Method for reducing cocoa component of cake while maintaining chocolate flavor

Abstract

The present invention relates to methods of reducing the cocoa component of a chocolate cake without impacting chocolate flavor. More particularly, the present invention relates to novel method of using cell wall modifying enzyme in making cocoa powder-based cake product to allow reduction of cocoa while maintaining chocolate flavor.

Description

[0001] METHOD FOR REDUCING COCOA COMPONENT OF CAKE WHILE MAINTAINING CHOCOLATE FLAVOR

[0002] CROSS REFERENCE TO REPLATED APPLICATIONS

[0003] This application claims benefit to U.S. Provisional Application Nos. 63 / 556,476, filed February 22, 2024, and 63 / 674,663, filed July 23, 2024, which is hereby incorporated by reference in its entirety.

[0004] TECHNICAL FIELD

[0005] The present invention relates to novel methods of making a cocoa powder containing cake product. More particularly, the present invention relates to an improved cocoa powder containing cake product making method by which the amount of cocoa powder in a recipe can be substantially reduced while maintaining the chocolatey flavor.

[0006] BACKGROUND

[0007] Cocoa bean is an important agriculture commodity which is used as a raw material in the food industry for the production of confectionary, beverages and bakery products. Cocoa beans undergo several processing conditions including, but not limited to, fermentation, grinding, roasting, milling to obtain the cocoa mass (cocoa liquor). An alkalization process (Dutching process) can also be implemented to modify color and flavor of final cocoa product. Cocoa powder is generated from the cocoa mass that is separated as cocoa cake and cocoa butter through a pressing process. The cocoa cake is further processed and milled into standardized particle size to produce the cocoa powder that is used as an ingredient in chocolate-based beverages and bakery products.

[0008] As the application and use of cocoa powder continues to grow, its demand has significantly increased resulting in substantial price increases over the years. To keep cocoa powder-based products affordable, manufacturers have had to reduce the amount of cocoa used which results in decreased chocolate flavor. Consumers find such products to be inferior. As a result, there is a continuing need for methods allowing for reduction of cocoa powder without loss of chocolate flavor. SUMMARY OF THE INVENTION

[0009] The present invention relates to the reduction of cocoa powder used in bakery products including cakes and brownies while maintaining desirable chocolate flavor. Surprisingly, it was found in accordance with the present invention that chocolate flavor can be maintained with reduced cocoa powder via the use of one or more cell-wall modifying enzymes. Without being bound by theory, the present invention includes the use of one or more cell wall modifying enzymes in the production of chocolate cake to allow reduced level of cocoa powder in the formulation or recipe. In accordance with the present invention, the amount of cocoa powder required in a shortened chocolate cake recipe for example, also called a brownie, can be reduced by 30% while the same chocolate flavor can be maintained.

[0010] In accordance with an aspect of the present invention, a method of preparing a chocolate cake having a reduced amount of cocoa in a recipe is presented, the method having the steps of: a. preparing a batter of the cake by adding: i. sugar; ii. flour; iii. cell wall modifying enzyme; iv egg; v. cocoa powder and; b. putting the batter in a suitable baking mold; and c. baking the cake; wherein the recipe contains an amount of cocoa which is reduced at least 5% w / w.

[0011] Optionally, the amount of cocoa is reduced by at least 10, 15, 20, 25, 30, 35, 40, 45 or 50% w / w. Optionally, the cake is shortened cake. Optionally, the cell wall modifying enzyme is one or more of a glycoside hydrolase, a carbohydrate esterase, a glycosyltransferase or a polysaccharide lyase. Optionally, the batter further includes one or more hydrocolloids. Optionally, the hydrocolloids are thickening / viscosity hydrocolloids or gelling agents. Optionally, the thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and the gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum. xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

[0012] In an aspect of the present invention, a method is presented of preparing a chocolate cake having a reduced amount of cocoa in a recipe, the method has the steps of: a. preparing a batter of the cake by adding: i. sugar; ii. flour; iii. an enzyme composition comprising one or more cell wall modifying enzymes; iv egg; v. cocoa powder and; b. putting the batter in a suitable baking mold; and c. baking the cake; wherein the recipe contains an amount of cocoa which is reduced at least 5% w / w.

[0013] Optionally, the one or more cell-wall modifying enzymes is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase. More preferably, the one or more cellwall modifying enzymes is a beta-glucanase.

[0014] Optionally, the amount of cocoa reduced is at least 10, 15, 20, 25, 30, 35, 40, 45 or 50% w / w.

[0015] Optionally, the cake is shortened cake.

[0016] Optionally, the batter further contains one or more hydrocolloids. Optionally, the one or more hydrocolloids are thickening / viscosity hydrocolloids or gelling agents. Optionally, the thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and said gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

[0017] In another aspect of the present invention, a method for increasing softness in a cake is presented having the steps: a. preparing a batter of the cake by adding: i. sugar; ii. flour; iii. an enzyme composition comprising one or more cell wall modifying enzymes; iv egg; v. cocoa powder and; b. putting the batter in a suitable baking mold ; and c. baking the cake; wherein the cake has increased softness compared to a cake prepared from batter not containing the one or more cell wall modifying enzymes.

[0018] Optionally, the softness is increased by at least 10, 20, 25, 30, 35, 40, 45 or 50%.

[0019] Optionally, the cake is shortened cake.

[0020] Optionally, the one or more cell-wall modifying enzymes is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase. Optionally, the one or more cell-wall modifying enzymes is a beta-glucanase. Optionally, the batter further contains one or more hydrocolloids. Optionally, the one or more hydrocolloids arc thickcning / viscosity hydrocolloids or gelling agents. Optionally, the thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and said gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

[0021] In another aspect of the present invention, a method of preparing a chocolate flavored food is provided having a reduced amount of cocoa in a recipe, the method comprising the steps of: (i). hydrating cocoa powder; (ii). treating the hydrated cocoa powder of step (i) with an enzyme composition having one or more cell wall modifying enzymes; (iii). preparing the chocolate flavored food with the treated cocoa of step (ii); wherein the recipe contains an amount of cocoa which is reduced at least 5% w / w.

