Method for enhancing the nutritional value of plant-based beverages employing a combination of phytase and xylanase

Abstract

The present invention relates to novel methods for increasing the beneficial health properties of plant-based beverages using enzymes. More particularly, the invention relates to increasing the available mineral content in plant-based beverages using a combination of a phytase and a xylanase.

Description

[0001] METHOD FOR ENHANCING THE NUTRITIONAL VALUE OF PLANT-BASED BEVERAGES EMPLOYING A COMBINATION OF PHYTASE AND XYLANASE

[0002] TECHNICAL FIELD

[0003] The present invention relates to novel methods for increasing the beneficial health properties of plant-based beverages using enzymes. More particularly, the invention relates to increasing the available mineral content in plant-based beverages using a combination of a phytase and a xylanase.

[0004] REFERENCE TO A SEQUENCE LISTING

[0005] The contents of the electronic submission of the text file Sequence Listing, named “IFF10179USPSP_SequenceListing.xml” was created on July 12, 2024, and is 6 KB in size, which is hereby incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Consumer demand for plant-based beverage alternatives is at an all-time high. Demand for plant-based beverages is driven by a multiplicity of reasons including interest in environmental sustainability, concerns for animal wellbeing and interest in health benefits. Moreover, plant-based beverages have long been used as dairy alternatives for vegans and vegetarians. Finally, those with lactose intolerance or allergies to dairy proteins also need plantbased beverages.

[0008] Despite the high demand for plant-based beverages, it is known that the nutritional value of plant-based beverages is still relatively poor as compared with their dairy counterparts. While plants typically used for preparing plant-based beverages such as oats are rich in phosphate and minerals such as calcium and magnesium, the bioavailability of these nutrients tends to be low.

[0009] One of the main anti-nutritional components of cereals and grains used in plant-based beverages is phytic acid (myo-inositol- (l,2,3,4,5,6)-hexakisphosphate, or IP6). Phytic acid is the primary way phosphorus is stored in plants. It is a strong chelator of minerals and proteins, forming insoluble and nondigestible complexes. This effect results in limited bioavailability of these nutrients in humans as well as monogastric animals lacking the phytase enzyme in their digestive system. In animal feed, exogenous phytase is supplemented to facilitate the breakdown of phytic acid and improve the bioavailability of nutrients. Phytase enzyme catalyzes the sequential dcphosphorylation of phytic acid (IP6) to its subsequent pcnta-(IP5), tctra-(IP4), tri- (IP3), di-(IP2) and mono (IP 1 )- esters of inositol in a stepwise manner.

[0010] The use of phytase to breakdown phytic acid in foods for human consumption has been described but relies on the use of organic solvents. See U.S. Patent No. 6,284,506. However, the use of organic solvents in the preparation of food and beverages for human consumption continues to be an issue due to concerns for toxic exposure as well as environmental pollution.

[0011] There is a continuing need for methods of preparing plant-based beverages with high nutritional value using phytase that do not employ organic solvents.

[0012] SUMMARY OF THE INVENTION

[0013] In an aspect of the present invention, a method is presented for increasing the nutritional value of a plant-based beverage, the method having the steps of: a.) providing a plant material having phytate; b.) suspending the plant material in an aqueous phase having water wherein the aqueous phase has no organic solvent to provide a solution; c.) adding a phytase and a xylanase to the solution; d.) incubating the solution for sufficient time to convert the phytate to inorganic phosphate; and e.) processing the solution into the plant-based beverage.

[0014] Optionally, the plant material is plant flour, plant protein isolate or plant protein concentrate. Optionally, the plant material is plant flour. Optionally, the plant flour is produced by a method having the steps of providing a plant component and grinding the plant component into plant flour. Optionally, the plant component is cereal, pseudo-cereal, legume, seed, fruit or nuts.

[0015] Optionally, the plant material is oat, rice, quinoa, soybean, pea, peanut, sesame, sunflower, walnut, cashew, almond or coconut. Optionally, the plant component is oat. Optionally, the oat is oat grain.

[0016] Optionally, the plant material is 1 to 60% wt / wt of the solution. Optionally, the plant material comprises 8 to 25% wt / wt of the solution. Optionally, the plant material is 10 to 20% wt / wt of the solution. Optionally, the plant material comprises 12 to 15% wt / wt of the solution.

[0017] Optionally, the phytase added in step c is in an amount from about 0.1 to about 1,000 FTU per gram of the plant material. Optionally, the phytase is added in an amount from about 1 to about 500 FTU per gram of the plant material. Optionally, the phytase added is in an amount from about 5 to about 100 FTU per gram of the plant material. Optionally, the phytase added is in an amount from about 10 to about 50 FTU per gram of the plant material.

[0018] Optionally, the xylanase added in step c. is in an amount from about 0.0005 to about 1000 GPU per gram of the plant material. Optionally, the xylanase is added in an amount from about 1 to about 500 GPU per gram of the plant material. Optionally, the xylanase added is in an amount from about 2 to about 100 GPU per gram of the plant material.

[0019] Optionally, the method has an additional step of adding one or more additional enzymes selected from the group consisting of a protease, a carboxypeptidase, a cellulase, a xylanase, a mannase, an amylase, a-galactosidase, a pectinase, a glucanase, an esterase and mixtures thereof.

[0020] Optionally, the additional step is performed at the same time as step c.

[0021] Optionally, the method has an additional step of adding one or more additional enzymes selected from the group consisting of a protease, a carboxypeptidase, a cellulase, a xylanase, a mannase, an amylase, a-galactosidase, a pectinase, a glucanase, an esterase and mixtures thereof.

[0022] Optionally, the additional step is performed at the same time as step c.

[0023] Optionally, the phytase is a polypeptide having at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO:2. Optionally, the phytase is a polypeptide according to SEQ ID NO:2.

[0024] Optionally, the xylanase is a polypeptide having at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO:4. Optionally, the xylanase is a polypeptide according to SEQ ID NO:4.

[0025] In another aspect of the present invention, a method is presented for increasing the nutritional value of a plant-based beverage, the method having the steps of: a.) providing a plant material having phytate; b.) suspending the plant material in an aqueous phase having essentially only water; c.) adding a phytase and a xylanase to the solution; d.) incubating the solution for sufficient time to convert the phytate to inorganic phosphate; and e.) processing the solution into the plant-based beverage.

[0026] Optionally, the aqueous phase has only water.

