Baking method for pulse protein-enriched bread using thermostable amyloglucosidase variant (EC 3.2.1.3)

Abstract

A method for producing a baked or partially baked product, comprising providing a dough comprising added pulse and / or legume protein and a mature thermostable variant of a parent glucoamylase that is at least 70% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10; and baking or partially baking the dough to produce the baked or partially baked product.

Description

[Technical Field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to a method of producing a baked or partially baked product, comprising providing a dough comprising added pulse and / or legume protein and a mature thermostable variant of a parent glucoamylase that is at least 70% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10; and baking or partially baking the dough to produce the baked or partially baked product. [Background technology]

[0003] The growing global interest in healthy foods has led to increased commercial interest in foods with high protein and / or fiber content, and high-protein baked products are no exception. However, as shown herein, simply adding flour from a high-protein crop, such as pulse flour, i.e., flour made from the edible seeds of legumes, to dough has a dramatic and detrimental effect on the volume of the resulting baked product. Technological solutions to counteract the negative impact on volume are highly desirable.

[0004] Parbaking is a technique in which bread or other dough products are partially (i.e., "par") baked and then typically cooled or frozen for storage. When a final baked product is desired, the cooled or frozen parbaked product is baked at normal baking temperatures, usually for 5 to 15 minutes; the resulting type of baked product is often called a "bake-off."

[0005] WO 2021 / 239267 discloses a method for producing a baked or partially baked edible product from a dough comprising added pulse and / or legume protein and at least one added lipase enzyme, wherein at least 2% (w / w) of the total flour content of pulse and / or legume protein is added. Summary of the Invention [Problem to be solved by the invention]

[0006] The inventors have found that thermostable variants of certain glucoamylases exhibit improved performance in baking with added pulses and / or legume proteins. One improved performance of the thermostable variants was that they increased the sweetness or flavor of the product, allowing for a reduction in the amount of sugar added in traditional recipes, and also, to some extent, were able to mask bitterness from other added pulses and / or legume proteins. [Means for solving the problem]

[0007] Thus, in a first aspect, the present invention relates to a method of producing a baked or partially baked product comprising providing a dough comprising added pulse and / or legume protein and a mature thermostable variant of a parent glucoamylase that is at least 70% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10; and baking or partially baking the dough to produce the baked or partially baked product.

[0008] Preferably, the mature thermostable variant of a parent glucoamylase of the present invention is at least 71% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10, such as at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, such as at least 79%, for example at least 80%, such as at least or 81%, such as at least 82%, for example at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99% identical. [Brief explanation of the drawings]

[0009] [Figure 1]1 shows a multiple alignment of the amino acid sequences of the following mature proteins: - wild-type AMG from Penicillium oxalicum (PoAMG) according to SEQ ID NO: 1 - PoAMG variant designated "AMG NL" according to SEQ ID NO: 2 - PoAMG variant designated "AMG anPAV498" according to SEQ ID NO: 3 - PoAMG variant designated "AMG JPO001" according to SEQ ID NO: 4 - PoAMG variant designated "AMG JPO124" according to SEQ ID NO: 5 - PoAMG variant designated "AMG JPO172" according to SEQ ID NO: 6 - wild-type AMG from Penicillium miczynskii (PoAMG) according to SEQ ID NO: 7 - wild-type AMG from Penicillium russellii (PoAMG) according to SEQ ID NO: 8 - wild-type AMG from Penicillium glabram (PoAMG) according to SEQ ID NO: 9 Wild-type AMG (PoAMG) from P. glabrum DETAILED DESCRIPTION OF THE INVENTION

[0010] definition Legumes: Legumes are plants of the Fabaceae (or Leguminosae) family, or the fruits or seeds (also called pulses) of such plants, especially in the mature, dried state. Well-known legumes include alfalfa, clover, beans, peas, chickpeas, lentils, lupine, mesquite, carob, soybeans, peanuts, and tamarind. Legumes produce a plant-specific type of fruit: a simple, dry fruit that develops from a simple carpel and usually has a cleft (opening along the seam) on both sides.

[0011] Pulses: The Food and Agriculture Organization of the United Nations (FAO) recognizes 11 types of pulses: dry beans, dry broad beans, dry peas, chickpeas, cow peas, pigeon peas, lentils, bambara groundnut, vetch, lupine and pulse NES (i.e. minor pulses including winged bean (Psophocarpus tetragonolobus), velvet pea, cawich (Mucuna pruriens var. utilis), yam bean (Pachyrhizus erosus)).

[0012] Pulse and / or legume protein: The term "pulse and / or legume protein" refers to pulse and / or legume proteins that are desirable components of pulse and / or legume flours; this term also includes processed flours and / or deflavored pulse and / or legume flours, where the processed flour has a higher protein content than unprocessed flour. Processed or deflavored pulse and / or legume flours may also be referred to as pulse and / or legume protein concentrates and / or isolates, respectively.

[0013] Deflavored pulse and / or legume flour or protein: In the context of the present invention, the term "deflavored" means that the flour or protein component has been processed to reduce off-flavors, such as bitterness.

[0014] Lipase activity: Triacylglycerol lipase activity (EC 3.1.1.3), i.e., hydrolysis activity on carboxylic acid ester bonds in triglycerides, such as tributyrin.

[0015] Phospholipase activity: Phospholipase activity (A1 or A2, EC 3.1.1.32 or 3.1.1.4), i.e., hydrolytic activity towards one or both carbonate ester bonds in phospholipids such as lecithin.

[0016] Galactolipase activity: Galactolipase activity (EC 3.1.1.26), i.e., hydrolysis activity against carboxylic acid ester bonds in galactolipids such as DGDG (digalactosyldiglyceride).

