Method for producing matairesinol using enzymes

Abstract

The present invention specifically relates to an enzyme combination comprising a glucosidase enzyme, a secoisolariciresinol dehydrogenase enzyme and a glucose dehydrogenase enzyme, which are capable of carrying out reactions to convert secoisolariciresinol diglucoside (SDG) into matairesinol with a high yield. The invention also relates to the uses of the above combination, and to methods for producing matairesinol.

Description

[0001] METHOD FOR THE PRODUCTION OF MATAIRESINOL USING ENZYMES

[0002] DESCRIPTION

[0003] The present invention relates to a method for producing matairesinol from lignans using enzymes. Therefore, the present invention falls within the field of biotechnology.

[0004] BACKGROUND OF THE INVENTION

[0005] Secoisolariciresinol diglucoside and matairesinol are lignans found in a variety of foods, including oilseeds, whole grains, vegetables, and fruits. These lignans are of interest due to their beneficial health effects, such as their potential anticancer, antiproliferative, and antioxidant properties, which are linked to the prevention of cardiometabolic, neurodegenerative, and cancerous diseases. While matairesinol is found in small concentrations and in non-bioavailable forms in some foods, it can be produced by the dehydrogenation of secoisolariciresinol (SECO), abundant in foods like flaxseed, in its glycosylated form (SDG). This conversion is mediated by the gut microbiota (Niemeyer, HB, et al. (2003). Journal of agricultural and food chemistry, 51(21), 6317-6325). However, this conversion is inefficient.

[0006] Since matairesinol is found in low concentrations in food and is produced inefficiently by the gut microbiota, food-derived matairesinol is unlikely to have a beneficial effect on people who consume these foods. In light of this, prior art documents exist that describe methods for the production of matairesinol.

[0007] Document W003057209A1 describes a method for the production of matairesinol from hydroxymatairesinol. The method described in this document is a chemical synthesis method involving the use of compounds such as alcohols, ammonium formate, cyclohexene, borohydrides, or silanes. However, because the methodology uses chemical compounds, it cannot be directly applied to food and is also costly.

[0008] In the document Xia, ZQ, et al. (2001). Journal of Biological Chemistry, 276( 6), 12614-12623, the production of matairesinol from secoisolariciresinol mediated by an enzyme, secoisolariciresinol dehydrogenase, is described.

[0009] However, there is a need in the state of the art to provide new efficient methodologies for the production of matairesinol from other starting products such as secoisolariciresinol diglucoside.

[0010] DESCRIPTION OF THE INVENTION

[0011] The present invention provides a combination of enzymes that overcomes the problems mentioned above. Specifically, the present invention relates to an enzyme combination comprising a glucosidase enzyme, a secoisolariciresinol dehydrogenase enzyme, and a glucose dehydrogenase enzyme, which are capable of carrying out the reactions that lead to the conversion of secoisolariciresinol diglucoside (SDG) to matairesinol in high yield. Specifically, the present inventors have demonstrated for the first time the secoisolariciresinol dehydrogenase activity of the enzyme SEQ ID NO: 2 from Lactonifactor longoviformis DSM17459T, and furthermore, its combination with a glucosidase enzyme and a glucose dehydrogenase enzyme allows the production of matairesinol from SDG. Remarkably, the enzyme combination of the invention results in a conversion of SDG to matairesinol of over 70%.Furthermore, the enzyme combination of the present invention does not require the use of chemical compounds to produce matairesinol, which also allows for its safe use in food.

[0012] Thus, in one respect, the present invention relates to a combination, hereinafter referred to as the “combination of the invention”, comprising:

[0013] - a glucosidase enzyme or a fragment thereof that maintains the function / activity of the complete enzyme,

[0014] - a secoisolariciresinol dehydrogenase enzyme comprising an amino acid sequence with at least 80% identity to the sequence SEQ ID NO: 2 that maintains the same function / activity as the enzyme of SEQ ID NO: 2 or a fragment thereof that maintains the function / activity of the complete enzyme, and

[0015] - a glucose dehydrogenase enzyme or a fragment thereof that maintains the function / activity of the complete enzyme, and where the glucosidase enzyme is a p-glucosidase.

[0016] From now on, glucosidase, secoisolariciresinol dehydrogenase and glucose dehydrogenase may also be referred to as “the glucosidase of the invention”, “the secoisolariciresinol dehydrogenase of the invention” and “the glucose dehydrogenase of the invention”, respectively.

[0017] As the expert in the field knows, the term “enzyme” refers to a protein or polypeptide that is capable of specifically catalyzing the conversion of a substrate into a product.

[0018] The present invention describes a combination of enzymes that catalyze a series of reactions that allow the conversion of secoisolariciresinol diglucoside (SDG) to matairesinol.

[0019] The first reaction that is part of the conversion of SDG to matairesinol is catalyzed by a glucosidase.

[0020] In the present invention, the term “glucosidase” refers to an enzyme that catalyzes the hydrolysis of O-glycosidic or S-glycosidic compounds, specifically catalyzing the hydrolysis reaction of the O- or S-glycosidic bond between two sugars or between a sugar and a non-sugar compound. Glucosidase enzymes are classified according to their classification number as EC. 3.2.1.

[0021] In the combination of the invention, the glucosidase enzyme is a p-glucosidase enzyme.

[0022] As used in the present invention, the term “P-glucosidase” refers to a glucosidase enzyme that catalyzes the hydrolysis of pi->4 linkages joining two glucose molecules or molecules with glucose substitutions. The classification number of the p-glucosidase enzyme is EC. 3.2.1. 21.

[0023] Thus, the glucosidase of the invention has p-glucosidase activity and catalyzes the hydrolysis reaction of the bonds that link the two glucose molecules to secoisolariciresinol in the SDG compound.

[0024] The CAS number for the compound secoisolariciresinol diglucoside (SDG) is CAS No.: 158932-33-3. The CAS number for the compound secoisolariciresinol (SECO) is CAS No.: 29388-59-8.

[0025] The reaction catalyzed by the glucosidase of the invention is described below:

[0026] Secoisolariciresinol diglucoside -> Secoisolariciresinol + 2 glucose

[0027] In another preferred embodiment of the combination of the invention, the glucosidase enzyme comprises an amino acid sequence with at least 80% identity to the sequence SEQ ID NO: 1. The amino acid sequence with at least 80% identity to the sequence SEQ ID NO: 1 maintains the same function / activity as the enzyme of SEQ ID NO: 1.

[0028] Amino acid sequence of Lactobacillus mucosae glucosidase enzyme INIA 425L, SEQ ID NO: 1 :

[0029] MTKVDLNFVEGLTLEERADLVSGTDFWFTAKVSGMDPMLMTDGPSGLRKQVNVHSA MDQSIEAVCFPCSALTASSFDDQMLEKLGEQLGTAARAEKIGVLLGPPGVNIKRSPLAG RNFEYFSEDPLAGRMGTAYVKGVQSTGVGVSVKHFATNSQTNSDRVDREVDE LREIYLAQFERIVKQAHPATIMCSYNKLNGVQVSQNQRLLTNILRDEWGYQGLVMSD WGAWVDHTAAIKAGLDLEMPGKGDASKEEIIQAVKEGRLQESTLNRSALRVLEMAER YGHPKTPAPAYDMEAQHEFARQLADDSIVLLKNEKQLASDKLASDPLEXPLEXPLGEF EGGGSSHVNAHRVVTPKEVMPENAVYAQGYRLDSDQPDEQLTAEAMALAKNSDQV VFFAGFPEAMESEGFDKHSISLPANQLLEQLLTVNPHVVVLQNGSAVEMPWEPET PAIVETYLAGEAVGEATWDILSGQVNPSGKLSETFPKRLADGRPTYVDRYV EGILMGYRYYDAKQLAVRFPFGHGLSYTTFAYQNLTIRENSDVTVSFDLENTGSQAG KEVAQVYVANHASQTIMPIDELLRDFVKVELQPGETKHLTLKLSRRAFAWYNSETWE ADNGQYEILVGSSSRDIRLRQEFELTIGTNPLGKITGESTYTYVELMQALM GLTATFDKFMSPELAPI FAN I PLRSL™ ADVDAETVH ELQKLN ELD

[0030] In another preferred embodiment of the combination of the invention, the glucosidase enzyme comprises an amino acid sequence with at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with the sequence SEQ ID NO: 1.D. This glucosidase enzyme maintains the same function / activity as the enzyme of SEQ ID NO: 1.

[0031] In another preferred embodiment of the combination of the invention, the glucosidase enzyme comprises, preferably consists of, the amino acid sequence SEQ ID NO: 1.