[0027] Optionally, the plant material is plant flour, plant protein isolate or plant protein concentrate. Optionally, the plant material is plant flour. Optionally, the plant flour is produced by a method having the steps of providing a plant component and grinding the plant component into plant flour. Optionally, the plant component is cereal, pseudo-cereal, legume, seed, fruit or nuts.

[0028] Optionally, the plant material is oat, rice, quinoa, soybean, pea, peanut, sesame, sunflower, walnut, cashew, almond or coconut. Optionally, the plant component is oat. Preferably, the oat is oat grain.

[0029] Optionally, the plant material is 1 to 60% wt / wt of the solution. Optionally, the plant material comprises 8 to 25% wt / wt of the solution. Optionally, the plant material is 10 to 20% wt / wt of the solution. Optionally, the plant material comprises 12 to 15% wt / wt of the solution.

[0030] Optionally, the phytase added in step c is in an amount from about 0.1 to about 1,000 FTU per gram of the plant material. Optionally, the phytase is added in an amount from about 1 to about 500 FTU per gram of the plant material. Optionally, the phytase added is in an amount from about 5 to about 100 FTU per gram of the plant material. Optionally, the phytase added is in an amount from about 10 to about 50 FTU per gram of the plant material.

[0031] Optionally, the xylanase added in step c. is in an amount from about 0.0005 to about 1000 GPU per gram of the plant material. Optionally, the xylanase is added in an amount from about 1 to about 500 GPU per gram of the plant material. Optionally, the xylanase added is in an amount from about 2 to about 100 GPU per gram of the plant material.

[0032] Optionally, the method has an additional step of adding one or more additional enzymes selected from the group consisting of a protease, a carboxypeptidase, a cellulase, a xylanase, a mannase, an amylase, a-galactosidase, a pectinase, a glucanase, an esterase and mixtures thereof.

[0033] Optionally, the additional step is performed at the same time as step c.

[0034] Optionally, the phytase is a polypeptide having at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO:2. Optionally, the phytase is a polypeptide according to SEQ ID NO:2.

[0035] Optionally, the xylanase is a polypeptide having at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO:4. Optionally, the xylanase is a polypeptide according to SEQ ID NO:4.

[0036] BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

[0037] SEQ ID NO:1 is the protein sequence of phytase precursor protein.

[0038] SEQ ID NO:2 is the protein sequence of phytase mature protein. SEQ ID N0:3 is the protein sequence of xylanase precursor protein.

[0039] SEQ ID NO:4 is the protein sequence of xylanase mature protein.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 shows released phosphate levels in oat beverage samples after treatment with phytase at different levels and with and without the addition of xylanase. A sample containing only xylanase was also evaluated. A control sample containing no enzyme was used a reference.

[0042] Figure 2 shows percent calcium and magnesium levels relative to the control. The control does not contain any enzyme. P-1000 contains phytase (100.13 FTU / g of the oat-based beverage) with and without xylanase (2.56 GPU / g of the oat-based beverage).

[0043] DETAILED DESCRIPTION OF THE INVENTION

[0044] The term “amino acid sequence” is synonymous with the terms “polypeptide,” “protein,” and “peptide,” and are used interchangeably. Where such amino acid sequences exhibit activity, they may be referred to as an “enzyme.” The conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino- to-carboxy terminal orientation (i.e., N— C).

[0045] “Phytic acid” refers to phosphate ester of myo-inositol containing six phosphate groups. The term “phytate” is used to indicate phytic acid that is complexed with minerals. Because phytic acid found in plants is mostly bound to minerals, the term “phytate” and “phytic acid” are used herein interchangeably.

[0046] The term “nucleic acid” encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation. A “vector” refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, and the like.

[0047] An “expression vector” refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

[0048] In addition to the specific amino acid sequences and polynucleotides mentioned herein, the present invention encompasses variants, homologues, derivatives, and fragments thereof.

[0049] The term "variant" is used to mean a nucleotide sequence or amino acid sequence which differs from a wild-type sequence.

[0050] For example, a variant may include substitutions, insertions, deletions, truncations, transversions and / or inversions at one or more position(s) relative to a wild-type sequence. Variants can be made using methods known in the art for example site scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution as well as using recombinant methods well known in the art. Polynucleotide sequences encoding variant amino acid sequences may readily be synthesized using methods known in the ail.

[0051] In some aspects, the variant is a naturally occurring nucleotide sequence or amino acid sequence which differs from a wild-type sequence. For example, the variant may be a natural genetic variant.

[0052] In some aspects, the variant is an engineered variant. For example, the variant may be engineered by recombinant methods.

[0053] The protein sequences of the instant invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0054] Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other as set forth in Table 1.

[0055] Table 1

[0056] The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e., like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e., from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.

[0057] Replacements may also be made by synthetic amino acids (e.g. unnatural amino acids) include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br- phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, B-alanine*, L-a-amino butyric acid*, L-g- amino butyric acid*, L-a-amino isobutyric acid*, L-e-amino caproic acid#, 7-amino heptanoic acid*, L-methionine sulfone#*, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L- hydroxyproline#, L- thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl- Phe*, pentamethyl-Phe*, L-Phe (4-amino)#, L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (l ,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid#and L-Phe (4- bcnzyl)*.

[0058] The notation * has been utilized for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative whereas # has been utilized to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.

[0059] Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl, or propyl groups in addition to amino acid spacers such as glycine or b-alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, “the peptoid form” is used to refer to variant amino acid residues wherein the a-carbon substituent group is on the residue’s nitrogen atom rather than the a-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.

[0060] The nucleotide sequences for use in the present invention may include within them synthetic or modified nucleotides. Several different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones and / or the addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art. Such modifications may be carried out to enhance the in vivo activity or life span of nucleotide sequences of the present invention.

[0061] The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein.

[0062] Other variants of the sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations. In addition, other homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridizing to the sequences shown in the sequence listing herein. Such sequences may be obtained by probing cDNA libraries or genomic DNA libraries made from other animal species and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.

[0063] Variants and strain / species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention. Conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants / homologues. Sequence alignments can be performed using computer software known in the ail. For example, the GCG Wisconsin PileUp program is widely used.

[0064] The primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.

[0065] Alternatively, such polynucleotides may be obtained by site directed mutagenesis of characterized sequences. This may be useful where for example silent codon sequence changes are required to optimize codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

[0066] The present invention employs, unless otherwise indicated, conventional techniques of biochemistry, molecular biology, microbiology, and recombinant DNA, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.

