Baking at low pH using a thermostable glucoamylase variant

Abstract

The present invention provides a method for producing a baked or par-baked product, comprising the steps of: a) preparing a dough comprising 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, added in an amount of 0.01 to 12.40 mg of enzyme protein (mgEP) per kg of flour, wherein the dough has a pH value in the range of 3.0 to 6.5; and b) baking or par-baking the dough to produce a baked or par-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 provides a method for producing a baked or par-baked product, comprising the steps of: a) preparing a dough comprising a mature thermostable variant of a parent glucoamylase (AMG) 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, added in an amount of 0.01 to 12.40 mg of enzyme protein (mgEP) per kg of flour, wherein the dough has a pH value in the range of 3.0 to 6.5; and b) Baking or semi-baking this dough to produce baked or semi-baked products. The present invention relates to a method comprising: [Background technology]

[0003] Sugar-containing baked goods (bread, biscuits, etc.) are one of the most popular product categories worldwide. The amount of sugar in a recipe will typically be 1-25% of the total flour weight.

[0004] However, due to rising market prices for sugar, limited availability of sugar in some parts of the world, and health concerns, there is a need for methods of producing baked goods with reduced amounts of added sugar without sacrificing, and potentially even improving, the quality of the baked goods.

[0005] WO 2019 / 238423 (Novozymes A / S, Denmark) discloses a method for producing dough with reduced amounts of added sugars, which involves adding a raw starch-degrading α-amylase and a glucoamylase to a dough ingredient.

[0006] WO 2022 / 090562 (Novozymes A / S, Denmark) discloses a method for producing baked or semi-baked products using mature thermostable variants of parent glucoamylases. Summary of the Invention [Problem to be solved by the invention]

[0007] The thermostabilized glucoamylase variants exhibit significantly improved freshness-keeping or anti-staling performance of baked or semi-baked goods. Another improved performance of the thermostabilized variants is that they increase the sweetness or sweet taste of the product, thereby allowing for a reduction in the amount of sugar added in traditional recipes. Here, another surprising effect of the thermostabilized glucoamylase variants is shown herein to be that they allow for a reduced enzyme dosage at a reduced dough pH in the range of 3.0 to 6.5. [Means for solving the problem]

[0008] Thus, in a first aspect, the present invention provides a method for producing a baked or semi-baked product, comprising the steps of: a) providing a dough comprising a mature thermostable variant of a parent glucoamylase having at least 70% sequence identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 10, added in an amount of 0.01 to 12.40 mg enzyme protein (mgEP) per kg flour, wherein the dough has a pH value in the range of 3.0 to 6.5, preferably in the range of 3.5 to 6.0, and even more preferably in the range of 4.0 to 5.5; and b) Baking or semi-baking this dough to produce baked or semi-baked products. The present invention relates to a method comprising:

[0009] Preferably, the mature thermostable variant of a parent glucoamylase of the present invention has at least 71% sequence identity 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 ...3% sequence identity 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 74% sequence identity 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 75% sequence identity 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 76% sequence identity 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 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%, 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%, for example at least 98%, such as at least 99%. [Brief explanation of the drawings]

[0010] [Figure 1-1]A multiple alignment of the amino acid sequences of the mature proteins is shown below. - the wild-type AMG (PoAMG) from Penicillium oxalicum according to SEQ ID NO: 1; - the PoAMG variant designated "AMG NL" according to SEQ ID NO: 2; - the PoAMG variant designated "AMG anPAV498" according to SEQ ID NO: 3; - the PoAMG variant designated "AMG JPO001" according to SEQ ID NO: 4; - the PoAMG variant designated "AMG JPO124" according to SEQ ID NO: 5; - the PoAMG variant designated "AMG JPO172" according to SEQ ID NO: 6; - the wild-type AMG (PoAMG) from Penicillium miczynskii according to SEQ ID NO: 7; - the wild-type AMG (PoAMG) from Penicillium russellii according to SEQ ID NO: 8; - the wild-type AMG (PoAMG) from Penicillium glabrum according to SEQ ID NO: 9. [Figure 1-2] A multiple alignment of the amino acid sequences of the mature proteins is shown below. - the wild-type AMG (PoAMG) from Penicillium oxalicum according to SEQ ID NO: 1; - the PoAMG variant designated "AMG NL" according to SEQ ID NO: 2; - the PoAMG variant designated "AMG anPAV498" according to SEQ ID NO: 3; - the PoAMG variant designated "AMG JPO001" according to SEQ ID NO: 4; - the PoAMG variant designated "AMG JPO124" according to SEQ ID NO: 5; - the PoAMG variant designated "AMG JPO172" according to SEQ ID NO: 6; - the wild-type AMG (PoAMG) from Penicillium miczynskii according to SEQ ID NO: 7; - the wild-type AMG (PoAMG) from Penicillium russellii according to SEQ ID NO: 8; - the wild-type AMG (PoAMG) from Penicillium glabrum according to SEQ ID NO: 9. [Figure 2]Figure 1 shows the pH-activity profile of a thermostable glucoamylase variant designated JPO-172. The pH-activity profile was determined at 40°C. Each data point represents the average of four measurements, and the error bars represent the standard deviation. pH 5 is set at 10%. The profile shows that the optimum pH is approximately pH 5, and that approximately 80% activity is observed between approximately pH 4 and 6. DETAILED DESCRIPTION OF THE INVENTION