[0032] Following the production of secoisolariciresinol, the enzyme secoisolariciresinol dehydrogenase catalyzes the following reaction. In the present invention, the term “secoisolariciresinol dehydrogenase” or “SDH” (used interchangeably) refers to an enzyme that catalyzes the conversion of secoisolariciresinol to matairesinol. The classification number of the enzyme secoisolariciresinol dehydrogenase is EC: 1.1.1.331.

[0033] The CAS number for matairesinol is CAS No.: 580-72-3.

[0034] The reaction catalyzed by the secoisolariciresinol dehydrogenase of the invention is described below:

[0035] Secoisolariciresinol + 2 NAD(P)+-> matairesinol + 2 NAD(P)H

[0036] The secoisolariciresinol dehydrogenase enzyme of the combination of the invention comprises an amino acid sequence with at least 80% identity to the sequence SEQ ID NO: 2. The enzyme with the amino acid sequence having at least 80% identity to the sequence SEQ ID NO: 2 maintains the same function / activity as the enzyme of SEQ ID NO: 2.

[0037] Amino acid sequence of the enzyme secoisolariciresinol dehydrogenase from Lactonifactor longoviformis DSM17459T, SEQ ID NO: 2:

[0038] MKDMLKGQIAIVAGGTSGMGEATAKLYAREGAWIIGGTNPKKGNRVLKEITDAGGEA RYYGPLNVASKKSCDDTVDAVIKEFGKIDILANFAGRSYDGASDMTPEERYQTTMDVN MTGTYN LAFSWPHM KEAKSGKI I LCSSNGAFN PTTPAYDYHMAKAACESLTI N LAM E LAPLGIRVNCIKPGPIVTPFWDELFPPEEKEAMEAAFHGIATREVPLNRMGTPDDIAGP ALFLASDLSAYITGLLLYVGGGMGYVYAHGQSAILGNVPLAGEK The enzyme secaisolariciresinol dehydrogenase from Lactonifactor longoviformis DSM17459T SEQ ID NO: 2 is also called EDIL2.

[0039] Surprisingly, the present inventors have demonstrated for the first time the secoisolariciresinol dehydrogenase activity of the enzyme SEQ ID NO: 2.

[0040] In another preferred embodiment of the combination of the invention, the secoisolariciresinol dehydrogenase enzyme comprises an amino acid sequence having at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the sequence SEQ ID NO: 2. Said secoisolariciresinol dehydrogenase enzyme maintains the same function / activity as the enzyme of SEQ ID NO: 2

[0041] In another preferred embodiment of the combination of the invention, the secoisolariciresinol dehydrogenase enzyme comprises, preferably consists of, the amino acid sequence SEQ ID NO: 2.

[0042] The enzyme secoisolariciresinol dehydrogenase is a type of enzyme that requires reducing power to catalyze the reaction it enacts. Thus, although the enzyme SEQ ID NO: 2 alone is capable of catalyzing the conversion of secoisolariciresinol to matairesinol, the present inventors provide an enzyme combination that also includes an enzyme that produces reducing power, thereby increasing the yield of the conversion of SDG to matairesinol.

[0043] In the present invention, the term “yield” in reference to an enzymatic reaction refers to the amount or proportion of a substrate that is converted into a given product, where preferably the yield is expressed as a % of a substrate that is finally converted into a given product by an enzymatic reaction.

[0044] Specifically, the combination of the invention comprises a glucose dehydrogenase enzyme, which provides reducing power in the conversion of SDG to matairesinol, thereby increasing the reaction yield. In the present invention, the term “glucose dehydrogenase” or “GDH” (used interchangeably) refers to an enzyme that catalyzes the conversion of glucose to glucuno-1,5-lactone by dehydrogenating a hydroxyl group of glucose, where reducing power is produced in the form of NAD(P)H. The classification number of the glucose dehydrogenase enzyme is EC 1.1.1.49.

[0045] Thus, the glucose dehydrogenase enzyme of the invention uses as its substrate the glucose released in the reaction catalyzed by the glucosidase that catalyzes the conversion of SDG to secoisolariciresinol. Therefore, the reactions catalyzed by the enzymes of the combination of the invention complement each other to efficiently produce matairesinol in high yield. Preferably, the glucose dehydrogenase of the invention is a glucose 6-phosphate dehydrogenase.

[0046] In a preferred embodiment of the combination of the invention, the glucose dehydrogenase enzyme comprises an amino acid sequence with at least 80% identity with SEQ ID NO: 3. The amino acid sequence with at least 80% identity with SEQ ID NO: 3 maintains the same function / activity as the enzyme of SEQ ID NO: 3.

[0047] Amino acid sequence of glucose dehydrogenase from Lactobacillus plantarum SEQ ID NO: 3:

[0048] MSNEKIALFTIFGGTGDLAQRKLYPSLFKLYQKGYLKDHFAVIGTARRPWTDDHYHEVI SDSLADLNADQETVTQFASHFYYQSHDVTDAEHYRTLKKLSEKLDAQYGLQGNRIFYL AMAPNFFGTIAQHLRSENILTDNGFNRVIIEKPFGHDYESAKELNDQLTATFNENQIYRI DHYLGKEMIQNITAIRFGNNIWESLWNNRYIDNVQITLSEKLGVEERAVYYDNSGALRD MVQNHILQILSLLTMDQPVEFTENDIDVEKVKALRSLRPLKPEEVATNFVRGQYGAGD DAKAYRDEDKISSDSNTDTFVAGKVMIDNYRWSGVPFYIRTGKRLADKFTRIDVVFKR PVVNIFNHEDVRANDDAATSLDPNILTINVEPTEGFELRMNAKAIGQGFATTPVKLNYS HDAEATAGSPEAYERLLHDALNGDATNFTHWQEVADSWKFVDVIQKYWDEHQPEFP NYRPGTMGPDAADELLRQDGHRWVYRN

[0049] In another preferred embodiment of the combination of the invention, the glucose dehydrogenase enzyme comprises an amino acid sequence having at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with the sequence SEQ ID NO: 3. Said amino acid sequence maintains the same function / activity as the enzyme of SEQ ID NO: 3.

[0050] In another preferred embodiment of the combination of the invention, the glucose dehydrogenase enzyme comprises, preferably consists of, the amino acid sequence SEQ ID NO: 3.

[0051] Preferably, the combination of the invention comprises:

[0052] - a p-glucosidase enzyme or a fragment thereof that maintains the function / activity of the complete enzyme, wherein the p-glucosidase enzyme comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 which is an amino acid sequence that maintains the same function / activity as the enzyme of SEQ ID NO: 1,

[0053] - a secoisolariciresinol dehydrogenase enzyme or a fragment thereof that maintains the function / activity of the complete enzyme, wherein the secoisolariciresinol dehydrogenase enzyme comprises an amino acid sequence with at least 80% identity to the sequence SEQ ID NO: 2, which is an amino acid sequence that maintains the same function / activity as the enzyme of SEQ ID NO: 2 and

[0054] - a glucose dehydrogenase enzyme or a fragment thereof that maintains the function / activity of the complete enzyme, wherein the glucose dehydrogenase enzyme comprises an amino acid sequence with at least 80% identity to the sequence SEQ ID NO: 3 which is an amino acid sequence that maintains the same function / activity as the enzyme of SEQ ID NO: 3.

[0055] More preferably, the combination of the invention comprises:

[0056] - a p-glucosidase enzyme or a fragment thereof that maintains the function / activity of the complete enzyme, wherein the p-glucosidase enzyme comprises an amino acid sequence with at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with SEQ ID NO: 1, which is an amino acid sequence that maintains the same function / activity as the enzyme of SEQ ID NO: 1,

[0057] - a secoisolariciresinol dehydrogenase enzyme or a fragment thereof maintaining the function / activity of the complete enzyme, wherein the secoisolariciresinol dehydrogenase enzyme comprises an amino acid sequence having at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with the sequence SEQ ID NO: 2, which is an amino acid sequence maintaining the same function / activity as the enzyme of SEQ ID NO: 2, and

[0058] - a glucose dehydrogenase enzyme or a fragment thereof maintaining the function / activity of the complete enzyme, wherein the glucose dehydrogenase enzyme comprises an amino acid sequence having at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with the sequence SEQ ID NO: 3 which is an amino acid sequence maintaining the same function / activity as the enzyme of SEQ ID NO: 3.

[0059] More preferably, the combination of the invention comprises:

[0060] - a p-glucosidase enzyme comprising, preferably consisting of, the amino acid sequence SEQ ID NO: 1 or a fragment thereof that maintains the function / activity of the complete enzyme,

[0061] - a secoisolariciresinol dehydrogenase enzyme comprising, preferably consisting of, the amino acid sequence SEQ ID NO: 2 or a fragment thereof that maintains the function / activity of the complete enzyme, and

[0062] - a glucose dehydrogenase enzyme comprising, preferably consisting of, the amino acid sequence SEQ ID NO: 3.