[0022] Optionally, the one or more cell-wall modifying enzymes is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase. Optionally, the one or more cell-wall modifying enzymes is a beta-glucanase.

[0023] Optionally, the amount of cocoa reduced is at least 10, 15, 20, 25, 30, 35, 40, 45 or 50% w / w.

[0024] Optionally, the chocolate flavored food is milk, including cow’s milk, raw milk, prepasteurize milk, whole milk, skimmed milk, lactase-treated milk, reduced-lactose milk, lactose- free milk, reconstituted milk, condensed milk, ultra-filtered milk, fresh-fermented beverages, neutral pH beverages, beverage powder, dairy protein powder, ice cream mix, ice cream, custard, custard mix, frozen dessert, frozen dessert mix, mousse, pudding, pudding powder, sorbet, dessert, milkshakes, Ryazhenka,, whey based drink, breads, buns, rolls, baguettes, artisan bread, bread sticks, breadcrumbs, muffins, cupcakes, wafers, cookies, biscuits, crackers, scones, naan, pies, cakes, fruit cakes, sweet doughs, laminated doughs, croissants, waffles, brownies, brioche, panettone, pancakes, chaiupas, nankatais, bars, tortillas, pizza, doughnuts, soft pretzels, hard pretzels, fillings, puddings, frostings, icings, glazes, and spreads. Optionally, the chocolate flavored food further comprises one or more hydrocolloids. Optionally, the one or more hydrocolloids arc thickcning / viscosity hydrocolloids or gelling agents. Optionally, the thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and said gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

[0025] In another aspect of the present invention, a method of preparing chocolate milk having improved cocoa perception is presented, having a reduced amount of cocoa in a recipe, the method having the steps of hydrating cocoa powder; treating cocoa powder with an enzyme composition comprising one or more cell wall-modifying enzymes; and mixing a milk with the enzyme composition treated cocoa of step ii.), wherein the recipe contains an amount of cocoa which is reduced at least 5% w / w.

[0026] Optionally, the one or more cell-wall modifying enzymes is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase. Optionally, the one or more cell-wall modifying enzymes is a beta-glucanase.

[0027] Optionally, the milk is cow’s milk. Optionally, the cow’s milk is raw milk, prepasteurize milk, whole milk, skimmed milk, lactase-treated milk, reduced-lactose milk, lactose- free milk, reconstituted milk, condensed milk or ultra-filtered milk.

[0028] Optionally, the milk comprises one or more hydrocolloids. Optionally, the one or more hydrocolloids are thickening / viscosity hydrocolloids or gelling agents. Optionally, the thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and said gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

[0029] Optionally, the method has the further the step of homogenizing and pasteurizing the milk.

[0030] Optionally, the step of contacting with the cocoa with an enzyme composition is performed after homogenizing and pasteurizing the milk.

[0031] In another aspect of the present invention, a method of preparing chocolate milk having improved cocoa perception is provided having the steps of: i.) hydrating cocoa powder; treating cocoa powder with an enzyme composition comprising one or more cell wall-modifying enzymes; and mixing a milk with the enzyme composition treated cocoa of step ii.).

[0032] Optionally, the one or more cell-wall modifying enzymes is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, a xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase. Optionally, the one or more cell-wall modifying enzymes is a beta-glucanase.

[0033] Optionally, the milk is cow’s milk. Optionally, the cow’s milk is raw milk, prepasteurize milk, whole milk, skimmed milk, lactase-treated milk, reduced-lactose milk, lactose- free milk, reconstituted milk, condensed milk or ultra-filtered milk.

[0034] Optionally, the milk comprises one or more hydrocolloids. Optionally, the one or more hydrocolloids are thickening / viscosity hydrocolloids or gelling agents. Optionally, the thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and said gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

[0035] Optionally, the method has the further the step of homogenizing and pasteurizing the milk.

[0036] Optionally, the step of contacting with the cocoa with an enzyme composition is performed after homogenizing and pasteurizing the milk.

[0037] In another aspect of the present invention, a method of increasing the theobromine content of cocoa is presented having the steps of: a. providing an aqueous slurry of cocoa; and b. adding one or more cell- wall modifying enzymes to said slurry to provide cocoa enriched in theobromine. Optionally, the cocoa is enriched at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold in theobromine.

[0038] Optionally, the method has the further step of drying the slurry of step b. to produce a theobromine enhanced cocoa powder.

[0039] Optionally, the one or more cell-wall modifying enzymes is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, a xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase. Optionally, the one or more cell- wall modifying enzymes is a beta-glucanase.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 shows texture analysis of brownie samples comparing the control (no cell wall modifying enzyme enzyme), 0.1% and 0.25% cell wall modifying enzyme. The main texture property analyzed was firmness measured in gram-force (g).

[0042] Figure 2 shows texture analysis of brownie samples comparing the control (100% cocoa powder with no added enzyme), 100% cocoa powder with enzyme addition, 70% cocoa powder with no added enzyme, and 70% cocoa powder with enzyme addition at day 10 and day 37. The main texture property analyzed was firmness measured in gram-force (g).

[0043] DETAILED DESCRIPTION OF THE INVENTION

[0044] Definitions

[0045] 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 (z.e., N— >C).

[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 arc presented in 5'-to-3' orientation.

[0047] 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.

[0048] 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.

[0049] In addition to the specific amino acid sequences and polynucleotides mentioned herein, the present invention encompasses variants, homologues, derivatives, and fragments thereof.

[0050] The term "variant" is used to mean a nucleotide sequence or amino acid sequence which differs from a wild-type sequence.

[0051] 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.

[0052] 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.

[0053] In some aspects, the variant is an engineered variant. For example, the variant may be engineered by recombinant methods.

[0054] 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.

[0055] 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.

[0056] Table 1

[0057] 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.

[0058] 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-norlcucinc*, L-norv aline*, p-nitro-L-phcnylalaninc*, 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- benzyl)*.

[0059] 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.

[0060] 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.