[0011] definition Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0012] For the purposes of the present invention, sequence homology between two amino acid sequences is preferably determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), 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 the alignment)

[0013] 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 occupying a position with a different amino acid; a deletion refers to the removal of an amino acid occupying a position; and an insertion refers to the addition of one or more amino acids adjacent to and immediately following the amino acid occupying a position. These amino acid changes can be minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 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 alter 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 are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0014] Strength increase: The term "increased dough strength" is defined herein as a characteristic of dough that generally has more elastic properties and / or requires more work input to mold and shape compared to a control.

[0015] Increased Elasticity: The term "increased fabric resilience" is defined herein as the property of a fabric that has an increased tendency to regain its original shape after being subjected to a specified physical strain compared to a control.

[0016] Increased fabric stability: The term "increased dough stability" is defined herein as a dough property that is less susceptible to mechanical abuse and therefore better maintains its shape and volume, as assessed by the cross-sectional height:width ratio of the loaf after standard proof and / or extended proof compared to a control.

[0017] Reduced dough stickiness: The term "reduced dough stickiness" is defined herein as the property of dough that has a reduced tendency to stick to a surface compared to a control, e.g., in a dough maker, as assessed empirically by a skilled test baker or measured, e.g., by a texture analyzer (e.g., TAXT2) as known in the art.

[0018] Improved stretchability: The term "improved fabric extensibility" is defined herein as the property of a fabric that can undergo increased strain or elongation without breaking compared to a control.

[0019] Improved machinability: The term "improved dough machinability" is defined herein as the property of the dough being generally less sticky and / or firmer and / or more elastic compared to a control.

[0020] Increase in volume of baked product: The term "baked product volume increase" is measured as the volume of a given loaf of bread compared to a control. This volume can be determined as known in the art.

[0021] Improved crumb structure of baked goods: The term "improved crumb structure of a baked good" is defined herein as the property of a baked good having finer cells and / or thinner cell walls in the crumb and / or a more uniform / homogeneous distribution of cells in the crumb compared to a control, typically as assessed visually by a skilled baker or by digital image analysis known in the art (e.g., C-cell, Calibre Control International Ltd, Appleton, Warrington, UK).

[0022] Improved softness of baked goods: The term "improved softness of baked goods" is the opposite of "firmness" and is defined herein as the property of a baked good that is easily compressed compared to a control, as assessed empirically by skilled test bakers or measured as known in the art, for example, by a texture analyzer (e.g., TAXT2 or TA-XT Plus from Stable Micro Systems Ltd, Surrey, UK).

[0023] Sensory characteristics of baked products: The sensory attributes may be evaluated using procedures well established in the baking industry, such as the use of a panel of trained taste testers.

[0024] Improved thermal stability: 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.

[0025] A first aspect of the present invention is a method for producing a baked or par-baked product, comprising the steps of: a) providing a dough comprising 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 semi-baking this dough to produce baked or semi-baked products. The present invention relates to a method comprising:

[0026] Another aspect of the invention relates to a method for reducing the amount of sugar in a dough in a method for producing a baked or par-baked product, and / or for reducing the amount of sugar in a dough in order to extend the shelf life of the baked or par-baked product, and / or for increasing the sweetness of the baked or par-baked product in a method as defined in the first aspect, whereby the baked or par-baked product after final baking has reduced initial hardness and / or increased initial springiness and / or has reduced gain in hardness and / or is more springy after 1, 7 or 14 days when cooled to room temperature, packed in a sealed container and stored at room temperature until analysed, compared to a control made without the addition of any glucoamylase.

[0027] Preferably, the mature thermostable variant of a parent glucoamylase of the present invention has at least 71% sequence identity 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 ...3% sequence identity 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 74% sequence identity 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 75% sequence identity 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 76% sequence identity 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 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%, 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%, for example at least 98%, such as at least 99%.

[0028] material As used herein, "dough" means any dough used to prepare baked goods, especially bread.

[0029] According to the present invention, the dough used to prepare the baked goods can be made from any suitable dough ingredients, including flour.