[0067] As used herein, “percent (%) sequence identity” means that a particular sequence has at least a certain percentage of amino acid residues identical to those in a specified reference sequence, when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithm are:

[0068] Gap opening penalty: 10.0

[0069] Gap extension penalty: 0.05

[0070] Protein weight matrix: BLOSUM series

[0071] DNA weight matrix: IUB

[0072] Delay divergent sequences %: 40

[0073] Gap separation distance: 8

[0074] DNA transitions weight: 0.50

[0075] List hydrophilic residues: GPSNDQEKR

[0076] Use negative matrix: OFF

[0077] Toggle Residue specific penalties: ON

[0078] Toggle hydrophilic penalties: ON

[0079] Toggle end gap separation penalty: OFF

[0080] Deletions are counted as non-identical residues, compared to a reference sequence.

[0081] Deletions occurring at either terminus are included. For example, a variant with five amino acid deletions of the C-terminus of the mature 617 residue polypeptide would have a percent sequence identity of 99% (612 / 617 identical residues x 100, rounded to the nearest whole number) relative to the mature polypeptide. Such a variant would be encompassed by a variant having “at least 99% sequence identity” to a mature polypeptide.

[0082] All references cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings of all references herein specifically referred to are incorporated by reference.

[0083] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0084] Other definitions are set forth below. Production of enzymes

[0085] The enzymes of the present invention can be produced in host cells, for example, by secretion or intracellular expression. A cultured cell material (e.g., a whole-cell broth) having an enzyme can be obtained following secretion of the enzyme into the cell medium. Optionally, the enzyme can be isolated from the host cells, or even isolated from the cell broth, depending on the desired purity of the final enzyme. Suitable host cells include bacterial, fungal (including yeast and filamentous fungi), and plant cells (including algae). Particularly useful host cells include Aspergillus niger, Aspergillus oryzae or Trichoderma reesei. Other host cells include bacterial cells, e.g., Bacillus subtilis or B. licheniformis, as well as Streptomyces, E. coli.

[0086] Vectors

[0087] A DNA construct comprising a nucleic acid encoding an enzyme can be constructed to be expressed in a host cell. Because of the well-known degeneracy in the genetic code, variant polynucleotides that encode an identical amino acid sequence can be designed and made with routine skill. It is also well-known in the art to optimize codon use for a particular host cell. Nucleic acids encoding enzymes of the present invention can be incorporated into a vector. Vectors can be transferred to a host cell using well-known transformation techniques, such as those disclosed below.

[0088] The vector may be any vector that can be transformed into and replicated within a host cell. For example, a vector comprising a nucleic acid encoding an enzyme can be transformed and replicated in a bacterial host cell as a means of propagating and amplifying the vector. The vector also may be transformed into an expression host, so that the encoding nucleic acids can be expressed as a functional enzyme. Host cells that serve as expression hosts can include filamentous fungi, for example. The Fungal Genetics Stock Center (FGSC) Catalogue of Strains lists suitable vectors for expression in fungal host cells. See FGSC, Catalogue of Strains, University of Missouri, al www.fgsc.net (last modified January 17, 2007). A representative vector is pJG153, a promoterless Cre expression vector that can be replicated in a bacterial host. See Harrison et al. (June 2011) Applied Environ. Microbiol. 77: 3916-22. pJG153can be modified with routine skill to comprise and express a nucleic acid encoding an enzyme.

[0089] A nucleic acid encoding an enzyme can be operably linked to a suitable promoter, which allows transcription in the host cell. The promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Exemplary promoters for directing the transcription of the DNA sequence encoding an enzyme, especially in a bacterial host, are the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA or celA promoters, the promoters of the Bacillus licheniformis a-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens a-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral a-amylase, A. niger acid stable a-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, or A. nidulans acetamidase. When a gene encoding an enzyme is expressed in a bacterial species such as E. coli, a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter. Examples of suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters, cbhl is an endogenous, inducible promoter from Trichoderma reesei. See Liu et al. (2008) “Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbhl) promoter optimization,” Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.

[0090] The coding sequence can be operably linked to a signal sequence. The DNA encoding the signal sequence may be the DNA sequence naturally associated with the enzyme gene to be expressed or from a different Genus or species. A signal sequence and a promoter sequence comprising a DNA construct or vector can be introduced into a fungal host cell and can be derived from the same source. For example, the signal sequence is the cbhl signal sequence that is operably linked to a cbhl promoter.

[0091] An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding a variant enzyme. Termination and poly adenylation sequences may suitably be derived from the same sources as the promoter.

[0092] The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBUO, pE194, pAMBl, and pIJ702.

[0093] The vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. lichenifonnis, or a gene that confers antibiotic resistance such as, e.g., ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and xxsC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, such as known in the art. See e.g., International PCT Application WO 91 / 17243.

[0094] Intracellular expression may be advantageous in some respects, e.g., when using certain bacteria or fungi as host cells to produce large amounts of enzyme for subsequent enrichment or purification. Extracellular secretion of enzyme into the culture medium can also be used to make a cultured cell material comprising the isolated enzyme.

[0095] The expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes. The expression vector normally comprises control nucleotide sequences such as a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes. Additionally, the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the enzyme to a host cell organelle such as a peroxisome, or to a particular host cell compartment. Such a targeting sequence includes but is not limited to the sequence, SKL. For expression under the direction of control sequences, the nucleic acid sequence of the enzyme is operably linked to the control sequences in proper manner with respect to expression.

[0096] The procedures used to ligate the DNA construct encoding an enzyme, the promoter, terminator, and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (see, e.g., Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nded., Cold Spring Harbor, 1989, and 3rded., 2001). Transformation and Culture of Host Cells

[0097] An isolated cell, cither comprising a DNA construct or an expression vector, is advantageously used as a host cell in the recombinant production of an enzyme according to the instant invention. The cell may be transformed with the DNA construct encoding the enzyme, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

[0098] Examples of suitable bacterial host organisms are Gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis', Streptomyces species such as Streptomyces murinus; lactic acid bacterial species including Lactococcus sp. such as Lactococcus lactis', Lactobacillus sp. including Lactobacillus reuteri', Leuconostoc sp.; Pediococcus sp.; and Streptococcus sp. Alternatively, strains of a Gram-negative bacterial species belonging to Enterobacteriaceae including E. coli, or to P seudomonadaceae can be selected as the host organism.