[0061] 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 ail. These include methylphosphonate and phosphoro thioate 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 canned out to enhance the in vivo activity or life span of nucleotide sequences of the present invention.

[0062] The present invention also encompasses the use of nucleotide sequences that are complementary to the sequences presented herein.

[0063] 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.

[0064] 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 art. For example, the GCG Wisconsin PileUp program is widely used.

[0065] 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.

[0066] 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.

[0067] 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. el 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: Essenticd 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.

[0068] 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:

[0069] Gap opening penalty: 10.0

[0070] Gap extension penalty: 0.05

[0071] Protein weight matrix: BLOSUM series

[0072] DNA weight matrix: IUB

[0073] Delay divergent sequences %: 40

[0074] Gap separation distance: 8

[0075] DNA transitions weight: 0.50

[0076] List hydrophilic residues: GPSNDQEKR

[0077] Use negative matrix: OFF

[0078] Toggle Residue specific penalties: ON

[0079] Toggle hydrophilic penalties: ON

[0080] Toggle end gap separation penalty: OFF

[0081] Deletions are counted as non-identical residues, compared to a reference sequence.

[0082] 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% (6121 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.

[0083] As used herein, the term “cell wall modifying enzyme” means an enzyme or enzymes capable of modifying carbohydrate polymers found in plant cell walls. Such enzymes include, but are not limited to, glycoside hydrolases, carbohydrate esterases, glycosyltransferases, and polysaccharide lyase. As used herein, “glycoside hydrolyases” are enzymes which hydrolyze the glycosidic bond between two or more carbohydrates or between a carbohydrate and a noncarbohydrate moiety. As used herein, “carbohydrate esterases” catalyze the de-0 or de-N- acylation of substituted saccharides. As used herein, “glycosyltransferases” are enzymes that catalyze the transfer of sugar moictics from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. As used herein, “polysaccharide lyases” are a group of enzymes that cleave uronic acid-containing polysaccharide chains via a P-elimination mechanism to generate an unsaturated hexenuronic acid residue and a new reducing end.

[0084] As used herein, “shortened cake” means a cake or sweet good characterized by high levels of sugar, fat, and water in the recipe. Shortened chocolate cake is also referred to as a brownie. A shortened cake batter is prepared by adding sugar, low protein flour, fat, egg, a liquid (commonly water or milk), and a baking powder (or a leavening system). Foam cakes typically utilize beating eggs to produce and entrap air bubbles in the cake, while shortened cakes typically rely on leavening agents to assist in the air bubble production. A chocolate cake or a brownie has cocoa powder added to the batter. This batter is placed into a baking mold and baked to a temperature high enough and held there long enough to gelatinize the starch, coagulate the egg protein, and evaporate water to the desired end moisture level.

[0085] As used herein “firmness” is a parameter used to describe the force applied by a probe traveling downward to a specific depth (7 mm) of deformation of the cake product with a specific speed (1 mm / s) in a single compression test. The unit of measurement for firmness is gram-force (g). “Softness” is used to describe a product that displays less resistance to deformation thereby requiring less force. Therefore, “softness” is used to describe texture property that is the opposite of firmness.”

[0086] In accordance with the instant invention, proteins, including enzymes, of the present invention exist in multiple forms. Proteins of the instant invention may be clipped or trimmed (i.e., removing amino acids) from the N-terminus and / or the C-terminus, resulting in a shorter protein. Proteins of the instant invention can also have internal deletions. Shorter proteins as described herein can have higher activity or lower activity than longer counterparts. Without being bound by theory, as used herein the term “pre -pro-protein” is a protein, including an enzyme, which has an N-terminal signal peptide that targets the protein for secretion. A pre-proprotein is sometimes referred to herein as “full length” or “full length protein”. The N-terminal signal peptide is cleaved off in the endoplasmic reticulum to yield a “pro-protein”. A proprotein, as used herein, is shorter in length than the full-length protein (it is missing the signal peptide) but longer than the mature protein. In general, a pro-protein is inactive or less active than the mature protein. A pro-protein can he activated or converted to a more active mature form by post-translational modification such as N- or C- terminal clipping. A pro-protcin which is an enzyme may be called a “proenzyme” or a “zymogen.” The clipped active protein (derived from the pro-protein) is also referred to herein as the mature protein. It is to be noted that the above terms are used for convenience and are not meant to override or determine the activities of a protein of the instant invention. It is also to be noted that any protein of the instant invention can have more than one variant described by the same term.

[0087] 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.

[0088] 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.

[0089] Other definitions are set forth below.

[0090] Production of enzymes

[0091] 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.

[0092] Vectors

[0093] 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.

[0094] 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, at 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.

[0095] 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 A0X1 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 ccllobiohydrolasc I gene (cbhl ) promoter optimization,” Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.

[0096] 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.

[0097] 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 polyadenylation sequences may suitably be derived from the same sources as the promoter.

[0098] 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.

[0099] 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. licheniformis, 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 xx.sC, 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.

[0100] 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.

[0101] 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.

[0102] 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).

[0103] Transformation and Culture of Host Cells

[0104] An isolated cell, either 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.

[0105] 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 Pseudomonadaceae can be selected as the host organism.

[0106] A suitable yeast host organism can be selected from the biotcchnologically 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 methylo trophic 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.

[0107] 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, egll, and egl2 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.

[0108] 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.

[0109] 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.

[0110] Uptake of DNA into the host Trichoderma 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 Trichoderma sp. strain. This fusion frequently leaves multiple copies of the plasmid DNA integrated into the host chromosome.

[0111] Usually, transformation of Trichoderma 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.

[0112] As used herein, Protein Identification (“JGI PID”) numbers for native Trichoderma genes reference Version 2 of the Trichoderma 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 / nar / gkr947). 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.

[0113] Expression

[0114] 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.

[0115] 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).

[0116] 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.

[0117] 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. 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.

[0118] 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.

[0119] 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.

[0120] Methods for Enriching and Purifying enzymes

[0121] Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used to prepare an enzyme polypeptide-containing solution.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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 linear 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-hydroxybenzoic 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] Enriched or purified enzymes can be made into a final product that is either liquid (solution, slurry) or solid (granular, powder).