[0063] In another preferred embodiment, the p-glucosidase, secoisolariciresinol dehydrogenase and glucose dehydrogenase enzymes of the combination of the invention are comprised in the same composition or in different compositions.

[0064] Preferably, the enzymes p-glucosidase, secoisolariciresinol dehydrogenase, and glucose dehydrogenase are contained in the same composition; or two of the enzymes p-glucosidase, secoisolariciresinol dehydrogenase, and glucose dehydrogenase are contained in the same composition, while the other is contained in a different composition; or each of the enzymes p-glucosidase, secoisolariciresinol dehydrogenase, and glucose dehydrogenase is contained in a different composition. Thus, the combination of the invention may also be referred to as the “combined product of the invention,” wherein said combination or combined product comprises the enzyme p-glucosidase, secoisolariciresinol dehydrogenase, and glucose dehydrogenase of the invention, which are preferably contained in the same composition or in different compositions.

[0065] Preferably, the composition or compositions comprising the enzyme -glucosidase, secoisolariciresinol dehydrogenase and glucose dehydrogenase of the invention are formulated as a solid, liquid, gel or powder, being preferably formulated as a liquid composition or compositions.

[0066] The enzymes of the combination of the invention can be immobilized on a support to facilitate their handling and use.

[0067] Thus, in another preferred embodiment of the combination of the invention, the enzymes p-glucosidase, secoisolariciresinol dehydrogenase and glucose dehydrogenase are immobilized on a support.

[0068] In the present invention, the term “support” refers to a material suitable for immobilizing reagents (e.g., enzymes or fragments thereof) to carry out a chemical reaction. Preferably, the support is a solid support. Examples of supports suitable for carrying out the present invention include, without limitation, gelatin, cellulose, pectin, synthetic polymers, inorganic materials, silica, zeolite, ceramics, agarose, glass, or activated carbon.

[0069] In preferred embodiments, the support is in the form of nanoparticles, having a particle size of less than 1000 nm, more preferably the support nanoparticles have a size between 10 nm and 1000 nm, more preferably between 10 nm and 500 nm, more preferably between 10 and 100 nm.

[0070] Preferably, the support is selected from a list consisting of agarose, cellulose, pectin, silica, zeolite, ceramic, glass, and carbon.

[0071] In the present invention, the enzymes of the combination of the invention may be immobilized on a support, where “immobilized” refers to the fact that the enzymes of the invention (or fragments thereof) are bound to the surface of the support. The bonding may be by covalent or non-covalent bonds, preferably by covalent bonds. The immobilization of enzymes or fragments thereof on a support may be carried out using techniques known to a person skilled in the art.

[0072] The support may comprise functional groups (e.g., aldehyde groups) to facilitate the immobilization of the enzymes of the invention through covalent bonds. Thus, the enzymes of the invention can be covalently bonded via amino groups of amino acid side chains in their sequence or at their N-terminus to functional groups present in the support.

[0073] The enzymes of the combination of the invention may be immobilized on the same support or on separate supports, preferably they are immobilized on the same support.

[0074] Preferably, in the present invention, each of the enzymes p-glucosidase, secoisolariciresinol dehydrogenase and glucose dehydrogenase are immobilized on a support in an amount of between 2 pg and 10 pg of enzyme (or fragment thereof) per milligram of support; more preferably they are immobilized in an amount of 4 pg / mg of support.

[0075] As used in the present invention, "identity" or "sequence identity" means the degree of similarity between two nucleotide or amino acid sequences obtained by optimal alignment of the two sequences. Depending on the number of common residues between the aligned sequences, a degree of identity expressed as a percentage will be obtained. The degree of identity between two amino acid sequences can be determined by conventional methods, for example, by standard sequence alignment algorithms known in the prior art, such as BLAST. BLAST programs, for example, BLASTN, BLASTX, TBLASTX, BLASTP, and TBLASTN, are publicly available on the website of the National Center for Biotechnology Information (NCBI).

[0076] The p-glucosidase, secoisolariciresinol dehydrogenase, and glucose dehydrogenase of the invention comprising an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 97, 98, or 99% identity with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, respectively, refer to functionally equivalent variants of the glucosidase of SEQ ID NO: 1, the secoisolariciresinol dehydrogenase of SEQ ID NO: 2, or the glucose dehydrogenase of SEQ ID NO: 3, respectively. Preferably, the term “functionally equivalent variant” in the present invention refers to a polypeptide or protein (in the present invention the protein is an enzyme) having the same activity as a reference polypeptide or protein but comprising an alteration in its sequence, i.e., a substitution, insertion and / or deletion, in one or more (e.g., positions).For example, a substitution means replacing the amino acid (occupying a position) with a different amino acid; a deletion means removing the amino acid occupying a position; and an insertion means adding an adjacent amino acid immediately after the amino acid occupying a position. Amino acid changes can be minor in nature, i.e., conservative amino acid deletions, substitutions, or insertions that do not significantly affect the folding and / or activity of the polypeptide / protein; small amino- or carboxy-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20–25 residues; or a small extension that facilitates purification by changing the net charge or other function, such as a polyhistidine tail, antigenic epitope, or binding domain.Examples of conservative substitutions are found within the groups of basic amino acids (arginine, lysine, and histidine), glutamic and aspartic acids, polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, threonine, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). In the field, amino acid substitutions are known that generally do not alter specific activity. Common examples of substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0077] Thus, when it is stated that an amino acid sequence with at least 80% identity with the sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, as the case may be, maintains the same function / activity as the enzyme of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively, it is indicated that said amino acid sequence maintains the same activity as the enzyme whose sequence is represented by SEQ ID NO: 1 (the glucosidase enzyme of Lactobacillus mucosae I NIA 425L), SEQ ID NO: 2 (secoisolariciresinol dehydrogenase of Lactonifactor longoviformis DSM17459T) or SEQ ID NO: 3 (glucose dehydrogenase of Lactobacillus plantarum), respectively, so that its function, for the purposes of the present invention, will also be that of the enzymes represented by each of those sequences.Therefore, stating that an enzyme comprising, for example, at least 80% identity with the sequence SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 maintains the same function / activity as the enzyme of that specific sequence is synonymous with saying that it is a functionally equivalent vahant of the enzyme in question, as defined in the preceding paragraph. Furthermore, the expression “same function / activity” can be simplified to “same activity,” since the coincidence in activity will result in parallelism in function as well.

[0078] Furthermore, the present invention also relates to the p-glucosidase, secoisolariciresinol dehydrogenase, and glucose dehydrogenase fragments of the invention. As used herein, the term "fragment" refers to a protein or polypeptide with one or more amino acids missing from the ends of its sequence, such as the amino and / or carboxyl terminus of the amino acid sequence, preferably where such fragment retains the same function as the complete protein or polypeptide. Therefore, the glucosidase, secoisolariciresinol dehydrogenase, and glucose dehydrogenase fragments of the invention retain the same function as their complete enzymes. Thus, the glucosidase, secoisolariciresinol dehydrogenase, and glucose dehydrogenase fragments of the invention are "functionally equivalent fragments."

[0079] Preferably, the p-glucosidase, secoisolariciresinol dehydrogenase, and glucose dehydrogenase fragments of the invention lack at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 amino acid residues in their amino-terminal and / or carboxy-terminal end with respect to the complete amino acid sequence of p-glucosidase, secoisolariciresinol dehydrogenase, and glucose dehydrogenase.

[0080] More preferably, the p-glucosidase, secoisolariciresinol dehydrogenase and glucose dehydrogenase fragments of the invention lack 1 to 20, more preferably 1 to 10 and more preferably 1 to 5 amino acid residues from the amino and / or carboxyl terminus with respect to the total complete amino acid sequence of p-glucosidase, secoisolariciresinol dehydrogenase and glucose dehydrogenase.

[0081] In another embodiment, the functionally equivalent fragment or functionally equivalent variants of p-glucosidase, secoisolariciresinol dehydrogenase, and glucose dehydrogenase of the invention maintain at least 60%, more preferably 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% of the respective enzymatic activities. Therefore, stating that a fragment maintains the function / activity of the complete enzyme does not imply that the level of activity is the same as that of the complete enzyme, but rather that the reaction catalyzed by the complete enzyme and by the fragment is the same.

[0082] Secoisolariciresinol dehydrogenase activity can be determined by detecting and quantifying the production of matairesinol from secoisolariciresinol using mass spectrometry techniques, preferably HPLC or HPLC-ESI / MS. More preferably, secoisolariciresinol activity is carried out using the methodology described in Gaya, P., et al. (2016a). Food Analytical Methods, 9, 537-54, or in Xia, ZQ, et al. (2001). Journal of Biological Chemistry, 276(16), 12614-12623.

