies accordingly to the method and/or mixture according to the invention, where applicable.

It is preferred for the method, mixture and/or use according to the invention that the, one, two or all fungi of the genus Lentinus is/are selected from Lentinus squarrosulus and Len- tinus cladopus.

It is preferred for the method, mixture and/or use according to the invention that the, one, two or all fungi of the genus Lentinula is/are selected from Lentinula edodes and Lentinula latent! a.

It is preferred for the method, mixture and/or use according to the invention that the or one fungus of the genus Hypsizygus is Hypsizygus tessulatus.

Short description of the sequences:

SEQ ID NO:1 Amino acid sequence encoding an O-methyltransferase from Lentinula edodes,

SEQ ID NO:2 Amino acid sequence encoding an O-methyltransferase from Lentinula edodes, NCBI accession number: GAW04926.1

SEQ ID NO:3 Amino acid sequence encoding an O-methyltransferase from Lentinula edodes, NCBI accession number: XP_046082518.1

SEQ ID NO:4 Amino acid sequence encoding an O-methyltransferase from Lentinula edodes, NCBI accession number: KAF8830229.1

SEQ ID NO:5 Amino acid sequence encoding an O-methyltransferase from Lentinula edodes, NCBI accession number: XP_046081978.1

SEQ ID NO:6 Amino acid sequence encoding an O-methyltransferase from Lentinula edodes, NCBI accession number: KAF8827526.1 SEQ ID NO:7 Amino acid sequence encoding an O-methyltransferase from Lentinula edodes, NCBI accession number: GAW00128

SEQ ID NO:8 Amino acid sequence encoding an O-methyltransferase from Lentinula edodes, NCBI accession number: XP_046089920.1 Further aspects and advantages of the invention result from the subsequent description of preferred examples.

Examples

Example 1 : Selection of fungi for producing an O-methylated phenolic substance from a phenolic substance

For some genera of fungi it was assumed or reported that these would inherit a gene encoding an O-methyltransferase. 11 fungi representing such genera were provided. The fungi were Pleurotus sapidus (Dikaryot) (PSA- 5), Pleurotus sapidus (Monokaryot) (PSA- MK57), Pleurotus floridanus, Flammulina velutipes, Phanerochaete chrysoporium, Spar- assis crispa, Clitocybe lignatilis, Lentinus squarrosulus, Lentinus cladopus, and Hypsizygus tessulatus and Lentinula edodes.

Each of the fungi was cultured on malt extract agar until mycelium covered approximately 70% of the agar.

After cultivation on agar, cultures were collected and further cultured by submerged cultivation in malt extract peptone medium for 72 h (Pleurotus sapidus (Dikaryot), Pleurotus sapidus (Monokaryot), Lentinus squarrosulus, Lentinus cladopus) or 48 h (Pleurotus floridanus, Flammulina velutipes, Phanerochaete chrysoporium) or 96 h (Hypsizygus tessulatus, Lentinula edodes) or 144 h (Sparassis crispa, Clitocybe lignatilis).

The obtained cultures were then centrifuged and the obtained biomass was lyophilized.

In a next step, a reaction mixture was obtained by mixing the following components:

The phosphate buffer was provided and the respective fungus biomass was admixed. Sub- seguently, the substrate and SAM were admixed to obtain the reaction mixture. The reaction mixture was reduced to 1 mL (10 mg fungus biomass in 1 mL buffer), wherein the concentration of the components was maintained. Substrates (i.e. phenolic substances) were selected from eriodictyol, naringenin, 3-hydrox- yphloretin, phloretin, sieboldin, protocatechu ic acid, protocatechu ic aldehyde, hydroxychavicol, and 4-ethylcatechol.

The different substrates were tested for the different fungi.

The reaction mixture was maintained at 24 °C for 20 h. Subsequently, it was tested by liquid-liquid extraction, RP-HPLC-DAD and LC-MS/MS whether the respective O-methyl- ated phenolic substances could be detected in the mixtures. In case the respective O- methylated phenolic substance could be detected at least for one substrate, it was assumed that the respective fungus was able to provide an O-methyltransferase and to produce an O-methylated phenolic substance from a phenolic substance.

The O-methylated phenolic substance of eriodictyol includes homoeriodictyol or hes- peretin. With regard to the reaction provided by a 4’-O-methyltransferase, the O-methylated phenolic substance of eriodictyol is hesperetin.

