Baking with thermostable amyloglucosidase (AMG) variants (EC 3.2. 1.3) and low addition of sugars

Abstract

The present invention relates to a method of producing a baked product, said method comprising adding to a dough a mature thermostable variant of a parent glucoamylase having at least 70% identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 10, adding to the dough less than 150% (baked%) sugar, and baking the dough, the invention also relates to the use of a mature thermostable variant of a parent glucoamylase having at least 70% identity 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% (baked%) sugar or sucrose is added to the dough, and the dough is baked.

Description

[0001] References to sequence listings

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Technical Field

[0003] The present invention relates to a method for producing a baked product having a reduced amount of added sugar compared to a standard recipe, the method comprising adding to a dough a mature thermostable variant of a parent glucoamylase having at least 70% identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 10, adding a reduced amount of sugar compared to a standard recipe, and baking the dough. Background Art

[0004] Recipes for different baked goods can vary greatly. One ingredient is sugar, which can affect the baking process as well as the final baked good.

[0005] -Dough properties: As sugar levels increase, the dough becomes softer

[0006] - Provides food for yeast. However, it also inhibits yeast activity, thereby reducing gas production

[0007] - Improves the crust color, making the final crust more golden in color.

[0008] -Affects the crumb of baked goods and keeps them soft and moist.

[0009] - Imparts sweetness to baked products.

[0010] There is a trend in the market to reduce the amount of sugar in food products. Removing sugar will affect the baking process and the final baked product. It makes the dough firmer, the crust produced during baking lighter in color, the product produced is less soft and moist, and makes the baked product less sweet.

[0011] Enzymes can improve some of these properties. Crust color can be improved by glucoamylases like The softness of the crumb can be improved by maltogenic amylases such as G. Novamyl G. Pro to improve.

[0012] Recipe changes can improve other parameters that are affected by sugar removal. Increasing water content can make the dough softer. The increase in yeast gassing ability when sugar is removed can be adjusted by reducing yeast levels. Summary of the invention

[0013] The inventors have found that mature thermostable glucoamylase variants exhibit higher enzyme activity or performance in baking recipes with lower initial concentrations of added sugar. An increase in enzyme activity may mean a lower enzyme dosage compared to recipes with higher initial concentrations of added sugar. Since glucoamylase produces glucose, the improved activity of the thermostable variants results in an increase in the sweetness or sweetness of the product. This discovery can reduce the amount of added sugar in traditional baking recipes without causing any significant loss in the sweetness of the final baked product, which has great commercial significance. At the same time, the recipe can be adjusted by adding more water and reducing the yeast level, thereby saving more costs.

[0014] Thus, in a first aspect, the present invention relates to a method for producing a baked product, the method comprising adding to a dough a mature thermostable variant of a parent glucoamylase having at least 70% identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 10, adding less than 150% (baked %) sugars to the dough, and baking the dough.

[0015] A second aspect of the invention relates to the use of a mature thermostable variant of a parent glucoamylase having at least 70% identity 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% (baked %) sugar or sucrose is added to the dough, and the dough is baked.

[0016] Preferably, the method of the first aspect or the use of the second aspect comprises adding a mature thermostable variant of a parent glucoamylase having at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 10.

[0017] Preferred embodiments of the invention relate to adding less than 120% (baked matter %) of sugar to the dough, preferably less than 110% (baked matter %), preferably less than 100% (baked matter %), preferably less than 90% (baked matter %), preferably less than 80% (baked matter %), preferably less than 70% (baked matter %), preferably less than 60% (baked matter %), preferably less than 50% (baked matter %), preferably less than 40% (baked matter %), preferably less than 30% (baked matter %), preferably less than 20% (baked matter %), preferably less than 10% (baked matter %), preferably less than 8% (baked matter %), preferably less than 6% (baked matter %), even more preferably less than 4% (baked matter %) and most preferably less than 2% (baked matter %) of sugar to the dough. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A multiple alignment of the amino acid sequences of the following mature proteins is shown:

[0019] - Wild-type AMG from Penicillium oxalicum (PoAMG) of SEQ ID NO: 1

[0020] - PoAMG variant denoted "AMG NL" of SEQ ID NO: 2

[0021] - A PoAMG variant of SEQ ID NO: 3 denoted "AMG anPAV498"

[0022] - PoAMG variant denoted "AMG JPO001" of SEQ ID NO:4

[0023] - A PoAMG variant of SEQ ID NO:5 designated as "AMG JPO124"

[0024] - A PoAMG variant of SEQ ID NO: 6 denoted "AMG JPO-172"

[0025] - Wild-type AMG from Penicillium miczynskii (PoAMG) of SEQ ID NO: 7

[0026] - Wild-type AMG from Penicillium russellii (PoAMG) of SEQ ID NO: 8

[0027] - Wild-type AMG from Penicillium glabrum (PoAMG) of SEQ ID NO: 9 DETAILED DESCRIPTION

[0028] definition

[0029] Baked products: As used herein, "baked products" means any type of baked products, including various bread types such as pan bread, toast bread, open bread, covered and uncovered pan bread, buns, hamburger buns, rolls, baguettes, brown bread, whole wheat bread, rich bread, bran bread, flat bread, tortillas, pita bread, Arabic bread, Indian flat bread, cookies, biscuits, cakes, brioche and any varieties thereof.

