Baking at low ph with thermostable glucoamylase variants

Abstract

The present invention relates to a method of producing a baked or partially baked product, said method comprising: a) providing a dough comprising 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, the variant being added in an amount of 0.01-12.40 mg zymoprotein (mgEP) / kg flour wherein said dough has a pH value in the range of 3.0-6.5; and b) baking or partially baking the dough to produce a baked or partially baked product.

Description

[0001] Reference to the Sequence Listing

[0002] This application contains a Sequence Listing in computer-readable form, which is hereby incorporated by reference. Field of the Invention

[0003] The present invention relates to a method for producing baked or partially baked products, the method comprising:

[0004] a) providing a dough comprising a mature thermostable variant of a parental glucoamylase (AMG) having at least 70% identity with SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10, the variant being added in an amount of 0.01 - 12.40 mg enzyme protein (mgEP) / kg flour, wherein the dough has a pH value in the range of 3.0 - 6.5; and

[0005] b) baking or partially baking the dough to produce a baked or partially baked product. Background of the Invention

[0006] Sugared baked products (bread, cookies, etc.) are one of the most popular product categories worldwide. The typical amount of formulated sugar is 1% - 25% of the total weight of the flour.

[0007] However, due to the rising market price of sugar, the insufficient supply of sugar in some parts of the world, and health concerns, there is a need for a method for producing baked products with reduced added sugar content without sacrificing the quality of the baked products and even potentially improving the quality of the baked products.

[0008] WO 2019 / 238423 (Novozymes A / S, Denmark) discloses methods for producing doughs with reduced added sugar content, which methods include adding raw starch-degrading α-amylase and glucoamylase to the dough ingredients.

[0009] WO 2022 / 090562 (Novozymes A / S, Denmark) discloses a method for producing baked or partially baked products with a mature thermostable variant of a parental glucoamylase. Summary of the Invention

[0010] Thermostable glucoamylase variants exhibit greatly improved performance in the preservation or anti-aging of baked or partially baked products. Another improved performance of the thermostable variants is that they increase the sweetness or sweetness of the product, thus reducing the amount of sugar added in traditional formulations. Now, another surprising effect of the thermostable glucoamylase variants is shown herein, which is to reduce the enzyme dosage at a reduced dough pH in the range of 3.0 - 6.5.

[0011] Accordingly, in a first aspect, the present invention relates to a method for producing a baked or partially baked product, the method comprising:

[0012] a) providing a dough comprising a mature thermostable variant of a parental glucoamylase having at least 70% sequence identity with SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10, the variant being added in an amount of 0.01 - 12.40 mg enzyme protein (mgEP) / kg flour, wherein the dough has a pH value in the range of 3.0 - 6.5, preferably in the range of 3.5 - 6.0, even more preferably in the range of 4.0 - 5.5; and

[0013] b) baking or partially baking the dough to produce a baked or partially baked product.

[0014] Preferably, the mature thermostable variant of the parental glucoamylase of the present invention has at least 71% sequence identity with SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10, for example at least 72%, for example at least 73%, for example at least 74%, for example at least 75%, for example at least 76%, for example at least 77%, for example at least 78%, for example at least 79%, for example at least 80%, for example at least 81%, for example 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% sequence identity with SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 shows a multiple alignment of the amino acid sequences of the following mature proteins:

[0016] - wild-type AMG (PoAMG) from Penicillium oxalicum of SEQ ID NO:1

[0017] - a variant of PoAMG designated "AMG NL" of SEQ ID NO:2

[0018] - The PoAMG variant designated "AMG anPAV498" of SEQ ID NO:3

[0019] - The PoAMG variant designated "AMG JPO001" of SEQ ID NO:4

[0020] - The PoAMG variant designated "AMG JPO124" of SEQ ID NO:5

[0021] - The PoAMG variant designated "AMG JPO172" of SEQ ID NO:6

[0022] - The wild - type AMG (PoAMG) from Penicillium miczynskii of SEQ ID NO:7

[0023] - The wild - type AMG (PoAMG) from Penicillium russellii of SEQ ID NO:8

[0024] - The wild - type AMG (PoAMG) from Penicillium glabrum of SEQ ID NO:9

[0025] Figure 2 The pH - activity curve of the thermostable glucoamylase variant designated JPO - 172 is shown. The pH - activity curve was determined at 40 °C. Each data point represents the average of four measurements and the error bars represent the standard deviation. pH 5 was set to 100%. As can be seen in this curve, the optimal pH is around pH 5 and approximately 80% activity is observed from about pH 4 to 6. Detailed Description

[0026] Definitions

[0027] Sequence identity: The degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0028] It should be noted that in the translation of the sentence "pH 5 was set to 100%", there may be some inaccuracies in the original Chinese expression. It is inferred that it may be "pH 5 was set as the reference value of 100%", but the translation is carried out according to the original text.For the purposes of the present invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) is used to determine the sequence identity between two amino acid sequences, which is implemented as in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277, preferably 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 (the EMBOSS version of BLOSUM62) substitution matrix. The output of "longest identity" marked with Needle (obtained using the non-abbreviated (-no brief) option) is used as the percentage of identity and is calculated as follows:

[0029] (Identical residues × 100) / (Alignment length - Total number of gaps in the alignment)

[0030] Variant: The term "variant" means a polypeptide that contains alterations (i.e., substitutions, insertions, and / or deletions) 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. Amino acid alterations can have 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 a methionine residue at the amino terminus; small linker peptides of up to 20 - 25 residues; or small extensions that facilitate purification by altering the net charge or another functionality (such as a polyhistidine segment, an epitope, or a binding domain). Examples of conservative substitutions are within the following groups: 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 R. L. 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.