[0030] The flour can be derived from any baking grain known in the art, such as wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, sorghum flour, potato flour, soy flour, and any combination thereof (e.g., wheat flour combined with one of the other flour sources, or rice flour combined with one of the other flour sources).

[0031] In a preferred embodiment, the flour is wheat flour.

[0032] In a preferred embodiment, at least 10% (w / w) or more of the total flour content is wheat flour, such as at least 15% or more of the total flour content is wheat flour, for example at least 20% or more of the total flour content is wheat flour, such as at least 25% or more of the total flour content is wheat flour, for example at least 30% or more of the total flour content is wheat flour, such as at least 35% or more of the total flour content is wheat flour, for example at least 40% or more of the total flour content is wheat flour, such as at least 45% or more of the total flour content is wheat flour, for example at least 50% or more of the total flour content is wheat flour, for example At least 55% or more of the total flour content is wheat flour, such as at least 60% or more of the total flour content is wheat flour, for example at least 65% or more of the total flour content is wheat flour, such as at least 70% or more of the total flour content is wheat flour, for example at least 75% or more of the total flour content is wheat flour, such as at least 80% or more of the total flour content is wheat flour, for example at least 85% or more of the total flour content is wheat flour, for example at least 90% or more of the total flour content is wheat flour, for example at least 95% or more of the total flour content is wheat flour, for example 100% of the total flour is wheat flour.

[0033] The dough of the present invention is typically a leavened dough or a dough that is subjected to leavening. The dough may be leavened in a variety of ways, for example, by adding dough ingredients such as chemical leavening agents (e.g., sodium bicarbonate) or by adding a leaven (which ferments the dough), but it is preferred that the dough be leavened by adding a suitable yeast culture, such as a culture (baker's yeast) of Saccharomyces cerevisiae (e.g., a commercially available strain of S. cerevisiae).

[0034] The dough of the present invention may typically contain some added sugar, but the method of the present invention allows the amount of added sugar to be reduced, usually resulting in a partially reduced sugar dough.

[0035] In one embodiment, the amount of added sugar is reduced by at least 10% (w / w) compared to the amount of sugar added to the dough in the original recipe, for example, the amount of added sugar is reduced by at least 20% (w / w) compared to the amount of sugar added to the dough in the original recipe, for example, the amount of added sugar is reduced by at least 30% (w / w) compared to the amount of sugar added to the dough in the original recipe, for example, the amount of added sugar is reduced by at least 40% (w / w) compared to the amount of sugar added to the dough in the original recipe, for example, the amount of added sugar is reduced by at least 50% (w / w) compared to the amount of sugar added to the dough in the original recipe. For example, the amount of added sugar is reduced by at least 60% (w / w) compared to the amount of sugar added to the dough in the original recipe, for example, the amount of added sugar is reduced by at least 70% (w / w) compared to the amount of sugar added to the dough in the original recipe, for example, the amount of added sugar is reduced by at least 80% (w / w) compared to the amount of sugar added to the dough in the original recipe, for example, the amount of added sugar is reduced by at least 90% (w / w) compared to the amount of sugar added to the dough in the original recipe, for example, the amount of added sugar is reduced by 100% (w / w) compared to the amount of sugar added to the dough in the original recipe.

[0036] The dough may also contain other common dough ingredients, such as proteins such as milk powder, gluten, and soybeans; eggs (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; salts such as sodium chloride, calcium acetate, sodium sulfate, or calcium sulfate; diluents such as silicon dioxide; and starches of various origins. Still other common ingredients include hydrocolloids such as CMC, guar gum, xanthan gum, and locust bean gum.

[0037] The dough ingredients may typically contain fats (triglycerides), and / or oils and / or shortening, in particular oils such as sunflower oil or rapeseed oil.

[0038] In a preferred embodiment, the dough of the present invention does not have any added emulsifiers, and preferably the dough of the present invention does not have any added SSL.

[0039] In a preferred embodiment, the pH of the dough of the present invention is adjusted to a range of 3.0 to 6.5 by the addition of a food-acceptable acid, preferably an organic acid such as acetic acid or citric acid, preferably to a range of 3.5 to 6.0, even more preferably to a range of 4.0 to 5.5, and most preferably the pH of the dough is adjusted by the addition of vinegar to the dough.

[0040] The dough may be prepared by applying any conventional mixing process, such as the continuous mixing process, the straight-dough process, or the sponge and dough method.

[0041] The present invention is particularly useful for preparing doughs and baked goods in industrial processes where the dough used to prepare the baked goods is prepared mechanically using automated or semi-automated equipment.

[0042] The process of preparing bread generally involves the successive steps of making dough, sheeting or dividing, shaping or rolling, and leavening the dough, which steps are known in the art.