[0030] Dough: As used herein, "dough" means any dough used to prepare bread. The dough used to prepare baked products can be made from any suitable dough ingredients, including flours derived from cereals, 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 sources; rice flour combined with one of the other flour sources). The dough of the present invention is typically a fermented dough or a dough to be subjected to fermentation. The dough can be fermented in various ways, such as by adding dough ingredients such as chemical leavening agents (e.g., sodium bicarbonate) or by adding a leavening agent (fermented dough), but preferably the dough is fermented by adding a suitable yeast culture such as a culture of Saccharomy cescerevisiae (baker's yeast) (e.g., a commercially available strain of Saccharomyces cerevisiae). The dough may also contain other conventional dough ingredients, for example: proteins, such as milk powder, gluten, and soy; eggs (whole eggs, egg yolks or egg whites); oxidants, such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammonium persulfate; amino acids, such as L-cysteine; sugars; salts, such as sodium chloride, calcium acetate, sodium sulfate, calcium sulfate; diluents (such as silicon dioxide); starches from different sources. Other conventional ingredients also include hydrocolloids, such as CMC, guar gum, xanthan gum, locust bean gum, etc. Modified starches may also be used. These dough ingredients may contain fats (triglycerides), such as granular fats or shortenings, but the present invention is particularly suitable for doughs to which less than 1% by weight of fat or shortening is added, and is particularly suitable for doughs made without adding fat or shortening. In a preferred embodiment, the dough ingredients include wheat flour; preferably, 10% (w / w) or more of the total flour content is wheat flour, 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 made using any conventional mixing process, such as a continuous mixing process, a straight-dough process, or a sponge and dough method.

[0031] Baking Matter %: Baking Matter % is a mathematical method widely used in baking to calculate the amount of macro, minor and micro ingredients. Baking Matter % is based on the total weight of flour that the recipe contains. Instead of dividing the weight of each ingredient by the total recipe weight, the baking matter is divided by the weight of each ingredient by the weight of the flour.

[0032] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0033] For the purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48: 443-453), as implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [Genetics Trend] 16: 276-277) (preferably version 5.0.0 or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the "longest identity" marked with Needle (obtained using the non-simplified (-no brief) option) is used as the identity percentage and is calculated as follows:

[0034] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0035] Variant: The term "variant" means a polypeptide comprising an alteration (i.e., a substitution, insertion, and / or deletion) at one or more (e.g., several) positions. Substitution means replacing the amino acid occupying a position with a different amino acid; deletion means removing the amino acid occupying a position; and insertion means adding one or more amino acids adjacent to and immediately following the amino acid occupying a position. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; typically small deletions of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function (such as a polyhistidine stretch, 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 small 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, by H. Neurath and RL Hill, 1979, The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0036] Improved thermostability: Thermostability improvement (Td) in °C is a measure of how much a variant is improved in thermostability relative to its parent glucoamylase under the same conditions, determined as exemplified herein.

[0037] As used herein, the term "cracked malt" is understood to mean the starch- or sugar-containing material, such as barley malt and adjuncts, which is the basis for beer production. Typically, cracked malt does not contain any added water.

[0038] 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) is a method that can be used to study the gradual process of starch gelatinization (describing the onset temperature and peak temperature (T o and T p The term "initial gelatinization temperature (To The term "peak gelatinization temperature (T p The term "end gelatinization temperature (T c )” is understood as the temperature at which gelatinization is terminated.

[0039] Glucoamylase

[0040] Glucoamylases are also called amyloglucosidases and glucan 1,4-alpha-glucosidases (EC 3.2.1.3), more commonly they are known as AMG.

[0041] According to the present invention, different types of amyloglucosidases can be used as parents for producing thermostable amyloglucosidase variants, for example, the amyloglucosidase can be a polypeptide encoded by a DNA sequence found in a fungal strain of Aspergillus, Rhizopus, Talaromyces (Rasamsonia) or Penicillium; preferably a DNA sequence found in a fungal strain of Penicillium, even more preferably a DNA sequence found in a fungal strain of Penicillium oxysporum, Penicillium oxicalum, Penicillium mickii, Penicillium roqueforti or Penicillium luminosporum. Preferably, the parent glucoamylase is from a species of Penicillium, preferably from Penicillium oxicalum, Penicillium mickii, Penicillium roqueforti or Penicillium luminosporum.

[0042] Examples of other suitable fungi include Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Rhizopus delemar, Rhizopus niveus, Rhizopus oryzae, and Talaromyces emersonii (Rasamsonia emersonii).