[0099] A suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as but not limited to yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces , Yarrowinia, Schizosaccharomyces species or a species of Saccharomyces, including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces such as, for example, 5. pombe species. A strain of the methylotrophic yeast species, Pichia pastoris, can be used as the host organism. Alternatively, the host organism can be a Hansenula species. Suitable host organisms among filamentous fungi include species of Aspergillus, e.g., Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans. Alternatively, strains of a Fusarium species, e.g., Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism. Other suitable strains include Thermomyces and Mucor species. In addition, Trichoderma sp. can be used as a host. A suitable procedure for transformation of Aspergillus host cells includes, for example, that described in EP 238023. An enzyme expressed by a fungal host cell can be glycosylated, i.e., will comprise a glycosyl moiety. The glycosylation pattern can be the same or different as present in the wild-type enzyme. The type and / or degree of glycosylation may impart changes in enzymatic and / or biochemical properties.

[0100] It may be advantageous to delete genes from expression hosts, where the gene deficiency can be cured by the transformed expression vector. Known methods may be used to obtain a fungal host cell having one or more inactivated genes. Gene inactivation may be accomplished by complete or partial deletion, by insertional inactivation or by any other means that renders a gene nonfunctional for its intended purpose, such that the gene is prevented from expression of a functional protein. Any gene from a Trichoderma sp. or other filamentous fungal host that has been cloned can be deleted, for example, cbhl, cbh2, eg IK and eg 12 genes. Gene deletion may be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art.

[0101] Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, e.g., lipofection mediated and DEAE-Dextrin mediated transfection; incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion. General transformation techniques are known in the art. See, e.g., Sambrook et al. (2001), supra. The expression of heterologous protein in Trichoderma is described, for example, in U.S. Patent No. 6,022,725. Reference is also made to Cao et al. (2000) Science 9:991-1001 for transformation of Aspergillus strains. Genetically stable transformants can be constructed with vector systems whereby the nucleic acid encoding an enzyme is stably integrated into a host cell chromosome. Transformants are then selected and purified by known techniques.

[0102] The preparation of Trichoderma sp. for transformation, for example, may involve the preparation of protoplasts from fungal mycelia. See Campbell et al. (1989) Curr. Genet. 16: 53- 56. The mycelia can be obtained from germinated vegetative spores. The mycelia are treated with an enzyme that digests the cell wall, resulting in protoplasts. The protoplasts are protected by the presence of an osmotic stabilizer in the suspending medium. These stabilizers include sorbitol, mannitol, potassium chloride, magnesium sulfate, and the like. Usually, the concentration of these stabilizers varies between 0.8 M and 1.2 M, e.g., a 1.2 M solution of sorbitol can be used in the suspension medium.

[0103] Uptake of DNA into the host Trichoclerma sp. strain depends upon the calcium ion concentration. Generally, between about 10-50 mM CaCh is used in an uptake solution. Additional suitable compounds include a buffering system, such as TE buffer (10 mM Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0 and polyethylene glycol. The polyethylene glycol is believed to fuse the cell membranes, thus permitting the contents of the medium to be delivered into the cytoplasm of the Trichoclerma sp. strain. This fusion frequently leaves multiple copies of the plasmid DNA integrated into the host chromosome.

[0104] Usually, transformation of Trichoclerma sp. uses protoplasts or cells that have been subjected to a permeability treatment, typically at a density of 105to 107 / mL, particularly 2xlO6 / mL. A volume of 100 pL of these protoplasts or cells in an appropriate solution (e.g., 1.2 M sorbitol and 50 mM CaCb) may be mixed with the desired DNA. Generally, a high concentration of PEG is added to the uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension; however, it is useful to add about 0.25 volumes to the protoplast suspension. Additives, such as dimethyl sulfoxide, heparin, spermidine, potassium chloride and the like, may also be added to the uptake solution to facilitate transformation. Similar procedures are available for other fungal host cells. See, e.g., U.S. Patent No. 6,022,725.

[0105] As used herein, Protein Identification (“JGI PID”) numbers for native Trichoderma genes reference Version 2 of the Trichoclerma reesei QM6a genome sequence assembly generated by the Department of Energy Joint Genome Institute (JGI). (The Genome Portal of the Department of Energy Joint Genome Institute, Grigoriev et al., Nucleic Acids Res 2012 Jan;40(Database issue):D26-32. doi: 10.1093 / nai7gkr947). The JGI assembled Scaffold sequences and annotated genes have also been deposited in GeneBank (The National Center for Biotechnology) under the nucleotide accession numbers GL985056.1 through GL985132.1.

[0106] Expression

[0107] A method of producing an enzyme of the instant invention may comprise cultivating a host cell as described above under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and / or culture medium.

[0108] The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of an enzyme. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).

[0109] An enzyme secreted from the host cells can be used in a whole broth preparation. In the present methods, the preparation of a spent whole fermentation broth of a recombinant microorganism can be achieved using any cultivation method known in the art resulting in the expression of an enzyme. Fermentation may, therefore, be understood as comprising shake flask cultivation, small- or large-scale fermentation (including continuous, batch, fed-batch, or solid- state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the enzyme to be expressed or isolated. The term “spent whole fermentation broth” is defined herein as unfractionated contents of fermentation material that includes culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It is understood that the term “spent whole fermentation broth” also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art.

[0110] An enzyme secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed using chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

[0111] The polynucleotide encoding an enzyme in a vector can be operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e., the vector is an expression vector. The control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators. The control sequences may comprise promoters.

[0112] Host cells may be cultured under suitable conditions that allow expression of an enzyme. Expression of the enzymes may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG or Sophorose. Polypeptides can also be produced recombinantly in an in vitro cell-free system, such as the TNT™ (Promega) rabbit reticulocyte system. An expression host also can be cultured in the appropriate medium for the host, under aerobic conditions. Shaking or a combination of agitation and aeration can be provided, with production occurring at the appropriate temperature for that host, e.g.. from about 25 °C to about 75°C (e.g., 30°C to 45°C), depending on the needs of the host and production of the desired enzyme. Culturing can occur from about 12 to about 100 hours or greater (and any hour value there between, e.g., from 24 to 72 hours). Typically, the culture broth is at a pH of about 4.0 to about 8.0, again depending on the culture conditions needed for the host relative to production of an enzyme.

[0113] Methods for Enriching and Purifying enzymes

[0114] Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used to prepare an enzyme polypeptide-containing solution.

[0115] After fermentation, a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques to obtain an enzyme solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultra-filtration, extraction, or chromatography, or the like, are generally used.

[0116] It is desirable to concentrate an enzyme polypeptide-containing solution to optimize recovery. Use of unconcentrated solutions requires increased incubation time to collect the enriched or purified enzyme precipitate.