[0141] Description of the Preferred Embodiments

[0142] In accordance with an aspect of the present invention, a method of preparing a chocolate cake having a reduced amount of cocoa in a recipe is presented, the method having the steps of: a. preparing a batter of the cake by adding: i. sugar; ii. flour; iii. cell wall modifying enzyme; iv egg; v. cocoa powder and; b. putting the batter in a suitable baking mold ; and c. baking the cake; wherein the recipe contains an amount of cocoa which is reduced at least 5% w / w.

[0143] Preferably, the amount of cocoa is reduced by at least 10, 15, 20, 25, 30, 35, 40, 45 or 50% w / w. Preferably, the cake is shortened cake. Preferably, the cell wall modifying enzyme is one or more of a glycoside hydrolase, a carbohydrate esterase, a glycosyltransferase or a polysaccharide lyase. Preferably, the batter further also includes one or more hydrocolloids. Preferably, the hydrocolloids are thickening / viscosity hydrocolloids or gelling agents. Preferably, the thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and the gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

[0144] In another aspect of the present invention, a method for increasing softness in a cake is presented having the steps of: a. preparing a batter of the cake by adding: i. sugar; ii. flour; iii. cell wall modifying enzyme; iv egg; v. cocoa powder and; b. putting the batter in a suitable baking mold; and c. baking the cake; wherein the cake has increased softness compared to a cake prepared from batter not containing the cell wall modifying enzyme. Preferably, the softness is increased by at least 10, 15, 20, 25, 30, 35, 40, 45 or 50%. Preferably, the cake is shortened cake. Preferably, the cell wall modifying enzyme is one or more of a glycoside hydrolase, a carbohydrate esterase, a glycosyltransferase or a polysaccharide lyase. Preferably, the batter further also includes one or more hydrocolloids. Preferably, the hydrocolloids are thickening / viscosity hydrocolloids or gelling agents. Preferably, the thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and the gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

[0145] In an aspect of the present invention, a method is presented of preparing a chocolate cake having a reduced amount of cocoa in a recipe, the method has the steps of: a. preparing a batter of the cake by adding: i. sugar" ii. flour; iii. an enzyme composition comprising one or more cell wall modifying enzymes; iv egg; v. cocoa powder and; b. putting the batter in a suitable baking mold; and c. baking the cake; wherein the recipe contains an amount of cocoa which is reduced at least 5% w / w.

[0146] Preferably, the one or more cell-wall modifying enzymes is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase. More preferably, the one or more cellwall modifying enzymes is a beta-glucanase.

[0147] Preferably, the amount of cocoa reduced is at least 10, 15, 20, 25, 30, 35, 40, 45 or 50% w / w.

[0148] Preferably, the cake is shortened cake.

[0149] Preferably, the batter further contains one or more hydrocolloids. Preferably, the one or more hydrocolloids are thickening / viscosity hydrocolloids or gelling agents. Preferably, the thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and said gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, mcthylhydroxypropylccllulosc and microcrystallinc cellulose.

[0150] In another aspect of the present invention, a method for increasing softness in a cake is presented having the steps: a. preparing a batter of the cake by adding: i. sugar; ii. flour; iii. an enzyme composition comprising one or more cell wall modifying enzymes; iv egg; v. cocoa powder and; b. putting the batter in a suitable baking mold ; and c. baking the cake; wherein the cake has increased softness compared to a cake prepared from batter not containing the one or more cell wall modifying enzymes.

[0151] Preferably, the softness is increased by at least 10, 20, 25, 30, 35, 40, 45 or 50%.

[0152] Preferably, the cake is shortened cake.

[0153] Preferably, the one or more cell-wall modifying enzymes is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase. More preferably, the one or more cellwall modifying enzymes is a beta-glucanase.

[0154] Preferably, the batter further contains one or more hydrocolloids. Preferably, the one or more hydrocolloids are thickening / viscosity hydrocolloids or gelling agents. Preferably, the xyloglucanase guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and said gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

[0155] In another aspect of the present invention, a method of preparing a chocolate flavored food is provided having a reduced amount of cocoa in a recipe, the method comprising the steps of: (i). hydrating cocoa powder; (ii). treating the hydrated cocoa powder of step (i) with an enzyme composition having one or more cell wall modifying enzymes; (iii). preparing the chocolate flavored food with the treated cocoa of step (ii); wherein the recipe contains an amount of cocoa which is reduced at least 5% w / w.

[0156] Preferably, the one or more cell-wall modifying enzymes is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase. More preferably, the one or more cellwall modifying enzymes is a beta-glucanase.

[0157] Preferably, the amount of cocoa reduced is at least 10, 15, 20, 25, 30, 35, 40, 45 or 50% w / w.

[0158] Preferably, the chocolate flavored food is milk, including cow’s milk, raw milk, prepasteurize milk, whole milk, skimmed milk, lactase-treated milk, reduced-lactose milk, lactose- free milk, reconstituted milk, condensed milk, ultra-filtered milk, fresh-fermented beverages, neutral pH beverages, beverage powder, dairy protein powder, ice cream mix, ice cream, custard, custard mix, frozen dessert, frozen dessert mix, mousse, pudding, pudding powder, sorbet, dessert, milkshakes, Ryazhenka,, whey based drink, breads, buns, rolls, baguettes, artisan bread, bread sticks, breadcrumbs, muffins, cupcakes, wafers, cookies, biscuits, crackers, scones, naan, pies, cakes, fruit cakes, sweet doughs, laminated doughs, croissants, waffles, brownies, brioche, panettone, pancakes, chaiupas, nankatais, bars, tortillas, pizza, doughnuts, soft pretzels, hard pretzels, fillings, puddings, frostings, icings, glazes, and spreads.