[0043] The following shows Figure 1 The % identity between the AMG amino acid sequences aligned in , and are also provided in the sequence listing:

[0044]

[0045] Thermostable variants of PoAMG have been generated (see Table 2 below).In preferred embodiments, the mature thermostable glucoamylase variants of the invention comprise one or more or all combinations of the amino acid substitutions listed in Table 2 below.

[0046] In a preferred embodiment, the mature variant of the present invention comprises at least one amino acid modification at one or more or all 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 positions corresponding to positions 1, 2, 4, 11, 65, 79 and 327 in SEQ ID NO: 1, preferably, the at least one amino acid modification comprises a substitution at one or more or all positions corresponding to R1A, P2N, P4S, P11F, T65A, K79V and Q327F in SEQ ID NO: 1; or preferably, the at least one amino acid modification comprises a substitution at one or more or all positions corresponding to SEQ ID NO: NO:1 comprises a substitution at one or more or all of positions 1, 6, 7, 31, 34, 79, 103, 132, 445, 447, 481, 566, 568, 594 and 595, preferably, the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to R1A, G6S, G7T, R31F, K34Y, K79V, S103N, A132P, D445N, V447S, S481P, D566T, T568V, Q594R and F595S in SEQ ID NO:1; or preferably, the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to SEQ ID NO:1. NO:1 comprises a substitution at one or more or all of positions 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595, preferably, the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO:1;or preferably, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595 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; or preferably, the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to SEQ ID NO: IDNO: 1 comprises a substitution at one or more or all of positions 1, 6, 7, 31, 34, 50, 79, 103, 132, 445, 447, 481, 484, 501, 539, 566, 568, 594 and 595, preferably, the at least one amino acid modification comprises a substitution at one or more or all of positions corresponding to R1A, G6S, G7T, R31F, K34Y, E50R, K79V, S103N, A132P, D445N, V447S, S481P, T484P, E501A, N539P, D566T, T568V, Q594R and F595S in SEQ ID NO: 1. ;

[0047] The improved thermostability (Td) of the variants in Table 2 are listed in Table 3, wherein the Td of the PoAMG variant denoted "anPAV498" (parent) is set to zero. In preferred embodiments, the mature thermostable variants of the invention have an improved thermostability (Td) of at least 5°C, preferably at least 6°C, 7°C or 8°C relative to their parent, preferably as determined as exemplified herein.

[0048] In another preferred embodiment, the mature thermostable variant of the present invention has a relative activity of at least 150, preferably at least 200, more preferably at least 250, most preferably at least 300 at 91°C compared to its parent.

[0049] Other enzymes

[0050] In a preferred embodiment of the first aspect, one or more additional enzymes are added to the dough, and the additional enzymes may be 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-maltotetraose hydrolase, glucanase, beta glucanase, galactoside. Polysaccharidase, α-galactosidase, β-galactosidase, glucose oxidase, α-glucosidase, β-glucosidase, haloperoxidase, hemicellulolytic enzyme, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinase, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, protease, pullulanase, raw starch degrading α-amylase, ribonuclease, transglutaminase and xylanase.

[0051] Lipase

[0052] In a preferred embodiment of the first aspect, one or more additional enzymes are added, the additional enzymes preferably being a mature lipolytic enzyme, preferably a mature lipolytic enzyme disclosed in WO 2018 / 150021 (Novozymes A / S), more preferably a mature polypeptide having lipolytic activity in WO 2018 / 150021 and having at least 65% sequence identity with amino acids 21 to 309 of SEQ ID NO: 1, or a polypeptide encoded by a polynucleotide in WO 2018 / 150021 having at least 65% sequence identity with the mature polypeptide coding sequence of SEQ ID NO: 2.

[0053] Raw starch degrading α-amylase

[0054] As used herein, "raw starch degrading α-amylase" refers to an enzyme that can directly degrade raw starch granules below the gelatinization temperature of starch.

[0055] Examples of raw starch degrading α-amylases include those disclosed in WO 2005 / 003311, U.S. Patent Publication No. 2005 / 0054071, and U.S. Patent No. 7,326,548. Examples also include those disclosed in Tables 1 to 5 of the Examples in U.S. Patent No. 7,326,548 and in U.S. Patent Publication No. 2005 / 0054071 (Table 3 on page 15), and enzymes disclosed in WO 2004 / 020499, WO 2006 / 06929, and WO 2006 / 066579.

[0056] In one embodiment, the raw starch degrading alpha-amylase is a GH13-1 amylase.

[0057] In an embodiment the raw starch degrading alpha-amylase has at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity to the raw starch degrading alpha-amylase in European Patent No. 2981170 (Novozymes) or shown in SEQ ID NO: 11 herein.

[0058] Amylase

[0059] α-Amylases (α-1,4-glucan-4-glucanohydrolases, EC.3.2.1.1) constitute a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glycosidic oligo- and polysaccharides.

