Thermostable amyloglucosidase (AMG) variants (EC 3.2.1.3) and baking with low added sugars

Abstract

The present invention relates to a method for producing a baked good, the method comprising adding 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, adding less than 150% (baker's %) of sugar to the dough, and baking the dough, as well as to the use of 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 for producing a baked good, wherein the variant is added to a dough, less than 150% (baker's %) of sugar or sucrose is added to the dough, and the dough is baked.

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 goods with a reduced amount of added sugar compared to standard recipes, comprising adding 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 dough, adding a reduced amount of sugar compared to standard recipes, and baking the dough. [Background technology]

[0003] The recipes for different baked goods can vary greatly. One ingredient is sugar, which affects the baking process and the final baked good. -Dough properties: Increased sugar levels make dough softer -Provides food for yeast. However, it also inhibits yeast activity, reducing gas production. -Improves crust coloration, giving the final bread a more golden crust color. - Affects the crumb of the baked goods, maintaining its softness and moistness. -It gives baked goods a sweet aroma.

[0004] There is a market trend to reduce the amount of sugar in food products. Removing sugar will affect the baking process and the final baked product, making the dough firmer, reducing the color of the crust during baking, producing a less soft and moist product, and making the baked product less sweet.

[0005] Enzymes can improve some of these properties: crust color can be improved with glucoamylases such as Goldcrust®, and crumb tenderness can be improved with maltogenic amylases such as Novamyl® G, Novamyl 3D® G, and Novamyl® Pro.

[0006] Recipe changes can improve other parameters affected by removing sugar: increasing the water content can make the dough softer; the increased gas-producing power of yeast when sugar is removed can be adjusted by lowering the yeast level. Summary of the Invention [Means for solving the problem]

[0007] The inventors have found that mature thermostable glucoamylase variants exhibit increased enzyme activity or performance in baking recipes with lower starting concentrations of added sugar. The increased enzyme activity may translate into lower enzyme dosages compared to recipes with higher starting concentrations of added sugar. The improved activity of the thermostable variants resulted in increased sweetness or flavor of the product, as glucoamylase produces glucose. This discovery allows for a reduction in the amount of added sugar in conventional baking recipes without a perceived decrease in sweetness in the final baked good, which is of great commercial interest. At the same time, the recipe can be adjusted by adding more water and lowering the yeast level, generating greater cost savings.

[0008] Thus, in a first aspect, the present invention relates to a method for producing a baked good, the method comprising adding 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, adding less than 150% (baker's %) sugars to the dough, and baking the dough.

[0009] A second aspect of the present invention relates to the use of 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 for the production of a baked product, wherein the variant is added to a dough, less than 150% (baker's %) of sugar or sucrose is added to the dough, and the dough is baked.

[0010] Preferably, the method of the first aspect or the use of the second aspect achieves a sequence identity of 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%, for example at least 84%, for example at least 85%, for example at least 86%, for example at least 87%, for example at least 88%, for example at least 89%, for example at least 90%, for example at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example at least 100%, such as at least 101%, for example at least 102%, for example at least 103%, for example at least 104%, for example at least 105%, for example at least 106%, for example at least 107%, for example at least 108%, for example at least 109%, for example at least 110%, for example at least 111%, for example at least 112%, for example at least 113%, for example at least 114%, for example at least 115%, for example at least 116%, for example at least 117%, for example at least 118%, for example at least 119%, for example at least 120%, for example at least 121%, for example at least 122%, for example at least 123%, for example at least 124%, for example at least 125%, for example at least 126%, for example at least 127%, for example at least 128 In some embodiments, the method further comprises adding a mature thermostable variant of the parent glucoamylase that is 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 to the parent glucoamylase.