[0159] Preferably, the chocolate flavored food further comprises one or more hydrocolloids. Preferably, the one or more hydrocolloids are thickening / viscosity hydrocolloids or gelling agents. Preferably, the thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and said gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

[0160] In another aspect of the present invention, a method of preparing chocolate milk having improved cocoa perception is presented, having a reduced amount of cocoa in a recipe, the method having the steps of hydrating cocoa powder; treating cocoa powder with an enzyme composition comprising one or more cell wall-modifying enzymes; and mixing a milk with the enzyme composition treated cocoa of step ii.), wherein the recipe contains an amount of cocoa which is reduced at least 5% w / w.

[0161] Preferably, the one or more cell-wall modifying enzymes is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, a xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase. More preferably, the one or more cellwall modifying enzymes is a beta-glucanase.

[0162] Preferably, the milk is cow’s milk. Preferably, the cow’s milk is raw milk, pre-pasteurize milk, whole milk, skimmed milk, lactase-treated milk, reduced-lactose milk, lactose-free milk, reconstituted milk, condensed milk or ultra-filtered milk.

[0163] Preferably, the milk comprises one or more hydrocolloids. Preferably, the one or more hydrocolloids are thickening / viscosity hydrocolloids or gelling agents. Preferably, the thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and said gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

[0164] Preferably, the method has the further the step of homogenizing and pasteurizing the milk.

[0165] Preferably, the step of contacting with the cocoa with an enzyme composition is performed after homogenizing and pasteurizing the milk.

[0166] In another aspect of the present invention, a method of preparing chocolate milk having improved cocoa perception is provided having the steps of: i.) hydrating cocoa powder; treating cocoa powder with an enzyme composition comprising one or more cell wall-modifying enzymes; and mixing a milk with the enzyme composition treated cocoa of step ii.).

[0167] Preferably, the one or more cell-wall modifying enzymes is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, a xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase. More preferably, the one or more cellwall modifying enzymes is a beta-glucanase.

[0168] Preferably, the milk is cow’s milk. Preferably, the cow’s milk is raw milk, pre-pasteurize milk, whole milk, skimmed milk, lactase-treated milk, reduced-lactose milk, lactose-free milk, reconstituted milk, condensed milk or ultra-filtered milk.

[0169] Preferably, the milk comprises one or more hydrocolloids. Preferably, the one or more hydrocolloids are thickening / viscosity hydrocolloids or gelling agents. Preferably, the thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and said gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

[0170] Preferably, the method has the further the step of homogenizing and pasteurizing the milk.

[0171] Preferably, the step of contacting with the cocoa with an enzyme composition is performed after homogenizing and pasteurizing the milk.

[0172] In another aspect of the present invention, a method of increasing the theobromine content of cocoa is presented having the steps of: a. providing an aqueous slurry of cocoa; and b. adding one or more cell-wall modifying enzymes to said slurry to provide cocoa enriched in theobromine.

[0173] Preferably, the cocoa is enriched at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold in theobromine.

[0174] Preferably, the method has the further step of drying the slurry of step b. to produce a theobromine enhanced cocoa powder.

[0175] Preferably, the one or more cell-wall modifying enzymes is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, a xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase. More preferably, the one or more cellwall modifying enzymes is a beta-glucanase.

[0176] 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. EXAMPLES

[0177] Example 1: Evaluation of different doses of the cell wall modifying enzyme in brownies

[0178] In this example, the use of cell wall modifying enzyme in baked brownie formulation was evaluated to determine its effect on the texture and sensory properties. The enzyme was obtained from International Flavors and Fragrances (IFF, New Century, Kansas) as POWERBake® 9000. There were three variables tested in this experiment which include the control (no added enzyme), and enzyme added at 0.1% and 0.25% on a flour weight basis (fwb). See Table 3 for percent addition of total formulation for the cell wall modifying enzyme. The processing conditions are listed in Table 2. For the samples containing the enzyme, the brownie batter was hydrated with the enzyme and incubated at ambient temperature for 60 min. Both the control and the enzyme containing batters were baked at the same time to reach an internal temperature of 204°F. After baking and cooling, brownies were held in a plastic cupcake clamshell over time and evaluated for texture and sensory after 10 days.

[0179] Table 2: Processing conditions for Brownie samples

[0180] Table 3: Formulation / Recipe for brownie (shortened cake) samples Results:

[0181] Sensory evaluation of brownie samples comparing the control against samples containing different levels of cell wall modifying enzyme:

[0182] Sensory evaluation was carried out with several trained flavorists to determine the effect of the enzyme on the eating quality and sensory properties of the brownies on day 5. The sensory evaluation is summarized in Table 4. A score was given to rate the fudginess perceived in the bite of the brownie. The scale ranges from + to +++, which would indicate low to high on the fudginess or chocolate flavor rating.

[0183] Table 4: Sensory results evaluating brownie samples with and without the addition of cell wall modifying enzyme

[0184] *Note: Sensory experience and comments are segmented into three stages of the tasting process: front of mouth or initial flavor, middle chew or during chewing, and lastly backend flavor or finish flavor that remains after chewing and swallowing*

[0185] Texture analysis of brownie samples comparing the control against samples containing different levels of cell wall modifying enzyme:

[0186] Texture was measured by a TA.XT Plus texture analyzer (Stable Micro Systems). Tests were conducted at ambient temperature. In this test, a cylindrical probe was used to puncture the brownies at a constant speed (1 mm / s) to a depth of 7 mm. The maximum force applied during this measurement was analyzed and reported in gram- force (g). Based on the texture analysis, the control was noticeably firmer than the sample containing the cell wall modifying enzyme at 0.25% (fwb) by day 10 (Figure 1). While the g enzyme at 0.1% (fwb) dose seems to be too low to make a substantial difference, the brownie samples seem to be softer than control at day 10.

[0187] Fiber analysis brownie samples comparing the control against samples containing different levels of cell wall modifying enzyme:

[0188] Brownies were sent to an external lab for fiber analysis to demonstrate the cell-wall breaking activity of the enzyme. As summarized in Table 5, Higher Molecular Weight Dietary Fiber (% HMWDF) and Total Dietary Fiber (%, TDF) decreased as the cell wall modifying enzyme levels increased. This result supports the texture analysis provided above in which the brownie samples containing the enzyme were softer than the control samples at day 10 due to the breakdown of the cell wall structure that binds the water in the formulation.