[0031] Increased strength: The term "increased strength of the dough" is defined herein as a property of the dough that generally has greater elastic properties and / or requires more work input to mold and shape compared to a control.

[0032] Increased elasticity: The term "increased elasticity of the dough" is defined herein as a property of the dough that has a higher tendency to recover its original shape after undergoing a certain physical stress compared to a control.

[0033] Increased stability of the dough: The term "increased stability of the dough" is defined herein as a property of the dough that is less susceptible to mechanical damage compared to a control, thus better maintaining its shape and volume, and is evaluated by the height:width ratio of the cross-section of the bread after normal and / or extended proofing.

[0034] Reduced stickiness of dough: The term "reduced stickiness of dough" is defined herein as a property of dough that, for example, has a lower tendency to adhere to surfaces compared to a control in a dough production machine and is evaluated empirically by a skilled test baker or measured using a texture analyzer known in the art (e.g., TAXT2).

[0035] Improved extensibility: The term "improved extensibility of dough" is defined herein as a property of dough that can withstand increased stress or stretching without breaking compared to a control.

[0036] Improved mechanical ability: The term "improved mechanical ability of dough" is defined herein as a property of dough that generally has less stickiness and / or is firmer and / or more elastic compared to a control.

[0037] Increased volume of baked product: The term "increased volume of baked product" is measured as the volume of a given loaf compared to a control. The volume can be determined using methods known in the art.

[0038] Improved crumb structure of baked product: The term "improved crumb structure of baked product" is defined herein as a property of a baked product that has finer and / or thinner cell walls in the crumb and / or a more uniform cell distribution in the crumb compared to a control, and is generally evaluated visually by a skilled baker or by digital image analysis known in the art (e.g., C-cell, Calibre Control International Ltd, Appleton, Warrington, UK).

[0039] Improved softness of baked product: The term "improved softness of baked product" is the opposite of "firmness" and is defined herein as a property of a baked product that is more easily compressible compared to a control and is evaluated empirically by a skilled test baker or measured, for example, using a texture analyzer known in the art (e.g., TAXT2 or TA-XT Plus from Stable Micro Systems Ltd, Surrey, UK).

[0040] Sensory attributes of baked product: Sensory attributes can be evaluated using procedures well established in the baking industry and can include, for example, using a panel of trained taste testers.

[0041] Improved thermal stability: The improvement in thermal stability (Td) in °C is a measure of how much the variant has improved in thermal stability relative to its parental glucoamylase under the same conditions, as determined as exemplified herein.

[0042] The first aspect of the present invention relates to a method for producing a baked or partially baked product, the method comprising:

[0043] a) providing a dough comprising a mature thermostable variant of a parental 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; and

[0044] b) baking or partially baking the dough to produce a baked or partially baked product.

[0045] Other aspects of the present invention relate to methods for increasing the sweetness of a baked or partially baked product, methods for reducing the amount of sugar in a dough in a method for producing a baked or partially baked product, and / or methods for extending the shelf life of a baked or partially baked product in a method for producing a baked or partially baked product, and in the method as defined in the first aspect, whereby compared to a control made without adding any glucoamylase, the finally fully baked baked or partially baked product, when cooled to room temperature, packaged in a sealed container and stored at room temperature until analysis, has reduced initial firmness and / or increased initial elasticity, and / or has reduced firmness increase and / or higher elasticity after 1, 7 or 14 days.

[0046] Preferably, the mature thermostable variant of the parental glucoamylase of the present invention has at least 71% sequence identity to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10, for example at least 72%, for example at least 73%, for example at least 74%, for example at least 75%, for example at least 76%, for example at least 77%, for example at least 78%, for example at least 79%, for example at least 80%, for example at least 81%, for example 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% sequence identity to SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:10.

[0047] Dough

[0048] As used herein, "dough" means dough for preparing a baked product, particularly bread.

[0049] According to the present invention, the dough for preparing a baked product can be made from any suitable dough ingredients containing flour.

[0050] The flour can be from any baking cereal known in the art, such as wheat flour, corn flour, rye flour, barley flour, oat flour, rice flour, sorghum flour, potato flour, soybean flour, and any combination thereof (for example, a combination of wheat flour and one of the other flour sources; or a combination of rice flour and one of the other flour sources).

[0051] In a preferred embodiment, the flour is wheat flour.

[0052] In a preferred embodiment, at least 10% (w / w) or more of the total flour content is wheat flour, for example, at least 15% or more of the total flour content is wheat flour, for example, at least 20% or more of the total flour content is wheat flour, for example, at least 25% or more of the total flour content is wheat flour, for example, at least 30% or more of the total flour content is wheat flour, for example, at least 35% or more of the total flour content is wheat flour, for example, at least 40% or more of the total flour content is wheat flour, for example, at least 45% or more of the total flour content is wheat flour, for example, at least 50% or more of the total flour content is wheat flour, for example, at least 55% or more of the total flour content is wheat flour, for example, at least 60% or more of the total flour content is wheat flour, for example, at least 65% or more of the total flour content is wheat flour, for example, at least 70% or more of the total flour content is wheat flour, for example, at least 75% or more of the total flour content is wheat flour, for example, at least 80% or more of the total flour content is wheat flour, for example, at least 85% or more of the total flour content is wheat flour, for example, at least 90% or more of the total flour content is wheat flour, for example, at least 95% or more of the total flour content is wheat flour, for example, 100% of the total flour content is wheat flour.