[0011] A preferred embodiment of the present invention relates to adding less than 120% (baker's%) sugar to the dough, preferably less than 110% (baker's%), preferably less than 100% (baker's%), preferably less than 90% (baker's%), preferably less than 80% (baker's%), preferably less than 70% (baker's%), preferably less than 60% (baker's%), preferably less than 50% (baker's%), preferably less than 40% (baker's%), preferably less than 30% (baker's%), preferably less than 20% (baker's%), preferably less than 10% (baker's%), preferably less than 8% (baker's%), preferably less than 6% (baker's%), even more preferably less than 4% (baker's%) and most preferably less than 2% (baker's%) sugar to the dough. [Brief explanation of the drawings]

[0012] [Figure 1A] 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 [Figure 1B]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

[0013] definition Baked Goods: As used herein, "baked goods" means any type of baked goods including bread types such as bread, toast, open breads, covered and open breads, buns, hamburger buns, rolls, baguettes, brown bread, whole wheat bread, rich bread, bran bread, flatbread, tortilla, pita, Arabic bread, Indian flatbread, cookies, biscuits, cakes, brioche and any variant thereof.

[0014] Dough: As used herein, "dough" refers to any dough used to prepare bread. Dough used to prepare baked goods may be prepared from any suitable dough ingredients, such as cereal-based flours, such as wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, or sorghum flour, potato flour, soy flour, and combinations thereof (e.g., wheat flour combined with one of the other flour ingredients; rice flour combined with one of the other flour ingredients). 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 various ways, such as by adding a chemical leavening agent (e.g., sodium bicarbonate) or by adding a leavening agent (which ferments the dough), but preferably the dough is leavened by adding a suitable yeast culture, such as a culture of Saccharomyces cerevisiae (baker's yeast), e.g., a commercially available S. cerevisiae strain. The dough may also contain other traditional dough ingredients, such as proteins such as milk powder, gluten, and soy; 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; sugars; salts such as sodium chloride, calcium acetate, sodium sulfate, and calcium sulfate; diluents such as silicon dioxide; and starches of different origins. Still other conventional ingredients include hydrocolloids such as CMC, guar gum, xanthan gum, locust bean gum, etc. Modified starches can also be used. The dough ingredients may include fat (triglycerides), such as granulated fat or shortening, although the present invention is particularly applicable to doughs to which less than 1% by weight of fat or shortening is added, and especially doughs made without added fat or shortening.In a preferred embodiment, the dough ingredients comprise wheat flour; preferably, 10% (w / w) or more of the total flour content is wheat flour, and preferably, 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. The dough can be prepared by applying any conventional mixing process, such as a continuous mixing process, a straight dough method, or a sponge dough method.

[0015] Baker's %: Baker's percent is a mathematical method widely used in baking to calculate the amounts of major, minor, and trace ingredients. It is based on the total weight of flour the formula contains. Instead of dividing the weight of each ingredient by the total weight of the formula, the baker divides each ingredient by the weight of the flour.

[0016] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0017] 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 open 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)

[0018] 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 consisting 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 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.

[0019] 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.

[0020] As used herein, the term "grist" is understood as the starch- or sugar-containing material that is the basis for beer production, e.g., barley malt and additives. Generally, grist does not contain any added water.

[0021] 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.

[0022] Glucoamylase Glucoamylases are also called amyloglucosidases and glucan 1,4-alpha-glucosidases (EC 3.2.1.3), more commonly they are referred to as AMG.

[0023] 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.

[0024] Other suitable examples of fungi include Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Rhizopus delemar, Rhizopus niveus, Rhizopus oryzae, and Talaromyces emersonii (Rasamsonia emersonii).

[0025] Below is the percent identity between the AMG amino acid sequences aligned in Figure 1 and also provided in the sequence listing.

[0026] [Table 1]

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Lipase In a preferred embodiment of the first aspect, one or more additional enzymes are added, said additional enzymes preferably being a mature lipolytic enzyme, preferably a mature lipolytic enzyme as disclosed in WO 2018 / 150021 (Novozymes A / S), more preferably a mature polypeptide having lipolytic activity and having at least 65% sequence identity to amino acids 21 to 309 of SEQ ID NO: 1 in WO 2018 / 150021 or a polypeptide encoded by a polynucleotide having at least 65% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 2 in WO 2018 / 150021.

[0033] 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.

[0034] 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), as well as the enzymes disclosed in WO 2004 / 020499, WO 2006 / 06929, and WO 2006 / 066579.