The O-methylated phenolic substance of naringenin is isosakuranetin.

The O-methylated phenolic substance of 3-hydroxyphloretin includes homoeriodictyol di- hydrochalcone or hesperetin dihydrochalcone. With regard to the reaction provided by a 4’- O-methyltransferase, the O-methylated phenolic substance of 3-hydroxyphloretin is hesperetin dihydrochalcone.

The O-methylated phenolic substance of phloretin includes isosakuranetin dihydrochalcone.

The O-methylated phenolic substance of sieboldin was homoeriodictyol dihydrochalcone and hesperetin dihydrochalcone, since the glucosyl group of sieboldin was removed. With regard to the reaction provided by a 4’-O-methyltransferase, the O-methylated phenolic substance of sieboldin was hesperetin dihydrochalcone (since the glucosyl group of sieboldin was removed).

The O-methylated phenolic substance of protocatechu ic acid includes vanillic acid and isovanillic acid. The O-methylated phenolic substance of protocatechu ic aldehyde includes vanillin and isovanillin.

The O-methylated phenolic substance of hydroxychavicol includes eugenol and chavibetol.

The O-methylated phenolic substance of 4-ethylcatechol includes 4-ethylguaiacol and 5- ethylguaiacol.

Although for the tested fungi it was assumed or reported that these would inherit a gene encoding an O-methyltransferase, it was surprisingly found that only Lentinus squarrosu- lus, Lentinus cladopus, and Hypsizygus tessulatus and Lentinula edodes were able to provide the O-methylated phenolic substance for at least one of the substrates.

For example, in the sample containing eriodictyol as substrate, the reaction mixture including Lentinula edodes also contained hesperetin after the reaction. Similarly, in the sample containing 3-hydroxyphloretin as substrate, the reaction mixture including Lentinula edodes also contained homoeriodictyol dihydrochalcone and hesperetin dihydrochalcone after the reaction.

Likewise, in the sample containing sieboldin as substrate, the reaction mixture including Lentinula edodes also contained hesperetin dihydrochalcone after the reaction.

Likewise, in the sample containing protocatechuic acid as substrate, the reaction mixture including Lentinula edodes also contained vanillic acid and isovanillic acid after the reaction.

Likewise, in the sample containing protocatechuic aldehyde as substrate, the reaction mixture including Lentinula edodes also contained vanillin and isovanillin after the reaction.

Likewise, in the sample containing hydroxychavicol as substrate, the reaction mixture including Lentinula edodes also contained chavibetol after the reaction.

Likewise, in the sample containing 4-ethylcatechol as substrate, the reaction mixture including Lentinula edodes also contained 4-ethylguaiacol and 5-ethylguaiacol after the reaction. However, Pleurotus sapidus (Dikaryot) (PSA- 5), Pleurotus sapidus (Monoka ryot) (PSA- MK57), Pleurotus floridanus, Flammulina velutipes, Phanerochaete chrysoporium, Spar- assis crispa and Clitocybe lignatilis surprisingly did not provide the O-methylated phenolic substance for any of the substrates.

Example 2: Selection of fungi for producing an O-methylated phenolic substance from a phenolic substance

Lyophilized biomass of Lentinula edodes was provided according to Example 1 .

3 mL of PO4³' buffer (50 mM, pH=7.5) including 1 mM DTT and 1 mM EDTA were provided for each approach.

In each approach, 20 mg lyophilized biomass per ml of the PO4^3- buffer were added.

Subseguently, the mixture was vigorously vortexed to extract possible O-methyltransfer- ase(s). The supernatant was collected.

The extract supernatant was further filtered with centrifugation to provide a retentate and a permeate.

The supernatant, the retentate and the permeate were each mixed with 200 pM substrate and 400 pM SAM. The obtained reaction mixture was maintained at 30 °C for 15 h or overnight. Subseguently, it was tested by liguid-liguid extraction, RP-HPLC-DAD and LC- MS/MS whether the respective O-methylated phenolic substances could be detected in the mixtures.

In the first approach, the substrate was eriodictyol. A positive signal for hesperetin could be detected in all samples.

In the second approach, the substrate was 3-hydroxyphloretin. A positive signal for hesperetin dihydrochalcone could be detected in all samples except for the permeate sample. A positive signal for homoeriodictyol dihydrochalcone could be detected in the supernatant sample.