[0043] As used herein, "baked goods" refers to any type of baked goods, such as types of bread, for example, toasted bread, toast bread, open bread, toasted bread with or without lid, buns, fino bread, hammam bread, samori bread, baguette, brioche hamburger bun, roll, brown bread, wholemeal bread, rich bread, bran bread, flatbread, tortilla, biscuit, and any variant thereof. According to the present invention, the baked goods may also be cakes or any pastry product as known in the art.

[0044] Raw starch-digesting alpha-amylase As used herein, "raw starch-degrading alpha-amylase" refers to an enzyme that can directly degrade raw granular starch below the gelatinization temperature of the starch.

[0045] Examples of raw starch-degrading alpha-amylases include those disclosed in WO 2005 / 003311, U.S. Patent Application Publication No. 2005 / 0054071, and U.S. Patent No. 7,326,548. Examples also include the enzymes disclosed in Tables 1-5 of the Examples in U.S. Patent Application Publication No. 2005 / 0054071 (Table 3 on page 15), and the enzymes disclosed in WO 2004 / 020499, WO 2006 / 06929, and WO 2006 / 066579.

[0046] In one embodiment, the native starch-degrading alpha-amylase is a GH13_1 amylase.

[0047] In one embodiment, the native amylolytic alpha-amylase enzyme is selected from the group consisting of those described in EP 2981170 (Novozymes A / S), has at least 70% sequence identity to the raw starch degrading alpha-amylase set forth in

[0048] In one embodiment, the raw starch-degrading alpha-amylase of the present invention may be added to flour or dough in an amount of 0.01 to 10 mg of enzyme protein per kg of flour, for example, 0.1 to 5 mg of enzyme protein per kg of flour.

[0049] Glucoamylase Glucoamylase is also called amyloglucosidase and glucan 1,4-alpha-glucosidase (EC 3.2.1.3), more commonly referred to as AMG.

[0050] According to the present invention, various types of amyloglucosidases can be used as parents for the production of thermostable amyloglucosidase variants, for example, the amyloglucosidase can be a polypeptide encoded by a DNA sequence found in a fungal strain of the genus Aspergillus, Rhizopusor, Talaromyces, or Penicillium, preferably a polypeptide encoded by a DNA sequence found in a fungal strain 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.

[0051] Other suitable examples of fungi include Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Rhizopus delemar, Rhizopus niveus, Rhizopus oryzae, and Talaromyces emersonii.

[0052] Below is shown the % identity between the AMG amino acid sequences aligned in Figure 1 and also provided in the Sequence Listing.

[0053] [Table 1]

[0054] In one embodiment, the glucoamylase of the present invention may be added to flour or dough in an amount of 0.01 to 1,000 mg enzyme protein (mg EP) per kg of flour, preferably 0.01 to 500 mg enzyme protein (mg EP) per kg of flour, and even more preferably 0.1 to 100 mg enzyme protein (mg EP) per kg of flour.

[0055] Thermostable variants of PoAMG have been generated (see Table 2 below). In preferred embodiments, the mature thermostable glucoamylase variants of the invention comprise one, more than one, or all of the combinations of amino acid substitutions listed in Table 2 below.

[0056] In a preferred embodiment, the mature variants of the invention comprise at least one amino acid modification at one, more than one, or all of the positions corresponding to positions 1, 2, 4, 6, 7, 11, 31, 34, 65, 79, 103, 132, 327, 445, 447, 481, 566, 568, 594, and 595 in SEQ ID NO:1, and preferably, the at least one amino acid modification comprises a substitution at one, more than one, or all of the positions corresponding to positions 1, 2, 4, 11, 65, 79, and 327 in SEQ ID NO:1. preferably, the 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, the 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; Preferably, the at least one amino acid modification comprises a substitution at one, more than one, or all of the positions corresponding to R1A, G6S, G7T, R31F, K34Y, K79V, S103N, A132P, D445N, V447S, S481P, D566T, T568V, Q594R, and F595S in SEQ ID NO:1, or preferably, the at least one amino acid modification comprises a substitution at one, more than one, or all of the positions corresponding to 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563 The at least one amino acid modification may include a substitution at one, more than one, or all of positions corresponding to 539, 566, 568, 594, and 595, and preferably the at least one amino acid modification includes a substitution at one, more than one, or all of 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.

[0057] 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) is set to zero. In preferred embodiments, a mature thermostable variant of the invention has a thermostability improvement (Td) over its parent of at least 3°C, preferably at least 4°C, 5°C, 6°C, 7°C, or 8°C, preferably determined as exemplified herein.