[0053] The dough of the present invention is typically a fermented dough or a dough to be fermented. 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 leavening agents (fermented dough), but preferably the dough is fermented by adding a suitable yeast culture such as a culture of Saccharomyces cerevisiae (baker's yeast) (e.g., a commercially available strain of Saccharomyces cerevisiae).

[0054] The dough of the present invention can typically contain a certain amount of added sugar, because although the amount of added sugar can be reduced according to the method of the present invention, normally only part of the sugar amount can be reduced.

[0055] In one embodiment, the amount of sugar added is at least 10% (w / w) less than the amount of sugar added to the dough in the original formulation. For example, the amount of sugar added is at least 20% (w / w) less than the amount of sugar added to the dough in the original formulation. For example, the amount of sugar added is at least 30% (w / w) less than the amount of sugar added to the dough in the original formulation. For example, the amount of sugar added is at least 40% (w / w) less than the amount of sugar added to the dough in the original formulation. For example, the amount of sugar added is at least 50% (w / w) less than the amount of sugar added to the dough in the original formulation. For example, the amount of sugar added is at least 60% (w / w) less than the amount of sugar added to the dough in the original formulation. For example, the amount of sugar added is at least 70% (w / w) less than the amount of sugar added to the dough in the original formulation. For example, the amount of sugar added is at least 80% (w / w) less than the amount of sugar added to the dough in the original formulation. For example, the amount of sugar added is at least 90% (w / w) less than the amount of sugar added to the dough in the original formulation. For example, the amount of sugar added is 100% (w / w) less than the amount of sugar added to the dough in the original formulation.

[0056] The dough may also contain other conventional dough ingredients, such as proteins, such as milk powder, gluten, and soybeans; eggs (whole eggs, egg yolks, or egg whites); oxidizing agents, such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA), or ammonium persulfate; amino acids, such as L-cysteine; salts, such as sodium chloride, calcium acetate, sodium sulfate, calcium sulfate; diluents (such as silica) and starches from different sources. Other commonly used ingredients also include hydrocolloids, such as CMC, guar gum, xanthan gum, locust bean gum, etc.

[0057] The dough ingredients may typically comprise fats (triglycerides) and / or oils and / or shortenings, particularly oils, such as sunflower oil or rapeseed oil.

[0058] In a preferred embodiment, no emulsifier is added to the dough of the present invention, and preferably no SSL is added to the dough of the present invention.

[0059] In a preferred embodiment, the pH of the dough of the present invention is adjusted to a range of 3.0 - 6.5, preferably a range of 3.5 - 6.0, even more preferably a range of 4.0 - 5.5, and most preferably the pH of the dough is adjusted by adding vinegar to the dough by adding a food-acceptable acid, preferably an organic acid, such as acetic acid or citric acid, to the dough.

[0060] The dough can be prepared by any conventional mixing process, such as a continuous mixing process, a straight-dough process, or a sponge and dough method.

[0061] The present invention is particularly useful for the preparation of dough and bakery products in an industrial process, wherein the dough for preparing bakery products is mechanically prepared using automated or semi-automated equipment.

[0062] The process for preparing bread generally involves the following sequential steps: making dough, sheeting or dividing, shaping or rolling the dough, and proofing, which steps are well known in the art.

[0063] As used herein, "bakery product" means any type of bakery product, including various types of breads such as pan bread, toast bread, open bread, pan bread with and without lids, buns, Fino bread, Hammam bread, Samoli bread, baguettes, brioche, hamburger buns, rolls, rye bread, whole wheat bread, rich bread, bran bread, flat bread, tortillas, cookies and any variety thereof. According to the present invention, the bakery product may also be a cake or any pastry product known in the art.

[0064] Raw starch-degrading α-amylase

[0065] As used herein, "raw starch-degrading α-amylase" refers to an enzyme that can directly degrade raw starch granules below the starch gelatinization temperature.

[0066] Examples of raw starch-degrading α-amylases include those disclosed in WO 2005 / 003311, US Patent Publication No. 2005 / 0054071 and US Patent No. 7,326,548. Examples also include those enzymes disclosed in Tables 1 to 5 of the examples in US Patent No. 7,326,548 and in US Patent Publication No. 2005 / 0054071 (Table 3 on page 15), as well as those disclosed in WO 2004 / 020499, WO 2006 / 06929 and WO 2006 / 066579.

[0067] In one embodiment, the raw starch-degrading α-amylase is a GH13_1 amylase.

[0068] In one embodiment, the raw starch-degrading α-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% sequence identity with the raw starch-degrading α-amylase shown in European Patent No. 2981170 (Novozymes A / S).

[0069] In one embodiment, the raw starch-degrading α-amylase according to the present invention can be added to flour or dough in an amount of 0.01 - 10 mg enzyme protein / kg flour, such as in an amount of 0.1 - 5 mg enzyme protein / kg flour.

[0070] Glucoamylase

[0071] Glucoamylase is also known as amyloglucosidase and glucan 1,4-α-glucosidase (EC 3.2.1.3), and more commonly they are referred to as AMG.