[0017] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal cleavage, glycosylation, phosphorylation, and the like.

[0018] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0019] For the purposes of the present invention, sequence homology between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), version 5.0.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output value of Needle, labeled "longest identity" (obtained using the -no brief option), is used as the percent identity, calculated as follows: (Identical residues × 100) / (length of alignment − total number of gaps in alignment)

[0020] Variant: The term "variant" refers to a polypeptide that contains modifications, i.e., substitutions, insertions, and / or deletions at one or more (e.g., several) positions. A substitution refers to the replacement of an amino acid at a position with another amino acid; a deletion refers to the removal of an amino acid at a position; and an insertion refers to the addition of one or more amino acids immediately adjacent to the amino acid at a position. Amino acid changes can be minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically 1-30 amino acids; small amino- or carboxyl-terminal extensions such as an amino-terminal methionine residue; small linker peptides of 20-25 residues or less; or small extensions that facilitate purification by altering net charge or another function, such as a polyhistidine tract, antigenic epitope, or binding domain. Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and low molecular weight amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that generally do not change specific activity are known in the art and are described, for example, in H. Neurath and RL Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions include Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0021] Thermostability Improvement: Thermostability improvement (Td) in °C is a measure of how much the variants have improved thermostability over their parent glucoamylase under the same conditions, determined as exemplified herein.

[0022] Improved crumb firmness of baked products: The term "improved crumb firmness" is defined herein as the property of a baked product that is more compressible compared to a baked product in which the enzyme solution according to the present invention has not been added to the dough. Crumb firmness is assessed empirically by a test beaker / sensory panel of persons skilled in the art or measured by using texture analyzers known in the art (e.g., TAXT2 or TA-XT Plus from Stable Micro Systems Ltd, Surrey, UK).

[0023] Improved flavor of baked products: The term "improved flavor of baked products" is assessed by a trained test panel and / or chemical analysis (e.g., headspace GC-MS analysis). Improved flavor of baked products includes a reduction in the off-flavor(s) of the baked products.

[0024] Improved anti-staling of baked products: The term "improved anti-staling of baked products" is defined herein as the property of a baked product in which the rate of deterioration of a quality parameter, e.g., softness and / or elasticity, during storage is reduced.

[0025] Volume of baked product: The term "volume of baked product" is defined herein as the measured volume of a given loaf of bread. This volume can be determined by the rapeseed displacement method.

[0026] Bread colour: The colour or whiteness of the baked or par-baked product is measured as the "colour L*" value in a C-cell (Calibre Instruments Ltd, Warrington, UK) using standard methods for collecting images and standard C-Cell software for data analysis.

[0027] Fabric according to the present invention The present invention relates to a dough for baked or par-baked products, the dough comprising added pulse and / or legume proteins.

[0028] The term "adding" is defined herein as the addition of the protein and / or enzyme of the present invention to dough, any ingredients for making dough, and / or any mixture of dough ingredients for making dough.

[0029] In other words, the proteins and / or enzymes may be added at any stage of dough preparation, and may be added in one, two, or more stages, and they may be added to the dough ingredients, which may be kneaded and processed as known in the art for baked and / or par-baked products.

[0030] The term "effective amount" is defined herein as an amount of an enzyme composition of the present invention sufficient to have a measurable effect on at least one desired property of the dough and / or baked product.

[0031] The term "dough" is defined herein as a mixture of flour and other baking ingredients that is stiff enough to be kneaded or rolled. In the context of the present invention, batter is encompassed by the term "dough"; preferably, dough of the present invention comprises wheat flour.

[0032] In a preferred embodiment, the dough ingredients comprise wheat flour; preferably, 2% (w / w) or more of the total flour content is wheat flour; preferably, 4% (w / w) or more of the total flour content is wheat flour, preferably at least 6%, at least 8%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or preferably at least 95% (w / w) of the flour is wheat flour.

[0033] The dough of the present invention comprises any cereal grain or flour obtained from other sources, including wheat, emmer, spelt, einkorn, barley, rye, oats, corn, sorghum, rice, millet, amaranth, quinoa, cassava, and any combination thereof.

[0034] In a preferred embodiment of the invention, the pulse and / or legume protein is added to the dough in the form of pulse and / or legume flour, processed pulse and / or legume flour, deflavored pulse and / or legume flour, or a protein concentrate and / or isolate made essentially from pulse and / or legume flour; preferably, the added pulse and / or legume protein comprises lentil protein, chickpea protein, pea protein and / or faba bean protein, or a protein concentrate and / or isolate thereof.

[0035] A preferred embodiment relates to a dough according to the first aspect to which at least 4% (w / w) of pulses and / or legume protein of the total flour is added, preferably at least 6% (w / w) of the total flour content of pulses and / or legume protein, more preferably at least 8% (w / w) of the total flour content of pulses and / or legume protein, even more preferably at least 10% (w / w) of the total flour content of pulses and / or legume protein, and most preferably at least 12% (w / w) of the total flour content of pulses and / or legume protein.

[0036] Preferably, the dough of the present invention also contains gluten.

[0037] The dough may also include other common dough ingredients, for example, proteins such as milk powder, gluten, dietary fiber sources (such as wheat, oat bran, beta-glucan and / or inulin), and eggs (either whole eggs, egg yolks, or egg whites); oxidizing agents such as ascorbic acid, potassium bromate, potassium iodide, azodicarbonamide (ADA), or ammonium persulfate; amino acids such as L-cysteine; sugars; salts such as sodium chloride, calcium acetate, sodium sulfate, or calcium sulfate, and / or emulsifiers.