[0058] In another preferred embodiment, a mature thermostable variant of the invention has a relative activity at 91°C compared to its parent of at least 150, preferably at least 200, more preferably at least 250, and most preferably at least 300.

[0059] Preferably, the mature thermostable variant glucoamylase enzyme is present in the dough in an amount of 0.01 to 1,000 mg enzyme protein (mg EP) per kg flour, preferably 0.01 to 500 mg enzyme protein (mg EP) per kg flour, and even more preferably 0.1 to 100 mg enzyme protein (mg EP) per kg flour.

[0060] amylase Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, EC.3.2.1.1) constitute a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glucoside oligosaccharides and polysaccharides.

[0061] A number of alpha-amylases are referred to as Termamyl™ and “Termamyl™-like alpha-amylases” and are known, for example, from WO 90 / 11352, WO 95 / 10603, WO 95 / 26397, WO 96 / 23873, and WO 96 / 23874.

[0062] Another group of alpha-amylases is referred to as Fungamyl™ and “Fungamyl™-like alpha-amylases,” which are alpha-amylases related to the alpha-amylase from Aspergillus oryzae disclosed in WO 01 / 34784.

[0063] Suitable commercially available alpha-amylase compositions according to the present invention include, for example, BAKEZYME P 300 (available from DSM), and FUNGAMYL 2500 SG, FUNGAMYL 4000 BG, FUNGAMYL 4000 SG, FUNGAMYL 800 L, FUNGAMYL ULTRA BG, and FUNGAMYL ULTRA SG (available from Novozymes A / S).

[0064] In one embodiment, the alpha-amylase of the present invention may be added to flour or dough in an amount of 0.01 to 1,000 mg enzyme protein (mg EP) per kg of flour, preferably 0.01 to 500 mg enzyme protein (mg EP) per kg of flour, and even more preferably 0.1 to 100 mg enzyme protein (mg EP) per kg of flour.

[0065] Additional enzymes Optionally, one or more additional enzymes may be used in conjunction with the enzyme composition of the present invention, such as alpha-amylase, maltogenic amylase, beta amylase, aminopeptidase, carboxypeptidase, catalase, cellulolytic enzymes, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1,4-alpha-maltotetrahydrolase, glucanase, galactanase, alpha-galactosidase ... Enzymes such as sidases, beta-galactosidase, glucose oxidase, alpha-glucosidase, beta-glucosidase, haloperoxidase, hemicellulolytic enzymes, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectolytic enzymes, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, proteolytic enzymes, ribonucleases, transglutaminases, and xylanases may be used.

[0066] The additional enzymes can be of any origin, including mammalian, plant, and microbial (bacterial, yeast, or fungal) origin.

[0067] Maltogenic alpha-amylases (EC 3.2.1.133) can be derived from Bacillus. A maltogenic alpha-amylase from B. stearothermophilus strain NCIB11837 is commercially available from Novozymes A / S under the trade name Novamyl®.

[0068] The maltogenic alpha-amylase may also be a variant of a maltogenic alpha-amylase from B. stearothermophilus, such as those disclosed in WO 99 / 43794, WO 2006 / 032281, or WO 2008 / 148845, e.g., Novamyl® 3D.

[0069] The anti-staling amylase for use in the present invention can also be an amylase from Pseudomonas saccharophilia (glucan 1,4-alpha-maltotetrahydrolase (EC 3.2.1.60)) or a variant thereof, such as any of the amylases disclosed in WO 99 / 50399, WO 2004 / 111217, or WO 2005 / 003339.

[0070] The glucose oxidase may be a fungal glucose oxidase, in particular an Aspergillus niger glucose oxidase (eg, GLUZYME®, available from Novozymes A / S).

[0071] The xylanase may be of microbial origin, for example derived from a bacterium or a fungus, for example derived from a strain of the genus Aspergillus, in particular derived from a strain of A. aculeatus, A. niger, A. awamori or A. tubingensis, or derived from a strain of the genus Trichoderma, for example derived from a strain of T. reesei, or derived from a strain of the genus Humicola, for example derived from a strain of H. insolens.

[0072] Suitable commercially available xylanase preparations for use in the present invention include PANZEA BG, PENTOPAN MONO BG, and PENTOPAN 500 BG (available from Novozymes A / S), GRINDAMYL POWERBAKE (available from Danisco), and BAKEZYME BXP 5000 and BAKEZYME BXP 5001 (available from DSM).

[0073] The protease may be derived from a Bacillus, for example, B. amyloliquefaciens. A suitable protease may be Neutrase® available from Novozymes A / S.