[0060] Termamylase is a type of α-amylase. TM , SC and "Termamyl TM -like α-amylases" and are known from, for example, WO 90 / 11352, WO 95 / 10603, WO 95 / 26397, WO 96 / 23873 and WO 96 / 23874.

[0061] Another group of α-amylases is called Fungamyl TM and "Fungamyl TM -like α-amylases", which are α-amylases related to the α-amylase derived from Aspergillus oryzae disclosed in WO 01 / 34784.

[0062] Protease

[0063] Suitable proteases include microbial proteases, such as fungal proteases and bacterial proteases. Preferred proteases are acidic proteases, i.e., proteases characterized by being able to hydrolyze proteins under acidic conditions below pH 7. The role of proteases is to shorten the total length of high molecular weight proteins in the mash to low molecular weight proteins. Low molecular weight proteins are essential 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 so as to provide sufficient free amino acids for yeast at the same time and leave enough high molecular weight proteins to stabilize the foam. On the one hand, the protease activity is provided by a proteolytic enzyme system having a suitable FAN producing activity, including endoproteases, exopeptidases or any combination thereof, preferably metalloproteases. 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 shown in SEQ ID NO: 6 as described in WO 9967370. In another aspect, the protease is available from Novozymes AG The amount of protease added can be 0.0001-1000AU / kg DS, preferably 1-100AU / kg DS and most preferably 5-25AU / kg dry weight grain. Proteolytic activity can be determined by using denatured 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 products is determined by using a phenol reagent, which can produce 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 under standard conditions (i.e., 25°C, pH 7.5 and 10min reaction time), so that a certain amount of TCA-soluble products is released per minute, and the color produced by the product and the phenol reagent is the same as one milliequivalent of tyrosine.

[0064] Enzyme composition

[0065] The mature thermostable variant glucoamylase of the invention and any one or more additional enzymes can be added in any suitable form, for example in the form of a liquid (particularly a stabilized liquid), or can be added as a substantially dry powder or granules.

[0066] For example, granules may be produced as disclosed in U.S. Pat. No. 4,106,991 and U.S. Pat. No. 4,661,452. Liquid enzyme preparations may be stabilized, for example, by adding sugars or sugar alcohols or lactic acid according to established procedures. Other enzyme stabilizers are well known in the art.

[0067] The one or more enzymes can be added in any suitable manner, such as as separate components (adding the enzymes separately or sequentially), or adding the enzymes together in one step or one composition.

[0068] 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 be composed of microparticle cores with a suitable particle size. The carrier can be soluble or insoluble, for example salt (such as NaCl or sodium sulfate), sugar (such as sucrose or lactose), sugar alcohol (such as sorbitol), starch, rice, corn residue, or soybean.

[0069] Examples

[0070] Example 1: Construction of PoAMG library

[0071] The PoAMG library was constructed as follows:

[0072] Design the forward or reverse primer with NNK or one or more desired mutations at one or more target sites, and these target sites have an overlap of 15bp each other. Use appropriate template plasmid DNA (for example, plasmid DNA containing JPO-0001 gene) to carry out inverse PCR under the following conditions, i.e., amplify the whole plasmid DNA sequence by reverse directional primer. Purify the obtained PCR fragment by QIAquick gel extraction kit [Qiagen (QIAGEN)], then introduce it into Escherichia coli ECOS competent Escherichia coli DH5α [Japan Gene Co., Ltd. (NIPPON GENE CO., LTD.)]. Extract plasmid DNA from Escherichia coli transformant by MagExtractor plasmid extraction kit [Toyo Company (TOYOBO)], then introduce it into Aspergillus niger competent cell.

[0073] PCR reaction mixture:

[0074] PrimeSTAR Max DNA Polymerase [TaKaRa]

[0075] Total 25 μl

[0076]

[0077]

[0078] PCR procedure:

[0079] 98℃ / 2min

[0080] 25x(98℃ / 10sec, 60℃ / 15sec, 72℃ / 2min)

[0081] 10℃ / maintain

[0082] Example 2: Screening for better thermal stability

[0083] The Bacillus subtilis library constructed as in Example 1 was fermented in a 96-well or 24-well MTP containing COVE liquid medium (2.0 g / L sucrose, 2.0 g / L isomaltose, 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) at 32° C. for 3 days. AMG activity in the culture supernatant was then measured at several temperatures by the pNPG assay as described below.

[0084] pNPG thermal stability assay:

[0085] The culture supernatant containing the desired enzyme was mixed with the same volume of pH 5.0 200mM NaOAc buffer. Twenty microliters of the mixture was distributed in a 96-well plate or 8-link PCR tubes and then heated for 30 minutes at various temperatures by a thermal cycler. Those samples were mixed with 10 μl of substrate solution containing 0.1% (w / v) pNPG [Wako Co., Ltd. (wako)] in pH 5.0 200mM NaOAc buffer and incubated at 70°C for 20 minutes for enzyme reaction. After the reaction, 60 μl of 0.1M Borax buffer was added to stop the reaction. 80 μl of the reaction supernatant was taken out and its OD was read by a photometer. 405 to evaluate enzyme activity.