In the third approach, the substrate was sieboldin. A positive signal for hesperetin dihydrochalcone and homoeriodictyol dihydrochalcone could be detected. In the fourth approach, the substrate was protocatechuic acid. A positive signal for vanillic acid and isovanillic acid could be detected.

In the fifth approach, the substrate was protocatechuic aldehyde. A positive signal for vanillin and isovanillin could be detected.

In the sixth approach, the substrate was hydroxychavicol. A positive signal for eugenol and chavibetol could be detected.

In the seventh approach, the substrate was 4-ethylcatechol. A positive signal for 4-ethyl- guaiacol and 5-ethylguaiacol could be detected.

Example 3: Influence of pH and reaction time

Lyophilized biomass of Lentinula edodes and a reaction mixture was provided, each according to Example 1 . The reaction was performed according to Example 1 .

3-hydroxyphloretin was used as substrate.

Different reaction times were applied and compared with each other. It was found that all tested reaction times (72 h to 120 h) provided a biotransformation of 3-hydroxyphloretin to homoeriodictyol dihydrochalcone and hesperetin dihydrochalcone, wherein the amount of hesperetin dihydrochalcone increased with time.

Likewise, different pH values were applied and compared with each other. It was found that all tested pH values (7.0 to 9.0) provided a biotransformation of 3-hydroxyphloretin to homoeriodictyol dihydrochalcone and hesperetin dihydrochalcone, wherein the amount of homoeriodictyol dihydrochalcone and hesperetin dihydrochalcone was particularly high at a pH value of 7.5 and 8.0.

Example 4: Cell free extract

Lentinula edodes biomass was provided according to Example 1 . In a subsequent step, a cell free extract was obtained by centrifugation.

A reaction mixture was provided according to Example 1 , wherein the fungus biomass was replaced by the supernatant as the cell free extract. 3-hydroxyphloretin was used as substrate.

It was found that the reaction with the cell free extract also provided a biotransformation of 3-hydroxyphloretin to homoeriodictyol dihydrochalcone and hesperetin dihydrochalcone. Furthermore, the cell free extract was found to be particularly advantageous, since partic- ularly high amounts of homoeriodictyol dihydrochalcone and hesperetin dihydrochalcone were obtained.

Example 5: Lentinula lateritia

The approach described in Example 4 was repeated with the fungus Lentinula lateritia.

Eriodictyol and 3-hydroxyphloretin were used as substrates. It was found that the reaction with the cell free extract also provided a biotransformation of eriodictyol to homoeriodictyol (2.3 ± 0.4 pM) and hesperetin (3.8 ± 0.6 pM).

It was further found that the reaction with the cell free extract also provided a biotransformation of 3-hydroxyphloretin to homoeriodictyol dihydrochalcone (4.7 ± 0.8 pM) and hesperetin dihydrochalcone (0.8 ± 0.1 pM).

Claims

1 . Method for producing an O-methylated phenolic substance from the corresponding phenolic substance, the method comprising the steps i) providing one or more phenolic substance(s), ii) providing one or more fungi, wherein the, one, two, three or more fungi is/are of a genus selected from the group consisting of Lentinus, Lentinula and Hyp- sizygus, iii) optionally: providing one or more methyl donors, iv) mixing the one or more phenolic substance(s) provided in step i), the one or more fungi provided in step ii) and, if present, the one or more methyl donor provided in step iii) to obtain a reaction mixture, and reacting the reaction mixture under conditions allowing the O-methylation of the phenolic substance(s).

2. Method according to claim 1 , wherein a methyl donor is provided in step iii), wherein the methyl donor is or comprises S-adenosylmethionine (SAM).

3. Method according to claims 1 or 2, wherein in the mixture obtained in step iv), the ratio of the amount of substance of the phenolic substance(s) and the amount of substance of the methyl donor(s) is in a range of from 10:1 to 1 :10, preferably in a range of from 5:1 to 1 :7.5, preferably in a range of from 2.5 to 1 to 1 :5, preferably in a range of from 1 :1 to 1 :2.5.