[0083] Glucose dehydrogenase activity can be determined using spectrophotometric techniques. More preferably, glucose dehydrogenase activity can be determined according to Pfeffer, DA, et al. (2020). PLoS medicine, 17(5), e1003084.

[0084] Glucosidase activity can be determined by a colorimetric assay with p-nitrophenyl-pd-glucopyranoside, preferably the glucosidase activity is determined according to Landete, JM, et al. (2014). Journal of Functional Foods, 7, 322-329.

[0085] The p-glucosidase, secoisolariciresinol dehydrogenase, and glucose dehydrogenase of the invention can be produced, without limitation, by any method of synthesis or recombination known to the skilled tradesperson; preferably, the enzymes of the invention are recombinantly expressed in a host cell, tissue, or organism comprising the nucleotide sequence encoding them. The host cell, tissue, or organism may be prokaryotic or eukaryotic, preferably a bacterial, animal, plant, fungal, or yeast cell. Examples of methods for isolating and / or purifying proteins or polypeptides include, without limitation, affinity chromatography, gel filtration, ion-exchange chromatography, or purification of labeled proteins.

[0086] In a preferred embodiment, the p-glucosidase enzyme is encoded by the nucleotide sequence SEQ ID NO: 4, the secoisolariciresinol dehydrogenase enzyme is encoded by the nucleotide sequence SEQ ID NO: 5 and / or the glucose dehydrogenase enzyme is encoded by the nucleotide sequence SEQ ID NO: 6.

[0087] SEQ ID NO: 4, nucleotide sequence that codes for the enzyme p-glucosidase:

[0088] ATGACAAAAGTAGACTTGAATTTTGTTGAGGGCTTGACGCTGGAAGAACGCGCCG ACCTGGTTTCAGGAACGGATTTTTGGTTTACGGCCAAGGTTTCAGGAATGGATCC TATGCTGATGACGGATGGTCCTTCTGGCTTGCGTAAACAGGTCAACGTGCACAGT GCTATGGATCAAAGTATCGAAGCCGTATGTTTTCCTTGTTCGGCATTGACCGCCA GCTCATTTGATGATCAGATGCTGGAAAAACTGGGCGAACAGTTGGGAACGGCTGC CCGTGCTGAAAAAATCGGCGTGCTGCTGGGGCCTGGGGTCAACATCAAGCGCAG TCCATTAGCCGGCCGTAATTTCGAATATTTCTCTGAAGATCCGCTGCTGGCCGGC CGGATGGGAACTGCCTACGTCAAGGGGGTTCAGAGCACTGGGGTTGGCGTCAGC GTTAAGCATTTTGCCACCAACAACCGTGAAGATCAGCGCTTTACCAATTCATCAGA TGTTGATGAACGAACGCTGCGTGAGATCTATCTGGCACAGTTTGAGCGCATCGTG AAGCAAGCGCACCCAGCCACGATCATGTGCTCTTATAATAAGCTTAATGGCGTTC AGGTATCGCAAAACCAGCGCCTTTTGACTAACATTCTGCGTGACGAATGGGGTTA TCAAGGTCTGGTCATGTCAGACTGGGGTGCCGTGGTGGTTGATCATACGGCCGC GATCAAGGCTGGCTTAGATCTGGAAATGCCGGGCAAGGGGGATGCCTCAAAAGA AGAAATCATTCAAGCCGTTAAAGAGGGCCGTCTTCAAGAGTCAACGCTGAACCGT TCTGCACTGCGCGTTTTAGAAATGGCTGAGCGTTATGGTCATCCAAAGACGCCAG CGCCAGCATATGATATGGAAGCTCAGCATGAGTTTGCCCGCCAGCTGGCTGATGACAGTATTGTCTTGTTGAAAAACGAAAAACAAGAGCTGCCATTGAGTGATTCCGCCA AACTGGCAGTAATTGGCGAGCTGGCCGAAAAGCCGCGTTTTGAAGGTGGTGGCA GCTCACATGTCAACGCCCATCGCGTAGTAACGCCTAAAGAGGTCATGCCTGAAAA TGCGGTTTACGCGCAAGGCTACCGTTTGGATAGTGATCAACCAGATGAACAATTG ACTGCTGAGGCAATGGCGCTAGCTAAAAACAGTGATCAAGTGGTCTTCTTTGCTG GTTTCCCTGAAGCTATGGAATCAGAAGGCTTTGACAAACATTCCATTAGTCTGCCA GCTAACCAGAATAAACTGCTCGAACAGCTGCTGACGGTCAATCCGCACGTGGTTG TCCTTCAAAACGGTTCAGCGGTTGAGATGCCTTGGGAACCAGAGACGCCAGCCAT TGTTGAGACTTATCTGGCTGGGGAGGCAGTCGGTGAAGCTACCTGGGATATTTTG

[0089] TCTGGCCAAGTCAATCCTTCAGGCAAGCTTTCCGAAACGTTCCCTAAGCGATTGG

[0090] CTGACAATCCAACCTATCCAACCTTTGGCAAGGATCGCAGACATGAAGTCTATCGT

[0091] GAAGGAATTTTGATGGGTTACCGCTACTACGATGCCAAGCAGCTGGCTGTACGCT

[0092] TCCCATTTGGCCACGGCTTGAGCTACACGACGTTTGCCTATCAAAACCTGACGAT

[0093] CAGGGAAAACAGTGATGACGTTACAGTCAGCTTTGATTTAGAGAATACAGGCAGT

[0094] CAAGCAGGCAAGGAAGTCGCTCAGGTCTATGTGGCTAATCACGCCAGCCAGACG

[0095] ATTATGCCAATTGATGAACTGCGCGATTTTGTTAAAGTTGAGCTGCAGCCGGGGG

[0096] AGACCAAGCATCTGACCCTTAAGCTCTCGCGGCGCGCGTTTGCCTGGTATAACTC

[0097] TGAAACTGAAACCTGGGAAGCCGATAATGGTCAATATGAGATTTTAGTCGGCAGC

[0098] TCGTCGCGTGATATTCGACTGCGGCAAGAGTTTGAGCTGACGATTGGGACGAACC

[0099] CGCTTGGTAAGATTACTGGAGAATCGACCTATGTTGGCGAACTGATGGAAAACGC

[0100] TGATGAACGAATCAAGCAGATCATTGCTGAAATGGGTCTGACGGCTACTTTTGATA

[0101] AGTTCATGTCGCCTGAATTGGCACCGATTTGCTAATATTCCCTTGCGTTCATTAA

[0102] CGATGGCGGATGTTGATGCTGAAACGGTTCATGAGTTGCAAAAATTAAATGAACTA GATTAG

[0103] SEQ ID NO: 5, nucleotide sequence encoding the enzyme secoisolariciresinol dehydrogenase:

[0104] ATGAAAGATATGTTGAAAGGTCAAATTGCTATTGTTGCAGGTGGCACATCAGGCAT

[0105] GGGTGAAGCTACTGCAAAATTATATGCTCGTGAAGGCGCAGTTGTTATTATTGGTG

[0106] GCACAAATCCAAAGAAAGGTAATCGTGTTTTGAAAGAAATTACTGATGCAGGTGGT

[0107] GAAGCACGTTATTATGGCCCATTGAATGTTGCTTCTAAAAAATCATGTGATGATAC

[0108] AGTTGATGCAGTTATTAAAATTTGGTAAAATTGATATTTTGGCTAATTTGCAGG

[0109] CCGTTCATATGGTGCTAGTGATATGACTCCAGAAGAACGTTATCAAACAACTA

[0110] TGGATGTTAATATGACAGGTACTTATATTGGCTTTTTCTGTTGTTCCACATATGA

[0111] AAGAAGCAAATCAGCAAAATTATTTTGTGTCCAAGTAATGGTGCATTTAATCCAA

[0112] CAACTCCAGCTTATGATTATCATATGGCAAAAGCTGCATGTGAAAGTTTGACAATT

[0113] AATTTGGCTATGGAATTGGCACCATTAGGCATTCGTGTTAATTTGTTAAACCAGG

[0114] TCCAATTGTTACTCCATTTGGGATGAATTGTTTCCACCAGAAGAAAAAGAAGCTA

[0115] TGGAAGCTGCATTTCATGGCATTGCAACACGTGAAGTTCCATTTGAATCGTATGGG

[0116] CACTCCAGATGATATTGCTGGTCCAGCATTGTTTTTAGCTAGTGATTTGTCTGCAT

[0117] ATATTACAGGTTTGTTGTTATATGTTGGTGGCGGTATGGGCTATGTTTATGCTCAT

[0118] GGTCAATCTGCAATTTTGGGCAATGTTCCATTAGCTGGTGAAAAATAA SEQ ID NO: 6, secuencia de nucleótidos que codifica la enzima glucosa deshidrogenasa:

[0119] ATGAGTAACGAAAAAATTGCCTTATTCACCATCTTTGGCGGTACTGGTGACCTCGC ACAGCGTAAACTGTACCCGTCACTATTTAAGCTCTATCAAAAGGGCTATCTCAAGG ACCATTTTGCCGTTATTGGGACGGCTCGGCGTCCTTGGACTGATGATCACTATCA TGAAGTTATTAGTGACTCCCTAGCTGATCTGAATGCTGATCAGGAAACGGTGACG

[0120] CAATTTGCGAGCCATTTTTATTATCAATCGCATGACGTTACTGATGCTGAACATTAC CGAACTTTGAAAAAATTATCAGAAAAGCTGGACGCCCAATATGGTTTACAAGGCAA CCGGATCTTCTACTTAGCAATGGCACCAAACTTCTTCGGTACGATTGCCCAACACT TACGTTCCGAAAATATTCTGACAGATAACGGTTTTAACCGCGTCATTATCGAAAAG

[0121] CCTTTTGGTCATGATTATGAAAGTGCCAAGGAACTTAACGACCAATTAACGGCTAC TTTTAACGAAAATCAGATTTATCGAATTGACCACTACCTTGGTAAGGAAATGATTCA AAATATCACGGCCATTCGTTTTGGCAATAATATTTGGGAATCATTATGGAACAACC GTTACATCGACAATGTCCAAATTACGTTAAGTGAAAAACTGGGTGTCGAAGAACG

[0122] GGCCGTTTACTATGATAACAGCGGTGCCTTGCGGGATATGGTCCAAAATCACATT TTGCAAATTTTGAGTCTGTTAACAATGGATCAACCAGTTGAATTCACTGAAAATGAT ATCGACGTGGAAAAGGTCAAGGCTTTACGGAGCTTACGACCACTTAAACCAGAAG AAGTCGCGACCAACTTTGTCCGTGGTCAATACGGTGCCGGCGATGATGCTAAAGC

[0123] TTACCGCGACGAAGATAAGATTTCATCAGATTCTAACACCGATACTTTCGTCGCTG GTAAAGTCATGATCGATAATTATCGGTGGTCCGGAGTGCCGTTTTACATCCGGAC TGGGAAACGCTTAGCCGATAAGTTCACGCGAATCGACGTGGTGTTCAAACGGCCA GTCGTCAATATCTTCAATCATGAAGACGTCCGAGCTAATGACGACGCAGCAACCA

[0124] GTCTCGATCCAAACATCTTGACAATCAACGTTGAACCGACTGAAGGCTTCGAGCT CCGCATGAATGCTAAAGCAATCGGCCAAGGCTTCGCAACCACCCCGGTCAAGTTG

[0125] AATTATTCTCACGATGCTGAAGCCACGGCTGGTAGCCCAGAAGCTTACGAACGTT TGCTTCACGATGCCTTAAATGGCGACGCAACCAACTTTACCCACTGGCAAGAGGT TGCCGATTCGTGGAAATTCGTTGACGTCATTCAAAAATACTGGGATGAACATCAAC CCGAATTTCCAAACTACCGCCCTGGTACAATGGGACCAGATGCGGCCGATGAATT

[0126] ATTACGTCAAGATGGTCACCGCTGGGTCTACCGCAACTAG

[0127] In the present invention, p-glucosidase, secoisolariciresinol dehydrogenase, and glucose dehydrogenase are preferably "isolated" enzymes, meaning a polypeptide or protein that has been identified and separated and / or recovered from a cell or cell culture from which it was expressed. No particular level of purification is required. For example, an isolated polypeptide may be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purposes of the invention, as are native or recombinant polypeptides that have been separated, fractionated, or purified partially or substantially by any suitable technique.

[0128] According to the results obtained by the present inventors, the combination of enzymes of the invention allows the production of matairesinol.

[0129] Thus, in another aspect, the invention relates to the use of the combination of the invention to produce matairesinol.

[0130] Furthermore, the present invention can be used in the food sector, enabling the production of foods containing matairesinol. These foods are of great interest because matairesinol exhibits beneficial health properties.

[0131] Therefore, in another aspect, the present invention relates to the use of the combination of the invention to produce a food composition comprising matairesinol, hereinafter referred to as “use to produce a food composition of the invention.” Preferably, the food composition is produced from a food composition comprising secoisolariciresinol and / or secoisolariciresinol diglucoside.

[0132] In the present invention, the term “food composition” refers to a composition comprising at least one edible ingredient, where said composition can be consumed alone as food or can be added as an ingredient to other food products. The edible ingredient may comprise proteins, lipids, amino acids, carbohydrates, vitamins, and / or combinations thereof. Examples of food compositions in the context of the present invention include, without limitation, beverages, plant-based beverages, food supplements, or nutraceutical compositions.

[0133] Thus, in a preferred embodiment of the use of the invention to produce a food composition, the food composition is selected from the list consisting of a beverage, a plant-based beverage, a food supplement, or a nutraceutical composition; preferably where the food composition comprises secoisolariciresinol diglucoside.

[0134] In the present invention, the term “beverage” refers to any liquid suitable for consumption.

[0135] In the present invention, the term “plant beverage” refers to any liquid suitable for consumption, where said liquid is made from or derived from a plant or group of plants. The term “plant beverage” also includes plant beverages mixed with other beverages of animal origin, such as milk. Examples of plant beverages include beverages made from soybeans, almonds, peas, oats, cashews, rice, and coconut. Preferably, the plant beverage also comprises flaxseed powder, a solution comprising flaxseed powder, a flaxseed extract, or a lignan extract.

[0136] As used in the present invention, the terms “food supplement,” “nutritional supplement,” or “dietary supplement,” used interchangeably, refer to a composition or product containing nutritive compounds such as vitamins, minerals, fiber, fatty acids, proteins, amino acids, carbohydrates, and / or other active compounds. Food supplements may be in the form of a pill, capsule, tablet, powder, or liquid.

[0137] The term “nutraceutical composition” in the present invention refers to a composition comprising nutritive compounds such as vitamins, minerals, fiber, fatty acids, proteins, amino acids, and / or carbohydrates, and further comprising at least one active compound (for example, matairesinol) capable of producing a beneficial effect on the individual consuming said composition. Beneficial effects refer to positive effects on the individual's health, whether by preventing or treating a disease. In the present invention, the nutraceutical composition comprises matairesinol as the active compound, which produces beneficial effects on the body of the individual to whom it is administered.

[0138] In another aspect, the present invention relates to a method for producing matairesinol, hereinafter referred to as the “method for producing matairesinol of the invention”, wherein said method comprises contacting secoisolariciresinol diglucoside, or a composition comprising secoisolariciresinol diglucoside, with the combination of the invention.

[0139] In a preferred embodiment of the method for producing matairesinol of the invention, the composition comprising secoisolariciresinol diglucoside is selected from the list consisting of a solution comprising secoisolariciresinol diglucoside, flax seeds, flax seed powder, a solution comprising flax seed powder, a flax seed extract, or a lignan extract.

[0140] Preferably, the enzymes of the combination of the invention can be contacted with secoisolariciresinol diglucoside, or a composition comprising secoisolariciresinol diglucoside, sequentially or simultaneously.

[0141] In another preferred embodiment of the method for producing matairesinol of the invention, secoisolariciresinol diglucoside, or a composition comprising secoisolariciresinol diglucoside, is contacted with the combination of the invention and maintained under conditions suitable for the production of matairesinol.

[0142] These suitable conditions preferably include suitable temperature and time conditions to produce matairesinol.

[0143] Preferably, in the method for producing matairesinol of the invention, secoisolariciresinol diglucoside is in an aqueous composition or solution, such that the reaction for producing matairesinol by the enzymes of the invention takes place in an aqueous medium. Preferably, this aqueous medium can be water or a buffer solution, for example, a phosphate buffer. More preferably, the aqueous solution or composition comprising secoisolariciresinol diglucoside is produced by mixing flaxseed powder, a flaxseed extract, or a lignan extract with water or a buffer solution.

[0144] In another preferred embodiment of the method for producing matairesinol of the invention, secoisolariciresinol diglucoside, or a composition comprising secoisolariciresinol diglucoside, is contacted with the combination of the invention and held for a period of time of between 2 and 6 hours, preferably at a temperature of between 35 and 42°C. In another preferred embodiment of the method for producing matairesinol of the invention, secoisolariciresinol diglucoside, or a composition comprising secoisolariciresinol diglucoside, is contacted with the combination of the invention and held for 4 hours at a temperature of 40°C.