[0038] In a preferred embodiment of the present invention, the dough of the present invention also comprises gluten.

[0039] The dough may contain fats (triglycerides) such as granulated fat or oil.

[0040] The dough of the present invention is typically a leavened dough or a dough that is subjected to leavening. The dough can be yeast-leavened in a variety of ways, such as by adding a chemical leavening agent, e.g., baking powder, sodium bicarbonate, or by adding yeast (to leaven the dough), but preferably the dough is yeast-leavened by adding a suitable yeast culture, such as Saccharomyces cerevisiae (baker's yeast), e.g., a commercially available culture of a strain of S. cerevisiae.

[0041] The dough of the present invention may comprise at least one added lipase enzyme, preferably a lipase and / or a phospholipase, preferably a mature lipase and / or a mature phospholipase.

[0042] Preferably, the at least one lipase enzyme added comprises a mature lipase having an amino acid sequence that is at least 70% identical to one or more of the sequences set forth in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, preferably at least 75% identical, at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or preferably at least 99% identical to one or more of the sequences set forth in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.

[0043] Preferably, the at least one added lipase enzyme is added in an amount in the range of 0 to 100 mg enzyme protein / kg flour, preferably in the range of 0 to 50 mg enzyme protein / kg flour; more preferably, in an amount in the range of 0 to 25 mg enzyme protein / kg flour; even more preferably, in an amount in the range of 0 to 10 mg enzyme protein / kg flour; even more preferably, in an amount in the range of 0 to 5 mg enzyme protein / kg flour; most preferably, in an amount in the range of 0 to 2.5 mg enzyme protein / kg flour.

[0044] Preferably, the dough of the first aspect also comprises at least one additional enzyme, preferably at least one mature α-amylase, more preferably a mature maltogenic α-amylase; preferably a mature maltogenic α-amylase derived from Bacillus stearothermophilus; more preferably a mature maltogenic α-amylase having an amino acid sequence at least 70% identical to that of SEQ ID NO: 6, preferably at least 75% identical, at least 80%, 85%, 90%, 92%, 94%, 96%, 98% or preferably at least 99% identical to that of SEQ ID NO: 11. The mature maltogenic α-amylase is preferably added in an amount ranging from 0 to 10,000 MANU / kg flour; preferably in the range of 0 to 7,500 MANU / kg flour; preferably in the range of 0 to 5,000 MANU / kg flour.

[0045] Preferably, the at least one additional enzyme comprises a mature alpha-amylase; preferably a mature fungal alpha-amylase; more preferably, a mature alpha-amylase derived from Aspergillus oryzae; preferably, said additional mature alpha-amylase is added in an amount in the range of 0-1,000 FAU / kg flour; preferably in the range of 0-500 FAU / kg flour; more preferably, in the range of 0-100 FAU / kg flour; even more preferably, in the range of 0-50 FAU / kg flour; and most preferably, in the range of 0-25 FAU / kg flour.

[0046] Preferably, the dough of the first aspect also comprises at least one additional added enzyme comprising at least one mature xylanase, preferably a GH5, GH8 and / or GH11 xylanase.

[0047] The present invention is particularly useful for preparing yeast-based dough, baked or parbaked products in industrialized processes using automated or semi-automated equipment to mechanically prepare the dough used to prepare the baked or parbaked product. The process of preparing bread generally involves the sequential steps of dough production (with an optional proofing step), sheeting or dividing, shaping or rolling, and fermentation of the dough, which are well known in the art. If an optional fermentation step is used, more flour is preferably added, and alkali may be added during the second fermentation step to neutralize any acids that have been or will be produced. In an industrial baking production process according to the present invention, one or more of these steps are carried out using automated or semi-automated equipment, such as:

[0048] Horizontal mixers: Roller bar mixers equipped with rotating arms typically have two speed settings, slow mixing at 35 rpm and fast mixing at 70 rpm in older models, but newer models often have variable speed settings ranging from 15 to 120 rpm.

[0049] Vertical Mixer: A spiral mixer is typically a mixer with a rotating bowl and a spiral that counteracts the rotation. Some spiral mixers can be bidirectional to provide better distribution of the ingredients.

[0050] The purpose of mixing is to achieve uniform blending and hydration of the dry ingredients, knead the dough to form the gluten network, and incorporate air into the dough. Two-speed mixing is typically used in both types of mixers: a slow speed to gather the dough without forcing it against the sides of the bowl, and a faster speed to aid in the formation of the gluten network.

[0051] In a preferred embodiment, the fabric comprises: a) mixing at a low mixing speed for at least 5 minutes, preferably in the range of 5 to 50 rpm, more preferably in the range of 10 to 40 rpm; more preferably at a low mixing speed for at least 10 minutes, even more preferably at a low mixing speed for at least 15 minutes; optionally, b) The dough is then mixed at a faster speed.

[0052] Parbaked products Parbaking is a technique in which bread or dough products are partially baked and then typically quickly cooled / frozen for storage.

[0053] The raw dough is baked as normal, but stopped at about 80% of the normal cooking time, then rapidly cooled.

[0054] Parbaked dough products are easy to transport and can be stored until needed. Parbaked dough products are stored in airtight containers that prevent moisture loss; they can be stored at room temperature; or in the refrigerator; or in the freezer.

[0055] The freezing process can lead to the formation of ice crystals and subsequent damage to the starch granules and leakage of amylose, so that bread before the second baking tends to have higher amounts of leaked amylose and unbound water than bread baked without the freezing process, two parameters known to increase the crumb stickiness.