[0072] According to the present invention, different types of glucoamylases can be used as parents for generating thermostable glucoamylase variants. For example, the glucoamylase can be a polypeptide encoded by a DNA sequence found in a fungal strain of the genus Aspergillus, Rhizopus, Talaromyces, or Penicillium; preferably a DNA sequence found in a fungal strain of the genus Penicillium, and even more preferably a DNA sequence found in a fungal strain of Penicillium oxysporum, Penicillium oxalicum, Penicillium meleagrinum, Penicillium roqueforti, or Penicillium glabrum. Preferably, the parent glucoamylase is from a species of the genus Penicillium, preferably from Penicillium oxalicum, Penicillium meleagrinum, Penicillium roqueforti, or Penicillium glabrum.

[0073] Examples of other suitable fungi include Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Rhizopus delemar, Rhizopus niveus, Rhizopus oryzae, and Talaromyces emersonii.

[0074] The following shows Figure 1 the % identity between the AMG amino acid sequences aligned in

[0075]

[0076]

[0077] In one embodiment, the glucoamylase according to the invention can be added to flour or dough in an amount of 0.01 - 1,000 mg enzyme protein (mgEP) / kg flour, preferably 0.01 - 500 mg enzyme protein (mgEP) / kg flour, and even more preferably 0.1 - 100 mg enzyme protein (mgEP) / kg flour.

[0078] Thermostable variants of PoAMG have been generated (see Table 2 below). In a preferred embodiment, the mature thermostable glucoamylase variant of the invention comprises one or more or all combinations of the amino acid substitutions listed in Table 2 below.

[0079] In a preferred embodiment, the mature variant of the present invention 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, 65, 79, 103, 132, 327, 445, 447, 481, 566, 568, 594, and 595 in SEQ ID NO:1; preferably, the at least one amino acid modification comprises substitutions 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 substitutions corresponding to one or more or all of the positions of R1A, P2N, P4S, P11F, T65A, K79V, and Q327F in SEQ ID NO:1; or preferably, the at least one amino acid modification comprises substitutions 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 substitutions corresponding to one or more or all of the positions of 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 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, preferably, the at least one amino acid modification comprises substitutions corresponding to one or more or all of the positions of 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.

[0080] The improvement in thermal stability (Td) of the variants in Table 2 is listed in Table 3, where the Td of the PoAMG variant designated "anPAV498" (parent) is set to zero. In a preferred embodiment, the mature thermostable variant of the present invention has an improvement in thermal stability (Td) of at least 3°C, preferably at least 4°C, 5°C, 6°C, 7°C, or 8°C relative to its parent, preferably as determined as exemplified herein.

[0081] In another preferred embodiment, the mature thermostable variant of the invention has a relative activity of at least 150, preferably at least 200, more preferably at least 250, and most preferably at least 300 at 91 °C compared to its parent.

[0082] Preferably, the mature thermostable variant glucoamylase is included in the dough in an amount of 0.01 - 1,000 mg enzyme protein (mgEP) / kg of flour, preferably in an amount of 0.01 - 500 mg enzyme protein (mgEP) / kg of flour, and even more preferably in an amount of 0.1 - 100 mg enzyme protein (mgEP) / kg of flour.

[0083] Amylase

[0084] α-Amylase (α-1,4-glucan-4-glucanohydrolase, EC. 3.2.1.1) constitutes a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4-glycosidic oligosaccharides and polysaccharides.

[0085] A number of α-amylases are known as Termamyl TM and "Termamyl TM -like α-amylases", and can be learned from, for example, WO90 / 11352, WO 95 / 10603, WO 95 / 26397, WO 96 / 23873, and WO 96 / 23874.

[0086] Another group of α-amylases is known as 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.

[0087] Suitable commercially available α-amylase compositions according to the invention include, for example, BAKEZYME P 300 (available from DSM) and FUNGAMYL 2500SG, FUNGAMYL 4000BG, FUNGAMYL 4000SG, FUNGAMYL800L, FUNGAMYL ULTRA BG, and FUNGAMYL ULTRA SG (available from Novozymes).

[0088] In one embodiment, the α-amylase according to the invention can be added to the flour or dough in an amount of 0.01 - 1,000 mg enzyme protein (mgEP) / kg of flour, preferably in an amount of 0.01 - 500 mg enzyme protein (mgEP) / kg of flour, and even more preferably in an amount of 0.1 - 100 mg enzyme protein (mgEP) / kg of flour.

[0089] Additional enzyme

[0090] Optionally, one or more additional enzymes (such as α - amylase, amylomaltase, β - amylase, aminopeptidase, carboxypeptidase, catalase, cellulolytic enzyme, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, dextran 1,4 - α - maltotetraohydrolase, dextranase, galactanase, α - galactosidase, β - galactosidase, glucose oxidase, α - glucosidase, β - glucosidase, haloperoxidase, hemicellulolytic enzyme, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinolytic enzyme, peptidylglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, and xylanase) can be used in combination with the enzyme composition according to the present invention.

[0091] One or more additional enzymes can be of any origin, including mammalian origin, plant origin, and microbial (bacterial, yeast, or fungal) origin.

[0092] Amylomaltase (EC 3.2.1.133) can be from the genus Bacillus. Amylomaltase from Bacillus stearothermophilus strain NCIB 11837 is commercially available from Novozymes under the trade name as follows.

[0093] The amylomaltase can also be a variant of the amylomaltase from Bacillus stearothermophilus, such as those disclosed in, for example, WO 99 / 43794, WO 2006 / 032281, or WO 2008 / 148845, such as 3D.