[0117] The enzyme containing solution is concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme containing solution may be achieved by any of the techniques discussed herein. Exemplary methods of enrichment and purification include but are not limited to rotary vacuum filtration and / or ultrafiltration.

[0118] The enzyme solution is concentrated into a concentrated enzyme solution until the enzyme activity of the concentrated enzyme polypeptide-containing solution is at a desired level.

[0119] Concentration may be performed using, e.g., a precipitation agent, such as a metal halide precipitation agent. Metal halide precipitation agents include but are not limited to alkali metal chlorides, alkali metal bromides and blends of two or more of these metal halides. Exemplary metal halides include sodium chloride, potassium chloride, sodium bromide, potassium bromide and blends of two or more of these metal halides. The metal halide precipitation agent, sodium chloride, can also be used as a preservative.

[0120] The metal halide precipitation agent is used in an amount effective to precipitate an enzyme. The selection of at least an effective amount and an optimum amount of metal halide effective to cause precipitation of the enzyme, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature, and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, after routine testing.

[0121] Generally, at least about 5% w / v (weight / volume) to about 25% w / v of metal halide is added to the concentrated enzyme solution, and usually at least 8% w / v. Generally, no more than about 25% w / v of metal halide is added to the concentrated enzyme solution and usually no more than about 20% w / v. The optimal concentration of the metal halide precipitation agent will depend, among others, on the nature of the specific enzyme polypeptide and on its concentration in the concentrated enzyme solution.

[0122] Another alternative way to precipitate the enzyme is to use organic compounds. Exemplary organic compound precipitating agents include: 4-hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid, alkyl esters of 4-hydroxybenzoic acid, and blends of two or more of these organic compounds. The addition of the organic compound precipitation agents can take place prior to, simultaneously with or subsequent to the addition of the metal halide precipitation agent, and the addition of both precipitation agents, organic compound, and metal halide, may be carried out sequentially or simultaneously.

[0123] Generally, the organic precipitation agents are selected from the group consisting of alkali metal salts of 4-hydroxybenzoic acid, such as sodium or potassium salts, and linear or branched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 12 carbon atoms, and blends of two or more of these organic compounds. The organic compound precipitation agents can be, for example, linear or branched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 10 carbon atoms, and blends of two or more of these organic compounds. Exemplary organic compounds are lineal' alkyl esters of 4- hydroxybenzoic acid, wherein the alkyl group contains from 1 to 6 carbon atoms, and blends of two or more of these organic compounds. Methyl esters of 4-hydroxybenzoic acid, propyl esters of 4-hydroxybenzoic acid, butyl ester of 4-hydroxybenzoic acid, ethyl ester of 4-hydroxybenzoic acid and blends of two or more of these organic compounds can also be used. Additional organic compounds also include but are not limited to 4-hydroxybenzoic acid methyl ester (named methyl PARABEN), 4-hydroxybcnzoic acid propyl ester (named propyl PARABEN), which also are both preservative agents. For further descriptions, see, e.g., U.S. Patent No. 5,281,526.

[0124] Addition of the organic compound precipitation agent provides the advantage of high flexibility of the precipitation conditions with respect to pH, temperature, enzyme concentration, precipitation agent concentration, and time of incubation.

[0125] The organic compound precipitation agent is used in an amount effective to improve precipitation of the enzyme by means of the metal halide precipitation agent. The selection of at least an effective amount and an optimum amount of organic compound precipitation agent, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature, and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, in light of the present disclosure, after routine testing.

[0126] Generally, at least about 0.01% w / v of organic compound precipitation agent is added to the concentrated enzyme solution and usually at least about 0.02% w / v. Generally, no more than about 0.3% w / v of organic compound precipitation agent is added to the concentrated enzyme solution and usually no more than about 0.2% w / v.

[0127] The concentrated polypeptide solution, containing the metal halide precipitation agent, and the organic compound precipitation agent, can be adjusted to a pH, which will, of necessity, depend on the enzyme to be enriched or purified. Generally, the pH is adjusted at a level near the isoelectric point of the enzyme. The pH can be adjusted at a pH in a range from about 2.5 pH units below the isoelectric point (pl) up to about 2.5 pH units above the isoelectric point.

[0128] The incubation time necessary to obtain an enriched or purified enzyme precipitate depends on the nature of the specific enzyme, the concentration of enzyme, and the specific precipitation agent(s) and its (their) concentration. Generally, the time effective to precipitate the enzyme is between about 1 to about 30 hours; usually it does not exceed about 25 hours. In the presence of the organic compound precipitation agent, the time of incubation can still be reduced to less about 10 hours and in most cases even about 6 hours.

[0129] Generally, the temperature during incubation is between about 4°C and about 50°C. Usually, the method is carried out at a temperature between about 10°C and about 45°C e.g., between about 20°C and about 40°C). The optimal temperature for inducing precipitation varies according to the solution conditions and the enzyme or precipitation agent(s) used. The overall recovery of enriched or purified enzyme precipitate, and the efficiency with which the process is conducted, is improved by agitating the solution comprising the enzyme, the added metal halide, and the added organic compound. The agitation step is done both during addition of the metal halide and the organic compound, and during the subsequent incubation period. Suitable agitation methods include mechanical stirring or shaking, vigorous aeration, or any similar technique.

[0130] After the incubation period, the enriched or purified enzyme is then separated from the dissociated pigment and other impurities and collected by conventional separation techniques, such as filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, press filtration, cross membrane microfiltration, cross flow membrane microfiltration, or the like. Further enrichment or purification of the enzyme precipitate can be obtained by washing the precipitate with water. For example, the enriched or purified enzyme precipitate is washed with water containing the metal halide precipitation agent, or with water containing the metal halide and the organic compound precipitation agents.

[0131] During fermentation, an enzyme polypeptide accumulates in the culture broth. For the isolation, enrichment, or purification of the desired enzyme, the culture broth is centrifuged or filtered to eliminate cells, and the resulting cell-free liquid is used for enzyme enrichment or purification. In one embodiment, the cell-free broth is subjected to salting out using ammonium sulfate at about 70% saturation; the 70% saturation-precipitation fraction is then dissolved in a buffer and applied to a column such as a Sephadex G-100 column and eluted to recover the enzyme-active fraction. For further enrichment or purification, a conventional procedure such as ion exchange chromatography may be used.

[0132] Enriched or purified enzymes can be made into a final product that is either liquid (solution, slurry) or solid (granular, powder).