[0189] Table 5: Fiber analysis results (External Lab)

[0190] Example 2: Evaluating the effect reducing of cocoa powder used in brownie formulation with the use of cell wall modifying enzyme

[0191] The sensory results from the first lab experiment led to further investigation to evaluate if the cell wall modifying enzyme can be used for the purpose of cocoa reduction in a brownie / cake-like application. The purpose of this lab experiment is to reduce the cocoa powder in the same formulation that was ran in example 1. The cocoa was reduced in 15% increments based on the flour weight. The processing conditions are listed in Table 6. The batters were baked at the same time to reach an internal temperature of 204°F. After baking and cooling, brownies were held in a plastic cupcake clamshell over time and evaluated for sensory. The tables below describe the brownie formulation, variables, and process.

[0192] Table 6: Processing conditions for Brownie samples

[0193] Table 7: Formulation of brownie samples

[0194] Sensory evaluation summary:

[0195] Similar to the previous sensory evaluation, trained flavorist evaluated the effect of the cell wall modifying enzyme on the overall chocolate flavor of the brownie samples on day 5. The summary shown in table 8 indicates overall notes when comparing the control sample and the variables with the cell wall modifying enzyme and incremental cocoa reduction. In addition, a score was given to rate the fudginess perceived in the bite of the brownie. The scale ranges from + to +++, which would indicate low to high on the fudginess rating. Table 8: Sensory Evaluation Results

[0196] *Note: Sensory experience and comments are segmented into three stages of the tasting process: front of mouth or initial flavor, middle chew or during chewing, and lastly backend flavor or finish flavor that remains after chewing and swallowing. Example 3: Cell wall modifying in enzyme in combination with hydrocolloids

[0197] In addition to the cell wall modifying enzyme, adding hydrocolloids to the batter system may positively impact the shelf life and overall eating quality of the products. Hydrocolloids include thickening / viscosity modification hydrocolloids that may positively impact the product. Thickening / viscosity hydrocolloids include: locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, mcthylhydroxypropylccllulosc, hydroxypropylccllulosc, cellulose, and gum karaya. Hydrocolloids also include gelling agents that may positively impact the product. Gelling agents include agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose, and microcrystalline cellulose. Some hydrocolloids fall into both classes depending on the application.

[0198] Example 4: Evaluation of the effect of cell wall modifying enzyme in brownie formulation with and without the reduction of cocoa powder

[0199] The purpose of this experiment was to further confirm the effect of the cell wall modifying enzyme on the texture and flavor attributes of brownie samples. The test consisted of four variables including the control (100% cocoa powder with no added enzyme), 100% cocoa powder with enzyme addition, 70% cocoa powder with no added enzyme, and 70% cocoa powder with enzyme addition. The product formulation is provided in Table 10. Sugar, com syrup and water were adjusted to account for the bulk loss of the 30% reduction of cocoa variables. The processing conditions are listed in Table 9. All brownie batter variables were hydrated with or without the enzyme and incubated at ambient temperature for 60 min. Both the control and the enzyme containing batters were baked at the same time to reach an internal temperature of 204°F. After baking and cooling, brownies were held in a plastic cupcake clamshell over time and evaluated for texture and sensory after 10 and 37 days.

[0200] Table 9: Processing conditions for Brownie samples

[0201]

[0202] Table 10: Formulation / Recipe for brownie (shortened cake) samples

[0203] Results:

[0204] Texture analysis of brownie samples comparing the control against samples containing wall modifying enzyme and reduced cocoa powder: Texture analysis data showed that there were observed differences in softness in the brownie samples. The 70% cocoa with no added enzyme was firmer than the 70% cocoa with enzyme addition. This trend was also seen on day 37 in the 70% cocoa variables. While there were no major differences in softness with the samples containing 100% cocoa samples at day 10, the texture difference was more apparent at day 37. The control (100% cocoa with no added enzyme) was noticeably firmer than the 100% cocoa sample containing the cell wall modifying enzyme at 0.25% (fwb) (Figure 2). Overall, the samples containing the cell modifying enzyme were softer than the samples without the enzyme at day 37.

[0205] Example 5: Application of cellulase wall-modifying enzyme in chocolate milk For many dairy producers using cocoa (e.g., manufacturers of chocolate flavored milks or chocolate ice cream), it is customary for many producers to heat cocoa powder to germinate spores, so they are in a vegetative state before pasteurization. This example investigated adding a cell wall-modifying enzyme during this time. Both treated and untreated cocoa slurries were processed in a similar manner (Table 11).

[0206] Table 11: Processing conditions for the cocoa slurry

[0207] A cell wall-modifying cellulase enzyme, which was obtained from International Flavors and Fragrances (IFF, New Century, Kansas, USA), was employed. Chocolate milks were blended with an overhead mixer for 10 min (ca. 800 rpm) to hydrate ingredients (Table 12) and subsequently treated by ultra-high temperature pasteurization (i.e., preheated to ca. 95 °C, homogenized at 2500 psi, heated to 143°C by direct steam injection for 4 s, and cooled to 4 °C). Table 12: Formulation of chocolate milks

[0208] The treated cocoa slurry described in Stage 1 of Table 12 was measured for theobromine levels by gas chromatography (Table 13) by IFF at the Union Beach, New Jersey, USA location. Samples of cocoa slurry were dilute 1:5 in water and further treated with two 30 mL extractions with an organic solvent, before being concentrated to 1 mL total volume. 1 L of the concentrate was injected into a gas chromatographer with a mass spectrometry detector with an OV 1 (non-polar) column, the run time was 65 min.

[0209] Table 13. Theobromine levels of cocoa slurries as measured by GC

[0210] Prepared chocolate milks were analyzed by a descriptive panel at IFF’s South Brunswick location (South Brunswick, New Jersey, USA). Nine panelists evaluated the chocolate milks described in Stage 2 of Table 12 as random samples in triplicate. Aggregate ratings of cocoa perception was rated on a scale of 1-15 for each chocolate milk and reported as an aggregate value, a post hoc analysis was conducted (Fisher’s LSD), significance was considered at 90% CL (Table 14).