[0094] The anti - aging amylase for use in the present invention can also be an amylase (dextran 1,4 - α - maltotetraohydrolase (EC 3.2.1.60)) from Pseudomonas saccharophilia or a variant thereof, such as any amylase disclosed in WO 99 / 50399, WO 2004 / 111217, or WO 2005 / 003339.

[0095] Glucose oxidase can be fungal glucose oxidase, especially glucose oxidase from Aspergillus niger (such as commercially available from Novozymes).

[0096] The xylanase can be of microbial origin, such as strains derived from bacteria or fungi such as Aspergillus spp. (in particular Aspergillus aculeatus, Aspergillus niger, Aspergillus awamori or Aspergillus tubigensis), strains derived from Trichoderma (e.g., Trichoderma reesei), or strains derived from Humicola (e.g., Humicola insolens).

[0097] Suitable commercially available xylanase preparations for use in the present invention include PANZEA BG, PENTOPAN MONO BG and PENTOPAN 500BG (available from Novozymes), GRINDAMYL POWERBAKE (available from Danisco), and BAKEZYME BXP 5000 and BAKEZYME BXP 5001 (available from DSM).

[0098] The protease can be from the genus Bacillus, such as Bacillus amyloliquefaciens. Suitable proteases can be those available from Novozymes

[0099] The phospholipase can have phospholipase A1, A2, B, C, D or lysophospholipase activity; it may or may not have lipase activity. It can be of animal origin, such as from pancreas, snake venom or bee venom, or it can be of microbial origin, such as from filamentous fungi, yeast or bacteria, such as Aspergillus or Fusarium, e.g., Aspergillus niger, Aspergillus oryzae or Fusarium oxysporum. Preferred lipase / phospholipases from Fusarium oxysporum are disclosed in WO 98 / 26057. Also, variants described in WO 00 / 32758 can be used.

[0100] Suitable phospholipase compositions are LIPOPAN F, LIPOPAN XTRA and LIPOPAN MAX (available from Novozymes) or PANAMORE GOLDEN and PANAMORE SPRING (available from DSM).

[0101] Preferably, one or more additional enzymes are added in an amount of 0.01 - 1,000 mg enzyme protein (mgEP) / kg flour, preferably 0.01 - 500 mg enzyme protein (mgEP) / kg flour, and even more preferably 0.1 - 100 mg enzyme protein (mgEP) / kg flour.

[0102] Enzyme composition

[0103] The mature thermostable variant glucoamylase of the present invention and any one or more additional enzymes can be added to flour or dough in any suitable form, such as, for example, in liquid (especially stabilized liquid) form, or they can be added to flour or dough as a substantially dry powder or granule.

[0104] Granules can be produced, for example, as disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452. Liquid enzyme preparations can be stabilized, for example, by adding sugars or sugar alcohols or lactic acid according to established procedures. Other enzyme stabilizers are well known in the art.

[0105] One or more enzymes can be added to bread dough ingredients in any suitable manner, such as added as separate components (adding the enzymes separately or sequentially), or adding these enzymes together in one step or in one composition.

[0106] Bread characteristics

[0107] The sensory quality or sensory attributes of bread can be measured as known in the art. The characteristics of bread can be referred to herein as sensory attributes, which include anti-staling (crumb firmness / hardness), crumb characteristics and mouthfeel, or more precisely, the attributes of bread detected in the mouth during eating (e.g., bread softness / resistance to the first bite, crumb moisture, crumb chewiness and adhesiveness, and crumb smoothness and melting characteristics).

[0108] In one embodiment, the sensory attribute obtained for the baked product by using the enzyme solution according to the present invention is increased sweetness.

[0109] In one embodiment, the sensory attribute obtained for the baked product by using the enzyme solution according to the present invention is increased crumb sweetness.

[0110] In a preferred embodiment of the present invention, compared to a control made without adding any glucoamylase, the fully baked or partially baked product after final baking, when cooled to room temperature, packaged in a sealed container and stored at room temperature until analysis, has reduced initial firmness and / or increased initial elasticity, and / or has reduced firmness increase and / or higher elasticity after 1, 7 or 14 days.

[0111] In another preferred embodiment, the fully baked or partially baked product after final full baking has at least the same sweetness or sweetness as a control product made with twice the amount of mature glucoamylase, the amino acid sequence of which is shown in SEQ ID NO: 10, preferably as determined as exemplified herein; preferably, the fully baked or partially baked product after final full baking has a higher sweetness or higher sweetness than a control product made with twice the amount of mature glucoamylase, the amino acid sequence of which is shown in SEQ ID NO: 10, preferably as determined as exemplified herein.

[0112] The inventions described and claimed herein are not limited to the scope of the specific embodiments disclosed herein, as these embodiments are intended to be illustrative of several aspects of the invention. Any equivalent embodiments, together with combinations of one or more of these embodiments, are intended to be included within the scope of the invention.

[0113] Multiple references are cited herein, the disclosures of which are incorporated by reference in their entirety. The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0114] Examples

[0115] Example 1: Construction of the PoAMG Library

[0116] The PoAMG library was constructed as follows:

[0117] Forward or reverse primers were designed to have NNK or one or more desired mutations at one or more target sites, which had 15 bp overlaps with each other. Reverse PCR was performed using appropriate template plasmid DNA (e.g., plasmid DNA containing the JPO-0001 gene) under the following conditions, i.e., the entire plasmid DNA sequence was amplified by a reverse-directed primer. The resulting PCR fragment was purified by the QIAquick Gel Extraction Kit [Qiagen], and then introduced into Escherichia coli ECOS competent Escherichia coli DH5α [NIPPON GENE CO., LTD.]. Plasmid DNA was extracted from the Escherichia coli transformants by the MagExtractor Plasmid Extraction Kit [TOYOBO], and then introduced into Aspergillus niger competent cells.