[0145] In another preferred embodiment of the method for producing matairesinol of the invention, the production of matairesinol is carried out at a pH of between 4.5 and 7.0.

[0146] In another aspect, the present invention relates to a method for obtaining a food composition comprising matairesinol, wherein said method comprises contacting the food composition (a food composition comprising secoisolariciresinol and / or secoisolariciresinol diglucoside) with the combination of the invention, hereinafter referred to as “method for obtaining a food composition of the invention”.

[0147] The combination of the invention can be brought into contact with the food composition during its manufacturing process or after it has already been manufactured. The enzymes of the combination of the invention can be brought into contact simultaneously or sequentially.

[0148] Preferably, when the enzymes of the composition of the invention are added sequentially, the p-glucosidase enzyme is added first, followed by the secoisolariciresinol dehydrogenase enzyme and the glucose dehydrogenase enzyme.

[0149] Preferably, the food composition of the method for obtaining a food composition of the invention comprises secoisolariciresinol diglucoside. The SDG comprising the food composition is converted to matairesinol, wherein said SDG may be added to the food composition or be intrinsically present in the food composition. The SDG may be added to the food composition by adding a composition comprising it, for example, but without limitation, a solution comprising secoisolariciresinol diglucoside, flaxseed, flaxseed powder, a solution comprising flaxseed powder, a flaxseed extract, or a lignan extract.

[0150] In another preferred embodiment of the method for obtaining a food composition of the invention, the food composition is selected from the list consisting of a beverage, a plant-based beverage, a food supplement, or a nutraceutical composition; preferably where the food composition comprises secoisolariciresinol diglucoside.

[0151] In another preferred embodiment of the method for obtaining a food composition of the invention, the combination of the invention is contacted with the food composition and maintained for a period of time of between 2 and 6 hours, preferably at a temperature of between 35 and 42°C.

[0152] In another preferred embodiment of the method for obtaining a food composition of the invention, the production of matairesinol is carried out at a pH of between 4.5 and 7.0.

[0153] As previously stated, for the development of the present invention, the inventors have surprisingly demonstrated the ability of the enzyme with SEQ ID NO: 2 to produce matairesinol.

[0154] Thus, in another aspect, the present invention relates to the use of a secoisolariciresinol dehydrogenase enzyme comprising an amino acid sequence with at least 80% identity to the sequence SEQ ID NO: 2 that maintains the same function / activity as the enzyme of SEQ ID NO: 2 or a fragment thereof that maintains the function / activity of the complete enzyme to produce matairesinol, hereinafter referred to as “use of the secoisolariciresinol dehydrogenase of the invention”.

[0155] In a preferred embodiment of the use of the secoisolariciresinol dehydrogenase of the invention, said enzyme comprises an amino acid sequence having at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with the sequence SEQ ID NO: 2 wherein the amino acid sequence maintains the same function / activity as the enzyme of SEQ ID NO: 2.

[0156] In a preferred embodiment of the use of the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol dehydrogenase comprises, preferably consists of, the amino acid sequence SEQ ID NO: 2.

[0157] The use of the secoisolariciresinol dehydrogenase of the invention preferably also comprises the use of a glucose dehydrogenase enzyme or a fragment thereof. Preferably, the glucose dehydrogenase is a glucose 6-phosphate dehydrogenase.

[0158] In another preferred embodiment of the use of the secoisolariciresinol dehydrogenase of the invention, the glucose dehydrogenase comprises an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with the sequence SEQ ID NO: 3 wherein the amino acid sequence maintains the same function / activity as the enzyme of SEQ ID NO: 3.

[0159] In another more preferred embodiment of the use of the secoisolariciresinol dehydrogenase of the invention, the glucose dehydrogenase comprises, preferably consists of, the sequence SEQ ID NO: 3.

[0160] In another aspect, the present invention relates to a method for producing matairesinol, wherein said method comprises contacting secoisolariciresinol, or a composition comprising secoisolariciresinol, with a secoisolariciresinol dehydrogenase enzyme comprising an amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 2, wherein the amino acid sequence maintains the same function / activity as the enzyme of SEQ ID NO: 2, or a fragment thereof maintaining the activity of the complete enzyme, hereinafter referred to as “method for producing matairesinol with the secoisolariciresinol dehydrogenase of the invention”.

[0161] In a preferred embodiment of the method for producing matairesinol with the secoisolariciresinol dehydrogenase of the invention, said enzyme comprises an amino acid sequence having at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with the sequence SEQ ID NO: 2 wherein the amino acid sequence maintains the same function / activity as the enzyme of SEQ ID NO: 2; more preferably comprising or consisting of the amino acid sequence SEQ ID NO: 2.

[0162] In another preferred embodiment, the method for producing matairesinol with the secoisolariciresinol dehydrogenase of the invention further comprises contacting secoisolariciresinol, or a composition comprising secoisolariciresinol, with a glucose dehydrogenase enzyme or a fragment thereof. Preferably, the glucose dehydrogenase is a glucose 6-phosphate dehydrogenase.

[0163] In another preferred embodiment, the method for producing matairesinol with the secoisolariciresinol dehydrogenase of the invention further comprises contacting secoisolariciresinol, or a composition comprising secoisolariciresinol, with a glucose dehydrogenase enzyme or a fragment thereof, wherein said glucose dehydrogenase comprises an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with the sequence SEQ ID NO: 3, wherein the amino acid sequence maintains the same function / activity as the enzyme of SEQ ID NO: 3; more preferably wherein the glucose dehydrogenase comprises or consists of the amino acid sequence SEQ ID NO: 3.

[0164] In another preferred embodiment of the method for producing matairesinol with the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol dehydrogenase and glucose dehydrogenase are contacted with the secoisolariciresinol, or a composition comprising it, simultaneously or sequentially.

[0165] Preferably, in the method for producing matairesinol with the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol dehydrogenase and the glucose dehydrogenase are comprised in the same composition or in different compositions; preferably said composition or compositions are formulated in the form of a solid, liquid, gel or powder.

[0166] In another preferred embodiment of the method for producing matairesinol with the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol dehydrogenase and / or glucose dehydrogenase enzymes are immobilized on a support, preferably the support being selected from the list consisting of agarose, cellulose, pectin, silica, zeolite, ceramic, glass, and carbon.

[0167] In another preferred embodiment of the method for producing matairesinol with the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol dehydrogenase and glucose dehydrogenase enzymes are immobilized on a support, wherein the support is made of agarose.

[0168] Preferably, the secoisolariciresinol dehydrogenase and glucose dehydrogenase enzymes are immobilized on the same support or on separate supports.

[0169] Preferably, in the method for producing matairesinol with the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol is in an aqueous composition or solution, such that the matairesinol production reaction takes place in an aqueous medium. Preferably, this aqueous medium can be water or a buffer solution, for example, a phosphate buffer.

[0170] In another preferred embodiment of the method for producing matairesinol with the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol dehydrogenase, or a fragment thereof that maintains the function / activity of the complete enzyme, is contacted with secoisolariciresinol, or a composition comprising secoisolariciresinol, and is maintained for a period of time between 2 and 6 hours, preferably at a temperature between 35 and 42°C.

[0171] In another preferred embodiment of the method for producing matairesinol with the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol dehydrogenase, or a fragment thereof that maintains the function / activity of the complete enzyme, is contacted with secoisolariciresinol, or a composition comprising secoisolariciresinol, and held for 4 hours, preferably at 40°C.

[0172] In another preferred embodiment of the method for producing matairesinol with the secoisolariciresinol dehydrogenase of the invention, the production of matairesinol is carried out at a pH of between 4.5 and 7.0.

[0173] In preferred embodiments, the methods for producing matairesinol of the invention further comprise a step of isolating and / or purifying the matairesinol. The isolation and / or purification of matairesinol can be carried out using methodologies known to the skilled trade, such as extraction with organic solvents, chromatography, or filtration. Preferably, the isolation and / or purification is carried out with an organic solvent, more preferably ethyl acetate.

[0174] Another aspect refers to a kit comprising the combination of the invention, henceforth the “kit of the invention”.

[0175] In another aspect, the present invention relates to the use of the kit of the invention to produce matairesinol or to obtain a food composition comprising matairesinol.

[0176] Preferably, the use of the kit of the invention is to produce a food composition comprising matairesinol.