[0211] Table 14. Trained panelists descriptive analysis

[0212] Attributes with the same letter are not significantly different, attributes with different letters are statistically different at 90% CL.

[0213] Results

[0214] The cell wall-modifying enzyme enhanced the amount of theobromine in the cocoa powder (Table 13). Theobromine is a molecule of interest for cocoa flavor. Untreated (without enzyme) chocolate milk with 20% reduced cocoa had the least amount of cocoa perception as compared to samples containing the enzyme with and without 20% cocoa reduction (Table 14). At the 20% reduced level of cocoa, milks made with cocoa treated with the cell wall-modifying enzyme had a significantly greater cocoa perception than the chocolate milk made without the treated cocoa (i.e., chocolate milk B2 had significantly greater cocoa perception than chocolate milk A2). 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 arc provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art 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

CLAIMS:What is claimed is:

1. A method of preparing a chocolate cake comprising a reduced amount of cocoa in a recipe, the method comprising the steps of: a. preparing a batter of the cake by adding:

1. sugar; ii. flour; iii. an enzyme composition comprising one or more cell wall modifying enzymes; iv egg; v. cocoa powder and; b. putting the batter in a suitable baking mold ; and c. baking the cake; wherein the recipe contains an amount of cocoa which is reduced at least 5% w / w.

2. The method of claim 1 wherein said one or more cell-wall modifying enzyme is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, a xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase.

3. The method of claim 2 wherein the one or more cell- wall modifying enzymes comprises a beta-glucanase.

4. The method of any of claims 1 to 3 wherein the amount of cocoa reduced is at least 10% w / w.

5. The method of claim 4 wherein the amount of cocoa reduced is at least 15% w / w.

6. The method of claim 5 wherein the amount of cocoa reduced is at least 20% w / w.

7. The method of claim 6 wherein the amount of cocoa reduced is at least 25% w / w.

8. The method of claim 7 wherein the amount of cocoa reduced is at least 30% w / w.

9. The method of claim 8 wherein the amount of cocoa reduced is at least 35% w / w.

10. The method of claim 9 wherein the amount of cocoa reduced is at least 40% w / w.

11. The method of claim 10 wherein the amount of cocoa reduced is at least 45% w / w.

12. The method of claim 11 wherein the amount of cocoa reduced is at least 50% w / w.

13. The method of any of claims 1 to 12, wherein the cake is shortened cake.

14. The method of any of claims 1 to 13 wherein the batter further comprises one or more hydrocolloids.

15. The method of claim 14 wherein said one or more hydrocolloids are thickening / viscosity hydrocolloids or gelling agents.

16. The method of claim 15 wherein said thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and said gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

17. A method for increasing softness in a cake comprising: a. preparing a batter of the cake by adding: i. sugar; ii. flour; iii. an enzyme composition comprising one or more cell wall modifying enzymes; iv egg;v. cocoa powder and; b. putting the batter in a suitable baking mold ; and c. baking the cake; wherein the cake has increased softness compared to a cake prepared from batter not containing the cell wall modifying enzyme.

18. The method of claim 17 wherein the softness is increased by at least 10%.

19. The method of claim 18 wherein the softness is increased by at least 20%.

20. The method of claim 19 wherein the softness is increased by at least 25%.

21. The method of claim 20 wherein the softness is increased by at least 30%.

22. The method of claim 21 wherein the softness is increased by at least 35%.

23. The method of claim 22 wherein the softness is increased by at least 40%.

24. The method of claim 23 wherein the softness is increased by at least 45%.

25. The method of claim 24 wherein the softness is increased by at least 50%.

26. The method of any of claims 16 to 25, wherein the cake is shortened cake.

27. The method of any of claims 16 to 26, wherein said one or more cell- wall modifying enzyme is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, a xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase.

28. The method of claim 27 wherein the one or more cell- wall modifying enzymes comprises a beta-glucanase.

29. The method of any of claims 16 to 28 wherein the batter further comprises one or more hydrocolloids.

30. The method of claim 29 wherein said one or more hydrocolloids are thickening / viscosity hydrocolloids or gelling agents.

31. The method of claim 30 wherein said thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and said gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

32. A method of preparing a chocolate flavored food comprising a reduced amount of cocoa in a recipe, the method comprising the steps of:(i). hydrating cocoa powder;(ii). treating the hydrated cocoa powder of step (i) with an enzyme composition comprising one or more cell wall modifying enzymes;(iii). preparing the chocolate flavored food with the treated cocoa of step (ii); wherein the recipe contains an amount of cocoa which is reduced at least 5% w / w.

33. The method of claim 32 wherein said one or more cell-wall modifying enzyme is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, a xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase.

34. The method of claim 33 wherein the one or more cell-wall modifying enzymes comprises a beta-glucanase.

35. The method of any of claims 32 to 34 wherein the amount of cocoa reduced is at least 10% w / w.

36. The method of claim 35 wherein the amount of cocoa reduced is at least 15% w / w.

37. The method of claim 36 wherein the amount of cocoa reduced is at least 20% w / w.

38. The method of claim 37 wherein the amount of cocoa reduced is at least 25% w / w.

39. The method of claim 38 wherein the amount of cocoa reduced is at least 30% w / w.

40. The method of claim 39 wherein the amount of cocoa reduced is at least 35% w / w.

41. The method of claim 40 wherein the amount of cocoa reduced is at least 40% w / w.

42. The method of claim 41 wherein the amount of cocoa reduced is at least 45% w / w.

43. The method of claim 42 wherein the amount of cocoa reduced is at least 50% w / w.

44. The method of any of claims 32 to 43, wherein the chocolate flavored food is milk, including cow’s milk, raw milk, pre-pasteurize milk, whole milk, skimmed milk, lactase-treated milk, reduced-lactose milk, lactose-free milk, reconstituted milk, condensed milk, ultra-filtered milk, fresh-fermented beverages, neutral pH beverages, beverage powder, dairy protein powder, ice cream mix, ice cream, custard, custard mix, frozen dessert, frozen dessert mix, mousse, pudding, pudding powder, sorbet, dessert, milkshakes, Ryazhenka, whey based drink, breads, buns, rolls, baguettes, artisan bread, bread sticks, breadcrumbs, muffins, cupcakes, wafers, cookies, biscuits, crackers, scones, naan, pies, cakes, fruit cakes, sweet doughs, laminated doughs, croissants, waffles, brownies, brioche, panettone, pancakes, chaiupas, nankatais, bars, tortillas, pizza, doughnuts, soft pretzels, hard pretzels, fillings, puddings, frostings, icings, glazes, and spreads.