[0133] Description of the Preferred Embodiments

[0134] In an aspect of the present invention, a method is presented for increasing the nutritional value of a plant-based beverage, the method having the steps of: a.) providing a plant material having phytate; b.) suspending the plant material in an aqueous phase having water wherein the aqueous phase has no organic solvent to provide a solution; c.) adding a phytase and a xylanase to the solution; d.) incubating the solution for sufficient time to convert the phytate to inorganic phosphate; and c.) processing the solution into the plant-based beverage.

[0135] Preferably, the plant material is plant flour, plant protein isolate or plant protein concentrate. More preferably, the plant material is plant flour. Preferably, the plant flour is produced by a method having the steps of providing a plant component and grinding the plant component into plant flour. Preferably, the plant component is cereal, pseudo-cereal, legume, seed, fruit or nuts.

[0136] Preferably, the plant material is oat, rice, quinoa, soybean, pea, peanut, sesame, sunflower, walnut, cashew, almond or coconut. More preferably, the plant component is oat. Preferably, the oat is oat grain.

[0137] Preferably, the plant material is 1 to 60% wt / wt of the solution. More preferably, the plant material comprises 8 to 25% wt / wt of the solution. Still more preferably, the plant material is f0 to 20% wt / wt of the solution, in the most preferred embodiments, the plant material comprises 12 to 15% wt / wt of the solution.

[0138] Preferably, the phytase added in step c is in an amount from about 0.1 to about 1,000 FTU per gram of the plant material. More preferably, the phytase is added in an amount from about 1 to about 500 FTU per gram of the plant material. Yet more preferably, the phytase added is in an amount from about 5 to about 100 FTU per gram of the plant material. In the most preferred embodiments, the phytase added is in an amount from about 10 to about 50 FTU per gram of the plant material.

[0139] Preferably, the xylanase added in step c. is in an amount from about 0.0005 to about 1000 GPU per gram of the plant material. More preferably, the xylanase is added is in an amount from about 1 to about 500 GPU per gram of the plant material. Still more preferably, the xylanase added is in an amount from about 2 to about 100 GPU per gram of the plant material.

[0140] Preferably, the method has an additional step of adding one or more additional enzymes selected from the group consisting of a protease, a carboxypeptidase, a cellulase, a xylanase, a mannase, an amylase, a-galactosidase, a pectinase, a glucanase, an esterase and mixtures thereof.

[0141] Preferably, the additional step is performed at the same time as step c.

[0142] Preferably, the phytase is a polypeptide having at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO:2. More preferably, the phytase is a polypeptide according to SEQ ID NO:2. Preferably, the xylanase is a polypeptide having at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO:4. More preferably, the xylanase is a polypeptide according to SEQ ID NO:4.

[0143] In another aspect of the present invention, a method is presented for increasing the nutritional value of a plant-based beverage, the method having the steps of: a.) providing a plant material having phytate; b.) suspending the plant material in an aqueous phase having essentially only water; c.) adding a phytase and a xylanase to the solution; d.) incubating the solution for sufficient time to convert the phytate to inorganic phosphate; and e.) processing the solution into the plant-based beverage.

[0144] Preferably, the aqueous phase has only water.

[0145] Preferably, the plant material is plant flour, plant protein isolate or plant protein concentrate. More preferably, the plant material is plant flour. Preferably, the plant flour is produced by a method having the steps of providing a plant component and grinding the plant component into plant flour. Preferably, the plant component is cereal, pseudo-cereal, legume, seed, fruit or nuts.

[0146] Preferably, the plant material is oat, rice, quinoa, soybean, pea, peanut, sesame, sunflower, walnut, cashew, almond or coconut. More preferably, the plant component is oat. Preferably, the oat is oat grain.

[0147] Preferably, the plant material is 1 to 60% wt / wt of the solution. More preferably, the plant material comprises 8 to 25% wt / wt of the solution. Still more preferably, the plant material is 10 to 20% wt / wt of the solution. In the most preferred embodiments, the plant material comprises 12 to 15% wt / wt of the solution.

[0148] Preferably, the phytase added in step c is in an amount from about 0.1 to about 1,000 FTU per gram of the plant material. More preferably, the phytase is added in an amount from about 1 to about 500 FTU per gram of the plant material. Yet more preferably, the phytase added is in an amount from about 5 to about 100 FTU per gram of the plant material. In the most preferred embodiments, the phytase added is in an amount from about 10 to about 50 FTU per gram of the plant material.

[0149] Preferably, the xylanase added in step c. is in an amount from about 0.0005 to about 1000 GPU per gram of the plant material. More preferably, the xylanase is added is in an amount from about 1 to about 500 GPU per gram of the plant material. Still more preferably, the xylanasc added is in an amount from about 2 to about 100 GPU per gram of the plant material.

[0150] Preferably, the method has an additional step of adding one or more additional enzymes selected from the group consisting of a protease, a carboxypeptidase, a cellulase, a xylanase, a mannase, an amylase, a-galactosidase, a pectinase, a glucanase, an esterase and mixtures thereof.

[0151] Preferably, the additional step is performed at the same time as step c.

[0152] The present disclosure is described in further detail in the following examples, which are not in any way intended to limit the scope of the disclosure as claimed. The attached figures are meant to be considered as integral parts of the specification and description of the disclosure. The following examples are offered to illustrate, but not to limit the claimed disclosure.

[0153] EXAMPLES

[0154] Materials and Methods

[0155] Oat samples that were dehulled, steam treated and rolled into flakes were used to prepare the oat-based beverages, obtained (Supplier: Lantmannen). Phytase enzyme - Purified Phyzyme XP (6200 FTU / g) and xylanase enzyme -AIM Xylanase AN 180 K (204500 GPU / g) enzyme were obtained from IFF (Brabrand, Denmark). The phytase enzyme was derived from an E. coli strain and expressed in a suitable Schiwsaccharmyces pompe host. The mature phytase which was used in the experiments discussed below is shown in SEQ ID NO:2. The phytase precursor is show in SEQ ID NO:1. The xylanase enzyme was derived from an Aspergillus niger strain and expressed in a suitable Bacillus subtilis host. The mature xylanase which was used in the experiments discussed below is shown in SEQ ID NO:4. The xylanase precursor is show in SEQ ID NO:3. Phosphate levels were determined using the reagent, Malachite Green (POPB_DP reagent from BioAssay Systems, USA).