[0056] If a final dough product is desired, the parbaked product is "finished" by baking at ambient temperature for an additional time, typically 5 to 15 minutes. The exact time will vary from product to product and must be determined by experimentation.

[0057] This produces a parbaked product in the following steps: a) The dough becomes the product, b) the product is calcined; c) the product is stored; d) The product is re-baked to form a par-baked product.

[0058] The product may be stored at ambient / room temperature or at low temperature, which typically means at temperatures below 5° C. In one embodiment, the product is stored in a freezer.

[0059] The process of the invention can be used for all kinds of parbaked products prepared from dough of either white, light or dark type, particularly of soft character.

[0060] Examples include bread, typically in the form of loaf or roll (particularly white, whole grain or rye bread), bread, flatbread, pita bread, tortillas, cakes, pancakes, biscuits, wafers, cookies, pie crust, pizza, and the like.

[0061] Glucoamylase Glucoamylases are also called amyloglucosidases and glucan 1,4-alpha-glucosidases (EC 3.2.1.3), more commonly referred to as AMG.

[0062] According to the present invention, different types of amyloglucosidases may be used as parents for the production of thermostable amyloglucosidase variants, for example, amyloglucosidases derived from DNA sequences found in fungal strains of the genus Aspergillus, Rhizopusor, Talaromyces (Rasamsonia), or Penicillium, preferably DNA sequences found in fungal strains of the genus Penicillium, even more preferably Penicillium oxysporum, Penicillium oxalicum, Penicillium miczynskii, Penicillium russellii, The parent glucoamylase may be a polypeptide encoded by a DNA sequence found in a fungal strain of Penicillium russellii, or Penicillium glabrum. Preferably, the parent glucoamylase is derived from a species of Penicillium, preferably Penicillium oxicalum, Penicillium miczynskii, Penicillium russellii, or Penicillium glabrum.

[0063] Other suitable examples of fungi include Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Rhizopus delemar, Rhizopus niveus, Rhizopus oryzae, and Talaromyces emersonii (Rasamsonia emersonii).

[0064] Below is the percent identity between the AMG amino acid sequences aligned in Figure 1 and also provided in the sequence listing.

[0065] [Table 1]

[0066] Thermostable variants of PoAMG have been produced (see Table 2 below). In a preferred embodiment, the mature thermostable glucoamylase variants of the invention comprise one or more or all of the combinations of amino acid substitutions listed in Table 2 below.

[0067] In a preferred embodiment, the mature variants of the invention comprise at least one amino acid modification at one or more or all of the positions corresponding to positions 1, 2, 4, 6, 7, 11, 31, 34, 50, 65, 79, 103, 132, 327, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595 in SEQ ID NO: 1, preferably the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 2, 4, 11, 65, 79 and 327 in SEQ ID NO: 1, or preferably at least At least one amino acid modification comprises a substitution at one or more or all of positions corresponding to R1A, P2N, P4S, P11F, T65A, K79V and Q327F in SEQ ID NO:1, or preferably at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to 1, 6, 7, 31, 34, 79, 103, 132, 445, 447, 481, 566, 568, 594 and 595 in SEQ ID NO:1, or preferably at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to R1A, G6S, G7T, R and / or at least one amino acid modification, which may include substitutions at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 594, and 595 in SEQ ID NO: 1, or preferably ... The amino acid modification may comprise a substitution at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO: 1, or preferably the at least one amino acid modification may comprise a substitution at one or more or all of the positions corresponding to 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628,The amino acid sequence may comprise a substitution at one or more or all of the positions corresponding to positions 594 and 595 in SEQ ID NO: 1, or preferably the at least one amino acid modification may comprise a substitution at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO: 1, or preferably the at least one amino acid modification may comprise a substitution at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 595 in SEQ ID NO: 1. The at least one amino acid modification may include a substitution at one or more or all of the positions corresponding to positions 0, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 594, and 595, and preferably the at least one amino acid modification includes a substitution at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R, and F595S in SEQ ID NO: 1.

[0068] The thermostability improvement (Td) of the variants in Table 2 is listed in Table 3, where the Td of the PoAMG variant designated "anPAV498" (parent) was set to zero. In preferred embodiments, a mature thermostable variant of the invention has a thermostability improvement (Td) over its parent of at least 5°C, preferably at least 6°C, 7°C, or 8°C, preferably determined as exemplified herein.

[0069] In another preferred embodiment, a mature thermostable variant of the invention has a relative activity at 91° C. of at least 150, preferably at least 200, more preferably at least 250, and most preferably at least 300 compared to its parent.

[0070] Sources of phospholipase enzymes The phospholipase enzyme may be derived from prokaryotic, particularly bacterial, or eukaryotic, such as fungal or animal, sources.

[0071] The phospholipase enzymes may be derived, for example, from the following genera or species: Thermomyces, T. lanuginosus (also known as Humicola lanuginosa), lanuginosa), Humicola, H. insolens, Fusarium, F. oxysporum, F. solani, F. heterosporum, Aspergillus, A. tubigensis, A. niger, A. oryzae, Rhizomucor, Candida, C. antarctica, C. rugosa, Penicillium, P. camembertii, Rhizopus, Rhizopus oryzae oryzae, Absidia, Dictyostelium, Mucor, Neurospora, Rhizopus, R. arrhizus, R. japonicus, Sclerotinia, Trichophyton, Whetzelinia, Bacillus, Citrobacter, Enterobacter bacter, Edwardsiella, Erwinia, Escherichia, E. coli, Klebsiella, Proteus, Providencia, Salmonella, Serratia, Shigella, Streptomyces, Yersinia, Pseudomonas, or P. cepacia.cepacia).