[0086] Table 1a. List of relative activities of PoAMG variants compared to their parents anPAV498 or JPO-0001 (anPAV498 with leader / propeptide)

[0087]

[0088]

[0089] name Relative activity at 80℃ / 75℃ (%) AnPav498 13％ JPO-009 16％ JPO-011 15％ JPO-012 15％ JPO-013 17％ JPO-020 20％

[0090] name Relative activity at 80℃ / 70℃ (%) JPO-001 10％ JPO-004 29％ JPO-009 13％ JPO-014 21％ JPO-020 16％ JPO-021 30％ JPO-052 33％

[0091] name Relative activity at 79℃ / 70℃ (%) JPO-001 23％ JPO-021 46％ JPO-022 39％ JPO-023 44％ JPO-025 51％ JPO-027 49％ JPO-029 37％

[0092] name Relative activity at 77℃ / 70℃ (%) JPO-001 72％ JPO-029 82％ JPO-047 80％ JPO-048 90％ JPO-049 84％ JPO-050 86％ JPO-064 87％

[0093] name Relative activity at 79℃ / 77℃ (%) JPO-001 36％ JPO-029 51％ JPO-047 45％ JPO-048 81％ JPO-049 53％ JPO-050 58％ JPO-064 65％

[0094]

[0095]

[0096] Table 1b. List of relative activities of PoAMG variants compared to their parent JPO-022

[0097] name Relative activity at 77℃ / 70℃ (%) JPO-022 60％ JPO-027 67％ JPO-042 8％ JPO-044 86％ JPO-045 67％ JPO-046 48％

[0098] name Relative activity at 77℃ / 70℃ (%) JPO-022 76％ JPO-023 75％ JPO-025 80％ JPO-027 84％ JPO-058 92％ JPO-059 88％ JPO-060 86％ JPO-061 83％ JPO-062 87％

[0099]

[0100]

[0101] Table 1c. List of relative activities of PoAMG variants compared to their parent JPO-063 at different temperatures

[0102] name Relative activity at 79℃ / 77℃ (%) JPO-063 91％ JPO-066 96％ JPO-071 89％ JPO-072 84％ JPO-074 103％ JPO-075 86％ JPO-076 92％ JPO-077 95％ JPO-078 88％ JPO-079 100％

[0103] name Relative activity at 84℃ / 80℃ (%) JPO-063 16％ JPO-065 26％ JPO-067 21％ JPO-070 12％ JPO-071 13％ JPO-074 32％ JPO-081 17％ JPO-082 24％ JPO-083 46％ JPO-084 26％ JPO-044 37％

[0104] name Relative activity at 82℃ / 70℃ (%) JPO-063 21％ JPO-093 43％ JPO-081 25％ JPO-088 39％ JPO-094 38％ JPO-096 38％ JPO-106 53％

[0105] name Relative activity at 83℃ / 80℃ (%) JPO-063 46％ JPO-051 44％ JPO-096 64％ JPO-106 88％ JPO-110 81％ JPO-111 100％ JPO-112 86％ JPO-113 83％ JPO-114 47％ JPO-115 90％

[0106] Table 1d. List of relative activities of PoAMG variants compared to their parent JPO-096

[0107]

[0108]

[0109] name Relative activity at 83℃ / 80℃ (%) JPO-051 20％ JPO-096 43％ JPO-109 51％ JPO-126 33％ JPO-129 48％ JPO-130 18％ JPO-131 51％ JPO-132 34％

[0110] Table 1e. List of relative activities of PoAMG variants compared to their parent JPO-129

[0111] name Relative activity at 84℃ / 80℃ (%) JPO-129 62％ JPO-156 51％ JPO-160 34％ JPO-161 41％ JPO-162 49％ JPO-163 21％ JPO-164 57％ JPO-165 77％

[0112] Table 1f. List of relative activities of PoAMG variants compared to their parent JPO-166

[0113]

[0114]

[0115] Table 2. Amino acid substitutions in variants of the PoAMG mature sequence

[0116]

[0117]

[0118]

[0119]

[0120]

[0121] Example 3: Fermentation of Aspergillus niger

[0122] Aspergillus niger strains were fermented in 500 ml baffled flasks containing 100 ml MU1 and 4 ml 50% urea on an orbital 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 precipitate.

[0123] Example 4: Purification of PoAMG (JPO-001) variants

[0124] PoAMG variants were purified by cation exchange chromatography. The respective peak fractions were combined individually and dialyzed against 20 mM sodium acetate buffer (pH 5.0), and the samples were then concentrated using a centrifugal filter device (Vivaspin Turbo 15, Sartorius). The enzyme concentration was determined by the A280 value.

[0125] Example 5: Thermal Stability Assay (TSA)

[0126] The purified enzyme was diluted to 0.5 mg / ml with 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 solution was transferred to a LightCycler 480 multiwell plate 384 (Roche Diagnostics) and the plate was sealed.

