Baking with thermostable AMG glucosidase variants (EC 3.2. 1.3) and with low or no emulsifier addition

Abstract

The present invention relates to a method of producing a baked product without or with a reduced amount of emulsifier compared to a standard formulation, 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, wherein no emulsifier is added or a reduced amount of emulsifier is added compared to a standard formulation, and baking the dough.

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 baked products without added emulsifiers or with reduced amounts of emulsifiers compared to standard recipes, said method comprising adding mature thermostable variants of certain parent glucoamylases. Background Art

[0004] Several properties of baked goods are important for consumer preference, especially in industrially produced bread. Industrial baked goods rely on the addition of one or more emulsifiers to obtain the correct properties.

[0005] DATEM (diacetyl tartaric acid esters of mono- and diglycerides) is an emulsifier used primarily in baking to strengthen the gluten network in the dough and improve the volume of the baked product. The DATEM consists of a mixed ester of mono- and diglycerides from edible sources, in which one or more hydroxyl groups of the glycerol have been esterified with diacetyl tartaric acid and fatty acids. In most commercial baked dough recipes, DATEM usually accounts for about 0.375% to 0.5% of the total weight of the flour.

[0006] DMG (distilled mono- and diglycerides) is a crumb-softening emulsifier widely used in bread baking to improve softness over time and to delay starch retrogradation. DMG also has an effect on the extensibility of the gluten network.

[0007] SSL (Sodium Stearoyl Lactylate) is another emulsifier derived from the sodium salts of lactic acid and stearic acid. SSL provides several functions:

[0008] - Improve the texture and shelf life of bread and rolls

[0009] - Produces softer and more pliable tortillas

[0010] - Improves baking elasticity by enhancing the gas retention capacity of the dough

[0011] - Gives the loaf greater side wall strength (prevents keyholing) - Gives the bread the desired elastic crumb

[0012] However, there is a growing trend among consumers to avoid baked products that contain emulsifiers or "E numbers" (commonly referred to colloquially by consumers as emulsifiers).

[0013] WO 2022 / 090562 discloses methods for producing baked goods using heat-stable AMG variants that provide many benefits such as extended shelf life, improved softness within the crust, etc.

[0014] And the use of lipolytic enzymes in baking has been known for many years. WO 98 / 26057 discloses lipases / phospholipases from Fusarium oxysporum and their use in baking. WO 2004 / 099400 discloses various lipolytic enzymes and their use in baking. WO 1999 / 053769 discloses the use of maltogenic alpha-amylases and phospholipases for improving the softness of baked products in the initial period after baking. WO 2018 / 150021 also discloses lipolytic enzymes suitable for baking. Summary of the invention

[0015] The inventors have unexpectedly discovered that adding a heat stable AMG variant to a dough and baking the dough to produce a baked product makes it possible to reduce the amount of emulsifier that would normally be included in a dough recipe, or even avoid adding any emulsifier.

[0016] Thus, in a first aspect, the present invention relates to a method for producing a baked product with no added emulsifier or a reduced amount of added emulsifier compared to a standard recipe, 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, wherein no emulsifier is added or a reduced amount of emulsifier is added compared to a standard recipe, and baking the dough.

[0017] In a second aspect, the present 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 producing a baked product with no added emulsifier or a reduced amount of emulsifier compared to a standard recipe, the method comprising adding the thermostable variant of the parent glucoamylase to a dough, wherein no emulsifier is added or a reduced amount of emulsifier is added compared to the standard recipe, and baking the dough.

[0018] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A multiple alignment of the amino acid sequences of the following mature proteins is shown:

[0020] - Wild-type AMG from Penicillium oxalicum (PoAMG) of SEQ ID NO: 1

[0021] - PoAMG variant denoted "AMG NL" of SEQ ID NO: 2

[0022] - A PoAMG variant of SEQ ID NO: 3 denoted "AMG anPAV498"

[0023] - PoAMG variant denoted "AMG JPO001" of SEQ ID NO:4

[0024] - A PoAMG variant of SEQ ID NO:5 designated as "AMG JPO124"

[0025] - A PoAMG variant of SEQ ID NO: 6 denoted "AMG JPO-172"

[0026] - Wild-type AMG from Penicillium miczynskii (PoAMG) of SEQ ID NO: 7

[0027] - Wild-type AMG from Penicillium russellii (PoAMG) of SEQ ID NO: 8

[0028] - Wild-type AMG from Penicillium glabrum (PoAMG) of SEQ ID NO: 9

[0029] Figure 2 Shown is a graph of the average sensory attribute scores for breads evaluated 1 day after baking as shown in Table 7 of Example 7; Note: JPO172 is JPO-172.

