Baking method using thermostable AMG variants and α-amylase

Abstract

A method for producing a baked or partially baked product with improved crumb softness and / or elasticity, comprising adding a mature α-amylase 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 to a dough, 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 for producing baked products with improved crumb softness and / or elasticity, comprising adding to dough a mature α-amylase 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 the dough. [Background technology]

[0003] WO 2019 / 238423 (Novozymes A / S, Denmark) discloses a method for producing dough with reduced amounts of added sugars, which involves adding raw starch-degrading α-amylase and glucoamylase to the dough ingredients.

[0004] WO 2022 / 090562 (Novozymes A / S) discloses a method for producing baked or par-baked products, comprising a first step of providing a dough containing the same mature thermostable variant of the parent glucoamylase disclosed herein, which has shown significantly improved performance in the freshness or anti-staling of the baked or par-baked products. Another improved performance of the thermostable variant is that it increases the sweetness or sweet taste of the products, which allows for a reduction in the amount of added sugar in traditional recipes. Summary of the Invention [Problem to be solved by the invention]

[0005] The baking method using the mature thermostable glucoamylase variant of WO 2022 / 090562 (Novozymes A / S) provides baking benefits that are comparable to or superior to those of currently commercially available α-amylase-based enzyme solutions. Given this performance, it was surprising to find that adding α-amylase to the dough could also further improve some properties of the baked product. [Means for solving the problem]

[0006] Thus, in a first aspect, the present invention relates to a method for producing baked or partially baked products with improved crumb softness and / or elasticity, the method comprising adding to a dough a mature α-amylase 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.

[0007] 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]

[0008] [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 JPO-172" 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

[0009] definition Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0010] 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)

[0011] 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.

[0012] 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.

[0013] The term "starch gelatinization" is understood as the irreversible order-disorder transition that starch undergoes when heated in the presence of water. Differential scanning calorimetry (DSC) can be used to determine the onset and peak temperatures (T o and T p The "gelatinization onset temperature (T)" is one technique available to study the gradual process of starch gelatinization. o The term "gelatinization peak temperature (T )" is understood as the temperature at which gelatinization begins. p The term "gelatinization end temperature (T )" is understood as the temperature at the endothermic peak. c ) is understood as the temperature at which gelatinization is complete.

[0014] 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.

[0015] Improved crumb firmness of the baked product: 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 hardness is assessed empirically by a test beaker / sensory panel of persons skilled in the art or measured by use of a texture analyzer known in the art (e.g., TAXT2 or TA-XT Plus from Stable Micro Systems Ltd, Surrey, UK).

[0016] Improved flavor of baked products: The term "improved flavor of baked products" is evaluated by a trained test panel and / or chemical analysis (e.g., headspace GC-MS analysis). Improved flavor of baked products includes a reduction in off-flavors of the baked products.

[0017] 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.

[0018] 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.

[0019] Bread colour: The colour or whiteness of the baked or par-baked product is measured as the "colour L*" value on a C-cell (Calibre Instruments Ltd, Warrington, UK) using standard methods for collecting images and standard C-Cell software for data analysis.

[0020] Fabric according to the present invention The present invention relates to a dough for baked or parbaked products.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] A preferred embodiment relates to a dough according to the first aspect, wherein 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.

[0029] Preferably, the dough of the present invention also contains gluten.

[0030] 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.

[0031] In a preferred embodiment of the present invention, the dough of the present invention also comprises gluten.

[0032] The dough may contain fats (triglycerides) such as granulated fat or oil.

[0033] The dough of the present invention is typically a leavened dough or a dough that is subjected to leavening.

[0034] Although the dough can be yeast-leavened in a variety of ways, such as by adding chemical leavening agents, e.g., baking powder, sodium bicarbonate, or by adding yeast (to leaven the dough), it is preferred that the dough be 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.

[0035] 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: 11, 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.

[0036] 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.

[0037] 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.

[0038] 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:

[0039] Horizontal Mixers: Roller bar mixers equipped with a rotating arm 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.

[0040] 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.

[0041] 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.