[0156] Example 1: Evaluation of the effect of phytase in combination with xylanase on the release of phosphates from oat-based beverage

[0157] In this experiment, 10% oat-based beverage was used to evaluate the effect of the enzymes. The rolled oat samples were first ground using a miniblender for 1.5 min, in 30 sec increments. 4g of the ground oat samples were combined with 36 g of purified water in 50 mL centrifuge tubes and thoroughly mixed using a vortex-mixer (5 see, 3x). The samples were then adjusted to pH 5 using 10% acetic acid (w / w). Following pH adjustment, phytase and xylanase were dosed as indicated in Table 1. Xylanase enzyme was diluted with purified water (1:99, w / w) prior to adding to the oat beverages as the activity was too high to start with. A control sample containing no enzyme was used as a reference. Additional water (purified) was added into the samples to adjust for volume differences between samples (Table 1).

[0158] Following the enzyme addition, the oat beverage samples were incubated at 37°C for 24 hr. The sample pH was adjusted to 6.5 using NaOH at the end of the incubation period. The samples were then frozen until further analysis.

[0159] Table 1: Enzyme levels added into the oat-based beverages per gram of oat-based beverage1

[0160] 'Enzyme activity: Phytase (6200 FTU / g) - and Xylanase - 2045 GPU / ml. Control sample was used as a reference with no enzyme addition.

[0161] Determination of phosphate levels:

[0162] Phosphate levels were determined using Malachite Green reagent. Quantification was carried out using a phosphate standard curve (0 - 40 pM). The oat beverage samples were thoroughly mixed, transferred into tubes (approx. 1 .5 mL), and centrifuged at 14,000 rpm for 10 min. 10 pL of each sample was then transferred into 96 well plates and diluted with purified water (400x). 50 L of the diluted samples were then mixed with 100 pL of the reagent and incubated for 30 min at ambient temperature. Absorbance was measured at 620nm.

[0163] Results: Evaluation of the effect of phytase in combination with xylanase on the release of phosphates from oat-based beverage

[0164] Phosphate levels:

[0165] The level of free phosphate levels ranged from 1.08 - 3.37 mM within the oat-beverages. The control sample contained the least amount of free phosphates (1.08mM) followed by the sample containing xylanase only (2.43 mM). As expected, the level of free phosphate increased with increased dose of phytase enzyme due to release of phosphate from IP6. Interestingly, the addition of xylanase and phytase together substantially increased the level of phosphates released as compared to the phytase alone at each dosage levels. Figure 1 shows released phosphate levels in oat beverage samples after treatment with phytase at different levels with and without the addition of xylanase.

[0166] Mineral Levels:

[0167] Mineral analysis was carried out using ICP from the following samples: control, xylanase alone, and phytase with and without xylanase (P-1000 and P-1000 + xyl). Mineral levels reported in Figure 2 are relative to the level found in the control (%). Figure 2 shows percent calcium and magnesium levels relative to the control. The control does not contain any enzyme. P-1000 contains phytase (100.13 FTU / g of the oat-based beverage) with and without xylanase (2.56 GPU / g of the oat-based beverage).

[0168] As shown in the Figure 2, all enzyme-treated oat-beverage samples had higher calcium and magnesium levels as compared to the control sample. Similar to the phosphate levels, the mineral content was higher in the samples containing both phytase and xylanase as compared to phytase alone. Example 2: The use of phytase in combination with xylanase in plant-based beverage and meat-alternatives

[0169] The use of phytase and xylanase to enhance the release of phosphates and increase accessible minerals may be implemented in other plant-based beverages. Plant-based beverages may be prepared from cereals (oats and rice), and pseudo-cereals (quinoa) as well as their protein components, legumes (soybeans and peas), seeds (peanuts, sesame, and sunflower), nuts (walnuts, cashew and almonds), and / or fatty fruits (coconut). Similar to beverages, the enzymes can be used in plant-based meat-alternatives to increase the release of phosphate and accessible minerals. The plant-based meat-alternatives may also be prepared from cereals, legumes, seeds, nuts and combination of these plant components.

[0170] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the ail without departing from the invention. Various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

What is claimed is:

1. A method for increasing the nutritional value of a plant-based beverage, said method comprising the steps of: a.) providing a plant material comprising phytate; b.) suspending the plant material in an aqueous phase comprising water wherein said aqueous phase has no organic solvent to provide a solution; c.) adding a phytase and a xylanase to the solution; d.) incubating the solution for sufficient time to convert the phytate to inorganic phosphate; and e.) processing the solution into the plant-based beverage.

2. The method of claim 1 wherein the plant material is plant flour, plant protein isolate or plant protein concentrate.

3. The method of claim 2 wherein the plant material is plant flour.

4. The method of claim 3 wherein the plant flour is produced by a method comprising providing a plant component and grinding said plant component into plant flour.

5. The method of claim 4 wherein the plant component comprises cereal, pseudo-cereal, legume, seed, fruit or nuts.

6. The method of any of the preceding claims wherein the plant material comprises oat, rice, quinoa, soybean, pea, peanut, sesame, sunflower, walnut, cashew, almond or coconut.

7. The method of claim 6 wherein the plant component comprises oat.

8. The method of claim 7 wherein the oat comprises oat grain.

9. The method of any of the preceding claims wherein the plant material comprises 1 to 60% wt / wt of the solution.

10. The method of claim 9 wherein the plant material comprises 8 to 25% wt / wt of the solution.

11. The method of claim 10 wherein the plant material comprises 10 to 20% wt / wt of the solution.

12. The method of claim 11 wherein the plant material comprises 12 to 15% wt / wt of the solution.

13. The method of any of the preceding claims wherein the phytase added in step c is in an amount from about 0.1 to about 1,000 FTU per gram of the plant material.

14. The method of claim 13 wherein the phytase is in added an amount from about 1 to about 500 FTU per gram of the plant material.

15. The method of claim 14 wherein the phytase is added in an amount from about 5 to about 100 FTU per gram of the plant material.

16. The method of claim 15 wherein the phytase is added in an amount from about 10 to about 50 FTU per gram of the plant material.

17. The method of any of the preceding claims wherein the xylanase added in step c. is added in an amount from about 0.0005 to about 1000 GPU per gram of the plant material.

18. The method claim 17 wherein the xylanase is added in an amount from about 1 to about 500 GPU per gram of the plant material.

19. The method claim 18 wherein the xylanase is added in an amount from about 2 to about 100 GPU per gram of the plant material.