[0030] Figure 3 Shown is a graph of the average sensory attribute scores for breads evaluated 1 day after baking as shown in Table 9 of Example 8; Note: JPO172 is JPO-172.

[0031] Figure 4 Shown is a graph of the average sensory attribute scores for breads evaluated 1 day after baking as shown in Table 11 of Example 9; Note: JPO172 is JPO-172. DETAILED DESCRIPTION

[0032] definition

[0033] 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.

[0034] Dough: As used herein, "dough" means any dough used to prepare a baked product bread. The dough used to prepare a baked product 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 it is preferred that 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.

[0035] 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.

[0036] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0037] 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:

[0038] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0039] 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.

[0040] 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.

[0041] 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 (T o The term "peak gelatinization temperature (T p The term "end gelatinization temperature (T c)” is understood as the temperature at which gelatinization is terminated.

[0042] A first aspect of the invention relates to a method for producing a baked product with no added emulsifier or a reduced amount of added emulsifier compared to a standard recipe, 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, wherein no emulsifier is added or a reduced amount of emulsifier is added compared to a standard recipe, and baking the dough.

[0043] 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 producing a baked product with no added emulsifier or with a reduced amount of emulsifier compared to a standard recipe, the method comprising adding the thermostable variant of the parent glucoamylase to a dough, wherein no emulsifier is added or a reduced amount of emulsifier is added compared to the standard recipe, and baking the dough.

[0044] Glucoamylase

[0045] Glucoamylases are also called amyloglucosidases and glucan 1,4-alpha-glucosidases (EC 3.2.1.3), more commonly they are known as AMG.

[0046] 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 the genus Aspergillus, Rhizopus, Rasamsonia or Penicillium; preferably a DNA sequence found in a fungal strain of the genus Penicillium, even more preferably a DNA sequence found in a fungal strain of Penicillium oxysporum, Penicillium oxalicum, Penicillium mikrokeri, Penicillium roqueforti or Penicillium luminosporum. Preferably, the parent glucoamylase is from a species of the genus Penicillium, preferably from Penicillium oxalicum, Penicillium mikrokeri, Penicillium roqueforti or Penicillium luminosporum.

[0047] Examples of other suitable fungi include Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Rhizopus delemar, Rhizopus niveus, Rhizopus oryzae, and Talaromyces emersonii (Rasamsonia emersonii).

[0048] The following shows Figure 1 The % identity between the AMG amino acid sequences aligned in , and are also provided in the sequence listing:

[0049]

[0050] 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.

[0051] 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. ;

[0052] 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.

[0053] 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.

[0054] Preferred embodiments relate to the first or second aspect of the invention, wherein no DMG, SSL and / or DATEM is added or a reduced amount of DMG, SSL and / or DATEM is added compared to a standard formulation.

[0055] Other enzymes

[0056] 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. Preferably, the one or more additional enzymes comprise a mature lipase and / or a phospholipase.

[0057] Lipase

[0058] 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.

[0059] Raw starch degrading α-amylase

[0060] 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.

[0061] 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.

[0062] In one embodiment the raw starch degrading alpha-amylase is a GH13-1 amylase.

[0063] 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 set forth in European Patent No. 2981170 (Novozymes AG).

[0064] Amylase

[0065] 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.

[0066] A 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.

[0067] Protease

[0068] Suitable proteases include microbial proteases, such as fungal proteases and bacterial proteases. Preferred proteases are acidic proteases, i.e., proteases characterized in 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 dough 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 to leave enough high molecular weight proteins to stabilize foam. On the one hand, 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.

[0069] Enzyme composition

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] Examples

[0075] Example 1: Construction of PoAMG library

[0076] The PoAMG library was constructed as follows:

[0077] 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.

[0078] PCR reaction mixture:

[0079] PrimeSTAR Max DNA Polymerase [TaKaRa]

[0080] Total 25 μl

[0081] 1.0μl template DNA (1ng / μl)

[0082] 9.5 μl HO

[0083] 12.5 μl 2x PrimeSTAR Max premix

[0084] 1.0 μl forward primer (5 μM)

[0085] 1.0 μl reverse primer (5 μM)

[0086] PCR procedure:

[0087] 98℃ / 2min

[0088] 25x(98℃ / 10sec, 60℃ / 15sec, 72℃ / 2min)

[0089] 10℃ / maintain

[0090] Example 2: Screening for better thermal stability

[0091] 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.