[0042] In a preferred embodiment, the fabric comprises: a) at least 5 minutes at a low mixing speed, preferably in the range of 5-50 rpm, more preferably in the range of 10-40 rpm; more preferably at least 10 minutes at a low mixing speed, even more preferably at least 15 minutes at a low mixing speed; and optionally, b) the dough is then mixed at a faster speed.

[0043] Parbaked products Parbaking is a technique in which bread or dough products are partially baked and then typically quickly cooled / frozen for storage.

[0044] The raw dough is baked as normal, but stopped at about 80% of the normal cooking time, then rapidly cooled.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] Glucoamylase Glucoamylases are also called amyloglucosidases and glucan 1,4-alpha-glucosidases (EC 3.2.1.3), more commonly referred to as AMG.

[0053] 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.

[0054] Other suitable examples of fungi include Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Rhizopus delemar, Rhizopus niveus, Rhizopus oryzae, and Talaromyces emersonii (Rasamsonia emersonii).

[0055] Below is the percent identity between the AMG amino acid sequences aligned in Figure 1 and also provided in the sequence listing.

[0056] [Table 1]

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] A preferred group of mature α-amylases is referred to as mature maltogenic α-amylases (EC 3.2.1.133), typically derived from Bacillus stearothermophilus. A preferred mature maltogenic α-amylase has an amino acid sequence which 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.

[0065] Also preferred are groups of mature 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 containing 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 and / or R376. As one such preferred variant, we refer herein to mature 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 mature 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, and / or N375. One such preferred variant, which we designate as mature sugar-tolerant maltogenic α-amylase 2 (ST-MAA2), has the following four substitutions: F194Y, D261G, T288P, and N375S.

[0066] Yet another group of preferred mature α-amylases are non-maltogenic maltotetrahydrolase exoamylases. WO 2004 / 111217 (Danisco A / S) discloses several preferred non-maltogenic thermostable variants of the Pseudomonas saccharophilia maltotetrahydrolase exoamylase 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. WO 2007 / 148224 (Danisco A / S) discloses more preferred non-maltogenic, anti-staling variants of Pseudomonas saccharophilia exoamylase having the amino acid sequence set forth in SEQ ID NO: 12, each of which contains an amino acid substitution at position 307 to lysine (K) or arginine (R). 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 such 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 in SEQ ID NO: 13 herein, which we refer to as HPL G+. Another preferred variant of 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.

[0067] 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).

[0068] Preferred 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 non-maltogenic α-amylases have one or more of the substitutions listed in the paragraph above.

[0069] Yet another group of preferred mature α-amylases are mature raw starch degrading α-amylases. 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.

[0070] Examples of mature raw starch-degrading alpha-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.

[0071] In one embodiment, the raw starch degrading alpha-amylase is a GH13_1 amylase.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] The enzymes 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.

[0077] 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]

[0078] 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.

[0079] 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)

[0080] PCR program: 98℃ / 2 minutes 25×(98℃ / 10 seconds, 60℃ / 15 seconds, 72℃ / 2 minutes) 10℃ / hold

[0081] 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.

[0082] 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.

[0083] Table 2

[0084] Table 3

[0085] Table 4

[0086] Table 5

[0087] Table 6

[0088] Table 7

[0089] Table 8

[0090] Table 9

[0091] Table 10

[0092] Table 11

[0093] Table 12

[0094] Table 13

[0095] Table 14

[0096] Table 15

[0097] Table 16

[0098] Table 17

[0099] Table 18

[0100] Table 19

[0101] Table 20

[0102] Table 21

[0103] Table 22

[0104] [Table 23]

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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

[0109] 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.

[0110] Example 6: PoAMG activity assay Maltodextrin (DE11) assay by GOD-POD method substrate solution 30g maltodextrin (MATSUTANI chemical industry Co., Ltd. pindex #2) 100 ml of 120 mM sodium acetate buffer, pH 5.0 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.