[0035] In one embodiment, the raw starch degrading alpha-amylase is a GH13_1 amylase.

[0036] 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 to the raw starch degrading alpha-amylase set forth in SEQ ID NO: 11 herein.

[0037] 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.

[0038] Several alpha-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.

[0039] Another group of alpha-amylases is referred to as Fungamyl™ and “Fungamyl™-like alpha-amylases,” which are alpha-amylases related to the alpha-amylase derived from Aspergillus oryzae disclosed in WO 01 / 34784.

[0040] Proteases Suitable proteases include microbial proteases, such as fungal and bacterial proteases. Preferred proteases are acidic proteases, i.e., proteases characterized by their ability to hydrolyze proteins under acidic conditions (pH less than 7). Proteases are responsible for reducing full-length high-molecular-weight proteins in mash to low-molecular-weight proteins. Low-molecular-weight proteins are necessary for yeast nutrition, while high-molecular-weight proteins ensure foam stability. Therefore, it is well known to those skilled in the art that proteases should be added in a balanced amount to simultaneously provide yeast with abundant free amino acids while leaving sufficient high-molecular-weight proteins to stabilize foam. In one embodiment, the protease activity is provided by a proteolytic enzyme system with suitable FAN-producing activity, including an endoprotease, an exopeptidase, or any combination thereof, preferably a metalloprotease. Preferably, the protease has at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% or even 100% identity to the amino acid sequence set forth in SEQ ID NO: 6 in WO 9967370. In another embodiment, the protease is Neutrase® available from Novozymes A / S. The protease may be added in an amount of 0.0001 to 1000 AU / kg DS, preferably 1 to 100 AU / kg DS, and most preferably 5 to 25 AU / kg dry weight of grain. Proteolytic activity may be determined using modified hemoglobin as a substrate. In the Anson hemoglobin method for determining proteolytic activity, denatured hemoglobin is digested and undigested hemoglobin is precipitated with trichloroacetic acid (TCA).The amount of TCA-soluble product is determined by using a phenol reagent that produces a blue color with tyrosine and tryptophan. One Anson unit (AU) is defined as the amount of enzyme that digests hemoglobin at an initial rate such that, under standard conditions (i.e., 25°C, pH 7.5, 10-minute reaction time), the amount of TCA-soluble product released per minute produces the same color as one milliequivalent of tyrosine in the phenol reagent.

[0041] 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.

[0042] 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 methods. Other enzyme stabilizers are well known in the art.

[0043] 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.

[0044] 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]

[0045] 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.

[0046] 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) PCR program: 98℃ / 2 minutes 25×(98℃ / 10 seconds, 60℃ / 15 seconds, 72℃ / 2 minutes) 10℃ / hold

[0047] 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.

[0048] 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.

[0049] [Table 2]

[0050] [Table 3]

[0051] [Table 4]

[0052] [Table 5]

[0053]

Table 6

[0054]

Table 7

[0055]

Table 8

[0056]

Table 9

[0057]

Table 10

[0058]

Table 11

[0059]

Table 12

[0060]

Table 13

[0061]

Table 14

[0062]

Table 15

[0063] [Table 16]

[0064] [Table 17]

[0065] [Table 18]

[0066] [Table 19]

[0067] [Table 20]

[0068] [Table 21]

[0069] [Table 22]

[0070] [Table 23]

[0071] [Table 24]

[0072] 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.

[0073] 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.

[0074] 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 LightCycler 480 Multiwell Plate 384 (Roche Diagnostics), and the plate was sealed.

[0075] 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: 465nm Emission wavelength 580nm

[0076] 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.

[0077] Example 6: PoAMG activity assay Maltodextrin (DE11) assay by GOD-POD method substrate solution 30g maltodextrin (Pindex #2, manufactured by MATSUTANI chemical industry Co., Ltd.) 100 ml of 120 mM sodium acetate buffer, pH 5.0 Glucose CII test kit (Wako Pure Chemical Industries, Ltd.)

[0078] 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 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 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.