[0074] The phospholipase may have phospholipase A1, A2, B, C, D, or lysophospholipase activity, with or without lipase activity. The phospholipase may be of animal origin, such as from the pancreas, snake venom, or bee venom, or of microbial origin, such as from a fungus, yeast, or bacterium, such as from Aspergillus or Fusarium, for example, from A. niger, A. oryzae, or F. oxysporum. A preferred lipase / phospholipase from Fusarium oxysporum is disclosed in WO 98 / 26057. Also, variants described in WO 00 / 32758 may be used.

[0075] Suitable phospholipase compositions are LIPOPAN F, LIPOPAN XTRA, and LIPOPAN MAX (available from Novozymes A / S), or PANAMORE GOLDEN and PANAMORE SPRING (available from DSM).

[0076] Preferably, the one or more additional enzymes are added in an amount of 0.01 to 1,000 mg enzyme protein (mg EP) per kg flour, preferably 0.01 to 500 mg enzyme protein (mg EP) per kg flour, and even more preferably 0.1 to 100 mg enzyme protein (mg EP) per kg flour.

[0077] Enzyme composition The mature thermostable variant glucoamylase of the present invention, and any additional enzymes, may be added to the flour or dough in any suitable form, such as in the form of a liquid (particularly a stabilized liquid), or may be added to the flour or dough as a substantially dry powder or granules.

[0078] Granules can be produced, for example, as disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452. Liquid enzyme preparations can be stabilized, for example, by adding sugars or sugar alcohols, or lactic acid, according to established procedures. Other enzyme stabilizers are known in the art.

[0079] The enzymes may be added to the dough ingredients in any suitable way, for example, as individual components (separate or sequential addition of enzymes) or the enzymes may be added together in one step or composition.

[0080] Pan Characteristics The organoleptic or sensory properties of bread may be measured as known in the art. Bread properties may be referred to herein as sensory properties, and include staling resistance (crumb firmness / hardness), crumb properties and mouthfeel, or more precisely, attributes of the bread as detected in the mouth during eating (e.g., bread softness / resistance at first bite, crumb moistness, crumb chewiness and gumminess, and crumb smoothness and melting properties).

[0081] In one embodiment, the sensory attribute of the baked goods is increased sweetness due to the use of the enzyme solution of the present invention.

[0082] In one embodiment, the sensory attribute of the baked goods is an increase in the sweetness of the crumb due to the use of an enzyme solution according to the present invention.

[0083] In preferred embodiments of the invention, the baked or par-baked products have reduced initial hardness and / or increased initial springiness after final baking and / or have reduced gain in hardness and / or increased springiness after 1, 7, or 14 days when cooled to room temperature, packed in a sealed container, and stored at room temperature until analyzed, compared to a control made without any added glucoamylase.

[0084] In another preferred embodiment, the baked or partially baked product has, after final baking, at least the same sweetness or sweet taste as a control product made using twice the amount of mature glucoamylase, the amino acid sequence of which is set forth in SEQ ID NO: 10, preferably as exemplified herein, and preferably, the baked or partially baked product has an increased sweetness or sweeter taste after final baking compared to a control product made using twice the amount of mature glucoamylase, the amino acid sequence of which is set forth in SEQ ID NO: 10, preferably as exemplified herein.

[0085] The invention described and claimed herein should not be limited in scope by the specific embodiments disclosed herein, as these are intended as illustrations of some aspects of the invention. Any equivalent embodiments are also intended to be within the scope of the invention, as are any combinations of one or more of the embodiments.

[0086] Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. [Example]

[0087] Example 1: Construction of the PoAMG library The PoAMG library was constructed as follows. Forward and reverse primers were designed with 15 bp overlapping each other, 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 the E. coli transformants using a MagExtractor plasmid extraction kit (TOYOBO) and then transformed into A. niger competent cells.

[0088] 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)

[0089] PCR program: 98℃ / 2 minutes 25×(98℃ / 10 seconds, 60℃ / 15 seconds, 72℃ / 2 minutes) 10℃ / hold

[0090] 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- 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 below.

[0091] pNPG Thermostability Assay: The culture supernatant containing the desired enzyme was mixed with an equal 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 a 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.

[0092] [Table 2]

[0093] [Table 3]

[0094] [Table 4]

[0095] [Table 5]

[0096] Table 6

[0097] Table 7

[0098] Table 8

[0099] Table 9

[0100] Table 10

[0101] Table 11

[0102] Table 12

[0103] Table 13

[0104] Table 14

[0105] Table 15

[0106] Example 3: Fermentation of Aspergillus niger Aspergillus niger strains were fermented in 500 ml baffled flasks containing 100 ml of MU1 containing 4 ml of 50% urea on a rotary shaker table 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.

[0107] 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 Turbo 15, Sartorius). The enzyme concentration was determined by the A280 value.

[0108] 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.

[0109] 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

[0110] 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, where the Td of the PoAMG variant designated as anPAV498 is set to 0.