[0072] Phospholipase enzymes can be produced in suitable host cells as known in the art. Phospholipases can also be obtained from bee or snake venom, or from mammalian pancreas, such as porcine pancreas.

[0073] WO 98 / 26057 discloses lipases / phospholipases from Fusarium oxysporum and their use in baking.

[0074] WO 2004 / 099400 discloses various phospholipase enzymes and their use in baking to reduce dough stickiness.

[0075] Suitable commercially available phospholipase preparations are Lipopan F™, Lipopan Xtra™ and Lipopan Prime™ (available from Novozymes A / S).

[0076] Other available phospholipases are, for example, Panamore™ available from DSM.

[0077] Commercially available lipase preparations are, for example, Lipopan F™ and Lipopan 50 BG™ available from Novozymes A / S.

[0078] α-amylase Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, EC.3.2.1.1) comprise a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glucoside oligosaccharides and polysaccharides.

[0079] Several α-amylases are called Termamyl™, Termamyl™ SC and "Termamyl™-like alpha-amylases" and are known from, for example, WO 90 / 11352, WO 95 / 10603, WO 95 / 26397, WO 96 / 23873 and WO 96 / 23874.

[0080] Another group of α-amylases is referred to as Fungamyl™ and “Fungamyl™-like α-amylases,” which are α-amylases related to the α-amylase from Aspergillus oryzae disclosed in WO 01 / 34784.

[0081] A preferred group of α-amylases is called maltogenic α-amylases (EC 3.2.1.133), typically derived from Bacillus stearothermophilus. Preferred maltogenic α-amylases have an amino acid sequence that is at least 70% identical to SEQ ID NO: 11 herein, such as at least 71%, such as at least 72%, for example at least 73%, such as at least 74%, for example at least 75%, such as at least 76%, for example at least 77%, such as at least 78%, for example at least 79%, such as at least 80%, for example at least 81%, such as at least 82%, for example at least 83%, such as at least 84%, for example at least 85%, such as at least 86%, for example at least 87%, for example at least 88%, such as at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99% identical to SEQ ID NO: 11 herein.

[0082] Also preferred are groups of sugar-tolerant maltogenic α-amylase variants such as those disclosed in WO 2006 / 032281 (Novozymes A / S), in which several sugar-tolerant variants are provided, each of which contains one or more amino acid changes that are substitutions, deletions or insertions adjacent to I15, R18, K44, N86, T87, G88, Y89, H90, Y92, W93, F188, T189, O190, P191, A192, F194, L196, O329, N371, O372, P373, N375 or R376. One such preferred variant presented by the inventors herein is the sugar-tolerant maltogenic α-amylase 1 (ST-MAA1), which has the following three substitutions: F188L, D261G and T288P. WO 2008 / 148845 also discloses several preferred sugar-tolerant maltogenic α-amylase variants, each of which contains two substitutions D261G and T288P and at least one further amino acid change that is a substitution or deletion of, or an insertion adjacent to, Y89, W93, P191, F194, Y360, or N375. One such preferred variant that we present is sugar-tolerant maltogenic α-amylase 2 (ST-MAA2), which has the following four substitutions: F194Y, D261G, T288P, and N375S.

[0083] Yet another group of preferred mature α-amylases are mature non-maltogenic maltotetrahydrolase exoamylases. WO 2004 / 111217 (Danisco A / S) discloses several preferred non-maltogenic thermostable variants of the exoamylase or maltotetrahydrolase of Pseudomonas saccharophilia having the amino acid sequence set forth in SEQ ID NO: 12, each of which contains one or more of the following substitutions: G69P, A141P, G223A, A268P, G313P, S399P, and G400P.

[0084] WO 2007 / 148224 (Danisco A / S) discloses more preferred mature non-maltogenic anti-staling variants of Pseudomonas saccharophilia exoamylase having the amino acid sequence shown in SEQ ID NO: 12, which variants contain an amino acid substitution at position 307 to lysine (K) or arginine (R), respectively.

[0085] WO 2010 / 133644 discloses other preferred mature non-maltogenic variants of Pseudomonas saccharophilia exoamylase having the amino acid sequence set forth in SEQ ID NO: 12, each of which contains one or more substitutions at positions including 42, 88, 205, 223, 235, 240, 311, 392, and 409. One example of such a preferred variant of Pseudomonas saccharophilia exoamylase is disclosed in SEQ ID NO: 31 of WO 2010 / 133644, the amino acid sequence of which is also set forth herein in SEQ ID NO: 13, which we refer to as: HPL G+.

[0086] Another preferred variant of the Pseudomonas saccharophilia exoamylase is disclosed in SEQ ID NO: 21 of WO 2007 / 148224, the amino acid sequence of which is also set forth in SEQ ID NO: 14 herein, which we refer to as: HPL G4.

[0087] Other preferred non-maltogenic α-amylases are disclosed in WO 2005003339 (Danisco A / S), WO 2005007818 (Danisco A / S) and WO 2022 / 216801 (DuPont Nutrition Biosciences ApS).

[0088] Preferred mature non-maltogenic α-amylases are at least 70% identical to SEQ ID NO: 12, 13 or 14 herein, such as at least 71%, for example at least 72%, such as at least 73%, for example at least 74%, such as at least 75%, for example at least 76%, such as at least 77%, for example at least 78%, such as at least 79%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84% to SEQ ID NO: 12, 13 or 14 herein. , for example at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99% identical; furthermore, preferred mature non-maltogenic α-amylases have one or more of the substitutions listed in the paragraph above.