[0111] [Table 24]

[0112] [Table 25]

[0113] [Table 26]

[0114] Example 7: JPO-172 in combination with maltogenic α-amylase Bread was baked using the straight baking method using the recipe in Table 4. Various treatments were performed according to Table 5, where JPO-172 is one of the thermostable glucoamylase variants identified in Table 2 above, having the amino acid sequence set forth in SEQ ID NO: 6, and MAA is an anti-starving maltogenic α-amylase (MAA) having the amino acid sequence set forth in SEQ ID NO: 11. The bread was baked in a covered baking pan so that all breads had the same volume. The ingredients were mixed in a spiral mixer at 17 rpm for 3 minutes and 35 rpm for 7 minutes to form a dough. The dough was allowed to rest for 5 minutes and then divided into 450 g pieces. The dough pieces were rolled, flattened, and placed in a baking pan. The filled baking pan was proofed for 55 minutes at 32°C and 86% relative humidity. The proofed dough was baked in a deck oven at 230°C for 28 minutes.

[0115] The bread was packed in sealed plastic bags 2 hours after baking and stored at room temperature until analysis.

[0116] [Table 27]

[0117] [Table 28]

[0118] 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.

[0119] 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).

[0120] 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.

[0121] The results from the texture analysis can be found in Table 6 (crumb hardness) and Table 7 (crumb elasticity). Breads without added maltogenic α-amylase had a soft and elastic crumb immediately after baking. However, during storage, the crumb became harder and less elastic with time.

[0122] The combination of JPO-172 at 25 mg EP / kg flour and 75 ppm MAA gave lower crumb hardness and higher crumb elasticity compared to 75 ppm MAA alone over the entire period tested. The combination of JPO-172 at 25 mg EP / kg flour and 75 ppm MAA was also less hard and more elastic than JPO-172 alone at 25 mg EP / kg flour.

[0123] The combination of 25 mg EP / kg flour JPO-172 and 37.5 ppm MAA gave lower crumb hardness and higher crumb elasticity compared to 37.5 ppm MAA alone over the entire period tested.

[0124] Conclusion: Combining JPO-172 and MAA made the bread crumb softer (less hard) than MAA alone and more elastic compared to both MAA and JPO-172 alone.

[0125] [Table 29]

[0126] [Table 30]

[0127] Example 8: Improved performance of JPO-172 and MAA compared to AMG NL and MAA Bread was baked using the straight baking method using the recipe according to Table 8. Different treatments were performed according to Table 9, where JPO-172 is a thermostable glucoamylase variant identified in Table 2 above, MAA is a maltogenic α-amylase of SEQ ID NO: 11, and AMG NL is another less thermostable glucoamylase variant identified in Table 2 above and having the amino acid sequence shown in SEQ ID NO: 2. Bread was baked in a covered baking pan so that all breads had the same volume. The ingredients were mixed in a spiral mixer at 17 rpm for 3 minutes and 35 rpm for 7 minutes to form a dough. The dough was rested for 5 minutes and divided into 450 g pieces. The dough pieces were rolled, flattened, and placed in a baking pan. The filled baking pan was proofed for 55 minutes at 32°C and 86% relative humidity. The proofed dough was baked in a deck oven at 230°C for 28 minutes.

[0128] The bread was packed in sealed plastic bags 2 hours after baking and stored at room temperature until analysis.

[0129] [Table 31]

[0130] [Table 32]

[0131] Bread texturing was carried out as in Example 7. Results from the texture analyzer can be found in Figure 10 (hardness) and Figure 11 (springiness).

[0132] The addition of MAA produced soft and elastic bread on day 1, and the higher the MAA dose, the less hard and more elastic it was on day 14.

[0133] Addition of 25 mg EP / kg flour of AMG NL together with 35 ppm MAA improved bread crumb softness (considered as lower hardness) and elasticity, but failed to reach the level of 70 ppm MAA at 14 days.

[0134] Bread with the combination of JPO-172 (25 mg EP / kg flour) and MAA (35 ppm) produced less hard and more elastic bread during the entire period tested (days 1-14) compared to 35 and 70 ppm MAA alone and the combination of AMG NL at 25 mg EP / kg flour + 35 ppm MAA.

[0135] Conclusion: The combination of JPO-172 and MAA was much more efficient in keeping breadcrumbs fresh (low hardness and high elasticity) compared to AMG NL and MAA.

[0136] [Table 33]

[0137] [Table 34]

[0138] Example 9: Improved Sensory Performance of JPO-172 and MAA Compared to AMG NL and MAA Firing procedure: The bread was baked by straight baking using the recipe according to Table 12. A different treatment was carried out according to Table 13 with the addition of AMG NL.