[0111] Example 6: PoAMG activity assay Maltodextrin (DE11) assay by GOD-POD method substrate solution Maltodextrin (MATSUTANI chemical industry Co., Ltd. pindex #2) 30g 120 mM sodium acetate buffer, pH 5.0 100 ml

[0112] Glucose CII test kit (Wako Pure Chemical Industries, Ltd.)

[0113] 20 μl of enzyme sample was mixed with 100 μl of substrate solution and incubated at a set temperature for 2 hours. The sample was cooled on an aluminum block for 3 hours, and then 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 this 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.

[0114] [Table 16]

[0115] [Table 17]

[0116] [Table 18]

[0117] Example 7: Baking with reduced doses of JPO-172 Breads were baked using the straight-through method using the recipe according to Table 4. Various treatments were performed according to Table 5 to compare the performance of JPO-172 to Novamyl® 3D (Novozymes, Denmark), the current best-performing commercial anti-staling baking maltogenic alpha-amylase product. Breads were baked in covered pans. The ingredients were mixed with the dough in a pin mixer at first speed for 1 minute, followed by thorough mixing at second speed. The dough was rested for 5 minutes, divided into 645g pieces, and rolled. The rounded pieces were rested for 10 minutes, sheeted, and placed in baking pans. The filled baking pans were proofed at 104-109°F and 85% relative humidity to allow rise. For covered breads, the dough was proofed until it was ¾ inch thick from the top. The leavened dough was baked in a rotary oven at approximately 227°C (440°F) for 17 minutes.

[0118] [Table 19]

[0119] [Table 20]

[0120] The bread was packed in sealed plastic bags 2 hours after baking and stored at room temperature until analysis.

[0121] 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 firmness (same as "hardness" and opposite to "softness") and springiness of the baked product. The standard method for measuring firmness and springiness is based on the force-deformation of the baked product. The force-deformation of the baked product can be performed using a 40 mm diameter cylindrical probe. The force on the cylindrical probe is recorded when a 25 mm thick bread slice is pressed down to a strain of 27% 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.

[0122] 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).

[0123] Elasticity (in %) is defined as the force recorded after 30 seconds of compression at 27% strain (corresponding to force at time = 36.75 seconds for a 25 mm thick bread slice) divided by the force required to press the probe 6.75 mm into the crumb (corresponding to force at time = 6.75 seconds for a 25 mm thick bread slice) multiplied by 100.

[0124] The results from the texture analysis can be found in Tables 6 and 7. Fresh bread is soft (less hard) and chewy. As the bread is stored, it becomes harder and less chewy. The higher the dose of JPO-172, the less hard and more chewy the bread is at days 7-21. However, for the period 1-21 days, the softest and most chewy bread at these conditions and doses was the bread containing 750 MANU / kg flour of Novamyl® 3D.

[0125] [Table 21]

[0126] [Table 22]

[0127] Example 8: Baking at lower pH and with reduced dosage of JPO-172 Breads were baked using the straight-through method using the recipe according to Table 8. Various treatments were performed according to Table 9. Breads were baked in covered pans. The ingredients were mixed with the dough in a pin mixer at first speed for 1 minute, followed by thorough mixing at second speed. The dough was allowed to rest for 5 minutes, divided into 645g pieces, and rolled. The rounded pieces were allowed to rest for 10 minutes, sheeted, and placed in baking pans. The filled baking pans were proofed at 104-109°F and 85% relative humidity to rise. For covered breads, the dough was proofed until it was 3 / 4 inch thick from the top. The proofed dough was baked in a rotary oven at approximately 227°C (440°F) for 17 minutes.

[0128] [Table 23]

[0129] [Table 24]

[0130] The bread was packed in sealed plastic bags 2 hours after baking and stored at room temperature until analysis.

[0131] Texture analysis was performed as in Example 7. The results from the texture analysis can be found in Tables 10 and 11. Fresh bread is soft (less hard) and chewy; as the bread is stored, it becomes harder and less chewy in the absence of anti-staling enzymes.

[0132] The inventors conclude that the addition of 2% vinegar lowered the pH of the dough, thereby allowing the JPO-172 dosage to be reduced to below 12 mg EP / mg flour, while still providing anti-staling efficacy equal to or greater than that of Novamyl 3D®.

[0133] [Table 25]

[0134] [Table 26]

[0135] Example 9: Activity of JPO-172 as a function of pH The pH activity profile is characteristic of an enzyme and is an important property for the use of enzymes in various applications. The pH profile of JPO-172 (within the pH range of 2-10) was determined at 40°C and incubation for 30 minutes.