[0177] Another aspect relates to the use of a secoisolariciresinol dehydrogenase enzyme comprising an amino acid sequence with at least 80% identity to the sequence SEQ ID NO: 2 or a fragment thereof, to produce a food composition comprising matairesinol, hereinafter referred to as “use to produce a food composition with the secoisolariciresinol dehydrogenase of the invention.” The amino acid sequence with at least 80% identity to the sequence SEQ ID NO: 2 maintains the same function / activity as the enzyme of SEQ ID NO: 2, and / or the fragment thereof maintains the function / activity of the complete enzyme of SEQ ID NO: 2. The food composition is produced from a food composition comprising secoisolariciresinol and / or glycosylated secoisolariciresinol.

[0178] In a preferred embodiment of the use for producing a food composition with the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol dehydrogenase enzyme comprises an amino acid sequence having at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with the sequence SEQ ID NO: 2, which is an amino acid sequence that maintains the same function / activity as the enzyme of SEQ ID NO: 2; more preferably comprising or consisting of the amino acid sequence SEQ ID NO: 2.

[0179] In another preferred embodiment of the use of the secoisolariciresinol dehydrogenase of the invention to produce a food composition, it further comprises the use of a glucose dehydrogenase enzyme or a fragment thereof. The glucose dehydrogenase enzyme fragment maintains the function / activity of the complete enzyme. Preferably, the glucose dehydrogenase is a glucose 6-phosphate dehydrogenase.

[0180] In another preferred embodiment of the use for producing a food composition with the secoisolariciresinol dehydrogenase of the invention, the glucose dehydrogenase comprises an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with the sequence SEQ ID NO: 3, wherein the amino acid sequence maintains the same activity as the enzyme of SEQ ID NO: 3.

[0181] In another preferred embodiment of the use for producing a food composition with the secoisolariciresinol dehydrogenase of the invention, the glucose dehydrogenase comprises, preferably consists of, the sequence SEQ ID NO: 3.

[0182] Another aspect of the present invention relates to a method for obtaining a food composition comprising matairesinol, wherein said method comprises contacting the food composition with a secoisolariciresinol dehydrogenase enzyme comprising an amino acid sequence with at least 80% identity to the sequence SEQ ID NO: 2 or a fragment thereof, hereinafter referred to as the “method for producing a food composition with the secoisolariciresinol dehydrogenase of the invention.” Preferably, the amino acid sequence with at least 80% identity to the sequence SEQ ID NO: 2 maintains the same function / activity as the enzyme of SEQ ID NO: 2 and / or the fragment thereof maintains the function / activity of the complete enzyme of SEQ ID NO: 2, and the food composition is produced from a food composition comprising secoisolariciresinol and / or glycosylated secoisolariciresinol.

[0183] In a preferred embodiment of the method for producing a food composition with the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol dehydrogenase enzyme comprises an amino acid sequence with at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with the sequence SEQ ID NO: 2 wherein the sequence maintains the same function / activity as the enzyme of SEQ ID NO: 2; more preferably comprising or consisting of the amino acid sequence SEQ ID NO: 2.

[0184] In another preferred embodiment of the method for producing a food composition with the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol dehydrogenase enzyme preferably comprises the amino acid sequence SEQ ID NO: 2. In another preferred embodiment of the method for producing a food composition with the secoisolariciresinol dehydrogenase of the invention, it further comprises contacting the food composition with a glucose dehydrogenase enzyme or a fragment thereof, wherein the fragment of the secoisolariciresinol dehydrogenase maintains the function / activity of the complete enzyme. Preferably, the glucose dehydrogenase is a glucose 6-phosphate dehydrogenase.

[0185] In another preferred embodiment of the method for producing a food composition with the secoisolariciresinol dehydrogenase of the invention, the glucose dehydrogenase comprises an amino acid sequence having at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity with the sequence SEQ ID NO: 3, wherein the amino acid sequence maintains the same function / activity as the enzyme of SEQ ID NO: 3.

[0186] In another preferred embodiment of the method for producing a food composition with the secoisolariciresinol dehydrogenase of the invention, the glucose dehydrogenase comprises, preferably consists of, the sequence SEQ ID NO: 3.

[0187] In another preferred embodiment of the method for producing a food composition with the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol dehydrogenase and glucose dehydrogenase enzymes are immobilized on a support, preferably where the support is selected from the list consisting of agarose, cellulose, pectin, silica, zeolite, ceramic, glass, and carbon.

[0188] In another preferred embodiment of the method for producing a food composition with the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol dehydrogenase and glucose dehydrogenase enzymes are immobilized on a support, wherein the support is made of agarose.

[0189] In another preferred embodiment of the method for producing a food composition with the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol dehydrogenase and glucose dehydrogenase enzymes are immobilized on the same support or on separate supports. Preferably, in the method for producing a food composition with the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol dehydrogenase and glucose dehydrogenase are contained in the same composition or in separate compositions; preferably said composition or compositions being formulated as a solid, liquid, gel, or powder.

[0190] In another preferred embodiment of the method for producing a food composition with the secoisolariciresinol dehydrogenase of the invention, the food composition is selected from the list consisting of a beverage, a plant-based beverage, a food supplement, or a nutraceutical composition.

[0191] In another preferred embodiment of the method for producing a food composition with the secoisolariciresinol dehydrogenase of the invention, the secoisolariciresinol dehydrogenase and / or glucose dehydrogenase can be contacted with the food composition during its manufacturing process or after it has been manufactured. The secoisolariciresinol dehydrogenase and / or glucose dehydrogenase enzymes can be contacted simultaneously or sequentially.

[0192] In another preferred embodiment of the method for producing a food composition with the secoisolariciresinol dehydrogenase of the invention, the food composition comprises secoisolariciresinol or secoisolariciresinol is added to it during its production process.

[0193] DESCRIPTION OF THE FIGURES

[0194] Figure 1. Schematic of enzymatic production of matairesinol.

[0195] EXAMPLES

[0196] The invention will then be illustrated by means of tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention.

[0197] I - MATERIALS AND METHODS

[0198] The strains L. plantarum DTA 365 and L. mucosae INIA 425L were grown in De Man-Rogosa-Sharpe (MRS) liquid medium (BD Biosciences) at 37°C for 24 hours, and Lactonifactor longoviformis DSM17459T in Bacto Brain Heart Infusion (BHI) medium (BD Biosciences) supplemented with L-cysteine ​​(0.05%), L-arginine (1% w / v), menadione (1 pg / ml), and hemin (5 pg / mL) at 37°C for 48 hours. All strains were grown under anaerobic conditions. After the respective incubation periods, the bacterial cultures were centrifuged separately, and the cell pellets were collected for genomic DNA extraction from each bacterial strain. The purified genomic DNA was then used to analyze the bacterial cultures. The nucleotide sequences of the gdh (SEQ ID NO: 6), glu425 (SEQ ID NO: 4) and edil 2 (SEQ ID NO: 5) genes were amplified by POR using specific primers from L. plantarum DTA 365, L. mucosae INIA 425L and Lactonifactor longoviformis DSM17459T.

[0199] Once amplified, the genes were cloned into an expression vector and transformed into Escherichia coli DH5a. Recombinant plasmids were purified from heterologous cells, and those whose sequence confirmed the correct insertion of the genes were propagated into E. coli BL21 (ADE3) cells.

[0200] Recombinant cells were cultured in 1 L of LB liquid medium supplemented with ampicillin, and gene expression was induced with isopropyl-pD-1-thiogalactopyranoside when the optical density reached 0.6 at 600 nm. After 18 h at 22°C, the cells were collected by centrifugation, washed twice, and lysed. Protein extracts were processed using an affinity column, and the recombinant enzymes (glucosidase -> SEQ ID NO: 1; secoisolariciresinol dehydrogenase -> SEQ ID NO: 2; glucose dehydrogenase -> SEQ ID NO: 3) were purified by increasing concentrations of imidazole. Finally, the purified enzymes were dialyzed using a membrane with a cutoff limit of 3500 daltons in sodium phosphate buffer, pH 7.0, at 4°C for 3 h.

[0201] The purified enzymes were incubated in the presence of agarose for immobilization in sodium phosphate buffer, pH 7.0 at room temperature for 6 h in the presence of sodium cyanoborohydride. Once the absence of enzyme in the suspension was confirmed, the support with the immobilized enzymes was incubated in Tris-HCl buffer, pH 7.4 at room temperature for 30 min to block binding sites on the support. Finally, the support was washed with 10 mL of sodium phosphate buffer, pH 7.4, and stored until use in the same buffer at 4 °C. Subsequently, the immobilized enzymes (4 pg of each immobilized enzyme / mg) were incubated separately or together, according to Table 1, with SECO (1 mM) and SECO (5 mM) purchased from Merck and SDG derived from commercial lignan extracts at concentrations of 1 mM and 5 mM. The incubation was carried out for 4 hours at 37°C in sodium phosphate buffer, pH 7.0.