[0092] pNPG thermal stability assay:

[0093] 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.

[0094] Table 1a. List of relative activities of PoAMG variants compared to their parents anPAV498 or JPO-0001 (anPAV498 with leader / propeptide)

[0095]

[0096]

[0097]

[0098]

[0099] 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％

[0100] 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％

[0101]

[0102]

[0103] name Relative activity at 79℃ / 77℃ (%) 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％

[0104] Table 1b. List of relative activities of PoAMG variants compared to their parent JPO-022

[0105] name Relative activity at 77℃ / 70℃ (%) JPO-022 60％ JPO-027 67％ JPO-042 8％ JPO-044 86％ JPO-045 67％ JPO-046 48％

[0106] 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％

[0107] name Relative activity at 79℃ / 77℃ (%) 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％

[0108] Table 1c. List of relative activities of PoAMG variants compared to their parent JPO-063 at different temperatures

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] Table 1d. List of relative activities of PoAMG variants compared to their parent JPO-096

[0116]

[0117]

[0118] Table 1e. List of relative activities of PoAMG variants compared to their parent JPO-129

[0119]

[0120] Table 1f. List of relative activities of PoAMG variants compared to their parent JPO-166

[0121]

[0122]

[0123] Table 2. Amino acid substitutions in variants of the PoAMG mature sequence

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] Example 3: Fermentation of Aspergillus niger

[0130] 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.

[0131] Example 4: Purification of PoAMG (JPO-001) variants

[0132] 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.

[0133] Example 5: Thermal Stability Assay (TSA)

[0134] 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.

[0135] TSA equipment parameters:

[0136] Instrument: LightCycler 480 Real-Time PCR System (Roche Applied Science)

[0137] Scan rate: 0.02℃ / sec

[0138] Scanning range: 37℃-96℃

[0139] Integration time: 1.0 sec

[0140] Excitation wavelength 465nm

[0141] Emission wavelength 580nm

[0142] 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.

[0143] Example 6: PoAMG activity assay

[0144] Determination of maltodextrin (DE11) by GOD-POD method

[0145] Substrate solution

[0146] 30g maltodextrin (pindex #2 from MATSUTANI chemical industry Co., Ltd.)

[0147] 100 ml 120 mM sodium acetate buffer, pH 5.0

[0148] Glucose CII test kit (Wako Pure Chemical Industries, Ltd., Japan)

[0149] 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.

[0150] Table 3.

[0151]

[0152]

[0153]

[0154] Example 7: DMG elimination using JPO-172 and optionally a phospholipase

[0155] Distilled mono- or diglycerides (DMG) are often added to dough to improve emulsion stability and soften the crumb (Pyler, EJ and Gortonla, LA, 2008, Baking, Science and Technology, Vol. 1, 4th Edition, pp. 437-452, Sosland Publishing Company, Kansas City, MO, ISBN 978-0-9820239-0-7).

[0156] Baking process:

[0157] Bread was baked in a direct fermentation baking process using the recipe according to Table 4. Different treatments were performed according to Table 5, with the addition of a baking lipase (as disclosed in WO 2018 / 150021) or an emulsifier DMG.

[0158] 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 15 minutes and divided into 320 g dough pieces. The dough pieces were rounded, sheeted and placed in a baking pan. The baking pan with the dough was proofed at 32° C. and 86% relative humidity for 58 min. The proofed dough was baked in a box oven at 230° C. for 28 min.

[0159] Table 4. Recipe

[0160]

[0161] Table 5. Treatment

[0162]

[0163] The breads were packaged in sealed plastic bags 2 hours after baking and stored at room temperature until evaluation.

[0164] Sensory evaluation method:

[0165] Each sensory assessor was provided with two slices of each type of bread (Day 1). An initial training session was held prior to the evaluation to define the attributes and procedures (Table 6) and the use of the intensity scale. The samples were provided in a blinded, 3-digit coded and random order. Five trained assessors participated in the evaluation. Two replicate sensory evaluations were performed. The intensity of the sensory attributes was assessed on a 1-9 point scale ranging from weak to very strong.

[0166] Table 6. Description of sensory attributes, procedures and evaluations

[0167]

[0168] result:

[0169] Table 7 and Figure 2 The data in show that monoglycerides increased the following sensory parameters compared to control bread: white crumb, moistness, softness, and softness within the crust. Springiness was reduced. JPO-172 improved moistness, softness, and softness within the crust at least to the extent of monoglycerides, and maintained springiness at least to the level of the control. Addition of JPO-172 and Lip182 to the dough additionally increased white crumb, matching that of distilled monoglycerides.