[0136] A 30 mM maltose (CAS No.: 6363-53-7) substrate solution was prepared in a buffer (0.1 M acetic acid; 0.1 M MES; 0.1 M HEPES; 0.1 M glycine) adjusted to pH 2-10 using HCl or NaOH. pH activity profiles were prepared by adding 15 μL of diluted enzyme sample (10 ppm, diluted in 20 mM MES, pH 5) or buffer to 135 μL of substrate solution in an Eppendorf tube. The pH of the reaction mixture was determined. The mixture was incubated at 40°C and 800 rpm for 30 minutes. After 30 minutes, the reaction was terminated by adding 16 μL of 0.5 M NaOH and placed on ice. The reaction mixture was diluted 10-fold with 20 mM MES, pH 5, in a microtiter plate. 40 μL of the diluted solution was mixed with 160 μL of the GOD-POD reaction mixture in a new microtiter plate and incubated for 30 minutes in the dark. After incubation, the absorbance at 420 nm was measured. The average of the reaction mixtures was subtracted from the average of the blank samples and is shown in Figure 2.

[0137] The pH activity profile is shown in Figure 2. All data points are the average of four measurements and are assigned a relative pH of 5, which was set at 100%. The profile shows that the optimum pH is approximately pH 5, and that approximately 80% activity is observed between approximately pH 4 and 6.

Claims

1. 1. A method for producing a baked or semi-baked product, comprising: a) providing a dough comprising 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 added in an amount of 0.01 to 12.40 mg enzyme protein (mgEP) per kg flour, wherein the dough has a pH value in the range of 3.0 to 6.5, preferably in the range of 3.5 to 6.0, and even more preferably in the range of 4.0 to 5.5; and b) baking or par-baking the dough to produce a baked or par-baked product. A method comprising:

2. 2. The method of claim 1, wherein the baked or semi-baked product is a type of bread, preferably toasted bread, toast bread, open bread, bun, fino bread, hammam bread, samori bread, baguette, brioche, hamburger bun, roll, brown bread, wholemeal bread, rich bread, bran bread, flatbread, tortilla or biscuit, cake or patisserie.

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

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

1.

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

1.

6. 5. The method of claim 4, wherein the 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 of SEQ ID NO:1, preferably wherein the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to R1A, G6S, G7T, R31F, K34Y, K79V, S103N, A132P, D445N, V447S, S481P, D566T, T568V, Q594R, and F595S of SEQ ID NO:

1.

7. 4. The method of any one of claims 1 to 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to 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 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.

8. 8. The method of any one of claims 1 to 7, wherein the mature thermostable variant has a thermostability improvement (Td) over its parent of at least 3°C, preferably at least 4°C, 5°C, 6°C, 7°C, or 8°C.

9. 9. The method of any one of claims 1 to 8, wherein the mature thermostable variant has a relative activity at 91°C compared to its parent of at least 150, preferably at least 200, more preferably at least 250, and most preferably at least 300.

10. The method according to any one of claims 1 to 9, wherein no emulsifier is added to the dough.

11. 11. The method of any one of claims 1 to 10, wherein the baked or par-baked product, after final baking, has reduced initial hardness and / or increased initial springiness and / or has reduced gain in hardness and / or is more springy after 1, 7 or 14 days when cooled to room temperature, packed in a sealed container and stored at room temperature until analyzed, compared to a control made without the addition of any glucoamylase.

12. 12. The method of any one of claims 1 to 11, wherein the baked or partially baked product has, after final baking, at least the same sweetness as a control product made with twice the amount of the mature glucoamylase whose amino acid sequence is set forth in SEQ ID NO:

10.

13. The dough may also contain an enzyme such as alpha-amylase, maltogenic amylase, raw starch degrading alpha-amylase, beta amylase, aminopeptidase, carboxypeptidase, catalase, cellulolytic enzymes, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1,4-alpha-maltotetrahydrolase, glucanase, galactanase, alpha-galactosidase, beta-galactosidase, glucose oxidase, alpha-glucosidase, beta-glucosidase, haloperoxidase, hemicellulolytic enzymes, invertase, laccase, lipase, mannanase, mannosidase, oleoside, hydroxybenzoate ...

13. The method of any one of claims 1 to 12, also comprising one or more additional enzymes selected from the group consisting of oxidases, pectinolytic enzymes, peptidoglutaminases, peroxidases, phospholipases, phytases, polyphenol oxidases, proteases, ribonucleases, transglutaminases and xylanases, preferably wherein the one or more additional enzymes are present in an amount of 0.1 to 1,000 mg enzyme protein (mg EP) per kg flour, preferably 0.01 to 500 mg enzyme protein (mg EP) per kg flour, even more preferably 0.1 to 100 mg enzyme protein (mg EP) per kg flour.

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