[0079] [Table 25]

[0080] [Table 26]

[0081] [Table 27]

[0082] Example 7: Effect of lowering sugar (sucrose) levels on the efficacy of JPO-172 in straight bread Breads were baked using the straight baking method using the recipe according to Table 4. Different treatments were made according to Table 5. Breads with lower levels of sucrose had 2.5% additional water so that all doughs had the same dough properties. Breads were baked in covered baking tins 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 rested for 5 minutes, divided into 450g pieces and rolled. The pieces were rolled out and placed in 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.

[0083] [Table 28]

[0084] [Table 29]

[0085] The bread was packed in sealed plastic bags 2 hours after baking and stored at room temperature until analysis.

[0086] Bread texture was evaluated with a texture analyzer (TA-XT plus, Stable microsystems, Godalmine, UK). Bread crumb texture properties were characterized by the firmness (same as "hardness" and opposite to "softness") and springiness of the baked goods. The standard method for measuring firmness and springiness is based on the force-deformation of baked goods. The force-deformation of baked goods 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 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.

[0087] 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).

[0088] 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.

[0089] The results from the texture analysis can be found in Tables 6 and 7. Bread without JPO-172 enzyme (control) is soft and elastic on day 1, and when the bread is stored for 14 days, it becomes harder and less elastic over time. When 46 mg EP JPO-172 enzyme per kg flour is added, the hardness on day 1 decreases from 496 g to 285 g, and the elasticity increases from 57.4% to 66.5%. When the bread with JPO-172 is stored for 14 days, it becomes harder and less elastic, however, not to the same extent as the bread without JPO-172.

[0090] The JPO-172 enzyme becomes even more efficient when the sucrose level in the bread is reduced from 8% to 2%. When 2% sucrose is present in the formula, a flour dosage of 31 mg EP / kg gives insignificantly different firmness and springiness from days 1 to 14 compared to 46 mg EP / kg flour when 8% sucrose is present in the formula. When 2% sucrose is present in the formula, a higher dosage of JPO-172 (38 mg EP / kg flour) results in reduced firmness on days 7 and 14 compared to 46 mg EP / kg flour when 8% sucrose is present in the formula.

[0091] [Table 30]

[0092] [Table 31]

[0093] Example 8. Effect of reducing sugar (sucrose) levels on the efficacy of JPO-172 in sponge bread The breads were baked using the sponge method using the recipe according to Table 8. Different treatments were made according to Table 9. Breads with lower levels of sucrose had additional water so that all doughs had the same dough properties. The breads were baked in covered baking tins so that all breads had the same volume. The sponge ingredients were mixed in a Pin mixer (Bjoern mixer) for 2 minutes at 50 rpm and 2 minutes at 150 rpm to form a sponge. The sponge was proofed for 3 hours at 27°C and 75% RH. The sponge and dough ingredients were placed in the bowl of a Pin mixer and mixed for 1 minute at 50 rpm and 3 minutes at 150 rpm. The dough was divided into rounded 400g pieces and allowed to rest for 5 minutes. The pieces were rolled out and placed in baking tins. The filled baking tins were proofed for 60 minutes at 43°C and 80% relative humidity. The proofed dough was baked in a rotary oven at 215°C for 20 minutes.

[0094] [Table 32]

[0095] [Table 33]

[0096] [Table 34]

[0097] The bread was packed in sealed plastic bags 2 hours after baking and stored at room temperature until analysis.

[0098] Bread texture was evaluated with a texture analyzer (TA-XT plus, Stable microsystems, Godalmine, UK). Bread crumb texture properties were characterized by the firmness (same as "hardness" and opposite to "softness") and springiness of the baked goods. The standard method for measuring firmness and springiness is based on the force-deformation of baked goods. The force-deformation of baked goods 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 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.

[0099] 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).

[0100] 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.

[0101] The results from the texture analysis can be found in Tables 10 and 11. Bread without freshness-keeping enzymes and 8% sucrose (Treatment 1 control) was soft and chewy on day 1, and when the bread was stored for 14 days, it became harder and less chewy with time.