[0170] Table 7. Average sensory attribute scores for breads evaluated 1 day after baking – Graphical representation of the data in the table is also available at Figure 2 Shown in.

[0171]

[0172] Example 8: DATEM elimination using thermostable AMG and optionally a phospholipase

[0173] Diacetyl tartaric acid mono- and diglycerides (DATEM) are added to dough for emulsion stability, to soften the crumb and to strengthen the dough (Pyler, EJ and Gortonla, LA, 2008, Baking, Science and Technology, Vol. 1, 4th Edition, pp. 437-452, Sosland Publishing Company, Kansas City, MO, ISBN 978-0-9820239-0-7).

[0174] Baking process:

[0175] Bread was baked in a direct fermentation baking process using the recipe according to Table 4. Different treatments were performed according to Table 8. 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 15 minutes and divided into 320 g dough pieces. The dough pieces were rounded, sheeted and placed in a baking pan. The baking pan with the dough was proofed at 32° C. and 86% relative humidity for 65 min. The proofed dough was baked in a box oven at 230° C. for 28 min.

[0176] formula:

[0177] The formulation was the same as described in Example 7, Table 4.

[0178] Table 8. Treatment

[0179]

[0180] The breads were packaged in sealed plastic bags 2 hours after baking and stored at room temperature until evaluation.

[0181] Sensory evaluation method:

[0182] Sensory evaluation was performed as described in Example 7, Table 6.

[0183] result:

[0184] Table 9 and Figure 3 The data in show that DATEM increased white crumb, moistness, tenderness, and softness within the crust compared to the control bread. Springiness was reduced. JPO-172 improved moistness, tenderness, and softness within the crust at least to the extent of DATEM, and maintained springiness at least to the extent of the control. Addition of JPO-172 and Lipopan Xtra additionally increased white crumb, matching DATEM.

[0185] Table 9. Mean sensory attribute scores for breads evaluated 1 day after baking – Graph also shown Figure 3 Available in.

[0186]

[0187] Example 9: SSL elimination using a combination of thermostable AMG and optionally a phospholipase and a lipase

[0188] Sodium stearoyl lactylate (SSL) is added to dough for emulsion stability, to soften the crumb and to strengthen the dough (Pyler, EJ and Gortonla, LA, 2008, Baking, Science and Technology, Vol. 1, 4th Edition, pp. 437-452, Sosland Publishing Company, Kansas City, MO, ISBN 978-0-9820239-0-7).

[0189] Baking process:

[0190] Bread was baked in a direct fermentation baking process using the recipe according to Table 4. Different treatments were performed according to Table 10. 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 15 minutes and divided into 320 g dough pieces. The dough pieces were rounded, sheeted and placed in a baking pan. The baking pan with 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 25 min.

[0191] formula:

[0192] The formulation was the same as described in Example 7, Table 4.

[0193] Table 10. Treatments:

[0194]

[0195] The breads were packaged in sealed plastic bags 2 hours after baking and stored at room temperature until evaluation.

[0196] Sensory evaluation method:

[0197] Sensory evaluation was performed as described in Example 7, Table 6.

[0198] result:

[0199] Table 11 and Figure 4 The data in show that SSL increased white crumb and decreased springiness compared to the control bread. JPO-172 improved moistness, tenderness, and tenderness within the crust at least to the extent of SSL, and maintained springiness at least to the level of the control. Addition of JPO-172, Lipopan Xtra, and Lipopan 50 additionally increased white crumb, matching SSL.

[0200] Table 11. Average sensory attribute scores for breads evaluated 1 day after baking – Graphical representation of the results is also in Figure 4 Available in.

[0201]

[0202]

Claims

1. A method for producing a baked product with no added emulsifier or a reduced amount of added emulsifier compared to a standard recipe, 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, wherein no emulsifier is added or an emulsifier is added in a reduced amount compared to the standard recipe, 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. according to the method described in any one of claims 1-2, wherein the mature thermostable variant 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.

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 process according to any one of claims 1 to 10, wherein no DMG, SSL and / or DATEM is added or a reduced amount of DMG, SSL and / or DATEM is added compared to the standard recipe.

12. The method according to any one of claims 1 to 11, 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.

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

14. 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 with no added emulsifier or with a reduced amount of emulsifier compared to a standard recipe, the method comprising adding the thermostable variant of the parent glucoamylase to dough, wherein no emulsifier is added or with a reduced amount of emulsifier compared to the standard recipe, and baking the dough.

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