[0118] PCR reaction mixture:

[0119] PrimeSTAR Max DNA Polymerase [TaKaRa]

[0120]

[0121] PCR program:

[0122] 98 °C / 2 min

[0123] 25 x (98 °C / 10 sec, 60 °C / 15 sec, 72 °C / 2 min)

[0124] 10 °C / hold

[0125] Example 2: Screening for better thermal stability

[0126] The Bacillus subtilis library constructed as in Example 1 was fermented in 96-well or 24-well MTPs 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 1M acetamide) at 32 °C for 3 days. Then, the AMG activity in the culture supernatant was measured by the pNPG assay described below at several temperatures.

[0127] pNPG thermal stability measurement:

[0128] The culture supernatant containing the desired enzyme was mixed with an equal volume of 200 mM NaOAc buffer at pH 5.0. Twenty microliters of this mixture was dispensed into 96-well plates or 8-strip PCR tubes and then heated in a thermal cycler at various temperatures for 30 min. Those samples were mixed with 10 μl of a substrate solution containing 0.1% (w / v) pNPG [Wako Pure Chemical Industries, Ltd.] in 200 mM NaOAc buffer at pH 5.0 and incubated at 70 °C for 20 min for the enzyme reaction. After the reaction, 60 μl of 0.1 M Borax buffer was added to stop the reaction. Eighty microliters of the reaction supernatant was taken out and its OD 405 value was read by a photometer to evaluate the enzyme activity.

[0129] Table 1a. List of relative activities of PoAMG variants compared to their parental anPAV498 or JPO-0001 (anPAV498 with leader peptide / propeptide)

[0130] Name Relative activity (%) at 80°C / 75°C anPAV498 17％ JPO-004 32％ JPO-005 15％ JPO-006 16％ JPO-007 3％

[0131]

[0132]

[0133] Name Relative activity (%) at 80°C / 70°C JPO-001 10％ JPO-004 29％ JPO-009 13％ JPO-014 21％ JPO-020 16％ JPO-021 30％ JPO-052 33％

[0134] Name Relative activity (%) at 79°C / 70°C JPO-001 23％ JPO-021 46％ JPO-022 39％ JPO-023 44％ JPO-025 51％ JPO-027 49％ JPO-029 37％

[0135]

[0136]

[0137] Name Relative activity (%) at 79°C / 77°C JPO-001 36％ JPO-029 51％ JPO-047 45％ JPO-048 81％ JPO-049 53％ JPO-050 58％ JPO-064 65％

[0138] Name Relative activity (%) at 79°C / 77°C JPO-001 41％ JPO-021 60％ JPO-022 48％ JPO-023 57％ JPO-025 56％ JPO-027 64％ JPO-029 66％ JPO-047 50％ JPO-048 72％ JPO-051 82％ JPO-058 73％ JPO-062 72％ JPO-063 85％ JPO-064 83％

[0139] Table 1b. List of relative activities of PoAMG variants compared to their parent JPO-022

[0140]

[0141]

[0142] Name Relative activity (%) at 77°C / 70°C 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％

[0143] Name Relative activity (%) at 79°C / 77°C JPO-022 49％ JPO-023 51％ JPO-025 52％ JPO-027 58％ JPO-058 69％ JPO-059 36％ JPO-060 41％ JPO-061 44％ JPO-062 57％

[0144] Table 1c. List of relative activities of PoAMG variants compared to their parent JPO-063

[0145] Name Relative activity (%) at 79°C / 77°C 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％

[0146] Name Relative activity (%) at 84°C / 80°C 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％

[0147]

[0148]

[0149] Name Relative activity (%) at 83°C / 80°C 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％

[0150] Table 1d. List of relative activities of PoAMG variants compared to their parent JPO-096

[0151] Name Relative activity (%) at 83°C / 70°C JPO-082 53％ JPO-088 70％ JPO-091 69％ JPO-092 65％ JPO-093 62％ JPO-094 74％ JPO-095 69％ JPO-096 67％ JPO-097 65％ JPO-098 65％

[0152] Name Relative activity (%) at 83°C / 80°C JPO-051 20％ JPO-096 43％ JPO-109 51％ JPO-126 33％ JPO-129 48％ JPO-130 18％ JPO-131 51％ JPO-132 34％

[0153] Table 1e. List of relative activities of PoAMG variants compared to their parent JPO-129

[0154] Name Relative activity (%) at 84°C / 80°C JPO-129 62％ JPO-156 51％ JPO-160 34％ JPO-161 41％ JPO-162 49％ JPO-163 21％ JPO-164 57％ JPO-165 77％

[0155] Table 1f. List of relative activities of PoAMG variants compared to their parent JPO-166

[0156] Name Relative activity (%) at 84°C / 75°C JPO-166 19％ JPO-167 66％ JPO-168 58％ JPO-169 53％ JPO-171 47％ JPO-172 98％

[0157] Table 2. Amino acid substitutions in variants of the mature sequence of PoAMG

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164] Example 3: Fermentation of Aspergillus niger

[0165] The Aspergillus niger strain was fermented in 500 ml baffled flasks on a rotary shaker at 220 rpm and 30 °C. These flasks contained 100 ml of MU1 and 4 ml of 50% urea. The culture broth was centrifuged (10,000 x g, 20 min) and the supernatant was carefully decanted from the pellet.