45. The method of any of claims 32 to 44 wherein the chocolate flavored food further comprises one or more hydrocolloids.

46. The method of claim 45 wherein said one or more hydrocolloids are thickening / viscosity hydrocolloids or gelling agents.

47. The method of claim 46 wherein said thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and said gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

48. A method of preparing chocolate milk having improved cocoa perception, comprising a reduced amount of cocoa in a recipe, the method comprising the steps of i.) hydrating cocoa powder; ii.) treating cocoa powder with an enzyme composition comprising one or more cell wallmodifying enzymes; and iii.) mixing a milk with the enzyme composition treated cocoa of step ii.), wherein the recipe contains an amount of cocoa which is reduced at least 5% w / w.

49. The method of claim 48 wherein said one or more cell-wall modifying enzyme is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, a xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase.

50. The method of claim 49 wherein the one or more cell-wall modifying enzymes comprises a beta-glucanase.

51. The method of any of claims 48 to 50 wherein the amount of cocoa reduced is at least 10% w / w.

52. The method of claim 51 wherein the amount of cocoa reduced is at least 15% w / w.

53. The method of claim 52 wherein the amount of cocoa reduced is at least 20% w / w.

54. The method of claim 53 wherein the amount of cocoa reduced is at least 25% w / w.

55. The method of claim 54 wherein the amount of cocoa reduced is at least 30% w / w.

56. The method of claim 55 wherein the amount of cocoa reduced is at least 35% w / w.

57. The method of claim 56 wherein the amount of cocoa reduced is at least 40% w / w.

58. The method of claim 57 wherein the amount of cocoa reduced is at least 45% w / w.

59. The method of claim 58 wherein the amount of cocoa reduced is at least 50% w / w.

60. The method of any of claims 48 to 59, wherein the milk is cow’s milk.

61. The method of claim 60 wherein the cow’s milk is raw milk, pre-pasteurize milk, whole milk, skimmed milk, lactase-treated milk, reduced-lactose milk, lactose-free milk, reconstituted milk, condensed milk, ultra-filtered milk62. The method of any of claims 48 to 61 wherein the milk comprises one or more hydrocolloids.

63. The method of claim 62 wherein said one or more hydrocolloids are thickening / viscosity hydrocolloids or gelling agents.

64. The method of claim 63 wherein said thickening / viscosity hydrocolloids are selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum,tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylccllulosc, cellulose and gum karaya and said gelling agents arc selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

65. The method of any of claims 48 to 64 further comprising the steps of homogenizing and pasteurizing the milk.

66. The method of claim 65 wherein the step of contacting with the cocoa with an enzyme composition is performed after homogenizing and pasteurizing the milk.

67. A method of preparing chocolate milk having improved cocoa perception comprising the steps of: i.) hydrating cocoa powder; ii.) treating cocoa powder with an enzyme composition comprising one or more cell wallmodifying enzymes; and iii.) mixing a milk with the enzyme composition treated cocoa of step ii.).

68. The method of claim 67 wherein said one or more cell-wall modifying enzyme is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, a xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase.

69. The method of claim 68 wherein the one or more cell-wall modifying enzymes comprises a beta-glucanase.

70. The method of any of claims 67 to 69, wherein the milk is cow’s milk.71 . The method of claim 70 wherein the cow’s milk is raw milk, pre-pasteurize milk, whole milk, skimmed milk, lactasc-trcatcd milk, rcduccd-lactosc milk, lactose-free milk, reconstituted milk, condensed milk, ultra-filtered milk72. The method of any of claims 67 to 72 wherein the milk comprises one or more hydrocolloids.

73. The method of claim 72 wherein said one or more hydrocolloids are thickening / viscosity hydrocolloids or gelling agents.

74. The method of claim 73 wherein said thickening / viscosity hydrocolloids arc selected from the group consisting of locust bean gum, guar gum, tara gum, konjac, gum arabic, xanthan gum, tragacanth, propylene glycol alginate, methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, cellulose and gum karaya and said gelling agents are selected from the group consisting of agar, carrageenan, pectin, konjac, gellan gum, xanthan gum, gelatin, sodium alginate, methylcellulose, methylhydroxypropylcellulose and microcrystalline cellulose.

75. The method of any of claims 67 to 74 further comprising the steps of homogenizing and pasteurizing the milk.

76. The method of claim 75 wherein the step of contacting with the cocoa with an enzyme composition is performed after homogenizing and pasteurizing the milk.

77. A method of increasing the theobromine content of cocoa comprising the steps of: a. providing an aqueous slurry of cocoa; b. adding one or more cell- wall modifying enzymes to said slurry to provide cocoa enriched in theobromine.

78. The method of claim 77 wherein said cocoa is enriched at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold in theobromine.

79. The method of claim 77 or claim 78 further comprising drying the slurry of step b. to produce a theobromine enhanced cocoa powder.

80. The method of any of claims 77 to 79 wherein said one or more cell-wall modifying enzyme is selected from the group consisting of a carbohydrate esterase, a glycosyltransferase, a polysaccharide lyase, a glycoside hydrolases including, a xylanase, a xyloglucanase, and a cellulase, such as cellobiohydrolases, beta-glucosidases, endo-glucanases, and beta-glucanase.

81. The method of claim 80 wherein the one or more cell-wall modifying enzymes comprises a beta-glucanase.

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