[0139] 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 15 minutes and divided into 450 g pieces. The dough pieces were rolled, flattened, and placed into covered baking tins. The filled baking tins were proofed for 55 minutes at 32°C and 86% relative humidity. The proofed dough was baked in a deck oven at 230°C for 28 minutes.

[0140] [Table 35]

[0141] [Table 36]

[0142] The breads were packed in sealed plastic bags 2 hours after baking and stored at room temperature until evaluation.

[0143] Sensory evaluation method: Each panelist was presented with two slices of each bread type on days 1, 7, and 14 after baking. An initial training session was held before each evaluation, defining the attributes and procedures on day 1 (Table 14) and aligning the use of the intensity scale on each evaluation day. Samples were presented blind, with three-digit codes, and in random order. Five trained panelists participated in the evaluation. Two sensory replicates were performed. The intensity of the sensory attributes was rated on a 1-9 point scale ranging from very little to very strong.

[0144] [Table 37]

[0145] result: The data in Table 15 show that at day 1, JPO-172 in combination with MAA was softer, sub-crust softer, and more foldable than Po-AMG PE001 in combination with MAA.

[0146] On days 7 and 14, JP-O172 in combination with MAA was more moisturizing in addition to the different attributes on day 1.

[0147] In conclusion, the combination of JPO-172 and MAA performed better on sensory freshness parameters than AMG NL combined with MAA.

[0148] [Table 38]

[0149] Example 10 JPO-172 in combination with sugar-tolerant maltogenic α-amylase 1 WO 2006 / 032281 discloses several sugar-tolerant variants of MAA shown in SEQ ID NO: 11, each of which contains an amino acid change that is a substitution, deletion, or adjacent insertion of I15, R18, K44, N86, T87, G88, Y89, H90, Y92, W93, F188, T189, O190, P191, A192, F194, L196, O329, N371, O372, P373, N375, or R376. As one such variant, the inventors designated sugar-tolerant maltogenic α-amylase 1 (ST-MAA1), which has the following three substitutions: F188L, D261G, and T288P.

[0150] Breads were baked using the straight baking method using the recipe in Table 16. Various treatments were performed according to Table 17, where JPO-172 is the mature thermostable glucoamylase identified in Table 2 above, and ST-MAA1 is the mature sugar-tolerant maltogenic α-amylase (ST-MAA) variant. Breads were baked in covered baking pans so that all breads had the same volume. The ingredients were mixed in a spiral mixer for 3 minutes at 17 rpm and 7 minutes at 35 rpm to form a dough. The dough was allowed to rest for 5 minutes and then divided into 450 g pieces. The dough pieces were rolled, flattened, and placed in baking pans. The filled baking pans were proofed for 55 minutes at 32°C and 86% relative humidity. The proofed dough was baked in a deck oven at 230°C for 28 minutes.

[0151] The bread was packed in sealed plastic bags 2 hours after baking and stored at room temperature until analysis.

[0152] [Table 39]

[0153] [Table 40]

[0154] Bread texture was performed as in Example 7. Results from texture analysis can be found in Table 18 (crumb hardness) and Table 19 (crumb elasticity). Bread without added ST-MAA1 had a soft, elastic crumb immediately after baking. However, during storage, the crumb became harder and less elastic over time.

[0155] The combination of 25 mg EP / kg flour JPO-172 and 20 ppm ST-MAA1 gave lower crumb hardness and higher crumb elasticity compared to 20 ppm ST-MAA1 alone at all times tested. The combination of 25 mg EP / kg flour JPO-172 and 20 ppm ST-MAA1 was also less hard and more elastic compared to 25 mg EP / kg flour JPO-172 alone on days 7 and 14.

[0156] The combination of 25 mg EP / kg flour JPO-172 and 40 ppm ST-MAA1 gave lower crumb hardness and higher crumb elasticity compared to 40 ppm ST-MAA1 alone on days 1 and 14. On day 7, the mixture was also more elastic and less hard. However, hardness was not significantly different compared to ST-MAA1 alone. The combination of 25 mg EP / kg flour JPO-172 and 40 ppm ST-MAA1 was also less hard and more elastic compared to 25 mg EP / kg flour JPO-172 alone on days 7 and 14.

