Baking with a thermostable AMG glucosidase variant (EC 3.2.1.3) and low or no emulsifier addition

Abstract

The present invention relates to a method for producing baked goods with no added emulsifier or with a reduced amount of added emulsifier compared to standard manufacturing methods, 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 (with no added emulsifier or with a reduced amount of added emulsifier compared to standard manufacturing methods) and baking the dough.

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 no added emulsifier or with a reduced amount of added emulsifier compared to standard manufacturing methods, which method comprises adding a mature thermostable variant of a particular parent glucoamylase. [Background technology]

[0003] Several properties of baked goods are important to consumer preference, especially in industrially produced bread, and industrial bakers have relied on the addition of one or more emulsifiers to achieve the appropriate properties.

[0004] DATEM (diacetyl tartaric acid esters of mono- and diglycerides) is an emulsifier primarily used in baking to strengthen the gluten network in dough and improve the volume of baked goods. It is composed of mixed esters of mono- and diglycerides derived from food sources, in which one or more of the hydroxyl groups of glycerol are esterified with diacetyl tartaric acid and fatty acids. DATEM typically accounts for approximately 0.375-0.5% of the total flour weight in most commercial baked dough formulas.

[0005] DMG (distilled mono- and diglycerides) is a crumb-softening emulsifier widely used in baking to improve softness over time and retard starch reversion. DMG also influences the extensibility of the gluten network.

[0006] SSL (sodium stearoyl lactylate) is another emulsifier derived from the sodium salt of lactic acid and stearic acid. SSL provides several functionalities: -Improve the shelf life of bread and bun texture -Produces softer, more flexible tortillas - Strengthens oven spring by increasing the dough's gas-holding capacity -Gives loaf of bread better sidewall strength (prevents keyholes) -Gives bread a desirable chewy crumb

[0007] However, as consumers often refer to emulsifiers in conversation, there is an increasing tendency for consumers to avoid consuming bakery products that contain emulsifiers or "E numbers."

[0008] WO 2022 / 090562 discloses a method for producing baked goods with a heat-stable AMG variant that offers several advantages, such as extended shelf life and improved internal softness.

[0009] The use of lipolytic enzymes in baking has also been known for many years. WO 98 / 26057 discloses lipases / phospholipases from Fusarium oxysporum and their use in baking. WO 2004 / 099400 discloses various lipolytic enzymes and their use in baking. WO 1999 / 053769 discloses the use of maltogenic alpha-amylases and phospholipases to improve the softness of baked goods in the early stages after baking. WO 2018 / 150021 also discloses lipolytic enzymes suitable for baking. Summary of the Invention [Means for solving the problem]

[0010] The inventors have surprisingly found that by adding a heat-stable AMG variant to a dough and baking the dough to produce a baked good, it is possible to reduce the amount of emulsifier that would typically be included in the dough recipe, or to avoid adding any emulsifier at all.

[0011] Thus, in a first aspect, the present invention relates to a method for producing a baked good with no added emulsifier or with a reduced amount of added emulsifier compared to standard recipes, 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 (with no added emulsifier or with a reduced amount of added emulsifier compared to standard recipes) and baking the dough.

[0012] In a second aspect, the present invention relates to the 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 good with no added emulsifier or with a reduced amount of added emulsifier compared to standard recipes, said method comprising adding said thermostable variant of a parent glucoamylase to dough (with no added emulsifier or with a reduced amount of added emulsifier compared to standard recipes) and baking the dough.

[0013] 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. [Brief explanation of the drawings]

[0014] [Figure 1-1] 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 [Figure 1-2] 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 2] Figure 1 shows a graphical representation of the mean sensory attribute scores of breads evaluated 1 day after baking as shown in Table 7 of Example 7; Note: JPO172 is JPO-172. [Figure 3] Figure 1 shows a graphical representation of the mean sensory attribute scores of breads evaluated 1 day after baking as shown in Table 9 of Example 8; Note: JPO172 is JPO-172. [Figure 4] Figure 1 shows a graphical representation of the mean sensory attribute scores of breads evaluated 1 day after baking as shown in Table 11 of Example 9; Note: JPO172 is JPO-172. DETAILED DESCRIPTION OF THE INVENTION

[0015] 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.

[0016] Dough: As used herein, "dough" refers to any dough used to prepare baked goods. 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.

[0017] 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.

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

[0019] For the purposes of the present invention, sequence homology between two amino acid sequences is 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)

[0020] Variant: The term "variant" refers to a polypeptide that contains modifications, i.e., substitutions, insertions, and / or deletions at one or more (e.g., several) positions. A substitution refers to the replacement of an amino acid at a position with another amino acid; a deletion refers to the removal of an amino acid at a position; and an insertion refers to the addition of one or more amino acids immediately adjacent to the amino acid at a position. Amino acid changes can be minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically 1-30 amino acids; small amino- or carboxyl-terminal extensions such as an amino-terminal methionine residue; small linker peptides of 20-25 residues or less; or small extensions that facilitate purification by altering net charge or another function, such as a polyhistidine tract, antigenic epitope, or binding domain. Examples of conservative substitutions are within the group 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.