[0127] TSA equipment parameters:

[0128] Instrument: LightCycler 480 Real-Time PCR System (Roche Applied Science)

[0129] Scan rate: 0.02℃ / sec

[0130] Scanning range: 37℃-96℃

[0131] Integration time: 1.0 sec

[0132] Excitation wavelength 465nm

[0133] Emission wavelength 580nm

[0134] The obtained fluorescence signal was normalized to the range of 0 and 1. Td was defined as the temperature at which the signal intensity was 0.5. The thermal stability improvements are listed in Table 3, where the PoAMG variant denoted anPAV498 had a Td of 0.

[0135] Example 6: PoAMG activity assay

[0136] Determination of maltodextrin (DE11) by GOD-POD method

[0137] Substrate solution

[0138] 30g maltodextrin (pindex #2 from MATSUTANI chemical industry Co., Ltd.)

[0139] 100 ml 120 mM sodium acetate buffer, pH 5.0

[0140] Glucose CII test kit (Wako Pure Chemical Industries, Ltd., Japan)

[0141] 20 μ l of enzyme sample was mixed with 100 μ l of substrate solution and incubated at set temperature for 2 hours. Sample was cooled on aluminum block for 3 min, then 10 ul of reaction solution was mixed with 590 ul of 1 M Tris-HCl (pH 8.0) to stop the reaction. 10 μ l of solution was mixed with 200 μ l of working solution of test kit and then left at room temperature for 15 min. Read absorbance at A505. Activity is listed in Table 3 as the relative activity of PoAMG variants represented as anPAV498.

[0142] Table 3.

[0143]

[0144]

[0145]

[0146]

[0147] Example 7: Effect of reducing sugar (sucrose) levels on the efficiency of JPO-172 in direct fermentation bread

[0148] Bread was baked in a direct fermentation baking process using the recipe according to Table 4. Different treatments were performed according to Table 5. Bread with a lower sucrose level had 2.5% additional water to give all doughs the same properties. The bread was baked in a covered baking tray to give all breads the same volume. The ingredients were mixed into a dough in a spiral mixer at 17 rpm for 3 min and 35 rpm for 7 min. The dough was left to rest for 5 minutes and divided into 450 g dough pieces and rounded. The dough pieces were sheeted and placed in a baking tray. The baking tray containing the dough was proofed at 32°C and 86% relative humidity for 55 min. The proofed dough was baked in a box oven at 230°C for 28 min.

[0149] Table 4. Recipe

[0150]

[0151]

[0152] Table 5 Treatment

[0153]

[0154] The breads were packaged in sealed plastic bags 2 hours after baking and stored at room temperature until analysis.

[0155] The texture of the bread was assessed using a texture analyser (TA-XT plus, Stable microsystems, Godalming, UK). Crumb texture properties are characterised by the firmness (same as "hardness" and opposite to "softness") and elasticity of the baked product. The standard method for measuring firmness and elasticity is based on the force-deformation of the baked product. The force-deformation of the baked product can be performed with a 40 mm diameter cylindrical probe. The force on the cylindrical probe is recorded as it is pressed down a 25 mm thick bread slice at a deformation rate of 1 mm / sec with a stress of 40%. The probe is then held in this position for 30 seconds while the force is recorded, after which the probe is returned to its initial position.

[0156] Firmness (in grams) is defined as the force required to compress the probe to 25% stress (corresponding to compression of 6.25 mm into a crumb slice having a thickness of 25 mm).

[0157] Elasticity (in %) is defined as the force recorded after compression for 30 seconds at 40% stress (corresponding to the force at time = 40s for a bread slice with a thickness of 25 mm) divided by the force required to press the probe 10 mm into the crumb (corresponding to the force at time = 10s for a bread slice with a thickness of 25 mm) multiplied by 100.

[0158] The results of the texture analysis can be seen in Tables 6 and 7. The bread without JPO-172 enzyme (control) was soft and elastic on the first day, and when the bread was stored for 14 days, it became harder and less elastic over time. When 46 mg EP JPO-172 enzyme / kg flour was added, the firmness on the first day decreased from 496 g to 285 g, and the elasticity increased from 57.4% to 66.5%. When the bread containing JPO-172 was stored for 14 days, it became harder and less elastic. However, it was far from the extent of the bread without JPO-172.

[0159] If the sucrose level in the bread was reduced from 8% to 2%, the JPO-172 enzyme became even more effective. A dose of 31 mg EP / kg flour when the formula contained 2% sucrose did not produce a significant difference in firmness and elasticity on days 1 to 14 compared to 46 mg EP / kg flour when the formula contained 8% sucrose. A higher dose of JPO-172 (38 mg EP / kg flour) when the formula contained 2% sucrose resulted in reduced firmness on days 7 and 14 compared to 46 mg EP / kg flour when the formula contained 8% sucrose.