[0102] The addition of freshness-preserving enzyme JPO-172 at a dosage of 100 mg EP / kg flour (doughs 2-5) reduces bread hardness and increases elasticity throughout the time period tested (days 1-14). By reducing the level of sucrose, freshness-preserving enzyme JPO-172 becomes more efficient and is able to reduce bread crumb hardness on days 7 and 14 by further improving freshness-preserving effect. The improvement in freshness-preserving enzyme efficiency by reducing sucrose levels in the recipe can also be seen as an increase in elasticity throughout the time range tested.

[0103] [Table 35]

[0104] [Table 36]

[0105] Example 9 Sweetness of bread due to recipe and reduced sugar levels in JPO-172 Breads were baked using the straight-through method using the recipe according to Table 12. Different treatments were made according to Table 13. Breads with lower levels of sucrose had 0.3% additional water / % sucrose reduction so that all doughs had the same dough properties. Because sugar inhibits yeast activity, breads with lower levels of sugar had 0.25% yeast reduction / % sugar so that all doughs had the same gas-producing power. 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 6 minutes at 35 rpm to form a dough. The dough was rested for 5 minutes, divided into 450g pieces, and rolled. The pieces were rolled out and placed in baking pans. The filled baking pans were proofed for 57 minutes at 32°C and 86% relative humidity. The proofed dough was baked in a deck oven at 215°C for 35 minutes.

[0106] [Table 37]

[0107] [Table 38]

[0108] The bread was allowed to cool at room temperature, packed in sealed plastic bags 2 hours after baking, and stored at room temperature until analysis.

[0109] Sensory evaluation method: The sweetness of the breads was evaluated in a sensory evaluation session two days after baking. Each panelist was provided with a slice of each bread type. Prior to the evaluation, a training session was held to define the sweetness attributes and procedures (Table 14) and the use of the intensity scale. Sweetness intensity was rated on a 1-9 point intensity scale ranging from almost none to very strong. A control sample with 8% sucrose served as a reference with a score of 5.0. The other samples were presented blindly with three-digit codes and in random order. Five trained panelists participated in the evaluation. Two sensory evaluations were performed in replicate. The significance of the sensory results was estimated using Student's t-test with a significance level of 0.05.

[0110] [Table 39]

[0111] Scores from the sensory evaluation of sweetness can be found in Table 15. Despite the dough with 0-2% added sucrose having less added sugar in the dough compared to the dough with 8% added sucrose, the final baked bread was perceived as sweet to the same extent as the bread with 8% sucrose in the dough.

[0112] [Table 40]

Claims

1. A method for producing a baked product, comprising adding 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, adding less than 150% (baker's %) of sugar to the dough, and baking the dough.

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 of claims 1-2, 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 in 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 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 the positions corresponding to R1A, P2N, P4S, P11F, T65A, K79V and Q327F in 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 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 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 in 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 1, 6, 7, 31, 34, 50, 103, 132, 445, 447, 481, 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 R1A, G6S, G7T, R31F, K34Y, E50R, S103N, A132P, D445N, V447S, S481P, E501A, N539P, D566T, T568V, Q594R and F595S in 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 1, 6, 7, 31, 34, 50, 103, 132, 445, 447, 481, 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 R1A, G6S, G7T, R31F, K34Y, E50R, S103N, A132P, D445N, V447S, S481P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO:

1.

8. 4. The method of claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to 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, 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, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO:

1.

9. 8. The method of claim 7, 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. The method of claim 8, 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.

11. 8. The method of claim 7, 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.

12. The method described in claim 8, 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.

13. The method also includes adding one or more additional enzymes, wherein the additional enzymes are 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, 8. The method of claim 7, wherein the enzyme is selected from the group consisting of 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, ribonuclease, transglutaminase, and xylanase.

14. The method also includes adding one or more additional enzymes, wherein the additional enzymes are 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, 9. The method of claim 8, wherein the enzyme is selected from the group consisting of 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, ribonuclease, transglutaminase, and xylanase.

15. 1. Use of a mature thermostable variant of a parent glucoamylase 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 for producing a baked product, wherein the variant is added to a dough, less than 150% (baker's %) of sugar or sucrose is added to the dough, and the dough is baked.