[0157] Conclusion: The combination of JPO-172 and ST-MAA1 improved the softness (reduced hardness) and elasticity of bread crumb compared to ST-MAA1 or JPO-172 alone.

[0158] [Table 41]

[0159] [Table 42]

[0160] Example 11: JPO-172 in combination with sugar-tolerant maltogenic α-amylase 2 WO 2008 / 148845 discloses several sugar-tolerant variants of MAA shown in SEQ ID NO: 11, each of which contains two substitutions D261G and T288P and at least one additional amino acid change that is a substitution or deletion of, or an insertion adjacent to, Y89, W93, P191, F194, Y360, or N375. As one such variant, the inventors designated sugar-tolerant maltogenic α-amylase 2 (ST-MAA2), which has the following four substitutions: F194Y, D261G, T288P, and N375S.

[0161] Bread was baked using the straight baking method using the recipe according to Table 20. Different treatments were performed according to Table 21, where JPO-172 is a thermostable glucoamylase variant identified in Table 2 above, and ST-MAA2 is another sugar-tolerant anti-staling maltogenic α-amylase having the amino acid sequence shown in SEQ ID NO: 13. Bread was baked in a covered baking pan so that all breads had the same volume. The ingredients were mixed in a spiral mixer at 17 rpm for 3 minutes and 35 rpm for 7 minutes to form a dough. The dough was rested for 5 minutes and divided into 450 g pieces. The dough pieces were rolled, flattened, and placed in a baking pan. The filled baking pan was proofed for 55 minutes at 32°C and 86% relative humidity. The proofed dough was baked in a deck oven at 230°C for 28 minutes.

[0162] The bread was packed in sealed plastic bags 2 hours after baking and stored at room temperature until analysis.

[0163] [Table 43]

[0164] [Table 44]

[0165] Bread texture was performed as in Example 7. Results from texture analysis can be found in Table 22 (crumb hardness) and Table 23 (crumb elasticity). Bread without added ST-MAA2 had a soft, elastic crumb immediately after baking. However, during storage, the crumb became harder and less elastic over time.

[0166] The combination of 25 mg EP / kg flour JPO-172 and 100 ppm ST-MAA2 gave lower crumb hardness and higher crumb elasticity compared to 100 ppm ST-MAA2 alone at all times tested. The combination of 25 mg EP / kg flour JPO-172 and 100 ppm ST-MAA2 was also less hard and more elastic than 25 mg EP / kg flour JPO-172 alone at day 14.

[0167] The combination of 25 mg EP / kg flour JPO-172 and 50 ppm ST-MAA2 gave lower crumb hardness and higher crumb elasticity compared to 50 ppm ST-MAA2 alone over the entire period tested. The combination of 25 mg EP / kg flour JPO-172 and 50 ppm ST-MAA2 was also less hard and more elastic than 25 mg EP / kg flour JPO-172 alone on days 1 and 14.

[0168] Conclusion: Combining JPO-172 and ST-MAA2 made bread softer (less hard) than JPO-172 or ST-MAA2 alone. Bread also became more chewy compared to ST-MAA2 alone.

[0169] [Table 45]

[0170] [Table 46]

[0171] Example 12 JPO-172 in combination with Veron® Maxima Bread was baked using the straight-bake method using the recipe in Table 24. JPO-172 is the thermostable glucoamylase variant identified in Table 2 above, and Veron® Maxima (AB Enzymes) is a commercially available anti-staling maltogenic α-amylase. Protein sequencing revealed a primary activity with the amino acid sequence of SEQ ID NO: 11 as well as Fusarium oxysporum lipase side activity. Various treatments were performed according to Table 25. Bread was baked in a covered baking pan to ensure all loaves had the same volume. The ingredients were mixed in a spiral mixer at 17 rpm for 3 minutes and 35 rpm for 7 minutes to form a dough. The dough was allowed to rest for 5 minutes and then divided into 450 g pieces. The pieces were rolled, flattened, and placed in the baking pan. The filled baking pan was proofed for 55 minutes at 32°C and 86% relative humidity. The leavened dough was baked in a deck oven at 230°C for 28 minutes.