[0089] Yet another group of preferred mature α-amylases are α-amylases that degrade raw starch. As used herein, "raw starch degrading α-amylase" refers to an enzyme that can directly degrade raw starch granules below the gelatinization temperature of the starch.

[0090] Examples of raw starch-degrading α-amylases include those disclosed in WO 2005 / 003311, US 2005 / 0054071, and US Pat. No. 7,326,548. Examples also include the enzymes disclosed in U.S. Pat. No. 7,326,548, Tables 1-5 of the Examples of U.S. Patent Application Publication No. 2005 / 0054071 (page 15, Table 3), as well as the enzymes disclosed in WO 2004 / 020499, WO 2006 / 06929 and WO 2006 / 066579, and the enzymes disclosed in the sequence listings and descriptions of WO 2006 / 069290 (Novozymes A / S) or WO 2013 / 006756 (Novozymes A / S), all of which are incorporated herein in their entireties.

[0091] In one embodiment, the raw starch degrading alpha-amylase is a GH13_1 amylase.

[0092] In one embodiment, the raw starch degrading alpha-amylase enzyme is selected from the group consisting of alpha-amylases described in EP 2981170 (Novozymes A / S) or the raw starch degrading alpha-amylase set forth in SEQ ID NO: 15 or 16 herein.

[0093] Additional enzymes In a preferred embodiment of the first aspect, one or more additional enzymes are added to the dough, said additional enzymes being selected from the group consisting of alpha-amylase, maltogenic amylase, raw starch-degrading alpha-amylase, beta amylase, aminopeptidase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1,4-alpha-maltotetrahydrolase, glucanase, beta glucanase, galactanase, alpha-galactosidase, The enzyme may be selected from the group consisting of: enzymes, beta-galactosidase, glucose oxidase, alpha-glucosidase, beta-glucosidase, haloperoxidase, hemicellulolytic enzymes, invertase, laccase, lipase, mannase, mannosidase, oxidase, pectolytic enzymes, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, protease, pullulanase, raw starch degrading alpha-amylase, ribonuclease, transglutaminase, and xylanase.

[0094] Enzyme composition The mature thermostable variant glucoamylase of the present invention as well as any additional enzymes may be added in any suitable form, such as, for example, in the form of a liquid, particularly a stabilized liquid, or it may be added as a substantially dry powder or granules.

[0095] Granules can be produced, for example, as disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452. Liquid enzyme preparations may be stabilized, for example, by adding sugars or sugar alcohols or lactic acid according to established procedures. Other enzyme stabilizers are well known in the art.

[0096] The enzyme(s) may be added in any suitable way, such as as individual components (separate or sequential addition of enzymes) or by adding the enzymes together in one step or composition.

[0097] Granules and agglomerated powders can be prepared by conventional methods, for example, by spraying the enzyme onto a carrier in a fluidized bed granulator. The carrier can consist of a particle core having a suitable particle size. The carrier can be soluble or insoluble, for example, a salt (such as NaCl or sodium sulfate), a sugar (such as sucrose or lactose), a sugar alcohol (such as sorbitol), starch, rice, corn grits, or soy. [Example]

[0098] Example 1: Construction of a PoAMG library The PoAMG library was constructed as follows. Forward and reverse primers were designed with a 15-bp overlap, carrying the NNK or desired mutation at the target site. Inverse PCR, which resulted in amplification of the entire plasmid DNA sequence using the reverse primer, was performed using an appropriate template plasmid DNA (e.g., plasmid DNA containing the JPO-0001 gene) under the following conditions. The resulting PCR fragment was purified using a QIAquick gel extraction kit (QIAGEN) and then transformed into Escherichia coli ECOS-competent E. coli DH5α (NIPPON GENE CO., LTD.). Plasmid DNA was extracted from E. coli transformants using a MagExtractor plasmid extraction kit (TOYOBO) and then transformed into A. niger competent cells.

[0099] PCR reaction mix: PrimeSTAR Max DNA Polymerase [TaKaRa] Total volume: 25 μl 1.0 μl template DNA (1 ng / μl) 9.5 μl H2O 12.5 μl 2x PrimeSTAR Max Premix 1.0 μl forward primer (5 μM) 1.0 μl reverse primer (5 μM)

[0100] PCR program: 98℃ / 2 minutes 25×(98℃ / 10 seconds, 60℃ / 15 seconds, 72℃ / 2 minutes) 10℃ / hold

[0101] Example 2: Screening for better thermostability The B. subtilis library constructed as in Example 1 was fermented for 3 days at 32°C in either 96-well or 24-well MTPs containing COVE liquid medium (2.0 g / L sucrose, 2.0 g / L iso-maltose, 2.0 g / L maltose, 4.9 mg / L, 0.2 ml / L 5N NaOH, 10 ml / L COVE salts, 10 ml / L 1 M acetamide). AMG activity in the culture supernatant was then measured at several temperatures by the pNPG assay described as follows.

[0102] pNPG Thermostability Assay: The culture supernatant containing the desired enzyme was mixed with the same volume of pH 5.0 200 mM NaOAc buffer. 20 microliters of this mixture was dispensed into either a 96-well plate or an 8-strip PCR tube, and then heated for 30 minutes in a thermal cycler at various temperatures. These samples were mixed with 10 μl of substrate solution containing 0.1% (w / v) pNPG [wako] in pH 5.0 200 mM NaOAc buffer and incubated at 70°C for 20 minutes for the enzyme reaction. After the reaction, 60 μl of 0.1 M borax buffer was added to stop the reaction. 80 microliters of the reaction supernatant was collected and its OD 405 The values ​​were read photometrically to assess the enzyme activity.