[0202] Finally, the lignans were extracted with ethyl acetate and the matairesinol and SECO produced were identified and quantified by HPLC-PAD according to Gaya, et al. (2016a). Food Analytical Methods, 9, 537-547.

[0203] II - RESULTS

[0204] First, a gene in the Lactonifactor longoviformis DSM17459T genome, called edi / 2, was identified. Its protein (EDIL2) is capable of transforming secoisolariciresinol (SECO) into matairesinol. This is the first time a SECO dehydrogenase of microbial origin has been identified. Subsequently, this dehydrogenase was cloned, purified, and characterized in our laboratory, and it was confirmed that this enzyme requires reducing power in the form of NADPH for its proper function.

[0205] The transformation of SECO into matairesinol has two major limitations: first, SECO is found primarily in glycosylated form (SDG) in food, and second, EDIL2 requires reducing power in the form of NADPH. Thus, initially, the combination of a glucosidase (GLu425) from Lactobacillus mucosae INI A 425L and EDIL2 enabled the transformation of SDG present in flaxseed or flaxseed extracts into matairesinol. Subsequently, the addition of a glucose-6-phosphate dehydrogenase (GDH) from Lactobacillus plantarum DTA 365, which generates the reducing power in the form of NADPH necessary for the proper functioning of EDIL2, allowed for the highly efficient transformation of SDG into matairesinol (Figure 1).The next step was to immobilize the three enzymes EDIL2, GLU425, and GDH on food-grade agarose support and apply them to food matrices containing lignan extracts or powdered flaxseed, achieving high concentrations of matairesinol, transforming between 70% and 90% of the SDG into this compound, depending on the food matrix (see Table 1). Table 1. Enzymatic production of matairesinol.

Claims

CLAIMS 1. A combination comprising: - a glucosidase enzyme or a fragment thereof that maintains the function / activity of the complete enzyme, - a secoisolariciresinol dehydrogenase enzyme comprising an amino acid sequence with at least 80% identity to the sequence SEQ ID NO: 2 that maintains the same function / activity as the enzyme of SEQ ID NO: 2, or a fragment thereof that maintains the function / activity of the complete enzyme, and - a glucose dehydrogenase enzyme or a fragment thereof that maintains the function / activity of the complete enzyme, wherein the glucosidase enzyme is a p-glucosidase.

2. Combination according to claim 1, wherein the p-glucosidase enzyme comprises an amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 1 and maintaining the same function / activity as the enzyme of SEQ ID NO:

1.

3. Combination according to any one of claims 1 or 2, wherein the glucose dehydrogenase enzyme comprises an amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 3 and maintaining the same function / activity as the enzyme of SEQ ID NO:

3.

4. Combination according to any one of claims 1 to 3, wherein the enzymes p-glucosidase, secoisolariciresinol dehydrogenase and glucose dehydrogenase are comprised in the same composition or in different compositions.

5. Combination according to any one of claims 1 to 4, wherein the enzymes p-glucosidase, secoisolariciresinol dehydrogenase and glucose dehydrogenase are immobilized on a support.

6. Combination according to claim 5, wherein the support is selected from the list consisting of agarose, cellulose, pectin, silica, zeolite, ceramic, glass, and carbon.

7. Combination according to claim 5 or 6, wherein the support is agarose.

8. Use of the combination according to any one of claims 1 to 7, to produce matairesinol.

9. Use of the combination according to any one of claims 1 to 7, to produce a food composition comprising matairesinol, from a food composition comprising secoisolariciresinol and / or secoisolariciresinol diglucoside.

10. A method for producing matairesinol wherein said method comprises contacting secoisolariciresinol diglucoside, or a composition comprising secoisolariciresinol diglucoside, with the combination according to any one of claims 1 to 7.

11. Method according to claim 10, wherein the composition comprising secoisolariciresinol diglucoside is selected from the list consisting of a solution comprising secoisolariciresinol diglucoside, flax seeds, flax seed powder, a solution comprising flax seed powder, a flax seed extract, or a lignan extract.

12. A method for obtaining a food composition comprising matairesinol, wherein said method comprises contacting a food composition comprising secoisolariciresinol and / or secoisolariciresinol diglucoside with the combination according to any one of claims 1 to 7.

13. Method according to claim 12, wherein the food composition is selected from a beverage, a plant-based beverage, a food supplement, or a nutraceutical composition.

14. Use of a secoisolariciresinol dehydrogenase enzyme comprising an amino acid sequence with at least 80% identity to the sequence SEQ ID NO: 2 that maintains the same function / activity as the enzyme of SEQ ID NO: 2, or a fragment thereof that maintains the function / activity of the complete enzyme, to produce matairesinol.

15. Use according to claim 14, wherein the enzyme secoisolariciresinol dehydrogenase comprises the amino acid sequence SEQ ID NO:

2.

16. Use according to claim 14 or 15, wherein it further comprises the use of a glucose dehydrogenase enzyme or a fragment thereof that maintains the function / activity of the complete enzyme.

17. Use according to claim 16, wherein the glucose dehydrogenase enzyme comprises an amino acid sequence having at least 80% identity with ID NO: 3 and maintaining the same function / activity as the enzyme of SEQ ID NO:

3.

18. Use according to claim 16 or 17, wherein the glucose dehydrogenase comprises the amino acid sequence SEQ ID NO:

3.

19. A method for producing matairesinol wherein said method comprises contacting secoisolariciresinol, or a composition comprising secoisolariciresinol, with a secoisolariciresinol dehydrogenase enzyme comprising an amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 2 that maintains the same function / activity as the enzyme of SEQ ID NO: 2 or a fragment thereof that maintains the function / activity of the complete enzyme.

20. Method according to claim 19, wherein the secoisolariciresinol dehydrogenase enzyme comprises the amino acid sequence SEQ ID NO:

2.

21. Method according to claim 19 or 20, further comprising contacting secoisolariciresinol, or a composition comprising secoisolariciresinol, with a glucose dehydrogenase enzyme or a fragment thereof maintaining the function / activity of the complete enzyme.

22. Method according to claim 21, wherein the glucose dehydrogenase enzyme comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 3 and maintaining the same function / activity as the enzyme of SEQ ID NO:

3.

23. Method according to claim 21 or 22, wherein the glucose dehydrogenase comprises the amino acid sequence SEQ ID NO:

3.

24. A kit comprising the combination according to any one of claims 1 to 7.

25. Use of the kit according to claim 24 to produce matairesinol or to obtain a food composition comprising matairesinol.

26. Use of a secoisolariciresinol dehydrogenase enzyme comprising an amino acid sequence with at least 80% identity to the sequence SEQ ID NO: 2 that maintains the same function / activity as the enzyme of SEQ ID NO: 2, or a fragment thereof that maintains the function / activity of the complete enzyme, to produce a food composition comprising matairesinol.

27. Use according to claim 26, wherein the secoisolariciresinol dehydrogenase enzyme comprises the amino acid sequence SEQ ID NO:

2.

28. Use according to claim 26 or 27, wherein it further comprises the use of a glucose dehydrogenase enzyme or a fragment thereof that maintains the function / activity of the complete enzyme.

29. Use according to claim 28, wherein the glucose dehydrogenase enzyme comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 3 and maintaining the same function / activity as the enzyme of SEQ ID NO:

3.

30. Use according to claim 28 or 29, wherein the glucose dehydrogenase comprises the amino acid sequence SEQ ID NO:

3.

31. A method for obtaining a food composition comprising matairesinol, wherein said method comprises contacting a food composition comprising secoisolariciresinol and / or secoisolariciresinol diglucoside with a secoisolariciresinol dehydrogenase enzyme comprising an amino acid sequence having at least 80% identity with the sequence SEQ ID NO: 2 that maintains the same function / activity as the enzyme of SEQ ID NO: 2 or a fragment thereof that maintains the function / activity of the complete enzyme.

32. Method according to claim 31, wherein the secoisolariciresinol dehydrogenase enzyme comprises the amino acid sequence SEQ ID NO:

2.

33. Method according to claim 31 or 32, further comprising contacting the food composition with a glucose dehydrogenase enzyme or a fragment thereof that maintains the function / activity of the complete enzyme.

34. Method according to claim 33, wherein the glucose dehydrogenase enzyme comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 3 and maintaining the same function / activity as the enzyme of SEQ ID NO:

3.

35. Method according to claim 33 or 34, wherein the glucose dehydrogenase comprises the amino acid sequence SEQ ID NO:

3.

36. Method according to any one of claims 31 to 35, wherein the food composition is selected from a beverage, a plant-based beverage, a food supplement, or a nutraceutical composition.

Images