[0172] The bread was packed in sealed plastic bags 2 hours after baking and stored at room temperature until analysis.

[0173] [Table 47]

[0174] [Table 48]

[0175] Bread texture was performed as in Example 7. Results from texture analysis can be found in Table 26 (crumb hardness) and Table 27 (crumb elasticity). Bread without Veron® Maxima added had a soft, elastic crumb immediately after baking. However, during storage, the crumb became harder and less elastic over time.

[0176] The combination of 25 mg EP / kg flour JPO-172 and 100 ppm Veron® Maxima gave lower crumb hardness and higher crumb elasticity compared to 100 ppm Veron® Maxima alone at all times tested. The combination of 25 mg EP / kg flour JPO-172 and 100 ppm Veron® Maxima was also less hard and more elastic compared to 25 mg EP / kg flour JPO-172 alone at days 7 and 14.

[0177] The combination of 25 mg EP / kg flour JPO-172 and 50 ppm Veron® Maxima gave lower crumb hardness and higher crumb elasticity compared to 50 ppm Veron® Maxima alone at all times tested. The combination of 25 mg EP / kg flour JPO-172 and 50 ppm Veron® Maxima was also less hard and more elastic compared to 25 mg EP / kg flour JPO-172 alone at days 7 and 14.

[0178] Conclusion: The combination of JPO-172 and Veron® Maxima is soft and elastic immediately after baking and can maintain its flexibility and elasticity better over time than Veron® Maxima or JPO-172 alone.

[0179] [Table 49]

[0180] [Table 50]

[0181] Example 13: JPO-172 in combination with non-maltogenic α-amylase (HPLG+) WO 2004 / 111217 discloses non-maltogenic thermostable variants of Pseudomonas saccharophilia exoamylase or maltotetrahydrolase 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.

[0182] WO 2007 / 148224 discloses non-maltogenic anti-staling variants of Pseudomonas saccharophilia exoamylase having the amino acid sequence shown in SEQ ID NO: 12, which contain an amino acid substitution at position 307 to lysine (K) or arginine (R), respectively.

[0183] WO 2010 / 133644 discloses non-maltogenic variants of Pseudomonas saccharophilia exoamylase having the amino acid sequence set forth in SEQ ID NO: 12, wherein the variants each contain one or more substitutions at positions including 42, 88, 205, 223, 235, 240, 311, 392, and 409. One such 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 in SEQ ID NO: 13 herein, which we refer to as :HPLG+.

[0184] Bread was baked by straight baking using the recipe according to Table 28. Various treatments were performed according to Table 29, where JPO-172 is a thermostable glucoamylase variant identified in Table 2 above having the amino acid sequence set forth in SEQ ID NO: 6, and HPLG+ is a non-maltogenic amylase variant set forth in SEQ ID NO: 13.

[0185] Breads were baked in covered baking tins so that all loaves had the same volume. 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 allowed to rest for 5 minutes and divided into 450g pieces. The pieces were rolled, flattened, and placed in baking tins. The filled baking tins were allowed to proof for 55 minutes at 32°C and 86% relative humidity. The proofed dough was baked in a deck oven at 230°C for 28 minutes.

[0186] The bread was packed in sealed plastic bags 2 hours after baking and stored at room temperature until analysis.

[0187] [Table 51]

[0188] [Table 52]

[0189] Bread texture was performed as in Example 7. Results from texture analysis can be found in Table 30 (crumb hardness) and Table 31 (crumb elasticity). Immediately after baking, the bread had a soft, elastic crumb. However, during storage, the crumb became harder and less elastic with time.

[0190] The combination of 25 ppm HPL G+ and 5 mg EP / kg flour of JPO-172 was less hard initially and over time than bread containing only 25 ppm HPL G+.

[0191] [Table 53]

[0192] [Table 54]

[0193] Example 14: JPO-172 in combination with a non-maltogenic α-amylase (HPLG4) Another 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 shown in SEQ ID NO: 14 herein, which we refer to as HPL G4.