[0103] Table 2

[0104] Table 3

[0105] Table 4

[0106] Table 5

[0107] Table 6

[0108] Table 7

[0109] Table 8

[0110] Table 9

[0111] Table 10

[0112] Table 11

[0113] Table 12

[0114] Table 13

[0115] Table 14

[0116] Table 15

[0117] Table 16

[0118] Table 17

[0119] Table 18

[0120] Table 19

[0121] Table 20

[0122] Table 21

[0123] Table 22

[0124] [Table 23]

[0125] Example 3: Fermentation of Aspergillus niger Aspergillus niger strains were fermented in 500 ml baffled flasks containing 100 ml MU1 with 4 ml 50% urea on a rotary shaker at 220 rpm and 30°C. The culture broth was centrifuged (10,000 × g, 20 min) and the supernatant was carefully decanted from the sediment.

[0126] Example 4: Purification of PoAMG (JPO-001) variants The PoAMG variants were purified by cation exchange chromatography. Each peak fraction was pooled and dialyzed against 20 mM sodium acetate buffer, pH 5.0, and then the sample was concentrated using a centrifugal filter unit (Vivaspin Turbo15, Sartorius). The enzyme concentration was determined by the A280 value.

[0127] Example 5: Thermal Stability Determination (TSA) The purified enzyme was diluted to 0.5 mg / ml in 50 mM sodium acetate buffer, pH 5.0, and mixed with an equal volume of SYPRO Orange (Invitrogen) diluted with Milli-Q water. 18 μl of the mixture was transferred to a LightCycler480 Multiwell Plate384 (Roche Diagnostics), and the plate was sealed.

[0128] TSA instrument parameters: Equipment: LightCycler 480 Real-Time PCR System (Roche Applied Science) Scanning speed: 0.02°C / sec Scanning range: 37~96℃ Integration time: 1.0 seconds Excitation wavelength: 465 nm Emission wavelength 580nm

[0129] The resulting fluorescent signals were normalized to a range of 0 and 1. Td was defined as the temperature at which the signal intensity was 0.5. The thermal stability improvements are listed in Table 3, with the Td of the PoAMG variant designated as anPAV498 being set to 0.

[0130] Example 6: PoAMG activity assay Maltodextrin (DE11) assay by GOD-POD method

[0131] substrate solution 30g maltodextrin (MATSUTANI chemical industry Co., Ltd. pindex #2) 100 ml of 120 mM sodium acetate buffer, pH 5.0

[0132] Glucose CII test kit (Wako Pure Chemical Industries, Ltd.) 20 μl of enzyme sample was mixed with 100 μl of substrate solution and incubated at the set temperature for 2 hours. The sample was cooled on an aluminum block for 3 minutes, and 10 μl of the reaction solution was mixed with 590 μl of 1 M Tris-HCl pH 8.0 to stop the reaction. 10 μl of the solution was mixed with 200 μl of the standard solution from the test kit and then left at room temperature for 15 minutes. The absorbance was read at A505. The activity is listed in Table 3 as the relative activity of the PoAMG variant designated anPAV498.

[0133] [Table 24]

[0134] [Table 25]

[0135] [Table 26]

[0136] Example 7: JPO172 in pulsed panning Bread was baked using the straight baking method with the recipe according to Table 4. Various treatments were carried out according to Table 5. Bread was baked in an open pan. The ingredients were mixed into a dough in a spiral mixer for 3 minutes at 17 rpm and 7 minutes at 35 rpm. The dough was rested for 10 minutes and divided into 320 g pieces. The pieces were rolled, flattened and placed in baking tins. The filled baking tins were proofed for 60 minutes at 32°C and 86% relative humidity. The proofed dough was baked in a deck oven at 180°C for 20 minutes.

[0137] The bread was packed in sealed plastic bags 2 hours after baking and stored at room temperature until analysis.

[0138] [Table 27]

[0139] [Table 28]

[0140] The texture of the bread was evaluated with a texture analyzer (TA-XT plus, Stable microsystems, Godalmine, UK). The crumb texture properties of the bread were characterized by the hardness (same as "hardness" and opposite to "softness") and elasticity of the baked product. The standard method for measuring hardness and elasticity is based on the force-deformation of the baked product. The force-deformation of the baked product can be performed using a cylindrical probe with a diameter of 40 mm. The force on the cylindrical probe is recorded when a 25 mm thick bread slice is pressed down to 40% strain at a deformation rate of 1 mm / s. The probe is then held in this position for 30 seconds while the force is recorded, and then the probe returns to its original position.

[0141] Hardness (in grams) is defined as the force required to compress the probe to 25% strain (corresponding to a 6.25 mm compression into a 25 mm thick breadcrumb slice).

[0142] Elasticity (in %) is defined as the force recorded after 30 seconds of compression at 40% strain (corresponding to the force at time = 40 seconds for a 25 mm thick bread slice) divided by the force required to press the probe 10 mm into the crumb (corresponding to the force at time = 10 seconds for a 25 mm thick bread slice) multiplied by 100.

[0143] The results from the texture analysis can be found in Table 6 (hardness) and Table 7 (elasticity). Bread containing 33 mg EP / kg flour of JPO172 and 40 ppm Lipopan Xtra was more elastic than bread containing 100 ppm Novamyl G and 40 ppm Lipopan Xtra. At the same time, the breads had equal hardness.

[0144] When additional vital wheat gluten was added to both treatments, bread containing 33 mg EP / kg flour, 40 ppm Lipopan Xtra and 6% vital wheat gluten was more elastic and had the same hardness over the entire period tested compared to bread containing 100 ppm Novamyl G, 40 ppm Lipopan Xtra and 6% vital wheat gluten.