4. Method according to any one of the preceding claims, wherein the, one, two, three or more or all phenolic substance(s) provided in step i) is/are selected from the group consisting of benzoic acids, phenylacetic acids, mandelic acids, cinnamic acids, dihydrocinnamic acids, chaicones, dihydrochalcones, flavanes, catechins, flavanones, flavones, 3-hydroxyflavones, anthocyanes, stilbenes, dihydrostilbenes, dihydroisocoumarins, isocoumarins, phenylpropanoids, flavanols, their glycosides, particularly O-glycosides, and mixtures thereof, preferably selected from the group consisting of flavanones, chaicones, dihydrochal- cones, phenylpropanoids, flavanols, dihydroisocoumarines, stilbenes, their glycosides, and mixtures thereof.

5. Method according to any one of the preceding claims, wherein the, one, two, three or more or all phenolic substance(s) provided in step i) is/are selected from the group consisting of naringenin, eriodictyol, homoeriodictyol, 3-hydroxyphloretin, phloretin, phloridzin, 3-hydroxyphloridzin, sieboldin, p-coumaric acid, caffeic acid, dihydrocaffeic acid, isoliquiritigenin, butein, liquiritigenin, resveratrol, piceatannol, afzelechin, epiafzelechin, catechin, epicatechin, quercetin, kaempferol, eriocitrin, trilobatin, rutin, luteolin, hydrangenol, thunberginol G, flavonols, protocatechu ic acid, protocate- chuic aldehyde, hydroxychavicol, 4-ethylcatechol, and their glycosides, preferably wherein the, one, two, three or more or all phenolic substance(s) provided in step i) is/are selected from the group consisting of naringenin, eriodictyol, homoeriodictyol, 3-hydroxyphloretin, phloretin, phloridzin, 3-hydroxyphloridzin, sieboldin, p- coumaric acid, caffeic acid, dihydrocaffeic acid, isoliquiritigenin, butein, liquiritigenin, resveratrol, piceatannol, afzelechin, epiafzelechin, catechin, epicatechin, quercetin, kaempferol, eriocitrin, trilobatin, rutin, luteolin, hydrangenol, thunberginol G, flavonols, and their glycosides.

6. Method according to any one of the preceding claims, wherein the, one, two, three or more or all fungi provided in step ii) is/are selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus, Lentinus cladopus, and Hypsizygus tessulatus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus and Lentinus cladopus.

7. Method according to any one of the preceding claims, wherein the conditions allowing the O-methylation of the phenolic substance(s) in step iv) include a pH in a range of from 6.5 to 9.5, preferably in a range of from 6.75 to 9, preferably in a range of from 7 to 8.5.

8. Method according to any one of the preceding claims, wherein the conditions allowing the O-methylation of the phenolic substance(s) in step iv) include a reaction time in the range of from 45 to 180 h, preferably in the range of from 60 to 150 h, preferably wherein the reaction is stopped by acidification, preferably by acidification with 4 M HCI.

9. Mixture comprising a) a fungus of a genus selected from the group consisting of Lentinus, Lentinula and Hypsizygus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus, Lentinus cladopus, and Hypsizygus tessula- tus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus and Lentinus cladopus, wherein the fungus expresses an O-methyltransferase, preferably an O-methyltransferase comprising or consisting of an amino acid sequence according to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or an amino acid sequence having at least 90%, preferably at least 91 %, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, sequence identity to the amino acid sequence according to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, and b) one or more compound(s) selected from the group consisting of naringenin, eriodictyol, homoeriodictyol, 3-hydroxyphloretin, phloretin, phloridzin, 3-hy- droxyphloridzin, sieboldin, p-coumaric acid, caffeic acid, dihydrocaffeic acid, isoliquiritigenin, butein, liquiritigenin, resveratrol, piceatannol, afzelechin, epiafzelechin, catechin, epicatechin, quercetin, kaempferol, eriocitrin, trilo- batin, rutin, luteolin, hydrangenol, thunberginol G, flavonols, protocatechu ic acid, protocatechu ic aldehyde, hydroxychavicol, 4-ethylcatechol, their glycosides, and mixtures thereof, preferably one or more compound(s) selected from the group consisting of naringenin, eriodictyol, homoeriodictyol, 3-hydroxyphloretin, phloretin, phlo- ridzin, 3-hydroxyphloridzin, sieboldin, p-coumaric acid, caffeic acid, dihydrocaffeic acid, isoliquiritigenin, butein, liquiritigenin, resveratrol, piceatannol, afzelechin, epiafzelechin, catechin, epicatechin, quercetin, kaempferol, eriocitrin, trilobatin, rutin, luteolin, hydrangenol, thunberginol G, flavonols, their glycosides, and mixtures thereof.