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

[0022] 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.

[0023] A first aspect of the present invention relates to a method for producing a baked good with no added emulsifier or with a reduced amount of added emulsifier compared to standard recipes, comprising adding 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 to a dough (with no added emulsifier or with a reduced amount of added emulsifier compared to standard recipes) and baking the dough.

[0024] A second aspect of the present invention relates to the 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 with no added emulsifier or with a reduced amount of added emulsifier compared to standard recipes, said method comprising adding said thermostable variant of a parent glucoamylase to dough (with no added emulsifier or with a reduced amount of added emulsifier compared to standard recipes) and baking the dough.

[0025] 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.

[0026] 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.

[0027] Other suitable examples of fungi include Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Rhizopus delemar, Rhizopus niveus, Rhizopus oryzae, and Talaromyces emersonii (Rasamsonia emersonii).

[0028] Below is the percent identity between the AMG amino acid sequences aligned in Figure 1 and also provided in the sequence listing.

[0029] [Table 1]

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] A preferred embodiment relates to the first or second aspect of the invention, wherein no DMG, SSL and / or DATEM is added or a reduced amount of DMG, SSL and / or DATEM is added compared to standard manufacturing processes.

[0035] 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 one or more additional enzymes may be selected from the group consisting of mature lipases and / or phospholipases, such as lactic acid bacteria, ...

[0036] Lipase In a preferred embodiment of the first aspect, one or more additional enzymes are added, said additional enzymes preferably being mature lipolytic enzymes, preferably the mature lipolytic enzymes 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.

[0037] 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.

[0038] 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.

[0039] In one embodiment, the raw starch degrading alpha-amylase is a GH13_1 amylase.

[0040] 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) to the raw starch degrading alpha-amylase set forth in

[0041] 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.

[0042] One 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.

[0043] 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 involved in reducing the full-length high-molecular-weight proteins in dough 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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]

[0048] 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.

[0049] 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

[0050] 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.

[0051] 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.

[0052] Table 1a. List of relative activities of PoAMG variants compared to their parent anPAV498 or JPO-0001 (anPAV498 w. leader / polypeptide).

[0053] [Table 2]

[0054] [Table 3]

[0055] [Table 4]

[0056] [Table 5]

[0057] [Table 6]

[0058] [Table 7]

[0059] [Table 8]

[0060] Table 1b. List of relative activities of PoAMG variants compared to their parent JPO-022

[0061] [Table 9]

[0062] [Table 10]

[0063] [Table 11]

[0064] Table 1c. List of relative activities of PoAMG variants compared to their parent JPO-063 at different temperatures.

[0065] [Table 12]

[0066] [Table 13]

[0067] [Table 14]

[0068] [Table 15]

[0069] Table 1d. List of relative activities of PoAMG variants compared to their parent JPO-096

[0070] [Table 16]

[0071] [Table 17]

[0072] Table 1e. List of relative activities of PoAMG variants compared to their parent JPO-129

[0073] [Table 18]

[0074] Table 1f. List of relative activities of PoAMG variants compared to their parent JPO-166

[0075] [Table 19]

[0076] Table 2. Amino acid substitutions in variants of the PoAMG mature sequence

[0077] [Table 20]

[0078] [Table 21]

[0079] [Table 22]

[0080] [Table 23]

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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

[0085] 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.

[0086] 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.) 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.

[0087] [Table 24]

[0088] [Table 25]

[0089] [Table 26]

[0090] Example 7: DMG removal with JPO-172 and optionally phospholipase Distilled diglycerides or monoglycerides (DMG) are often added to dough to improve emulsion stability and result in a softer crumb (Pyler, EJ and Gorton, LA, 2008, Baking, Science and Technology, Vol. 1, 4 th Edition, p437-452, Sosland Publishing Company, Kansas City, MO, ISBN978-0-9820239-0-7).

[0091] Firing procedure: Bread was baked by straight baking using the recipe according to Table 4. Different treatments were made according to Table 5, adding baking lipase (as disclosed in WO 2018 / 150021) or emulsifier DMG.

[0092] 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 320 g pieces. The pieces were rolled, flattened, and placed in baking tins. The filled baking tins were proofed for 58 minutes at 32°C and 86% relative humidity. The proofed dough was baked in a deck oven at 230°C for 28 minutes.

[0093] [Table 27]

[0094] [Table 28]

[0095] The breads were packed in sealed plastic bags 2 hours after baking and stored at room temperature until evaluation.

[0096] 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 6) 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.