[0166] Example 4: Purification of the PoAMG (JPO - 001) variant

[0167] The PoAMG variant was purified by cation exchange chromatography. The respective peak fractions were combined separately and dialyzed against 20 mM sodium acetate buffer (pH 5.0), and then the samples were concentrated using a centrifugal filtration device (Vivaspin Turbo 15, Sartorius). The enzyme concentration was determined by the A280 value.

[0168] Example 5: Thermal stability assay (TSA)

[0169] 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 multi - well plate 384 (Roche Diagnostics) and the plate was sealed. For TSA Equipment parameters:

[0170] Instrument: LightCycler 480 real - time PCR system (Roche Applied Science)

[0171] Scanning rate: 0.02 °C / sec

[0172] Scanning range: 37 °C - 96 °C

[0173] Integration time: 1.0 sec

[0174] Excitation wavelength: 465 nm

[0175] Emission wavelength: 580 nm

[0176] 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 improvement in thermal stability is listed in Table 3, where the Td of the PoAMG variant designated as anPAV498 was 0.

[0177] Example 6: PoAMG Activity Assay

[0178] Determination of maltodextrin (DE11) by the GOD-POD method

[0179] Substrate solution

[0180] 30 g of maltodextrin (pindex #2 from MATSUTANI chemical industry Co., Ltd.)

[0181] 100 ml of 120 mM sodium acetate buffer, pH 5.0

[0182] Glucose CII test kit (Wako Pure Chemical Industries, Ltd.)

[0183] 20 μl of the enzyme sample was mixed with 100 μl of the substrate solution and incubated at the set temperature for 2 hours. The sample was cooled on an aluminum block for 3 min, 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 working solution of the test kit and then left standing at room temperature for 15 min. The absorbance was read at A505. The activity is listed in Table 3 as the relative activity of the PoAMG variant designated as anPAV498.

[0184] Table 3.

[0185]

[0186]

[0187]

[0188]

[0189] Example 7: Baking with a Reduced Dose of JPO-172

[0190] Bake the bread using the formulation according to Table 4 with a direct fermentation baking process. Conduct different treatments according to Table 5, where the performance of JPO - 172 is compared with that of the currently best - performing anti - aging commercial baking maltogenic α - amylase product 3D (Novozymes A / S, Denmark). Bake the bread in a covered baking pan. Mix the ingredients in a planetary mixer at the first speed for 1 min to form a dough, and then mix at the second speed until the dough is fully formed. Let the dough rest for 5 minutes, divide it into 645 g dough pieces, and round them. Let the round dough pieces rest for 10 min, roll them out, and place them in the baking pans. Let the baking pans with the dough proof at 104 - 109°F and 85% relative humidity until the dough reaches a certain height. For a covered pan, let the dough proof until the dough is 3 / 4” from the top. Bake the proofed dough in a rotary oven at about 227°C (440°F) for 17 minutes.

[0191] Table 4. Formulation

[0192]

[0193]

[0194] Table 5. Treatments

[0195]

[0196] Pack the bread in a sealed plastic bag 2 hours after baking and store it at room temperature until analysis.

[0197] Evaluate the texture of the bread using a texture analyzer (TA - XT plus, Stable Micro Systems, Godalming, UK). The crumb texture characteristics are characterized by the firmness (the 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 carried out using a 40 mm diameter cylindrical probe. When the cylindrical probe is pressed down on a 25 mm thick bread slice at a deformation speed of 1 mm / sec with 27% stress, record the force on the cylindrical probe. Then, hold the probe in this position for 30 seconds while recording the force, and then the probe returns to its initial position.

[0198] Firmness (in grams) is defined as the force required to compress the probe to 25% stress (corresponding to compressing 6.25 mm into a bread crumb slice with a thickness of 25 mm).

[0199] Elasticity (in %) is defined as the force recorded after compressing for 30 seconds at 27% stress (corresponding to the force at time = 36.75 s for a bread slice with a thickness of 25 mm) divided by the force required to press the probe 6.75 mm into the crumb (corresponding to the force at time = 6.75 s for a bread slice with a thickness of 25 mm), multiplied by 100.

[0200] The results of the texture analysis are shown in Tables 6 and 7. Fresh bread is soft (low firmness) and elastic. When bread is stored, it becomes harder and less elastic. The higher the dose of JPO-172, the lower the firmness and the higher the elasticity of the bread on days 7 - 21. However, under these conditions and at these doses, the softest and most elastic bread during the 1 - 21 day period is the bread with 3D 750 MANU / kg of flour.

[0201] Table 6. Firmness (g) of bread crumb at different time points.

[0202]

[0203] Table 7. Elasticity (%) of bread crumb at different time points.

[0204]

[0205] Example 8: Baking with a reduced dose of JPO-172 at a lower pH

[0206] Bread was baked using a direct fermentation baking process according to the formulation in Table 8. Different treatments were carried out according to Table 9. The bread was baked in a covered baking pan. The ingredients were mixed in a planetary mixer at the first speed for 1 min to form a dough, and then at the second speed until the dough was fully formed. The dough was left to rest for 5 minutes, divided into 645 g dough pieces and rounded. The round dough pieces were left to rest for 10 min, sheeted, and placed in the baking pans. The baking pans containing the dough were proofed at 104 - 109°F and 85% relative humidity until the dough reached a certain height. For a covered pan, the dough was proofed until the dough was 3 / 4” from the top. The proofed dough was baked in a rotary oven at approximately 227°C (440°F) for 17 minutes.