10. Mixture, preferably a mixture according to claim 9, comprising a) an O-methyltransferase of a fungus of a genus selected from the group consisting of Lentinus, Lentinula and Hypsizygus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus, Lentinus cladopus, and Hypsizygus tessula- tus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus and Lentinus cladopus, preferably an O-methyltransferase comprising or consisting of an amino acid sequence according to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, or an amino acid sequence having at least 90%, preferably at least 91 %, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, sequence identity to the amino acid sequence according to SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8, and b) one or more compound(s) selected from the group consisting of naringenin, eriodictyol, homoeriodictyol, 3-hydroxyphloretin, phloretin, phloridzin, 3-hy- droxyphloridzin, sieboldin, p-coumaric acid, caffeic acid, dihydrocaffeic acid, isoliquiritigenin, butein, liquiritigenin, resveratrol, piceatannol, afzelechin, epiafzelechin, catechin, epicatechin, quercetin, kaempferol, eriocitrin, trilobatin, rutin, luteolin, hydrangenol, thunberginol G, flavonols, protocatechu ic acid, protocatechu ic aldehyde, hydroxychavicol, 4-ethylcatechol, their glycosides, and mixtures thereof, preferably one or more compound(s) selected from the group consisting of naringenin, eriodictyol, homoeriodictyol, 3-hydroxyphloretin, phloretin, phloridzin, 3-hydroxyphloridzin, sieboldin, p-coumaric acid, caffeic acid, dihydrocaffeic acid, isoliquiritigenin, butein, liquiritigenin, resveratrol, piceatannol, afzelechin, epiafzelechin, catechin, epicatechin, quercetin, kaempferol, eriocitrin, trilobatin, rutin, luteolin, hydrangenol, thunberginol G, flavonols, their glycosides, and mixtures thereof, wherein the mixture is obtained or obtainable by a method according to any one of claims 1 to 8.

11 . Use of a mixture according to claim 9 or 10 for producing an O-methylated phenolic substance, preferably wherein the O-methylated phenolic substance is selected from the group consisting of Ferulic acid, Dihydroferulic acid, Homoeriodictyol dihydro- chalcone, Homobutein, Homoeriodictyol, 2,3-Dihydro-7-hydroxy-2-(4-hydroxy-3- methoxyphenyl)-4H-1-benzopyran-4-one, Isorhapontigenin, 3'-O-Methyl-(-)-epicate- chin, 3,4-Dihydro-8-hydroxy-3-(4-hydroxy-3-methoxyphenyl)-1 H-2-benzopyran-1- one, Isorhamnetin, 1-[2,6-dihydroxy-4-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2- yl]oxyphenyl]-3-(4-hydroxy-3-methoxyphenyl)propan-1-one, 5-hydroxy-2-(4-hy- droxy-3-methoxyphenyl)-7-[3,4,5-trihydroxy-6-[(3,4,5-trihydroxy-6-methyloxan-2- yl)oxymethyl]oxan-2-yl]oxy-2,3-dihydrochromen-4-one, Narcissin, p-Methox- ycinnamic acid, Isoferulic acid, Dihydroisoferulic acid, Isosakuranetin dihydrochal- cone, Hesperetin dihydrochalcone, 2',4'-Dihydroxy-4-methoxychalcone, (2E)-1-(2,4- Dihydroxyphenyl)-3-(3-hydroxy-4-methoxyphenyl)-2-propen-1-one, Isosakuranetin, Hesperetin, 2,3-Dihydro-7-hydroxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one, 2,3-Dihydro-7-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, Desoxyrhapontigenin, Rhapontigenin, (2R,3R)-3,4-Dihydro-2-(4-methoxyphenyl)- 2H-1-benzopyran-3,5,7-triol, 4'-O-Methyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3- (4-methoxyphenyl)-1 H-2-benzopyran-1-one, stilbene derivatives, dihydrostilbene derivatives, Phyllodulcin, Tamarixetin, Kaempferid, Hesperetin dihydrochalcone glucoside, Hesperidin, 1-[4-(p-D-glucopyranosyloxy)-2,6-dihydroxyphenyl]-3-(4-meth- oxyphenyl)-1 -Propanone, 3-[[6-0-(6-deoxy-a-L-mannopyranosyl)-p-D-glucopyra- nosyl]oxy]-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-Benzopyran-4-one, 3',4'-Dimethoxycinnamic acid, 3-(3,4-Dimethoxyphenyl)-1-(2,4,6-trihydroxyphenyl)-