[0160] Table 6. Crumb firmness at different time points (g) The letters after the numbers indicate the significance level using Student's t-test, where the significance level is 0.05.

[0161] deal with Day 1 Day 7 Day 14 Control (8% sucrose) 496A 1094A 1579A 46mgEP / kg flour JPO-172 (8% sucrose) 285B 614BC 913C 27mgEP / kg flour JPO-172 (2% sucrose) 284B 640B 1040B 31mgEP / kg flour JPO-172 (2% sucrose) 269B 654B 885C 35mgEP / kg flour JPO-172 (2% sucrose) 267B 565C 894C 38mgEP / kg flour JPO-172 (2% sucrose) 230B 481D 779D

[0162] Table 7. Crumb elasticity at different time points (%) The letters after the numbers indicate the significance level using Student's t-test, where the significance level is 0.05.

[0163] deal with Day 1 Day 7 Day 14 Control (8% sucrose) 57.4B 45.8E 43.7D 46mgEP / kg flour JPO-172 (8% sucrose) 66.5A 61.0C 56.8B 27mgEP / kg flour JPO-172 (2% sucrose) 67.6A 58.6D 53.0C 31mgEP / kg flour JPO-172 (2% sucrose) 68.1A 61.2C 56.7B 35mgEP / kg flour JPO-172 (2% sucrose) 67.9A 62.6B 58.8A 38mgEP / kg flour JPO-172 (2% sucrose) 68.2A 64.3A 59.6A

[0164] Example 8. Effect of reducing sugar (sucrose) levels on the efficiency of JPO-172 in mid-fermented bread

[0165] Breads were baked with a medium fermentation baking process using the recipe according to Table 8. Different treatments were performed according to Table 9. Breads with lower sucrose levels had additional water to give all doughs the same properties. Breads were baked in covered baking pans to give all breads the same volume. The starter ingredients were mixed in a pin mixer ( Mix the starter in a pin mixer at 50 rpm for 2 min and 150 rpm for 2 min to form a starter. The starter was fermented at 27° C. and 75 RH% for 3 hours. The starter and dough ingredients were placed in the bowl of a pin mixer and mixed at 50 rpm for 1 min and 150 rpm for 3 min. The dough was divided into 400 g dough pieces, rounded, and left to stand for 5 minutes. The dough pieces were sheeted and placed in a baking pan. The baking pan containing the dough was proofed at 43° C. and 80% relative humidity for 60 min. The proofed dough was baked in a rotary oven at 215° C. for 20 min.

[0166] Table 8. Types of fermentation in the formula

[0167]

[0168]

[0169] Table 9. Treatment

[0170]

[0171] The breads were packaged in sealed plastic bags 2 hours after baking and stored at room temperature until analysis.

[0172] The texture of bread was assessed using a texture analyzer (TA-XT plus, Stable Microsystems, Godalming, UK). Crumb texture properties are characterized by the firmness (same as "hardness" and opposite to "softness") and elasticity of the baked product. The standard method for measuring firmness and elasticity is based on the force-deformation of baked products. The force-deformation of baked products can be performed with a 40 mm diameter cylindrical probe. The force on the cylindrical probe is recorded while it is pressed down by 40% stress on a 25 mm thick bread slice at a deformation rate of 1 mm / sec. The probe is then held in this position for 30 seconds while the force is recorded, after which the probe is returned to its initial position.

[0173] Firmness (in grams) is defined as the force required to compress the probe to 25% stress (corresponding to compression of 6.25 mm into a crumb slice having a thickness of 25 mm).

[0174] Elasticity (in %) is defined as the force recorded after compression for 30 seconds at 40% stress (corresponding to the force at time = 40s for a bread slice with a thickness of 25 mm) divided by the force required to press the probe 10 mm into the crumb (corresponding to the force at time = 10s for a bread slice with a thickness of 25 mm) multiplied by 100.

[0175] The results of the texture analysis can be seen in Tables 10 and 11. The bread without preservative enzyme and 8% sucrose (Treatment 1 Control) was soft and elastic on day 1, and when the bread was stored for 14 days, it became harder and less elastic over time.

[0176] The addition of the preservative enzyme JPO-172 at a dose of 100 mg EP / kg flour (doughs 2-5) made the bread less hard and more elastic over the entire study time interval (days 1-14). By reducing the sucrose level, the preservative enzyme JPO-172 became more effective and could even further improve the preservation effect, making the bread crumb less hard on days 7 and 14. Improving the efficiency of the preservative enzyme by reducing the sucrose level in the recipe can also be seen as increasing elasticity over the entire study time frame.

[0177] Table 10. Crumb firmness at different time points (g) The letters after the numbers indicate the significance level using Student's t-test, where the significance level is 0.05.