[0207] Table 8. Formulation

[0208]

[0209]

[0210] Table 9. Treatments

[0211]

[0212] The bread was packaged in a sealed plastic bag 2 hours after baking and stored at room temperature until analysis.

[0213] Texture analysis was performed as in Example 7. The results of the texture analysis are shown in Tables 10 and 11. Fresh bread is soft (low firmness) and elastic. When bread is stored, in the absence of anti-staling enzymes, it becomes harder and less elastic.

[0214] We conclude that adding 2% vinegar, thus lowering the pH of the dough, allows the dose of JPO-172 to be reduced to below 12 mg EP / kg flour while still providing an anti-staling effect equivalent to or better than 3D.

[0215] Table 10. Firmness (g) of bread crumb at different time points.

[0216]

[0217] Table 11. Elasticity (%) of bread crumb at different time points.

[0218]

[0219]

[0220] Example 9. Activity of JPO-172 as a function of pH

[0221] The pH-activity curve is a characteristic property of an enzyme and is important for its use in different applications. The pH curve of JPO-172 was determined at 40 °C and 30-minute incubation (in the range of pH 2 - 10).

[0222] Prepare a 30 mM maltose (CAS No.: 6363-53-7) substrate solution in buffer (0.1 M acetic acid; 0.1 M MES; 0.1 M HEPES; 0.1 M glycine) and adjust to pH 2 - 10 using HCl or NaOH. Prepare the pH activity curve by adding 15 μL of the diluted enzyme sample (10 ppm, diluted in 20 mM MES, pH 5) or buffer to 135 μL of the substrate solution in an Eppendorf tube. Measure the pH of the reaction mixture. Incubate the mixture at 40 °C and 800 rpm for 30 minutes. After 30 minutes, terminate the reaction by adding 16 μL of 0.5 M NaOH and place on ice. Dilute the reaction mixture 10-fold with 20 mM MES (pH 5) in a microtiter plate. Mix 40 μL of the diluted solution with 160 μL of the GOD-POD reaction mixture in a new microtiter plate and incubate for 30 minutes in the absence of light. After incubation, measure the absorbance at 420 nm. Subtract the average value of the blank sample from the average value of the reaction mixture, as Figure 2 shown.

[0223] The pH-activity curve is shown in Figure 2 . All data points are the average of four measurements and are given relative to pH 5, which is set to 100%. It can be seen from this curve that the optimal pH is around pH 5 and approximately 80% activity is observed from about pH 4 to 6.

Claims

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

2. The method according to claim 1, wherein the baked or partially baked product is a type of bread, preferably molded bread, toast bread, open - face bread, bun, fino bread, hammam bread, samori bread, baguette, brioche, hamburger bun, roll, rye bread, whole - wheat bread, high - fat and high - sugar bread, bran bread, flatbread, tortilla, or a cookie, cake or pastry.

3. The method according to claim 1 or 2, wherein the parental glucoamylase is from a species of Penicillium, preferably from Penicillium oxalicum, Penicillium meleagrinum, Penicillium roqueforti or Penicillium glabrum.

4. The method according to any one of claims 1 - 3, 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, 65, 79, 103, 132, 327, 445, 447, 481, 566, 568, 594 and 595 in SEQ ID NO:

1.

5. The method according to claim 4, wherein the at least one amino acid modification comprises substitutions 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 substitutions at one or more or all of the positions corresponding to R1A, P2N, P4S, P11F, T65A, K79V and Q327F in SEQ ID NO:

1.

6. The method according to claim 4, wherein the at least one amino acid modification comprises substitutions 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 substitutions 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.

7. The method according to any one of claims 1-3, 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.

8. The method according to any one of claims 1-7, wherein the mature thermostable variant has an improvement in thermal stability (Td) of at least 3 °C, preferably at least 4 °C, 5 °C, 6 °C, 7 °C or 8 °C relative to its parent.

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

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

11. The method according to any one of claims 1-10, wherein the baked or partially baked product after final full baking has a reduced initial firmness and / or increased initial elasticity, and / or has a reduced firmness increase and / or higher elasticity after 1, 7 or 14 days, compared to a control made without adding any glucoamylase, when cooled to room temperature, packaged in a sealed container and stored at room temperature until analysis.

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

10.

13. The method according to any one of claims 1-12, wherein the dough further comprises one or more additional enzymes selected from the group consisting of: α-amylase, amylomaltase, raw starch-degrading α-amylase, β-amylase, aminopeptidase, carboxypeptidase, catalase, cellulolytic enzyme, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, glucan 1,4-α-maltotetraohydrolase, glucanase, galactanase, α-galactosidase, β-galactosidase, glucose oxidase, ɑ-glucosidase, β-glucosidase, haloperoxidase, hemicellulolytic enzyme, invertase, laccase, lipase, mannanase, mannosidase, oxidase, pectinolytic enzyme, peptidylglutaminase, peroxidase, phospholipase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, and xylanase; preferably, the one or more additional enzymes are included in an amount of 01-1,000 mg enzyme protein (mgEP) / kg of flour, preferably 0.01-500 mg enzyme protein (mgEP) / kg of flour, and even more preferably 0.1-100 mg enzyme protein (mgEP) / kg of flour.

Images