1 -propanone, (2E)-1-(2,4-Dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-2-propen-1- one, 2-(3,4-Dimethoxyphenyl)-2,3-dihydro-5,7-dihydroxy-4H-1-benzopyran-4-one,

2-(3,4-Dimethoxyphenyl)-2,3-dihydro-7-hydroxy-4H-1-benzopyran-4-one, 5-[(1 E)-2- (3,4-Dimethoxyphenyl)ethenyl]-1 ,3-benzenediol, (2R,3R)-2-(3,4-Dimethoxyphenyl)- 3,4-dihydro-2H-1-benzopyran-3,5,7-triol, Thunberginol H, Dillenetin, Calomelanone, vanillin, isovanillin, vanillic acid, isovanillic acid, homovanillic acid, isohomovanillic acid, vanillomandelic acid, isovanillomandelic acid, sinapic acid, eugenol, chavibetol, 4-ethylguaiacol, 5-ethylguaiacol, their glycosides, and mixtures thereof, preferably wherein the O-methylated phenolic substance is selected from the group consisting of Ferulic acid, Dihydroferulic acid, Homoeriodictyol dihydrochalcone, Ho- mobutein, Homoeriodictyol, 2,3-Dihydro-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)- 4H-1-benzopyran-4-one, Isorhapontigenin, 3'-O-Methyl-(-)-epicatechin, 3,4-Dihydro- 8-hydroxy-3-(4-hydroxy-3-methoxyphenyl)-1 H-2-benzopyran-1-one, Isorhamnetin,

1-[2,6-dihydroxy-4-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyphenyl]-3-(4- hydroxy-3-methoxyphenyl)propan-1-one, 5-hydroxy-2-(4-hydroxy-3-methoxy- phenyl)-7-[3,4,5-trihydroxy-6-[(3,4,5-trihydroxy-6-methyloxan-2-yl)oxymethyl]oxan-

2-yl]oxy-2,3-dihydrochromen-4-one, Narcissin, p-Methoxycinnamic acid, Isoferulic acid, Dihydroisoferulic acid, Isosakuranetin dihydrochalcone, Hesperetin dihydrochalcone, 2',4'-Dihydroxy-4-methoxychalcone, (2E)-1-(2,4-Dihydroxyphenyl)-3-(3- hydroxy-4-methoxyphenyl)-2-propen-1-one, Isosakuranetin, Hesperetin, 2,3-Dihy- dro-7-hydroxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one, 2,3-Dihydro-7-hy- droxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, Desoxyrhaponti- genin, Rhapontigenin, (2R,3R)-3,4-Dihydro-2-(4-methoxyphenyl)-2H-1-benzopyran- 3,5,7-triol, 4'-O-Methyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-methoxyphenyl)- 1 H-2-benzopyran-1-one, stilbene derivatives, dihydrostilbene derivatives, Phyllodul- cin, Tamarixetin, Kaempferid, Hesperetin dihydrochalcone glucoside, Hesperidin, 1- [4-(p-D-glucopyranosyloxy)-2,6-dihydroxyphenyl]-3-(4-methoxyphenyl)-1 -Propanone, 3-[[6-0-(6-deoxy-a-L-mannopyranosyl)-p-D-glucopyranosyl]oxy]-5,7-dihy- droxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-Benzopyran-4-one, 3',4'-Dimethox- ycinnamic acid, 3-(3,4-Dimethoxyphenyl)-1 -(2, 4, 6-trihydroxyphenyl)-1 -propanone, (2E)-1-(2,4-Dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-2-propen-1-one, 2-(3,4-Di- methoxyphenyl)-2,3-dihydro-5,7-dihydroxy-4H-1-benzopyran-4-one, 2-(3,4-Di- methoxyphenyl)-2,3-dihydro-7-hydroxy-4H-1-benzopyran-4-one, 5-[(1 E)-2-(3,4-Di- methoxyphenyl)ethenyl]-1 ,3-benzenediol, (2R,3R)-2-(3,4-Dimethoxyphenyl)-3,4-di- hydro-2H-1-benzopyran-3,5,7-triol, Thunberginol H, Dillenetin, Calomelanone, vanillic acid, isovanillic acid, homovanillic acid, isohomovanillic acid, vanillomandelic acid, isovanillomandelic acid, sinapic acid, their glycosides, and mixtures thereof.