[0097] Table 6. Description of sensory attributes, procedures, and evaluations

[0098] [Table 29]

[0099] result: The data in Table 7 and Figure 2 show that monoglycerides increased the sensory parameters white crumb, moistness, softness, and inner softness compared to the control bread. Springiness decreased. JPO-172 improved moistness, softness, and inner softness at least to the extent of monoglycerides, while maintaining springiness at least to the level of the control. Addition of JPO-172 and Lip182 to the dough further increased white crumb, comparable to distilled monoglycerides.

[0100] Table 7. Mean sensory attribute scores of breads evaluated 1 day after baking - A graphical representation of the data in the table is also shown in Figure 2.

[0101] [Table 30]

[0102] Example 8: DATEM Removal with Heat-Stabilized AMG and Optionally Phospholipase Diacetyl tartaric acid esters of mono- and diglycerides (DATEM) are added to dough for emulsion stability, crumb softening, and dough strengthening (Pyler, EJ and Gorton, LA, 2008, Baking, Science and Technology, Vol. 1, 4 th Edition, p437-452, Sosland Publishing Company, Kansas City, MO, ISBN978-0-9820239-0-7).

[0103] Firing procedure: Bread was baked by straight baking using the recipe according to Table 4. Different treatments were made according to Table 8. 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 320 g pieces. The pieces were rolled, flattened and placed in baking tins. The filled baking tins were proofed for 65 minutes at 32°C and 86% relative humidity. The proofed dough was baked in a deck oven at 230°C for 28 minutes.

[0104] Manufacturing Method: The preparation was the same as that described in Example 7, Table 4.

[0105] [Table 31]

[0106] The breads were packed in sealed plastic bags 2 hours after baking and stored at room temperature until evaluation.

[0107] Sensory evaluation method: Sensory evaluation was performed as described in Example 7, Table 6.

[0108] result: The data in Table 9 and Figure 3 show that DATEM increased white crumb, moistness, softness, and internal softness compared to the control bread. Springiness decreased. JPO-172 improved moistness, softness, and internal softness at least to the extent of DATEM, while retaining springiness at least to the extent of the control. The addition of JPO-172 and Lipopan Xtra further increased white crumb, comparable to DATEM.

[0109] Table 9. Mean sensory attribute scores for breads evaluated 1 day after baking - graphical representation is also provided in Figure 3.

[0110] [Table 32]

[0111] Example 9: SSL Removal with Thermostabilized AMG and Optionally a Combination of Phospholipase and Lipase Sodium stearoyl lactylate (SSL) is added to dough for emulsion stability, crumb softening, and dough reinforcement (Pyler, EJ and Gorton, LA, 2008, Baking, Science and Technology, Vol. 1, 4 th Edition, p437-452, Sosland Publishing Company, Kansas City, MO, ISBN978-0-9820239-0-7).

[0112] Firing procedure: Bread was baked by straight baking using the recipe according to Table 4. Different treatments were made according to Table 10. 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 15 minutes and divided into 320 g pieces. The pieces were rolled, flattened 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 25 minutes.

[0113] Manufacturing Method: The preparation was the same as that described in Example 7, Table 4.

[0114] [Table 33]

[0115] The breads were packed in sealed plastic bags 2 hours after baking and stored at room temperature until evaluation.

[0116] Sensory evaluation method: Sensory evaluation was performed as described in Example 7, Table 6.

[0117] result: The data in Table 11 and Figure 4 show that SSL increased white crumb and decreased springiness compared to the control bread. JPO-172 improved moistness, softness, and inner softness at least to the extent of SSL, and maintained springiness at least to the level of the control. The addition of JPO-172 and Lipopan Xtra and Lipopan 50 further increased white crumb, comparable to SSL.

[0118] Table 11. Mean sensory attribute scores for breads evaluated 1 day after baking - a graphical representation of the results is also provided in Figure 4.

[0119] [Table 34]

Claims

1. A method for producing a baked good without the addition of an emulsifier or with a reduced amount of added emulsifier compared to standard production methods, 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 dough (without added emulsifier or with a reduced amount of added emulsifier compared to standard production methods), and baking the dough, wherein the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to positions 50, 132, 481, 48 and 595 in SEQ ID NO:

1.

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. 2. The method of claim 1, wherein the mature thermostable 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. 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 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. 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. 10. The method of claim 1, wherein no DMG, SSL and / or DATEM is added or a lower amount of DMG, SSL and / or DATEM is added compared to standard manufacturing processes.

12. 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, 2. The method of claim 1, 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.

13. 13. The method of claim 12, wherein the one or more additional enzymes comprise a mature lipase or a phospholipase.

14. 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 baked goods with no added emulsifier or with a reduced amount of added emulsifier compared to standard manufacturing methods, said method comprising adding said thermostable variant of a parent glucoamylase to dough (with no added emulsifier or with a reduced amount of added emulsifier compared to standard manufacturing methods) and baking the dough.

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