[0178] deal with Day 1 Day 7 Day 14 2% sucrose 100mgEP / kg JPO-172 199 B 260C 323D 3% sucrose 100mgEP / kg JPO 172 197B 304B 378C 4% sucrose 100mgEP / kg JPO 172 201B 293B 411BC 5% sucrose 100mgEP / kg JPO 172 191B 313B 428B 8% sucrose (control) 231A 600A 875A

[0179] Table 11 Elasticity of bread crumb at different time points (%) The letters after the numbers indicate the significance level using Student's t-test, where the significance level is 0.05.

[0180] deal with Day 1 Day 7 Day 14 2% sucrose 100mgEP / kg JPO-172 62.5A 59.8A 57.8A 3% sucrose 100mgEP / kg JPO 172 61.9B 58.8B 57.0B 4% sucrose 100mgEP / kg JPO 172 61.5C 58.1C 55.7C 5% sucrose 100mgEP / kg JPO 172 61.2C 57.1D 54.6D 8% sucrose (control) 52.5D 48.0E 44.4E

[0181] Example 9 Sweetness of bread with reduced sugar level in the formulation and containing JPO-172

[0182] Bread was baked in a direct fermentation baking process using the recipe according to Table 12. Different treatments were performed according to Table 13. Breads with lower sucrose levels had 0.3% additional water / reduced sucrose% to have the same properties for all doughs. Since sugar inhibits yeast activity, breads with lower sugar levels had 0.25% less yeast / sugar% to have the same gas production capacity for all doughs. The bread was baked in a covered baking tray to have the same volume for all breads. The ingredients were mixed into dough in a spiral mixer at 17 rpm for 3 min and 35 rpm for 6 min. The dough was left to rest for 5 minutes and was divided into 450 g dough pieces and rounded. The dough pieces were sheeted and placed in baking trays. The baking trays containing the dough were proofed at 32°C and 86% relative humidity for 57 min. The proofed dough was baked in a box oven at 215°C for 35 min.

[0183] Table 12 Formulation

[0184]

[0185] Table 13 Treatment

[0186]

[0187] The breads were allowed to cool at room temperature and were packaged in sealed plastic bags 2 hours after baking and stored at room temperature until analysis.

[0188] Sensory evaluation method:

[0189] The sweetness of the bread was evaluated in a sensory session on the second day after baking. Each sensory assessor was provided with 1 slice of each type of bread. A training session was held before the evaluation to define the sweetness attributes and procedures (Table 14) and the use of the intensity scale. The intensity of sweetness was evaluated on a 1-9 point intensity scale from weak to very strong. A control sample containing 8% sucrose was used as a reference and scored 5.0. The other samples were provided in a blinded, 3-digit coded and random order. Five trained assessors participated in the evaluation. Two repeated sensory evaluations were performed. The significance of the sensory results was evaluated using the Student's t-test at a significance level of 0.05.

[0190] Table 14 Sensory attributes

[0191]

[0192] The scores for the sensory evaluation of sweetness can be found in Table 15. Although the doughs with 0-2% sucrose had less added sugar in the dough compared to the dough with 8% sucrose, the final baked bread felt just as sweet as the bread with 8% sucrose added to the dough.

[0193] Table 15. Mean sensory scores for sweetness. Letters following the sensory scores indicate statistical significance using Student's t-test with a = 0.05. Levels with the same unconnected letters are significantly different.

[0194]

Claims

1. A method for producing a baked product, the method comprising adding a mature thermostable variant of a parent glucoamylase having at least 70% identity 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% (baked %) sugar to the dough, and baking the dough.

2. The method according to claim 1, wherein the parent glucoamylase is from a species of the genus Penicillium, preferably from Penicillium oxalicum, Penicillium miekii, Penicillium roqueforti or Penicillium luminosporum.

3. The method of any one 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. The method according to claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 2, 4, 11, 65, 79 and 327 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. The method of claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 79, 103, 132, 445, 447, 481, 566, 568, 594 and 595 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. The method of claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 50, 103, 132, 445, 447, 481, 501, 539, 566, 568, 594 and 595 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. The method of claim 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of the positions corresponding to positions 1, 6, 7, 31, 34, 50, 103, 132, 445, 447, 481, 501, 539, 566, 568, 594 and 595 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. The method according to any one of claims 1 to 3, wherein the at least one amino acid modification comprises a substitution at one or more or all of 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, 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. The method according to any one of claims 1 to 8, wherein the mature thermostable variant has an improvement in thermostability (Td) of at least 5°C, preferably at least 6°C, 7°C or 8°C relative to its parent.

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

11. The method according to any one of claims 1 to 10, comprising further adding one or more additional enzymes 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-maltotetraose hydrolase, dextran Enzymes, beta-glucanase, galactanase, alpha-galactosidase, beta-galactosidase, glucose oxidase, alpha-glucosidase, beta-glucosidase, haloperoxidase, hemicellulolytic enzyme, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinase, peptidoglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, protease, pullulanase, ribonuclease, transglutaminase and xylanase.

12. Use of a mature thermostable variant of a parent glucoamylase having at least 70% identity 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 and less than 150% (baked %) sugar or sucrose is added to the dough and the dough is baked.

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