20. The method of any or the preceding claims comprising an additional step of adding one or more additional enzymes selected from the group consisting of a protease, a carboxypeptidase, a cellulase, a xylanase, a mannase, an amylase, a-galactosidase, a pectinase, a glucanase, an esterase and mixtures thereof.

21. The method of claim 20 wherein the additional step is performed at the same time as step c.

22. The method of any of the preceding claims wherein the phytase comprises a polypeptide having at least 80% sequence identity to SEQ ID NO:2.

23. The method of any of claim 22 wherein the phytase comprises a polypeptide having at least 85% sequence identity to SEQ ID NO:2.

24. The method of claim 23 wherein the phytase comprises a polypeptide having at least 90% sequence identity to SEQ ID NO:2.

25. The method of claim 24 wherein the phytase comprises a polypeptide having at least 95% sequence identity to SEQ ID NO:2.

26. The method of claim 25 wherein the phytase comprises a polypeptide having at least 98% sequence identity to SEQ ID NO:2.

27. The method of claim 26 wherein the phytase comprises a polypeptide having at least 99% sequence identity to SEQ ID NO:2.

28. The method of claim 27 wherein the phytase comprises a polypeptide according to SEQ ID NO:2.

29. The method of any of the preceding claims wherein the xylanase comprises a polypeptide having at least 80% sequence identity to SEQ ID NO:4.

30. The method of claim 29 wherein the xylanase comprises a polypeptide having at least 85% sequence identity to SEQ ID NO:4.

31. The method of claim 30 wherein the xylanase comprises a polypeptide having at least 90% sequence identity to SEQ ID NO:4.

32. The method of claim 31 wherein the xylanase comprises a polypeptide having at least 95% sequence identity to SEQ ID NO:4.

33. The method of claim 32 wherein the xylanase comprises a polypeptide having at least 98% sequence identity to SEQ ID NO:4.

34. The method of claim 33 wherein the xylanase comprises a polypeptide having at least 99% sequence identity to SEQ ID NO:4.

35. The method of claim 34 wherein the xylanase comprises a polypeptide according to SEQ ID NO:4.

36. A method for increasing the nutritional value of a plant-based beverage, said method comprising the steps of: a.) providing a plant material comprising phytate; b.) suspending the plant material in an aqueous phase consisting essentially of water; c.) adding a phytase and a xylanase to the solution; d.) incubating the solution for sufficient time to convert the phytate to inorganic phosphate; and e.) processing the solution into the plant-based beverage.

37. The method of claim 36 wherein the aqueous phase consists of water.

38. The method of claim 36 or 37 wherein the plant material is plant flour, plant protein isolate or plant protein concentrate.

39. The method of claim 38 wherein the plant material is plant flour.

40. The method of claim 39 wherein the plant flour is produced by a method comprising providing a plant component and grinding said plant component into plant flour.

41. The method of claim 36 wherein the plant component comprises cereal, pseudo-cereal, legume, seed, fruit or nuts.

42. The method of any of claims 36 to 41 wherein the plant material comprises oat, rice, quinoa, soybean, pea, peanut, sesame, sunflower, walnut, cashew, almond or coconut.

43. The method of claim 42 wherein the plant component comprises oat.

44. The method of claim 43 wherein the oat comprises oat grain.

45. The method of any of claims 36 to 44 wherein the plant material comprises 1 to 60% wt / wt of the solution.

46. The method of claim 45 wherein the plant material comprises 8 to 25% wt / wt of the solution.

47. The method of claim 46 wherein the plant material comprises 10 to 20% wt / wt of the solution.

48. The method of claim 47 wherein the plant material comprises 12 to 15% wt / wt of the solution.

49. The method of any claims 36 to 48 wherein the phytase added in step c is in an amount from about 0.1 to about 1,000 FTU per gram of the plant material.

50. The method of claim 49 wherein the phytase added is in an amount from about 1 to about 500 FTU per gram of the plant material.

51. The method of claim 50 wherein the phytase added is in an amount from about 5 to about 100 FTU per gram of the plant material.

52. The method of claim 51 wherein the phytase added is in an amount from about 10 to about 50 FTU per gram of the plant material.

53. The method of claims any of claims 36 to 52 wherein the xylanase added in step c. is in an amount from about 0.0005 to about 1000 GPU per gram of the plant material.

54. The method claim 53 wherein the xylanase added is in an amount from about 1 to about 500 GPU per gram of the plant material.

55. The method claim 54 wherein the xylanase added is in an amount from about 2 to about 100 GPU per gram of the plant material.

56. The method of any of claims 36 to 55 comprising an additional step of adding one or more additional enzymes selected from the group consisting of a protease, a carboxypeptidase, a cellulase, a xylanase, a mannase, an amylase, a-galactosidase, a pectinase, a glucanase, an esterase and mixtures thereof.

57. The method of claim 56 wherein the additional step is performed at the same time as step c.

58. The method of any of claims 36 to 57 wherein the phytase comprises a polypeptide having at least 80% sequence identity to SEQ ID NO:2.

59. The method of claim 58 wherein the phytase comprises a polypeptide having at least 85% sequence identity to SEQ ID NO:2.

60. The method of claim 59 wherein the phytase comprises a polypeptide having at least 90% sequence identity to SEQ ID NO:2.

61. The method of claim 60 wherein the phytase comprises a polypeptide having at least 95% sequence identity to SEQ ID NO:2.

62. The method of claim 61 wherein the phytase comprises a polypeptide having at least 98% sequence identity to SEQ ID NO:2.

63. The method of claim 62 wherein the phytase comprises a polypeptide having at least 99% sequence identity to SEQ ID NO:2.

64. The method of claim 63 wherein the phytase comprises a polypeptide according to SEQ ID NO:2.

65. The method of any of claims 36 to 64 wherein the xylanase comprises a polypeptide having at least 80% sequence identity to SEQ ID NO:4.

66. The method of claim 65 wherein the xylanase comprises a polypeptide having at least 85% sequence identity to SEQ ID NO:4.

67. The method of claim 66 wherein the xylanase comprises a polypeptide having at least 90% sequence identity to SEQ ID NO:4.

68. The method of claim 67 wherein the xylanase comprises a polypeptide having at least 95% sequence identity to SEQ ID NO:4.

69. The method of claim 68 wherein the xylanase comprises a polypeptide having at least 98% sequence identity to SEQ ID NO:4.

70. The method of claim 69 wherein the xylanase comprises a polypeptide having at least 99% sequence identity to SEQ ID NO:4.

71. The method of claim 70 wherein the xylanase comprises a polypeptide according to SEQ ID NO:4.

Images