2. Use of one or more fungi for producing an O-methylated phenolic substance, wherein the, one, two, three or more fungi is/are selected from a genus selected from the group consisting of Lentinus, Lentinula and Hypsizygus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus, Lentinus cladopus, and Hypsizygus tessulatus, preferably selected from the group consisting of Lentinula edodes, Lentinula lateritia, Lentinus squarrosulus and Lentinus cladopus, preferably wherein the O-methylated phenolic substance is selected from the group consisting of Ferulic acid, Dihydroferulic acid, Homoeriodictyol dihydrochalcone, Ho- mobutein, Homoeriodictyol, 2,3-Dihydro-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)- 4H-1-benzopyran-4-one, Isorhapontigenin, 3'-O-Methyl-(-)-epicatechin, 3,4-Dihydro- 8-hydroxy-3-(4-hydroxy-3-methoxyphenyl)-1 H-2-benzopyran-1-one, Isorhamnetin,

2-yl]oxy-2,3-dihydrochromen-4-one, Narcissin, p-Methoxycinnamic acid, Isoferulic acid, Dihydroisoferulic acid, Isosakuranetin dihydrochalcone, Hesperetin dihydrochalcone, 2',4'-Dihydroxy-4-methoxychalcone, (2E)-1-(2,4-Dihydroxyphenyl)-3-(3- hydroxy-4-methoxyphenyl)-2-propen-1-one, Isosakuranetin, Hesperetin, 2,3-Dihy- dro-7-hydroxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one, 2,3-Dihydro-7-hy- droxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one, Desoxyrhaponti- genin, Rhapontigenin, (2R,3R)-3,4-Dihydro-2-(4-methoxyphenyl)-2H-1-benzopyran- 3,5,7-triol, 4'-O-Methyl-(-)-epicatechin, 3,4-Dihydro-8-hydroxy-3-(4-methoxyphenyl)- 1 H-2-benzopyran-1-one, stilbene derivatives, dihydrostilbene derivatives, Phyllodul- cin, Tamarixetin, Kaempferid, Hesperetin dihydrochalcone glucoside, Hesperidin, 1- [4-(p-D-glucopyranosyloxy)-2,6-dihydroxyphenyl]-3-(4-methoxyphenyl)-1 -Propanone, 3-[[6-0-(6-deoxy-a-L-mannopyranosyl)-p-D-glucopyranosyl]oxy]-5,7-dihy- droxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-Benzopyran-4-one, 3',4'-Dimethox- ycinnamic acid, 3-(3,4-Dimethoxyphenyl)-1 -(2, 4, 6-trihydroxyphenyl)-1 -propanone, (2E)-1-(2,4-Dihydroxyphenyl)-3-(3,4-dimethoxyphenyl)-2-propen-1-one, 2-(3,4-Di- methoxyphenyl)-2,3-dihydro-5,7-dihydroxy-4H-1-benzopyran-4-one, 2-(3,4-Di- methoxyphenyl)-2,3-dihydro-7-hydroxy-4H-1-benzopyran-4-one, 5-[(1 E)-2-(3,4-Di- methoxyphenyl)ethenyl]-1 ,3-benzenediol, (2R,3R)-2-(3,4-Dimethoxyphenyl)-3,4-di- hydro-2H-1-benzopyran-3,5,7-triol, Thunberginol H, Dillenetin, Calomelanone, vanillin, isovanillin, vanillic acid, isovanillic acid, homovanillic acid, isohomovanillic acid, vanillomandelic acid, isovanillomandelic acid, sinapic acid, eugenol, chavibetol, 4- ethylguaiacol, 5-ethylguaiacol, their glycosides, and mixtures thereof, preferably wherein the O-methylated phenolic substance is selected from the group consisting of Ferulic acid, Dihydroferulic acid, Homoeriodictyol dihydrochalcone, Ho- mobutein, Homoeriodictyol, 2,3-Dihydro-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)- 4H-1-benzopyran-4-one, Isorhapontigenin, 3'-O-Methyl-(-)-epicatechin, 3,4-Dihydro- 8-hydroxy-3-(4-hydroxy-3-methoxyphenyl)-1 H-2-benzopyran-1-one, Isorhamnetin,