[0194] Breads were baked in a straight dough mini-bake machine using the recipe according to Table 32. Different treatments were carried out according to Table 33. Breads were baked in covered baking pans so that all breads had the same volume. The ingredients were mixed into a dough in a spin mixer at 90 rpm for 4 minutes. The dough was rested for 5 minutes and divided into 17 g dough pieces. The dough pieces were rolled by hand and placed in baking pans. The molds with dough were placed on the conveyor belt of a mini-tunnel proofer and proofed for 55 minutes at 36°C and 80% relative humidity. After passing through the proofing tunnel, the dough was transferred to a mini-tunnel oven. The proofed dough was baked in the mini-tunnel oven at 210°C for 12 minutes.

[0195] The bread was packed in sealed plastic bags 25 minutes after baking and stored at room temperature until analysis.

[0196] [Table 55]

[0197] [Table 56]

[0198] 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 spherical probe with a diameter of 25 mm. The force on the spherical 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.

[0199] 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).

[0200] 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.

[0201] The results from the bread evaluation can be found in Table 34 (hardness) and Table 35 (springiness). Mixing HPL G4 with JPO-172 produced softer bread than HPL G4 alone, and this was true for both the low dosage of 15 ppm HPL G4 and the high dosage of 25 ppm HPL G4.

[0202] [Table 57]

[0203] [Table 58]

[0204] Example 15: Sensory effects of adding raw starch-degrading α-amylase (RSDA) to JPO-172 Firing procedure: Bread was baked using a straight dough baking process using the method described in Example 9 and the recipe described in Table 12, where JPO-172 is a thermostable glucoamylase variant identified in Table 2 above, and RSDA is a mature thermostable hybrid raw starch-degrading α-amylase whose amino acid sequence is disclosed in SEQ ID NO: 4 of EP 1 576 152, incorporated herein in its entirety. The mature amino acid sequence of RSDA is also shown in SEQ ID NO: 15 herein. Different treatments were performed according to Table 36 with the addition of RSDA.

[0205] [Table 59]

[0206] The breads were packed in sealed plastic bags 2 hours after baking and stored at room temperature until evaluation.

[0207] Sensory evaluation method: Each panelist was provided with two slices of each bread type (day 1). Prior to the evaluation, an initial training session was held that defined the attributes and procedures (Table 14) and the use of the intensity scale. Samples were presented blind, with three-digit codes, and in a randomized order. Five trained panelists participated in the evaluation. Two sensory replicates were performed. The intensity of the sensory attributes was rated on a 1-9 point scale ranging from very little to very strong.

[0208] result: The data in Table 37 show that JPO-172 alone increased the moist, soft, soft subcrust, and foldable properties compared to the control bread. However, in some applications, lower springiness is a desirable property.

[0209] RSDA is an enzyme that can reduce the elasticity of the bread crumb, as can be seen in Table 37. However, RSDA also reduced the moist, soft, and soft subcrust parameters compared to the control, which is typically undesirable.

[0210] The combination of JPO-172 and RSDA resulted in bread with desirable properties of being moister, softer, sub-crusted and softer, more foldable, and less elastic compared to the control.

[0211] [Table 60]

Claims

1. 1. A method for producing a baked or partially baked product with improved crumb softness and / or elasticity, comprising adding to a dough a mature α-amylase 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; the mature thermostable variant comprises at least one amino acid modification; and The at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 50, 132, 481, 484, 539, 595 of SEQ ID NO: 1; method.

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. 2. The method of claim 1, 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 the mature α-amylase is a maltogenic α-amylase, a non-maltogenic α-amylase, or a raw starch degrading α-amylase.

12. 12. The method of claim 11, wherein the mature α-amylase is a maltogenic α-amylase, preferably comprising or having an amino acid sequence at least 80% identical to SEQ ID NO: 11, or wherein the mature α-amylase is a sugar-tolerant maltogenic α-amylase variant, preferably comprising or having an amino acid sequence at least 80% identical to SEQ ID NO:

11.

13. 12. The method of claim 11, wherein the mature α-amylase is a non-maltogenic α-amylase, preferably comprising or having an amino acid sequence at least 80% identical to SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:

14.

14. 12. The method of claim 11, wherein the mature α-amylase is a raw starch-degrading α-amylase, preferably comprising or having an amino acid sequence at least 80% identical to SEQ ID NO:15 or SEQ ID NO:

16.

15. 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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