[0145] The addition of JPO172 alone at 33 mg EP / kg flour was also able to improve elasticity, but the bread was much harder than the combination of JPO172 at 33 mg EP / kg flour and 40 ppm Lipopan Xtra.

[0146] Conclusion: The combination of JPO172 and Lipopan Xtra was able to produce bread with a combination of low hardness and high elasticity over the entire period tested.

[0147] [Table 29]

[0148] Table 30

Claims

1. 1. A method for producing a calcined or partially calcined product, comprising: a) providing a dough comprising added pulse and / or legume proteins and a mature thermostable variant of a parent glucoamylase that is at least 70% identical to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:10; and b) baking or partially baking said dough to produce said baked or partially baked product.

2. 2. The method of claim 1, wherein the parent glucoamylase is derived from a species of the genus Penicillium, preferably from Penicillium oxycalum, Penicillium miczynskii, Penicillium russellii, or Penicillium glabrum.

3. 3. The method of any one of claims 1 to 2, wherein the mature variant comprises at least one amino acid modification at one or more or all of the positions corresponding to positions 1, 2, 4, 6, 7, 11, 31, 34, 50, 65, 79, 103, 132, 327, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595 of SEQ ID NO:

1.

4. 4. The method of claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 2, 4, 11, 65, 79 and 327 of SEQ ID NO: 1, preferably the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, P2N, P4S, P11F, T65A, K79V and Q327F of SEQ ID NO:

1.

5. 4. The method of claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 79, 103, 132, 445, 447, 481, 566, 568, 594 and 595 of SEQ ID NO: 1, preferably the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, K79V, S103N, A132P, D445N, V447S, S481P, D566T, T568V, Q594R and F595S of SEQ ID NO:

1.

6. 4. The method of claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 50, 103, 132, 445, 447, 481, 501, 539, 566, 568, 594 and 595 of SEQ ID NO: 1, preferably the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, S103N, A132P, D445N, V447S, S481P, E501A, N539P, D566T, T568V, Q594R and F595S of SEQ ID NO:

1.

7. 4. The method of claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 50, 103, 132, 445, 447, 481, 501, 539, 566, 568, 594 and 595 of SEQ ID NO: 1, preferably the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, S103N, A132P, D445N, V447S, S481P, E501A, N539P, D566T, T568V, Q594R and F595S of SEQ ID NO:

1.

8. 4. The method of claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595 of SEQ ID NO: 1, preferably wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R and F595S of SEQ ID NO:

1.

9. 2. The method of claim 1, wherein the mature thermostable variant has a thermostability improvement (Td) over its parent of at least 5°C, preferably at least 6°C, 7°C or 8°C.

10. 2. The method of claim 1, wherein the mature thermostable variant has a relative activity at 91°C of at least 150, preferably at least 200, more preferably at least 250, and most preferably at least 300 compared to its parent.

11. 2. The method of claim 1, wherein at least 2% (w / w) of the total flour content of pulses and / or legume protein is added, preferably at least 4% (w / w) of the total flour content of pulses and / or legume protein is added, preferably at least 6% (w / w) of the total flour content of pulses and / or legume protein is added, more preferably at least 8% (w / w) of the total flour content of pulses and / or legume protein, even more preferably at least 10% (w / w) of the total flour content of pulses and / or legume protein, and most preferably at least 12% (w / w) of the total flour content of pulses and / or legume protein.

12. The method of claim 1 , wherein the dough also comprises gluten.

13. 2. The method of claim 1, wherein the dough also comprises at least one added lipase enzyme, preferably wherein the at least one added lipase enzyme comprises a lipase and / or a phospholipase, preferably a mature lipase and / or a mature phospholipase, preferably wherein the at least one added lipase enzyme comprises a mature lipase having an amino acid sequence at least 70% identical to one or more of the sequences set forth in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:

21.

14. 14. The method of claim 13, wherein the at least one added lipase enzyme is added in an amount in the range of 0 to 100 mg enzyme protein / kg flour, preferably in the range of 0 to 50 mg enzyme protein / kg flour; more preferably in an amount in the range of 0 to 25 mg enzyme protein / kg flour; even more preferably in an amount in the range of 0 to 10 mg enzyme protein / kg flour; even more preferably in an amount in the range of 0 to 5 mg enzyme protein / kg flour; and most preferably in an amount in the range of 0 to 2.5 mg enzyme protein / kg flour.

15. The fabric comprising: a) mixed for at least 5 minutes at a low mixing speed, preferably in the range of 5 to 50 rpm, more preferably in the range of 10 to 40 rpm; more preferably at least 10 minutes at a low mixing speed, even more preferably at least 15 minutes at a low mixing speed; optionally 10. The method of claim 1, wherein b) the dough is then mixed at a faster speed.

16. It also includes adding one or more additional enzymes, wherein the additional enzymes are selected from the group consisting of α-amylase, maltogenic amylase, raw starch-degrading α-amylase, β-amylase, aminopeptidase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1,4-α-maltotetrahydrolase, glucanase, β-glucanase, galactanase, α-galactosidase, β-galactosidase, and β-galactosidase.

2. The method of claim 1, wherein the enzyme is selected from the group consisting of: hydroxylase, glucose oxidase, α-glucosidase, β-glucosidase, haloperoxidase, hemicellulolytic enzyme, invertase, laccase, lipase, mannase, mannosidase, oxidase, pectin degrading enzyme, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, protease, pullulanase, ribonuclease, transglutaminase, and xylanase.

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